Science.gov

Sample records for air brazing rab

  1. Diffusion barriers in modified air brazes

    DOEpatents

    Weil, Kenneth Scott; Hardy, John S; Kim, Jin Yong; Choi, Jung-Pyung

    2013-04-23

    A method for joining two ceramic parts, or a ceramic part and a metal part, and the joint formed thereby. The method provides two or more parts, a braze consisting of a mixture of copper oxide and silver, a diffusion barrier, and then heats the braze for a time and at a temperature sufficient to form the braze into a bond holding the two or more parts together. The diffusion barrier is an oxidizable metal that forms either a homogeneous component of the braze, a heterogeneous component of the braze, a separate layer bordering the braze, or combinations thereof. The oxidizable metal is selected from the group Al, Mg, Cr, Si, Ni, Co, Mn, Ti, Zr, Hf, Pt, Pd, Au, lanthanides, and combinations thereof.

  2. Diffusion barriers in modified air brazes

    DOEpatents

    Weil, Kenneth Scott; Hardy, John S.; Kim, Jin Yong; Choi, Jung-Pyung

    2010-04-06

    A method for joining two ceramic parts, or a ceramic part and a metal part, and the joint formed thereby. The method provides two or more parts, a braze consisting of a mixture of copper oxide and silver, a diffusion barrier, and then heats the braze for a time and at a temperature sufficient to form the braze into a bond holding the two or more parts together. The diffusion barrier is an oxidizable metal that forms either a homogeneous component of the braze, a heterogeneous component of the braze, a separate layer bordering the braze, or combinations thereof. The oxidizable metal is selected from the group Al, Mg, Cr, Si, Ni, Co, Mn, Ti, Zr, Hf, Pt, Pd, Au, lanthanides, and combinations thereof.

  3. Microstructural Development and Mechanical Properties for Reactive Air Brazing of ZTA to Ni Alloys using Ag-CuO Braze Alloys

    SciTech Connect

    Prevost, Erica; DeMarco, A.Joseph; MacMichael, Beth; Joshi, Vineet V.; Meier, Alan; Hoffman, John W.; Walker, William J.

    2014-12-01

    Reactive air brazing (RAB) is a potential joining technique to join metal alloys to ceramics for a variety of applications. In the current study, nickel (Ni) alloys were heat treated to form an oxide layer prior to RAB joining to zirconia toughened alumina (ZTA). The Ni alloys evaluated were Nicrofer 6025 HT, Inconel 600, Inconel 693, Haynes 214 and Inconel 601. The ZTA studied had compositions of 0 to 15 wt% zirconia and 0 to 14 wt% glass. Four point-bend tests were performed to evaluate the joint strength of ZTA/ZTA and ZTA/nickel alloys brazed with Ag-2wt% CuO braze alloys. It was determined that the joint strength is not a function of the ZTA composition, but that the strength is a strong function of the chemistry and microstructure of the oxide layer formed on the nickel alloy. It was determined that an increase in the aluminum content of the Ni alloy resulted in an increase of the thickness of alumina in the oxide layer and was directly proportional to the bond strength with the exception of Inconel 601 which exhibited relatively high joint strengths even though it had a relatively low aluminum content.

  4. Brazing.

    SciTech Connect

    Cadden, Charles H.

    2005-04-01

    The use of a lower-melting-point molten metal to join metallic components is perhaps the earliest example of processing which employs metallurgical bonding principles, having roots as far back as 4200 BC (Peaslee 2003). More than 6000 years later, brazing occupies a prominent position in our suite of joining processes where it offers cost and/or performance advantages in the fabrication of many structures. More precisely, brazing can be described as the use of a molten filler metal to wet the closely fitting faying surfaces of a joint, leading to formation of metallurgical bonds between the filler metal and substrates. Historically, brazing processes employ filler metals whose solidus temperature exceeds 723 K, as opposed to soldering processes which use lower-melting-point temperature filler materials. In the past several decades, technological advances have facilitated a broadening of applications for brazing while simultaneously contradicting some of the traditional perceptions of the process. However, many of those tenets remain appropriate for the majority of brazing processes and products. Accordingly, this article provides a brief description of traditional brazing and some important factors to be considered when designing and producing brazed structures. An additional section describes the technical advances in the field.

  5. The Effect of Braze Interlayer Thickness on the Mechanical Strength of Alumina Brazed with Ag-CuO Braze Alloys

    SciTech Connect

    Erskine, Kevin M.; Meier, Alan; Joshi, Vineet V.; Pilgrim, Steven M.

    2014-12-01

    The effect of braze interlayer thickness on the strength of alumina brazed with silver-copper oxide reactive air braze (RAB) alloys was evaluated using a four point bend test configuration. The brazed samples had an average fracture strength of 180 MPa or approximately 60 percent of the average monolithic alumina strength. The joint strength values obtained exceeded the yield strength and ultimate tensile strength of the silver interlayer indicating strong ceramic to metal adhesion and the development of a triaxial stress state in the braze interlayer. The average fracture strength was relatively constant (190 ± 60 MPa) in the thickness range of 0.030 mm to 0.230 mm for all test conditions. The braze fracture strength then decreased down to 100 ± 30 MPa as the braze thickness increased from 0.230 mm to 0.430 mm indicating a loss of triaxial constraint with increasing interlayer thickness. In addition, four different fracture modes were observed.

  6. INVESTIGATION OF A NOVEL AIR BRAZING COMPOSITION FOR HIGH-TEMPERATURE, OXIDATION-RESISTANT CERAMIC JOINING

    SciTech Connect

    Weil, K. Scott; Hardy, John S.; Darsell, Jens T.

    2004-01-30

    One of the challenges in developing a useful ceramic joining technique is in producing a joint that offers good strength under high temperature and highly oxidizing operating conditions. Unfortunately many of the commercially available active metal ceramic brazing alloys exhibit oxidation behaviors which are unacceptable for use in a high temperature application. We have developed a new approach to ceramic brazing, referred to as air brazing, that employs an oxide wetting agent dissolved in a molten noble metal solvent, in this case CuO in Ag, such that acceptable wetting behavior occurs on a number of ceramic substrates. In an effort to explore how to increase the operating temperature of this type of braze, we have investigated the effect of ternary palladium additions on the wetting characteristics of our standard Ag-CuO air braze composition

  7. Effects of two ergonomic improvements in brazing coils of air-handler units.

    PubMed

    Loo, H S; Yeow, Paul H P

    2015-11-01

    The research aims to address the physically loading task and quality and productivity problems in the brazing of coils of air-handler units. Eight operators participated in two intervention studies conducted in a factory in Malaysia to compare the status quo brazing with (1) the use of a new twin-brazing torch that replaced the single-brazing gun and (2) brazing in a sitting position. The outcome measures are related to quality, productivity, monetary costs, body postures and symptoms. After baseline, Interventions I and II were applied for 3 months respectively. The results show a 58.9% quality improvement, 140% productivity increase and 113 times ROI. There was also a reduction in poor work postures e.g. in the raising of the arms and shoulders; bending, twisting and extending of the neck; and bending of left and right wrists, and the back. This research can be replicated in other factories that share similar processes.

  8. Effects of two ergonomic improvements in brazing coils of air-handler units.

    PubMed

    Loo, H S; Yeow, Paul H P

    2015-11-01

    The research aims to address the physically loading task and quality and productivity problems in the brazing of coils of air-handler units. Eight operators participated in two intervention studies conducted in a factory in Malaysia to compare the status quo brazing with (1) the use of a new twin-brazing torch that replaced the single-brazing gun and (2) brazing in a sitting position. The outcome measures are related to quality, productivity, monetary costs, body postures and symptoms. After baseline, Interventions I and II were applied for 3 months respectively. The results show a 58.9% quality improvement, 140% productivity increase and 113 times ROI. There was also a reduction in poor work postures e.g. in the raising of the arms and shoulders; bending, twisting and extending of the neck; and bending of left and right wrists, and the back. This research can be replicated in other factories that share similar processes. PMID:26154237

  9. WETTING AND REACTIVE AIR BRAZING OF BSCF FOR OXYGEN SEPARATION DEVICES

    SciTech Connect

    LaDouceur, Richard M.; Meier, Alan; Joshi, Vineet V.

    2014-10-13

    Reactive air brazes Ag-CuO and Ag-V2O5 were evaluated for brazing Ba0.5Sr0.5Co0.8Fe0.2O(3-δ) (BSCF). BSCF has been determined in previous work to have the highest potential mixed ionic/electronic conducting (MIEC) ceramic material based on the design and oxygen flux requirements of an oxy-fuel plant such as an integrated gasification combined cycle (IGCC) used to facilitate high-efficiency carbon capture. Apparent contact angles were observed for Ag-CuO and Ag-V2O5 mixtures at 1000 °C for isothermal hold times of 0, 10, 30, and 60 minutes. Wetting apparent contact angles (θ<90°) were obtained for 1%, 2%, and 5% Ag-CuO and Ag-V2O5 mixtures, with the apparent contact angles between 74° and 78° for all compositions and furnace dwell times. Preliminary microstructural analysis indicates that two different interfacial reactions are occurring: Ag-CuO interfacial microstructures revealed the same dissolution of copper oxide into the BSCF matrix to form copper-cobalt-oxygen rich dissolution products along the BSCF grain boundaries and Ag-V2O5 interfacial microstructures revealed the infiltration and replacement of cobalt and iron with vanadium and silver filling pores in the BSCF microstructure. The Ag-V2O5 interfacial reaction product layer was measured to be significantly thinner than the Ag-CuO reaction product layer. Using a fully articulated four point flexural bend test fixture, the flexural fracture strength for BSCF was determined to be 95 ± 33 MPa. The fracture strength will be used to ascertain the success of the reactive air braze alloys. Based on these results, brazes were fabricated and mechanically tested to begin to optimize the brazing parameters for this system. Ag-2.5% CuO braze alloy with a 2.5 minute thermal cycle achieved a hermetic seal with a joint flexural strength of 34 ± 15 MPa and Ag-1% V2O5 with a 30 minute thermal cycle had a joint flexural strength of 20 ± 15 MPa.

  10. Air Brazing: A New Method of Ceramic-Ceramic and Ceramic-Metal Joining

    SciTech Connect

    Weil, K. Scott; Darsell, Jens T.; Kim, Jin Yong

    2011-10-01

    A new method of ceramic-ceramic and ceramic-metal joining has emerged over the past several years. Referred to as air brazing, the technique was originally designed and developed for use in fabricating high-temperature solid-state electrochemical devices such as planar SOFCs and oxygen and hydrogen concentrators. The primary advantage of air brazing is that a predominantly metallic joint can be formed directly in air without need of an inert cover gas or the use of surface reactive fluxes. The resulting bond is hermetic, offers excellent room temperature strength, and is inherently resistant to oxidation at high temperature. The key to developing a successful filler metal composition for air brazing is to identify a metal oxide wetting agent that is mutually soluble in a molten noble metal solvent. One particular oxide-metal combination that appears readily suited for this purpose is CuOx-Ag, a system originally of interest in the development of silver clad cuprate-based superconductors. Studies of the equilibrium phases studies in this system indicate that there are two invariant points in the pseudobinary CuOx-Ag phase diagram around which new braze compositions can be developed: 1) a monotectic reaction at 969±1°C, where CuO and a Ag-rich liquid L1 coexist with a second CuOx-rich liquid phase L2 at a composition of xAg/(xAg + xCu) = 0.10±0.03 Ag and 2) a eutectic reaction at 942±1°C, where CuO and Ag coexist with L1 at a composition of xAg/(xAg + xCu) = 0.99±0.005. Specifically, near-eutectic Ag-CuO filler metal compositions have shown good promise in joining electrochemically active ceramics such as yttria-stabilized zirconia, lanthanum strontium manganite, and barium strontium cobalt ferrite, as well as alumina and magnesia. More recently it has been found that various ternary additions can further improve the wetting characteristics of these filler metals, increase their potential operating temperatures, and/or increase the resulting strength of the

  11. Brazing technique

    DOEpatents

    Harast, Donald G.

    1986-01-01

    A method of brazing comprises sand blasting the surfaces of the components to be brazed with particles of a brazing material to clean the surfaces and to embed brazing material in the surfaces, applying the brazing material to the surfaces, and heating the brazing material to form a brazement between the components.

  12. Investigation of Pd-Modified Ag-CuO Air Braze Filler Metals

    SciTech Connect

    Darsell, Jens T.; Hardy, John S.; Kim, Jin Yong; Weil, K. Scott

    2006-01-10

    Palladium was added as a ternary component to a series of silver - copper oxide alloys in an effort to increase the use temperature of these materials for potential ceramic air brazing applications. Large portions of the silver component of the Ag-CuO system were substituted by palladium forming the following alloys: (100-y)[(100-z)Pd - (z)Ag] - (y)CuOx where y = 0 - 34 mol% CuOx, z = 50 - 100 mol% silver, and x = 0, 0.5, and 1, denoting copper metal, Cu2O, or CuO. From differential scanning calorimetry, it was determined that the addition of palladium causes an increase in the solidus and liquidus temperatures of the resulting Pd-Ag-CuO brazes. In general, the liquidus was found to increase by approximately 220°C for the (100-y)(25Pd - 75Ag) - (z)CuOx filler metal compositions relative to comparable Ag-CuOx alloys. Likewise, the solidus was found to increase for these alloys, respectively by 185°C and 60°C, respectively for CuOx contents of y = 0 - 1mol% and 4 - 10 mol%. For the (100-y)(50Pd - 50Ag) - (y)CuOx alloys, the solidus increased between 280 - 390°C over a copper oxide compositional range of x = 0 to 8 mol%. It was determined from sessile drop experiments that palladium causes an increase in the wetting angle for all of the samples tested. Alloy compositions of (100-y)(25Pd - 75Ag) - (y)CuOx displayed increased wetting angles of 5-20° relative to comparable binary compositions. (100-y)(50Pd - 50Ag) - (y)CuOx alloys exhibited an increase in contact angle of 10-60° and compositions containing less than 10 mol% CuOx were not able to wet the substrate. Scanning electron microscopy indicates that the microstructure of the braze consists of Ag-Pd solid solution with CuOx precipitates. In general, a reaction layer consisting of CuAlO2 forms adjacent to the alumina substrate. However, the formation of this layer is apparently hindered by the addition of large amounts of palladium, causing poor wetting behavior, as denoted by substantial porosity found along

  13. Atomic structure and thermophysical properties of molten silver-copper oxide air braze alloys

    NASA Astrophysics Data System (ADS)

    Hardy, John Steven

    The Ag-CuOx materials system is the basis for a family of filler alloys used in a recently developed ceramic-metal joining technique referred to as air brazing, which is a brazing process that can be carried out in ambient air rather than under the vacuum or inert to reducing gas conditions required for conventional brazing methods. This research was conducted to elucidate the atomic coordination and selected thermophysical properties of these materials as a function of temperature when they are in the salient liquid state in air, since this is when the critical steps of wetting and spreading occur in the joining process. A series of alloys was selected spanning the entire length of the phase diagram including the pure end members, Ag and CuOx; alloys that form the two constituent single phase liquids; and alloys for which the two liquid phases coexist in the miscibility gap of the phase diagram. The oxygen content of the liquid alloys in air was measured using thermogravimetry. The oxidative weight gain of 99.999% pure metallic precursors was measured while simultaneously accounting for the concurrent silver volatility using a method that was developed in the course of the study. The surface tension and mass density were measured using the maximum bubble pressure method. The number density was calculated based on the information gained from the oxygen content and mass density measurements. For compositions that were amenable to laser heating, containerless high energy x-ray scattering measurements of the liquid atomic coordination were performed using a synchrotron beamline, an aerodynamic levitator, and laser heating. For the remaining compositions x-ray scattering measurements were performed in a beamline-compatible furnace. The two liquid phases that form in this materials system have distinct atomic coordinations characterized by an average of nearly two-fold coordinated ionic metal-oxygen pairs in the CuOx-rich liquid and nearly eight-fold coordinated atomic

  14. Structural Design and Preliminary Evaluation of a Lightweight, Brazed, Air-Cooled Turbine Rotor Assembly

    NASA Technical Reports Server (NTRS)

    Meyer, Andre J., Jr.; Morgan, William C.

    1958-01-01

    A lightweight turbine rotor assembly was devised, and components were evaluated in a full-scale jet engine. Thin sheet-metal airfoils were brazed to radial fingers that were an integral part of a number of thin disks composing the turbine rotor. Passages were provided between the disks and in the blades for air cooling. The computed weight of the assembly was 50 percent less than that of a similar turbine of normal construction used in a conventional turbojet engine. Two configurations of sheet-metal test blades simulating the manner of attachment were fabricated and tested in a turbojet engine at rated speed and temperature. After 8-1/2 hours of operation pieces broke loose from the tip sections of the better blades. Severe cracking produced by vibration was determined as the cause of failure. Several methods of overcoming the vibration problem are suggested.

  15. BRAZING ALLOYS

    DOEpatents

    Donnelly, R.G.; Gilliland, R.G.; Slaughter, G.M.

    1962-02-20

    A brazing alloy is described which, in the molten state, is characterized by excellent wettability and flowability and is capable of forming a corrosion-resistant brazed joint. At least one component of said joint is graphite and the other component is a corrosion-resistant refractory metal. The brazing alloy consists essentially of 40 to 90 wt % of gold, 5 to 35 wt% of nickel, and 1 to 45 wt% of tantalum. (AEC)

  16. Brazing method

    DOEpatents

    McCormick, James T.; Ferry, Paul B.; Hall, John C.

    1981-10-06

    There is disclosed a positive cathode electrode structure formed by brazing a thin porous membrane to a backing material by preselecting a predetermined area of the thin porous membrane and thereafter providing a braze flow barrier throughout the remainder of the membrane and electrolessly plating a nickel-phosphide alloy on the backing material, or in this case the honeycomb structure. The preselected area of the thin porous membrane is placed in intimate contact with the electrolessly plated portion of the backing material and heated to elevated temperatures in the absence of oxygen to form a brazed joint limited to a preselected area. If the braze flow barrier is provided by application of a liquid organic solvent, then the organic solvent is driven off by maintaining the thin porous membrane at elevated temperatures for an extended period of time prior to the brazing operation.

  17. BRAZING OF POROUS ALUMINA TO MONOLITHIC ALUMINA WITH Ag-CuO and Ag-V2O5 ALLOYS

    SciTech Connect

    Lamb, M. C.; Camardello, Sam J.; Meier, Alan; Weil, K. Scott; Hardy, John S.

    2005-01-31

    The feasibility of joining porous alumina (Al{sub 2}O{sub 3}) bodies to monolithic Al{sub 2}O{sub 3} using Ag-CuO and Ag-V{sub 2}O{sub 5} alloys via reactive air brazing (RAB) was examined for a nanoporous filter application. Brazing for these systems is complicated by the conflicting requirements of satisfactory wetting to fill the braze gap, while minimizing the infiltration of the porous body. By varying the firing time, temperature, and initial powder size, porous bodies with a range of pore microstructures were fabricated. The wettability was evaluated via sessile drop testing on monolithic substrates and porous body infiltration. Porous Al{sub 2}O{sub 3}/monolithic Al{sub 2}O{sub 3} brazed samples were fabricated, and the microstructures were evaluated. Both systems exhibited satisfactory wetting for brazing, but two unique types of brazing behavior were observed. In the Ag-CuO system, the braze alloy infiltrated a short distance into the porous body. For these systems, the microstructures indicated satisfactory filling of the brazed gap and a sound joint regardless of the processing conditions. The Ag-V{sub 2}O{sub 5} alloys brazed joints exhibited a strong dependence on the amount of V{sub 2}O{sub 5} available. For Ag-V{sub 2}O{sub 5} alloys with large V{sub 2}O{sub 5} additions, the braze alloy aggressively infiltrated the porous body and significantly depleted the Ag from the braze region resulting in poor bonding and large gaps within the joint. With small additions of V{sub 2}O{sub 5}, the Ag infiltrated the porous body until the V{sub 2}O{sub 5} was exhausted and the Ag remaining at the braze interlayer bonded with the Al{sub 2}O{sub 3}. Based on these results, the Ag-CuO alloys have the best potential for brazing porous Al{sub 2}O{sub 3} to monolithic Al{sub 2}O{sub 3}.

  18. Laser Brazing of High Temperature Braze Alloy

    NASA Technical Reports Server (NTRS)

    Gao, Y. P.; Seaman, R. F.; McQuillan, T. J.; Martiens, R. F.

    2000-01-01

    The Space Shuttle Main Engine (SSME) consists of 1080 conical tubes, which are furnace brazed themselves, manifolds, and surrounding structural jacket making almost four miles of braze joints. Subsequent furnace braze cycles are performed due to localized braze voids between the coolant tubes. SSME nozzle experiences extremely high heat flux (180 mW/sq m) during hot fire. Braze voids between coolant tubes may result in hot combustion gas escape causing jacket bulges. The nozzle can be disqualified for flight or result in mission failure if the braze voids exceed the limits. Localized braze processes were considered to eliminate braze voids, however, damage to the parent materials often prohibited use of such process. Being the only manned flight reusable rocket engine, it has stringent requirement on the braze process. Poor braze quality or damage to the parent materials limits the nozzle service life. The objective of this study was to develop a laser brazing process to provide quality, localized braze joints without adverse affect on the parent materials. Gold (Au-Cu-Ni-Pd-Mn) based high temperature braze alloys were used in both powder and wire form. Thin section iron base superalloy A286 tube was used as substrate materials. Different Laser Systems including CO2 (10.6 micrometers, 1kW), ND:YAG (1.06 micrometers, 4kW). and direct diode laser (808nm. 150W) were investigated for brazing process. The laser process variables including wavelength. laser power, travel speed and angle of inclination were optimized according to bead geometry and braze alloy wetting at minimum heat input level, The properties of laser brazing were compared to that of furnace brazing. Microhardness profiles were used for braze joint property comparison between laser and furnace brazing. The cooling rate of laser brazing was compared to furnace brazing based on secondary dendritic arm spacing, Both optical and Scanning Electron Microscope (SEM) were used to evaluate the microstructures of

  19. BRAZING ALLOYS

    DOEpatents

    Donnelly, R.G.; Gilliland, R.G.; Slaughter, G.M.

    1963-02-26

    A brazing alloy which, in the molten state, is characterized by excellent wettability and flowability, said alloy being capable of forming a corrosion resistant brazed joint wherein at least one component of said joint is graphite and the other component is a corrosion resistant refractory metal, said alloy consisting essentially of 20 to 50 per cent by weight of gold, 20 to 50 per cent by weight of nickel, and 15 to 45 per cent by weight of molybdenum. (AEC)

  20. Improved brazing technique

    DOEpatents

    Harast, D.G.

    1984-01-27

    A method of brazing comprises sand blasting the surfaces of the components to be brazed with particles of a brazing material to clean the surfaces and to embed brazing material in the surfaces, applying the brazing material to the surfaces, and heating the brazing material to form a brazement between the components.

  1. Development of Brazing Technology for Use in High- Temperature Gas Separation Equipment

    SciTech Connect

    Weil, K.S.; Hardy, J.S.; Kim, J.Y.

    2003-04-23

    -to-metal braze and consequently lead to the development of the novel reactive air brazing (RAB) concept. The goal in RAB is to reactively modify one or both oxide faying surfaces with an oxide compound dissolved in a molten noble metal alloy such that the newly formed surface is readily wetted by the remaining liquid filler material. In many respects, this concept is similar to active metal brazing, except that joining can be conducted in air and the final joint will be resistant to oxidation at high temperature. Potentially, there are a number of metal oxide-noble metal systems that can be considered for RAB, including Ag-CuO, Ag-V2O5, and Pt-Nb2O5. Our current interest is in determining whether the Ag-CuO system is suitable for air brazing functional ceramic-to-metal joints such as those needed in practical electrochemical devices. In a series of studies, the wetting behavior of the Ag-CuO braze was investigated with respect to a number of potential hydrogen separation, oxygen separation, and fuel cell electrolyte membrane materials and heat resistant metal systems, including: alumina, (La0.6Sr0.4)(Co0.2Fe0.8)O3, (La0.8Sr0.2)FeO3, YSZ, fecralloy, and Crofer-22APU. Selected findings from these studies as well as from our work on joint strength and durability during high-temperature exposure testing will be discussed.

  2. Trends in wetting behavior for Ag–CuO braze alloys on Ba0.5Sr0.5Co0.8Fe0.2O(3-δ) at elevated temperatures in air

    SciTech Connect

    Joshi, Vineet V.; Meier, Alan; Darsell, Jens; Weil, K. Scott; Bowden, Mark

    2013-06-21

    Ba0.5Sr0.5Co0.80.2O(3-δ)(BSCF) is a potential oxygen separation membrane material for advanced coal based power plants. For this application, BSCF must be joined to a metal. In the current study, Ag-CuO, a reactive air brazing (RAB) alloy was evaluated for brazing BSCF. In-situ contact angle tests were performed on BSCF using Ag-CuO binary mixtures at 950 and 1000°C and the interfacial microstructures were evaluated. Wetting contact angles (θ< 90°) were obtained at short times at 950°C and the contact angles remained constant at 1000°C for 1, 2 and 8 mol% CuO contents. Microstructural analysis revealed the dissolution of copper oxide into the BSCF matrix to form copper-cobalt-oxygen rich dissolution products along the BSCF grain boundary. The formation of a thick interfacial reaction product layer and ridging at the sessile drop triple point indicate that the reaction kinetics are very rapid and that it will require careful process control to obtain the desired thin but continuous interfacial product layer.

  3. The Effect of Palladium Additions on the Solidus/Liquidus Temperatures and Wetting Properties of Ag-CuO Based Air Brazes

    SciTech Connect

    Darsell, Jens T.; Weil, K. Scott

    2007-05-16

    As a means of increasing the use temperature of ceramic-ceramic and ceramic-metal air brazes, palladium was investigated as possible ternary addition to the currently employed silver - copper oxide system. The silver component was directly substituted with palladium to form the following series of alloys: (100-y)[(100-z)Pd - (z)Ag] - (y)CuOx where y = 0 - 34 mol% CuOx, z = 50 - 100 mol% silver, and x = 0, 0.5, and 1, denoting copper metal, Cu2O, or CuO. From differential scanning calorimetry, it was determined that the addition of palladium causes an increase in the solidus and liquidus temperatures of the resulting Pd-Ag-CuO brazes. In general, the liquidus was found to increase by approximately 220°C for the (100-y)(25Pd - 75Ag) - (z)CuOx filler metal compositions relative to comparable Ag-CuOx alloys. Likewise, the solidus was found to increase for these alloys, respectively by 185°C and 60°C, respectively for CuOx contents of y = 0 - 1mol% and 4 - 10 mol%. For the (100-y)(50Pd - 50Ag) - (y)CuOx alloys, the solidus increased between 280 - 390°C over a copper oxide compositional range of x = 0 to 8 mol%. It was determined from sessile drop experiments conducted on alumina substrates that in all cases the palladium causes an increase in the wetting angle relative to the corresponding binary braze. Alloy compositions of (100-y)(25Pd - 75Ag) - (y)CuOx displayed increased wetting angles of 5-20° relative to comparable binary compositions. (100-y)(50Pd - 50Ag) - (y)CuOx alloys exhibited an increase in contact angle of 10-60° and compositions containing less than 10 mol% CuOx were not able to wet the substrate. Scanning electron microscopy indicated that the microstructure of the braze consists of discrete CuOx precipitates in an alloyed silver-palladium matrix. In both the binary and ternary filler metal formulations, a reaction layer consisting of CuAlO2 was observed along the interface with the alumina substrate. This reaction product appears to be beneficial

  4. BRAZE BONDING OF COLUMBIUM

    DOEpatents

    Heestand, R.L.; Picklesimer, M.L.

    1962-07-31

    A method of brazing niobium parts together is described. The surfaces of the parts to be brazed together are placed in abutting relationship with a brazing alloy disposed adjacent. The alloy consists essentially of, by weight, 12 to 25% niobium, 0.5 to 5% molybdenum, and the balance zirconium, The alloy is heated to at least its melting point to braze the parts together. The brazed joint is then cooled. The heating, melting and cooling take place in an inert atmosphere. (AEC)

  5. Fluxless aluminum brazing

    DOEpatents

    Werner, W.J.

    1974-01-01

    This invention relates to a fluxless brazing alloy for use in forming brazed composites made from members of aluminum and its alloys. The brazing alloy consists of 35-55% Al, 10--20% Si, 25-60% Ge; 65-88% Al, 2-20% Si, 2--18% In; 65--80% Al, 15-- 25% Si, 5- 15% Y. (0fficial Gazette)

  6. Interaction behaviors at the interface between liquid Al-Si and solid Ti-6Al-4V in ultrasonic-assisted brazing in air.

    PubMed

    Chen, Xiaoguang; Yan, Jiuchun; Gao, Fei; Wei, Jinghui; Xu, Zhiwu; Fan, Guohua

    2013-01-01

    Power ultrasonic vibration (20 kHz, 6 μm) was applied to assist the interaction between a liquid Al-Si alloy and solid Ti-6Al-4V substrate in air. The interaction behaviors, including breakage of the oxide film on the Ti-6Al-4V surface, chemical dissolution of solid Ti-6Al-4V, and interfacial chemical reactions, were investigated. Experimental results showed that numerous 2-20 μm diameter-sized pits formed on the Ti-6Al-4V surface. Propagation of ultrasonic waves in the liquid Al-Si alloy resulted in ultrasonic cavitation. When this cavitation occurred at or near the liquid/solid interface, many complex effects were generated at the small zones during the bubble implosion, including micro-jets, hot spots, and acoustic streaming. The breakage behavior of oxide films on the solid Ti-6Al-4V substrate, excessive chemical dissolution of solid Ti-6Al-4V into liquid Al-Si, abnormal interfacial chemical reactions at the interface, and phase transformation between the intermetallic compounds could be wholly ascribed to these ultrasonic effects. An effective bond between Al-Si and Ti-6Al-4V can be produced by ultrasonic-assisted brazing in air.

  7. Exothermic brazing units

    NASA Technical Reports Server (NTRS)

    Mc Caig, J. C.

    1971-01-01

    Units are used for repairing and assembling stainless steel tubing. Heat generated by chemical reaction is used to melt brazing alloy and bonded area is not contaminated by the reactants or by-products of the reaction.

  8. METHOD OF BRAZING

    DOEpatents

    Patriarca, P.; Slaughter, G.M.

    1962-11-27

    A method of joining metal surfaces is given. Surfaces having nickel or iron as the base metal are joined together with a brazing composition consisting of 80% nickel, 10% phosphorus, and 10% chromium. (AEC)

  9. METHOD OF BRAZING BERYLLIUM

    DOEpatents

    Hanks, G.S.; Keil, R.W.

    1963-05-21

    A process is described for brazing beryllium metal parts by coating the beryllium with silver (65- 75 wt%)-aluminum alloy using a lithium fluoride (50 wt%)-lithium chloride flux, and heating the coated joint to a temperature of about 700 un. Concent 85% C for about 10 minutes. (AEC)

  10. Induction brazing manual

    NASA Technical Reports Server (NTRS)

    1971-01-01

    Manual presents standards and techniques which are known or are particular to specific industry, and is useful as guide in closing tolerance brazing. Material and equipment specifications, tool setting tables, and quality control data and instructions are included. Since similar standards are available, manual is supplementary reference.

  11. Phylogeny and evolution of Rab7 and Rab9 proteins

    PubMed Central

    Mackiewicz, Paweł; Wyroba, Elżbieta

    2009-01-01

    Background An important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network. Results Although Rab7 and Rab9 proteins are quite small and show heterogeneous rates of substitution in different lineages, we found a phylogenetic signal and inferred evolutionary relationships between them. Rab7 proteins evolved before radiation of main eukaryotic supergroups while Rab9 GTPases diverged from Rab7 before split of choanoflagellates and metazoans. Additional duplication of Rab9 and Rab7 proteins resulting in several isoforms occurred in the early evolution of vertebrates and next in teleost fishes and tetrapods. Three Rab7 lineages emerged before divergence of monocots and eudicots and subsequent duplications of Rab7 genes occurred in particular angiosperm clades. Interestingly, several Rab7 copies were identified in some representatives of excavates, ciliates and amoebozoans. The presence of many Rab copies is correlated with significant differences in their expression level. The diversification of analysed Rab subfamilies is also manifested by non-conserved sequences and structural features, many of which are involved in the interaction with regulators and effectors. Individual sites discriminating different subgroups of Rab7 and Rab9 GTPases have been identified. Conclusion Phylogenetic reconstructions of Rab7 and Rab9 proteins were performed by a variety of methods. These Rab GTPases show diversification both at the phylogenetic

  12. Brazed aluminum, Plate-fin heat exchangers for OTEC

    SciTech Connect

    Foust, H.D.

    1980-12-01

    Brazed aluminum plate-fin heat exchangers have been available for special applications for over thirty years. The performance, compactness, versatility, and low cost of these heat exchangers has been unequaled by other heat exchanger configuration. The application of brazed aluminum has been highly limited because of necessary restrictions for clean non-corrosive atmospheres. Air and gas separation have provided ideal conditions for accepting brazed aluminum and in turn have benefited by the salient features of these plate-fin heat exchangers. In fact, brazed aluminum and cryogenic gas and air separation have become nearly synonymous. Brazed aluminum in its historic form could not be considered for a seawater atmosphere. However, technology presents a new look of significant importance to OTEC in terms of compactness and cost. The significant technological variation made was to include one-piece hollow extensions for the seawater passages. Crevice corrosion sites are thereby entirely eliminated and pitting corrosion attack will be controlled by an integral and sacrificial layer of a zinc-aluminum alloy. This paper on brazed aluminum plate-fin heat exchangers for OTEC will aquaint the reader with the state-of-art and variations suggested to qualify this form of aluminum for seawater use. In order to verify the desirable cost potential for OTEC, Trane teamed with Westinghouse to perform an OTEC system analysis with this heat exchanger. These results are very promising and reported in detail elsewhere.

  13. Method for controlling brazing

    DOEpatents

    Hosking, F. Michael; Hall, Aaron C.; Givler, Richard C.; Walker, Charles A.

    2006-08-01

    A method for making a braze joint across a discontinuity in a work piece using alternating current. A filler metal is pre-placed at a location sufficiently close to the discontinuity such that, when an alternating current is applied across a work piece to heat the work piece and melt the filler metal, the filler metal is drawn into the discontinuity. The alternating current is maintained for a set residence time, generally less than 10 seconds and more particularly less than 3 seconds. The alternating current is then altered, generally by reducing the current and/or voltage such that the filler metal can solidify to form a braze joint of desired quality and thickness.

  14. Failure Assessment of Brazed Structures

    NASA Technical Reports Server (NTRS)

    Flom, Yuri

    2012-01-01

    Despite the great advances in analytical methods available to structural engineers, designers of brazed structures have great difficulties in addressing fundamental questions related to the loadcarrying capabilities of brazed assemblies. In this chapter we will review why such common engineering tools as Finite Element Analysis (FEA) as well as many well-established theories (Tresca, von Mises, Highest Principal Stress, etc) don't work well for the brazed joints. This chapter will show how the classic approach of using interaction equations and the less known Coulomb-Mohr failure criterion can be employed to estimate Margins of Safety (MS) in brazed joints.

  15. Vacuum Brazing of Accelerator Components

    NASA Astrophysics Data System (ADS)

    Singh, Rajvir; Pant, K. K.; Lal, Shankar; Yadav, D. P.; Garg, S. R.; Raghuvanshi, V. K.; Mundra, G.

    2012-11-01

    Commonly used materials for accelerator components are those which are vacuum compatible and thermally conductive. Stainless steel, aluminum and copper are common among them. Stainless steel is a poor heat conductor and not very common in use where good thermal conductivity is required. Aluminum and copper and their alloys meet the above requirements and are frequently used for the above purpose. The accelerator components made of aluminum and its alloys using welding process have become a common practice now a days. It is mandatory to use copper and its other grades in RF devices required for accelerators. Beam line and Front End components of the accelerators are fabricated from stainless steel and OFHC copper. Fabrication of components made of copper using welding process is very difficult and in most of the cases it is impossible. Fabrication and joining in such cases is possible using brazing process especially under vacuum and inert gas atmosphere. Several accelerator components have been vacuum brazed for Indus projects at Raja Ramanna Centre for Advanced Technology (RRCAT), Indore using vacuum brazing facility available at RRCAT, Indore. This paper presents details regarding development of the above mentioned high value and strategic components/assemblies. It will include basics required for vacuum brazing, details of vacuum brazing facility, joint design, fixturing of the jobs, selection of filler alloys, optimization of brazing parameters so as to obtain high quality brazed joints, brief description of vacuum brazed accelerator components etc.

  16. Brazing graphite to graphite

    DOEpatents

    Peterson, George R.

    1976-01-01

    Graphite is joined to graphite by employing both fine molybdenum powder as the brazing material and an annealing step that together produce a virtually metal-free joint exhibiting properties similar to those found in the parent graphite. Molybdenum powder is placed between the faying surfaces of two graphite parts and melted to form molybdenum carbide. The joint area is thereafter subjected to an annealing operation which diffuses the carbide away from the joint and into the graphite parts. Graphite dissolved by the dispersed molybdenum carbide precipitates into the joint area, replacing the molybdenum carbide to provide a joint of virtually graphite.

  17. Brazing titanium to stainless steel

    NASA Technical Reports Server (NTRS)

    Batista, R. I.

    1980-01-01

    Titanium and stainless-steel members are usually joined mechanically for lack of any other effective method. New approach using different brazing alloy and plating steel member with nickel resolves problem. Process must be carried out in inert atmosphere.

  18. Thermally stable diamond brazing

    DOEpatents

    Radtke, Robert P.

    2009-02-10

    A cutting element and a method for forming a cutting element is described and shown. The cutting element includes a substrate, a TSP diamond layer, a metal interlayer between the substrate and the diamond layer, and a braze joint securing the diamond layer to the substrate. The thickness of the metal interlayer is determined according to a formula. The formula takes into account the thickness and modulus of elasticity of the metal interlayer and the thickness of the TSP diamond. This prevents the use of a too thin or too thick metal interlayer. A metal interlayer that is too thin is not capable of absorbing enough energy to prevent the TSP diamond from fracturing. A metal interlayer that is too thick may allow the TSP diamond to fracture by reason of bending stress. A coating may be provided between the TSP diamond layer and the metal interlayer. This coating serves as a thermal barrier and to control residual thermal stress.

  19. Protective coating for salt-bath brazing

    NASA Technical Reports Server (NTRS)

    Francisco, A. C.; Gyorgak, C. A.

    1971-01-01

    Ceramic coating, consisting of graphite, enameler's clay, and algin binder, applied to materials prior to salt bath brazing facilitates brazing process and results in superior joints. Alternate coating materials and their various proportions are given.

  20. Brazeability of powder aluminum brazing filler metals with non-corrosive flux

    SciTech Connect

    Takemoto, T.; Matsunawa, A.; Ujie, T.

    1994-12-31

    Various brazed aluminum products, mainly heat exchangers, have been widely used especially in automobiles and electric power industries. They have been produced by using brazing sheet; however, recently the demand to braze the complicated shape is increasing, leading to the necessity for setting brazing filler metal at braze parts instead of using brazing sheet. Therefore, the present work aimed to investigated the brazeability of aluminum powder filler metal in nitrogen gas atmosphere using KAIF{sub 4}-K{sub 3}AIF{sub 6} system noncorrosive brazing flux. By considering the applicability of filler metal, brazing pastes were made of powder filler metal, flux and organic binder. AI-Si powder brazing filler metals were made by automization. T-type specimen was made by A3003 base metal with thickness of 2 mm. in the present experiment, fillet formabiltity, the percentage of the length of formed fillet against the length of vertical member wall at each side, was adopted to evaluate brazeability. The shape of the atomized powder depended on atomize atmosphere and atomizing gas. Sound fillet formation was achieved on the full length of both sides of the vertical member under the condition of appropriate surface treatment and sufficient flux content. Decrease in flux content gave partial fillet formation at the opposite side of the paste set side. Further decrease brought the partial fillet formation at the paste set side also. The paste made of air-atomized powder required more flux content to achieve 100% fillet formation at the opposite side. On the other hand, argon-atomized powders formed fillet in full length using paste with less flux content. powders sorted to remove fine particles and powders with low oxygen content were found to be suitable for brazing filler metal powders, because they required less flux content to obtain 100% fillet formation under the same amount of paste.

  1. Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface.

    PubMed

    Pereira-Leal, José B; Strom, Molly; Godfrey, Richard F; Seabra, Miguel C

    2003-01-31

    Rab proteins are a large family of monomeric GTPases with 60 members identified in the human genome. Rab GTPases require an isoprenyl modification to their C-terminus for membrane association and function in the regulation of vesicular trafficking pathways. This reaction is catalysed by Rab geranylgeranyl transferase, which recognises as protein substrate any given Rab in a 1:1 complex with Rab Escort Protein (REP). REP is therefore able to bind many distinct Rab proteins but the molecular basis for this activity is still unclear. We recently identified conserved motifs in Rabs termed RabF motifs, which we proposed to mediate a conserved mode of interaction between Rabs and REPs. Here, we tested this hypothesis. We first used REP1 as a bait in the yeast two-hybrid system and isolated strictly full-length Rabs, suggesting that REP recognises multiple regions within and properly folded Rabs. We introduced point mutations in Rab3a as a model Rab and assessed the ability of the mutants to interact with REP using the yeast two-hybrid system and an in vitro prenylation assay. We identified several residues that affect REP:Rab binding in the RabF1, RabF3, and RabF4 regions (which include parts of the switch I and II regions), but not other RabF regions. These results support the hypothesis that Rabs bind REP via conserved RabF motifs and provide a molecular explanation for the preferential recognition of the GDP-bound conformation of Rab by REP. PMID:12535645

  2. Silver-hafnium braze alloy

    DOEpatents

    Stephens, Jr., John J.; Hosking, F. Michael; Yost, Frederick G.

    2003-12-16

    A binary allow braze composition has been prepared and used in a bonded article of ceramic-ceramic and ceramic-metal materials. The braze composition comprises greater than approximately 95 wt % silver, greater than approximately 2 wt % hafnium and less than approximately 4.1 wt % hafnium, and less than approximately 0.2 wt % trace elements. The binary braze alloy is used to join a ceramic material to another ceramic material or a ceramic material, such as alumina, quartz, aluminum nitride, silicon nitride, silicon carbide, and mullite, to a metal material, such as iron-based metals, cobalt-based metals, nickel-based metals, molybdenum-based metals, tungsten-based metals, niobium-based metals, and tantalum-based metals. A hermetic bonded article is obtained with a strength greater than 10,000 psi.

  3. Weld-brazing - a new joining process. [combination resistance spot welding and brazing of titanium alloys

    NASA Technical Reports Server (NTRS)

    Bales, T. T.; Royster, D. M.; Arnold, W. E., Jr.

    1972-01-01

    A joining process designated weld brazing which combines resistance spot welding and brazing has been developed. Resistance spot welding is used to position and align the parts as well as to establish a suitable faying surface gap for brazing. Fabrication is then completed by capillary flow of the braze alloy into the joint. The process has been used successfully to fabricate Ti-6Al-4V titanium alloy joints using 3003 aluminum braze alloy. Test results obtained on single overlap and hat-stiffened structural specimens show that weld brazed joints are superior in tensile shear, stress rupture, fatigue, and buckling than joint fabricated by spotwelding or brazing. Another attractive feature of the process is that the brazed joints is hermetically sealed by the braze material.

  4. Field installed brazed thermocouple feedthroughs for high vacuum experiments

    NASA Astrophysics Data System (ADS)

    Anderson, P. M.; Messick, C.

    1983-12-01

    In order to reduce the occurrence of vacuum leaks and to increase the availability of the DIII vacuum vessel for experimental operation, effort was applied to developing a vacuum-tight brazed feedthrough system for sheathed thermocouples, stainless steel sheathed conductor cables and tubes for cooling fluids. This brazed technique is a replacement for elastomer O ring sealed feedthroughs that have proven vulnerable to leaks caused by thermal cycling, etc. To date, about 200 feedthroughs were used. Up to 91 were grouped on a single conflat flange mounted in a bulkhead connector configuration which facilitates installation and removal. Investigation was required to select a suitable braze alloy, flux and installation procedure. Braze alloy selection was challenging since the alloy was required to have: (1) melting temperature in excess of the 250 C (482 F) bakeout temperature; (2) no high vapor pressure elements; (3) good wetting properties when used in air with acceptable flux; and (4) good wettability to 300 series stainless steel and Inconel.

  5. Strength testing of Ti-vapor-coated silicon nitride braze joints

    SciTech Connect

    Santella, M.L.

    1994-09-01

    Sintered silicon nitride was vacuum brazed to itself at 1130{degree}C with a Au-25Ni-25Pd wt % filler metal. Wetting was obtained by coating the Si{sub 3}N{sub 4} surfaces with titanium prior to brazing by electron beam evaporation. The brazed joints were virtually free of porosity. Metallographic analysis showed that Ti reacted with the Si{sub 3}N{sub 4}, to form a TiN reaction layer during brazing. Small amounts of Si and Ti dissolved in the filler metal layers but they did not appear to influence the mechanical properties of the braze layer. Flexure bars were made from the brazed coupons and tested at room temperature, 600C, 700C, and 800C in air. At 700C and below, fracture of the test bars occurred in the Si{sub 3}N{sub 4}, either near the brazed surfaces or at some distance into the monolithic material. The measured strength of joint specimens decreased slightly with increasing test temperature, and generally exceeded the intrinsic braze filler metal strength in this temperature range. It was also found that lapping the Si{sub 3}N{sub 4}, prior to Ti coating reduced the number of near-surface flaws and produced joints with higher average strength and lower scatter than those left in a ground condition. Specimens tested at 800C had very low strengths, and this behavior was related to the microstructure at the brazed Si{sub 3}N{sub 4} surfaces.

  6. Simulation based analysis of laser beam brazing

    NASA Astrophysics Data System (ADS)

    Dobler, Michael; Wiethop, Philipp; Schmid, Daniel; Schmidt, Michael

    2016-03-01

    Laser beam brazing is a well-established joining technology in car body manufacturing with main applications in the joining of divided tailgates and the joining of roof and side panels. A key advantage of laser brazed joints is the seam's visual quality which satisfies highest requirements. However, the laser beam brazing process is very complex and process dynamics are only partially understood. In order to gain deeper knowledge of the laser beam brazing process, to determine optimal process parameters and to test process variants, a transient three-dimensional simulation model of laser beam brazing is developed. This model takes into account energy input, heat transfer as well as fluid and wetting dynamics that lead to the formation of the brazing seam. A validation of the simulation model is performed by metallographic analysis and thermocouple measurements for different parameter sets of the brazing process. These results show that the multi-physical simulation model not only can be used to gain insight into the laser brazing process but also offers the possibility of process optimization in industrial applications. The model's capabilities in determining optimal process parameters are exemplarily shown for the laser power. Small deviations in the energy input can affect the brazing results significantly. Therefore, the simulation model is used to analyze the effect of the lateral laser beam position on the energy input and the resulting brazing seam.

  7. Rab5 and Rab4 Regulate Axon Elongation in the Xenopus Visual System

    PubMed Central

    Konopacki, Filip A.; Zivraj, Krishna H.; Holt, Christine E.

    2014-01-01

    The elongation rate of axons is tightly regulated during development. Recycling of the plasma membrane is known to regulate axon extension; however, the specific molecules involved in recycling within the growth cone have not been fully characterized. Here, we investigated whether the small GTPases Rab4 and Rab5 involved in short-loop recycling regulate the extension of Xenopus retinal axons. We report that, in growth cones, Rab5 and Rab4 proteins localize to endosomes, which accumulate markers that are constitutively recycled. Fluorescence recovery after photo-bleaching experiments showed that Rab5 and Rab4 are recruited to endosomes in the growth cone, suggesting that they control recycling locally. Dynamic image analysis revealed that Rab4-positive carriers can bud off from Rab5 endosomes and move to the periphery of the growth cone, suggesting that both Rab5 and Rab4 contribute to recycling within the growth cone. Inhibition of Rab4 function with dominant-negative Rab4 or Rab4 morpholino and constitutive activation of Rab5 decreases the elongation of retinal axons in vitro and in vivo, but, unexpectedly, does not disrupt axon pathfinding. Thus, Rab5- and Rab4-mediated control of endosome trafficking appears to be crucial for axon growth. Collectively, our results suggest that recycling from Rab5-positive endosomes via Rab4 occurs within the growth cone and thereby supports axon elongation. PMID:24403139

  8. TBC-2 regulates RAB-5/RAB-7-mediated endosomal trafficking in Caenorhabditis elegans.

    PubMed

    Chotard, Laëtitia; Mishra, Ashwini K; Sylvain, Marc-André; Tuck, Simon; Lambright, David G; Rocheleau, Christian E

    2010-07-01

    During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7-positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(-) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(-) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

  9. Surface preparation for high purity alumina ceramics enabling direct brazing in hydrogen atmospheres

    DOEpatents

    Cadden, Charles H.; Yang, Nancy Yuan Chi; Hosking, Floyd M.

    2001-01-01

    The present invention relates to a method for preparing the surface of a high purity alumina ceramic or sapphire specimen that enables direct brazing in a hydrogen atmosphere using an active braze alloy. The present invention also relates to a method for directly brazing a high purity alumina ceramic or sapphire specimen to a ceramic or metal member using this method of surface preparation, and to articles produced by this brazing method. The presence of silicon, in the form of a SiO.sub.2 -containing surface layer, can more than double the tensile bond strength in alumina ceramic joints brazed in a hydrogen atmosphere using an active Au-16Ni-0.75 Mo-1.75V filler metal. A thin silicon coating applied by PVD processing can, after air firing, produce a semi-continuous coverage of the alumina surface with a SiO.sub.2 film. Room temperature tensile strength was found to be proportional to the fraction of air fired surface covered by silicon-containing films. Similarly, the ratio of substrate fracture versus interface separation was also related to the amount of surface silicon present prior to brazing. This process can replace the need to perform a "moly-manganese" metallization step.

  10. Fluxless Brazing of Large Structural Panels

    NASA Technical Reports Server (NTRS)

    Beuyukian, C. S.

    1982-01-01

    Fluxless brazing is used in fabricating aluminum structural panels that withstand high internal pressure. Aluminum sheet of structural thickness with 4045 aluminum/silicon-braze-alloy cladding is brazed to corrugated "fin stock" having channels 0.001 inch (0.03mm) high by same width. Process is carried out in an inert (argon) atmosphere in a retort furnace. Filler bars are used in some channels to prevent fin stock from collapsing as pressure is applied.

  11. Brazing of beryllium for structural applications

    NASA Technical Reports Server (NTRS)

    Vogan, J. W.

    1972-01-01

    Progress made in fabricating a beryllium compression tube structure and a stiffened beryllium panel. The compression tube was 7.6cm in diameter and 30.5cm long with titanium end fittings. The panel was 203cm long and stiffened with longitudinal stringers. Both units were assembled by brazing with BAg-18 braze alloy. The detail parts were fabricated by hot forming 0.305cm beryllium sheet and the brazing parameters established.

  12. Thermal resilient multiple jaw braze fixture

    DOEpatents

    Ney, Robert; Perrone, Alex J.

    1995-07-11

    A braze fixture has side walls forming a cavity with an opening to receive a stack of parts to be brazed. Sidewalls of the housing have a plurality of bearing receiving openings into which bearing rods or jaws are inserted to align the stacked elements of the workpiece. The housing can also have view ports to allow a visual check of the alignment. Straps or wires around the fixture are selected to have thermal characteristics similar to the thermal characteristics of the workpiece undergoing brazing. The straps or wires make physical contact with the bearing rods thereby causing bearing rods to maintain the workpiece in proper alignment throughout the entire brazing cycle.

  13. Susceptor heating device for electron beam brazing

    DOEpatents

    Antieau, Susan M.; Johnson, Robert G. R.

    1999-01-01

    A brazing device and method are provided which locally apply a controlled amount of heat to a selected area, within a vacuum. The device brazes two components together with a brazing metal. A susceptor plate is placed in thermal contact with one of the components. A serrated pedestal supports the susceptor plate. When the pedestal and susceptor plate are in place, an electron gun irradiates an electron beam at the susceptor plate such that the susceptor plate is sufficiently heated to transfer heat through the one component and melt the brazing metal.

  14. Intelligent control of vacuum aluminum brazing

    SciTech Connect

    Zhong, G.; Pai, D.M.; Badgley, S.

    1995-06-01

    Vacuum brazing is a versatile modern day metal joining method. It usually includes vacuum producing, heating and residual gas analyzing systems. When used to join aluminum parts, the quality of the brazed joint is highly dependent on the residual gases in the vacuum, especially the residual oxygen and water vapor in the vacuum chamber which affect the formation of oxides. These vacuum environment brazing contaminants are reduced during the out-gassing processes of degassing and desorption during heating under vacuum. Exceeding the boundary limits of these contaminants causes unacceptable flow of braze filler in the joints during the brazing process. Identification of the quality control boundary for vacuum brazing of aluminum alloy makes computer control of vacuum brazing quality for aluminum alloys possible. The present study utilizes a residual gas analyzer (RGA) to monitor the partial pressures of residual gases during brazing. These data are transported to computer through the RS-232 interface port on the RGA, and used in real time to monitor the brazing quality by an algorithm based on the quality control boundary. If the quality is bad, the computer will send a ``hold`` signal to the heating system via another Rs-232 port until the environment is reestablished within the acceptable boundary.

  15. Brazing applications: An automotive perspective

    SciTech Connect

    Winterbottom, W.L.

    1994-12-31

    Currently the US auto industry consumes 20% of the nation`s steel production, 50% of lead and cast iron, one-third of its zinc and 10% of its aluminum. Overall, more than 23 million tons of materials are used by the industry each year. But stricter environmental and CAFE laws could change those numbers markedly. Experts predict that without downsizing, weight reductions of 1000 pounds-per-vehicle may be needed to achieve proposed improvements in CAFE. If there is to be wide scale application of advanced materials such as metal matrix composites and ceramics in harsh automotive environments, alternate joining methods to the standard welding process will certainly be required. Quality and cost competitiveness in the automotive market place are forcing the manufacturing operations to focus on improving process robustness and efficiency. The extent to which braze methodology is capable of contributing to satisfying these needs will determine its utility in the automotive industry. The features of the braze process that determine its versatility and reliability in meeting the material and processing needs of the automotive industry will be used in presenting the current status and projections of future developments in brazing applications.

  16. Brazed Joints Design and Allowables: Discuss Margins of Safety in Critical Brazed Structures

    NASA Technical Reports Server (NTRS)

    FLom, Yury

    2009-01-01

    This slide presentation tutorial discusses margins of safety in critical brazed structures. It reviews: (1) the present situation (2) definition of strength (3) margins of safety (4) design allowables (5) mechanical testing (6) failure criteria (7) design flowchart (8) braze gap (9) residual stresses and (10) delayed failures. This presentation addresses the strength of the brazed joints, the methods of mechanical testing, and our ability to evaluate the margins of safety of the brazed joints as it applies to the design of critical and expensive brazed assemblies.

  17. Distinct Actions of Rab3 and Rab27 GTPases on Late Stages of Exocytosis of Insulin

    PubMed Central

    Cazares, Victor A.; Subramani, Arasakumar; Saldate, Johnny J.; Hoerauf, Widmann; Stuenkel, Edward L.

    2014-01-01

    Rab GTPases associated with insulin containing secretory granules are key in targeting, docking and assembly of molecular complexes governing pancreatic β-cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase activating protein overexpression in β-cells from wild-type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP-bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool and central to regulating the size of the readily releasable pool. By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the readily releasable pool with secretory granules. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release-ready secretory granules in β-cells, they also direct unique kinetic and functional properties of the exocytotic pathway. PMID:24909540

  18. Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells.

    PubMed

    Loraine, A E; Yalovsky, S; Fabry, S; Gruissem, W

    1996-04-01

    Rab proteins attach to membranes along the secretory pathway where they contribute to distinct steps in vesicle-mediated transport. To bind membranes, Rab proteins in fungal and animal cells must be isoprenylated by the enzyme Rab geranylgeranyl transferase (Rab GGTase). We have isolated three tomato (Lycopersicon esculentum, M.) cDNAs (LeRab 1A, B, and C) encoding Rab-like proteins and show here that all three are substrates for a Rab GGTase-like activity in plant cells. The plant enzyme is similar to mammalian Rab GGTase in that the plant activity (a) is enhanced by detergent and (b) is inhibited by mutant Rab lacking a prenylation consensus sequence. LeRab1B contains a rare prenylation target motif and was the best substrate for the plant, but not the yeast, Rab GGTase. LeRab1A, B, and C are functional homologs of the Saccharomyces cerevisiae Rab protein encoded by YPT1 and are differentially expressed in tomato. LeRab1A mRNA, but not that of LeRab1B or C, is induced by ethylene in tomato seedlings and is also upregulated in ripening fruit. The increase in LeRab1A mRNA expression in ripe fruit may be linked to increased synthesis and export of enzymes like polygalacturonase, pectin esterase, and other enzymes important in fruit softening.

  19. Method for brazing together planar and nonplanar metal members

    DOEpatents

    Hammersand, Fred G.; Witkowski, Anthony J.

    1985-01-01

    The invention relates to a method and apparatus for brazing two metal members together, at least one of which is nonplanar, in a brazing furnace using a substantially pure brazing material. The method comprises the steps of utilizing a brazing fixture to hold the two metal members in tangential relation to one another along a portion of each member so that a cavity is formed adjacent to the contacting portions. A braze material is then positioned within the cavity. The braze fixture, the metal members, and the braze material are then placed in a brazing furnace. A heat shield is then placed over the braze fixture, the metal members, and the braze material to shield the braze material from direct furnace radiation. The furnace temperature is linearly increased at a rate of about 180.degree. C. per hour until a temperature of 350.degree. C. is achieved. Heat is transferred by conduction from the metal members to the braze material to cause the braze material to melt. Some material from the metal members slowly diffuses into the braze material forming a braze joint. The furnace is rapidly cooled to room temperature using nitrogen gas. The brazed assemblies made according to this method are superior to assemblies formed by heliarc welding.

  20. Braze alloy spreading on steel

    NASA Technical Reports Server (NTRS)

    Siewert, T. A.; Heine, R. W.; Lagally, M. G.

    1978-01-01

    Scanning electron microscopy (SEM) and Auger electron microscopy (AEM) were employed to observe elemental surface decomposition resulting from the brazing of a copper-treated steel. Two types of steel were used for the study, stainless steel (treated with a eutectic silver-copper alloy), and low-carbon steel (treated with pure copper). Attention is given to oxygen partial pressure during the processes; a low enough pressure (8 x 10 to the -5th torr) was found to totally inhibit the spreading of the filler material at a fixed heating cycle. With both types of steel, copper treatment enhanced even spreading at a decreased temperature.

  1. Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP).

    PubMed

    Lall, Patrick; Lindsay, Andrew J; Hanscom, Sara; Kecman, Tea; Taglauer, Elizabeth S; McVeigh, Una M; Franklin, Edward; McCaffrey, Mary W; Khan, Amir R

    2015-07-24

    Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis.

  2. 31 Flavors of Drosophila Rab proteins

    SciTech Connect

    Zhang, Jun; Schulze, Karen L.; Hiesinger, P. Robin; Suyama, Kaye; Wang, Stream; Fish, Matthew; Acar, Melih; Hoskins, Roger A.; Bellen, HugoJ.; Scott, Matthew P.

    2007-04-03

    Rab proteins are small GTPases that play important roles intransport of vesicle cargo and recruitment, association of motor andother proteins with vesicles, and docking and fusion of vesicles atdefined locations. In vertebrates, more than 75 Rab genes have beenidentified, some of which have been intensively studied for their rolesin endosome and synaptic vesicle trafficking. Recent studies of thefunctions of certain Rab proteins have revealed specific roles inmediating developmental signal transduction. We have begun a systematicgenetic study of the 33 Rab genes in Drosophila. Most of the fly proteinsare clearly related to specific vertebrate proteins. We report here thecreation of a set of transgenic fly lines that allow spatially andtemporally regulated expression of Drosophila Rab proteins. We generatedfluorescent protein-tagged wild-type, dominant-negative, andconstitutively active forms of 31 Drosophila Rab proteins. We describeDrosophila Rab expression patterns during embryogenesis, the subcellularlocalization of some Rab proteins, and comparisons of the localization ofwild-type, dominant-negative, and constitutively active forms of selectedRab proteins. The high evolutionary conservation and low redundancy ofDrosophila Rab proteins make these transgenic lines a useful toolkit forinvestigating Rab functions in vivo.

  3. RabGEFs are a major determinant for specific Rab membrane targeting.

    PubMed

    Blümer, Julia; Rey, Juliana; Dehmelt, Leif; Mazel, Tomáš; Wu, Yao-Wen; Bastiaens, Philippe; Goody, Roger S; Itzen, Aymelt

    2013-02-01

    Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct membrane, but it is unclear how this specific membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal targeting machinery for recruiting Rabs from the cytosol to the correct membrane using the Rab-GEF pairs Rab5A-Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific mistargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab membrane targeting.

  4. Theory and modeling of active brazing.

    SciTech Connect

    van Swol, Frank B.; Miller, James Edward; Lechman, Jeremy B.; Givler, Richard C.

    2013-09-01

    Active brazes have been used for many years to produce bonds between metal and ceramic objects. By including a relatively small of a reactive additive to the braze one seeks to improve the wetting and spreading behavior of the braze. The additive modifies the substrate, either by a chemical surface reaction or possibly by alloying. By its nature, the joining process with active brazes is a complex nonequilibrium non-steady state process that couples chemical reaction, reactant and product diffusion to the rheology and wetting behavior of the braze. Most of the these subprocesses are taking place in the interfacial region, most are difficult to access by experiment. To improve the control over the brazing process, one requires a better understanding of the melting of the active braze, rate of the chemical reaction, reactant and product diffusion rates, nonequilibrium composition-dependent surface tension as well as the viscosity. This report identifies ways in which modeling and theory can assist in improving our understanding.

  5. Failure Assessment Diagram for Titanium Brazed Joints

    NASA Technical Reports Server (NTRS)

    Flom, Yury; Jones, Justin S.; Powell, Mollie M.; Puckett, David F.

    2011-01-01

    The interaction equation was used to predict failure in Ti-4V-6Al joints brazed with Al 1100 filler metal. The joints used in this study were geometrically similar to the joints in the brazed beryllium metering structure considered for the ATLAS telescope. This study confirmed that the interaction equation R(sub sigma) + R(sub Tau) = 1, where R(sub sigma) and R(sub Tau)are normal and shear stress ratios, can be used as conservative lower bound estimate of the failure criterion in ATLAS brazed joints as well as for construction of the Failure Assessment Diagram (FAD).

  6. Direct metal brazing to cermet feedthroughs

    DOEpatents

    Hopper, Jr., Albert C.

    1984-12-18

    An improved method for brazing metallic components to a cermet surface in an alumina substrate eliminates the prior art metallized layer over the cermet via and adjoining alumina surfaces. Instead, a nickel layer is applied over the cermet surface only and metallic components are brazed directly to this nickel coated cermet surface. As a result, heretofore unachievable tensile strength joints are produced. In addition, cermet vias with their brazed metal components can be spaced more closely in the alumina substrate because of the elimination of the prior art metallized alumina surfaces.

  7. Direct metal brazing to cermet feedthroughs

    DOEpatents

    Not Available

    1982-07-29

    An improved method for brazing metallic components to a cermet surface in an alumina substrate eliminates the prior art metallized layer over the cermet via and adjoining alumina surfaces. Instead, a nickel layer is applied over the cermet surface only and metallic components are brazed directly to this nickel coated cermet surface. As a result, heretofore unachievable tensile strength joints are produced. In addition, cermet vias with their brazed metal components can be spaced more closely in the alumina substrate because of the elimination of the prior art metallized alumina surfaces.

  8. Infrared repair brazing of 403 stainless steel with a nickel-based braze alloy

    NASA Astrophysics Data System (ADS)

    Shiue, R. K.; Wu, S. K.; Hung, C. M.

    2002-06-01

    Martensitic stainless steel (403SS) is extensively used for intermediate and low-pressure steam turbine blades in fossil-fuel power plants. The purpose of this investigation is to study the repair of shallow cracks on the surface of 403SS steam turbine blades by infrared repair brazing using rapid thermal cycles. A nickel-based braze alloy (NICROBRAZ LM) is used as filler metal. The braze alloy after brazing is primarily comprised of borides and an FeNi3 matrix with different amounts of alloying elements, especially B and Si. As the brazing temperature increases, more Fe atoms are dissolved into the molten braze. Some boron atoms diffuse into the 403SS substrate primarily via grain boundary diffusion and form B-Cr-Fe intermetallic precipitates along the grain boundaries. The LM filler metal demonstrates better performance than 403SS in both microhardness and wear tests. It is also noted that specimens brazed in a vacuum have less porosity than those brazed in an Ar atmosphere. The shear strength of the joint is around 300 MPa except for specimens brazed in short time periods, e.g., 5 seconds in Ar flow and 30 seconds in vacuum. The fractographs mainly consist of brittle fractures and no ductile dimple fractures observed in the scanning electron microscope (SEM) examination.

  9. Boron-deoxidized copper withstands brazing temperatures

    NASA Technical Reports Server (NTRS)

    Schmidt, E. H.

    1966-01-01

    Boron-deoxidized high-conductivity copper is used for fabrication of heat transfer components that are brazed in a hydrogen atmosphere. This copper has high strength and ductility at elevated temperatures and does not exhibit massive intergranular failure.

  10. Zebrafish Rab5 proteins and a role for Rab5ab in nodal signalling

    PubMed Central

    Kenyon, Emma J.; Campos, Isabel; Bull, James C.; Williams, P. Huw; Stemple, Derek L.; Clark, Matthew D.

    2015-01-01

    The RAB5 gene family is the best characterised of all human RAB families and is essential for in vitro homotypic fusion of early endosomes. In recent years, the disruption or activation of Rab5 family proteins has been used as a tool to understand growth factor signal transduction in whole animal systems such as Drosophila melanogaster and zebrafish. In this study we have examined the functions for four rab5 genes in zebrafish. Disruption of rab5ab expression by antisense morpholino oligonucleotide (MO) knockdown abolishes nodal signalling in early zebrafish embryos, whereas overexpression of rab5ab mRNA leads to ectopic expression of markers that are normally downstream of nodal signalling. By contrast MO disruption of other zebrafish rab5 genes shows little or no effect on expression of markers of dorsal organiser development. We conclude that rab5ab is essential for nodal signalling and organizer specification in the developing zebrafish embryo. PMID:25478908

  11. Zebrafish Rab5 proteins and a role for Rab5ab in nodal signalling.

    PubMed

    Kenyon, Emma J; Campos, Isabel; Bull, James C; Williams, P Huw; Stemple, Derek L; Clark, Matthew D

    2015-01-15

    The RAB5 gene family is the best characterised of all human RAB families and is essential for in vitro homotypic fusion of early endosomes. In recent years, the disruption or activation of Rab5 family proteins has been used as a tool to understand growth factor signal transduction in whole animal systems such as Drosophila melanogaster and zebrafish. In this study we have examined the functions for four rab5 genes in zebrafish. Disruption of rab5ab expression by antisense morpholino oligonucleotide (MO) knockdown abolishes nodal signalling in early zebrafish embryos, whereas overexpression of rab5ab mRNA leads to ectopic expression of markers that are normally downstream of nodal signalling. By contrast MO disruption of other zebrafish rab5 genes shows little or no effect on expression of markers of dorsal organiser development. We conclude that rab5ab is essential for nodal signalling and organizer specification in the developing zebrafish embryo.

  12. Brazing of Stainless Steels to Yttria Stabilized Zirconia (YSZ) for Solid Oxide Fuel Cells

    NASA Technical Reports Server (NTRS)

    Shpargel, Tarah P.; Needham, Robert J.; Singh, M.; Kung, Steven C.

    2005-01-01

    Recently, there has been a great deal of interest in research, development, and commercialization of solid oxide fuel cells. Joining and sealing are critical issues that will need to be addressed before SOFC's can truly perform as expected. Ceramics and metals can be difficult to join together, especially when the joint must withstand up to 900 C operating temperature of the SOFC's. The goal of the present study is to find the most suitable braze material for joining of yttria stabilized zirconia (YSZ) to stainless steels. A number of commercially available braze materials TiCuSil, TiCuNi, Copper-ABA, Gold-ABA, and Gold-ABA-V have been evaluated. The oxidation behavior of the braze materials and steel substrates in air was also examined through thermogravimetric analysis. The microstructure and composition of the brazed regions have been examined by optical and scanning electron microscopy and EDS analysis. Effect of braze composition and processing conditions on the interfacial microstructure and composition of the joint regions will be presented.

  13. Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo.

    PubMed

    Sorvina, Alexandra; Shandala, Tetyana; Brooks, Douglas A

    2016-01-01

    The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.

  14. Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo

    PubMed Central

    Sorvina, Alexandra; Shandala, Tetyana; Brooks, Douglas A.

    2016-01-01

    ABSTRACT The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens. PMID:27190105

  15. Measurement of Rab35 activity with the GTP-Rab35 trapper RBD35.

    PubMed

    Kobayashi, Hotaka; Etoh, Kan; Marubashi, Soujiro; Ohbayashi, Norihiko; Fukuda, Mitsunori

    2015-01-01

    Small GTPase Rab35 functions as a molecular switch for membrane trafficking, specifically for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane trafficking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 trapper, and we named it RBD35 (Rab-binding domain specific for Rab35). Because RBD35 specifically interacts with the GTP-bound active form of Rab35 and does not interact with any of the other 59 Rab proteins identified in humans and mice, RBD35 is a useful tool for measuring the level of active Rab35 by pull-down assays and for inhibiting the function of Rab35 by overexpression. In this chapter, we describe the assay procedures for analyzing Rab35 with RBD35.

  16. Brazing of Stainless Steels to Yttria Stabilized Zirconia (YSZ) Using Silver -Base Brazes

    NASA Technical Reports Server (NTRS)

    Singh, Mrityunjay; Shpargel, Tarah P.; Asthana, Rajiv

    2005-01-01

    Three silver-base brazes containing either noble metal palladium (Palcusil-10 and Palcusil-15) or active metal titanium (Ticusil) were evaluated for high-temperature oxidation resistance, and their effectiveness in joining yttria stabilized zirconia (YSZ) to a corrosion-resistant ferritic stainless steel. Thermogravimetric analysis (TGA), and optical- and scanning electron microscopy (SEM) coupled with energy dispersive spectrometry (EDS) were used to evaluate the braze oxidation behavior and the structure and chemistry of the YSZ/braze/steel joints. The effect of the braze type and processing conditions on the interfacial microstructure and composition of the joint regions is discussed with reference to the chemical changes that occur at the interface. It was found that chemical interdiffusion of the constituents of YSZ, steel and the brazes led to compositional changes and/or interface reconstruction, and metallurgically sound joints.

  17. Ultrasonics permits brazing complex stainless steel assembly without flux

    NASA Technical Reports Server (NTRS)

    Baker, W. H.

    1967-01-01

    Ultrasonic vibration of an assembly of stainless steel instrumentation tubes ensures brazing without flux. Vibration with an ultrasonic transducer permits the brazing material to flow down each tube in contact with a seal plug installed in a pressure vessel wall.

  18. Refractory metals welded or brazed with tungsten inert gas equipment

    NASA Technical Reports Server (NTRS)

    Wisner, J. P.

    1965-01-01

    Appropriate brazing metals and temperatures facilitate the welding or brazing of base metals with tungsten inert gas equipment. The highest quality bond is obtained when TIG welding is performed in an inert atmosphere.

  19. High-strength braze joints between copper and steel

    NASA Technical Reports Server (NTRS)

    Kuhn, R. F.

    1967-01-01

    High-strength braze joints between copper and steel are produced by plating the faying surface of the copper with a layer of gold. This reduces porosity in the braze area and strengthens the resultant joint.

  20. Are There Rab GTPases in Archaea?

    PubMed Central

    Surkont, Jaroslaw; Pereira-Leal, Jose B.

    2016-01-01

    A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins. PMID:27034425

  1. Small GTPases Rab8a and Rab11a Are Dispensable for Rhodopsin Transport in Mouse Photoreceptors

    PubMed Central

    Ying, Guoxin; Gerstner, Cecilia D.; Frederick, Jeanne M.; Boye, Sanford L.; Hauswirth, William W.; Baehr, Wolfgang

    2016-01-01

    Rab11a and Rab8a are ubiquitous small GTPases shown as required for rhodopsin transport in Xenopus laevis and zebrafish photoreceptors by dominant negative (dn) disruption of function. Here, we generated retina-specific Rab11a (retRab11a) and Rab8a (retRab8a) single and double knockout mice to explore the consequences in mouse photoreceptors. Rhodopsin and other outer segment (OS) membrane proteins targeted correctly to OS and electroretinogram (ERG) responses in all three mutant mouse lines were indistinguishable from wild-type (WT). Further, AAV (adeno-associated virus)-mediated expression of dnRab11b in retRab11a-/- retina, or expression of dnRab8b in retRab8a-/- retina did not cause OS protein mislocalization. Finally, a retRab8a-/- retina injected at one month of age with AAVs expressing dnRab11a, dnRab11b, dnRab8b, and dnRab10 (four dn viruses on Rab8a-/- background) and harvested three months later exhibited normal OS protein localization. In contrast to results obtained with dnRab GTPases in Xenopus and zebrafish, mouse Rab11a and Rab8a are dispensable for proper rhodopsin and outer segment membrane protein targeting. Absence of phenotype after expression of four dn Rab GTPases in a Rab8a-/- retina suggests that Rab8b and Rab11b paralogs maybe dispensable as well. Our data thus demonstrate significant interspecies variation in photoreceptor membrane protein and rhodopsin trafficking. PMID:27529348

  2. Evidence for sequential action of Rab5 and Rab7 GTPases in prevacuolar organelle partitioning.

    PubMed

    Bottanelli, Francesca; Gershlick, David C; Denecke, Jürgen

    2012-02-01

    GTPases of the Rab5 and Rab7 families were shown to control vacuolar sorting but their specific subcellular localization is controversial in plants. Here, we show that both the canonical as well as the plant-specific Rab5 reside at the newly discovered 'late prevacuolar compartment' (LPVC) while Rab7 partitions to the vacuolar membrane when expressed at low levels. Higher expression levels of wild-type Rab5 GTPases but not Rab7 lead to dose-dependent inhibition of biosynthetic vacuolar transport. In the case of Ara6, this included aberrant co-localization with markers for earlier post-Golgi compartments including the trans-Golgi network. However, nucleotide-free mutants of all three GTPases (Rha1, Ara6 and Rab7) cause stronger dose-dependent inhibition of vacuolar sorting. In addition, nucleotide-free Rha1 led to a later maturation defect and co-localization of markers for the prevacuolar compartment (PVC) and the LPVC. The corresponding Rab7 mutant strongly inhibited vacuolar delivery without merging of PVC and LPVC markers. Evidence for functional differentiation of the Rab5 family members is underlined by the fact that mutant Rha1 expression can be suppressed by increasing wild-type Rha1 levels while mutant Ara6 specifically titrates the nucleotide exchange factor Vps9. A model describing the sequential action of Rab5 and Rab7 GTPases is presented in the light of the current observations.

  3. RAB-6.1 and RAB-6.2 Promote Retrograde Transport in C. elegans

    PubMed Central

    Zhang, Donglei; Dubey, Jyoti; Koushika, Sandhya P.; Rongo, Christopher

    2016-01-01

    Retrograde transport is a critical mechanism for recycling certain membrane cargo. Following endocytosis from the plasma membrane, retrograde cargo is moved from early endosomes to Golgi followed by transport (recycling) back to the plasma membrane. The complete molecular and cellular mechanisms of retrograde transport remain unclear. The small GTPase RAB-6.2 mediates the retrograde recycling of the AMPA-type glutamate receptor (AMPAR) subunit GLR-1 in C. elegans neurons. Here we show that RAB-6.2 and a close paralog, RAB-6.1, together regulate retrograde transport in both neurons and non-neuronal tissue. Mutants for rab-6.1 or rab-6.2 fail to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. Loss of both rab-6.1 and rab-6.2 results in an additive effect on GLR-1 retrograde recycling, indicating that these two C. elegans Rab6 isoforms have overlapping functions. MIG-14 (Wntless) protein, which undergoes retrograde recycling, undergoes a similar degradation in intestinal epithelia in both rab-6.1 and rab-6.2 mutants, suggesting a broader role for these proteins in retrograde transport. Surprisingly, MIG-14 is localized to separate, spatially segregated endosomal compartments in rab-6.1 mutants compared to rab-6.2 mutants. Our results indicate that RAB-6.1 and RAB-6.2 have partially redundant functions in overall retrograde transport, but also have their own unique cellular- and subcellular functions. PMID:26891225

  4. Cell type-specific and light-dependent expression of Rab1 and Rab6 GTPases in mammalian retinas

    PubMed Central

    Huang, Wei; Wu, Guangyu; Wang, Guo-Yong

    2010-01-01

    The Ras-like Rab1 and Rab6 GTPases modulate protein traffic along the early secretory pathway and are involved in the regulation of maturation of rhodopsin in the outer retina. However, Rab GTPases have not been studied in the inner retinas. Here, we analyzed the anatomatic distribution and expression of Rab1 and Rab6 in the mouse and rat retinas by immunohistochemistry and immunoblotting. We found that Rab1 was specifically expressed in the rod bipolar cells, while Rab6 was expressed in a different cell type(s) from rod bipolar cells in the inner retina. We also demonstrated that expression of Rab1 and Rab6 was increased with light. These data provided the first evidence implicating that Rab1 and Rab6 may be involved in the regulation of the retinal adaptation. PMID:20003598

  5. 46 CFR 56.30-30 - Brazed joints.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Pressure Vessel Code (incorporated by reference; see 46 CFR 56.01-2) are satisfied at the highest....75). Brazed socket-type joints shall be made with suitable brazing alloys. The minimum socket depth shall be sufficient for the intended service. Brazing alloy shall either be end-fed into the socket...

  6. 46 CFR 56.30-30 - Brazed joints.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Pressure Vessel Code (incorporated by reference; see 46 CFR 56.01-2) are satisfied at the highest....75). Brazed socket-type joints shall be made with suitable brazing alloys. The minimum socket depth shall be sufficient for the intended service. Brazing alloy shall either be end-fed into the socket...

  7. 46 CFR 56.30-30 - Brazed joints.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Pressure Vessel Code (incorporated by reference; see 46 CFR 56.01-2) are satisfied at the highest....75). Brazed socket-type joints shall be made with suitable brazing alloys. The minimum socket depth shall be sufficient for the intended service. Brazing alloy shall either be end-fed into the socket...

  8. 46 CFR 56.30-30 - Brazed joints.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Pressure Vessel Code (incorporated by reference; see 46 CFR 56.01-2) are satisfied at the highest....75). Brazed socket-type joints shall be made with suitable brazing alloys. The minimum socket depth shall be sufficient for the intended service. Brazing alloy shall either be end-fed into the socket...

  9. 46 CFR 56.30-30 - Brazed joints.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Pressure Vessel Code (incorporated by reference; see 46 CFR 56.01-2) are satisfied at the highest....75). Brazed socket-type joints shall be made with suitable brazing alloys. The minimum socket depth shall be sufficient for the intended service. Brazing alloy shall either be end-fed into the socket...

  10. 32 CFR 202.10 - RAB adjournment and dissolution.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Commander is considering dissolving the RAB, the Installation Commander shall: (i) Consult with EPA, state, tribal and local government representatives, as appropriate, regarding dissolving the RAB. (ii) Notify the RAB community co-chair and members in writing of the intent to dissolve the RAB and the...

  11. PpRab1, a Rab GTPase from maritime pine is differentially expressed during embryogenesis.

    PubMed

    Gonçalves, Sónia; Cairney, John; Rodríguez, María Pérez; Cánovas, Francisco; Oliveira, Margarida; Miguel, Célia

    2007-09-01

    Rab-related small GTP-binding proteins are known to be involved in the regulation of the vesicular transport system in eukaryotic cells. We report the characterization of a previously isolated full-length cDNA PpRab1 from Pinus pinaster. Amino acid sequence analysis revealed the presence of G1-G5 conserved domains of the GTPase Ras superfamily and a double cysteine motif in the C-terminal, characteristic of Rab proteins. The PpRab1 protein shows high sequence similarity to several Rab1 GTP-binding proteins in plants. Phylogenetic analysis showed that, within the Ras superfamily, PpRab1 is more closely related to the Rab family and within this, PpRab1 protein was found to cluster with Arabidopsis subfamily AtRABE, whose members are known to regulate ER-to-Golgi membrane trafficking steps. PpRab1 transcripts were expressed at constitutively high levels for the initial stages of zygotic embryo development, and then their relative abundance decreased as embryo matures. The PpRab1 transcript is not embryo-specific as it was found in roots, cotyledons and hypocotyls. An increase in PpRab1 expression level was observed when seeds are germinated and collected at successive time points of development. In situ RT-PCR analysis revealed an expression signal in early zygotic embryos. In view of the proposed roles of Rab1 GTP-binding protein, the possible function of the protein encoded by PpRab1 in embryogenesis is discussed.

  12. ZIRCONIUM-TITANIUM-BERYLLIUM BRAZING ALLOY

    DOEpatents

    Gilliland, R.G.; Patriarca, P.; Slaughter, G.M.; Williams, L.C.

    1962-06-12

    A new and improved ternary alloy is described which is of particular utility in braze-bonding parts made of a refractory metal selected from Group IV, V, and VI of the periodic table and alloys containing said metal as a predominating alloying ingredient. The brazing alloy contains, by weight, 40 to 50 per cent zirconium, 40 to 50 per cent titanium, and the balance beryllium in amounts ranging from 1 to 20 per cent, said alloy having a melting point in the range 950 to 1400 deg C. (AEC)

  13. Characterization of exothermic brazing components Skylab experiment M552

    NASA Technical Reports Server (NTRS)

    Pattee, H. E.; Monroe, R. E.

    1973-01-01

    Information developed to characterize flight and ground based samples from the Exothermic Brazing Experiment is detailed. Included is information developed from metallographic observation, chemical analysis, and measurements of component dimensions. Comparisons of the flight and ground based specimens showed that good quality brazes were obtained. Effects of the zero gravity processing were noted on liquid metal flow and braze alloy-base metal reactions. Unusual metallurgical structures exhibited in the nickel brazes made in Skylab were the result of composition variations apparently related to the time-temperature cycle characteristic of this braze.

  14. Methods to Predict Stresses in Cutting Inserts Brazed Using Iron-Carbon Brazing Alloy

    NASA Astrophysics Data System (ADS)

    Konovodov, V. V.; Valentov, A. V.; Retuynskiy, O. Yu; Esekuev, Sh B.

    2016-04-01

    This work describes a method for predicting residual and operating stresses in a flat-form tool insert made of tungsten free carbides brazed using iron-carbon alloy. According to the studies’ results it is concluded that the recommendations relating to the limitation of a melting point of tool brazing alloys (950-1100°C according to different data) are connected with a negative impact on tools as a composite made of dissimilar materials rather than on hard alloys as a tool material. Due to the cooling process stresses inevitably occur in the brazed joint of dissimilar materials, and these stresses increase with the higher solidification temperature of the brazing alloy.

  15. Molecular and biochemical analyses of OsRab7, a rice Rab7 homolog.

    PubMed

    Nahm, Min Yeop; Kim, Sam Woong; Yun, Daejin; Lee, Sang Yeol; Cho, Moo Je; Bahk, Jeong Dong

    2003-12-01

    Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells.

  16. Active Metal Brazing and Characterization of Brazed Joints in Titanium to Carbon-Carbon Composites

    NASA Technical Reports Server (NTRS)

    Singh, M.; Shpargel, T. P.; Morscher, G. N.; Asthana, R.

    2006-01-01

    The Ti-metal/C-C composite joints were formed by reactive brazing with three commercial brazes, namely, Cu-ABA, TiCuNi, and TiCuSiI. The joint microstructures were examined using optical microscopy and scanning electron microscopy (SEM) coupled with energy dispersive spectrometry (EDS). The results of the microstructure analysis indicate solute redistribution across the joint and possible metallurgical bond formation via interdiffusion, which led to good wetting and spreading. A tube-on-plate tensile test was used to evaluate joint strength of Ti-tube/ C-C composite joints. The load-carrying ability was greatest for the Cu-ABA braze joint structures. This system appeared to have the best braze spreading which resulted in a larger braze/C-C composite bonded area compared to the other two braze materials. Also, joint loadcarrying ability was found to be higher for joint structures where the fiber tows in the outer ply of the C-C composite were aligned perpendicular to the tube axis when compared to the case where fiber tows were aligned parallel to the tube axis.

  17. Brazed bipolar plates for PEM fuel cells

    DOEpatents

    Neutzler, Jay Kevin

    1998-01-01

    A liquid-cooled, bipolar plate separating adjacent cells of a PEM fuel cell comprising corrosion-resistant metal sheets brazed together so as to provide a passage between the sheets through which a dielectric coolant flows. The brazement comprises a metal which is substantially insoluble in the coolant.

  18. Brazed bipolar plates for PEM fuel cells

    DOEpatents

    Neutzler, J.K.

    1998-07-07

    A liquid-cooled, bipolar plate separating adjacent cells of a PEM fuel cell comprises corrosion-resistant metal sheets brazed together so as to provide a passage between the sheets through which a dielectric coolant flows. The brazement comprises a metal which is substantially insoluble in the coolant. 6 figs.

  19. Nickel-chromium-silicon brazing filler metal

    DOEpatents

    Martini, Angelo J.; Gourley, Bruce R.

    1976-01-01

    A brazing filler metal containing, by weight percent, 23-35% chromium, 9-12% silicon, a maximum of 0.15% carbon, and the remainder nickel. The maximum amount of elements other than those noted above is 1.00%.

  20. Welding/brazing for Space Station repair

    NASA Astrophysics Data System (ADS)

    Dickinson, David W.; Babel, H. W.; Conaway, H. R.; Hooper, W. H.

    Viewgraphs on welding/brazing for space station repair are presented. Topics covered include: fabrication and repair candidates; debris penetration of module panel; welded repair patch; mechanical assembly of utility fluid line; space station utility systems; Soviet aerospace fabrication - an overview; and processes under consideration.

  1. Thermal and Fluid Flow Brazing Simulations

    SciTech Connect

    HOSKING, FLOYD MICHAEL; GIANOULAKIS,STEVEN E.; GIVLER,RICHARD C.; SCHUNK,P. RANDALL

    1999-12-15

    The thermal response of fixtured parts in a batch-type brazing furnace can require numerous, time-consuming development runs before an acceptable furnace schedule or joint design is established. Powerful computational simulation tools are being developed to minimize the required number of verification experiments, improve furnace throughput, and increase product yields. Typical furnace simulations are based on thermal, fluid flow, and structural codes that incorporate the fundamental physics of the brazing process. The use of massively parallel computing to predict furnace and joint-level responses is presented. Measured and computed data are compared. Temperature values are within 1-270 of the expected peak brazing temperature for different loading conditions. Sensitivity studies reveal that the thermal response is more sensitive to the thermal boundary conditions of the heating enclosure than variability y in the materials data. Braze flow simulations predict fillet geometry and free surface joint defects. Dynamic wetting conditions, interfacial reactions, and solidification structure add a high degree of uncertainty to the flow results.

  2. Gold-nickel-titanium brazing alloy

    DOEpatents

    Mizuhara, Howard

    1995-01-03

    A brazing alloy in accordance with this invention has the following composition, by weight: 91 to 99 gold, 0.5 to 7% nickel; 0.10 to 2% titanium. Alternatively, with palladium present, the composition is as follows, by weight: 83 to 96% gold; 3 to 10% palladium; 0.5 to 5% nickel; 0.10 to 2% titanium.

  3. Gold-nickel-titanium brazing alloy

    DOEpatents

    Mizuhara, Howard

    1990-07-03

    A brazing alloy in accordance with this invention has the following composition, by weight: 91 to 99% gold, 0.5 to 7% nickel; 0.10 to 2% titanium. Alternatively, with palladium present, the composition is as follows, by weight: 83 to 96% gold; 3 to 10% palladium; 0.5 to 5% nickel; 0.10 to 2% titanium.

  4. Thousands of Rab GTPases for the Cell Biologist

    PubMed Central

    Diekmann, Yoan; Seixas, Elsa; Gouw, Marc; Tavares-Cadete, Filipe; Seabra, Miguel C.; Pereira-Leal, José B.

    2011-01-01

    Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform

  5. Thousands of rab GTPases for the cell biologist.

    PubMed

    Diekmann, Yoan; Seixas, Elsa; Gouw, Marc; Tavares-Cadete, Filipe; Seabra, Miguel C; Pereira-Leal, José B

    2011-10-01

    Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform

  6. Thousands of rab GTPases for the cell biologist.

    PubMed

    Diekmann, Yoan; Seixas, Elsa; Gouw, Marc; Tavares-Cadete, Filipe; Seabra, Miguel C; Pereira-Leal, José B

    2011-10-01

    Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform

  7. Identification of an Oncogenic RAB Protein

    PubMed Central

    Wheeler, Douglas B.; Zoncu, Roberto; Root, David E.; Sabatini, David M.; Sawyers, Charles L.

    2015-01-01

    In an shRNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small GTPase—a protein previously implicated in endomembrane trafficking—as a new regulator of the PI3K pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and co-purifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive PDGFRα to LAMP2-positive endomembranes in the absence of ligand, suggesting there may be latent oncogenic potential in dysregulated endomembrane trafficking. PMID:26338797

  8. Identification of an oncogenic RAB protein.

    PubMed

    Wheeler, Douglas B; Zoncu, Roberto; Root, David E; Sabatini, David M; Sawyers, Charles L

    2015-10-01

    In a short hairpin RNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small guanosine triphosphatase (GTPase)-a protein previously implicated in endomembrane trafficking-as a regulator of the phosphatidylinositol 3'-OH kinase (PI3K) pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and copurifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive platelet-derived growth factor receptor α to LAMP2-positive endomembranes in the absence of ligand, suggesting that there may be latent oncogenic potential in dysregulated endomembrane trafficking.

  9. 49 CFR 178.50 - Specification 4B welded or brazed steel cylinders.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... brazed until complete penetration by the brazing material of the brazed joint is secured. Depth of... of the testing machine may not exceed 1/8 inch per minute during yield strength determination....

  10. 49 CFR 178.55 - Specification 4B240ET welded or brazed cylinders.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... brazed until complete penetration of the joint by the brazing material is secured. Brazed joints may be... of the testing machine may not exceed 1/8 inch per minute during yield strength determination....

  11. 49 CFR 178.50 - Specification 4B welded or brazed steel cylinders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., and be thoroughly brazed until complete penetration by the brazing material of the brazed joint is... of the testing machine may not exceed 1/8 inch per minute during yield strength determination....

  12. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

    PubMed Central

    Rojas, Raul; van Vlijmen, Thijs; Mardones, Gonzalo A.; Prabhu, Yogikala; Rojas, Adriana L.; Mohammed, Shabaz; Heck, Albert J.R.; Raposo, Graça; van der Sluijs, Peter; Bonifacino, Juan S.

    2008-01-01

    The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes. PMID:18981234

  13. Applying Taguchi Methods To Brazing Of Rocket-Nozzle Tubes

    NASA Technical Reports Server (NTRS)

    Gilbert, Jeffrey L.; Bellows, William J.; Deily, David C.; Brennan, Alex; Somerville, John G.

    1995-01-01

    Report describes experimental study in which Taguchi Methods applied with view toward improving brazing of coolant tubes in nozzle of main engine of space shuttle. Dr. Taguchi's parameter design technique used to define proposed modifications of brazing process reducing manufacturing time and cost by reducing number of furnace brazing cycles and number of tube-gap inspections needed to achieve desired small gaps between tubes.

  14. Evaluation of Brazed Joints Using Failure Assessment Diagram

    NASA Technical Reports Server (NTRS)

    Flom, Yury

    2012-01-01

    Fitness-for service approach was used to perform structural analysis of the brazed joints consisting of several base metal / filler metal combinations. Failure Assessment Diagrams (FADs) based on tensile and shear stress ratios were constructed and experimentally validated. It was shown that such FADs can provide a conservative estimate of safe combinations of stresses in the brazed joints. Based on this approach, Margins of Safety (MS) of the brazed joints subjected to multi-axial loading conditions can be evaluated..

  15. GDP dissociation inhibitor domain II required for Rab GTPase recycling.

    PubMed

    Gilbert, P M; Burd, C G

    2001-03-16

    Rab GTPases are localized to distinct subsets of organelles within the cell, where they regulate SNARE-mediated membrane trafficking between organelles. One factor required for Rab localization and function is Rab GDP dissociation inhibitor (GDI), which is proposed to recycle Rab after vesicle fusion by extracting Rab from the membrane and loading Rab onto newly formed transport intermediates. GDI is composed of two domains; Rab binding is mediated by Domain I, and the function of Domain II is not known. In this study, Domain II of yeast GDI, encoded by the essential GDI1/SEC19 gene, was targeted in a genetic screen to obtain mutants that might lend insight into the function of this domain. In one gdi1 mutant, the cytosolic pools of all Rabs tested were depleted, and Rab accumulated on membranes, suggesting that this mutant Gdi1 protein has a general defect in extraction of Rab from membranes. In a second gdi1 mutant, the endosomal/vacuolar Rabs Vps21/Ypt51p and Ypt7p accumulated in the cytosol bound to Gdi1p, but localization of Ypt1p and Sec4p were not significantly affected. Using an in vitro assay which reconstitutes Gdi1p-mediated membrane loading of Rab, this mutant Gdi1p was found to be defective in loading of Vps21p but not Ypt1p. Loading of Vps21p by loading-defective Gdi1p was restored when acceptor membranes prepared from a deletion strain lacking Vps21p were used. These results suggest that membrane-associated Rab may regulate recruitment of GDI-Rab from the cytosol, possibly by regulating a GDI-Rab receptor. We conclude that Domain II of Gdi1p is essential for Rab loading and Rab extraction, and confirm that each of these activities is required for Gdi1p function in vivo.

  16. Several braze filler metals for joining an oxide-dispersion-strengthened nickel-chromium-aluminum alloy

    NASA Technical Reports Server (NTRS)

    Gyorgak, C. A.

    1975-01-01

    An evaluation was made of five braze filler metals for joining an aluminum-containing oxide dispersion-strengthened (ODS) alloy, TD-NiCrAl. All five braze filler metals evaluated are considered suitable for joining TD-NiCrAl in terms of wettability and flow. Also, the braze alloys appear to be tolerant of slight variations in brazing procedures since joints prepared by three sources using three of the braze filler metals exhibited similar brazing characteristics and essentially equivalent 1100 C stress-rupture properties in a brazed butt-joint configuration. Recommendations are provided for brazing the aluminum-containing ODS alloys.

  17. Evaluation of segmented and brazed mirror assemblies

    NASA Astrophysics Data System (ADS)

    Bath, David A.; Spain, David; Ness, Eric; Williams, Steve; Bougoin, Michel

    2005-08-01

    Direct Sintered Silicon Carbide (SSiC) is a promising material for mirror optics due to its low density, high stiffness and high thermal stability. In order to make large mirror optics (over 1 meter diameter), processing limitations to create monolithic structures of this size class require that smaller segments need to be fabricated and then joined in a post sintering operation. Fabrication of segmented nullset 300mm lightweighted concave mirrors to demonstrate different fabrication methods is presented here. The mirrors are comprised of 6 radial segments joined by means of silicon braze technology and are coated with a SSiC Chemical Vapor Deposition (CVD) layer for improved surface finish to reduce straylight scatter. Evaluation of conventional pitch lap polishing of brazed and coated optic surfaces has shown no degradation to surface figure and surface roughness.

  18. The story of laser brazing technology

    NASA Astrophysics Data System (ADS)

    Hoffmann, Peter; Dierken, Roland

    2012-03-01

    This article gives an overview on the development of laser brazing technology as a new joining technique for car body production. The story starts with fundamental research work at German institutes in 1993, continues with the first implementations in automobile production in 1998, gives examples of applications since then and ends with an outlook. Laser brazing adapted design of joints and boundary conditions for a safe processing are discussed. Besides a better understanding for the sensitivity of the process against joint irregularities and misalignment, the key to successful launch was an advanced system technology. Different working heads equipped with wire feeding device, seam tracking system or tactile sensors for an automated teaching are presented in this paper. Novel laser heads providing a two beam technology will allow improved penetration depth of the filler wire and a more ecological processing by means of energy consumption.

  19. Composition and method for brazing graphite to graphite

    DOEpatents

    Taylor, Albert J.; Dykes, Norman L.

    1984-01-01

    The present invention is directed to a brazing material for joining graphite structures that can be used at temperatures up to about 2800.degree. C. The brazing material formed of a paste-like composition of hafnium carbide and uranium oxide with a thermosetting resin. The uranium oxide is converted to uranium dicarbide during the brazing operation and then the hafnium carbide and uranium dicarbide form a liquid phase at a temperature about 2600.degree. C. with the uranium diffusing and vaporizing from the joint area as the temperature is increased to about 2800.degree. C. so as to provide a brazed joint consisting essentially of hafnium carbide. This brazing temperature for hafnium carbide is considerably less than the eutectic temperature of hafnium carbide of about 3150.degree. C. The brazing composition also incorporates the thermosetting resin so that during the brazing operation the graphite structures may be temporarily bonded together by thermosetting the resin so that machining of the structures to final dimensions may be completed prior to the completion of the brazing operation. The resulting brazed joint is chemically and thermally compatible with the graphite structures joined thereby and also provides a joint of sufficient integrity so as to at least correspond with the strength and other properties of the graphite.

  20. Ultrasonic scanning system for inspection of brazed tube joints

    NASA Technical Reports Server (NTRS)

    Haynes, J. L.; Maurer, N. A.

    1972-01-01

    An ultrasonic scanning system used to inspect and evaluate in-place brazed tube joints is considered. The system was designed, developed, and built especially for nondestructive testing and was selected because of its known response to brazing defects not associated with material density changes. The scan system is capable of scanning brazed joints in union, tee, elbow and cross configuration of 3/16-inch through 5/8-inch diameters. It is capable of detecting brazed defects as small as 0.008 by 0.010-inch, which exceeds the 0.015-inch diameter defect resolution required by specification.

  1. 32 CFR 203.14 - RAB/TRC reporting requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... RESTORATION ACTIVITIES § 203.14 RAB/TRC reporting requirements. The community point of contact of the RAB or... of the TAPP project, a summary of services and products obtained, and a statement regarding...

  2. 32 CFR 203.14 - RAB/TRC reporting requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... RESTORATION ACTIVITIES § 203.14 RAB/TRC reporting requirements. The community point of contact of the RAB or... of the TAPP project, a summary of services and products obtained, and a statement regarding...

  3. 32 CFR 203.14 - RAB/TRC reporting requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... RESTORATION ACTIVITIES § 203.14 RAB/TRC reporting requirements. The community point of contact of the RAB or... of the TAPP project, a summary of services and products obtained, and a statement regarding...

  4. The multiple roles of Rab9 in the endolysosomal system

    PubMed Central

    Kucera, Ana; Bakke, Oddmund; Progida, Cinzia

    2016-01-01

    ABSTRACT The small GTPase Rab9 has long been described as a protein that mediates endosome-to-trans-Golgi Network (TGN) transport, and specifically mannose-6-phospate receptor (MPR) recycling. However, studies have challenged this view by showing that Rab9 also is connected to sorting pathways toward the endolysosomal compartments. We recently characterized the spatio-temporal dynamics of Rab9 and, by using live cell imaging, we showed that it enters the endosomal pathway together with CI-MPR at the transition stage between early, Rab5-positive, and late, Rab7a-positive, endosomes. More so, the Rab9 constitutively active mutant, Rab9Q66L, accumulates on late endosomes and promotes carrier formation at the TGN. Here, we discuss our findings in light of previous reports on Rab9 in the retrograde transport pathway. PMID:27574541

  5. The multiple roles of Rab9 in the endolysosomal system.

    PubMed

    Kucera, Ana; Bakke, Oddmund; Progida, Cinzia

    2016-01-01

    The small GTPase Rab9 has long been described as a protein that mediates endosome-to-trans-Golgi Network (TGN) transport, and specifically mannose-6-phospate receptor (MPR) recycling. However, studies have challenged this view by showing that Rab9 also is connected to sorting pathways toward the endolysosomal compartments. We recently characterized the spatio-temporal dynamics of Rab9 and, by using live cell imaging, we showed that it enters the endosomal pathway together with CI-MPR at the transition stage between early, Rab5-positive, and late, Rab7a-positive, endosomes. More so, the Rab9 constitutively active mutant, Rab9Q66L, accumulates on late endosomes and promotes carrier formation at the TGN. Here, we discuss our findings in light of previous reports on Rab9 in the retrograde transport pathway. PMID:27574541

  6. Small RAB GTPases Regulate Multiple Steps of Mitosis.

    PubMed

    Miserey-Lenkei, Stéphanie; Colombo, María I

    2016-01-01

    GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis.

  7. Small RAB GTPases Regulate Multiple Steps of Mitosis

    PubMed Central

    Miserey-Lenkei, Stéphanie; Colombo, María I.

    2016-01-01

    GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis. PMID:26925400

  8. Copper-phosphorus alloys offer advantages in brazing copper

    SciTech Connect

    Rupert, W.D.

    1996-05-01

    Copper-phosphorus brazing alloys are used extensively for joining copper, especially refrigeration and air-conditioning copper tubing and electrical conductors. What is the effect of phosphorus when alloyed with copper? The following are some of the major effects: (1) It lowers the melt temperature of copper (a temperature depressant). (2) It increases the fluidity of the copper when in the liquid state. (3) It acts as a deoxidant or a fluxing agent with copper. (4) It lowers the ductility of copper (embrittles). There is a misconception that silver improves the ductility of the copper-phosphorus alloys. In reality, silver added to copper acts in a similar manner as phosphorus. The addition of silver to copper lowers the melt temperature (temperature depressant) and decreases the ductility. Fortunately, the rate and amount at which silver lowers copper ductility is significantly less than that of phosphorus. Therefore, taking advantage of the temperature depressant property of silver, a Ag-Cu-P alloy can be selected at approximately the same melt temperature as a Cu-P alloy, but at a lower phosphorus content. The lowering of the phosphorus content actually makes the alloy more ductile, not the silver addition. A major advantage of the copper-phosphorus alloys is the self-fluxing characteristic when joining copper to copper. They may also be used with the addition of a paste flux on brass, bronze, and specialized applications on silver, tungsten and molybdenum. Whether it is selection of the proper BCuP alloy or troubleshooting an existing problem, the suggested approach is a review of the desired phosphorus content in the liquid metal and how it is being altered during application. In torch brazing, a slight change in the oxygen-fuel ratio can affect the joint quality or leak tightness.

  9. Roles for the Recycling Endosome, Rab8, and Rab11 in hantavirus release from epithelial cells

    PubMed Central

    Rowe, Regina K.; Suszko, Jason W.; Pekosz, Andrew

    2008-01-01

    Hantavirus structural proteins are believed to localize to intracellular membranes often identified as Golgi membranes, in virus-infected cells. After virus budding into the Golgi luminal space, virus-containing vesicles are transported to the plasma membrane via trafficking pathways that are not well defined. Using the New World hantavirus, Andes virus, we have investigated the role of various Rab proteins in the release of hantavirus particles from infected cells. Rabs 8 and 11 were found to colocalize with Andes virus proteins in virus infected cells and when expressed from cDNA, implicating the recycling endosome as an organelle important for hantavirus infection. Small interfering RNA-mediated downregulation of Rab11a alone or Rab11a and Rab11b together resulted in a decrease in infectious virus particle secretion from infected cells. Downregulation of Rab8a did not alter infectious virus release but reduction of both isoforms did. These data implicate the recycling endosome and the Rab proteins associated with vesicular transport to or from this intracellular organelle as an important pathway for hantavirus trafficking to the plasma membrane. PMID:18951604

  10. Localization of Rab proteins to peroxisomes: a proteomics and immunofluorescence study.

    PubMed

    Gronemeyer, Thomas; Wiese, Sebastian; Grinhagens, Sören; Schollenberger, Lukas; Satyagraha, Ari; Huber, Lukas A; Meyer, Helmut E; Warscheid, Bettina; Just, Wilhelm W

    2013-02-14

    A proteomics screen was initiated to identify Rab proteins regulating transport to and away from peroxisomes. Mass spectrometry-based protein correlation profiling of rat liver organelles and immunofluorescence analysis of the peroxisome candidate Rab proteins revealed Rab6, Rab10, Rab14 and Rab18 to associate with the peroxisomal membrane. While Rab14 localized to peroxisomes predominantly in its dominant-active form, other Rab proteins associated with peroxisomes in both their GTP- and GDP-bound state. In summary, our data suggest that Rab6, Rab10, Rab14 and Rab18 associate with the peroxisomal compartment and similar as previously shown for Rab8, Rab18 in its GDP-bound state favors peroxisome proliferation.

  11. An Ag based brazing system with a tunable thermal expansion for the use as sealant for solid oxide cells

    NASA Astrophysics Data System (ADS)

    Kiebach, Ragnar; Engelbrecht, Kurt; Grahl-Madsen, Laila; Sieborg, Bertil; Chen, Ming; Hjelm, Johan; Norrman, Kion; Chatzichristodoulou, Christodoulos; Hendriksen, Peter Vang

    2016-05-01

    An Ag-Al2TiO5 composite braze was developed and successfully tested as seal for solid oxide cells. The thermo-mechanical properties of the Ag-Al2TiO5 system and the chemical compatibility between this composite braze and relevant materials used in stacks were characterized and the leak rates as a function of the operation temperature were measured. The thermal expansion coefficient in the Ag-Al2TiO5 system can be tailored by varying the amount of the ceramic filler. The brazing process can be carried out in air, the joining partners showed a good chemical stability and sufficient low leak rates were demonstrated. Furthermore, the long-term stability of the Ag-Al2TiO5 composite braze was studied under relevant SOFC and SOEC conditions. The stability of brazed Crofer/Ag-Al2TiO5/NiO-YSZ assemblies in reducing atmosphere and in pure oxygen was investigated over 500 h at 850 °C. Additionally, a cell component test was performed to investigate the durability of the Ag-Al2TiO5 seal when exposed to dual atmosphere. The seals performed well over 900 h under electrolysis operation conditions (-0.5 A cm2, 850 °C), and no cell degradation related to the Ag-Al2TiO5 sealing was found, indicating that the developed braze system is applicable for the use in SOFC/SOEC stacks.

  12. RAB GTPases and RAB-interacting proteins and their role in the control of cognitive functions.

    PubMed

    D'Adamo, Patrizia; Masetti, Michela; Bianchi, Veronica; Morè, Lorenzo; Mignogna, Maria Lidia; Giannandrea, Maila; Gatti, Silvia

    2014-10-01

    A RAS-related class of small monomeric G proteins, the RAB GTPases, is emerging as of key biological importance in compartment specific directional control of vesicles formation, transport and fusion. Thanks to human genetic observation and to the consequent dedicated biochemical work, substantial progress has been made on the understanding of the role played by RAB GTPases and their effector proteins on neuronal development and the shaping of cognitive functions. This review is highlighting these initial elements to broaden the current scope of research on developmental cognitive deficits and take the point of view of RAB GTPases control on membrane transport in neurons and astrocytes.

  13. Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes*

    PubMed Central

    Bultema, Jarred J.; Boyle, Judith A.; Malenke, Parker B.; Martin, Faye E.; Dell'Angelica, Esteban C.; Cheney, Richard E.; Di Pietro, Santiago M.

    2014-01-01

    Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion. PMID:25324551

  14. Silver-palladium braze alloy recovered from masking materials

    NASA Technical Reports Server (NTRS)

    Cierniak, R.; Colman, G.; De Carlo, F.

    1966-01-01

    Method for recovering powdered silver-palladium braze alloy from an acrylic spray binder and rubber masking adhesive used in spray brazing is devised. The process involves agitation and dissolution of masking materials and recovery of suspended precious metal particles on a filter.

  15. Failure Assessment Diagram for Brazed 304 Stainless Steel Joints

    NASA Technical Reports Server (NTRS)

    Flom, Yory

    2011-01-01

    Interaction equations were proposed earlier to predict failure in Albemet 162 brazed joints. Present study demonstrates that the same interaction equations can be used for lower bound estimate of the failure criterion in 304 stainless steel joints brazed with silver-based filler metals as well as for construction of the Failure Assessment Diagrams (FAD).

  16. Split glass tube assures quality in electron beam brazing

    NASA Technical Reports Server (NTRS)

    Kressin, W. J.

    1966-01-01

    Sealed enclosure of heat-resistant glass tubing and silicone rubber molds provide good visibility for electron beam brazing of metal tubes in an inert gas atmosphere. The glass tubing and rubber molds, which are bonded together, are easily applied to and removed from the brazing area by operation of a clamp.

  17. Reactive Boride Brazing on Low-Alloy Automotive Grade Steel

    NASA Astrophysics Data System (ADS)

    Palanisamy, B.; Upadhyaya, A.

    2011-11-01

    Brazing is a widely used process to improve the performance of steels used in automotive applications. The substrate material is often exposed to harsh conditions in these applications and may affect the service life of the component. Reactive boride brazing aims to improve the mechanical properties of the substrate material by forming a ceramic-metal composite coating in a single-step process in situ. In this study, sintered Ancor 4300 low-alloy steel is used as the substrate with chromium-rich braze and chromium-lean braze materials. The mechanical properties of the brazed samples were studied in detail using microindentation hardness measurements and the transverse rupture test. The results indicate that the brazed superlayer has a 10 times higher hardness. There was a significant improvement in the transverse rupture strength of the steel brazed with the chromium-rich boride as compared to the pure substrate material. In an effort to reduce processing time, green compacts of the substrate were also directly brazed and yielded favorable results.

  18. Tube dimpling tool assures accurate dip-brazed joints

    NASA Technical Reports Server (NTRS)

    Beuyukian, C. S.; Heisman, R. M.

    1968-01-01

    Portable, hand-held dimpling tool assures accurate brazed joints between tubes of different diameters. Prior to brazing, the tool performs precise dimpling and nipple forming and also provides control and accurate measuring of the height of nipples and depth of dimples so formed.

  19. Evaluation of Margins of Safety in Brazed Joints

    NASA Technical Reports Server (NTRS)

    Flom, Yury; Wang, Len; Powell, Mollie M.; Soffa, Matthew A.; Rommel, Monica L.

    2009-01-01

    One of the essential steps in assuring reliable performance of high cost critical brazed structures is the assessment of the Margin of Safety (MS) of the brazed joints. In many cases the experimental determination of the failure loads by destructive testing of the brazed assembly is not practical and cost prohibitive. In such cases the evaluation of the MS is performed analytically by comparing the maximum design loads with the allowable ones and incorporating various safety or knock down factors imposed by the customer. Unfortunately, an industry standard methodology for the design and analysis of brazed joints has not been developed. This paper provides an example of an approach that was used to analyze an AlBeMet 162 (38%Be-62%Al) structure brazed with the AWS BAlSi-4 (Al-12%Si) filler metal. A practical and conservative interaction equation combining shear and tensile allowables was developed and validated to evaluate an acceptable (safe) combination of tensile and shear stresses acting in the brazed joint. These allowables are obtained from testing of standard tensile and lap shear brazed specimens. The proposed equation enables the assessment of the load carrying capability of complex brazed joints subjected to multi-axial loading.

  20. Composition and method for brazing graphite to graphite

    DOEpatents

    Taylor, A.J.; Dykes, N.L.

    1982-08-10

    A brazing material is described for joining graphite structures that can be used up to 2800/sup 0/C. The brazing material is formed of a paste-like composition of hafnium carbide and uranium oxide with a thermosetting resin. The uranium oxide is converted to uranium dicarbide during the brazing operation and then the hafnium carbide and uranium dicarbide form a liquid phase at a temperature about 2600/sup 0/C with the uranium diffusing and vaporizing from the joint area as the temperature is increased to about 2800/sup 0/C so as to provide a brazed joint consisting essentially of hafnium carbide. The resulting brazed joint is chemically and thermally compatible with the graphite structures.

  1. Brazing of the Tore Supra actively cooled Phase III Limiter

    SciTech Connect

    Nygren, R.E.; Walker, C.A.; Lutz, T.J.; Hosking, F.M.; McGrath, R.T.

    1993-12-31

    The head of the water-cooled Tore Supra Phase 3 Limiter is a bank of 14 round OFHC copper tubes, curved to fit the plasma radius, onto which several hundred pyrolytic graphite (PG) tiles and a lesser number of carbon fiber composite tiles are brazed. The small allowable tolerances for fitting the tiles to the tubes and mating of compound curvatures made the brazing and fabrication extremely challenging. The paper describes the fabrication process with emphasis on the procedure for brazing. In the fixturing for vacuum furnace brazing, the tiles were each independently clamped to the tube with an elaborate set of window frame clamps. Braze quality was evaluated with transient heating tests. Some rebrazing was necessary.

  2. Flaw Tolerance in Lap Shear Brazed Joints. Part 1

    NASA Technical Reports Server (NTRS)

    Flom, Yury; Wang, Li-Qin

    2003-01-01

    Furnace brazing is a joining process used in the aerospace and other industries to produce strong permanent and hermetic structural joints. As in any joining process, brazed joints have various imperfections and defects. At the present time, our understanding of the influence of the internal defects on the strength of the brazed joints is not adequate. The goal of this 3-part investigation is to better understand the properties and failure mechanisms of the brazed joints containing defects. This study focuses on the behavior of the brazed lap shear joints because of their importance in manufacturing aerospace structures. In Part 1, an average shear strength capability and failure modes of the single lap joints are explored. Stainless steel specimens brazed with pure silver are tested in accordance with the AWS C3.2 standard. Comparison of the failure loads and the ultimate shear strength with the Finite Element Analysis (FEA) of the same specimens as a function of the overlap widths shows excellent correlation between the experimental and calculated values for the defect-free lap joints. A damage zone criterion is shown to work quite well in understanding the failure of the braze joints. In Part 2, the findings of the Part 1 will be verified on the larger test specimens. Also, various flaws will be introduced in the test specimens to simulate lack of braze coverage in the lap joints. Mechanical testing and FEA will be performed on these joints to verify that behavior of the flawed ductile lap joints is similar to joints with a reduced braze area. Finally, in Part 3, the results obtained in Parts 1 and 2 will be applied to the actual brazed structure to evaluate the load-carrying capability of a structural lap joint containing discontinuities. In addition, a simplified engineering procedure will be offered for the laboratory testing of the lap shear specimens.

  3. Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae.

    PubMed

    Chen, Ming-Chyuan; Cheng, Ying-Min; Hong, Min-Chang; Fang, Lee-Shing

    2004-11-19

    The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts. PMID:15485657

  4. The C. elegans rab family: identification, classification and toolkit construction.

    PubMed

    Gallegos, Maria E; Balakrishnan, Sanjeev; Chandramouli, Priya; Arora, Shaily; Azameera, Aruna; Babushekar, Anitha; Bargoma, Emilee; Bokhari, Abdulmalik; Chava, Siva Kumari; Das, Pranti; Desai, Meetali; Decena, Darlene; Saramma, Sonia Dev Devadas; Dey, Bodhidipra; Doss, Anna-Louise; Gor, Nilang; Gudiputi, Lakshmi; Guo, Chunyuan; Hande, Sonali; Jensen, Megan; Jones, Samantha; Jones, Norman; Jorgens, Danielle; Karamchedu, Padma; Kamrani, Kambiz; Kolora, Lakshmi Divya; Kristensen, Line; Kwan, Kelly; Lau, Henry; Maharaj, Pranesh; Mander, Navneet; Mangipudi, Kalyani; Menakuru, Himabindu; Mody, Vaishali; Mohanty, Sandeepa; Mukkamala, Sridevi; Mundra, Sheena A; Nagaraju, Sudharani; Narayanaswamy, Rajhalutshimi; Ndungu-Case, Catherine; Noorbakhsh, Mersedeh; Patel, Jigna; Patel, Puja; Pendem, Swetha Vandana; Ponakala, Anusha; Rath, Madhusikta; Robles, Michael C; Rokkam, Deepti; Roth, Caroline; Sasidharan, Preeti; Shah, Sapana; Tandon, Shweta; Suprai, Jagdip; Truong, Tina Quynh Nhu; Uthayaruban, Rubatharshini; Varma, Ajitha; Ved, Urvi; Wang, Zeran; Yu, Zhe

    2012-01-01

    Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB). PMID:23185324

  5. Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila

    PubMed Central

    Chan, Chih-Chiang; Scoggin, Shane; Wang, Dong; Cherry, Smita; Dembo, Todd; Greenberg, Ben; Jin, Eugene Jennifer; Kuey, Cansu; Lopez, Antonio; Mehta, Sunil Q.; Perkins, Theodore J.; Brankatschk, Marko; Rothenfluh, Adrian; Buszczak, Michael; Hiesinger, P. Robin

    2012-01-01

    Summary Background Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization and function of Rab GTPases in an organism with a brain. Results We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knock-ins in large genomic fragments that are additionally designed to generated mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knock-out of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression. Conclusions Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila. PMID:22000105

  6. The C. elegans Rab Family: Identification, Classification and Toolkit Construction

    PubMed Central

    Gallegos, Maria E.; Balakrishnan, Sanjeev; Chandramouli, Priya

    2012-01-01

    Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB). PMID:23185324

  7. Rab'ing tumor cell migration and invasion: focal adhesion disassembly driven by Rab5.

    PubMed

    Torres, Vicente A

    2014-01-01

    The small GTPase Rab5 has been extensively studied in the context of endocytic trafficking because it is critical in the regulation of early endosome dynamics. In addition to this canonical role, evidence obtained in recent years implicates Rab5 in the regulation of cell migration. This novel role of Rab5 is based not only on an indirect relationship between cell migration and endosomal trafficking as separate processes, but also on the direct regulation of signaling proteins implicated in cell migration. However, the precise mechanisms underlying this connection have remained elusive. Recent studies have shown that the activation of Rab5 is a critical event for maintaining the dynamics of focal adhesions, which is fundamental in regulating not only cell migration but also tumor cell invasion.

  8. Crystal structure of inactive form of Rab3B

    SciTech Connect

    Zhang, Wei; Shen, Yang; Jiao, Ronghong; Liu, Yanli; Deng, Lingfu; Qi, Chao

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer This is the first structural information of human Rab3B. Black-Right-Pointing-Pointer To provides a structural basis for the GDP/GTP switch in controlling the activity of Rab3. Black-Right-Pointing-Pointer The charge distribution of Rab3B indicates its unique roles in vesicular trafficking. -- Abstract: Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 A resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

  9. Multiple Roles of the Small GTPase Rab7.

    PubMed

    Guerra, Flora; Bucci, Cecilia

    2016-01-01

    Rab7 is a small GTPase that belongs to the Rab family and controls transport to late endocytic compartments such as late endosomes and lysosomes. The mechanism of action of Rab7 in the late endocytic pathway has been extensively studied. Rab7 is fundamental for lysosomal biogenesis, positioning and functions, and for trafficking and degradation of several signaling receptors, thus also having implications on signal transduction. Several Rab7 interacting proteins have being identified leading to the discovery of a number of different important functions, beside its established role in endocytosis. Furthermore, Rab7 has specific functions in neurons. This review highlights and discusses the role and the importance of Rab7 on different cellular pathways and processes. PMID:27548222

  10. Multiple Roles of the Small GTPase Rab7

    PubMed Central

    Guerra, Flora; Bucci, Cecilia

    2016-01-01

    Rab7 is a small GTPase that belongs to the Rab family and controls transport to late endocytic compartments such as late endosomes and lysosomes. The mechanism of action of Rab7 in the late endocytic pathway has been extensively studied. Rab7 is fundamental for lysosomal biogenesis, positioning and functions, and for trafficking and degradation of several signaling receptors, thus also having implications on signal transduction. Several Rab7 interacting proteins have being identified leading to the discovery of a number of different important functions, beside its established role in endocytosis. Furthermore, Rab7 has specific functions in neurons. This review highlights and discusses the role and the importance of Rab7 on different cellular pathways and processes. PMID:27548222

  11. Rab GTPase mediated procollagen trafficking in ascorbic acid stimulated osteoblasts.

    PubMed

    Nabavi, Noushin; Pustylnik, Sofia; Harrison, Rene E

    2012-01-01

    Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.

  12. Rab7 induces clearance of α-synuclein aggregates.

    PubMed

    Dinter, Elisabeth; Saridaki, Theodora; Nippold, Markus; Plum, Sarah; Diederichs, Leonie; Komnig, Daniel; Fensky, Luisa; May, Caroline; Marcus, Katrin; Voigt, Aaron; Schulz, Jörg B; Falkenburger, Björn H

    2016-09-01

    Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, we hypothesized that over-expressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis, we expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila melanogaster. In HEK293 cells, EGFP-Rab7-decorated vesicles contain α-synuclein. Rab7 over-expression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Time-lapse microscopy confirmed that particles frequently disappeared with Rab7 over-expression. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 over-expression, presumably representing autolysosomes. Rab7 over-expression reduced apoptosis and the percentage of dead cells in trypan blue staining. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. These beneficial effects were not produced by Rab7 missense mutations causing Charcot Marie Tooth neuropathy, or by the related GTPases Rab5, Rab9, or Rab23. Using mass spectrometry, we identified Rab7 in neuromelanin granules purified from human substantia nigra, indicating that Rab7 might be involved in the biogenesis of these possibly protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease. Cells over-expressing aggregation-prone A53T alpha-synuclein develop cytoplasmic aggregates mimicking changes observed in

  13. The Rab GTPase RabA4d regulates pollen tube tip growth in Arabidopsis thaliana.

    PubMed

    Szumlanski, Amy L; Nielsen, Erik

    2009-02-01

    During reproduction in flowering plants, pollen grains form a tube that grows in a polarized fashion through the female tissues to eventually fertilize the egg cell. These highly polarized pollen tubes have a rapid rate of growth that is supported by a tip-focused delivery of membrane and cell wall components. To gain a better understanding of how this growth is regulated, we investigated the function RABA4D, a member of the Arabidopsis thaliana RabA4 subfamily of Rab GTPase proteins. Here, we show that RABA4D was expressed in a pollen-specific manner and that enhanced yellow fluorescent protein (EYFP)-RabA4d-labeled membrane compartments localized to the tips of growing pollen tubes. Mutant pollen in which the RABA4D gene was disrupted displayed bulged pollen tubes with a reduced rate of growth in vitro and displayed altered deposition of some cell wall components. Expression of EYFP-RabA4d restored wild-type phenotypes to the raba4d mutant pollen tubes, while expression of EYFP-RabA4b did not rescue the raba4d phenotype. In vivo, disruption of RABA4D resulted in a male-specific transmission defect with mutant raba4d pollen tubes displaying aberrant growth in the ovary and reduced guidance at the micropyle. We propose that RabA4d plays an important role in the regulation of pollen tube tip growth.

  14. Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

    PubMed Central

    Liu, Ou; Grant, Barth D.

    2015-01-01

    The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions. PMID:26393361

  15. Molecular mechanism of constitutively active Rab11A was revealed by crystal structure of Rab11A S20V.

    PubMed

    Choi, Jae Young; Shin, Young-Cheul; Yoon, Jong Hwan; Kim, Chang Min; Lee, Jun Hyuck; Jeon, Ju-Hong; Park, Hyun Ho

    2016-03-01

    Rab11A is a small GTP-binding protein involved in the regulation of vesicle trafficking during recycling of endosomes. Substitution of S20 to V (S20V) at Rab11A inhibits the GTP hydrolysis activity of Rab11A. This mutation is known to be constitutively in an active form. Here, we report the crystal structure of the human Rab11A S20V mutant form complexed with GTP at a resolution of 2.4 Å. Without adding any substrate, Rab11A contained non-hydrolyzed natural substrate GTP in the nucleotide binding pocket with Mg(2+). In our observations, substituted V20 of Rab11A was found to interfere with proper localization of the water molecule, which mediated GTP hydrolysis, resulting in GTP being locked in an active form of Rab11A S20V.

  16. Brazing copper to dispersion-strengthened copper

    SciTech Connect

    Ryding, D.G.; Allen, D.; Lee, R.

    1996-08-01

    The Advanced Photon Source (APS) is a state-of-the-art synchrotron light source that will produce intense x-ray beams, which will allow the study of smaller samples and faster reactions and processes at a greater level of detail that has been possible to date. The beam is produced by using third-generation insertion devices in a 7 GeV electron/positron storage ring that is 1100 meters in circumference. The heat load from these intense high power devices is very high and certain components must sustain total heat loads of 3 to 15 kW and heat fluxes of 30 W/mm{sup 2}. Because the beams will cycle on and off many times, thermal shock and fatigue will be a problem. High heat flux impinging on a small area causes a large thermal gradient that results in high stress. GlidCop{reg_sign}, a dispersion strengthened copper, is the desired material because of its high thermal conductivity and superior mechanical properties as compared to copper and its alloys. GlidCop is not amenable to joining by fusion welding, and brazing requires diligence because of high diffusivity. Brazing procedures were developed using optical and scanning electron microscopy.

  17. Cathodic ARC surface cleaning prior to brazing

    SciTech Connect

    Dave, V. R.; Hollis, K. J.; Castro, R. G.; Smith, F. M.; Javernick, D. A.

    2002-01-01

    Surface cleanliness is one the critical process variables in vacuum furnace brazing operations. For a large number of metallic components, cleaning is usually accomplished either by water-based alkali cleaning, but may also involve acid etching or solvent cleaning / rinsing. Nickel plating may also be necessary to ensure proper wetting. All of these cleaning or plating technologies have associated waste disposal issues, and this article explores an alternative cleaning process that generates minimal waste. Cathodic arc, or reserve polarity, is well known for welding of materials with tenacious oxide layers such as aluminum alloys. In this work the reverse polarity effect is used to clean austenitic stainless steel substrates prior to brazing with Ag-28%Cu. This cleaning process is compared to acid pickling and is shown to produce similar wetting behavior as measured by dynamic contact angle experiments. Additionally, dynamic contact angle measurements with water drops are conducted to show that cathodic arc cleaning can remove organic contaminants as well. The process does have its limitations however, and alloys with high titanium and aluminum content such as nickel-based superalloys may still require plating to ensure adequate wetting.

  18. Rene 95 brazed joint metallurgical program

    NASA Technical Reports Server (NTRS)

    Gay, C.; Givens, J.; Mastrorroco, S.; Sterman, A.

    1972-01-01

    This metallurgical program was specifically conducted for the establishment of material properties required for the design of the LF460 fan. The LF460 lift fan is an advanced 18:1 high thrust to weight single stage design. It has a turbine attached to the outer flowpath of the fan blade tip which minimizes the axial depth of the fan. Advanced lightweight attachment designs are employed in this concept to achieve minimum mass polar moments of inertia which are required for good aircraft flight response control. The design features which are unique to this advanced LF460 lift fan are the 0.010 inch thin Udimet 700 alloy integral tip turbine design, minimum weight braze attachment of the turbine to the fan blade, and the high strength and elevated temperature capability of the Rene'95 alloy for the fan blade. The data presented in this report show that the LF460 fan rotor design is feasible and that the design stresses and margins of safety were more than adequate. Prior to any production application, however, additional stress rupture/shear lap joints should be run in order to establish a firm 1200 F stress rupture curve for the CM50 braze metal.

  19. Coating system to permit direct brazing of ceramics

    DOEpatents

    Cadden, Charles H.; Hosking, F. Michael

    2003-01-01

    This invention relates to a method for preparing the surface of a ceramic component that enables direct brazing using a non-active braze alloy. The present invention also relates to a method for directly brazing a ceramic component to a ceramic or metal member using this method of surface preparation, and to articles produced by using this brazing method. The ceramic can be high purity alumina. The method comprises applying a first coating of a silicon-bearing oxide material (e.g. silicon dioxide or mullite (3Al.sub.2 O.sub.3.2SiO.sub.2) to the ceramic. Next, a thin coating of active metal (e.g. Ti or V) is applied. Finally, a thicker coating of a non-active metal (e.g. Au or Cu) is applied. The coatings can be applied by physical vapor deposition (PVD). Alternatively, the active and non-active metals can be co-deposited (e.g. by sputtering a target made of mullite). After all of the coatings have been applied, the ceramic can be fired at a high temperature in a non-oxidizing environment to promote diffusion, and to enhance bonding of the coatings to the substrate. After firing, the metallized ceramic component can be brazed to other components using a conventional non-active braze alloy. Alternatively, the firing and brazing steps can be combined into a single step. This process can replace the need to perform a "moly-manganese" metallization step.

  20. Identification and characterization of a member of Rab subfamily, Rab8, from Clonorchis sinensis.

    PubMed

    Liang, Pei; He, Lei; Yu, Jinyun; Xie, Zhizhi; Chen, Xueqing; Mao, Qiang; Liang, Chi; Huang, Yan; Lu, Gang; Yu, Xinbing

    2015-05-01

    The Rabs act as a binary molecular switch that utilizes the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins. It regulates a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, and intracellular membrane trafficking in eukaryotes. The Rab8 from Clonorchis sinensis (CsRab8) was composed of 199 amino acids. The deduced amino acid sequence shared above 50% identities with other species from trematode, tapeworm, mammal, insecta, nematode, and reptile, respectively. The homologous analysis of sequences showed the conservative domains: G1 box (GDSGVGKS), G2 box (T), G3 box (DTAG), G4 box (GNKCDL), and G5 box. In addition, the structure modeling had also shown other functional domains: GTP/Mg(2+) binding sites, switch I region, and switch II region. A phylogenic tree analysis indicated that the CsRab8 was clustered with the Rab from Schistosoma japonicum, and trematode and tapeworm came from the same branch, which was different from an evolutional branch built by other species, such as mammal animal, insecta, nematode, and reptile. The recombinant CsRab8 protein was expressed in Escherichia coli and the purified protein was a soluble molecule by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. CsRab8 was identified as a component of excretory/secretory products of C. sinensis by western blot analysis. The transcriptional level of CsRab8 at metacercaria stage was the highest at the four stages and higher by 56.49-folds than that at adult worm, 1.23-folds than that at excysted metacercaria, and 2.69-folds than that at egg stage. Immunohistochemical localization analysis showed that CsRab8 was specifically distributed in the tegument, vitellarium, eggs, and testicle of adult worms, and detected on the vitellarium and tegument of metacercaria. Combined with the results, CsRab8 is indispensable for survival and development of parasites, especially for regulating

  1. UNC-108/Rab2 Regulates Postendocytic Trafficking in Caenorhabditis elegans

    PubMed Central

    Chun, Denise K.; McEwen, Jason M.; Burbea, Michelle

    2008-01-01

    After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking. PMID:18434599

  2. Cloning of two genes encoding Rab7 in Paramecium.

    PubMed

    Surmacz, Liliana; Wiejak, Jolanta; Wyroba, Elzbieta

    2006-01-01

    Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.

  3. Relationship between the expression of Rab family GTPases and neuropeptide hormones in the brain of Bombyx mori.

    PubMed

    Uno, Tomohide; Sakamoto, Kazuki; Isoyama, Yuri; Hiragaki, Susumu; Uno, Yuichi; Kanamaru, Kengo; Yamagata, Hiroshi; Takagi, Michihiro; Mizoguchi, Akira; Takeda, Makio

    2013-02-01

    Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects.

  4. Braze system and method for reducing strain in a braze joint

    DOEpatents

    Cadden, Charles H.; Goods, Steven H.; Prantil, Vincent C.

    2004-05-11

    A system for joining a pair of structural members having widely differing coefficients of thermal expansion is disclosed. A mechanically "thick" foil is made by dispersing a refractory metal powder, such as molybdenum, niobium, tantalum, or tungsten into a quantity of a liquid, high expansion metal such as copper, silver, or gold, casting an ingot of the mixture, and then cutting sections of the ingot about 1 mm thick to provide the foil member. These foil members are shaped, and assembled between surfaces of structural members for joining, together with a layer of a braze alloy on either side of the foil member capable of wetting both the surfaces of the structural members and the foil. The assembled body is then heated to melt the braze alloy and join the assembled structure. The foil member subsequently absorbs the mechanical strain generated by the differential contraction of the cooling members that results from the difference in the coefficients of thermal expansion of the members.

  5. Effect of filler metal composition on the strength of yttria stabilized zirconia joints brazed with Pd-Ag-CuOx

    SciTech Connect

    Darsell, Jens T.; Weil, K. Scott

    2008-09-08

    The Ag-CuOx system is of interest to be used to be used as an air braze filler metal for joining high temperature electrochemical devices. Previous work has shown that the melting temperatures can be increased by adding palladium to Ag-CuOx and it is expected that this may aid high temperature stability. This work compares the room temperature bend strength of joints made between yttria-stabilized zirconia (YSZ) air brazed using Ag-CuOx without palladium and with 5 and 15mol% palladium additions. It has been found that in general palladium decreases joint strength, especially in low copper oxide compositions filler metals. At high copper oxide contents, brittle fracture through both copper oxide rich phases and the YSZ limits joint strength.

  6. Rab GTPases bind at a common site within the angiotensin II type I receptor carboxyl-terminal tail: evidence that Rab4 regulates receptor phosphorylation, desensitization, and resensitization.

    PubMed

    Esseltine, Jessica L; Dale, Lianne B; Ferguson, Stephen S G

    2011-01-01

    The human angiotensin II type 1 receptor (AT₁R) is a member of the G protein-coupled receptor (GPCR) superfamily and represents an important target for cardiovascular therapeutic intervention. Agonist-activation of the AT₁R induces β-arrestin-dependent endocytosis to early endosomes in which the receptor resides as a protein complex with the Rab GTPase Rab5. In the present study, we examined whether other Rab GTPases that regulate receptor trafficking through endosomal compartments also bind to the AT₁R. We find that Rab4, Rab7, and Rab11 all bind to the last 10 amino acid residues of the AT₁R carboxyl-terminal tail. Rab11 binds AT₁R more effectively than Rab5, whereas Rab4 binds less effectively than Rab5. Alanine scanning mutagenesis reveals that proline 354 and cysteine 355 contribute to Rab protein binding, and mutation of these residues does not affect G protein coupling. We find that the Rab GTPases each compete with one another for receptor binding and that although Rab4 interacts poorly with the AT₁R, it effectively displaces Rab11 from the receptor. In contrast, Rab11 overexpression does not prevent Rab4 binding to the AT₁R. Overexpression of wild-type Rab4, but not Rab11, facilitates AT₁R dephosphorylation, and a constitutively active Rab4-Q67L mutant reduces AT₁R desensitization and promotes AT₁R resensitization. Taken together, our data indicate that multiple Rab GTPases bind to a motif localized to the distal end of the AT₁R tail and that increased Rab4 activity may contribute to the regulation AT₁R desensitization and dephosphorylation.

  7. Photograph of John Fedak, marine coppersmith, brazing a funner splice ...

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

    Photograph of John Fedak, marine coppersmith, brazing a funner splice in the process of making a funnel. - Naval Base Philadelphia-Philadelphia Naval Shipyard, Pipe Coppersmith Shop, League Island, Philadelphia, Philadelphia County, PA

  8. Low vapor pressure braze alloys for thermionic energy converters

    NASA Technical Reports Server (NTRS)

    Bair, V. L.

    1976-01-01

    The evaluation of cesium diode electrode materials called for braze fillers with very low vapor pressures and a wide range of melting points. Binary alloys of low vapor pressure refractory metals were chosen to fill this need. These alloys of Th, Zr, Hf, Ru, Nb, Ir, Mo, Ta, Os, Re, and W have reported melting point minima or eutectics from 1,510 K to above 3,000 K. Preliminary data are compiled on the use of several of these braze alloys. Melting points and surface wetting on a Ta base are given. Results of brazing Ir, LaB6, Nb, Re, W, and Zr-22 wt % ZrO2 materials into Ta and Nb-1% Zr bases are presented. Current braze usage is summarized.

  9. Failure Assessment of Stainless Steel and Titanium Brazed Joints

    NASA Technical Reports Server (NTRS)

    Flom, Yury A.

    2012-01-01

    Following successful application of Coulomb-Mohr and interaction equations for evaluation of safety margins in Albemet 162 brazed joints, two additional base metal/filler metal systems were investigated. Specimens consisting of stainless steel brazed with silver-base filler metal and titanium brazed with 1100 Al alloy were tested to failure under combined action of tensile, shear, bending and torsion loads. Finite Element Analysis (FEA), hand calculations and digital image comparison (DIC) techniques were used to estimate failure stresses and construct Failure Assessment Diagrams (FAD). This study confirms that interaction equation R(sub sigma) + R(sub tau) = 1, where R(sub sigma) and R(sub t u) are normal and shear stress ratios, can be used as conservative lower bound estimate of the failure criterion in stainless steel and titanium brazed joints.

  10. Emerging nexus between RAB GTPases, autophagy and neurodegeneration.

    PubMed

    Jain, Navodita; Ganesh, Subramaniam

    2016-05-01

    The RAB class of small GTPases includes the major regulators of intracellular communication, which are involved in vesicle generation through fusion and fission, and vesicular trafficking. RAB proteins also play an imperative role in neuronal maintenance and survival. Recent studies in the field of neurodegeneration have also highlighted the process of autophagy as being essential for neuronal maintenance. Here we review the emerging roles of RAB proteins in regulating macroautophagy and its impact in the context of neurodegenerative diseases. PMID:26985808

  11. Method For Brazing And Thermal Processing

    DOEpatents

    Milewski, John O.; Dave, Vivek R.; Christensen, Dane; Carpenter, II, Robert W.

    2005-07-12

    The present invention includes a method for brazing of two objects or heat treatment of one object. First, object or objects to be treated are selected and initial conditions establishing a relative geometry and material characteristics are determined. Then, a first design of an optical system for directing heat energy onto the object or objects is determined. The initial conditions and first design of the optical system are then input into a optical ray-tracing computer program. The program is then run to produce a representative output of the heat energy input distribution to the object or objects. The geometry of the object or objects, material characteristics, and optical system design are then adjusted until an desired heat input is determined.

  12. Cryogenic performance of slotted brazed Rene 41 honeycomb panels

    NASA Technical Reports Server (NTRS)

    Hepler, A. K.; Swegle, A. R.

    1982-01-01

    Two brazed Rene 41 honeycomb panels that would incorporate a frame element were designed, fabricated and tested. The panels were representative of the lower surface of an advanced space transportation vehicle. The first panel was a two span panel supported by a center frame and on edges parallel to it. The second panel was a two span panel supported by a center frame and on edges parallel to it. The second panel was a three span panel supported on two frames and on edges parallel to the frames. Each panel had its outer skin slotted to reduce the thermal stresses of the panel skins. The first panel was tested under simulated boost conditions that included liquid hydrogen exposure of the frame and inner skin and radiant heat to 478K on the outer skins. The first panel was tested to investigate the effect of thermal stresses in skins and core caused by the panel being restrained by a cold integral frame and to observe the effects of frost formation and possible liquid air development in and around outer skin slots.

  13. Active Metal Brazing of Carbon-Carbon Composites to Titanium

    NASA Technical Reports Server (NTRS)

    Singh, M.; Shpargel, T. P.; Morscher, G.; Asthana, R.

    2004-01-01

    The Ti-metal/C-C composite joints were formed by reactive brazing with three commercial brazes, namely, Cu-ABA, TiCuNi, and TiCuSil. The joint microstructures were examined using optical microscopy, and scanning electron microscopy (SEM) coupled with energy dispersive spectrometry (EDS). The results of the microstructure analysis indicate solute redistribution across the joint which led to good wetting, spreading, and metallurgical bond formation via interdiffusion.

  14. Reactive Brazing of Carbon-Carbon Composites to Titanium

    NASA Technical Reports Server (NTRS)

    Shpargel, Tarah; Singh, M.; Morscher, Gregory; Asthana, Rajiv

    2004-01-01

    The Ti-metal/C-C composite joints were formed by reactive brazing with three commercial brazes, namely, Cu-ABA, TiCuNi, and TiCuSil. The joint microstructures were examined using optical microscopy, and scanning electron microscopy (SEM) coupled with energy dispersive spectrometry (EDS). The results of the microstructure analysis indicate solute redistribution across the joint and possible metallurgical bond formation via interdiffusion, which led to good wetting and spreading.

  15. Ceramic-to-metal stator vane assembly with braze

    DOEpatents

    Chase, Donna J.; Fang, Ho T.; Irwin, Craig W.; Schienle, James L.

    1995-01-01

    A stator vane assembly for a gas turbine engine that includes a plurality of circumferentially spaced ceramic vanes, each of which has an inner and outer ceramic shroud, and a ceramic post extending from one of the shrouds, and a metallic platform having a plurality of circumferentially spaced recesses. The posts are inserted into a metallic sleeve and then brazed. The brazed sleeves are then mounted in the recesses. A method for assembling these components to form the stator assembly is also described.

  16. Improved Assembly for Gas Shielding During Welding or Brazing

    NASA Technical Reports Server (NTRS)

    Gradl, Paul; Baker, Kevin; Weeks, Jack

    2009-01-01

    An improved assembly for inert-gas shielding of a metallic joint is designed to be useable during any of a variety of both laser-based and traditional welding and brazing processes. The basic purpose of this assembly or of a typical prior related assembly is to channel the flow of a chemically inert gas to a joint to prevent environmental contamination of the joint during the welding or brazing process and, if required, to accelerate cooling upon completion of the process.

  17. Mutation of melanosome protein RAB38 in chocolate mice

    PubMed Central

    Loftus, Stacie K.; Larson, Denise M.; Baxter, Laura L.; Antonellis, Anthony; Chen, Yidong; Wu, Xufeng; Jiang, Yuan; Bittner, Michael; Hammer, John A.; Pavan, William J.

    2002-01-01

    Mutations of genes needed for melanocyte function can result in oculocutaneous albinism. Examination of similarities in human gene expression patterns by using microarray analysis reveals that RAB38, a small GTP binding protein, demonstrates a similar expression profile to melanocytic genes. Comparative genomic analysis localizes human RAB38 to the mouse chocolate (cht) locus. A G146T mutation occurs in the conserved GTP binding domain of RAB38 in cht mice. Rab38cht/Rab38cht mice exhibit a brown coat similar in color to mice with a mutation in tyrosinase-related protein 1 (Tyrp1), a mouse model for oculocutaneous albinism. The targeting of TYRP1 protein to the melanosome is impaired in Rab38cht/Rab38cht melanocytes. These observations, and the fact that green fluorescent protein-tagged RAB38 colocalizes with end-stage melanosomes in wild-type melanocytes, suggest that RAB38 plays a role in the sorting of TYRP1. This study demonstrates the utility of expression profile analysis to identify mammalian disease genes. PMID:11917121

  18. Crystal structure of inactive form of Rab3B

    SciTech Connect

    Zhang, Wei; Shen, Yang; Jiao, Ronghong; Liu, Yanli; Deng, Lingfu; Qi, Chao

    2012-06-28

    Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 {angstrom} resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

  19. Rab Proteins and the Compartmentalization of the Endosomal System

    PubMed Central

    Wandinger-Ness, Angela; Zerial, Marino

    2014-01-01

    Of the approximately 70 human Rab GTPases, nearly three-quarters are involved in endocytic trafficking. Significant plasticity in endosomal membrane transport pathways is closely coupled to receptor signaling and Rab GTPase-regulated scaffolds. Here we review current literature pertaining to endocytic Rab GTPase localizations, functions, and coordination with regulatory proteins and effectors. The roles of Rab GTPases in (1) compartmentalization of the endocytic pathway into early, recycling, late, and lysosomal routes; (2) coordination of individual transport steps from vesicle budding to fusion; (3) effector interactomes; and (4) integration of GTPase and signaling cascades are discussed. PMID:25341920

  20. Electron Beam Welder Used to Braze Sapphire to Platinum

    NASA Technical Reports Server (NTRS)

    Forsgren, Roger C.; Vannuyen, Thomas

    1998-01-01

    A new use for electron beam brazing was recently developed by NASA Lewis Research Center's Manufacturing Engineering Division. This work was done to fabricate a fiberoptic probe (developed by Sentec Corporation) that could measure high temperatures less than 600 deg C of vibrating machinery, such as in jet engine combustion research. Under normal circumstances, a sapphire fiber would be attached to platinum by a ceramic epoxy. However, no epoxies can adhere ceramic fibers to platinum under such high temperatures and vibration. Also, since sapphire and platinum have different thermal properties, the epoxy bond is subjected to creep over time. Therefore, a new method had to be developed that would permanently and reliably attach a sapphire fiber to platinum. Brazing a sapphire fiber to a platinum shell. The fiber-optic probe assembly consists of a 0.015-in.-diameter sapphire fiber attached to a 0.25-in.-long, 0.059-in.-diameter platinum shell. Because of the small size of this assembly, electron beam brazing was chosen instead of conventional vacuum brazing. The advantage of the electron beam is that it can generate a localized heat source in a vacuum. Gold reactive braze was used to join the sapphire fiber and the platinum. Consequently, the sapphire fiber was not affected by the total heat needed to braze the components together.

  1. Annular beam shaping system for advanced 3D laser brazing

    NASA Astrophysics Data System (ADS)

    Pütsch, Oliver; Stollenwerk, Jochen; Kogel-Hollacher, Markus; Traub, Martin

    2012-10-01

    As laser brazing benefits from advantages such as smooth joints and small heat-affected zones, it has become established as a joining technology that is widely used in the automotive industry. With the processing of complex-shaped geometries, recent developed brazing heads suffer, however, from the need for continuous reorientation of the optical system and/or limited accessibility due to lateral wire feeding. This motivates the development of a laser brazing head with coaxial wire feeding and enhanced functionality. An optical system is designed that allows to generate an annular intensity distribution in the working zone. The utilization of complex optical components avoids obscuration of the optical path by the wire feeding. The new design overcomes the disadvantages of the state-of-the-art brazing heads with lateral wire feeding and benefits from the independence of direction while processing complex geometries. To increase the robustness of the brazing process, the beam path also includes a seam tracking system, leading to a more challenging design of the whole optical train. This paper mainly discusses the concept and the optical design of the coaxial brazing head, and also presents the results obtained with a prototype and selected application results.

  2. Microstructural and Mechanical Characterization of Actively Brazed Alumina Specimens

    SciTech Connect

    Hosking, F.M.; Cadden, C.H.; Stephens, J.J.; Glass, S.J.; Yang, N.Y.C.; Vianco, P.V.; Walker, C.A.

    1999-08-26

    Alumina (94 and 99.8% grade compositions) was brazed directly to itself with gold-based active brazing alloys (ABA's) containing vanadium additions of 1,2 and 3 weight percent. The effects of brazing conditions on the joint properties were investigated. Wetting behavior, interfacial reactions, microstructure, hermeticity and tensile strength were determined. Wetting was fair to good for the ABA and base material combinations. Microanalysis identified a discontinuous Al-V-O spinel reaction product at the alumina-braze interface. Tensile strength results for 94% alumina were uniformly good and generally not sensitive to the vanadium concentration, with tensile values of 85-105 MPa. There was more variability in the 99.8% alumina strength results, with values ranging from 25-95 MPa. The highest vanadium concentration (3 wt. %) yielded the highest joint strength for the brazed 99.8% alumina. Failures in the 99.8% alumina samples occurred at the braze-alumina interface, while the 94% alumina specimens exhibited fracture of the ceramic substrate.

  3. Brazing of Stainless Steel to Yttria-Stabilized Zirconia Using Gold-Based Brazes for Solid Oxide Fuel Cell Applications

    NASA Technical Reports Server (NTRS)

    Singh, M.; Shpargel, T. P.; Asthana, R.

    2007-01-01

    Two gold-base active metal brazes (gold-ABA and gold-ABA-V) were evaluated for oxidation resistance to 850 C, and used to join yttria-stabilized zirconia (YSZ) to a corrosion-resistant ferritic stainless steel for possible use in solid oxide fuel cells. Thermogravimetric analysis and optical microscopy and scanning electron microscopy coupled with energy-dispersive spectroscopy were used to evaluate the braze oxidation behavior, and microstructure and composition of the YSZ/braze/steel joints. Both gold-ABA and gold-ABA-V exhibited nearly linear oxidation kinetics at 850 C, with gold-ABA-V showing faster oxidation than gold-ABA. Both brazes produced metallurgically sound YSZ/steel joints due to chemical interactions of Ti and V with the YSZ and steel substrates.

  4. Brazing open cell reticulated copper foam to stainless steel tubing with vacuum furnace brazed gold/indium alloy plating

    DOEpatents

    Howard, Stanley R.; Korinko, Paul S.

    2008-05-27

    A method of fabricating a heat exchanger includes brush electroplating plated layers for a brazing alloy onto a stainless steel tube in thin layers, over a nickel strike having a 1.3 .mu.m thickness. The resultant Au-18 In composition may be applied as a first layer of indium, 1.47 .mu.m thick, and a second layer of gold, 2.54 .mu.m thick. The order of plating helps control brazing erosion. Excessive amounts of brazing material are avoided by controlling the electroplating process. The reticulated copper foam rings are interference fit to the stainless steel tube, and in contact with the plated layers. The copper foam rings, the plated layers for brazing alloy, and the stainless steel tube are heated and cooled in a vacuum furnace at controlled rates, forming a bond of the copper foam rings to the stainless steel tube that improves heat transfer between the tube and the copper foam.

  5. Similarities of Drosophila rab GTPases based on expression profiling: completion and analysis of the rab-Gal4 kit.

    PubMed

    Jin, Eugene Jennifer; Chan, Chih-Chiang; Agi, Egemen; Cherry, Smita; Hanacik, Elizabeth; Buszczak, Michael; Hiesinger, P Robin

    2012-01-01

    We recently generated rab-Gal4 lines for 25 of 29 predicted Drosophila rab GTPases. These lines provide tools for the expression of reporters, mutant rab variants or other genes, under control of the regulatory elements of individual rab loci. Here, we report the generation and characterization of the remaining four rab-Gal4 lines. Based on the completed 'rab-Gal4 kit' we performed a comparative analysis of the cellular and subcellular expression of all rab GTPases. This analysis includes the cellular expression patterns in characterized neuronal and non-neuronal cells and tissues, the subcellular localization of wild type, constitutively active and dominant negative rab GTPases and colocalization with known intracellular compartment markers. Our comparative analysis identifies all Rab GTPases that are expressed in the same cells and localize to the same intracellular compartments. Remarkably, similarities based on these criteria are typically not predicted by primary sequence homology. Hence, our findings provide an alternative basis to assess potential roles and redundancies based on expression in developing and adult cell types, compartment identity and subcellular localization.

  6. A Paired RNAi and RabGAP Overexpression Screen Identifies Rab11 as a Regulator of β-Amyloid Production

    PubMed Central

    Udayar, Vinod; Buggia-Prévot, Virginie; Guerreiro, Rita L.; Siegel, Gabriele; Rambabu, Naresh; Soohoo, Amanda L.; Ponnusamy, Moorthi; Siegenthaler, Barbara; Bali, Jitin; Guerreiro, Rita; Brás, José; Sassi, Celeste; Gibbs, J. Raphael; Hernandez, Dena; Lupton, Michelle K.; Brown, Kristelle; Morgan, Kevin; Powell, John; Singleton, Andrew; Hardy, John; Simons, Mikael; Ries, Jonas; Puthenveedu, Manojkumar A.; Hardy, John; Thinakaran, Gopal; Rajendran, Lawrence

    2014-01-01

    Summary Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides, which are generated from amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aβ levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aβ production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled β-secretase endosomal recycling to the plasma membrane and thus affected Aβ production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aβ levels and show how systems biology approaches can unravel the molecular complexity underlying AD. PMID:24373285

  7. Warburg Micro syndrome is caused by RAB18 deficiency or dysregulation

    PubMed Central

    Handley, Mark T.; Carpanini, Sarah M.; Mali, Girish R.; Sidjanin, Duska J.; Aligianis, Irene A.; Jackson, Ian J.; FitzPatrick, David R.

    2015-01-01

    RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary ‘RAB3GAP’ complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20. PMID:26063829

  8. Warburg Micro syndrome is caused by RAB18 deficiency or dysregulation.

    PubMed

    Handley, Mark T; Carpanini, Sarah M; Mali, Girish R; Sidjanin, Duska J; Aligianis, Irene A; Jackson, Ian J; FitzPatrick, David R

    2015-06-01

    RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary 'RAB3GAP' complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20. PMID:26063829

  9. Online monitoring of the laser brazing of titanium overlap joints

    NASA Astrophysics Data System (ADS)

    Schmitt, R.; Vielhaber, K.; Donst, D.; Klocke, F.

    2007-06-01

    Image processing and thermography for its own are very versatile and established measurement techniques for many years. However, the combination of these two measurement technologies opens a new field of applications. The online monitoring of the laser-brazing of titanium overlap joints is such a new application. The laser brazing process for overlap joining of formed titanium sheets for the production of heat exchangers is presently being investigated at the Fraunhofer IPT. In comparison to conventional furnace brazing the laser brazing technology decreases substantially the heat impact and thus reduces the thermal material damage in the parts due to local selective heating in a laser beam focal spot. Even though the process is stable, errors in the brazing seam such as pores or unacceptable material oxidation can occur. To ensure a high quality an online process monitoring or even process control is necessary. But since the surface remains unchanged during this brazing process no geometrical inspection of the surface can be conducted. Therefore today's quality assurance performs x-ray or destructive testing. This paper demonstrates how the use of thermography in combination with image processing allows a machine integrated online monitoring of the laser brazing process. First the basic principals are presented which cover the fields of heat coupling, heat transmission and heat distribution as well as the temperature emission of light and the spectral properties of the laser beam shaping optic and so lead to the optical set-up. Then analysis algorithms are derived which characterize the process, detect process failures and make a seam tracking possible.

  10. 49 CFR 178.51 - Specification 4BA welded or brazed steel cylinders.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... skirt or flange of the head and must be thoroughly brazed until complete penetration by the brazing... must be set at the calculated corresponding strain. (iv) Cross-head speed of the testing machine...

  11. Capillary brazing under microgravity (TEXUS-II) and 1g conditions

    NASA Astrophysics Data System (ADS)

    Frieler, K.; Phlippovich, N.; Stickler, R.; Bathke, W.

    Experiments of vacuum brazing under both microgravity and 1-g conditions show the effect of hydrostatic pressure on ga-filling. The absence of buoyancy forces under microgravity affects the microstructure of the solidified braze in the joint.

  12. 32 CFR 202.9 - Conducting RAB meetings.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... under the installation's Community Relations Plan, a public library, or in the offices of the...) MISCELLANEOUS RESTORATION ADVISORY BOARDS Operating Requirements § 202.9 Conducting RAB meetings. (a) Public participation. RAB meetings shall be open to the public. (1) The installation co-chair shall prepare and...

  13. Rab proteins: The key regulators of intracellular vesicle transport

    SciTech Connect

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  14. RAB24 facilitates clearance of autophagic compartments during basal conditions

    PubMed Central

    Ylä-Anttila, Päivi; Mikkonen, Elisa; Happonen, Kaisa E; Holland, Petter; Ueno, Takashi; Simonsen, Anne; Eskelinen, Eeva-Liisa

    2015-01-01

    RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions. PMID:26325487

  15. 32 CFR 202.4 - Composition of a RAB.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 2 2011-07-01 2011-07-01 false Composition of a RAB. 202.4 Section 202.4 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS RESTORATION ADVISORY BOARDS General Requirements § 202.4 Composition of a RAB. (a) Membership....

  16. 32 CFR 202.4 - Composition of a RAB.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 2 2014-07-01 2014-07-01 false Composition of a RAB. 202.4 Section 202.4 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS RESTORATION ADVISORY BOARDS General Requirements § 202.4 Composition of a RAB. (a) Membership....

  17. 32 CFR 202.4 - Composition of a RAB.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 2 2013-07-01 2013-07-01 false Composition of a RAB. 202.4 Section 202.4 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS RESTORATION ADVISORY BOARDS General Requirements § 202.4 Composition of a RAB. (a) Membership....

  18. 32 CFR 202.4 - Composition of a RAB.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 2 2012-07-01 2012-07-01 false Composition of a RAB. 202.4 Section 202.4 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS RESTORATION ADVISORY BOARDS General Requirements § 202.4 Composition of a RAB. (a) Membership....

  19. Two Rab2 interactors regulate dense-core vesicle maturation.

    PubMed

    Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M

    2014-04-01

    Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1, and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

  20. Substrate Effects on the High Temperature Oxidation Behavior of a Gold-Based Braze Filler Metal

    SciTech Connect

    Weil, K. Scott; Rice, Joseph P.

    2005-06-30

    Oxidation testing was conducted on a commercial gold-based braze alloy, Gold ABA, and on zirconia and stainless steel joining couples prepared using this braze filler metal. Preliminary results reveal that both substrates play a significant role in determining the overall oxidation resistance of the brazed joint.

  1. Inert-gas welding and brazing enclosure fabricated from sheet plastic

    NASA Technical Reports Server (NTRS)

    Wisner, J. P.

    1965-01-01

    Custom-fabricated plastic bag maintains an inert-gas atmosphere for welding and brazing certain metals. The bag fits over part of the workpieces and the welding and brazing tools. It is also used for metal brazing and fusion plating which require an inert-gas atmosphere.

  2. bMERB domains are bivalent Rab8 family effectors evolved by gene duplication.

    PubMed

    Rai, Amrita; Oprisko, Anastasia; Campos, Jeremy; Fu, Yangxue; Friese, Timon; Itzen, Aymelt; Goody, Roger S; Gazdag, Emerich Mihai; Müller, Matthias P

    2016-01-01

    In their active GTP-bound form, Rab proteins interact with proteins termed effector molecules. In this study, we have thoroughly characterized a Rab effector domain that is present in proteins of the Mical and EHBP families, both known to act in endosomal trafficking. Within our study, we show that these effectors display a preference for Rab8 family proteins (Rab8, 10, 13 and 15) and that some of the effector domains can bind two Rab proteins via separate binding sites. Structural analysis allowed us to explain the specificity towards Rab8 family members and the presence of two similar Rab binding sites that must have evolved via gene duplication. This study is the first to thoroughly characterize a Rab effector protein that contains two separate Rab binding sites within a single domain, allowing Micals and EHBPs to bind two Rabs simultaneously, thus suggesting previously unknown functions of these effector molecules in endosomal trafficking. PMID:27552051

  3. Design, synthesis, and characterization of peptide-based rab geranylgeranyl transferase inhibitors.

    PubMed

    Tan, Kui-Thong; Guiu-Rozas, Ester; Bon, Robin S; Guo, Zhong; Delon, Christine; Wetzel, Stefan; Arndt, Sabine; Alexandrov, Kirill; Waldmann, Herbert; Goody, Roger S; Wu, Yao-Wen; Blankenfeldt, Wulf

    2009-12-24

    Rab geranylgeranyl transferase (RabGGTase) catalyzes the attachment of geranylgeranyl isoprenoids to Rab guanine triphosphatases, which are key regulators in vesicular transport. Because geranylgeranylation is required for proper function and overexpression of Rabs has been observed in various cancers, RabGGTase may be a target for novel therapeutics. The development of selective inhibitors is, however, difficult because two related enzymes involved in other cellular processes exist in eukaryotes and because RabGGTase recognizes protein substrates indirectly, resulting in relaxed specificity. We report the synthesis of a peptidic library based on the farnesyl transferase inhibitor pepticinnamin E. Of 469 compounds investigated, several were identified as selective for RabGGTase with low micromolar IC(50) values. The compounds were not generally cytotoxic and inhibited Rab isoprenylation in COS-7 cells. Crystal structure analysis revealed that selective inhibitors interact with a tunnel unique to RabGGTase, implying that this structural motif is an attractive target for improved RabGGTase inhibitors.

  4. bMERB domains are bivalent Rab8 family effectors evolved by gene duplication.

    PubMed

    Rai, Amrita; Oprisko, Anastasia; Campos, Jeremy; Fu, Yangxue; Friese, Timon; Itzen, Aymelt; Goody, Roger S; Gazdag, Emerich Mihai; Müller, Matthias P

    2016-08-23

    In their active GTP-bound form, Rab proteins interact with proteins termed effector molecules. In this study, we have thoroughly characterized a Rab effector domain that is present in proteins of the Mical and EHBP families, both known to act in endosomal trafficking. Within our study, we show that these effectors display a preference for Rab8 family proteins (Rab8, 10, 13 and 15) and that some of the effector domains can bind two Rab proteins via separate binding sites. Structural analysis allowed us to explain the specificity towards Rab8 family members and the presence of two similar Rab binding sites that must have evolved via gene duplication. This study is the first to thoroughly characterize a Rab effector protein that contains two separate Rab binding sites within a single domain, allowing Micals and EHBPs to bind two Rabs simultaneously, thus suggesting previously unknown functions of these effector molecules in endosomal trafficking.

  5. Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

    PubMed Central

    Iversen, Tore-Geir; Skretting, Grethe; Llorente, Alicia; Nicoziani, Paolo; van Deurs, Bo; Sandvig, Kirsten

    2001-01-01

    The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway. PMID:11452006

  6. Endosome to Golgi transport of ricin is independent of clathrin and of the Rab9- and Rab11-GTPases.

    PubMed

    Iversen, T G; Skretting, G; Llorente, A; Nicoziani, P; van Deurs, B; Sandvig, K

    2001-07-01

    The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.

  7. Graphite-to-304SS Braze Joining by Active Metal-Brazing Technique: Improvement of Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Ray, Ajoy K.; Kar, Abhijit; Kori, S. A.; Pathak, L. C.; Sonnad, A. N.

    2013-01-01

    In the present investigation, an attempt has been made to improve the mechanical strength of graphite-stainless steel-brazed joint. Due to high capillary action, the liquid filler alloy usually tends to percolate into the pores of graphite causing severe stress in the graphite near the joint interface resulting in poor joint strength of 10-15 MPa. In the present investigation, a thin coating of SiC was applied on graphite before the joining process to avoid the penetration of liquid filler alloy into the pores of the graphite. Active filler alloy Ag-Cu-Ti was used to braze the substrates. The brazing was carried out at 850, 900, 950, and 1000 °C. The characterization of the interfaces of the brazed joints was carried out using scanning electron microscopy attached with energy dispersive spectroscopy and x-ray diffraction analysis. From the correlation between the microstructural and mechanical properties, shear strength of approximately 35 MPa for graphite-304SS-brazed joint produced at 900 °C was demonstrated. After the shear tests, the fracture surfaces were analyzed by SEM-EDS.

  8. Improved Air-Treatment Canister

    NASA Technical Reports Server (NTRS)

    Boehm, A. M.

    1982-01-01

    Proposed air-treatment canister integrates a heater-in-tube water evaporator into canister header. Improved design prevents water from condensing and contaminating chemicals that regenerate the air. Heater is evenly spiraled about the inlet header on the canister. Evaporator is brazed to the header.

  9. Development of the copper-tin diffusion-brazing process

    SciTech Connect

    Sangha, S.P.S.; Jacobson, D.M.; Peacock, A.T.

    1998-10-01

    The copper-tin diffusion-brazing process has been studied with the objective of applying it to the joining of plasma-facing beryllium tiles to copper-based heat sinks in a nuclear-fusion reactor. The process is silver-free -- an essential requirement for this application -- and can be carried out at temperatures below 700 C (1,292 F). This approach produces thin joints of essentially pure copper of high thermal conductance with the requisite strength. Satisfactory conditions for achieving robust joints under the constraints demanded by the nuclear-fusion application have been established. The roles of the process parameters -- thickness of the filler metal tin, the compressive loading applied to the components during the brazing cycle and the brazing temperature -- have been assessed.

  10. Rab3-GEF Controls Active Zone Development at the Drosophila Neuromuscular Junction1,2,3

    PubMed Central

    Bae, Haneui; Chen, Shirui; Roche, John P.; Ai, Minrong; Wu, Chunlai

    2016-01-01

    Abstract Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. Here we identify Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca2+ channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. We show that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. Our results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse. PMID:27022630

  11. Distinct and opposing roles for Rab27a/Mlph/MyoVa and Rab27b/Munc13-4 in mast cell secretion.

    PubMed

    Singh, Rajesh K; Mizuno, Koichi; Wasmeier, Christina; Wavre-Shapton, Silene T; Recchi, Chiara; Catz, Sergio D; Futter, Clare; Tolmachova, Tanya; Hume, Alistair N; Seabra, Miguel C

    2013-02-01

    Mediator release from mast cells is a critical step in allergic and inflammatory disease. However, the processes regulating the latter stages of granule release are yet to be fully understood. Rab27 small GTPases regulate release of secretory lysosomes in a variety of cells, including mast cell granules. In the present study, using murine bone marrow-derived mast cells (BMMC) from Rab27-deficient mutant mice, we found that, in contrast to Rab27b, Rab27a primarily plays an inhibitory role in regulating degranulation. Immunofluorescence analysis revealed that resting Rab27a-deficient (ashen) BMMCs display abnormal cortical F-actin distribution. Actin disassembly prior to IgE cross-linking increased wild-type BMMC secretion to ashen levels, suggesting that changes in the integrity of cortical F-actin underlie the ashen phenotype. Comparison of the secretory impairment of Rab27b knockout and Rab27a/b double knockout BMMCs highlighted a secondary positive role for Rab27a in enhancing degranulation. Rab27 is known to interact with actin via its effectors melanophilin (Mlph) and myosin Va (MyoVa) in other cell types. To better understand the differing roles of Rab27 proteins, we analysed the secretory phenotype of BMMCs derived from mice lacking Rab27 effector proteins. These experiments revealed that the phenotype of BMMCs deficient in Mlph (leaden) and BMMCs deficient in MyoVa (dilute) resembles the hyper-secretion of ashen BMMCs, while Munc13-4-deficient (jinx) BMMCs phenocopy the Rab27b knockout and double Rab27a/b knockout secretory impairment. We conclude that Rab27a and Rab27b regulate distinct steps in the BMMC degranulation pathway, with Rab27a/Mlph/MyoVa regulating cortical actin stability upstream of Rab27a/b/Munc13-4-dependent granule exocytosis.

  12. ApRab3, a biosynthetic Rab protein, accumulates on the maturing phagosomes and symbiosomes in the tropical sea anemone, Aiptasia pulchella.

    PubMed

    Hong, Ming-Cheng; Huang, Yung-Sen; Lin, Wen-Wen; Fang, Lee-Shing; Chen, Ming-Chyuan

    2009-03-01

    Symbiosome biogenesis and function are central to the endosymbiotic interaction between symbiotic dinoflagellates and their host cnidarians. To understand these important organelles, we have been conducting studies to identify and characterize symbiosome-associated proteins of the Rab family, key regulatory components of vesicular trafficking and membrane fusion in eukaryotic cells. Our prior studies have implicated three endocytic Rab proteins in the regulation of symbiosome biogenesis. Here, we show that ApRab3 is a new member of the Rab3 subfamily, associating with symbiosomes and accumulating on the maturing phagosomes in the A. pulchella digestive cells. ApRab3 is 78% identical to human Rab3C, and contains all Rab 3-specific signature motifs. EGFP-ApRab3-labeled vesicular structures tended to either align along the cell peripheral, or aggregate at one side of the nucleus. ApRab3 specifically co-distributed with the TGN marker, WGA, but not other organelle-specific markers tested. Immunofluorescence staining with a specific peptide antibody showed similar results. Significantly, an expression of a constitutively active mutant caused the enlargement and random dispersion of EGFP-ApRab3-decorated compartments in PC12 cells. Together, these data suggest that ApRab3 is a new member of the Rab3 subfamily, participating in the biosynthetic trafficking pathway, and symbiosome biogenesis involves an interaction with ApRab3-positive vesicles. PMID:19110066

  13. Laser beam active brazing of metal ceramic joints

    NASA Astrophysics Data System (ADS)

    Haferkamp, Heinz; Bach, Friedrich W.; von Alvensleben, Ferdinand; Kreutzburg, K.

    1996-04-01

    The use of engineering ceramics is becoming more and more important. Reasons for this are the specific properties of these materials, such as high strength, corrosion resistance and wear resistance. To apply the advantages of ceramics, joining techniques of metal ceramic parts are required. In this paper, joining of metal ceramic joints by laser beam brazing is presented. This joining technique is characterized by local heat input, and the minimal thermal stress of the brazed components. During the investigations, an Nd:YAG laser and a vacuum chamber were applied. The advantages of Nd:YAG lasers are the simple mechanical construction, and laser beam guidance via quartz glass fibers, which leads to high handling flexibility. In addition, most of the materials show a high absorption rate for this kind of radiation. As materials, ceramic Al2O3 with a purity of 99.4% and metals such as X5CrNi189 and Fe54Ni29Co17 were used. As a filler material, commercially available silver and silver- copper brazes with chemically active elements like titanium were employed. During this study, the brazing wetting behavior and the formation of diffusion layers in dependence on processing parameters were investigated. The results have shown that high brazing qualities can be achieved by means of the laser beam brazing process. Crack-free joining of metal ceramic parts is currently only possible by the use of metals such as Fe54Ni29Co17 because of its low thermal expansion coefficient, which reduces thermal stresses within the joining zone.

  14. LIFE CYCLE DESIGN OF AIR INTAKE MANIFOLDS; PHASE I: 2.0 L FORD CONTOUR AIR INTAKE MANIFOLD

    EPA Science Inventory

    The project team applied the life cycle design methodology to the design analysis of three alternative air intake manifolds: a sand cast aluminum, brazed aluminum tubular, and nylon composite. The design analysis included a life cycle inventory analysis, environmental regulatory...

  15. Air

    MedlinePlus

    ... do to protect yourself from dirty air . Indoor air pollution and outdoor air pollution Air can be polluted indoors and it can ... this chart to see what things cause indoor air pollution and what things cause outdoor air pollution! Indoor ...

  16. Rab6 functions in polarized transport in Drosophila photoreceptors

    PubMed Central

    Satoh, Takunori; Nakamura, Yuri; Satoh, Akiko K.

    2016-01-01

    ABSTRACT Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport. Although the functions of Rab6 in the Golgi apparatus are well known, its function in polarized transport is unexpected. The mutant phenotype and localization of Rab6 strongly indicate that Rab6 regulates transport between the trans-Golgi network (TGN) and recycling endosomes (REs): basolateral cargos are segregated at the TGN before Rab6 functions, but cargos going to multiple apical domains are sorted at REs. Both the medial-Golgi resident protein Metallophosphoesterase (MPPE) and the TGN marker GalT::CFP exhibit diffused co-localized distributions in Rab6-deficient cells, suggesting they are trapped in the retrograde transport vesicles returning to trans-Golgi cisternae. Hence, we propose that Rab6 regulates the fusion of retrograde transport vesicles containing medial, trans-Golgi resident proteins to the Golgi cisternae, which causes Golgi maturation to REs. PMID:27116570

  17. Numerical simulation of filler metal droplets spreading in laser brazing

    NASA Astrophysics Data System (ADS)

    Chen, Yanbin; Feng, Xiaosong; Li, Liqun

    2007-11-01

    A finite element model was constructed using a commercial software Fidap to analyze the Cu-base filler metal droplet spreading process in laser brazing, in which the temperature distribution, droplet geometry, and fluid flow velocity were calculated. Marangoni and buoyancy convection and gravity force were considered, and the effects of laser power and spot size on the spreading process were evaluated. Special attention was focused on the free surface of the droplet, which determines the profile of the brazing spot. The simulated results indicate that surface tension is the dominant flow driving force and laser spot size determines the droplet spreading domain.

  18. Radioisotope tracer studies in the NASA Skylab ethothermic brazing experiment M-552

    NASA Technical Reports Server (NTRS)

    Braski, D. N.; Adair, H. L.; Kobisk, E. H.

    1974-01-01

    The first use of radioisotope tracer for mapping flow patterns during brazing of metal components in a space environment (near-zero gravity) proved successful. A nickel ferrule was brazed to a nickel tube with Lithobraze BT (71.8% Ag, 28% Cu, 0.2% Li) which contained a trace amount of radioactive Ag-110. Mapping of the flow of the braze alloy in the annulus formed between the tube and the concentric ferrule was determined by counting the radiation intensity as a function of position in the braze joint. Significant information concerning the thermal history of the braze was determined.

  19. Arabidopsis Rab Geranylgeranyltransferases Demonstrate Redundancy and Broad Substrate Specificity in Vitro.

    PubMed

    Shi, Wan; Zeng, Qin; Kunkel, Barbara N; Running, Mark P

    2016-01-15

    Posttranslational lipid modifications mediate the membrane attachment of Rab GTPases, facilitating their function in regulating intracellular vesicular trafficking. In Arabidopsis, most Rab GTPases have two C-terminal cysteines and potentially can be double-geranylgeranylated by heterodimeric Rab geranylgeranyltransferases (Rab-GGTs). Genes encoding two putative α subunits and two putative β subunits of Rab-GGTs have been annotated in the Arabidopsis thaliana genome, but little is known about Rab-GGT activity in Arabidopsis. In this study, we demonstrate that four different heterodimers can be formed between putative Arabidopsis Rab-GGT α subunits RGTA1/RGTA2 and β subunits RGTB1/RGTB2, but only RGTA1·RGTB1 and RGTA1·RGTB2 exhibit bona fide Rab-GGT activity, and they are biochemically redundant in vitro. We hypothesize that RGTA2 function might be disrupted by a 12-amino acid insertion in a conserved motif. We present evidence that Arabidopsis Rab-GGTs may have preference for prenylation of C-terminal cysteines in particular positions. We also demonstrate that Arabidopsis Rab-GGTs can not only prenylate a great variety of Rab GTPases in the presence of Rab escort protein but, unlike Rab-GGT in yeast and mammals, can also prenylate certain non-Rab GTPases independently of Rab escort protein. Our findings may help to explain some of the phenotypes of Arabidopsis protein prenyltransferase mutants. PMID:26589801

  20. Interfacial Microstructure Evolution and Shear Strength of Titanium Sandwich Structures Fabricated by Brazing

    NASA Astrophysics Data System (ADS)

    Wang, Wentao; Fan, Minyu; Li, Jinlong; Tao, Jie

    2016-03-01

    The corrugated sandwich structure, consisting of a CP Ti (commercially pure titanium) core between two Ti-6Al-4V face sheets, was brazed using pasty Ti-37.5Zr-15Cu-10Ni as filler alloy, at the temperature of 870°C for 5, 10, 20, and 30 min. The effect of brazing time on the microstructure and elemental distribution of the brazed joints was examined by means of SEM, EDS, and XRD analyses. It was found that various intermetallic phases were formed in the brazed joints, following a brazing time of 5 min, and their contents were decreased by the increment of brazing time, while prolonged brazing time resulted in a fine, acicular Widmanstätten microstructure throughout the entire joint. In addition, shear testing was performed in the brazed corrugated specimens in order to indirectly assess the quality of the joints. The debonding between CP Ti and Ti-6Al-4V was observed in the specimen brazed for 5 min and the fracture of the CP Ti corrugated core occurred after 30 min of brazing time. Additionally, when brazed for 10 min or 20 min, brittle intermetallic compounds in the joints and the grain growth of the base metal were controllable. Therefore, the sandwich structures failed without debonding in the joints or fracture within the base metal, demonstrating a good combination of strength and ductility.

  1. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants.

    PubMed

    Maringer, Katherine; Saheb, Entsar; Bush, John

    2014-01-01

    Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA), and dominant negative (DN) forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell-cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

  2. High Temperature Strength of YSZ Joints Brazed with Palladium Silver Copper Oxide Filler Metals

    SciTech Connect

    Darsell, Jens T.; Weil, K. Scott

    2010-06-09

    The Ag-CuOx system is being investigated as potential filler metals for use in air brazing high-temperature electrochemical devices such as solid oxide fuel cells and gas concentrators. The current study examines the effects of palladium addition on the high temperature joint strength of specimens prepared from yttria stabilized zirconia (YSZ) bars brazed with the binary Ag-CuOx, and 15Pd-Ag-CuO. It was found that while the binary Ag-CuOx system exhibits stronger room temperature strength than the 15Pd system the strength is reduced to values equivalent of the 15Pd system at 800°C. The 15Pd system exhibits a lower ambient temperature strength that is retained at 800°C. In both systems the failure mechanism at high temperature appears to be peeling of the noble metal component from the oxide phases and tearing through the noble metal phase whereas sufficient adhesion is retained at lower temperatures to cause fracture of the YSZ substrate.

  3. C. elegans Rassf homolog, rasf-1, is functionally associated with rab-39 Rab GTPase in oxidative stress response.

    PubMed

    Takenaka, Motohiko; Inoue, Hideki; Takeshima, Atsushi; Kakura, Tomonori; Hori, Toshiyuki

    2013-03-01

    The Ras association domain family (Rassf) is one of the Ras effectors, which can bind to several GTP-charged Ras-like GTPases. The Rassf proteins are widely conserved beyond species from nematode to human. To explore the novel functions of Rassf proteins, we took advantage of nematode C. elegans as a model animal with only one Rassf homolog, T24F1.3 (rasf-1). The rasf-1-mutant as well as rasf-1-knockdown animals were found to be more sensitive to oxidative stress of arsenite than in wild type, indicating that rasf-1 is involved in oxidative stress response. We next screened for proteins that interact with RASF-1 by the yeast two-hybrid system and identified RAB-39 Rab GTPase as an interacting partner of RASF-1. We not only confirmed specific binding between these molecules but also demonstrated that RASF-1 binds to GTP-bound form but not GDP-bound form of RAB-39. Importantly, rab-39 mutant animals were also sensitive to oxidative stress, which was dependent on rasf-1 according to the epistasis analysis. Moreover, Rassf1 and Rab39, mammalian homologs of rasf-1 and rab-39, respectively, were shown to interact with each other in vitro. These results indicate that the RASF-1 functionally interacts with RAB-39 and that the interaction between their homologs is conserved in mammals.

  4. Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

    PubMed

    Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

    2016-02-12

    The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. PMID:26637356

  5. Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

    PubMed

    Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

    2016-02-12

    The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.

  6. Cloning and characterization of ApRab4, a recycling Rab protein of Aiptasia pulchella, and its implication in the symbiosome biogenesis.

    PubMed

    Hong, Ming-Cheng; Huang, Yung-Sen; Song, Pa-Ching; Lin, Wen-Wen; Fang, Lee-Shing; Chen, Ming-Chyuan

    2009-01-01

    The biogenesis of Symbiodinium symbiosome in the host cells of the sea anemone, Aiptasia pulchella, involves retention of ApRab5 on and exclusion of ApRab11 from the organelle. One predicted consequence of this differential Rab association is the constant membrane fusion of symbiosomes with endocytic vesicles in the absence of parallel membrane retrieval and the subsequent formation of spacious symbiosomes, which nevertheless, contradicts the common perception. To solve this discrepancy, we determined whether membrane fusion occurs between symbiosomes and endocytic vesicles, and whether ApRab11-independent recycling is involved in symbiosome biogenesis. By using the biotin-avidin detection system, we found evidence for symbiosome-endocytic vesicle fusion. Cloning and characterization of ApRab4, an A. pulchella homolog of Rab4, showed that ApRab4 is associated with both the early endocytic and the perinuclear recycling compartments, and its normal function is required for the organization of the recycling compartments. Immunostaining localized ApRab4 to the symbiosome membrane, partially overlapping with ApRab5-decorated microdomains. Significantly, a treatment that impaired Symbiodinium photosynthesis also abolished symbiosome association of ApRab4. Furthermore, ApRab4 was quickly recruited to newly formed phagosomes, but prolonged association only occurred in those harboring live zooxanthelllae. We propose that ApRab4 retention on the symbiosome is an essential part of the mechanism for the biogenesis of Symbiodinium symbiosome. PMID:19459008

  7. 46 CFR 56.75-20 - Brazing qualification.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Brazing qualification. 56.75-20 Section 56.75-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING PIPING SYSTEMS AND... and Pressure Vessel Code (incorporated by reference; see 46 CFR 56.01-2) and part 57 of...

  8. More About Brazing Or Welding NiAl Without Filler

    NASA Technical Reports Server (NTRS)

    Moore, Thomas J.; Kalinowski, Joseph M.

    1996-01-01

    Two reports present additional information about two processes for joining, brazing, or welding workpieces made of nickel aluminide alloys, without use of filler metal. Joining processes involve uniform heating in vacuum-controlled furnace. Eliminates internal thermal gradients in workpieces joined and greatly reduces tendency toward cracking.

  9. Brazed boron-silicon carbide/aluminum structural panels

    NASA Technical Reports Server (NTRS)

    Arnold, W. E., Jr.; Bales, T. T.; Brooks, T. G.; Lawson, A. G.; Mitchell, P. D.; Royster, D. M.; Wiant, R.

    1978-01-01

    Fluxless brazing process minimizes degradation of mechanical properties composite material of silicon carbide coated boron fibers in an aluminum matrix. Process is being used to fabricate full-scale Boron-Silicon Carbide/Aluminum-Titanium honeycomb core panels for flight testing and ground testing.

  10. Braze alloy holds bonding strength over wide temperature range

    NASA Technical Reports Server (NTRS)

    1966-01-01

    Copper-based quaternary alloys of the solid solution type is used for vacuum furnace brazing of large stainless steel components at a maximum temperature of 1975 deg F. The alloy has high bonding strength and good ductility over a temperature range extending from the cryogenic region to approximately 800 deg F.

  11. Low-energy gamma ray inspection of brazed aluminum joints

    NASA Technical Reports Server (NTRS)

    Brown, J. A.

    1967-01-01

    Americium 241 serves as a suitable radioisotope /gamma ray source/ and exposure probe for radiographic inspection of brazed aluminum joints in areas of limited accessibility. The powdered isotope is contained in a sealed capsule mounted at the end of a spring-loaded pushrod in the probe assembly.

  12. Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

    PubMed Central

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Kim, Hyo Jeong; Ko, Myoung Seok; Seol, Wongi; Joe, Yeonsoo; Chung, Hun Taeg; Lee, Byung Ju; Moon, Chang Hoon; Cho, Wha Ja; Park, Jeong Woo

    2016-01-01

    The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling. PMID:26582392

  13. 32 CFR 202.9 - Conducting RAB meetings.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... applicable, it is recommended that the meeting also be announced on the installation's Web site. (2) Each RAB... all participants, including persons with disabilities. (3) Presentation materials and readable...

  14. 32 CFR 202.9 - Conducting RAB meetings.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... applicable, it is recommended that the meeting also be announced on the installation's Web site. (2) Each RAB... all participants, including persons with disabilities. (3) Presentation materials and readable...

  15. 32 CFR 202.9 - Conducting RAB meetings.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... applicable, it is recommended that the meeting also be announced on the installation's Web site. (2) Each RAB... all participants, including persons with disabilities. (3) Presentation materials and readable...

  16. 32 CFR 202.9 - Conducting RAB meetings.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... applicable, it is recommended that the meeting also be announced on the installation's Web site. (2) Each RAB... all participants, including persons with disabilities. (3) Presentation materials and readable...

  17. Rapidly evolving Rab GTPase paralogs and reproductive isolation in Drosophila.

    PubMed

    Hutter, Pierre

    2007-01-01

    Alterations at the X-linked Hmr gene of Drosophila melanogaster can fully restore viability and partially restore fertility in hybrid flies from crosses between D. melanogaster and any of its three most closely related species. Although more than one gene is expected to be involved in these barriers to reproduction, a single DNA-binding protein was recently identified as HMR. The Hmr gene was shown to evolve unusually fast, a feature that supports its role in causing genetic incompatibility in a hybrid genotype. The current treatment of hybrid genetics focuses not only on Hmr but also on the Rab9D gene, which lies only 1kb from Hmr. Rab9D is proposed also to influence hybrid viability. This gene has remained tightly linked to Hmr for about 10 million years, but it has diverged even more than Hmr with regard to D. melanogaster and its most closely related species. Furthermore, the 197-amino acid RAB9D protein contains four amino acid substitutions in the D. melanogaster-rescuing mutant Hmr1. Rab9D is shown to have evolved under very strong positive selection and to be the most recent member of a cluster of six paralogs that encode small RAB GTPases. Four of the six paralogs are unique to D. melanogaster in which they have diverged considerably, their encoded proteins sharing less than 50% amino acid identities with proteins from their orthologs in the closest species. Only two Rab orthologs are present in these sibling species and none is present in the genomes of more distantly related Drosophila species. Rapidly evolving Rab paralogs near the Hmr locus probably developed functional specialization of redundant proteins involved in trafficking macromolecules between cytoplasm and nucleus. Positive selection acting on duplicates of these Rab genes appears to participate in reproductive isolation.

  18. α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

    PubMed

    Castillo-Badillo, Jean A; Sánchez-Reyes, Omar B; Alfonzo-Méndez, Marco A; Romero-Ávila, M Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  19. α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

    PubMed Central

    Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  20. α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

    PubMed

    Castillo-Badillo, Jean A; Sánchez-Reyes, Omar B; Alfonzo-Méndez, Marco A; Romero-Ávila, M Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  1. RAB26 coordinates lysosome traffic and mitochondrial localization.

    PubMed

    Jin, Ramon U; Mills, Jason C

    2014-03-01

    As they mature, professional secretory cells like pancreatic acinar and gastric chief cells induce the transcription factor MIST1 (also known as BHLHA15) to substantially scale up production of large secretory granules in a process that involves expansion of apical cytoplasm and redistribution of lysosomes and mitochondria. How a scaling factor like MIST1 rearranges cellular architecture simply by regulating expression levels of its transcriptional targets is unknown. RAB26 is a MIST1 target whose role in MIST1-mediated secretory cell maturation is also unknown. Here, we confirm that RAB26 expression, unlike most Rabs which are ubiquitously expressed, is tissue specific and largely confined to MIST1-expressing secretory tissues. Surprisingly, functional studies showed that RAB26 predominantly associated with LAMP1/cathepsin D lysosomes and not directly with secretory granules. Moreover, increasing RAB26 expression - by inducing differentiation of zymogen-secreting cells or by direct transfection - caused lysosomes to coalesce in a central, perinuclear region. Lysosome clustering in turn caused redistribution of mitochondria into distinct subcellular neighborhoods. The data elucidate a novel function for RAB26 and suggest a mechanism for how cells could increase transcription of key effectors to reorganize subcellular compartments during differentiation.

  2. Low-melting-point titanium-base brazing alloys—part 2: Characteristics of brazing Ti-21Ni-14Cu on Ti-6Al-4v substrate

    NASA Astrophysics Data System (ADS)

    Chang, E.; Chen, C.-H.

    1997-12-01

    Filler metal of a low-melting-point (917 °C) Ti-21Ni-14Cu was brazed onto the substrate of Ti-6Al-4V alloy at 960 °C for 2,4, and 8 h to investigate the microstructural evolution and electrochemical characteristics of the brazed metal as a function of the period of brazing time. Optical microscopy, scanning and transmission electron microscopy, and x-ray diffractometry were used to characterize the microstructure and phase of the brazed metal; also, the potentiostat was used for corrosion study. Experimental results indicate that diffusion of copper and nickel from the filler metal into the equiaxed a plus intergranular β structure of Ti-6Al-4V substrate causes the lamellar Widmanstätten structure to form. The intermetallic Ti2Ni phase existing in the prior filler metal diminishes, while the Ti2Cu phase can be identified for the metal brazed at 960 °C for 2 h, but the latter phase decreases with time. Advantage might be taken from the evidence of faster diffusion of nickel than copper along the β phase to the substrate. In deaerated Hank’s solution, corrosion potential, corrosion current density, and critical potential for active-to-passive transition decrease while the passivation range broadens with the period of brazing time. However, all the brazed metals, immersed for different periods in oxygen-saturated Hank’s solution, show similar corrosion behavior, irrespective of the brazing time.

  3. Low-melting-point titanium-base brazing alloys. Part 2: Characteristics of brazing Ti-21Ni-14Cu on Ti-6Al-4V substrate

    SciTech Connect

    Chang, E.; Chen, C.H.

    1997-12-01

    Filler metal of a low-melting-point (917 C) Ti-21Ni-14Cu was brazed onto the substrate of Ti-6Al-4V alloy at 960 C for 2, 4, and 8 h to investigate the microstructural evolution and electrochemical characteristics of the brazed metal as a function of the period of brazing time. Optical microscopy, scanning and transmission electron microscopy, and x-ray diffractometry were used to characterize the microstructure and phase of the brazed metal; also, the potentiostat was used for corrosion study. Experimental results indicate that diffusion of copper and nickel from the filler metal into the equiaxed {alpha} plus intergranular {beta} structure of Ti-6Al-4V substrate causes the lamellar Widmanstaetten structure to form. The intermetallic Ti{sub 2}Ni phase existing in the prior filler metal diminishes, while the Ti{sub 2}Cu phase can be identified for the metal brazed at 960 C for 2 h, but the latter phase decreases with time. Advantage might be taken from the evidence of faster diffusion of nickel than copper along the {beta} phase to the substrate. In deaerated Hank`s solution, corrosion potential, corrosion current density, and critical potential for active-to-passive transition decrease while the passivation range broadens with the period of brazing time. However, all the brazed metals, immersed for different periods in oxygen-saturated Hank`s solution, show similar corrosion behavior, irrespective of the brazing time.

  4. Maturation of late Golgi cisternae into RabE(RAB11) exocytic post-Golgi carriers visualized in vivo.

    PubMed

    Pantazopoulou, Areti; Pinar, Mario; Xiang, Xin; Peñalva, Miguel A

    2014-08-15

    The mechanism(s) by which proteins traverse and exit the Golgi are incompletely understood. Using Aspergillus nidulans hyphae, we show that late Golgi cisternae undergo changes in composition to gradually lose Golgi identity while acquiring post-Golgi RabE(RAB11) identity. This behavior of late Golgi cisternae is consistent with the cisternal maturation model. Post-Golgi RabE(RAB11) carriers travel to, and accumulate at, the apex, indicating that fusion is rate limiting for exocytosis. These carriers, which are loaded with kinesin, dynein, and MyoE(MYO5), move on a microtubule-based bidirectional conveyor belt relaying them to actin, which ultimately focuses exocytosis at the apex. Dynein drags RabE(RAB11) carriers away if engagement of MyoE(MYO5) to actin cables fails. Microtubules seemingly cooperating with F-actin capture can sustain secretion if MyoE(MYO5) is absent. Thus, filamentous fungal secretion involving post-Golgi carriers is remarkably similar, mechanistically, to the transport of melanosomes in melanocyte dendrites, even though melanosome biogenesis involves lysosomes rather than Golgi. PMID:24943841

  5. The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis.

    PubMed

    Kwon, Soon Il; Cho, Hong Joo; Jung, Jin Hee; Yoshimoto, Kohki; Shirasu, Ken; Park, Ohkmae K

    2010-10-01

    The tracheary elements (TEs) of the xylem serve as the water-conducting vessels of the plant vascular system. To achieve this, TEs undergo secondary cell wall thickening and cell death, during which the cell contents are completely removed. Cell death of TEs is a typical example of developmental programmed cell death that has been suggested to be autophagic. However, little evidence of autophagy in TE differentiation has been provided. The present study demonstrates that the small GTP binding protein RabG3b plays a role in TE differentiation through its function in autophagy. Differentiating wild type TE cells were found to undergo autophagy in an Arabidopsis culture system. Both autophagy and TE formation were significantly stimulated by overexpression of a constitutively active mutant (RabG3bCA), and were inhibited in transgenic plants overexpressing a dominant negative mutant (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid insensitive mutant bri1-301, and an autophagy mutant atg5-1. Taken together, our results suggest that autophagy occurs during TE differentiation, and that RabG3b, as a component of autophagy, regulates TE differentiation.

  6. The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh.

    PubMed

    Lång, V; Palva, E T

    1992-12-01

    We have isolated a rab-related (responsive to ABA) gene, rab18 from Arabidopsis thaliana. The gene encodes a hydrophilic, glycine-rich protein (18.5 kDa), which contains the conserved serine- and lysine-rich domains characteristic of similar RAB proteins in other plant species. The rab18 mRNA accumulates in plants exposed to low temperature, water stress or exogenous ABA but not in plants subjected to heat shock. This stress-related accumulation of the rab18 mRNA is markedly decreased in the ABA-synthesis mutant aba-1, the ABA-response mutant abi-1 or in wild-type plants treated with the carotenoid synthesis inhibitor, fluridone. Exogenous ABA treatment can induce the rab18 mRNA in the aba-1 mutant but not in the abi-1 mutant. These results provide direct genetic evidence for the ABA-dependent regulation of the rab18 gene in A. thaliana.

  7. Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

    PubMed Central

    Casanova, James E.; Wang, Xiaoye; Kumar, Ravindra; Bhartur, Sheela G.; Navarre, Jennifer; Woodrum, Julie E.; Altschuler, Yoram; Ray, Greg S.; Goldenring, James R.

    1999-01-01

    Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways. PMID:9880326

  8. Dissimilar joint characteristics of SiC and WC-Co alloy by laser brazing

    NASA Astrophysics Data System (ADS)

    Nagatsuka, K.; Sechi, Y.; Nakata, K.

    2012-08-01

    SiC and WC-Co alloys were joined by laser brazing with an active braze metal. The braze metal based on eutectic Ag-Cu alloy with additional Ti as an active element ranging from 0 to 2.8 mass% was sandwiched by the SiC block and WC-Co alloy plate. The brazing was carried out by selective laser beam irradiation on the WC-Co alloy plate. The content of Ti in the braze metal was required to exceed 0.6 mass% in order to form a brazed joint with a measurable shear strength. The shear strength increased with increasing Ti content up to 2.3 mass%Ti and decreased with a higher content.

  9. The influence of metallic brazing materials on the strain formation of internally water-cooled X-ray optics.

    PubMed

    Oberta, P; Kittler, M; Áč, V; Hrdý, J; Iragashi, N; Scheinost, A C; Uchida, Y

    2015-03-01

    A study of metallic brazing material for internally cooled optics is presented. The study shows the influence of the different material properties on the final quality of the bond in terms of diffracted wavefront distortion, i.e. enlargement of the rocking curve. By choosing the proper brazing material and applying the proper brazing conditions, the influence of the brazing material can be fully eliminated. Furthermore the degradation of some brazing material due to the extreme working conditions of the optics is presented. Measurement results from ESRF and KEK confirm the importance of the proper brazing material choice.

  10. The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin.

    PubMed Central

    Papini, E; Satin, B; Bucci, C; de Bernard, M; Telford, J L; Manetti, R; Rappuoli, R; Zerial, M; Montecucco, C

    1997-01-01

    The VacA cytotoxin, produced by toxigenic strains of Helicobacter pylori, induces the formation of large vacuoles highly enriched in the small GTPase rab7. To probe the role of rab7 in vacuolization, HeLa cells were transfected with a series of rab mutants and exposed to VacA. Dominant-negative mutants of rab7 effectively prevented vacuolization, whereas homologous rab5 and rab9 mutants were only partially inhibitory or ineffective, respectively. Expression of wild-type or GTPase-deficient rab mutants synergized with VacA in inducing vacuolization. In vitro fusion of late endosomes was enhanced by active rab7 and inhibited by inactive rab7, consistent with vacuole formation by merging of late endosomes in a process that requires functional rab7. Taken together, the effects of overexpressed rab proteins described here indicate that continuous membrane flow along the endocytic pathway is necessary for vacuole growth. PMID:9009263

  11. Flight service evaluation of two aluminum-brazed titanium spoilers

    NASA Technical Reports Server (NTRS)

    Boyer, R. R.

    1984-01-01

    The long-term service evaluation of two aluminum-brazed titanium (ABTi) honeycomb flight spoilers was concluded. The two spoilers had about 7.5 years of commercial flight experience on All Nippon Airways Model 737 aircraft. All Nippon Airways was selected because Japan has one of the most severe marine-industrial environments in the world. The results indicated that both flight spoilers still had the same load-carrying capability as when they were originally installed. No direct evidence of any corrosion was observed on either spoiler. Another significant accomplishment of this effort was the development of a braze design for efficiently distributing point loads from the fittings and skin into the honeycomb core.

  12. Effects of service environments on aluminum-brazed titanium (ABTi)

    NASA Technical Reports Server (NTRS)

    Cotton, W. L.

    1978-01-01

    Aluminum brazed titanium (ABTi) structures were evaluated during prolonged exposure to extreme environments: elevated temperature exposure to airline service fluids, hydraulic fluid, and seawater, followed by laboratory corrosion tests. Solid-face and perforated face honeycomb sandwich panel specimens, stressed panel assemblies, and faying surface brazed joints were tested. The corrosion resistance of ABTi is satisfactory for commercial airline service. Unprotected ABTi proved inherently resistant to attack by all of the extreme service aircraft environments except: seawater at 700 K (800 F) and above, dripping phosphate ester hydraulic fluid at 505 K (450 F), and a marine environment at ambient temperature. The natural oxides and deposits present on titanium surfaces in airline service provide protection against hot salt corrosion pitting. Coatings are required to protect titanium dripping phosphate ester fluid at elevated temperatures and to protect exposed acoustic honeycomb parts against corrosion in a marine environment.

  13. Influence of joint topology on the formation of brazed joints

    NASA Astrophysics Data System (ADS)

    Sekulic, Dusan P.; Zellmer, Benjamin P.; Nigro, Nicholas

    2001-09-01

    This paper discusses the influence of joint topology on the formation of brazed joints. For the purposes of this study, the joint topology is defined by dimensionless parameters that characterize: (i) the geometry of the mating surfaces (shape and configuration), (ii) the gap between bonded parts (clearance and tolerances), (iii) the joint orientation in the gravity field and (iv) the volume of the joint. The influence of these parameters is presented for several joints with a topology that is typically found in the manufacture of compact heat exchangers. The results were obtained by employing a method that is based on a variational principle and minimization of the potential energy of the molten aluminium liquid metal just prior to the onset of solidification. The method, which was verified by comparison with experimental data obtained from controlled atmosphere brazing of aluminium alloys, is discussed in an accompanying paper.

  14. Optical Measurement of the Connection State in Laser Brazing

    NASA Astrophysics Data System (ADS)

    Tenner, Felix; Ramoser, Stephan; Dobler, Michael; Zalevsky, Zeev; Schmidt, Michael

    The laser brazing of steel sheets is widely applied in car-body manufacturing. The critical value for the strength of the joint is the minimal connection width between the two sheets. However, this value is depending on the feed rate and power of the laser and the feed rate and preheating current of the filler wire used. Furthermore, the wetting behavior of the brazing solder is affected by the surface properties of the joining partners and is thus prone to errors. Currently, mostly destructive testing is used to evaluate the connection state. Therefore, we studied a novel optical measurement technique which is capable of remotely measuring oscillations of the joining partners. In the proposed study, we show how the oscillations are connected to the process behavior and the fusion area and how the setup might be applied as a post- and in-process measurement system.

  15. New hermetic sealing material for vacuum brazing of stainless steels

    NASA Astrophysics Data System (ADS)

    Hildebrandt, S.; Wiehl, G.; Silze, F.

    2016-03-01

    For vacuum brazing applications such as in vacuum interrupter industry Hermetic Sealing Materials (HSM) with low partial pressure are widely used. AgCu28 dominates the hermetic sealing market, as it has a very good wetting behavior on copper and metallized ceramics. Within recent decades wetting on stainless steel has become more and more important. However, today the silver content of HSMs is more in focus than in the past decades, because it has the biggest impact on the material prices. Umicore Technical Materials has developed a new copper based HSM, CuAg40Ga10. The wettability on stainless steel is significantly improved compared to AgCu28 and the total silver content is reduced by almost 44%. In this article the physical properties of the alloy and its brazed joints will be presented compared to AgCu28.

  16. Low vapor pressure braze alloys for thermionic energy converters

    NASA Technical Reports Server (NTRS)

    Bair, V. L.

    1976-01-01

    Preliminary results in the use of some low-vapor-pressure braze alloys are reported; these are binary alloys of refractory metals (Th, Zr, Hf, Ru, Nb, Ir, Mo, Ta, Os, Re, W) with vapor pressures below 0.1 nanotorr at 1500 K or 10 microtorr at 2000 K. The melting point minima or eutectics of the alloys range from 1510 K to above 3000 K. Melting points and surface wetting on a Ta base are given. Results are presented on brazing of Ir, LaB6, Nb, Re, W, and ZrO2 (with 22 wt % Zr) into a Ta base or a Nb-1% Zr base. The results are applicable in electrode screening programs for thermionic cesium diodes.

  17. Separation and Sealing of a Sample Container Using Brazing

    NASA Technical Reports Server (NTRS)

    Bar-Cohen, Yoseph; Rivellini, Tommaso P.; Wincentsen, James E.; Gershman, Robert

    2007-01-01

    A special double-wall container and a process for utilizing the container are being developed to enable (1) acquisition of a sample of material in a dirty environment that may include a biological and/or chemical hazard; (2) sealing a lid onto the inner part of the container to hermetically enclose the sample; (3) separating the resulting hermetic container from the dirty environment; and (4) bringing that hermetic container, without any biological or chemical contamination of its outer surface, into a clean environment. The process is denoted S(exp 3)B (separation, seaming, and sealing using brazing) because sealing of the sample into the hermetic container, separating the container from the dirty environment, and bringing the container with a clean outer surface into the clean environment are all accomplished simultaneously with a brazing operation.

  18. Rab11-FIP1A regulates early trafficking into the recycling endosomes.

    PubMed

    Schafer, Jenny C; McRae, Rebecca E; Manning, Elizabeth H; Lapierre, Lynne A; Goldenring, James R

    2016-01-15

    The Rab11 family of small GTPases, along with the Rab11-family interacting proteins (Rab11-FIPs), are critical regulators of intracellular vesicle trafficking and recycling. We have identified a point mutation of Threonine-197 site to an Alanine in Rab11-FIP1A, which causes a dramatic dominant negative phenotype when expressed in HeLa cells. The normally perinuclear distribution of GFP-Rab11-FIP1A was condensed into a membranous cisternum with almost no GFP-Rab11-FIP1A(T197A) remaining outside of this central locus. Also, this condensed GFP-FIP1A(T197A) altered the distribution of proteins in the Rab11a recycling pathway including endogenous Rab11a, Rab11-FIP1C, and transferrin receptor (CD71). Furthermore, this condensed GFP-FIP1A(T197A)-containing structure exhibited little movement in live HeLa cells. Expression of GFP-FIP1A(T197A) caused a strong blockade of transferrin recycling. Treatment of cells expressing GFP-FIP1A(T197A) with nocodazole did not disperse the Rab11a-containing recycling system. We also found that Rab5 and EEA1 were accumulated in membranes by GFP-Rab11-FIP1A but Rab4 was unaffected, suggesting that a direct pathway may exist from early endosomes into the Rab11a-containing recycling system. Our study of a potent inhibitory trafficking mutation in Rab11-FIP1A shows that Rab11-FIP1A associates with and regulates trafficking at an early step in the process of membrane recycling.

  19. The Rab1 GTPase of Sciaenops ocellatus modulates intracellular bacterial infection.

    PubMed

    Hu, Yong-hua; Deng, Tian; Sun, Li

    2011-12-01

    The Rab family proteins belong to the Ras-like GTPase superfamily and play important roles in intracellular membrane trafficking. To date no studies on fish Rab have been documented, though rab-like sequences have been found in a number of teleosts. In this study, we identified and analyzed a Rab homologue, SoRab1, from red drum, Sciaenops ocellatus. The cDNA of SoRab1 contains a 5'- untranslated region (UTR) of 358 bp, an open reading frame (ORF) of 612 bp, and a 3'-UTR of 265 bp. The ORF encodes a putative protein of 203 residues, which shares 92-99% overall sequence identities with the Rab1 from fish, human, and mouse. SoRab1 possesses a typical Rab1 GTPase domain with the conserved G box motifs and the switch I and switch II regions. Recombinant SoRab1 purified from Escherichia coli exhibits apparent GTPase activity. Quantitative real time RT-PCR analysis showed that SoRab1 expression was detected in a number of tissues, with the lowest expression found in blood and highest expression found in muscle. Bacterial and lipopolysaccharide challenges significantly upregulated SoRab1 expression in liver, kidney, and spleen in time-dependent manners. Transient overexpression of SoRab1 in primary hepatocytes reduced intracellular bacterial infection, whereas interference with SoRab1 expression by RNAi enhanced intracellular bacterial invasion. These results provide the first indication that a fish Rab1 GTPase, SoRab1, regulates intracellular bacterial infection and thus is likely to play a role in bacteria-induced host immune defense. PMID:21889593

  20. Braze bonding of diamond, CBN, sapphire and other non-metallic hard materials for industrial devices

    SciTech Connect

    Beck, A.

    1995-12-31

    The nature of braze bonding by chemical redox reactions causing a diffusion zone on the surface of the nonmetal with the braze alloy is explained briefly. Some technical and economic advantages for tool and device manufacturing are discussed. Samples of monocrystal devices and grinding tools with braze bonded diamond grit demonstrate the potential of this technology particularly for miniaturization. New applications with other hard materials are shown. Wearing tests have proved that the lifetime of tools with brazed on diamonds is several times as long as of conventional ones with formlocked diamonds. The reason for this is explained.

  1. The use of Ni-Cr-Si-Be filler metals for brazing of stainless steels

    NASA Astrophysics Data System (ADS)

    Ivannikov, A.; Fedotov, V.; Suchkov, A.; Penyaz, M.; Fedotov, I.; Tarasov, B.

    2016-04-01

    Nanocrystalline ribbon filler metal-alloys of system Ni-Cr-Si-Be are produced by the rapidly quenching of the melt method. By these filler metals carried out hight temperature vacuum brazing of austenitic steels (12Kh18N10T and Kh18N8G2) and austenitic-ferritic class EI-811 (12Kh21N5T). The basic laws of structure-phase state foundation of brazed joints are determined, features of the interaction of the molten filler metal to the brazed materials are identified, the optimal temperature and time parameters of the brazing process are determined.

  2. Brazing SiC/SiC Composites to Metals

    NASA Technical Reports Server (NTRS)

    Steffier, Wayne S.

    2004-01-01

    Experiments have shown that active brazing alloys (ABAs) can be used to join SiC/SiC composite materials to metals, with bond strengths sufficient for some structural applications. The SiC/SiC composite coupons used in the experiments were made from polymerbased SiC fiber preforms that were chemical-vapor-infiltrated with SiC to form SiC matrices. Some of the metal coupons used in the experiments were made from 304 stainless steel; others were made from oxygen-free, high-conductivity copper. Three ABAs were chosen for the experiments: two were chosen randomly from among a number of ABAs that were on hand at the time; the third ABA was chosen because its titanium content (1.25 percent) is less than those of the other two ABAs (1.75 and 4.5 percent, respectively) and it was desired to evaluate the effect of reducing the titanium content, as described below. The characteristics of ABAs that are considered to be beneficial for the purpose of joining SiC/SiC to metal include wettability, reactivity, and adhesion to SiC-based ceramics. Prior to further development, it was verified that the three chosen ABAs have these characteristics. For each ABA, suitable vacuum brazing process conditions were established empirically by producing a series of (SiC/SiC)/ABA wetting samples. These samples were then sectioned and subjected to scanning electron microscopy (SEM) and energy-dispersive x-ray spectrometry (EDS) for analysis of their microstructures and compositions. Specimens for destructive mechanical tests were fabricated by brazing of lap joints between SiC/SiC coupons 1/8-in. (.3.2- mm) thick and, variously, stainless steel or copper tabs. The results of destructive mechanical tests and the SEM/EDS analysis were used to guide the development of a viable method of brazing the affected materials.

  3. A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis.

    PubMed

    Zheng, Huanquan; Camacho, Luísa; Wee, Edmund; Batoko, Henri; Legen, Julia; Leaver, Christopher J; Malhó, Rui; Hussey, Patrick J; Moore, Ian

    2005-07-01

    The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1(d)[NI]) of Arabidopsis thaliana RAB-E1(d) and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1(d)[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1(d)[NI] caused less intracellular secGFP accumulation than RAB-D2(a)[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2(a)[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1(d)[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)-tagged Arabidopsis RAB-F2(b). Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1(d)[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1(d), YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1(d)[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway.

  4. A Rab-E GTPase Mutant Acts Downstream of the Rab-D Subclass in Biosynthetic Membrane Traffic to the Plasma Membrane in Tobacco Leaf EpidermisW⃞

    PubMed Central

    Zheng, Huanquan; Camacho, Luísa; Wee, Edmund; Batoko, Henri; Legen, Julia; Leaver, Christopher J.; Malhó, Rui; Hussey, Patrick J.; Moore, Ian

    2005-01-01

    The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1d[NI]) of Arabidopsis thaliana RAB-E1d and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1d[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1d[NI] caused less intracellular secGFP accumulation than RAB-D2a[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2a[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1d[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)–tagged Arabidopsis RAB-F2b. Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1d[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1d, YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1d[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway. PMID:15972698

  5. Laser Brazing with Beam Scanning: Experimental and Simulative Analysis

    NASA Astrophysics Data System (ADS)

    Heitmanek, M.; Dobler, M.; Graudenz, M.; Perret, W.; Göbel, G.; Schmidt, M.; Beyer, E.

    Laser beam brazing with copper based filler wire is a widely established technology for joining zinc-coated steel plates in the body-shop. Successful applications are the divided tailgate or the zero-gap joint, which represents the joint between the side panel and the roof-top of the body-in-white. These joints are in direct view to the customer, and therefore have to fulfil highest optical quality requirements. For this reason a stable and efficient laser brazing process is essential. In this paper the current results on quality improvement due to one dimensional laser beam deflections in feed direction are presented. Additionally to the experimental results a transient three-dimensional simulation model for the laser beam brazing process is taken into account. With this model the influence of scanning parameters on filler wire temperature and melt pool characteristics is analyzed. The theoretical predictions are in good accordance with the experimental results. They show that the beam scanning approach is a very promising method to increase process stability and seam quality.

  6. Ultrasonic guided wave inspection of Inconel 625 brazed lap joints

    NASA Astrophysics Data System (ADS)

    Comot, Pierre; Bocher, Philippe; Belanger, Pierre

    2016-04-01

    The aerospace industry has been investigating the use of brazing for structural joints, as a mean of reducing cost and weight. There therefore is a need for a rapid, robust, and cost-effective non-destructive testing method for evaluating the structural integrity of the joints. The mechanical strength of brazed joints depends mainly on the amount of brittle phases in their microstructure. Ultrasonic guided waves offer the possibility of detecting brittle phases in joints using spatio-temporal measurements. Moreover, they offer the opportunity to inspect complex shape joints. This study focused on the development of a technique based on ultrasonic guided waves for the inspection of Inconel 625 lap joints brazed with BNi-2 filler metal. A finite element model of a lap joint was used to optimize the inspection parameters and assess the feasibility of detecting the amount of brittle phases in the joint. A finite element parametric study simulating the input signal shape, the center frequency, and the excitation direction was performed. The simulations showed that the ultrasonic guided wave energy transmitted through, and reflected from, the joints was proportional to the amount of brittle phases in the joint.

  7. Dispersed metal-toughened ceramics and ceramic brazing

    SciTech Connect

    Moorhead, A.J.; Tiegs, T.N.; Lauf, R.J.

    1983-01-01

    An alumina (Al/sub 2/O/sub 3/) based material that contains approximately 1 vol % finely dispersed platinum or chromium was developed for use in high temperature thermal-shock resistant electrical insulators. The work at ORNL is divided into two areas: (1) development of DMT ceramics; and (2) development of brazing filler metals suitable for making ceramic-to-ceramic and ceramic-to-metal brazements. The DMT ceramics and brazements are intended for service at elevated temperatures and at high stress levels in the dirty environments of advanced heat engines. The development and characterization of DMT ceramics includes processing (powder preparation, densification and heat treatment) and detailed measurement of mechanical and physical properties (strength, fracture toughness, and thermal conductivity). The brazing work includes: (1) the formulation and melting of small quantities of experimental brazing filler metals; (2) evaluation of the wetting and bonding behavior of these filler metals on Al/sub 2/O/sub 3/, partially stabilized zirconia and ..cap alpha..-SiC in a sessile drop apparatus; and (3) determine the short-term strength and fracture toughness of brazements.

  8. Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

    SciTech Connect

    Mesa, Rosana; Magadan, Javier; Barbieri, Alejandro; Lopez, Cecilia; Stahl, Philip D.; Mayorga, Luis S. . E-mail: lmayorga@fcm.uncu.edu.ar

    2005-04-01

    The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN)

  9. Identification and function of 11 Rab GTPases in giant freshwater prawn Macrobrachium rosenbergii.

    PubMed

    Huang, Ying; Ren, Qian

    2015-03-01

    Rab GTPases, members of the Ras-like GTPase superfamily, are central elements in endocytic membrane trafficking. However, little is known of the Rab genes in the giant freshwater prawn Macrobrachium rosenbergii. In this study, 11 Rab genes were identified from M. rosenbergii. All MrRabs have a RAB domain. Phylogenetic analysis showed that these 11 MrRabs were divided into different groups. The MrRab genes were ubiquitously expressed in heart, hemocytes, hepatopancreas, gills, stomach, and intestines. Real-time polymerase chain reaction revealed that the MrRab genes were significantly upregulated by white spot syndrome virus (WSSV) in the prawns, indicating that MrRabs might play an important role in innate immune response against WSSV. Moreover, after challenge with Vibrio parahaemolyticus, the expression levels of all MrRabs in the hepatopancreas were also upregulated, which might indicated the involvement of MrRabs in prawns antibacterial immunity. In all, these preliminary results showed that MrRabs were involved in innate immunity of M. rosenbergii.

  10. Cell-specific abnormal prenylation of Rab proteins in platelets and melanocytes of the gunmetal mouse.

    PubMed

    Zhang, Qing; Zhen, Lijie; Li, Wei; Novak, Edward K; Collinson, Lucy M; Jang, Elliott K; Haslam, Richard J; Elliott, Rosemary W; Swank, Richard T

    2002-05-01

    The mutant gunmetal mouse exhibits reduced rates of platelet synthesis, abnormalities of platelet alpha and dense granules and hypopigmentation. Several of these features resemble those of human alpha/delta platelet storage pool disease, grey platelet syndrome and Hermansky-Pudlak syndrome. Gunmetal mice have reduced levels of Rab geranylgeranyltransferase (RabGGTase), which adds lipophilic prenyl groups to the carboxyl terminus of Rab proteins. The degree of prenylation and the subcellular distribution of several Rab proteins were evaluated in mutant platelets, melanocytes and other tissues. Significant deficits in prenylation and membrane binding of most Rabs were observed in platelets and melanocytes. In contrast, minimal alterations in Rab prenylation were apparent in several other gunmetal tissues despite the fact that RabGGTase activity was equally diminished in these tissues. The mutant tissue-specific effects are probably due to increased concentrations of Rab proteins in platelets and melanocytes. These experiments show that Rab proteins are differentially sensitive to levels of RabGGTase activity and that normal platelet synthesis, platelet organelle function and normal pigmentation are highly sensitive to the degree of prenylation and membrane association of Rab proteins. Further, the tissue-specific effects of the gunmetal mutation suggest that RabGGTase is a potential target for therapy of thrombocytosis.

  11. An essential role for Rab27a GTPase in eosinophil exocytosis

    PubMed Central

    Kim, John Dongil; Willetts, Lian; Ochkur, Sergei; Srivastava, Nutan; Hamburg, Rudolf; Shayeganpour, Anooshirvan; Seabra, Miguel C.; Lee, James J.; Moqbel, Redwan; Lacy, Paige

    2013-01-01

    Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab-related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a-deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a+ structures, some of which colocalized with CD63+ crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a-dependent eosinophil degranulation in airway inflammation. PMID:23986549

  12. A role for Rab23 in the trafficking of Kif17 to the primary cilium.

    PubMed

    Lim, Yi Shan; Tang, Bor Luen

    2015-08-15

    The small GTPase Rab23 is an antagonist of sonic hedgehog (Shh) signaling during mouse development. Given that modulation of Shh signaling depends on the normal functioning of the primary cilium, and overexpression of Evi5L, a putative Rab23 GTPase-activating protein (GAP), leads to reduced ciliogenesis, Rab23 could have a role at the primary cilium. Here, we found that wild-type Rab23 and the constitutively active Rab23 Q68L mutant were enriched at the primary cilium. Therefore, we tested the role of Rab23 in the ciliary targeting of known cargoes and found that ciliary localization of the kinesin-2 motor protein Kif17 was disrupted in Rab23-depleted cells. Co-immunoprecipitation and affinity-binding studies revealed that Rab23 exists in a complex with Kif17 and importin β2 (the putative Kif17 ciliary import carrier), implying that Kif17 needs to bind to regulatory proteins like Rab23 for its ciliary transport. Although a ciliary-cytoplasmic gradient of nuclear Ran is necessary to regulate the ciliary transport of Kif17, Rab23 and Ran appear to have differing roles in regulating the ciliary entry of Kif17. Our findings have uncovered a hitherto unknown effector of Rab23 and demonstrate how Rab23 could mediate the transport of Kif17 to the primary cilium.

  13. 48 CFR 1352.271-72 - Additional Item Requirements (AIR)-growth work

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Clauses 1352.271-72 Additional Item Requirements (AIR)—growth work As prescribed in 48 CFR 1371.103... Cleaning/Water Blasting, Tank Cleaning, Welding, Burning, Brazing, Blacksmithing, Machining (inside...

  14. 48 CFR 1352.271-72 - Additional Item Requirements (AIR)-growth work

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Clauses 1352.271-72 Additional Item Requirements (AIR)—growth work As prescribed in 48 CFR 1371.103... Cleaning/Water Blasting, Tank Cleaning, Welding, Burning, Brazing, Blacksmithing, Machining (inside...

  15. The Rab GTPase RabG3b positively regulates autophagy and immunity-associated hypersensitive cell death in Arabidopsis.

    PubMed

    Kwon, Soon Il; Cho, Hong Joo; Kim, Sung Ryul; Park, Ohkmae K

    2013-04-01

    A central component of the plant defense response to pathogens is the hypersensitive response (HR), a form of programmed cell death (PCD). Rapid and localized induction of HR PCD ensures that pathogen invasion is prevented. Autophagy has been implicated in the regulation of HR cell death, but the functional relationship between autophagy and HR PCD and the regulation of these processes during the plant immune response remain controversial. Here, we show that a small GTP-binding protein, RabG3b, plays a positive role in autophagy and promotes HR cell death in response to avirulent bacterial pathogens in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing a constitutively active RabG3b (RabG3bCA) displayed accelerated, unrestricted HR PCD within 1 d of infection, in contrast to the autophagy-defective atg5-1 mutant, which gradually developed chlorotic cell death through uninfected sites over several days. Microscopic analyses showed the accumulation of autophagic structures during HR cell death in RabG3bCA cells. Our results suggest that RabG3b contributes to HR cell death via the activation of autophagy, which plays a positive role in plant immunity-triggered HR PCD.

  16. Characterization of RAB-like4, the first identified RAB-like protein from Trypanosoma cruzi with GTPase activity.

    PubMed

    Ramos, Fabiane Pereira; Araripe, Júlia Rolão; Urményi, Turán Péter; Silva, Rosane; Cunha e Silva, Narcisa Leal; Leite Fontes, Carlos Frederico; da Silveira, José Franco; Rondinelli, Edson

    2005-08-01

    RAB proteins, which belong to the RAS superfamily, regulate exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. Few RAB proteins have been identified in parasites. Molecular markers for cellular compartments are important to studies concerning about the protein traffic in Trypanosoma cruzi, the causal agent of Chagas disease. In this work, we describe the characterization of TcRABL4, the first RAB-like gene identified in T. cruzi (GenBank Accession No.: ), present as a single-copy gene. TcRABL4 contains all five consensus RAB motifs but lacks cysteine residues at the C terminus, which are essential to isoprenylation, an absolute prerequisite for membrane association of these proteins. TcRABL4 is a functional GTPase that is able to bind and hydrolyze GTP, and its gene is transcribed as a single 1.2 kb mRNA in epimastigotes. TcRABL4 appears to be differentially regulated in the three cell forms of the parasite, and the protein is not associated to membranes, unlike other RAB proteins. It is possible that TcRABL4 may be a member of a novel family of small GTPases.

  17. HIGH TEMPERATURE BRAZING ALLOY FOR JOINT Fe-Cr-Al MATERIALS AND AUSTENITIC AND FERRITIC STAINLESS STEELS

    DOEpatents

    Cost, R.C.

    1958-07-15

    A new high temperature brazing alloy is described that is particularly suitable for brazing iron-chromiumaluminum alloys. It consists of approximately 20% Cr, 6% Al, 10% Si, and from 1.5 to 5% phosphorus, the balance being iron.

  18. Plasmodium falciparum Rab1A Localizes to Rhoptries in Schizonts

    PubMed Central

    Morse, David; Webster, Wesley; Kalanon, Ming; Langsley, Gordon; McFadden, Geoffrey I.

    2016-01-01

    Over-expression of a GFP-PfRab1A fusion protein in Plasmodium falciparum schizonts produces a punctate pattern of fluorescence typical of rhoptries, secretory organelles involved in host cell invasion. The GFP-positive bodies were purified by a combination of differential and density gradient centrifugation and their protein content determined by MS/MS sequencing. Consistent with the GFP rhoptry-like pattern of transgenic parasites, four of the 19 proteins identified have been previously described to be rhoptry-associated and another four are ER or ER-associated proteins. Confirmation that GFP-PfRab1A decorates rhoptries was obtained by its co-localization with Rap1 and Ron4 in late phase schizonts. We conclude that PfRab1A potentially regulates vesicular traffic from the endoplasmic reticulum to the rhoptries in Apicomplexa parasites. PMID:27348424

  19. Rice Rab11 is required for JA-mediated defense signaling

    SciTech Connect

    Hong, Min Ji; Lee, Yun mi; Son, Young Sim; Im, Chak Han; Yi, Young Byung; Rim, Yeong Gil; Bahk, Jeong Dong; Heo, Jae Bok

    2013-05-17

    Highlights: •OsRab11 interacts with OsOPR8. •OsOPR8 is localized in the cytosol and peroxisome. •OsRab11 enhances the NADPH consumption by OsOPR8. •Transgenic Arabidopsis overexpressing OsRab11 represents a pathogen-resistant phenotype. -- Abstract: Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8.

  20. Role for Rab10 in Methamphetamine-Induced Behavior.

    PubMed

    Vanderwerf, Scott M; Buck, David C; Wilmarth, Phillip A; Sears, Leila M; David, Larry L; Morton, David B; Neve, Kim A

    2015-01-01

    Lipid rafts are specialized, cholesterol-rich membrane compartments that help to organize transmembrane signaling by restricting or promoting interactions with subsets of the cellular proteome. The hypothesis driving this study was that identifying proteins whose relative abundance in rafts is altered by the abused psychostimulant methamphetamine would contribute to fully describing the pathways involved in acute and chronic effects of the drug. Using a detergent-free method for preparing rafts from rat brain striatal membranes, we identified density gradient fractions enriched in the raft protein flotillin but deficient in calnexin and the transferrin receptor, markers of non-raft membranes. Dopamine D1- and D2-like receptor binding activity was highly enriched in the raft fractions, but pretreating rats with methamphetamine (2 mg/kg) once or repeatedly for 11 days did not alter the distribution of the receptors. LC-MS analysis of the protein composition of raft fractions from rats treated once with methamphetamine or saline identified methamphetamine-induced changes in the relative abundance of 23 raft proteins, including the monomeric GTP-binding protein Rab10, whose abundance in rafts was decreased 2.1-fold by acute methamphetamine treatment. Decreased raft localization was associated with a selective decrease in the abundance of Rab10 in a membrane fraction that includes synaptic vesicles and endosomes. Inhibiting Rab10 activity by pan-neuronal expression of a dominant-negative Rab10 mutant in Drosophila melanogaster decreased methamphetamine-induced activity and mortality and decreased caffeine-stimulated activity but not mortality, whereas inhibiting Rab10 activity selectively in cholinergic neurons had no effect. These results suggest that activation and redistribution of Rab10 is critical for some of the behavioral effects of psychostimulants.

  1. Role for Rab10 in Methamphetamine-Induced Behavior

    PubMed Central

    Vanderwerf, Scott M.; Buck, David C.; Wilmarth, Phillip A.; Sears, Leila M.; David, Larry L.; Morton, David B.; Neve, Kim A.

    2015-01-01

    Lipid rafts are specialized, cholesterol-rich membrane compartments that help to organize transmembrane signaling by restricting or promoting interactions with subsets of the cellular proteome. The hypothesis driving this study was that identifying proteins whose relative abundance in rafts is altered by the abused psychostimulant methamphetamine would contribute to fully describing the pathways involved in acute and chronic effects of the drug. Using a detergent-free method for preparing rafts from rat brain striatal membranes, we identified density gradient fractions enriched in the raft protein flotillin but deficient in calnexin and the transferrin receptor, markers of non-raft membranes. Dopamine D1- and D2-like receptor binding activity was highly enriched in the raft fractions, but pretreating rats with methamphetamine (2 mg/kg) once or repeatedly for 11 days did not alter the distribution of the receptors. LC-MS analysis of the protein composition of raft fractions from rats treated once with methamphetamine or saline identified methamphetamine-induced changes in the relative abundance of 23 raft proteins, including the monomeric GTP-binding protein Rab10, whose abundance in rafts was decreased 2.1-fold by acute methamphetamine treatment. Decreased raft localization was associated with a selective decrease in the abundance of Rab10 in a membrane fraction that includes synaptic vesicles and endosomes. Inhibiting Rab10 activity by pan-neuronal expression of a dominant-negative Rab10 mutant in Drosophila melanogaster decreased methamphetamine-induced activity and mortality and decreased caffeine-stimulated activity but not mortality, whereas inhibiting Rab10 activity selectively in cholinergic neurons had no effect. These results suggest that activation and redistribution of Rab10 is critical for some of the behavioral effects of psychostimulants. PMID:26291453

  2. Trypanosoma cruzi: TcRAB7 protein is localized at the Golgi apparatus in epimastigotes.

    PubMed

    Araripe, Júlia R; Cunha e Silva, Narcisa L; Leal, Simone T; de Souza, Wanderley; Rondinelli, Edson

    2004-08-20

    In mammalian cells, the Rab7 protein is a key element of late endocytic membrane traffic. Several results suggest that it is involved in the transport from early to late endosome or from late endosome to lysosome. We have previously characterized a Rab7 gene homologue (TcRAB7) in Trypanosoma cruzi. Now, using an affinity-purified antibody specific to TcRAB7 protein we have determined that it is localized at the Golgi apparatus of the parasite. Our results indicate that the T. cruzi Rab7 homologue may function in a different route than its counterparts in mammalian cells.

  3. Identification of the Rab5 Binding Site in p110β - Assays for PI3Kβ binding to Rab5

    PubMed Central

    Salamon, Rachel S.; Dbouk, Hashem A.; Collado, Denise; Lopiccolo, Jaclyn; Bresnick, Anne R.; Backer, Jonthan M.

    2015-01-01

    Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110β, and p110δ), p110β has unique properties. Unlike the other family members, p110β directly binds to Gβγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110β binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for defining their role in p110β signaling. This chapter describes the approach that was used to identify the Rab5 binding site in p110β, and discusses methods for the analysis of p110β-Rab5 interactions. PMID:25800850

  4. Recent experience in the fabrication and brazing of ceramic beam tubes for kicker magnets at FNAL

    SciTech Connect

    Ader, C.R.; Jensen, C.; Reilly, R.; Snee, D.; Wilson, J.H.; /Fermilab

    2008-06-01

    Ceramic beam tubes are utilized in numerous kicker magnets in different accelerator rings at Fermi National Accelerator Laboratory. Kovar flanges are brazed onto each beam tube end, since kovar and high alumina ceramic have similar expansion curves. The tube, kovar flange, end piece, and braze foil (titanium/incusil) alloy brazing material are stacked in the furnace and then brazed in the furnace at 1000 C. The ceramic specified is 99.8% Alumina, Al{sub 2}O{sub 3}, a strong recrystallized high-alumina fabricated by slip casting. Recent experience at Fermilab with the fabrication and brazing of these tubes has brought to light numerous problems including tube breakage and cracking and also the difficulty of brazing the tube to produce a leak-tight joint. These problems may be due to the ceramic quality, voids in the ceramic, thinness of the wall, and micro-cracks in the ends which make it difficult to braze because it cannot fill tiny surface cracks which are caused by grain pullout during the cutting process. Solutions which are being investigated include lapping the ends of the tubes before brazing to eliminate the micro-cracks and also metallization of the tubes.

  5. Substrate Effects on the High Temperature Oxidation Behavior of a Gold-Based Braze Filler Metal

    SciTech Connect

    Weil, K. Scott; Rice, Joseph P.

    2005-06-01

    Oxidation testing was conducted on a commercial gold-based braze alloy, Gold ABA®, and on zirconia/stainless steel couples joined using this filler metal. Preliminary results reveal that both substrates play a significant role in determining the overall oxidation behavior of the brazed joint.

  6. High expression of small GTPase Rab3D promotes cancer progression and metastasis

    PubMed Central

    Yang, Jian; Liu, Wei; Lu, Xin'an; Fu, Yan; Li, Lin; Luo, Yongzhang

    2015-01-01

    Rab GTPases control exocytic and endocytic membrane trafficking such as exosomes release. As a secretory small GTPase, Rab3D is a vital regulator for protein secretion. However, the role of Rab3D in cancer was never systematically studied. The aim of this study is to examine its function and mechanism in cancer, especially metastasis. We detected protein levels of Rab3D in nine cancer cell lines and twelve types of clinical cancer specimens. Subsequently, we established in vitro migration and in vivo orthotopic metastatic mouse models to study the role of Rab3D in tumor metastasis. Here, we reported that the expression levels of Rab3D were dysregulated in cancer cells and highly correlated with tumor malignancies in the clinical samples. Increased expressions of Rab3D led to tumor invasion in vitro and lung metastasis in vivo, whereas Rab3D knockdown suppressed the tumor cell motility. Mechanistic studies revealed that Rab3D activated intracellular the AKT/GSK3β signaling to induce the EMT process. In addition, it also regulated the extracellular secretion of Hsp90α to promote tumor cell migration and invasion. These results prove that Rab3D is a key molecule to regulate tumor metastasis, suggesting that blocking the Rab3D function can be a potential therapeutic approach for cancer metastasis. PMID:25823663

  7. [Alterations in recruitment and activation of Rab proteins during mycobacterial infection].

    PubMed

    Castaño, Diana; Rojas, Mauricio

    2010-01-01

    At the phagosome level, Mycobacterium spp. alters activation and recruitment of several "Ras gene from rat brain" proteins, commonly known as Rab. Mycobacterial phagosomes have a greater and sustained expression of Rab5, Rab11, Rab14 and Rab22a, and lowered or no expression of Rab7, Rab9 and Rab6. This correlates with increased fusion of the phagosomes with early and recycling endosomes acquiring some features of early phagosomes, allowing the bacteria to gain access to nutrients and preventing the activation of anti-mycobacterial mechanisms. The expression of constitutively active mutants of Rab from the early stage endosomes prevents the maturation of phagosomes containing latex beads or heat-inactivated mycobacteria. Silencing of these mutants by interference RNA or dominant negative forms induces the maturation of mycobacterial phagosomes. The mechanisms have not been established by which mycobacteria alter the expression of these GTPases and thereby shift the phagolysosomal maturation. The problem can be explained by alterations in the recruitment of proteins that interact with Rab, such as phosphoinositide 3-kinases and early endosomal antigen 1. Identifying the mechanisms used by Mycobacterium spp. to disrupt the cycle of Rab activation will be essential to understand the pathophysiology of mycobacterial infections and usefully to potential drug targets.

  8. Identification of Rab41/6d Effectors Provides an Explanation for the Differential Effects of Rab41/6d and Rab6a/a' on Golgi Organization.

    PubMed

    Liu, Shijie; Majeed, Waqar; Kudlyk, Tetyana; Lupashin, Vladimir; Storrie, Brian

    2016-01-01

    Unexpectedly, members of the Rab VI subfamily exhibit considerable variation in their effects on Golgi organization and trafficking. By fluorescence microscopy, neither depletion nor overexpression of the GDP-locked form of Rab6a/a', the first trans Golgi-associated Rab protein discovered, affects Golgi ribbon organization while, on the other hand, both Rab41/6d depletion and overexpression of GDP-locked form cause Golgi fragmentation into a cluster of punctate elements, suggesting that Rab41/6d has an active role in maintenance of Golgi ribbon organization. To establish a molecular basis for these differences, we screened for Rab41/6d interacting proteins by yeast two-hybrid assay. 155 non-repetitive hits were isolated and sequenced, and after searching in NCBI database, 102 different proteins and protein fragments were identified. None of these hits overlapped with any published Rab6a/a' effector. Eight putative Rab41 interactors involved in membrane trafficking were found. Significantly, these exhibited a preferential interaction with GTP- vs. GDP-locked Rab41/6d. Of the 8 hits, the dynactin 6, syntaxin 8, and Kif18A plasmids were the only ones expressing the full-length protein. Hence, these 3 proteins were selected for further study. We found that depletion of dynactin 6 or syntaxin 8, but not Kif18A, resulted in a fragmented Golgi apparatus that displayed a Rab41/6d knockdown phenotype, i.e., the Golgi apparatus was disrupted into a cluster of punctate Golgi elements. Co-immunoprecipation experiments verified that the interaction of dynactin 6 and syntaxin 8 with GTP-locked Rab41/6d was stronger than that with wild type Rab41/6d and least with the GDP-locked form. In contrast, co-immunoprecipitation interaction with Rab6a was greatest with the GDP-locked Rab6a, suggestive of a non-physiological interaction. In conclusion, we suggest that dynactin 6, a subunit of dynactin complex, the minus-end-directed, dynein motor, provides a sufficient molecular basis to

  9. Identification of Rab41/6d Effectors Provides an Explanation for the Differential Effects of Rab41/6d and Rab6a/a' on Golgi Organization.

    PubMed

    Liu, Shijie; Majeed, Waqar; Kudlyk, Tetyana; Lupashin, Vladimir; Storrie, Brian

    2016-01-01

    Unexpectedly, members of the Rab VI subfamily exhibit considerable variation in their effects on Golgi organization and trafficking. By fluorescence microscopy, neither depletion nor overexpression of the GDP-locked form of Rab6a/a', the first trans Golgi-associated Rab protein discovered, affects Golgi ribbon organization while, on the other hand, both Rab41/6d depletion and overexpression of GDP-locked form cause Golgi fragmentation into a cluster of punctate elements, suggesting that Rab41/6d has an active role in maintenance of Golgi ribbon organization. To establish a molecular basis for these differences, we screened for Rab41/6d interacting proteins by yeast two-hybrid assay. 155 non-repetitive hits were isolated and sequenced, and after searching in NCBI database, 102 different proteins and protein fragments were identified. None of these hits overlapped with any published Rab6a/a' effector. Eight putative Rab41 interactors involved in membrane trafficking were found. Significantly, these exhibited a preferential interaction with GTP- vs. GDP-locked Rab41/6d. Of the 8 hits, the dynactin 6, syntaxin 8, and Kif18A plasmids were the only ones expressing the full-length protein. Hence, these 3 proteins were selected for further study. We found that depletion of dynactin 6 or syntaxin 8, but not Kif18A, resulted in a fragmented Golgi apparatus that displayed a Rab41/6d knockdown phenotype, i.e., the Golgi apparatus was disrupted into a cluster of punctate Golgi elements. Co-immunoprecipation experiments verified that the interaction of dynactin 6 and syntaxin 8 with GTP-locked Rab41/6d was stronger than that with wild type Rab41/6d and least with the GDP-locked form. In contrast, co-immunoprecipitation interaction with Rab6a was greatest with the GDP-locked Rab6a, suggestive of a non-physiological interaction. In conclusion, we suggest that dynactin 6, a subunit of dynactin complex, the minus-end-directed, dynein motor, provides a sufficient molecular basis to

  10. A modification switch on a molecular switch: Phosphoregulation of Rab7 during endosome maturation.

    PubMed

    Shinde, Swapnil Rohidas; Maddika, Subbareddy

    2016-07-01

    Rab GTPases, the highly conserved members of Ras GTPase superfamily are the pivotal regulators of vesicle-mediated trafficking. Rab GTPases, each with a specific subcellular localization, exert tremendous control over various aspects of vesicular transport, identity and dynamics. Several lines of research have established that GDI, GEFs and GAPs are the critical players to orchestrate Rab GTPase activity and function. The importance of post translational modifications in Rab GTPase functional regulation is poorly or not yet been addressed except for prenylation. Our recent study has revealed a novel dephosphorylation dependent regulatory mechanism for Rab7 activity and function. We have shown the importance of PTEN mediated dephosphorylation of Rab7 on highly conserved S72 and Y183 residues, which is essential for its GDI mediated membrane targeting and further activation by GEF. In conclusion, our study highlighted the importance of a phosphorylation/dephosphorylation switch in controlling timely Rab7 localization and activity on endosomes. PMID:27070490

  11. Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22

    PubMed Central

    Woller, Barbara; Luiskandl, Susan; Popovic, Milica; Prieler, Barbara E.M.; Ikonge, Gloria; Mutzl, Michaela; Rehmann, Holger; Herbst, Ruth

    2011-01-01

    RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis. PMID:21419809

  12. Conservation and function of Rab small GTPases in Entamoeba: annotation of E. invadens Rab and its use for the understanding of Entamoeba biology.

    PubMed

    Nakada-Tsukui, Kumiko; Saito-Nakano, Yumiko; Husain, Afzal; Nozaki, Tomoyoshi

    2010-11-01

    Entamoeba invadens is a reptilian enteric protozoan parasite closely related to the human pathogen Entamoeba histolytica and a good model organism of encystation. To understand the molecular mechanism of vesicular trafficking involved in the encystation of Entamoeba, we examined the conservation of Rab small GTPases between the two species. E. invadens has over 100 Rab genes, similar to E. histolytica. Most of the Rab subfamilies are conserved between the two species, while a number of species-specific Rabs are also present. We annotated all E. invadens Rabs according to the previous nomenclature [Saito-Nakano, Y., Loftus, B.J., Hall, N., Nozaki, T., 2005. The diversity of Rab GTPases in Entamoeba histolytica. Experimental Parasitology 110, 244-252]. Comparative genomic analysis suggested that the fundamental vesicular traffic machinery is well conserved, while there are species-specific protein transport mechanisms. We also reviewed the function of Rabs in Entamoeba, and proposed the use of the annotation of E. invadens Rab genes to understand the ubiquitous importance of Rab-mediated membrane trafficking during important biological processes including differentiation in Entamoeba.

  13. Diversification of the duplicated Rab1a genes in a hypoxia-tolerant fish, common carp (Cyprinus carpio).

    PubMed

    Zhao, Zi-Xia; Cao, Ding-Chen; Xu, Jian; Xu, Ru; Li, Jiong-Tang; Zhang, Yan; Xu, Peng; Sun, Xiao-Wen

    2015-10-01

    Common carp is a widely cultivated fish with longer than 2,000 years domestication history, due to its strong environmental adaptabilities, especially hypoxia tolerance. The common carp genome has experienced a very recent whole genome duplication (WGD) event. Among a large number of highly similar duplicated genes, a pair of Ras-associated binding-GTPase 1a (Rab1a) genes were found fast diverging. Four analogous Rab1a genes were identified in the common carp genome. Comparisons of gene structures and sequences indicated Rab1a-1 and Rab1a-2 was a pair of fast diverging duplicates, while Rab1a-3 and Rab1a-4 was a pair of less diverged duplicates. All putative Rab1a proteins shared conserved GTPase domain, which enabled the proteins serve as molecular switches for vesicular trafficking. Rab1a-1 and Rab1a-2 proteins varied in their C-terminal sequences, which were generally considered to encode the membrane localization signals. Differential expression patterns were observed between Rab1a-1 and Rab1a-2 genes. In blood, muscle, spleen, and heart, the mRNA level of Rab1a-1 was higher than that of Rab1a-2. In liver and intestine, the mRNA level of Rab1a-2 was higher. Expression of Rab1a-1 and Rab1a-2 showed distinct hypoxia responses. Under severe hypoxia, Rab1a-1 expression was down-regulated in blood, while Rab1a-2 expression was up-regulated in liver. Compared with the less diverged Rab1a-3/4 gene pair, common carp Rab1a-1/2 gene pair exhibited strong characteristics of sub-functionalization, which might contribute to a sophisticated and efficient Ras-dependent regulating network for the hypoxia-tolerant fish. PMID:26129846

  14. The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes

    PubMed Central

    Huang, Bernice; Hubber, Andree; McDonough, Justin A.; Roy, Craig R.; Scidmore, Marci A.; Carlyon, Jason A.

    2010-01-01

    Summary Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV’s selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A, and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase-deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A, and GFP-Rab11A localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV. PMID:20345488

  15. Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease.

    PubMed

    Knowles, Byron C; Roland, Joseph T; Krishnan, Moorthy; Tyska, Matthew J; Lapierre, Lynne A; Dickman, Paul S; Goldenring, James R; Shub, Mitchell D

    2014-07-01

    Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea. PMID:24892806

  16. Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease

    PubMed Central

    Knowles, Byron C.; Roland, Joseph T.; Krishnan, Moorthy; Tyska, Matthew J.; Lapierre, Lynne A.; Dickman, Paul S.; Goldenring, James R.; Shub, Mitchell D.

    2014-01-01

    Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea. PMID:24892806

  17. Distinct biochemical and functional properties of two Rab5 homologs from the rice blast fungus Magnaporthe oryzae.

    PubMed

    Qi, Yaoyao; Marlin, M Caleb; Liang, Zhimin; Berry, William L; Janknecht, Ralf; Zhou, Jie; Wang, Zonghua; Lu, Guodong; Li, Guangpu

    2014-10-10

    Rab5 is a key regulator of early endocytosis by promoting early endosomal fusion and motility. In this study, we have unexpectedly found distinct properties of the two Rab5 homologs (MoRab5A and MoRab5B) from Magnaporthe oryzae, a pathogenic fungus in plants whose infection causes rice blast disease. Like mammalian Rab5, MoRab5A and MoRab5B can bind to several Rab5 effectors in a GTP-dependent manner, including EEA1, Rabenosyn-5, and Rabaptin-5. However, MoRab5A shows distinct binding characteristics in the sense that both the wild-type and the GTP hydrolysis-defective constitutively active mutant bind the effectors equally well in GST pull-down assays, suggesting that MoRab5A is defective in GTP hydrolysis and mostly in the GTP-bound conformation in the cell. Indeed, GTP hydrolysis assays indicate that MoRab5A GTPase activity is dramatically lower than MoRab5B and human Rab5 and is insensitive to RabGAP5 stimulation. We have further identified a Pro residue in the switch I region largely responsible for the distinct MoRab5A properties by characterization of MoRab5A and MoRab5B chimeras and mutagenesis. The differences between MoRab5A and MoRab5B extend to their functions in the cell. Although they both target to early endosomes, only MoRab5B closely resembles human Rab5 in promoting early endosome fusion and stimulating fluid phase endocytosis. In contrast, MoRab5A correlates with another related early endosomal Rab, Rab22, in terms of the presence of the switch I Pro residue and the blocked GTPase activity. Our data thus identify MoRab5B as the Rab5 ortholog and suggest that MoRab5A specializes to perform a non-redundant function in endosomal sorting.

  18. Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4*

    PubMed Central

    Bastin, Guillaume; Heximer, Scott P.

    2013-01-01

    RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartments. Here, we examined Rab-mediated control of RGS4 internalization and recycling. Wild type and constitutively active Rab5 decreased RGS4 PM levels while increasing its endosomal targeting. Rab5, however, did not appreciably affect the PM localization or function of the M1 muscarinic receptor (M1R)/Gq signaling cascade. RGS4-containing endosomes co-localized with subsets of Rab5-, transferrin receptor-, and Lamp1/Lysotracker-marked compartments suggesting RGS4 traffics through PM recycling or acidified endosome pathways. Rab7 activity promoted TGN association, whereas Rab7(dominant negative) trapped RGS4 in late endosomes. Furthermore, RGS4 was found to co-localize with an endosomal pool marked by Rab11, the protein that mediates recycling/sorting of proteins to the PM. The Cys-12 residue in RGS4 appeared important for its Rab11-mediated trafficking to the PM. Rab11(dominant negative) decreased RGS4 PM levels and increased the number of RGS4-containing endosomes. Inhibition of Rab11 activity decreased RGS4 function as an inhibitor of M1R activity without affecting localization and function of the M1R/Gq signaling complex. Thus, both Rab5 activation and Rab11 inhibition decreased RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/Gq signaling complex. This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. Thus, Rab GTPases may be novel molecular targets for the selective regulation of M1R-mediated signaling via their specific effects on RGS4 trafficking and function. PMID:23733193

  19. Comprehensive analysis reveals dynamic and evolutionary plasticity of Rab GTPases and membrane traffic in Tetrahymena thermophila.

    PubMed

    Bright, Lydia J; Kambesis, Nichole; Nelson, Scott Brent; Jeong, Byeongmoon; Turkewitz, Aaron P

    2010-10-14

    Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways.

  20. Identification and characterization of Rab7 from orange-spotted grouper, Epinephelus coioides.

    PubMed

    Fu, Jing; Huang, Youhua; Cai, Jia; Wei, Shina; Ouyang, Zhengliang; Ye, Fuzhou; Huang, Xiaohong; Qin, Qiwei

    2014-01-01

    Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. During the virus-host co-evolution, host Rab7 was also exploited by virus to complete their life cycle. To date, however, the roles of fish Rab7 in virus infection remained largely unknown. Here, we cloned and characterized a Rab7 gene from grouper, Epinephelus coioides (Ec-Rab7). The full-length Ec-Rab7 cDNA was composed of 1182 bp and encoded a polypeptide of 207 amino acids which shared 99% identity with that from Anoplopoma fimbria or Oreochromis niloticus. Ec-Rab7 contained five conserved domains of Rab GTPase family including GTP-binding or GTPase regions as well as an effector site. RT-PCR analysis revealed that Ec-Rab7 ubiquitously expressed in all detected tissues and its transcript in spleen was up-regulated after challenge with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-Rab7 was distributed in the cytoplasm as spots and mostly colocalized with lysosomes. Notably, the ectopic expressed Ec-Rab7 partly aggregated into the viral factories in cells infected by SGIV. Furthermore, overexpression of Ec-Rab7 accelerated the occurrence of cytopathic effect (CPE) induced by SGIV infection and promoted viral gene transcription. In addition, far western blotting assay revealed that Ec-Rab7 might interact with viral proteins, including SGIV VP69 and VP101. Taken together, our data suggested that Ec-Rab7 might be potentially involved in SGIV replication.

  1. Interfacial segregation of Ti in the brazing of diamond grits onto a steel substrate using a Cu-Sn-Ti brazing alloy

    NASA Astrophysics Data System (ADS)

    Li, Wen-Chung; Lin, Shun-Tian; Liang, Cheng

    2002-07-01

    Diamond grits were brazed onto a steel substrate using a prealloyed Cu-10Sn-15Ti (wt pct) brazing alloy at 925 °C and 1050 °C. Due to the relatively high concentration of Ti in the brazing alloy, the braze matrix exhibited a composite structure, composed of β-(Cu,Sn), a Cu-based solid solution, and various intermetallic compounds with different morphologies. The reaction of Ti with diamond yielded a continuous TiC layer on the surfaces of the diamond grits. On top of the TiC growth front, an intermetallic compound, composed of Sn and Ti, nucleated and grew into a randomly interwoven fine lacey structure. An interfacial structure developed as the interwoven fine lacey phase was semicoherently bonded to the TiC layer, with the Cu-based braze matrix filling its interstices. The thickness of such a composite layer was increased linearly with the square root of isothermal holding time at 925 °C, complying with the law of a diffusion-controlled process. However, at 1050 °C, the segregation behavior of Ti and Sn to the interfaces between the TiC layer and the braze matrix diminished, due to the increased solubility of Ti in the Cu-based liquid phase. The enhanced dissolution of Ti in the Cu-based liquid phase at 1050 °C also caused the precipitation of rod-like CuTi with an average diameter of about 0.2 µm during cooling. SnTi3 was the predominant intermetallic compound and existed in three different forms in the braze matrix. It existed as interconnected grains of large size which either floated to the surface of the braze matrix or grew into faceted grains. It also exhibited a nail-like structure with a mean diameter of about 1 µm for the rod section and a lamellar structure arising from a eutectic reaction during cooling.

  2. Microstructure and corrosion behavior of porous coatings on titanium alloy by vacuum-brazed method.

    PubMed

    Lee, T M; Chang, E; Yen, C H

    2006-05-01

    The microstructural evolution and electrochemical characteristics of brazed porous-coated Ti-6Al-4V alloy were analyzed and compared with respect to the conventionally 1300 degrees C sintering method. The titanium filler metal of low-melting-point (934 degrees C) Ti-15Cu-15Ni was used to braze commercially pure (CP) titanium beads onto the substrate of Ti-6Al-4V alloy at 970 degrees C for 2 and 8 h. Optical microscopy, scanning and transmission electron microscopy, and X-ray diffractometry (XRD) were used to characterize the microstructure and phase of the brazed metal; also, the potentiostat was used for corrosion study. Experimental results indicate that the bead/substrate contact interface of the 970 degrees C brazed specimens show larger contact area and higher radius curvature in comparison with 1300 degrees C sintering method. The microstructure of brazed specimens shows the Widmanstätten structure in the brazed zone and equiaxed alpha plus intergranular beta in the Ti-6Al-4V substrate. The intermetallic Ti2Ni phase existing in the prior filler metal diminishes, while the Ti2Cu phase can be identified for the substrate at 970 for 2 h, but the latter phase decrease with time. In Hank's solution at 37 degrees C, the corrosion rates of the 1300 degrees C sintering and the 970 degrees C brazed samples are similar at corrosion potential (E(corr)) in potentiodynamic test, and the value of E(corr) for the brazed sample is noble to the sintering samples. The current densities of the brazed specimens do not exceed 100 microA/cm2 at 3.5 V (SCE). These results suggest that the vacuum-brazed method exhibits the potentiality to manufacture the porous-coated specimens for biomedical application.

  3. Evolution of Microstructure in Brazed Joints of Austenitic-Martensitic Stainless Steel with Pure Silver Obtained with Ag-27Cu-5Sn Brazing Filler Material

    NASA Astrophysics Data System (ADS)

    Gangadharan, S.; Sivakumar, D.; Venkateswaran, T.; Kulkarni, Kaustubh

    2016-10-01

    Brazing of an austenitic-martensitic stainless steel (AMSS) with pure silver was carried out at 1053 K, 1073 K, and 1093 K (780 °C, 800 °C, and 820 °C) with Ag-27Cu-5Sn (wt pct) as brazing filler material (BFM). Wettability of the liquid BFM over base AMSS surface was found to be poor. Application of nickel coating to the steel was observed to enhance the wettability and to enable the formation of a good bond between BFM and the steel. The mechanism responsible for enhanced metallurgical bonding of the BFM with AMSS in the presence of nickel coating was explained based on diffusional interactions and uphill diffusion of iron, chromium and nickel observed in the brazed microstructure. Good diffusion-assisted zone was observed to form on silver side at all three temperatures. Four phases were encountered within the joint including silver solid solution, copper solid solution, Cu3Sn intermetallic and Ni-Fe solid solution. The Cu3Sn intermetallic was present in small amounts in the joints brazed at 1053 K and 1073 K (780 °C and 800 °C). The joint formed at 1093 K (820 °C) exhibited the absence of Cu3Sn, fewer defects and larger diffusion-assisted zone. Hardness of base AMSS was found to reduce during brazing due to austenite reversion and post-brazing sub-zero treatment for 2.5 hours was found suitable to recover the hardness.

  4. Design data for brazed Rene 41 honeycomb sandwich

    NASA Technical Reports Server (NTRS)

    Hepler, A. K.; Arnquist, J.; Koetje, E. L.; Esposito, J. J.; Lindsay, V. E. J.; Swegle, A. R.

    1981-01-01

    Strength data, creep data and residual strength data after cyclic thermal exposure were obtained at temperatures from 78 K to 1144 K (-320 F to 1600 F). The influences of face thickness, core depth, core gage, cell size and thermal/stress exposure conditions on the mechanical design properties were investigated. A braze alloy and process was developed that is adequate to fully develop the strength of the honeycomb core while simultaneously solution treating and aging the Rene 41 fact sheets. New test procedures and test specimen configurations were developed to avoid excessive thermal stresses during cyclic thermal exposure.

  5. Optineurin mediates a negative regulation of Rab8 by the GTPase-activating protein TBC1D17.

    PubMed

    Vaibhava, Vipul; Nagabhushana, Ananthamurthy; Chalasani, Madhavi Latha Somaraju; Sudhakar, Cherukuri; Kumari, Asha; Swarup, Ghanshyam

    2012-11-01

    Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).

  6. [Isolation, phylogenetic analysis and developmental expression parttern of AmphiRab23b in amphioxus].

    PubMed

    Li, Jian-Wei; Lin, Yu-Shuang; Chen, Dong-Yan; Zhang, Hong-Wei

    2009-12-01

    The hedgehog (Hh) pathway plays an important role during the embryonic development and is related to the progression of cancers. Rab23 is a crucial functional molecule in Hh pathway. However, there is no report about amphioxus Rab23 up to now except the annotations of two isoforms in the genome of Florida lancelet (Branchiostoma floridae). Here a 2062 bp full-length cDNA sequence of the Rab23, AmphiRab23b, was isolated from Chinese amphioxus (Branchiostoma belcheri), which included the UTRs and an open reading frame of 714 bp, encoding a protein of 237 amino acids. Phylogenetic analysis suggested that AmphiRab23b falled outside the vertebrate clade. But sequence analysis indicated that this putative AmphiRab23b protein contained a specific Rab23_lke domain, which implied that Rab23 gene was functional conservative during evolution. And its developmental expression pattern showed that AmphiRab23b was expressed in the differentiating neural plate and alimentary canal, as the same as the expression pattern of the homologous vertebrate genes, which suggested that AmphiRab23b may function in the development of nervous system and alimentary canal.

  7. Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

    PubMed Central

    Mahanty, Sarmistha; Ravichandran, Keerthana; Chitirala, Praneeth; Prabha, Jyothi; Jani, Riddhi Atul; Setty, Subba Rao gangi

    2016-01-01

    Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky–Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes. PMID:26527546

  8. Yersinia pestis Requires Host Rab1b for Survival in Macrophages

    PubMed Central

    Connor, Michael G.; Pulsifer, Amanda R.; Price, Christopher T.; Abu Kwaik, Yousef; Lawrenz, Matthew B.

    2015-01-01

    Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV) and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH. PMID:26495854

  9. Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

    PubMed Central

    Edinger, Robert S.; Silvis, Mark R.; Gallo, Luciana I.; Liang, Xiubin; Apodaca, Gerard; Fizzell, Raymond A.; Johnson, John P.

    2012-01-01

    Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na+) transport. The greatest reduction in Na+ transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na+ transport in mpkCCD cells. PMID:22129970

  10. Interferon-γ-inducible Rab20 regulates endosomal morphology and EGFR degradation in macrophages.

    PubMed

    Pei, Gang; Schnettger, Laura; Bronietzki, Marc; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel

    2015-09-01

    Little is known about the molecular players that regulate changes in the endocytic pathway during immune activation. Here we investigate the role of Rab20 in the endocytic pathway during activation of macrophages. Rab20 is associated with endocytic structures, but the function of this Rab GTPase in the endocytic pathway remains poorly characterized. We find that in macrophages, Rab20 expression and endosomal association significantly increase after interferon-γ (IFN-γ) treatment. Moreover, IFN-γ and Rab20 expression induce a dramatic enlargement of endosomes. These enlarged endosomes are the result of homotypic fusion promoted by Rab20 expression. The expression of Rab20 or the dominant-negative mutant Rab20T19N does not affect transferrin or dextran 70 kDa uptake. However, knockdown of Rab20 accelerates epidermal growth factor (EGF) trafficking to LAMP-2-positive compartments and EGF receptor degradation. Thus this work defines a function for Rab20 in the endocytic pathway during immune activation of macrophages.

  11. bMERB domains are bivalent Rab8 family effectors evolved by gene duplication

    PubMed Central

    Rai, Amrita; Oprisko, Anastasia; Campos, Jeremy; Fu, Yangxue; Friese, Timon; Itzen, Aymelt; Goody, Roger S; Gazdag, Emerich Mihai; Müller, Matthias P

    2016-01-01

    In their active GTP-bound form, Rab proteins interact with proteins termed effector molecules. In this study, we have thoroughly characterized a Rab effector domain that is present in proteins of the Mical and EHBP families, both known to act in endosomal trafficking. Within our study, we show that these effectors display a preference for Rab8 family proteins (Rab8, 10, 13 and 15) and that some of the effector domains can bind two Rab proteins via separate binding sites. Structural analysis allowed us to explain the specificity towards Rab8 family members and the presence of two similar Rab binding sites that must have evolved via gene duplication. This study is the first to thoroughly characterize a Rab effector protein that contains two separate Rab binding sites within a single domain, allowing Micals and EHBPs to bind two Rabs simultaneously, thus suggesting previously unknown functions of these effector molecules in endosomal trafficking. DOI: http://dx.doi.org/10.7554/eLife.18675.001 PMID:27552051

  12. Rab18 Dynamics in Adipocytes in Relation to Lipogenesis, Lipolysis and Obesity

    PubMed Central

    Pulido, Marina R.; Diaz-Ruiz, Alberto; Jiménez-Gómez, Yolanda; Garcia-Navarro, Socorro; Gracia-Navarro, Francisco; Tinahones, Francisco; López-Miranda, José; Frühbeck, Gema; Vázquez-Martínez, Rafael; Malagón, Maria M.

    2011-01-01

    Lipid droplets (LDs) are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K), the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER) is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed. PMID:21829560

  13. RAB-7 antagonizes LET-23 EGFR signaling during vulva development in Caenorhabditis elegans.

    PubMed

    Skorobogata, Olga; Rocheleau, Christian E

    2012-01-01

    The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(-) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.

  14. Crystal Structures of Human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating Protein (RabGAP) Domains Reveal Critical Elements for GLUT4 Translocation

    SciTech Connect

    S Park; W Jin; S Shoelson

    2011-12-31

    We have solved the x-ray crystal structures of the RabGAP domains of human TBC1D1 and human TBC1D4 (AS160), at 2.2 and 3.5 {angstrom} resolution, respectively. Like the yeast Gyp1p RabGAP domain, whose structure was solved previously in complex with mouse Rab33B, the human TBC1D1 and TBC1D4 domains both have 16 {alpha}-helices and no {beta}-sheet elements. We expected the yeast Gyp1p RabGAP/mouse Rab33B structure to predict the corresponding interfaces between cognate mammalian RabGAPs and Rabs, but found that residues were poorly conserved. We further tested the relevance of this model by Ala-scanning mutagenesis, but only one of five substitutions within the inferred binding site of the TBC1D1 RabGAP significantly perturbed catalytic efficiency. In addition, substitution of TBC1D1 residues with corresponding residues from Gyp1p did not enhance catalytic efficiency. We hypothesized that biologically relevant RabGAP/Rab partners utilize additional contacts not described in the yeast Gyp1p/mouse Rab33B structure, which we predicted using our two new human TBC1D1 and TBC1D4 structures. Ala substitution of TBC1D1 Met{sup 930}, corresponding to a residue outside of the Gyp1p/Rab33B contact, substantially reduced catalytic activity. GLUT4 translocation assays confirmed the biological relevance of our findings. Substitutions with lowest RabGAP activity, including catalytically dead RK and Met{sup 930} and Leu{sup 1019} predicted to perturb Rab binding, confirmed that biological activity requires contacts between cognate RabGAPs and Rabs beyond those in the yeast Gyp1p RabGAP/mouse Rab33B structure.

  15. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

    PubMed Central

    Maringer, Katherine; Saheb, Entsar; Bush, John

    2014-01-01

    Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA), and dominant negative (DN) forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses. PMID:25157910

  16. A Compendium of Brazed Microstructures For Fission Power Systems Applications

    NASA Technical Reports Server (NTRS)

    Locci, Ivan E.; Bowman, Cheryl L.

    2012-01-01

    NASA has been supporting design studies and technology development for fission-based power systems that could provide power to an outpost on the Moon, Mars, or an asteroid. Technology development efforts have included fabrication and evaluation of components used in a Stirling engine power conversion system. This investigation is part of the development of several braze joints crucial for the heat exchanger transfer path from a hot-side heat exchanger to a Stirling engine heat acceptor. Dissimilar metal joints are required to impart both mechanical strength and thermal path integrity for a heater head of interest. Preliminary design work for the heat exchanger involved joints between low carbon stainless steel to Inconel 718, where the 316L stainless steel would contain flowing liquid metal NaK while Inconel 718, a stronger alloy, would be used as structural reinforcement. This paper addressed the long-term microstructural stability of various braze alloys used to join 316L stainless steel heater head to the high conductivity oxygen-free copper acceptor to ensure the endurance of the critical metallic components of this sophisticated heat exchanger. The bonding of the 316L stainless steel heater head material to a copper heat acceptor is required to increase the heat-transfer surface area in contact with flowing He, which is the Stirling engine working fluid.

  17. Mechanical design of ceramic beam tube braze joints for NOvA kicker magnets

    SciTech Connect

    Ader, C.R.; Reilly, R.E.; Wilson, J.H.; /Fermilab

    2010-05-01

    The NO?A Experiment will construct a detector optimized for electron neutrino detection in the existing NuMI neutrino beam. The NuMI beam line is capable of operating at 400 kW of primary beam power and the upgrade will allow up to 700 kW. Ceramic beam tubes are utilized in numerous kicker magnets in different accelerator rings at Fermi National Accelerator Laboratory. Kovar flanges are brazed onto each beam tube end, since kovar and high alumina ceramic have similar expansion curves. The tube, kovar flange, end piece, and braze foil alloy brazing material are stacked in the furnace and then brazed. The most challenging aspect of fabricating kicker magnets in recent years have been making hermetic vacuum seals on the braze joints between the ceramic and flange. Numerous process variables can influence the robustness of conventional metal/ceramic brazing processes. The ceramic-filler metal interface is normally the weak layer when failure does not occur within the ceramic. Differences between active brazing filler metal and the moly-manganese process will be discussed along with the applicable results of these techniques used for Fermilab production kicker tubes.

  18. Brazed Diamond Micropowder Bur Fabricated by Supersonic Frequency Induction Heating for Precision Machining

    NASA Astrophysics Data System (ADS)

    Ma, Bojiang; Lou, Jianpeng; Pang, Qian

    2014-04-01

    The common brazed diamond micropowder bur fabricated in a vacuum furnace produces an even brazing alloy surface. The small brazed diamond grits show low outcropping from the brazing alloy surface, and the chip space between them is small. The bur shows a low grinding efficiency and poor heat dissipation. In this study, a brazed diamond micropowder bur was fabricated by supersonic frequency induction heating. The method afforded a fluctuant surface on the brazing alloy. The brazed diamond grits with an outcropping height distributed uniformly on the fluctuant surface. The fluctuant surface showed a certain chip space. These characteristics of the tool increased the grinding efficiency and decreased the temperature of the grinding arc area. The roughness R a of the ceramic tile surface trimmed by the tool cylinder was between 0.09 and 0.12 μm. In the first 90 min, the decrease in the weight of the ceramic tile ground by the tool cylinder was higher than that ground by the tool fabricated in a vacuum furnace. When the ceramic tile was cylindrically ground, the temperature of the grinding arc area measured using a thermocouple remained below 70 °C.

  19. Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.

    PubMed

    Saito-Nakano, Yumiko; Yasuda, Tomoyoshi; Nakada-Tsukui, Kumiko; Leippe, Matthias; Nozaki, Tomoyoshi

    2004-11-19

    In mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation. Here, we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba histolytica, EhRab5 and EhRab7A, are involved in steps that are distinct from those known for mammals. EhRab5 and EhRab7A were localized to independent small vesicular structures at steady state. Priming with red blood cells induced the formation of large vacuoles associated with both EhRab5 and EhRab7A ("prephagosomal vacuoles (PPV)") in the amoeba within an incubation period of 5-10 min. PPV emerged de novo physically and distinct from phagosomes. PPV were gradually acidified and matured by fusion with lysosomes containing a digestive hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore. After EhRab5 dissociated from PPV, 5-10 min later, the EhRab7A-PPV fused with phagosomes, and EhRab7A finally dissociated from the phagosomes. Immunoelectron and light micrographs showed that PPV contained small vesicle-like structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the mechanism was similar to multivesicular body formation in PPV generation. In contrast to Rab5 from other organisms, EhRab5 was involved exclusively in phagocytosis, but not in endocytosis. Overexpression of wild-type EhRab5 enhanced phagocytosis and the transport of amoebapore to phagosomes. Conversely, expression of an EhRab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis. Altogether, we propose that the amoebic Rab5 plays an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for packaging of lysosomal hydrolases, prior to the targeting to phagosomes. PMID:15347665

  20. Regulation of β2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor.

    PubMed

    Lachance, Véronik; Cartier, Andréane; Génier, Samuel; Munger, Sandra; Germain, Pascale; Labrecque, Pascale; Parent, Jean-Luc

    2011-11-25

    Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the β(2)-adrenergic receptor (β(2)AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that β(2)AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L(339)L(340) motif in the β(2)AR C terminus known to be involved in the transport of the receptor from the endoplasmic reticulum to the cell surface. Modulation of the cellular levels of RGGTA protein by overexpression or siRNA-mediated knockdown of the endogenous protein demonstrated that RGGTA has a positive role in the maturation and anterograde trafficking of the β(2)AR, which requires the interaction of RGGTA with the β(2)AR L(339)L(340) motif. Furthermore, the β(2)AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the β(2)AR was dependent on the RGGTA-interacting L(339)L(340) motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted β(2)AR maturation, suggesting that RGGTA may have functions independent of Rab geranylgeranylation. We demonstrate for the first time an interaction between a transmembrane receptor and RGGTA which regulates the maturation and anterograde transport of the receptor, as well as geranylgeranylation of Rab GTPases.

  1. Analyzing the Functions of Rab11-Effector Proteins During Cell Division

    PubMed Central

    Prekeris, Rytis

    2015-01-01

    Recycling endosomes recently have emerged as major regulators of cytokinesis and abscission steps of cell division. Rab11-endosomes in particular were shown to transport proteins to the mitotic ingression furrow and play a key role in establishing the abscission site. Rab11 GTPase function by binding and activations various effector proteins, such as Rab11 family interacting proteins (FIPs). FIPs appear to be at the core of many Rab11 functions, with FIP3 playing a role in targeting of the Rab11-endosomes during mitosis. Here we summarize the newest finding regarding the roles and regulation of FIP3 and Rab11 complex, as well as describe the methods developed to analyze membrane and cytoskeleton dynamics during abscission step of cytokinesis. PMID:26360025

  2. Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic.

    PubMed

    Poteryaev, Dmitry; Fares, Hanna; Bowerman, Bruce; Spang, Anne

    2007-01-24

    The small rab-GTPase RAB-7 acts in endosome and endosome to lysosome traffic. We identified SAND-1 as a protein required for RAB-7 function based on similarities between SAND-1 and RAB-7 RNAi phenotypes. Although the initial uptake of yolk protein in oocytes, or of soluble secreted (ss) GFP in coelomocytes, appeared normal, further transport along the endocytic traffic route was delayed in the absence of SAND-1 function, and yolk proteins failed to reach yolk granules efficiently. Moreover, in coelomocytes, ssGFP and BSA-Texas-Red were endocytosed but not transported to lysosomes. We show that SAND-1 is essential for RAB-7 function at the transition from early to late endosomes, but not for RAB-7 function at lysosomes.

  3. Analyzing the functions of Rab11-effector proteins during cell division.

    PubMed

    Prekeris, Rytis

    2015-01-01

    Recycling endosomes recently have emerged as major regulators of cytokinesis and abscission steps of cell division. Rab11-endosomes in particular were shown to transport proteins to the mitotic ingression furrow and play a key role in establishing the abscission site. Rab11 GTPase functions by binding and activating various effector proteins, such as Rab11 family interacting proteins (FIPs). FIPs appear to be at the core of many Rab11 functions, with FIP3 playing a role in targeting of the Rab11-endosomes during mitosis. Here we summarize the newest finding regarding the roles and regulation of FIP3 and Rab11 complex, as well as describe the methods developed to analyze membrane and cytoskeleton dynamics during abscission step of cytokinesis. PMID:26360025

  4. Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster.

    PubMed

    Mavor, Lauren M; Miao, Hui; Zuo, Zhongyuan; Holly, Ryan M; Xie, Yi; Loerke, Dinah; Blankenship, J Todd

    2016-03-01

    One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminate in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events are unclear. Here, we show that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. We additionally use CRISPR/Cas9 technology to N-terminally tag Rab8, which is then used to address endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. We also demonstrate that Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome protein Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, whereas GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through interactions with the exocyst tethering complex. PMID:26839362

  5. Rab3A deletion selectively reduces spontaneous neurotransmitter release at the mouse neuromuscular synapse.

    PubMed

    Sons, Michèle S; Plomp, Jaap J

    2006-05-17

    Rab3A is a synaptic vesicle-associated GTP-binding protein thought to be involved in modulation of presynaptic transmitter release through regulation of vesicle trafficking and membrane fusion. Electrophysiological studies at central nervous system synapses of Rab3A null-mutant mice have indicated that nerve stimulation-evoked transmitter release and its short- and long-term modulation are partly dependent on Rab3A, whereas spontaneous uniquantal release is completely independent of it. Here, we studied the acetylcholine (ACh) release at the neuromuscular junction (NMJ) of diaphragm and soleus muscles from Rab3A-deficient mice with intracellular microelectrode methods. Surprisingly, we found 20-40% reduction of spontaneous ACh release but completely intact nerve action potential-evoked release at both high- and low-rate stimulation and during recovery from intense release. The ACh release induced by hypertonic medium was also unchanged, indicating that the pool of vesicles for immediate release is unaltered at the Rab3A-deficient NMJ. These results indicate a selective role of Rab3A in spontaneous transmitter release at the NMJ which cannot or only partly be taken over by the closely related Rab3B, Rab3C, or Rab3D isoforms when Rab3A is deleted. It has been hypothesized that Rab3A mutation underlies human presynaptic myasthenic syndromes, in which severely reduced nerve action potential-evoked ACh release at the NMJ causes paralysis. Our observation that Rab3A deletion does not reduce evoked ACh release at any stimulation rate at the mouse NMJ, argues against this hypothesis. PMID:16631140

  6. Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster.

    PubMed

    Mavor, Lauren M; Miao, Hui; Zuo, Zhongyuan; Holly, Ryan M; Xie, Yi; Loerke, Dinah; Blankenship, J Todd

    2016-03-01

    One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminate in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events are unclear. Here, we show that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. We additionally use CRISPR/Cas9 technology to N-terminally tag Rab8, which is then used to address endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. We also demonstrate that Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome protein Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, whereas GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through interactions with the exocyst tethering complex.

  7. Rab25 upregulation correlates with the proliferation, migration, and invasion of renal cell carcinoma

    SciTech Connect

    Li, Yuanyuan; Jia, Qingzhu; Zhang, Qian; Wan, Ying

    2015-03-20

    Renal cell carcinoma (RCC) is a common urological cancer with a poor prognosis. A recent cohort study revealed that the median survival of RCC patients was only 1.5 years and that <10% of the patients in the study survived up to 5 years. In tumor development, Rab GTPase are known to play potential roles such as regulation of cell proliferation, migration, invasion, communication, and drug resistance in multiple tumors. However, the correlation between Rabs expression and the occurrence, development, and metastasis of RCC remains unclear. In this study, we analyzed the transcriptional levels of 52 Rab GTPases in RCC patients. Our results showed that high levels of Rab25 expression were significantly correlated with RCC invasion classification (P < 0.01), lymph-node metastasis (P < 0.001), and pathological stage (P < 0.01). Conversely, in 786-O and A-498 cells, knocking down Rab25 protein expression inhibited cell proliferation, migration, and invasion. Our results also demonstrated that Rab25 is a target gene of let-7d, and further suggested that Rab25 upregulation in RCC is due to diminished expression of let-7d. These findings indicate that Rab25 might be a novel candidate molecule involved in RCC development, thus identifying a potential biological therapeutic target for RCC. - Highlights: • The transcriptional levels of 52 Rab GTPases were analyzed in renal cell carcinoma (RCC). • High levels of Rab25 expression were significantly correlated with clinicopathological factors of RCC. • Knockdown of Rab25 protein expression reduced RCC cells proliferation, migration, and invasion. • Rab25 is a target gene of let-7d in RCC.

  8. Insights into endosomal maturation of human holo-transferrin in the enteric parasite Entamoeba histolytica: essential roles of Rab7A and Rab5 in biogenesis of giant early endocytic vacuoles.

    PubMed

    Verma, Kuldeep; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2015-12-01

    The pathogenic amoeba Entamoeba histolytica is one of the causative agents of health hazards in tropical countries. It causes amoebic dysentery, colitis and liver abscesses in human. Iron is one of the essential nutritional resources for survival and chronic infection caused by the amoeba. The parasite has developed multiple ways to import, sequester and utilize iron from various iron-binding proteins from its host. In spite of its central role in pathogenesis, the mechanism of iron uptake by the parasite is largely unknown. Here, we carried out a systematic study to understand the role of some of the amoebic homologues of mammalian endocytic Rab GTPases (Rab5 and Rab21, Rab7A and Rab7B) in intracellular transport of human holo-transferrin by the parasite. Flow cytometry and quantitative microscopic image analysis revealed that Rab5 and Rab7A are required for the biogenesis of amoebic giant endocytic vacuoles (GEVs) and regulate the early phase of intracellular trafficking of transferrin. Rab7B is involved in the late phase, leading to the degradation of transferrin in the amoebic lysosome-like compartments. Using time-lapse fluorescence imaging in fixed trophozoites, we determined the kinetics of the vesicular transport of transferrin through Rab5-, Rab7A- and Rab7B-positive compartments. The involvement of Rab7A in the early phase of endocytosis by the parasite marks a significant divergence from its host in terms of spatiotemporal regulation by the Rab GTPases. PMID:26096601

  9. Regulation of liver metabolism by the endosomal GTPase Rab5.

    PubMed

    Zeigerer, Anja; Bogorad, Roman L; Sharma, Kirti; Gilleron, Jerome; Seifert, Sarah; Sales, Susanne; Berndt, Nikolaus; Bulik, Sascha; Marsico, Giovanni; D'Souza, Rochelle C J; Lakshmanaperumal, Naharajan; Meganathan, Kesavan; Natarajan, Karthick; Sachinidis, Agapios; Dahl, Andreas; Holzhütter, Hermann-Georg; Shevchenko, Andrej; Mann, Matthias; Koteliansky, Victor; Zerial, Marino

    2015-05-12

    The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases. PMID:25937276

  10. Identifying a Rab effector on the macroautophagy pathway.

    PubMed

    Wang, Juan; Cervantes, Serena; Davis, Saralin; Ferro-Novick, Susan

    2015-01-01

    Rab GTPases are key regulators of membrane traffic. The Rab GTPase Ypt1 is essential for endoplasmic reticulum (ER)-Golgi traffic, intra-Golgi traffic, and the macroautophagy pathway. To identify effectors on the macroautophagy pathway, known autophagy-related genes (Atg genes) required for macroautophagy were tagged with GFP and screened for mislocalization in the ypt1-2 mutant. At the pre-autophagosomal structure (PAS), the localization of the serine/threonine kinase Atg1 was affected in the ypt1-2 mutant. We then used an in vitro binding assay to determine if Atg1 and Ypt1 physically interact with each other and co-immunoprecipitation experiments were performed to address if Atg1 preferentially interacts with the GTP-bound form of Ypt1.

  11. Active Metal Brazing and Characterization of Brazed Joints in C-C and C-SiC Composites to Copper-Clad-Molybdenum System

    NASA Technical Reports Server (NTRS)

    Singh, M.; Asthana, R.

    2008-01-01

    Carbon/carbon composites with CVI and resin-derived matrices, and C/SiC composites reinforced with T-300 carbon fibers in a CVI SiC matrix were joined to Cu-clad Mo using two Ag-Cu braze alloys, Cusil-ABA (1.75% Ti) and Ticusil (4.5% Ti). The brazed joints revealed good interfacial bonding, preferential precipitation of Ti at the composite/braze interface, and a tendency toward delamination in resin-derived C/C composite. Extensive braze penetration of the inter-fiber channels in the CVI C/C composites was observed. The Knoop microhardness (HK) distribution across the C/C joints indicated sharp gradients at the interface, and a higher hardness in Ticusil than in Cusil-ABA. For the C/SiC composite to Cu-clad-Mo joints, the effect of composite surface preparation revealed that ground samples did not crack whereas unground samples cracked. Calculated strain energy in brazed joints in both systems is comparable to the strain energy in a number of other ceramic/metal systems. Theoretical predictions of the effective thermal resistance suggest that such joined systems may be promising for thermal management applications.

  12. Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

    SciTech Connect

    Silva, Patricio; Soto, Nicolás; Díaz, Jorge; Mendoza, Pablo; Díaz, Natalia; Quest, Andrew F.G.; Torres, Vicente A.

    2015-08-21

    The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5 is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.

  13. Involvement of Rab6 in the regulation of phagocytosis against virus infection in invertebrates.

    PubMed

    Ye, Ting; Tang, Wen; Zhang, Xiaobo

    2012-10-01

    Phagocytosis, which is of fundamental importance for innate and adaptive immunity in animals, is driven by organization of the actin cytoskeleton. To date, however, the molecular events involved in the regulation of phagocytosis through reorganization of actin by small G proteins remains to be elucidated. To address this issue, the molecular mechanism of Rab6 in phagocytosis against virus infection in invertebrates was characterized in this study. The results showed that the Rab6 obtained from shrimp could interact with actin to regulate shrimp hemocyte phagocytosis through induction of the rearrangement of actin to protect against white spot syndrome virus (WSSV) infection. The Rab6 protein in Drosophila melanogaster shared the same mechanism of action as that of Rab6 in shrimp, indicating that the function of Rab6 in phagocytosis was conserved in invertebrates. By comparison with the early marker (Rab5) and late marker (LAMP1) of phagosomes, Rab6 was critically involved in the regulation of actin organization throughout the entire phagocytosis process. The presence of the evolutionarily conserved amino acid sequences of Rab6 in invertebrates and vertebrates indicated a conserved mechanism of Rab6 function in phagocytosis of animals. Therefore, our findings presented novel molecular events in the regulation of phagocytosis by small G proteins.

  14. The small GTPase Rab33A participates in regulation of amylase release from parotid acinar cells.

    PubMed

    Imai, Akane; Tsujimura, Maiko; Yoshie, Sumio; Fukuda, Mitsunori

    2015-06-01

    Amylase is released from exocrine parotid acinar cells via typical exocytosis. Exocytosis of amylase-containing granules occurs through several steps, including formation, maturation, and transport of granules. These steps are thought to be regulated by members of the small GTPase Rab family. We previously demonstrated that Rab27 and its effectors mediate amylase release from parotid acinar cells, but the functional involvement of other Rab proteins in exocrine granule exocytosis remains largely unknown. Here, we studied isoproterenol (IPR)-induced amylase release from parotid acinar cells to investigate the possible involvement of Rab33A, which was recently suggested to regulate exocytosis in hippocampal neurons and PC12 cells. Rab33A was endogenously expressed in parotid acinar cells and present in secretory granules and the Golgi body. Functional ablation of Rab33A with anti-Rab33A antibody or a dominant-negative Rab33A-T50N mutant significantly reduced IPR-induced amylase release. Our results indicated that Rab33A is a novel component of IPR-stimulated amylase secretion from parotid acinar cells.

  15. Rab from the white shrimp Litopenaeus vannamei: characterization and its regulation upon environmental stress.

    PubMed

    Wang, Lei; Wang, Xiao-Rong; Liu, Jin; Chen, Chu-Xian; Liu, Yuan; Wang, Wei-Na

    2015-10-01

    With the destruction of the ecological environment, shrimp cultivation in China has been seriously affected by outbreaks of infectious diseases. Rab, which belong to small GTPase Ras superfamily, can regulate multiple steps in eukaryotic vesicle trafficking including vesicle budding, vesicle tethering, and membrane fusion. Knowledge of Rab in shrimp is essential to understanding regulation and detoxification mechanisms of environmental stress. In this study, we analyzed the functions of Rab from the Pacific white shrimp, Litopenaeus vannamei. Full-length cDNA of Rab was obtained, which was 751 bp long, with open reading frame encoding 206 amino acids. In this study, for the first time, the gene expression of Rab of L. vannamei was analyzed by quantitative real-time PCR after exposure to five kinds of environmental stresses (bacteria, pH, Cd, salinity and low temperature). The results demonstrate that Rab is sensitive and involved in bacteria, pH, and Cd stress responses and Rab is more sensitive to bacteria than other stresses. Therefore we infer that Rab may have relationship with the anti-stress mechanism induced by environment stress in shrimp and Rab could be used as critical biomarkers for environmental quality assessment.

  16. Regulation of Cancer Cell Behavior by the Small GTPase Rab13.

    PubMed

    Ioannou, Maria S; McPherson, Peter S

    2016-05-01

    The members of the Rab family of GTPases are master regulators of cellular membrane trafficking. With ∼70 members in humans, Rabs have been implicated in all steps of membrane trafficking ranging from vesicle formation and transport to vesicle docking/tethering and fusion. Vesicle trafficking controls the localization and levels of a myriad of proteins, thus regulating cellular functions including proliferation, metabolism, cell-cell adhesion, and cell migration. It is therefore not surprising that impairment of Rab pathways is associated with diseases including cancer. In this review, we highlight evidence supporting the role of Rab13 as a potent driver of cancer progression. PMID:27044746

  17. Role of Rab27 in synaptic transmission at the squid giant synapse.

    PubMed

    Yu, Eunah; Kanno, Eiko; Choi, Soonwook; Sugimori, Mutsuyuki; Moreira, Jorge E; Llinás, Rodolfo R; Fukuda, Mitsunori

    2008-10-14

    Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.

  18. Role of Rab27 in synaptic transmission at the squid giant synapse

    PubMed Central

    Yu, Eunah; Kanno, Eiko; Choi, Soonwook; Sugimori, Mutsuyuki; Moreira, Jorge E.; Llinás, Rodolfo R.; Fukuda, Mitsunori

    2008-01-01

    Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca2+ current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca2+-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse. PMID:18840683

  19. Rab10 associates with primary cilia and the exocyst complex in renal epithelial cells

    PubMed Central

    Babbey, Clifford M.; Bacallao, Robert L.

    2010-01-01

    Rab10, a mammalian homolog of the yeast Sec4p protein, has previously been associated with endocytic recycling and biosynthetic membrane transport in cultured epithelia and with Glut4 translocation in adipocytes. Here, we report that Rab10 associates with primary cilia in renal epithelia in culture and in vivo. In addition, we find that Rab10 also colocalizes with exocyst proteins at the base of nascent cilia, and physically interacts with the exocyst complex, as detected with anti-Sec8 antibodies. These data suggest that membrane transport to the primary cilum may be mediated by interactions between Rab10 and an exocyst complex located at the cilium base. PMID:20576682

  20. The late endocytic Rab39a GTPase regulates the interaction between multivesicular bodies and chlamydial inclusions.

    PubMed

    Gambarte Tudela, Julian; Capmany, Anahi; Romao, Maryse; Quintero, Cristian; Miserey-Lenkei, Stephanie; Raposo, Graca; Goud, Bruno; Damiani, Maria Teresa

    2015-08-15

    Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating the interaction between MVBs and chlamydial inclusions remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles - mainly MVBs - that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development. PMID:26163492

  1. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    SciTech Connect

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K.; James, John; Prescott, Alan; Haga, Ismar R.; Beard, Philippa M.

    2015-01-15

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

  2. The Rab11 Pathway Is Required for Influenza A Virus Budding and Filament Formation▿

    PubMed Central

    Bruce, Emily A.; Digard, Paul; Stuart, Amanda D.

    2010-01-01

    Influenza A virus buds through the apical plasma membrane, forming enveloped virus particles that can take the shape of pleomorphic spheres or vastly elongated filaments. For either type of virion, the factors responsible for separation of viral and cell membranes are not known. We find that cellular Rab11 (a small GTP-binding protein involved in endocytic recycling) and Rab11-family interacting protein 3 ([FIP3] which plays a role in membrane trafficking and regulation of actin dynamics) are both required to support the formation of filamentous virions, while Rab11 is additionally involved in the final budding step of spherical particles. Cells transfected with Rab11 GTP-cycling mutants or depleted of Rab11 or FIP3 content by small interfering RNA treatment lost the ability to form virus filaments. Depletion of Rab11 resulted in up to a 100-fold decrease in titer of spherical virus released from cells. Scanning electron microscopy of Rab11-depleted cells showed high densities of virus particles apparently stalled in the process of budding. Transmission electron microscopy of thin sections confirmed that Rab11 depletion resulted in significant numbers of abnormally formed virus particles that had failed to pinch off from the plasma membrane. Based on these findings, we see a clear role for a Rab11-mediated pathway in influenza virus morphogenesis and budding. PMID:20357086

  3. Characterization of in vivo functions of Nicotiana benthamiana RabE1.

    PubMed

    Ahn, Chang Sook; Han, Jeong-A; Pai, Hyun-Sook

    2013-01-01

    We characterized the gene expression, subcellular localization, and in vivo functions of a Nicotiana benthamiana small GTPase belonging to the RabE family, designated NbRabE1. The NbRabE1 promoter drove strong β-glucuronidase reporter expression in young tissues containing actively dividing cells and in stomata guard cells. GFP fusion proteins of NbRabE1 and its dominant-negative and constitutively active mutants were all localized to the Golgi apparatus and the plasma membrane but showed different affinities for membrane attachment. Virus-induced gene silencing of NbRabE1 resulted in pleiotropic phenotypes, including growth arrest, premature senescence, and abnormal leaf development. At the cellular level, the leaves in which NbRabE1 was silenced contained abnormal stomata that lacked pores or contained incomplete ventral walls, suggesting that NbRabE1 deficiency leads to defective guard cell cytokinesis. Ectopic expression of the dominant-negative mutant of NbRabE1 in Arabidopsis thaliana resulted in retardation of shoot and root growth accompanied by defective root hair formation. These developmental defects are discussed in conjunction with proposed functions of RabE GTPases in polarized secretory vesicle trafficking.

  4. Effect of Rab23 on the proliferation and apoptosis in breast cancer.

    PubMed

    Liu, Yali; Zeng, Chao; Bao, Nandi; Zhao, Jie; Hu, Yuzhen; Li, Chengxin; Chi, Sumin

    2015-10-01

    Rab23, as a negative regulatory molecule of the Hedgehog (Hh) signaling pathway, may be a new target for treating carcinoma. In the present study, we aimed to determine whether Rab23 is expressed in breast cancer cells and whether Rab23 affects the viability and proliferation of breast cancer cells. We evaluated Rab23 expression in several breast cancer cell lines including MDA-MB-231, Bcap37 and MCF-7 by reverse transcription-PCR (RT-PCR), western blotting and immunofluorescence in vitro. We assessed cell growth and proliferation by 3-(4,5-dimethylthiazol‑2-y1)‑3,5-diphenyltetrazolium bromide (MTT), colony formation and bromodeoxyuridine (BrdU) incorporation assays. The distribution of the cell cycle and the rate of apoptosis were assessed using flow cytometry (FCM). In addition, we determined the mechanisms by which Rab23 regulates the Hh pathway by detecting the level of Gli molecules by RT-PCR. We found that Rab23 mRNA and protein levels were expressed in breast cancer cells, and the expression of Rab23 in MDA-MB-231 cells was higher than that in the MCF-7 cells. Rab23 protein was primarily expressed and localized in the cytoplasm surrounding the nucleus. The MTT assay showed that the absorbance value at A(490 nm) of the Rab23‑transfected group was reduced in comparison with the control group. The number of colonies formed in the breast cancer cells was significantly reduced and BrdU labeling was weakened in the group transfected with Rab23. The results of FCM showed that overexpression of Rab23 protein caused cell cycle arrest in the G1 phase and a decrease in the S phase population as well as induction of apoptosis. Furthermore, Rab23 decreased Gli1 and Gli2 mRNA levels when compared with the control group. Our results indicate that Rab23 is expressed in breast cancer cells, and ectopic expression of Rab23 inhibits the growth and proliferation as well as induces cell apoptosis in breast cancer cells. These effects may be due to the inhibition by Rab23

  5. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules

    SciTech Connect

    Darchen, F.; Hammel, F.; Monteils, M.P.; Scherman, D. ); Zahraoui, A.; Tavitian, A. )

    1990-08-01

    The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells, Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine {beta}-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.

  6. Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34.

    PubMed

    Kasmapour, Bahram; Gronow, Achim; Bleck, Christopher K E; Hong, Wanjin; Gutierrez, Maximiliano Gabriel

    2012-12-11

    Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through size-selective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria. PMID:23197834

  7. Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34

    PubMed Central

    Kasmapour, Bahram; Gronow, Achim; Bleck, Christopher K. E.; Hong, Wanjin; Gutierrez, Maximiliano Gabriel

    2012-01-01

    Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through size-selective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria. PMID:23197834

  8. Aberrant expression of RAB1A in human tongue cancer

    PubMed Central

    Shimada, K; Uzawa, K; Kato, M; Endo, Y; Shiiba, M; Bukawa, H; Yokoe, H; Seki, N; Tanzawa, H

    2005-01-01

    This study was designed to identify specific gene expression changes in tongue squamous cell carcinomas (TSCCs) compared with normal tissues using in-house cDNA microarray that comprised of 2304 full-length cDNAs from a cDNA library prepared from normal oral tissues, primary oral cancers, and oral cancer cell lines. The genes identified by our microarray system were further analysed at the mRNA or protein expression level in a series of clinical samples by real-time quantitative reverse transcriptase–polymerase chain reaction (qRT–PCR) analysis and imuunohositochemistry. The microarray analysis identified a total of 16 genes that were significantly upregulated in common among four TSCC specimens. Consistent with the results of the microarray, increased mRNA levels of selected genes with known molecular functions were found in the four TSCCs. Among genes identified, Rab1a, a member of the Ras oncogene family, was further analysed for its protein expression in 54 TSCCs and 13 premalignant lesions. We found a high prevalence of Rab1A-overexpression not only in TSCCs (98%) but also in premalignant lesions (93%). Thus, our results suggest that rapid characterisation of the target gene(s) for TSCCs can be accomplished using our in-house cDNA microarray analysis combined with the qRT–PCR and immunohistochemistry, and that the Rab1A is a potential biomarker of tongue carcinogenesis. PMID:15870709

  9. Stress level evaluation of thin films under thermal loading from a brazing process

    SciTech Connect

    Wang, Z.; Kuzay, T.M.

    1996-06-01

    X-ray front end beamline windows are made of thin beryllium foil that is commonly brazed or diffusion bonded onto a copper frame. In the brazing process, due to differences in the thermal expansion coefficient of the beryllium and copper materials, the beryllium film ends up in a state of very high level compression stress after cooling from 700{degrees}C (the brazing temperature) to room temperature. This makes the thin Be foil deform into a dome-shaped structure due to the usual asymmetrical geometry of the window. This paper studies the brazing process using a finite element method and explains the reason for such phenomena. Recommendations are offered for possible improvement in the bonding process of beryllium to the window block.

  10. Studies and research on the microstructure of brazed aluminum alloys in the repair process

    NASA Astrophysics Data System (ADS)

    Dimitescu, A.; Amza, Gh; Niţoi, D. F.; Amza, C. Gh; Apostolescu, Z.

    2016-08-01

    In aeronautical industry, in recent years, brazing joints got a growing spread. Therefore, it is necessary a detailed study on the microstructure of the assembly base and filler material brazed in conditions of reconditioning operation [1]. The methods of destructive examination are not associated with any particular type of test piece but lay down the general principles of the types of testing described. It is emphasized that a satisfactory examination method can only be developed and used after taking into account all the relevant factors regarding the equipment to be used and the characteristics of the test pieces being examined. [1, 2]. Brazing joints are most often systematically designed to be resistant to shearing, and the size of the joint influences the shear strength more than the tensile strength. Studies and researches on the microstructure may be necessary to determine the effects of brazing process or of any subsequent heat treatment on the characteristics of the joint.

  11. Accelerator drift-tube braze-joint failures in the PIGMI APF cavity

    SciTech Connect

    Hansborough, L.D.; Levinson, L.S.; Reiswig, R.D.; Wilkerson, L.C.

    1981-01-01

    During the assembly of the Alternating Phase Focusing cavity for the PIGMI Prototype proton accelerator, recurring failures of drift-tube braze joints occurred. In the fabrication technique, a torch braze was used to attach the stems to both the drift-tube body and the stem termination; all materials used were stainless steel. The assemblies were copper plated, using bright-acid-leveling copper plating. Some braze joints, although satisfactorily tension-tested before plating, later failed at a relatively low loading. A detailed investigation of one drift tube indicated that residual copper-plating solution in the cooling passages acted to dissolve the braze solution over a period of weeks, leading to an eventual joint failure.

  12. The active metal brazing of TZM-Mo and SiN ceramics

    NASA Astrophysics Data System (ADS)

    Stephens, J. J.; Vianco, P. T.; Hosking, F. M.

    1996-01-01

    The active metal brazing of ceramics holds the opportunity to design metal-ceramic brazing processes without conventional metallization and nickel-plating steps. Because of their intermediate thermal coefficient of expansion (TCE) properties, molybdenum and the TZM-Mo alloy (Mo-0.5Ti-0.08Zr) are attractive candidates for joining to silicon- nitride ceramics. The recently developed Fe-Ni-Co-based Thermo-Span™ alloy, with reduced TCE from room temperature up to ≈400°C, is also an attractive candidate material for 700°C joint service applications. This article discusses the wetting, solid-state aging, and mechanical behavior of unalloyed molybdenum, TZM-Moalloy, Thermo-Span alloy, and silicon-nitride ceramic brazes made with three different active-metal braze alloys.

  13. Structure of Rab11-FIP3-Rabin8 reveals simultaneous binding of FIP3 and Rabin8 effectors to Rab11.

    PubMed

    Vetter, Melanie; Stehle, Ralf; Basquin, Claire; Lorentzen, Esben

    2015-09-01

    The small GTPase Rab11 and its effectors FIP3 and Rabin8 are essential to membrane-trafficking pathways required for cytokinesis and ciliogenesis. Although effector binding is generally assumed to be sequential and mutually exclusive, we show that Rab11 can simultaneously bind FIP3 and Rabin8. We determined crystal structures of human Rab11-GMPPNP-Rabin8 and Rab11-GMPPNP-FIP3-Rabin8. The structures reveal that the C-terminal domain of Rabin8 adopts a previously undescribed fold that interacts with Rab11 at an unusual effector-binding site neighboring the canonical FIP3-binding site. We show that Rab11-GMPPNP-FIP3-Rabin8 is more stable than Rab11-GMPPNP-Rabin8, owing to direct interaction between Rabin8 and FIP3 within the dual effector-bound complex. The data allow us to propose a model for how membrane-targeting complexes assemble at the trans-Golgi network and recycling endosomes, through multiple weak interactions that create high-avidity complexes.

  14. Flight/ground sample comparison relating to flight experiment M552, exothermic brazing

    NASA Technical Reports Server (NTRS)

    Heine, R. W.; Adams, C. M.; Siewert, T. A.

    1973-01-01

    Comparisons were made between Skylab and ground-based specimens of nickel and stainless steel which were vacuum brazed using silver-copper-lithium alloy with various joint configurations. It was established that the absence of gravity greatly extends the scope of brazing since capillary flow can proceed without gravity interference. There was also evidence of enhanced transport, primarily in that liquid silver copper alloy dissolves nickel to a much greater extent in the zero gravity environment.

  15. Some possible filler alloys with low vapor pressures for refractory-metal brazing

    NASA Technical Reports Server (NTRS)

    Morris, J. F.

    1973-01-01

    A compilation of eutectics and melting-point minima for binary combinations of metals having vapor pressures below 10 to the minus 10th power torr at 1500 degrees K and .00005 torr at 2000 degree K is presented. These compositions and others near them on their phase diagrams are potential special brazing fillers for refractory metals. Some possible problems and advantages for fusion bonds of such mixtures are indicated. Evaluations of brazing fillers containing refractory metals are reported.

  16. Characterization of Brazed Joints of C-C Composite to Cu-clad-Molybdenum

    NASA Technical Reports Server (NTRS)

    Singh, M.; Asthana, R.

    2008-01-01

    Carbon-carbon composites with either pitch+CVI matrix or resin-derived matrix were joined to copper-clad molybdenum using two active braze alloys, Cusil-ABA (1.75% Ti) and Ticusil (4.5% Ti). The brazed joints revealed good interfacial bonding, preferential precipitation of Ti at the composite/braze interface, and a tendency toward de-lamination in resin-derived C-C composite due to its low inter-laminar shear strength. Extensive braze penetration of the inter-fiber channels in the pitch+CVI C-C composites was observed. The relatively low brazing temperatures (<950 C) precluded melting of the clad layer and restricted the redistribution of alloying elements but led to metallurgically sound composite joints. The Knoop microhardness (HK) distribution across the joint interfaces revealed sharp gradients at the Cu-clad-Mo/braze interface and higher hardness in Ticusil (approx.85-250 HK) than in Cusil-ABA (approx.50-150 HK). These C-C/Cu-clad-Mo joints with relatively low thermal resistance may be promising for thermal management applications.

  17. Chemical elements diffusion in the stainless steel components brazed with Cu-Ag alloy

    NASA Astrophysics Data System (ADS)

    Voiculescu, I.; Geanta, V.; Vasile, I. M.; Binchiciu, E. F.; Winestoock, R.

    2016-06-01

    The paper presents the study of diffusion of chemical elements through a brazing joint, between two thin components (0.5mm) made of stainless steel 304. An experimental brazing filler material has been used for brazing stainless steel component and then the diffusion phenomenon has been studied, in terms of chemical element displacement from the brazed separation interface. The filler material is in the form of a metal rod coated with ceramic slurry mixture of minerals, containing precursors and metallic powders, which can contribute to the formation of deposit brazed. In determining the distance of diffusion of chemical elements, on both sides of the fusion line, were performed measurements of the chemical composition using electron microscopy SEM and EDX spectrometry. Metallographic analysis of cross sections was performed with the aim of highlight the microstructural characteristics of brazed joints, for estimate the wetting capacity, adherence of filler metal and highlight any imperfections. Analyzes performed showed the penetration of alloying elements from the solder (Ag, Cu, Zn and Sn) towards the base material (stainless steel), over distances up to 60 microns.

  18. JOINING OF MOLYBDENUM DISILICIDE TO STAINLESS STEEL USING AMORPHOUS METAL BRAZES-RESIDUAL STRESS ANALYSIS

    SciTech Connect

    VAIDYA, RAJENDRA U; KAUTZ, DOUGLAS D.; GALLEGOS, DAVID E.

    2007-01-30

    Molybdenum disilicide (MoSi{sub 2})/stainless steel 316 L jOints were produced by high temperature brazing using a cobalt-based metallic-glass (METGLAS{trademark} 2714A). Successful joining was completed in two different ways; either by feeding excess braze into the braze gap upon heating or by constraining the MoSi{sub 2}/stainiess steel assembly with an alumina (Al{sub 2}O{sub 3}) fixture during the heating cycle. These steps were necessary to ensure the production of a high quality void free joint. Residual stress measurements were completed on these joints. Indentation results show higher tensile residual stresses in the stainless steel for the joint with the external constraint, in comparison to the unconstrained state. In contrast, the compressive residual stresses In the MoSi{sub 2} (as measured by X-ray diffraction) were lower in the constrained state relative to the unconstrained state. These results and a lack of residual stress balance indicate that the stress state in the braze is significantly different under the two joining conditions and the volume of the braze plays an important role in the development of the residual stresses. Push-out tests carried out on these joints gave higher joint strengths in the unconstrained as compared to the constrained condition. The results of this study have important implications on the selection of the appropriate joining process (use of constraint versus extra braze).

  19. Laser brazing of inconel 718 alloy with a silver based filler metal

    NASA Astrophysics Data System (ADS)

    Khorram, A.; Ghoreishi, M.; Torkamany, M. J.; Bali, M. M.

    2014-03-01

    In the presented study laser brazing of an inconel 718 alloy with silver based filler metal using 400 W pulsed Nd:YAG laser is investigated. Laser brazing was performed with varying laser frequency, pulse width, process speed and gap distance. The effect of preheating on wetting and spreading also was studied. Brazing geometrical images were observed using an optical microscope. The composition analysis and microstructure of the filler metal and brazed joints were examined using X-ray diffraction analyzer (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Micro-hardness and tensile test were performed for investigation of mechanical properties. The experimental observations show that filler metal consist of α-Ag solid solution, ά-Cu solid solution surround by the α-Ag solid solution and eutectic structure. Phases of the brazed joint are similar to the filler metal. The results indicate that the filler metal has adequate wetting and spreading on inconel 718 and the wetting angle depends on the heat input significantly. Interdiffusion occurs in laser brazing and the average thickness of reaction layer is approximately 2.5 μm. Whenever the gap is big, it is needed to use longer pulse width in order to have a better melting flow. Preheating has significant influence on wetting and spreading of the filler metal.

  20. Mechanical properties of Inconel 718 and Nickel 201 alloys after thermal histories simulating brazing and high temperature service

    NASA Technical Reports Server (NTRS)

    James, W. F.

    1985-01-01

    An experimental investigation was made to evaluate two nickel base alloys (Nickel-201 and Inconel-718) in three heat treated conditions. These conditions were: (1) annealed; (2) after thermal exposure simulating a braze cycle; and (3) after a thermal exposure simulating a braze cycle plus one operational lifetime of high temperature service. For the Nickel-201, two different braze cycle temperatures were evaluated. A braze cycle utilizing a lower braze temperature resulted in less grain growth for Nickel-201 than the standard braze cycle used for joining Nickel-201 to Inconel-718. It was determined, however, that Nickel-201, was marginal for temperatures investigated due to large grain growth. After the thermal exposures described above, the mechanical properties of Nickel-201 were degraded, whereas similar exposure on Inconel-718 actually strengthened the material compared with the annealed condition. The investigation included tensile tests at both room temperature and elevated temperatures, stress-rupture tests, and metallographic examination.

  1. Braze alloy process and strength characterization studies for 18 nickel grade 200 maraging steel with application to wind tunnel models

    NASA Technical Reports Server (NTRS)

    Bradshaw, James F.; Sandefur, Paul G., Jr.; Young, Clarence P., Jr.

    1991-01-01

    A comprehensive study of braze alloy selection process and strength characterization with application to wind tunnel models is presented. The applications for this study include the installation of stainless steel pressure tubing in model airfoil sections make of 18 Ni 200 grade maraging steel and the joining of wing structural components by brazing. Acceptable braze alloys for these applications are identified along with process, thermal braze cycle data, and thermal management procedures. Shear specimens are used to evaluate comparative shear strength properties for the various alloys at both room and cryogenic (-300 F) temperatures and include the effects of electroless nickel plating. Nickel plating was found to significantly enhance both the wetability and strength properties for the various braze alloys studied. The data are provided for use in selecting braze alloys for use with 18 Ni grade 200 steel in the design of wind tunnel models to be tested in an ambient or cryogenic environment.

  2. Subcellular localization and functional analysis of the Arabidopsis GTPase RabE.

    PubMed

    Speth, Elena Bray; Imboden, Lori; Hauck, Paula; He, Sheng Yang

    2009-04-01

    Membrane trafficking plays a fundamental role in eukaryotic cell biology. Of the numerous known or predicted protein components of the plant cell trafficking system, only a relatively small subset have been characterized with respect to their biological roles in plant growth, development, and response to stresses. In this study, we investigated the subcellular localization and function of an Arabidopsis (Arabidopsis thaliana) small GTPase belonging to the RabE family. RabE proteins are phylogenetically related to well-characterized regulators of polarized vesicle transport from the Golgi apparatus to the plasma membrane in animal and yeast cells. The RabE family of GTPases has also been proposed to be a putative host target of AvrPto, an effector protein produced by the plant pathogen Pseudomonas syringae, based on yeast two-hybrid analysis. We generated transgenic Arabidopsis plants that constitutively expressed one of the five RabE proteins (RabE1d) fused to green fluorescent protein (GFP). GFP-RabE1d and endogenous RabE proteins were found to be associated with the Golgi apparatus and the plasma membrane in Arabidopsis leaf cells. RabE down-regulation, due to cosuppression in transgenic plants, resulted in drastically altered leaf morphology and reduced plant size, providing experimental evidence for an important role of RabE GTPases in regulating plant growth. RabE down-regulation did not affect plant susceptibility to pathogenic P. syringae bacteria; conversely, expression of the constitutively active RabE1d-Q74L enhanced plant defenses, conferring resistance to P. syringae infection. PMID:19233904

  3. Rab4GTPase modulates CFTR function by impairing channel expression at plasma membrane

    SciTech Connect

    Saxena, Sunil K. . E-mail: ssaxena@stevens.edu; Kaur, Simarna; George, Constantine

    2006-03-03

    Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein-protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein-protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function.

  4. Hypoxia promotes Rab5 activation, leading to tumor cell migration, invasion and metastasis.

    PubMed

    Silva, Patricio; Mendoza, Pablo; Rivas, Solange; Díaz, Jorge; Moraga, Carolina; Quest, Andrew F G; Torres, Vicente A

    2016-05-17

    Hypoxia, a common condition of the tumor microenvironment, is associated with poor patient prognosis, tumor cell migration, invasion and metastasis. Recent evidence suggests that hypoxia alters endosome dynamics in tumor cells, leading to augmented cell proliferation and migration and this is particularly relevant, because endosomal components have been shown to be deregulated in cancer. The early endosome protein Rab5 is a small GTPase that promotes integrin trafficking, focal adhesion turnover, Rac1 activation, tumor cell migration and invasion. However, the role of Rab5 and downstream events in hypoxia remain unknown. Here, we identify Rab5 as a critical player in hypoxia-driven tumor cell migration, invasion and metastasis. Exposure of A549 human lung carcinoma, ZR-75, MDA-MB-231 and MCF-7 human breast cancer and B16-F10 mouse melanoma cells to hypoxia increased Rab5 activation, followed by its re-localization to the leading edge and association with focal adhesions. Importantly, Rab5 was required for hypoxia-driven cell migration, FAK phosphorylation and Rac1 activation, as shown by shRNA-targeting and transfection assays with Rab5 mutants. Intriguingly, the effect of hypoxia on both Rab5 activity and migration was substantially higher in metastatic B16-F10 cells than in poorly invasive B16-F0 cells. Furthermore, exogenous expression of Rab5 in B16-F0 cells predisposed to hypoxia-induced migration, whereas expression of the inactive mutant Rab5/S34N prevented the migration of B16-F10 cells induced by hypoxia. Finally, using an in vivo syngenic C57BL/6 mouse model, Rab5 expression was shown to be required for hypoxia-induced metastasis. In summary, these findings identify Rab5 as a key mediator of hypoxia-induced tumor cell migration, invasion and metastasis.

  5. Synaptic Function of Rab11Fip5: Selective Requirement for Hippocampal Long-Term Depression

    PubMed Central

    Ahmad, Mohiuddin; Jurado, Sandra; Malenka, Robert C.

    2015-01-01

    Postsynaptic AMPA-type glutamate receptors (AMPARs) are among the major determinants of synaptic strength and can be trafficked into and out of synapses. Neuronal activity regulates AMPAR trafficking during synaptic plasticity to induce long-term changes in synaptic strength, including long-term potentiation (LTP) and long-term depression (LTD). Rab family GTPases regulate most membrane trafficking in eukaryotic cells; particularly, Rab11 and its effectors are implicated in mediating postsynaptic AMPAR insertion during LTP. To explore the synaptic function of Rab11Fip5, a neuronal Rab11 effector and a candidate autism-spectrum disorder gene, we performed shRNA-mediated knock-down and genetic knock-out (KO) studies. Surprisingly, we observed robust shRNA-induced synaptic phenotypes that were rescued by a Rab11Fip5 cDNA but that were nevertheless not observed in conditional KO neurons. Both in cultured neurons and acute slices, KO of Rab11Fip5 had no significant effect on basic parameters of synaptic transmission, indicating that Rab11Fip5 is not required for fundamental synaptic operations, such as neurotransmitter release or postsynaptic AMPAR insertion. KO of Rab11Fip5 did, however, abolish hippocampal LTD as measured both in acute slices or using a chemical LTD protocol in cultured neurons but did not affect hippocampal LTP. The Rab11Fip5 KO mice performed normally in several behavioral tasks, including fear conditioning, but showed enhanced contextual fear extinction. These are the first findings to suggest a requirement for Rab11Fip5, and presumably Rab11, during LTD. PMID:25972173

  6. Hypoxia promotes Rab5 activation, leading to tumor cell migration, invasion and metastasis

    PubMed Central

    Silva, Patricio; Mendoza, Pablo; Rivas, Solange; Díaz, Jorge; Moraga, Carolina; Quest, Andrew F.G.; Torres, Vicente A.

    2016-01-01

    Hypoxia, a common condition of the tumor microenvironment, is associated with poor patient prognosis, tumor cell migration, invasion and metastasis. Recent evidence suggests that hypoxia alters endosome dynamics in tumor cells, leading to augmented cell proliferation and migration and this is particularly relevant, because endosomal components have been shown to be deregulated in cancer. The early endosome protein Rab5 is a small GTPase that promotes integrin trafficking, focal adhesion turnover, Rac1 activation, tumor cell migration and invasion. However, the role of Rab5 and downstream events in hypoxia remain unknown. Here, we identify Rab5 as a critical player in hypoxia-driven tumor cell migration, invasion and metastasis. Exposure of A549 human lung carcinoma, ZR-75, MDA-MB-231 and MCF-7 human breast cancer and B16-F10 mouse melanoma cells to hypoxia increased Rab5 activation, followed by its re-localization to the leading edge and association with focal adhesions. Importantly, Rab5 was required for hypoxia-driven cell migration, FAK phosphorylation and Rac1 activation, as shown by shRNA-targeting and transfection assays with Rab5 mutants. Intriguingly, the effect of hypoxia on both Rab5 activity and migration was substantially higher in metastatic B16-F10 cells than in poorly invasive B16-F0 cells. Furthermore, exogenous expression of Rab5 in B16-F0 cells predisposed to hypoxia-induced migration, whereas expression of the inactive mutant Rab5/S34N prevented the migration of B16-F10 cells induced by hypoxia. Finally, using an in vivo syngenic C57BL/6 mouse model, Rab5 expression was shown to be required for hypoxia-induced metastasis. In summary, these findings identify Rab5 as a key mediator of hypoxia-induced tumor cell migration, invasion and metastasis. PMID:27121131

  7. Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a.

    PubMed

    Cogli, Laura; Progida, Cinzia; Bramato, Roberta; Bucci, Cecilia

    2013-06-01

    Intermediate filaments are cytoskeletal elements important for cell architecture. Recently it has been discovered that intermediate filaments are highly dynamic and that they are fundamental for organelle positioning, transport and function thus being an important regulatory component of membrane traffic. We have identified, using the yeast two-hybrid system, vimentin, a class III intermediate filament protein, as a Rab7a interacting protein. Rab7a is a member of the Rab family of small GTPases and it controls vesicular membrane traffic to late endosomes and lysosomes. In addition, Rab7a is important for maturation of phagosomes and autophagic vacuoles. We confirmed the interaction in HeLa cells by co-immunoprecipitation and pull-down experiments, and established that the interaction is direct using bacterially expressed recombinant proteins. Immunofluorescence analysis on HeLa cells indicate that Rab7a-positive vesicles sometimes overlap with vimentin filaments. Overexpression of Rab7a causes an increase in vimentin phosphorylation at different sites and causes redistribution of vimentin in the soluble fraction. Consistently, Rab7a silencing causes an increase of vimentin present in the insoluble fraction (assembled). Also, expression of Charcot-Marie-Tooth 2B-causing Rab7a mutant proteins induces vimentin phosphorylation and increases the amount of vimentin in the soluble fraction. Thus, modulation of expression levels of Rab7a wt or expression of Rab7a mutant proteins changes the assembly of vimentin and its phosphorylation state indicating that Rab7a is important for the regulation of vimentin function.

  8. Rab GTPases regulate endothelial cell protein C receptor-mediated endocytosis and trafficking of factor VIIa.

    PubMed

    Nayak, Ramesh C; Keshava, Shiva; Esmon, Charles T; Pendurthi, Usha R; Rao, L Vijaya Mohan

    2013-01-01

    Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa.

  9. Hypoxia promotes Rab5 activation, leading to tumor cell migration, invasion and metastasis.

    PubMed

    Silva, Patricio; Mendoza, Pablo; Rivas, Solange; Díaz, Jorge; Moraga, Carolina; Quest, Andrew F G; Torres, Vicente A

    2016-05-17

    Hypoxia, a common condition of the tumor microenvironment, is associated with poor patient prognosis, tumor cell migration, invasion and metastasis. Recent evidence suggests that hypoxia alters endosome dynamics in tumor cells, leading to augmented cell proliferation and migration and this is particularly relevant, because endosomal components have been shown to be deregulated in cancer. The early endosome protein Rab5 is a small GTPase that promotes integrin trafficking, focal adhesion turnover, Rac1 activation, tumor cell migration and invasion. However, the role of Rab5 and downstream events in hypoxia remain unknown. Here, we identify Rab5 as a critical player in hypoxia-driven tumor cell migration, invasion and metastasis. Exposure of A549 human lung carcinoma, ZR-75, MDA-MB-231 and MCF-7 human breast cancer and B16-F10 mouse melanoma cells to hypoxia increased Rab5 activation, followed by its re-localization to the leading edge and association with focal adhesions. Importantly, Rab5 was required for hypoxia-driven cell migration, FAK phosphorylation and Rac1 activation, as shown by shRNA-targeting and transfection assays with Rab5 mutants. Intriguingly, the effect of hypoxia on both Rab5 activity and migration was substantially higher in metastatic B16-F10 cells than in poorly invasive B16-F0 cells. Furthermore, exogenous expression of Rab5 in B16-F0 cells predisposed to hypoxia-induced migration, whereas expression of the inactive mutant Rab5/S34N prevented the migration of B16-F10 cells induced by hypoxia. Finally, using an in vivo syngenic C57BL/6 mouse model, Rab5 expression was shown to be required for hypoxia-induced metastasis. In summary, these findings identify Rab5 as a key mediator of hypoxia-induced tumor cell migration, invasion and metastasis. PMID:27121131

  10. Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

    PubMed Central

    Sanford, J C; Pan, Y; Wessling-Resnick, M

    1995-01-01

    Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue. Images PMID:7749197

  11. Insulin and AMPK regulate FA translocase/CD36 plasma membrane recruitment in cardiomyocytes via Rab GAP AS160 and Rab8a Rab GTPase.

    PubMed

    Samovski, Dmitri; Su, Xiong; Xu, Yingcheng; Abumrad, Nada A; Stahl, Philip D

    2012-04-01

    The FA translocase cluster of differentiation 36 (CD36) facilitates FA uptake by the myocardium, and its surface recruitment in cardiomyocytes is induced by insulin, AMP-dependent protein kinase (AMPK), or contraction. Dysfunction of CD36 trafficking contributes to disordered cardiac FA utilization and promotes progression to disease. The Akt substrate 160 (AS160) Rab GTPase-activating protein (GAP) is a key regulator of vesicular trafficking, and its activity is modulated via phosphorylation. Our study documents that AS160 mediates insulin or AMPK-stimulated surface translocation of CD36 in cardiomyocytes. Knock-down of AS160 redistributes CD36 to the surface and abrogates its translocation by insulin or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). Conversely, overexpression of a phosphorylation-deficient AS160 mutant (AS160 4P) suppresses the stimulated membrane recruitment of CD36. The AS160 substrate Rab8a GTPase is shown via overexpression and knock-down studies to be specifically involved in insulin/AICAR-induced CD36 membrane recruitment. Our findings directly demonstrate AS160 regulation of CD36 trafficking. In myocytes, the AS160 pathway also mediates the effect of insulin, AMPK, or contraction on surface recruitment of the glucose transporter GLUT4. Thus, AS160 constitutes a point of convergence for coordinating physiological regulation of CD36 and GLUT4 membrane recruitment.

  12. Projected Life of the SLAC Linac Braze Joints: Braze integrity and corrosion of cooling water hardware on accelerator sections

    SciTech Connect

    Glesener, W.F.; Garwin, E.L.; /SLAC

    2006-07-17

    The objective of this study was to ascertain the condition of braze joints and cooling water hardware from an accelerator section after prolonged use. Metallographic analysis was used to examine critical sites on an accelerator section that had been in use for more than 30 years. The end flange assembly showed no internal operational damage or external environmental effects. The cavity cylinder stack showed no internal operational damage however the internal surface was highly oxidized. The internal surface of the cooling water tubing was uniformly corroding at a rate of about 1 mil per year and showed no evidence of pitting. Tee fitting internal surfaces are corroding at non-uniform rates due to general corrosion and pitting. Remaining service life of the cooling water jacket is estimated to be about 20 years or year 2027. At this time, water supply pressure will exceed allowable fitting pressure due to corrosion of tubing walls.

  13. Marked amplification and diversification of products of ras genes from rat brain, Rab GTPases, in the ciliates Tetrahymena thermophila and Paramecium tetraurelia.

    PubMed

    Saito-Nakano, Yumiko; Nakahara, Tohru; Nakano, Kentaro; Nozaki, Tomoyoshi; Numata, Osamu

    2010-01-01

    Small GTPase Rab (products of ras genes from rat brain) is a widely conserved molecular switch among eukaryotes and regulates membrane trafficking pathways. It is generally considered that the number of Rab encoded in the genome correlates with multicellularity; however, we found that unicellular ciliates Tetrahymena thermophila (Tt) and Paramecium tetraurelia (Pt) possess many more Rab genes in their genome than the 64 HsRab genes in the human genome. We succeeded in isolating 86 cDNA clones of 88 TtRab genes in the Tetrahymena genome. By comparing the amino acid sequence of Rab in humans and the budding yeast Saccharomyces cerevisiae, 42 TtRab belonged to subfamilies functionally characterized and designated as conventional Rab, while the remaining 44 TtRab were considered to be species-specific. To examine the diversity of Rab in ciliates, we searched for Rab genes in the genome database of P. tetraurelia. Overall, 229 PtRab genes were found and categorized as 157 conventional and 72 species-specific PtRab, respectively. Among them, nine PtRab genes showed high homology to seven TtRab, suggesting the conservation of ciliate-specific Rab. These data suggested that the range of Rab is markedly amplified and diversified in ciliates, which may support the elaborate cellular structures and vigorous phagocytosis of those organisms.

  14. Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and Thyroglobulin Internalization in a Thyroid Cell Line.

    PubMed

    Mascia, Anna; Gentile, Flaviana; Izzo, Antonella; Mollo, Nunzia; De Luca, Maria; Bucci, Cecilia; Nitsch, Lucio; Calì, Gaetano

    2016-08-01

    Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc.

  15. Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells.

    PubMed

    Sun, Yi; Chiu, Tim T; Foley, Kevin P; Bilan, Philip J; Klip, Amira

    2014-04-01

    Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase-dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)-MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP-MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP-MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA-mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane.

  16. Ubiquitylation and activation of a Rab GTPase is promoted by a β₂AR-HACE1 complex.

    PubMed

    Lachance, Véronik; Degrandmaison, Jade; Marois, Sébastien; Robitaille, Mélanie; Génier, Samuel; Nadeau, Stéphanie; Angers, Stéphane; Parent, Jean-Luc

    2014-01-01

    We and others have shown that trafficking of G-protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Here, we show by overexpression and depletion studies that HACE1, a HECT-domain-containing ubiquitin ligase, promotes the recycling of the β₂-adrenergic receptor (β₂AR), a prototypical G-protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the β₂AR in conjunction with HACE1 triggered ubiquitylation of Rab11a, as observed by western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitylated on Lys145. A Rab11a-K145R mutant failed to undergo β₂AR-HACE1-induced ubiquitylation and inhibited the HACE1-mediated recycling of the β₂AR. Rab11a, but not Rab11a-K145R, was activated by β₂AR-HACE1, indicating that ubiquitylation of Lys145 is involved in activation of Rab11a. Co-expression of β₂AR-HACE1 also potentiated ubiquitylation of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases through their ubiquitylation, with associated functional effects demonstrated on Rab11a. This suggests a new pathway whereby a cargo protein, such as a G-protein-coupled receptor, can regulate its own trafficking by inducing the ubiquitylation and activation of a Rab GTPase.

  17. Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells

    PubMed Central

    Sun, Yi; Chiu, Tim T.; Foley, Kevin P.; Bilan, Philip J.; Klip, Amira

    2014-01-01

    Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase–dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)–MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP–MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP–MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA–mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane. PMID:24478457

  18. Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif.

    PubMed

    Yalovsky, S.; Loraine, A. E.; Gruissem, W.

    1996-04-01

    Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity.

  19. The GTPase RAB20 is a HIF target with mitochondrial localization mediating apoptosis in hypoxia.

    PubMed

    Hackenbeck, Thomas; Huber, Regina; Schietke, Ruth; Knaup, Karl X; Monti, Juliana; Wu, Xiaoqing; Klanke, Bernd; Frey, Benjamin; Gaipl, Udo; Wullich, Bernd; Ferbus, Didier; Goubin, Gérard; Warnecke, Christina; Eckardt, Kai-Uwe; Wiesener, Michael S

    2011-01-01

    Hypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress. PMID:21056597

  20. Rab3 is involved in cellular immune responses of the cotton bollworm, Helicoverpa armigera.

    PubMed

    Li, Jie; Song, Cai-Xia; Li, Yu-Ping; Li, Li; Wei, Xiu-Hong; Wang, Jia-Lin; Liu, Xu-Sheng

    2015-06-01

    Rab3, a member of the Rab GTPase family, has been found to be involved in innate immunity. However, the precise function of this GTPase in innate immunity remains unknown. In this study, we identified a Rab3 gene (Ha-Rab3) from the cotton bollworm, Helicoverpa armigera and studied its roles in innate immune responses. Expression of Ha-Rab3 was upregulated in the hemocytes of H. armigera larvae after the injection of Escherichia coli or chromatography beads. The dsRNA-mediated knockdown of Ha-Rab3 gene in H. armigera larval hemocytes led to significant reduction in the phagocytosis and nodulation activities of hemocytes against E. coli, significant increase in the bacterial load in larval hemolymph, and significant reduction in the encapsulation activities of hemocytes toward invading chromatography beads. Furthermore, Ha-Rab3 knockdown significantly suppressed spreading of plasmatocytes. These results suggest that Ha-Rab3 plays important roles in H. armigera cellular immune responses, possibly by mediating spreading of hemocytes.

  1. Rab3 is involved in cellular immune responses of the cotton bollworm, Helicoverpa armigera.

    PubMed

    Li, Jie; Song, Cai-Xia; Li, Yu-Ping; Li, Li; Wei, Xiu-Hong; Wang, Jia-Lin; Liu, Xu-Sheng

    2015-06-01

    Rab3, a member of the Rab GTPase family, has been found to be involved in innate immunity. However, the precise function of this GTPase in innate immunity remains unknown. In this study, we identified a Rab3 gene (Ha-Rab3) from the cotton bollworm, Helicoverpa armigera and studied its roles in innate immune responses. Expression of Ha-Rab3 was upregulated in the hemocytes of H. armigera larvae after the injection of Escherichia coli or chromatography beads. The dsRNA-mediated knockdown of Ha-Rab3 gene in H. armigera larval hemocytes led to significant reduction in the phagocytosis and nodulation activities of hemocytes against E. coli, significant increase in the bacterial load in larval hemolymph, and significant reduction in the encapsulation activities of hemocytes toward invading chromatography beads. Furthermore, Ha-Rab3 knockdown significantly suppressed spreading of plasmatocytes. These results suggest that Ha-Rab3 plays important roles in H. armigera cellular immune responses, possibly by mediating spreading of hemocytes. PMID:25662061

  2. Rab GTPases as regulators of endocytosis, targets of disease and therapeutic opportunities.

    PubMed

    Agola, J O; Jim, P A; Ward, H H; Basuray, S; Wandinger-Ness, A

    2011-10-01

    Rab GTPases are well-recognized targets in human disease, although are underexplored therapeutically. Elucidation of how mutant or dysregulated Rab GTPases and accessory proteins contribute to organ specific and systemic disease remains an area of intensive study and an essential foundation for effective drug targeting. Mutation of Rab GTPases or associated regulatory proteins causes numerous human genetic diseases. Cancer, neurodegeneration and diabetes represent examples of acquired human diseases resulting from the up- or downregulation or aberrant function of Rab GTPases. The broad range of physiologic processes and organ systems affected by altered Rab GTPase activity is based on pivotal roles in responding to cell signaling and metabolic demand through the coordinated regulation of membrane trafficking. The Rab-regulated processes of cargo sorting, cytoskeletal translocation of vesicles and appropriate fusion with the target membranes control cell metabolism, viability, growth and differentiation. In this review, we focus on Rab GTPase roles in endocytosis to illustrate normal function and the consequences of dysregulation resulting in human disease. Selected examples are designed to illustrate how defects in Rab GTPase cascades alter endocytic trafficking that underlie neurologic, lipid storage, and metabolic bone disorders as well as cancer. Perspectives on potential therapeutic modulation of GTPase activity through small molecule interventions are provided.

  3. Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles.

    PubMed

    Nonet, M L; Staunton, J E; Kilgard, M P; Fergestad, T; Hartwieg, E; Horvitz, H R; Jorgensen, E M; Meyer, B J

    1997-11-01

    Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterized Caenorhabditis elegans rab-3 mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore, rab-3 animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed in rab-3 mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.

  4. Regulation of the abscisic acid-responsive gene rab28 in maize viviparous mutants.

    PubMed

    Pla, M; Gómez, J; Goday, A; Pagès, M

    1991-12-01

    We have isolated a new maize gene, rab28, that responds to abscisic acid (ABA) treatment. This gene has been characterized by determining the sequence of the cDNA and corresponding genomic copy, and by mapping the start site of its transcript. The rab 28 gene encodes a protein of predicted molecular weight 27713 Da which shows strong homology with the Lea D-34 protein identified in cotton. The proximal promoter region contains the conserved ABA-response element, CACGTGG, reported in other plant genes to be responsible for ABA induction. rab 28 mRNA has been identified as ABA-inducible in embryos and young leaves. It is also induced by water-stress in leaves of wild-type plants. Regulation of the rab 28 gene was studied in maize viviparous mutants. The results obtained with the ABA-insensitive vp1 mutant show that rab 28 transcripts do not accumulate to a significant level during embryogenesis. Surprisingly, induction of rab 28 mRNA can be achieved in young embryos by exogenous ABA treatment. Moreover, water-stressed or ABA-treated seedlings of vp1 contain significant levels of rab 28 mRNA which is not detectable in well-watered seedlings. Regulation of the rab 28 gene in excised young embryos of ABA-deficient vp2 mutants, in which influences of the maternal environment are absent, closely resembles that found in non-mutant excised young embryos. The significance of these results is discussed.

  5. RAB5 activation is required for multiple steps in Arabidopsis thaliana root development.

    PubMed

    Inoue, Takeshi; Kondo, Yuki; Naramoto, Satoshi; Nakano, Akihiko; Ueda, Takashi

    2013-10-01

    Rab GTPases regulate the tethering and fusion of transport vesicles to target membranes in membrane trafficking by acting as a molecular switch, cycling between GDP- and GTP-bound states. RAB5 is a member of the Rab GTPase family, the members of which have been shown to perform various functions in the endocytic pathway, including the regulation of endosomal fusion and motility in animal cells. RAB5-mediated endosomal trafficking has also been found to play important roles in various higher order plant functions, which include the regulation of the polar transport of auxin and responses to environmental conditions. The regulatory mechanisms and functions of plant RAB5 have also been investigated at the molecular and cellular levels. However, the significance of RAB5 activity at the tissue and organ levels has hardly been investigated thus far. In the present study, we examined the effect of a mutation in VPS9a, which encodes the sole guanine nucleotide exchange factor for all RAB5s in the vegetative stages of Arabidopsis thaliana. We found that multiple developmental processes were impaired in the mutant plants, including the growth and pattern formation of the roots and establishment of auxin maxima. Our results indicate that RAB5 plays distinctive pivotal roles in the development of plants.

  6. GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

    PubMed

    Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

    2015-01-01

    Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

  7. RAB-11 Permissively Regulates Spindle Alignment by Modulating Metaphase Microtubule Dynamics in Caenorhabditis elegans Early Embryos

    PubMed Central

    Zhang, Haining; Squirrell, Jayne M.

    2008-01-01

    Alignment of the mitotic spindle along a preformed axis of polarity is crucial for generating cell diversity in many organisms, yet little is known about the role of the endomembrane system in this process. RAB-11 is a small GTPase enriched in recycling endosomes. When we depleted RAB-11 by RNAi in Caenorhabditis elegans, the spindle of the one-cell embryo failed to align along the axis of polarity in metaphase and underwent violent movements in anaphase. The distance between astral microtubules ends and the anterior cortex was significantly increased in rab-11(RNAi) embryos specifically during metaphase, possibly accounting for the observed spindle alignment defects. Additionally, we found that normal ER morphology requires functional RAB-11, particularly during metaphase. We hypothesize that RAB-11, in conjunction with the ER, acts to regulate cell cycle–specific changes in astral microtubule length to ensure proper spindle alignment in Caenorhabditis elegans early embryos. PMID:18385514

  8. Distinct expression, localization and function of two Rab7 proteins encoded by paralogous genes in a free-living model eukaryote.

    PubMed

    Osińska, Magdalena; Wiejak, Jolanta; Wypych, Emilia; Bilski, Henryk; Bartosiewicz, Rafał; Wyroba, Elżbieta

    2011-01-01

    Rab7 GTPases are involved in membrane trafficking in the late endosomal/lysosomal pathway. In Paramecium octaurelia Rab7a and Rab7b are encoded by paralogous genes. Antipeptide antibodies generated against divergent C-termini recognize Rab7a of 22.5 kDa and Rab7b of 25 kDa, respectively. In 2D gel electrophoresis two immunoreactive spots were identified for Rab7b at pI about 6.34 and about 6.18 and only one spot for Rab7a of pI about 6.34 suggesting post-translational modification of Rab7b. Mass spectrometry revealed eight identical phosphorylated residues in the both proteins. ProQ Emerald staining and ConA overlay of immunoprecipitated Rab7b indicated its putative glycosylation that was further supported by a faster electrophoretic mobility of this protein upon deglycosylation. Such a post-translational modification and substitution of Ala(140) in Rab7a for Ser(140) in Rab7b may result in distinct targeting to the oral apparatus where Rab7b associates with the microtubular structures as revealed by STED confocal and electron microscopy. Rab7a was mapped to phagosomal compartment. Absolute qReal-Time PCR analysis revealed that expression of Rab7a was 2.6-fold higher than that of Rab7b. Upon latex internalization it was further 2-fold increased for Rab7a and only slightly for Rab7b. Post-transcriptional gene silencing of rab7a suppressed phagosome formation by 70 % and impaired their acidification. Ultrastructural analysis with double immunogold labeling revealed that this effect was due to the lack of V-ATPase recruitment to phagolysosomes. No significant phenotype changes were noticed in cells upon rab7b silencing. In conclusion, Rab7b acquired a new function, whereas Rab7a can be assigned to the phagolysosomal pathway.

  9. Functional analysis of novel Rab GTPases identified in the proteome of purified Legionella-containing vacuoles from macrophages.

    PubMed

    Hoffmann, Christine; Finsel, Ivo; Otto, Andreas; Pfaffinger, Gudrun; Rothmeier, Eva; Hecker, Michael; Becher, Dörte; Hilbi, Hubert

    2014-07-01

    The opportunistic pathogen Legionella pneumophila employs the Icm/Dot type IV secretion system and ∼300 different effector proteins to replicate in macrophages and amoebae in a distinct 'Legionella-containing vacuole' (LCV). LCVs from infected RAW 264.7 macrophages were enriched by immuno-affinity separation and density gradient centrifugation, using an antibody against the L. pneumophila effector SidC, which specifically binds to the phosphoinositide PtdIns(4)P on the pathogen vacuole membrane. The proteome of purified LCVs was determined by mass spectro-metry (data are available via ProteomeXchange with identifier PXD000647). The proteomics analysis revealed more than 1150 host proteins, including 13 small GTPases of the Rab family. Using fluorescence microscopy, 6 novel Rab proteins were confirmed to localize on pathogen vacuoles harbouring wild-type but not ΔicmT mutant L. pneumophila. Individual depletion of 20 GTPases by RNA interference indicated that endocytic GTPases (Rab5a, Rab14 and Rab21) restrict intracellular growth of L. pneumophila, whereas secretory GTPases (Rab8a, Rab10 and Rab32) implicated in Golgi-endosome trafficking promote bacterial replication. Upon silencing of Rab21 or Rab32, fewer LCVs stained positive for Rab4 or Rab9, implicated in secretory or retrograde trafficking respectively. Moreover, depletion of Rab8a, Rab14 or Rab21 significantly decreased the number of SidC-positive LCVs, suggesting that PtdIns(4)P is reduced under these conditions. L. pneumophila proteins identified in purified LCVs included proteins putatively implicated in phosphorus metabolism and as many as 60 Icm/Dot-translocated effectors, which are likely required early during infection. Taken together, the phagocyte and Legionella proteomes of purified LCVs lay the foundation for further hypothesis-driven investigations of the complex process of pathogen vacuole formation.

  10. Regulation of β2-Adrenergic Receptor Maturation and Anterograde Trafficking by an Interaction with Rab Geranylgeranyltransferase

    PubMed Central

    Lachance, Véronik; Cartier, Andréane; Génier, Samuel; Munger, Sandra; Germain, Pascale; Labrecque, Pascale; Parent, Jean-Luc

    2011-01-01

    Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the β2-adrenergic receptor (β2AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that β2AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L339L340 motif in the β2AR C terminus known to be involved in the transport of the receptor from the endoplasmic reticulum to the cell surface. Modulation of the cellular levels of RGGTA protein by overexpression or siRNA-mediated knockdown of the endogenous protein demonstrated that RGGTA has a positive role in the maturation and anterograde trafficking of the β2AR, which requires the interaction of RGGTA with the β2AR L339L340 motif. Furthermore, the β2AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the β2AR was dependent on the RGGTA-interacting L339L340 motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted β2AR maturation, suggesting that RGGTA may have functions independent of Rab geranylgeranylation. We demonstrate for the first time an interaction between a transmembrane receptor and RGGTA which regulates the maturation and anterograde transport of the receptor, as well as geranylgeranylation of Rab GTPases. PMID:21990357

  11. Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling.

    PubMed

    Binnington, Beth; Nguyen, Long; Kamani, Mustafa; Hossain, Delowar; Marks, David L; Budani, Monique; Lingwood, Clifford A

    2016-02-01

    Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol and cardiac risk, but many other effects are seen. We now show an effect of these drugs to induce profound changes in the step-wise synthesis of glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased several-fold in all cell lines tested, demonstrating a widespread effect. Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer; GlcNAcβ1-3Galβ1-4GlcCer) synthesis was observed in 70%. Western blot showed that GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase (Lc3S) activities were increased; however, transcript was elevated for Lc3S only. Supplementation with the isoprenoid precursor, geranylgeranyl pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl transferase, or geranylgeranyl transferase I, was sufficient to replicate statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab prenylation-dependent mechanism. While total cholesterol was unaffected, the trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS partially relocated by statins. GSL-dependent vesicular retrograde transport of Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling. These in vitro studies indicate a previously unreported link between Rab prenylation and regulation of GCS activity and GlcCer metabolism. PMID:26405105

  12. Aberrant amino acid signaling promotes growth and metastasis of hepatocellular carcinomas through Rab1A-dependent activation of mTORC1 by Rab1A

    PubMed Central

    Yang, Yang; Zhang, Mei-Yin; Rao, Hui-Lan; Wang, Hui-Yun; Zheng, X.F. Steven

    2015-01-01

    mTORC1 is a master regulator of cell growth and proliferation, and an established anticancer drug target. Aberrant mTORC1 signaling is common in hepatocellular carcinoma (HCC), but the underlying mechanism remains elusive. Rab1A is a newly identified mTORC1 activator that mediates an alternative amino acid (AA) signaling branch to Rag GTPases. Because liver is a physiological hub for nutrient sensing and metabolic homeostasis, we investigated the possible role of Rab1A in HCC. We found that Rab1A is frequently overexpressed in HCC, which enhances hyperactive AA-mTORC1 signaling, promoting malignant growth and metastasis of HCC in vitro and in vivo. Moreover, aberrant Rab1A expression is closely associated with poor prognosis. Strikingly, aberrant Rab1A overexpression leads to increased rapamycin sensitivity, indicating that inappropriate activation of AA signaling is a cancer-driving event in HCC. Our findings further suggest that Rab1A is a valuable biomarker for prognosis and personalized mTORC1-targeted therapy in liver cancer. PMID:26308575

  13. A Role for Na+,K+-ATPase α1 in Regulating Rab27a Localisation on Melanosomes

    PubMed Central

    Booth, Antonia E. G.; Tarafder, Abul K.; Hume, Alistair N.; Recchi, Chiara; Seabra, Miguel C.

    2014-01-01

    The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation. PMID:25051489

  14. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    PubMed

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

  15. A GDP-bound of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments

    PubMed Central

    1994-01-01

    Rab1 is a small GTPase regulating vesicular traffic between early compartments of the secretory pathway. To explore the role of rab1 we have analyzed the function of a mutant (rab1a[S25N]) containing a substitution which perturbs Mg2+ coordination and reduces the affinity for GTP, resulting in a form which is likely to be restricted to the GDP-bound state. The rab1a(S25N) mutant led to a marked reduction in protein export from the ER in vivo and in vitro, indicating that a guanine nucleotide exchange protein (GEP) is critical for the recruitment of rab1 during vesicle budding. The mutant protein required posttranslational isoprenylation for inhibition and behaved as a competitive inhibitor of wild-type rab1 function. Both rab1a and rab1b (92% identity) were able to antagonize the inhibitory activity of the rab1a(S25N) mutant, suggesting that these two isoforms are functionally interchangeable. The rab1 mutant also inhibited transport between Golgi compartments and resulted in an apparent loss of the Golgi apparatus, suggesting that Golgi integrity is coupled to rab1 function in vesicular traffic. PMID:8163542

  16. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    PubMed

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D. PMID:26845357

  17. A role for Na+,K+-ATPase α1 in regulating Rab27a localisation on melanosomes.

    PubMed

    Booth, Antonia E G; Tarafder, Abul K; Hume, Alistair N; Recchi, Chiara; Seabra, Miguel C

    2014-01-01

    The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation. PMID:25051489

  18. Neovascularization in the pulmonary endothelium is regulated by the endosome: Rab4-mediated trafficking and p18-dependent signaling.

    PubMed

    Chichger, Havovi; Braza, Julie; Duong, Huetran; Stark, Myranda; Harrington, Elizabeth O

    2015-10-01

    Neovascularization, the formation of new blood vessels, requires multiple processes including vascular leak, migration, and adhesion. Endosomal proteins, such as Rabs, regulate trafficking of key signaling proteins involved in neovascularization. The novel endosome protein, p18, enhances vascular endothelial (VE)-cadherin recycling from early endosome to cell junction to improve pulmonary endothelial barrier function. Since endothelial barrier integrity is vital in neovascularization, we sought to elucidate the role for endosome proteins p18 and Rab4, Rab7, and Rab9 in the process of vessel formation within the pulmonary vasculature. Overexpression of wild-type p18 (p18(wt)), but not the nonendosomal-binding mutant (p18(N39)), significantly increased lung microvascular endothelial cell migration, adhesion, and both in vitro and in vivo tube formation. Chemical inhibition of mTOR or p38 attenuated the proneovascularization role of p18(wt). Similar to the effect of p18(wt), overexpression of prorecycling wild-type (Rab4(WT)) and endosome-anchored (Rab4(Q67L)) Rab4 enhanced neovascularization processes, whereas molecular inhibition of Rab4, by using the nonendosomal-binding mutant (Rab4(S22N)) attenuated VEGF-induced neovascularization. Unlike p18, Rab4-induced neovascularization was independent of mTOR or p38 inhibition but was dependent on p18 expression. This study shows for the first time that neovascularization within the pulmonary vasculature is dependent on the prorecycling endocytic proteins Rab4 and p18.

  19. Spatial-Temporal Study of Rab1b Dynamics and Function at the ER-Golgi Interface

    PubMed Central

    Martinez, Hernán; García, Iris A.; Sampieri, Luciana

    2016-01-01

    The GTPase Rab1b is involved in ER to Golgi transport, with multiple Rab1b effectors (located at ERES, VTCs and the Golgi complex) being required for its function. In this study, we performed live-cell dual-expression studies to analyze the dynamics of Rab1b and some effectors located at the ERES-Golgi interface. Rab1b occupied widely distributed mobile punctate and tubular structures, displaying a transient overlaps with its effectors and showing that these overlaps occurred at the same time in spatially distinct steps of ER to Golgi transport. In addition, we assessed Rab1b dynamics during cargo sorting by analyzing the concentration at ERES of a Golgi protein (SialT2-CFP) during Brefeldin A washout (BFA WO). Rab1b was associated to most of the ERES structures, but at different times during BFA WO, and recurrently SialT2-CFP was sorted in the ERES-Rab1b positive structures. Furthermore, we reveal for first time that Rab1b localization time at ERES depended on GBF1, a Rab1b effector that acts as the guanine nucleotide exchange factor of Arf1, and that Rab1b membrane association/dissociation dynamics at ERES was dependent on the GBF1 membrane association and activity, which strongly suggests that GBF1 activity modulates Rab1b membrane cycling dynamic. PMID:27500526

  20. Spatial-Temporal Study of Rab1b Dynamics and Function at the ER-Golgi Interface.

    PubMed

    Martinez, Hernán; García, Iris A; Sampieri, Luciana; Alvarez, Cecilia

    2016-01-01

    The GTPase Rab1b is involved in ER to Golgi transport, with multiple Rab1b effectors (located at ERES, VTCs and the Golgi complex) being required for its function. In this study, we performed live-cell dual-expression studies to analyze the dynamics of Rab1b and some effectors located at the ERES-Golgi interface. Rab1b occupied widely distributed mobile punctate and tubular structures, displaying a transient overlaps with its effectors and showing that these overlaps occurred at the same time in spatially distinct steps of ER to Golgi transport. In addition, we assessed Rab1b dynamics during cargo sorting by analyzing the concentration at ERES of a Golgi protein (SialT2-CFP) during Brefeldin A washout (BFA WO). Rab1b was associated to most of the ERES structures, but at different times during BFA WO, and recurrently SialT2-CFP was sorted in the ERES-Rab1b positive structures. Furthermore, we reveal for first time that Rab1b localization time at ERES depended on GBF1, a Rab1b effector that acts as the guanine nucleotide exchange factor of Arf1, and that Rab1b membrane association/dissociation dynamics at ERES was dependent on the GBF1 membrane association and activity, which strongly suggests that GBF1 activity modulates Rab1b membrane cycling dynamic.

  1. Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome-lysosome fusion.

    PubMed

    Jean, Steve; Cox, Sarah; Nassari, Sonya; Kiger, Amy A

    2015-03-01

    Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation-induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome-lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF-Rab-effector pathway. These results identify starvation-responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.

  2. Corrosion Behavior of Aluminum-Steel Weld-Brazing Joint

    NASA Astrophysics Data System (ADS)

    Shi, Yu; Li, Jie; Zhang, Gang; Huang, Jiankang; Gu, Yufen

    2016-05-01

    Dissimilar metals of 1060 aluminum and galvanized steel were joined with a lap joint by pulsed double-electrode gas metal arc weld brazing with aluminum-magnesium and aluminum-silicon filler metals. The corrosion behavior of the weld joints was investigated with immersion corrosion and electrochemical corrosion tests, and the corrosion morphology of the joints was analyzed with scanning electron microscopy (SEM). Galvanic corrosion was found to occur when the samples were immersed in corrosive media, and the corrosion rate of joints was increased with increased heat input of the workpiece. Comparison of the corrosion properties of weld joints with different filler wires indicated that the corrosion rate of weld joints with aluminum-silicon filler wire was larger than that of weld joints with aluminum-magnesium filler wire. Results also showed that the zinc-rich zone of weld joints was prone to corrosion. The corrosion behavior of zinc-rich zone was analyzed with SEM equipped with an energy-dispersive x-ray spectroscopy analysis system based on the test results.

  3. Hermetic diamond capsules for biomedical implants enabled by gold active braze alloys.

    PubMed

    Lichter, Samantha G; Escudié, Mathilde C; Stacey, Alastair D; Ganesan, Kumaravelu; Fox, Kate; Ahnood, Arman; Apollo, Nicholas V; Kua, Dunstan C; Lee, Aaron Z; McGowan, Ceara; Saunders, Alexia L; Burns, Owen; Nayagam, David A X; Williams, Richard A; Garrett, David J; Meffin, Hamish; Prawer, Steven

    2015-01-01

    As the field of biomedical implants matures the functionality of implants is rapidly increasing. In the field of neural prostheses this is particularly apparent as researchers strive to build devices that interact with highly complex neural systems such as vision, hearing, touch and movement. A retinal implant, for example, is a highly complex device and the surgery, training and rehabilitation requirements involved in deploying such devices are extensive. Ideally, such devices will be implanted only once and will continue to function effectively for the lifetime of the patient. The first and most pivotal factor that determines device longevity is the encapsulation that separates the sensitive electronics of the device from the biological environment. This paper describes the realisation of a free standing device encapsulation made from diamond, the most impervious, long lasting and biochemically inert material known. A process of laser micro-machining and brazing is described detailing the fabrication of hermetic electrical feedthroughs and laser weldable seams using a 96.4% gold active braze alloy, another material renowned for biochemical longevity. Accelerated ageing of the braze alloy, feedthroughs and hermetic capsules yielded no evidence of corrosion and no loss of hermeticity. Samples of the gold braze implanted for 15 weeks, in vivo, caused minimal histopathological reaction and results were comparable to those obtained from medical grade silicone controls. The work described represents a first account of a free standing, fully functional hermetic diamond encapsulation for biomedical implants, enabled by gold active alloy brazing and laser micro-machining.

  4. Hermetic diamond capsules for biomedical implants enabled by gold active braze alloys.

    PubMed

    Lichter, Samantha G; Escudié, Mathilde C; Stacey, Alastair D; Ganesan, Kumaravelu; Fox, Kate; Ahnood, Arman; Apollo, Nicholas V; Kua, Dunstan C; Lee, Aaron Z; McGowan, Ceara; Saunders, Alexia L; Burns, Owen; Nayagam, David A X; Williams, Richard A; Garrett, David J; Meffin, Hamish; Prawer, Steven

    2015-01-01

    As the field of biomedical implants matures the functionality of implants is rapidly increasing. In the field of neural prostheses this is particularly apparent as researchers strive to build devices that interact with highly complex neural systems such as vision, hearing, touch and movement. A retinal implant, for example, is a highly complex device and the surgery, training and rehabilitation requirements involved in deploying such devices are extensive. Ideally, such devices will be implanted only once and will continue to function effectively for the lifetime of the patient. The first and most pivotal factor that determines device longevity is the encapsulation that separates the sensitive electronics of the device from the biological environment. This paper describes the realisation of a free standing device encapsulation made from diamond, the most impervious, long lasting and biochemically inert material known. A process of laser micro-machining and brazing is described detailing the fabrication of hermetic electrical feedthroughs and laser weldable seams using a 96.4% gold active braze alloy, another material renowned for biochemical longevity. Accelerated ageing of the braze alloy, feedthroughs and hermetic capsules yielded no evidence of corrosion and no loss of hermeticity. Samples of the gold braze implanted for 15 weeks, in vivo, caused minimal histopathological reaction and results were comparable to those obtained from medical grade silicone controls. The work described represents a first account of a free standing, fully functional hermetic diamond encapsulation for biomedical implants, enabled by gold active alloy brazing and laser micro-machining. PMID:25890743

  5. Joining of Cf/SiC Composite and TC4 Using Ag-Al-Ti Active Brazing Alloy

    NASA Astrophysics Data System (ADS)

    Xiong, Jin Hui; Huang, Ji Hua; Zhang, Hua; Zhao, Xing Ke

    2011-08-01

    Carbon fiber reinforced SiC (Cf/SiC) composite was successfully joined to TC4 with Ag-Al-Ti alloy powder by brazing. Microstructures of the brazed joints were investigated by scanning electron microscope, energy dispersive spectrometer, and x-ray diffraction. The mechanical properties of the brazed joints were measured by mechanical testing machine. The results showed that the brazed joint mainly consists of TiC, Ti3SiC2, Ti5Si3, Ag, TiAl, and Ti3Al reaction products. TiC + Ti3SiC2/Ti5Si3 + TiAl reaction layers are formed near Cf/SiC composite while TiAl/Ti3Al/Ti + Ti3Al reaction layers are formed near TC4. The thickness of reaction layers of the brazed joint increases with the increased brazing temperature or holding time. The maximum room temperature and 500 °C shear strengths of the joints brazed at brazing temperature 930 °C for holding time 20 min are 84 and 40 MPa, respectively.

  6. 75 FR 52037 - Welding, Cutting and Brazing Standard; Extension of the Office of Management and Budget's (OMB...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-24

    ... Order No. 5-2007 (67 FR 31160). Signed at Washington, DC, on August 16, 2010. David Michaels, Assistant... Occupational Safety and Health Administration Welding, Cutting and Brazing Standard; Extension of the Office of... the information collection requirements contained in the Welding, Cutting and Brazing Standard (29...

  7. Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs.

    PubMed

    Bello, Oscar Daniel; Cappa, Andrea Isabel; de Paola, Matilde; Zanetti, María Natalia; Fukuda, Mitsunori; Fissore, Rafael A; Mayorga, Luis S; Michaut, Marcela A

    2016-09-10

    Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs. PMID:27423421

  8. Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs.

    PubMed

    Bello, Oscar Daniel; Cappa, Andrea Isabel; de Paola, Matilde; Zanetti, María Natalia; Fukuda, Mitsunori; Fissore, Rafael A; Mayorga, Luis S; Michaut, Marcela A

    2016-09-10

    Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs.

  9. RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

    PubMed

    Singh, Chabungbam Orville; Xin, Hu-hu; Chen, Rui-ting; Wang, Mei-xian; Liang, Shuang; Lu, Yan; Cai, Zi-zheng; Zhang, Deng-pan; Miao, Yun-gen

    2015-06-01

    Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.

  10. Osmotic regulation of Rab-mediated organelle docking.

    PubMed

    Brett, Christopher L; Merz, Alexey J

    2008-07-22

    Osmotic gradients across organelle and plasma membranes modulate the rates of membrane fission and fusion; sufficiently large gradients can cause membrane rupture [1-6]. Hypotonic gradients applied to living yeast cells trigger prompt (within seconds) swelling and fusion of Saccharomyces cerevisiae vacuoles, whereas hypertonic gradients cause vacuoles to fragment on a slower time scale [7-11]. Here, we analyze the influence of osmotic strength on homotypic fusion of isolated yeast vacuoles. Consistent with previously reported in vivo results, we find that decreases in osmolyte concentration increase the rate and extent of vacuole fusion in vitro, whereas increases in osmolyte concentration prevent fusion. Unexpectedly, our results reveal that osmolytes regulate fusion by inhibiting early Rab-dependent docking or predocking events, not late events. Our experiments reveal an organelle-autonomous pathway that may control organelle surface-to-volume ratio, size, and copy number: Decreasing the osmolyte concentration in the cytoplasmic compartment accelerates Rab-mediated docking and fusion. By altering the relationship between the organelle surface and its enclosed volume, fusion in turn reduces the risk of membrane rupture. PMID:18619842

  11. Osmotic regulation of Rab-mediated organelle docking.

    PubMed

    Brett, Christopher L; Merz, Alexey J

    2008-07-22

    Osmotic gradients across organelle and plasma membranes modulate the rates of membrane fission and fusion; sufficiently large gradients can cause membrane rupture [1-6]. Hypotonic gradients applied to living yeast cells trigger prompt (within seconds) swelling and fusion of Saccharomyces cerevisiae vacuoles, whereas hypertonic gradients cause vacuoles to fragment on a slower time scale [7-11]. Here, we analyze the influence of osmotic strength on homotypic fusion of isolated yeast vacuoles. Consistent with previously reported in vivo results, we find that decreases in osmolyte concentration increase the rate and extent of vacuole fusion in vitro, whereas increases in osmolyte concentration prevent fusion. Unexpectedly, our results reveal that osmolytes regulate fusion by inhibiting early Rab-dependent docking or predocking events, not late events. Our experiments reveal an organelle-autonomous pathway that may control organelle surface-to-volume ratio, size, and copy number: Decreasing the osmolyte concentration in the cytoplasmic compartment accelerates Rab-mediated docking and fusion. By altering the relationship between the organelle surface and its enclosed volume, fusion in turn reduces the risk of membrane rupture.

  12. Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes.

    PubMed

    Kobayashi, Shintaro; Suzuki, Tadaki; Kawaguchi, Akira; Phongphaew, Wallaya; Yoshii, Kentaro; Iwano, Tomohiko; Harada, Akihiro; Kariwa, Hiroaki; Orba, Yasuko; Sawa, Hirofumi

    2016-03-18

    West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane. PMID:26817838

  13. Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation

    PubMed Central

    Russo, Ashley J.; Mathiowetz, Alyssa J.; Hong, Steven; Welch, Matthew D.; Campellone, Kenneth G.

    2016-01-01

    Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity. PMID:26823012

  14. Rab43 regulates the sorting of a subset of membrane protein cargo through the medial Golgi

    PubMed Central

    Cox, John V.; Kansal, Rita; Whitt, Michael A.

    2016-01-01

    To evaluate the role of cytoplasmic domains of membrane-spanning proteins in directing trafficking through the secretory pathway, we generated fluorescently tagged VSV G tsO45 with either the native G tail (G) or a cytoplasmic tail derived from the chicken AE1-4 anion exchanger (GAE). We previously showed that these two proteins progressed through the Golgi with distinct kinetics. To investigate the basis for the differential sorting of G and GAE, we analyzed the role of several Golgi-associated small GTP-binding proteins and found that Rab43 differentially regulated their transport through the Golgi. We show that the expression of GFP-Rab43 arrested the anterograde transport of GAE in a Rab43-positive medial Golgi compartment. GFP-Rab43 expression also inhibited the acquisition of endoH-resistant sugars and the surface delivery of GAE, as well as the surface delivery of the AE1-4 anion exchanger. In contrast, GFP-Rab43 expression did not affect the glycosylation or surface delivery of G. Unexpectedly, down-regulation of endogenous Rab43 using small interfering RNA resulted in an increase in the accumulation of GAE on the cell surface while having minimal effect on the surface levels of G. Our data demonstrate that Rab43 regulates the sorting of a subset of membrane-spanning cargo as they progress through the medial Golgi. PMID:27053659

  15. Purification and characterization of Ras related protein, Rab5a from Tinospora cordifolia.

    PubMed

    Amir, Mohd; Wahiduzzaman; Dar, Mohammad Aasif; Haque, Md Anzarul; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-01-01

    Ras related protein (Rab5a) is one of the most important member of the Rab family which regulates the early endosome fusion in endocytosis, and it also helps in the regulation of the budding process. Here, for the first time we report a simple and reproducible method for the purification of the Rab5a from a medicinal plant Tinospora cordifolia. We have used weak cation-exchange (CM-Sepharose-FF) followed by gel-filtration chromatography. A purified protein of 22-kDa was observed on SDS-PAGE which was identified as Rab5a using MALDI-TOF/MS. Our purification procedure is fast and simple with high yield. The purified protein was characterized using circular dichroism for the measurement of secondary structure followed by GdmCl- and urea-induced denaturation to calculate the values of Gibbs free energy change (ΔGD), ΔGD°, midpoint of the denaturation Cm, i.e. molar GdmCl [GdmCl] and molar urea [Urea] concentration at which ΔGD=0; and m, the slope (=∂ΔGD/∂[d]) values. Furthermore, thermodynamic properties of Rab5a were also measured by differential scanning calorimeter. Here, using isothermal calorimeteric measurements we further showed that Rab5a binds with the GTP. This is a first report on the purification and biophysical characterization of Rab5a protein from T. cordifolia. PMID:26517959

  16. Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion

    PubMed Central

    Charrasse, Sophie; Comunale, Franck; De Rossi, Sylvain; Echard, Arnaud; Gauthier-Rouvière, Cécile

    2013-01-01

    Cadherins are homophilic cell–cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell–cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell–cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell–cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell–cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2–positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell–cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion. PMID:23197472

  17. Distinct Sets of Rab6 Effectors Contribute to ZW10- and COG-Dependent Golgi Homeostasis

    PubMed Central

    Majeed, Waqar; Liu, Shijie; Storrie, Brian

    2014-01-01

    The organization of the Golgi apparatus is determined in part by the interaction of Rab proteins and their diverse array of effectors. Here, we used multiple approaches to identify and characterize a small subset of effectors that mimicked the effects of Rab6 on Golgi ribbon organization. In a visual-based, candidate-protein screen, we found that the individual depletion of any of three Rab6 effectors, myosin IIA (MyoIIA), Kif20A, and Bicaudal D (BicD), was sufficient to suppress Golgi ribbon fragmentation/dispersal coupled to retrograde tether proteins in a manner paralleling Rab6. MyoIIA and Kif20A depletion were pathway selective and suppressed ZW10-dependent Golgi ribbon fragmentation/dispersal only while BicD depletion, like Rab6, suppressed both ZW10- and COG-dependent Golgi ribbon fragmentation. The MyoIIA effects could be produced in short term assays by the reversible myosin inhibitor, blebbistatin. At the electron microscope level, the effects of BicD-depletion mimicked many of those of Rab6-depletion: longer and more continuous Golgi cisternae and a pronounced accumulation of coated vesicles. Functionally, BicD-depleted cells were inhibited in transport of newly synthesized VSV-G protein to the cell surface. In sum, our results indicate small, partially overlapping subsets of Rab6 effectors are differentially important to two tether-dependent pathways essential to Golgi organization and function. PMID:24575842

  18. A Bacterial Pathogen Targets a Host Rab-Family GTPase Defense Pathway with a GAP.

    PubMed

    Spanò, Stefania; Gao, Xiang; Hannemann, Sebastian; Lara-Tejero, María; Galán, Jorge E

    2016-02-10

    Cell-autonomous defense mechanisms are potent strategies that protect individual cells against intracellular pathogens. The Rab-family GTPase Rab32 was previously shown to restrict the intracellular human pathogen Salmonella Typhi, but its potential broader role in antimicrobial defense remains unknown. We show that Rab32 represents a general cell-autonomous, antimicrobial defense that is counteracted by two Salmonella effectors. Mice lacking Rab-32 or its nucleotide exchange factor BLOC-3 are permissive to S. Typhi infection and exhibit increased susceptibility to S. Typhimurium. S. Typhimurium counters this defense pathway by delivering two type III secretion effectors, SopD2, a Rab32 GAP, and GtgE, a specific Rab32 protease. An S. Typhimurium mutant strain lacking these two effectors exhibits markedly reduced virulence, which is fully restored in BLOC-3-deficient mice. These results demonstrate that a cell-autonomous, Rab32-dependent host defense pathway plays a central role in the defense against vacuolar pathogens and describe a mechanism evolved by a bacterial pathogen to counter it.

  19. Visualizing Directional Rab7 and TrkA Cotrafficking in Axons by pTIRF Microscopy

    PubMed Central

    Zhang, Kai; Chowdary, Praveen D.; Cui, Bianxiao

    2016-01-01

    Rab7 GTPase is known to regulate protein degradation and intracellular signaling via endocytic sorting and is also known to be involved in peripheral neurodegeneration. Mutations in the GTP-binding pocket of Rab7 cause Charcot–Marie–Tooth type 2B (CMT-2B) neuropathy. It has been suggested that the CMT-2B-associated Rab7 mutants may disrupt retrograde survival signaling by degrading the signaling endosomes carrying the nerve growth factor (NGF) and its TrkA receptor. Studying the cotrafficking of Rab7 and retrograde-TrkA endosomes in axons is therefore important to understand how Rab7 mutants affect the NGF signaling in neurons. However, tracking the axonal transport of Rab7 and TrkA with conventional microscopy and assigning the transport directionality in mass neuronal cultures pose some practical challenges. In this chapter, we describe the combination of a single-molecule imaging technique, pseudo-total internal reflection fluorescence (pTIRF) microscopy, with microfluidic neuron cultures that enables the simultaneous tracking of fluorescently labeled Rab7- and TrkA-containing endosomes in axons. PMID:25800854

  20. The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

    PubMed

    Binotti, Beyenech; Pavlos, Nathan J; Riedel, Dietmar; Wenzel, Dirk; Vorbrüggen, Gerd; Schalk, Amanda M; Kühnel, Karin; Boyken, Janina; Erck, Christian; Martens, Henrik; Chua, John J E; Jahn, Reinhard

    2015-01-01

    Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.

  1. Role of Arabidopsis RabG3b and autophagy in tracheary element differentiation.

    PubMed

    Kwon, Soon Il; Cho, Hong Joo; Park, Ohkmae K

    2010-11-01

    The vascular system of plants consists of two conducting tissues, xylem and phloem, which differentiate from procambium cells. Xylem serves as a transporting system for water and signaling molecules and is formed by sequential developmental processes, including cell division/expansion, secondary cell wall deposition, vacuole collapse and programmed cell death (PCD). PCD during xylem differentiation is accomplished by degradation of cytoplasmic constituents, and it is required for the formation of hollow vessels, known as tracheary elements (TEs). Our recent study revealed that the small GTPase RabG3b acts as a regulator of TE differentiation through its autophagic activation. By using an Arabidopsis in vitro cell culture system, we showed that autophagy is activated during TE differentiation. Overexpression of a constitutively active RabG3b (RabG3bCA) significantly enhances both autophagy and TE differentiation, which are consistently suppressed in transgenic plants overexpressing a dominant negative form (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid-insensitive mutant bri1-301 and an autophagy mutant atg5-1. On the basis of our results, we propose that RabG3b functions as a component of autophagy and regulates TE differentiation by activating the process of PCD.

  2. Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene.

    PubMed

    Amara, Imen; Capellades, Montserrat; Ludevid, M Dolors; Pagès, Montserrat; Goday, Adela

    2013-06-15

    Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants.

  3. Spatial and Functional Aspects of ER-Golgi Rabs and Tethers

    PubMed Central

    Saraste, Jaakko

    2016-01-01

    Two conserved Rab GTPases, Rab1 and Rab2, play important roles in biosynthetic-secretory trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus in mammalian cells. Both are expressed as two isoforms that regulate anterograde transport via the intermediate compartment (IC) to the Golgi, but are also required for transport in the retrograde direction. Moreover, Rab1 has been implicated in the formation of autophagosomes. Rab1 and Rab2 have numerous effectors or partners that function in membrane tethering, but also have other roles. These include the coiled-coil proteins p115, GM130, giantin, golgin-84, and GMAP-210, as well as the multisubunit COG (conserved oligomeric Golgi) and TRAPP (transport protein particle) tethering complexes. TRAPP also acts as the GTP exchange factor (GEF) in the activation of Rab1. According to the traditional view of the IC elements as motile, transient structures, the functions of the Rabs could take place at the two ends of the ER-Golgi itinerary, i.e., at ER exit sites (ERES) and/or cis-Golgi. However, there is considerable evidence for their specific association with the IC, including its recently identified pericentrosomal domain (pcIC), where many of the effectors turn out to be present, thus being able to exert their functions at the pre-Golgi level. The IC localization of these proteins is of particular interest based on the imaging of Rab1 dynamics, indicating that the IC is a stable organelle that bidirectionally communicates with the ER and Golgi, and is functionally linked to the endosomal system via the pcIC. PMID:27148530

  4. Crystallization of an engineered RUN domain of Rab6-interacting protein 1/DENND5

    SciTech Connect

    Fernandes, Humberto; Franklin, Edward; Khan, Amir R.

    2011-08-29

    Effectors of the Rab small GTPases are large multi-domain proteins which have proved difficult to express in soluble form in Escherichia coli. Generally, effectors are recruited to a distinct subcellular compartment by active (GTP-bound) Rabs, which are linked to membranes by one or two prenylated Cys residues at their C-termini. Following recruitment via their Rab-binding domain (RBD), effectors carry out various aspects of vesicle formation, transport, tethering and fusion through their other domains. Previously, successful purification of the RUN-PLAT tandem domains (residues 683-1061) of the 1263-residue Rab6-interacting protein 1 (R6IP1) required co-expression with Rab6, as attempts to solubly express the effector alone were unsuccessful. R6IP1 is also known as DENN domain-containing protein 5 (DENND5) and is expressed as two isoforms, R6IP1A/B (DENND5A/B), which differ by 24 amino acids at the N-terminus. Here, a deletion in R6IP1 was engineered to enable soluble expression and to improve the quality of the crystals grown in complex with Rab6. A large 23-residue loop linking two -helices in the RUN1 domain was removed and replaced with a short linker. This loop resides on the opposite face to the Rab6-binding site and is not conserved in the RUN-domain family. In contrast to wild-type R6IP1-Rab6 crystals, which took several weeks to grow to full size, the engineered R6IP1 (RPdel)-Rab6 crystals could be grown in a matter of days.

  5. Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

    SciTech Connect

    B McCray; E Skordalakes; J Taylor

    2011-12-31

    Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

  6. Thermal analysis of brazing seal and sterilizing technique to break contamination chain for Mars sample return

    NASA Astrophysics Data System (ADS)

    Bao, Xiaoqi; Badescu, Mircea; Bar-Cohen, Yoseph

    2015-03-01

    The potential to return Martian samples to Earth for extensive analysis is in great interest of the planetary science community. It is important to make sure the mission would securely contain any microbes that may possibly exist on Mars so that they would not be able to cause any adverse effects on Earth's environment. A brazing sealing and sterilizing technique has been proposed to break the Mars-to-Earth contamination chain. Thermal analysis of the brazing process was conducted for several conceptual designs that apply the technique. Control of the increase of the temperature of the Martian samples is a challenge. The temperature profiles of the Martian samples being sealed in the container were predicted by finite element thermal models. The results show that the sealing and sterilization process can be controlled such that the samples' temperature is maintained below the potentially required level, and that the brazing technique is a feasible approach to break the contamination chain.

  7. Nondestructive test of brazed cooling tubes of prototype bolometer camera housing using active infrared thermography

    NASA Astrophysics Data System (ADS)

    Tahiliani, Kumudni; Pandya, Santosh P.; Pandya, Shwetang; Jha, Ratneshwar; Govindarajan, J.

    2011-01-01

    The active infrared thermography technique is used for assessing the brazing quality of an actively cooled bolometer camera housing developed for steady state superconducting tokamak. The housing is a circular pipe, which has circular tubes vacuum brazed on the periphery. A unique method was adopted to monitor the temperature distribution on the internal surface of the pipe. A stainless steel mirror was placed inside the pipe and the reflected IR radiations were viewed using an IR camera. The heat stimulus was given by passing hot water through the tubes and the temperature distribution was monitored during the transient phase. The thermographs showed a significant nonuniformity in the brazing with a contact area of around 51%. The thermography results were compared with the x-ray radiographs and a good match between the two was observed. Benefits of thermography over x-ray radiography testing are emphasized.

  8. Effect of Composite Substrates on the Mechanical Behavior of Brazed Joints in Metal-Composite System

    NASA Technical Reports Server (NTRS)

    Singh, M.; Morscher, Gregory N.; Shpargel, Tarah; Asthana, Rajiv

    2006-01-01

    Advanced composite components are being considered for a wide variety of demanding applications in aerospace, space exploration, and ground based systems. A number of these applications require robust integration technologies to join dissimilar materials (metalcomposites) into complex structural components. In this study, three types of composites (C-C, C-SiC, and SiC-SiC) were vacuum brazed to commercially pure Ti using the active metal braze alloy Cusil-ABA (63Ag-35.3Cu-1.75Ti). Composite substrates with as fabricated and polished surfaces were used for brazing. The microstructure and composition of the joint, examined using scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (EDS), showed sound metallurgical bonding in all systems. The butt strap tensile (BST) test was performed on bonded specimens at room and elevated temperatures. Effect of substrate composition, interlaminar properties, and surface roughness on the mechanical properties and failure behavior of joints will be discussed.

  9. Effects of Different Braze Materials and Composite Substrates on Composite/Ti Joints

    NASA Technical Reports Server (NTRS)

    Morscher, Gregory N.; Singh, Mrityunjay; Asthana, Rajiv; Shpargel, Tarah

    2007-01-01

    An ever increasing number of applications require robust joining technologies of dissimilar materials. In this study, three types of ceramic composites (C-C, C-SiC, and SiC-SiC) were vacuum brazed to commercially pure Ti using the Cusil-ABA (63 Ag - 35.5 Cu - 1.75 Ti) active metal braze alloy. The study also compared composite specimens as-fabricated and after surface grinding/polishing. A butt-strap tensile shear strength test was used to evaluate the joined structures at room temperature, 270 and 500 C. The elevated temperatures represent possible use temperatures for some heat rejection type applications. Joint strength will be discussed in light of braze wetting and spreading properties, composite properties, and test temperature.

  10. A novel RAB7 mutation associated with ulcero-mutilating neuropathy.

    PubMed

    Houlden, Henry; King, Rosalind H M; Muddle, John R; Warner, Thomas T; Reilly, Mary M; Orrell, Richard W; Ginsberg, Lionel

    2004-10-01

    There are two known autosomal dominant genes for the hereditary ulcero-mutilating neuropathies: SPTLC1 (hereditary sensory neuropathy type 1) and RAB7 (Charcot-Marie-Tooth disease type 2B). We report a family with autosomal dominant ulcero-mutilating neuropathy, developing in the teens and characterized by ulcers, amputations, sensory involvement in the feet but no motor features. Sequencing the RAB7 gene showed a novel heterozygous A to C mutation, changing asparagine to threonine at codon 161. The mutation is situated adjacent to a previously identified valine to methionine mutation at codon 162, implying a hotspot for mutations in the highly conserved C terminus of RAB7. PMID:15455439

  11. Aromatic acid metabolites of Escherichia coli K-12 can induce the marRAB operon.

    PubMed

    Chubiz, Lon M; Rao, Christopher V

    2010-09-01

    MarR is a key regulator of the marRAB operon involved in antibiotic resistance and solvent stress tolerance in Escherichia coli. We show that two metabolic intermediates, 2,3-dihydroxybenzoate and anthranilate, involved in enterobactin and tryptophan biosynthesis, respectively, can activate marRAB transcription. We also found that a third intermediate involved in ubiquinone biosynthesis, 4-hydroxybenzoate, activates marRAB transcription in the absence of TolC. Of the three, however, only 2,3-dihydroxybenzoate directly binds MarR and affects its activity. PMID:20639340

  12. Basic principles of creating a new generation of high- temperature brazing filler alloys

    NASA Astrophysics Data System (ADS)

    Kalin, B. A.; Suchkov, A. N.

    2016-04-01

    The development of new materials is based on the formation of a structural-phase state providing the desired properties by selecting the base and the complex of alloying elements. The development of amorphous filler alloys for a high-temperature brazing has its own features that are due to the limited life cycle and the production method of brazing filler alloys. The work presents a cycle of analytical and experimental materials science investigations including justification of the composition of a new amorphous filler alloy for brazing the products from zirconium alloys at the temperature of no more than 800 °C and at the unbrazing temperature of permanent joints of more than 1200 °C. The experimental alloys have been used for manufacture of amorphous ribbons by rapid quenching, of which the certification has been made by X-ray investigations and a differential-thermal analysis. These ribbons were used to obtain permanent joints from the spacer grid cells (made from the alloy Zr-1% Nb) of fuel assemblies of the thermal nuclear reactor VVER-440. The brazed samples in the form of a pair of cells have been exposed to corrosion tests in autoclaves in superheated water at a temperature of 350 °C, a pressure of 160 MPa and duration of up to 6,000 h. They have been also exposed to destructive tests using a tensile machine. The experimental results obtained have made it possible to propose and patent a brazing filler alloy of the following composition: Zr-5.5Fe-(2.5-3.5)Be-1Nb-(5-8)Cu-2Sn-0.4Cr-(0.5-1.0)Ge. Its melting point is 780 °C and the recommended brazing temperature is 800°C.

  13. Development of the activated diffusion brazing process for fabrication of finned shell to strut turbine blades

    NASA Technical Reports Server (NTRS)

    Wilbers, L. G.; Berry, T. F.; Kutchera, R. E.; Edmonson, R. E.

    1971-01-01

    The activated diffusion brazing process was developed for attaching TD-NiCr and U700 finned airfoil shells to matching Rene 80 struts obstructing the finned cooling passageways. Creep forming the finned shells to struts in combination with precise preplacement of brazing alloy resulted in consistently sound joints, free of cooling passageway clogging. Extensive tensile and stress rupture testing of several joint orientation at several temperatures provided a critical assessment of joint integrity of both material combinations. Trial blades of each material combination were fabricated followed by destructive metallographic examination which verified high joint integrity.

  14. Development of brazing process for W-EUROFER joints using Cu-based fillers

    NASA Astrophysics Data System (ADS)

    de Prado, J.; Sánchez, M.; Ureña, A.

    2016-02-01

    A successful joint between W and EUROFER using high temperature brazing technique has been achieved for structural application in future fusion power plants. Cu-based powder alloy mixed with a polymeric binder has been used as filler. Microstructural analysis of the joints revealed that the joint consisted mainly of primary phases and acicular structures in a Cu matrix. Interaction between EUROFER and filler took place at the interface giving rise to several Cu-Ti-Fe rich layers. A loss of hardness at the EUROFER substrate close to the joint due to a diffusion phenomenon during brazing cycle was measured; however, the joints had an adequate shear strength value.

  15. Combined in-situ dilatometer and contact angle studies of interfacial reaction kinetics in brazing.

    SciTech Connect

    Dave, V. R.; Javernick, D. A.; Thoma, D. J.; Cola, M. J.; Hollis, K. J.; Smith, F. M.; Dauelsberg, L. B.

    2001-01-01

    Multi-component dissimilar material braze joints as shown in Figure 1 consisting of dissimilar base materials, filler materials and wetting agents are of tantamount importance in a wide variely of applications. This work combines dilatometry and contact angle measurements to characterize in-situ the multiple interfacial reaction pathways that occur in such systems. Whereas both of these methods are commonly used tools in metallurgical investigation, their combined use within the context of brazing studies is new and offers considerable additional insight. Applications are discussed to joints made between Beryllium and Monel with TiH{sub 2} as the wetting agent and Cu-28%Ag as the filler material.

  16. Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen.

    PubMed

    Levin, Rebecca S; Hertz, Nicholas T; Burlingame, Alma L; Shokat, Kevan M; Mukherjee, Shaeri

    2016-08-16

    TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPase-activating protein (GAP) enzymes, and is exclusive to membrane-localized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection. PMID:27482120

  17. IL4/PGE{sub 2} induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

    SciTech Connect

    Wainszelbaum, Marisa J.; Proctor, Brandon M.; Pontow, Suzanne E.; Stahl, Philip D. . E-mail: pstahl@cellbiology.wustl.edu; Barbieri, M. Alejandro

    2006-07-15

    The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E{sub 2} (PGE{sub 2}) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.

  18. Arabidopsis RAB geranylgeranyl transferase beta-subunit mutant is constitutively photomorphogenic, and has shoot growth and gravitropic defects.

    PubMed

    Hála, Michal; Soukupová, Hana; Synek, Lukás; Zárský, Viktor

    2010-05-01

    RAB GTPases are important directional regulators of intracellular vesicle transport. Membrane localization of RAB GTPases is mediated by C-terminal double geranylgeranylation. This post-translational modification is catalyzed by the alpha-beta-heterodimer catalytic core of RAB geranylgeranyl transferase (RAB-GGT), which cooperates with the RAB escort protein (REP) that presents a nascent RAB. Here, we show that RAB-geranylgeranylation activity is significantly reduced in two homozygous mutants of the major Arabidopsis beta-subunit of RAB-GGT (AtRGTB1), resulting in unprenylated RAB GTPases accumulation in the cytoplasm. Both endocytosis and exocytosis are downregulated in rgtb1 homozygotes defective in shoot growth and morphogenesis. Root gravitropism is normal in rgtb1 roots, but is significantly compromised in shoots. Mutants are defective in etiolation and show constitutive photomorphogenic phenotypes that cannot be rescued by brassinosteroid treatment, similarly to the det3 mutant that is also defective in the secretory pathway. Transcriptomic analysis revealed an upregulation of specific RAB GTPases in etiolated wild-type plants. Taken together, these data suggest that the downregulation of the secretory pathway is interpreted as a photomorphogenic signal in Arabidopsis.

  19. [SapM-induced fusion blocking of autophagosome-lysosome is depended on interaction with Rab7].

    PubMed

    Hu, Dong; Wang, Wan; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Ni, Shengfa; Xu, Congjing; Tie, Baoxian; Zhang, Rongbo; Wu, Jing

    2016-09-01

    Objective To study the role of Rab7 in the blockage of autophagosome-lysosome fusion induced by secretory acid phosphatase (SapM), a virulence factor of mycobacterium tuberculosis. Methods The Raw264.7 cells were transfected with siRab7, and the P62 was detected using Western blotting. After transfected with mCherry-SapM, the co-localization of SapM and Rab7 in Raw264.7 cells was detected by immunofluorescence cytochemistry and the interaction of SapM with Rab7 was determined by co-immunoprecipitation. SapM mutants including SapM(δ ARCA), SapM(δ FRED) and SapM(δ CT) were used to transfect Raw264.7 cells, and their associations with Rab7 were analyzed. Results The treatment of siRab7 induced a significant increase of P62 in these cells. Immunofluorescence cytochemistry showed the intracellular co-localization of SapM and Rab7. Co-immunoprecipitation showed that SapM and Rab7 were precipitated by each other. Only SapM(δ CT) failed to interact with Rab7 among the three SapM mutants. Conclusion The inhibition of autophagosome-lysosome fusion induced by SapM is dependent on the interaction between SapM and Rab7. PMID:27609571

  20. Depletion of Rab32 decreases intracellular lipid accumulation and induces lipolysis through enhancing ATGL expression in hepatocytes.

    PubMed

    Li, Qing; Wang, Jun; Wan, Ying; Chen, Dongfeng

    2016-03-18

    Nonalcoholic fatty liver disease (NAFLD) is a disease caused by the accumulation of lipids in hepatocytes. To date, however, the pathogenesis of NAFLD is still unclear. Recent studies have shown that Rab GTPases, a major protein family in vesicle trafficking, are associated with intracellular lipid accumulation. Here, we show that Rab32, the only Rab GTPase located in mitochondria, participates in hepatic steatosis. Ablation of Rab32 can decrease intracellular lipid accumulation in hepatocytes (HepG2, L02). Further studying the possible mechanism, we found that knockdown of Rab32 can enhance lipolysis instead of lipogenesis via inducing the expression of adipose triglyceride lipase (ATGL), a key enzyme on the surface of lipid droplets which has been proved to be significant in controlling intracellular lipid accumulation. Co-immunoprecipitation shows that Rab32 and ATGL are not directly associated. These findings suggest that knockdown of Rab32 indirectly affects lipolysis through increasing the expression of ATGL. Taken together, our study reveals that Rab32 can participate in regulating intracellular lipid accumulation and that knockdown of Rab32 can decrease intracellular lipid accumulation in hepatocytes. We also demonstrated that ablation of Rab32 can induce intracellular lipolysis by enhancing the expression of ATGL. PMID:26882978

  1. Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

    PubMed

    Inada, Noriko; Betsuyaku, Shigeyuki; Shimada, Takashi L; Ebine, Kazuo; Ito, Emi; Kutsuna, Natsumaro; Hasezawa, Seiichiro; Takano, Yoshitaka; Fukuda, Hiroo; Nakano, Akihiko; Ueda, Takashi

    2016-09-01

    RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens. PMID:27318282

  2. Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

    PubMed

    Inada, Noriko; Betsuyaku, Shigeyuki; Shimada, Takashi L; Ebine, Kazuo; Ito, Emi; Kutsuna, Natsumaro; Hasezawa, Seiichiro; Takano, Yoshitaka; Fukuda, Hiroo; Nakano, Akihiko; Ueda, Takashi

    2016-09-01

    RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.

  3. Rab11a differentially modulates epidermal growth factor-induced proliferation and motility in immortal breast cells.

    PubMed

    Palmieri, Diane; Bouadis, Amina; Ronchetti, Ruban; Merino, Maria J; Steeg, Patricia S

    2006-11-01

    The development of cancer prevention strategies depends on the elucidation of molecular pathways underlying oncogenesis. In a previous proteomic study of matched normal breast ducts and Ductal Carcinoma in Situ (DCIS), we identified overexpression of Rab11a in DCIS. Rab11a is not well studied in cancer, but is known to regulate the recycling of internalized cell surface proteins and receptors from the early endosome through the trans-Golgi network. Using immunohistochemistry, we confirmed our observation, noting increased Rab11a expression in 19 of 22 (86%) DCIS cases compared to matched normal breast epithelium. To study the function of Rab11a, immortal, nontumorigenic MCF10A breast cells were stimulated with ligands to the EGF receptor (EGFR) after transfection with empty vector (control), Rab11a, or a S25N dominant-negative (DN) Rab11a. Using an iodinated ligand:receptor recycling assay, transfection of Rab11a accelerated, while DN-Rab11a postponed EGFR recycling in vitro. The signaling and in vitro phenotypic consequences of Rab11a expression and function were studied. Transfection of DN-Rab11a increased Erk1/2 activation downstream of EGF, but exerted no effect on the Akt pathway. Expression of DN-Rab11a inhibited MCF10A proliferation by 50-60%, and also inhibited anchorage-dependent colonization. Notably, DN-Rab11a transfection increased motility toward EGFR ligands. The data provide a first demonstration that Rab11a modulates EGFR recycling, and promotes the proliferation but inhibits the motility of an immortal breast line, consistent with the DCIS phenotype.

  4. Rab5 is required in metastatic cancer cells for Caveolin-1-enhanced Rac1 activation, migration and invasion.

    PubMed

    Díaz, Jorge; Mendoza, Pablo; Ortiz, Rina; Díaz, Natalia; Leyton, Lisette; Stupack, Dwayne; Quest, Andrew F G; Torres, Vicente A

    2014-06-01

    Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85α (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding p85α-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1-Rab5-Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion.

  5. Mechanical Property and Corrosion Resistance Evaluations of Ti-6Al-7Nb Alloy Brazed with Bulk Metallic Glasses

    SciTech Connect

    Miura, E.; Kato, H.; Ogata, Toshiaki; Nishiyama, Nobuyuki; Specht, Eliot D; Shiraishi, Takanobu; Inoue, A.; Hisatsune, K.

    2007-01-01

    Exploitation of metallic glass as new brazing filler for Ti-based biomedical alloy was attempted. Ti-6Al-7Nb was used as a brazed material, and candidates of bulk metallic glass brazing filler were Cu60Hf25Ti15, Mg65Cu25Gd10, Zr55Cu30Al10Ni5 and Pd40Cu30P20Ni10. Convergence infrared-ray brazing was conducted for brazing Ti-6Al-7Nb/metallic glass in Ar atmosphere. After brazing, hardness measurement, X-ray tomography, cross-sectional observation, artificial saliva immersion test and tensile test were performed to evaluate brazability, mechanical property and corrosion resistance of the obtained brazing joints. The results of brazing using these metallic glass fillers show that all the metallic glasses were brazable to Ti-6Al-7Nb except for Mg65Cu25Gd10. Mg65Cu25Gd10, Cu60Hf25Ti15 and their joints collapsed rapidly during immersion test. Zr55Cu30Al10Ni5 joint was the best in terms of degradation resistance; however, tensile strength was inferior to the conventional one. Pd40Cu30Ni10P20 filler and Zr55Cu30Al10Ni5 filler and their joints did not show any collapse or tarnish during the immersion test. Pd40Cu30Ni10P20 joint showed the excellent properties in terms of both corrosion resistance and tensile strength, which were superior to a joint brazed using Ti-15Cu-25Ni conventional filler. X-ray tomograph indicates that fracture tends to occur in the vicinity of the brazing interface after tensile test. The brazed metallic glass fillers were fully crystallized, excluding Pd40Cu30Ni10P20 filler. Pd40Cu30Ni10P20 brazed filler contained mapleleaf like primary dendrite, peritectoid and a few microns interfacial reaction layer in glassy matrix. The results indicated that Pd40Cu30Ni10P20 is promising brazing filler for dental or biomaterial devices.

  6. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

    PubMed

    D'Costa, Vanessa M; Braun, Virginie; Landekic, Marija; Shi, Rong; Proteau, Ariane; McDonald, Laura; Cygler, Miroslaw; Grinstein, Sergio; Brumell, John H

    2015-09-01

    Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target. PMID:26299973

  7. PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7

    PubMed Central

    Shinde, Swapnil Rohidas; Maddika, Subbareddy

    2016-01-01

    Tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase that negatively regulates growth factor-induced survival signalling. Here, we demonstrate that PTEN attenuates epidermal growth factor receptor (EGFR) signalling by promoting late endosome maturation by virtue of its protein phosphatase activity. Loss of PTEN impairs the transition of ligand-bound EGFR from early to late endosomes. We unveil Rab7, a critical GTPase for endosome maturation, as a functional PTEN interacting partner. PTEN dephosphorylates Rab7 on two conserved residues S72 and Y183, which are necessary for GDP dissociation inhibitor (GDI)-dependent recruitment of Rab7 on to late endosomes and subsequent maturation. Thus, our findings reveal PTEN-dependent endosome maturation through phosphoregulation of Rab7 as an important route of controlling EGFR signalling. PMID:26869029

  8. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

    PubMed

    D'Costa, Vanessa M; Braun, Virginie; Landekic, Marija; Shi, Rong; Proteau, Ariane; McDonald, Laura; Cygler, Miroslaw; Grinstein, Sergio; Brumell, John H

    2015-09-01

    Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

  9. PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7.

    PubMed

    Shinde, Swapnil Rohidas; Maddika, Subbareddy

    2016-01-01

    Tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase that negatively regulates growth factor-induced survival signalling. Here, we demonstrate that PTEN attenuates epidermal growth factor receptor (EGFR) signalling by promoting late endosome maturation by virtue of its protein phosphatase activity. Loss of PTEN impairs the transition of ligand-bound EGFR from early to late endosomes. We unveil Rab7, a critical GTPase for endosome maturation, as a functional PTEN interacting partner. PTEN dephosphorylates Rab7 on two conserved residues S72 and Y183, which are necessary for GDP dissociation inhibitor (GDI)-dependent recruitment of Rab7 on to late endosomes and subsequent maturation. Thus, our findings reveal PTEN-dependent endosome maturation through phosphoregulation of Rab7 as an important route of controlling EGFR signalling. PMID:26869029

  10. A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus

    PubMed Central

    1994-01-01

    The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225- 237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta- COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during

  11. Deficient Rab11 activity underlies glucose hypometabolism in primary neurons of Huntington's disease mice

    SciTech Connect

    Li, Xueyi; Valencia, Antonio; McClory, Hollis; Sapp, Ellen; Kegel, Kimberly B.; DiFiglia, Marian

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Primary Huntington's disease neurons are impaired in taking up glucose. Black-Right-Pointing-Pointer Rab11 modulates glucose uptake in neurons. Black-Right-Pointing-Pointer Increasing Rab11 activity attenuates the glucose uptake defect in disease neurons. Black-Right-Pointing-Pointer We provide a novel mechanism for glucose hypometabolism in Huntington's disease. -- Abstract: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Positron emission tomography studies have revealed a decline in glucose metabolism in the brain of patients with HD by a mechanism that has not been established. We examined glucose utilization in embryonic primary cortical neurons of wild-type (WT) and HD knock-in mice, which have 140 CAG repeats inserted in the endogenous mouse huntingtin gene (HD{sup 140Q/140Q}). Primary HD{sup 140Q/140Q} cortical neurons took up significantly less glucose than did WT neurons. Expression of permanently inactive and permanently active forms of Rab11 correspondingly altered glucose uptake in WT neurons, suggesting that normal activity of Rab11 is needed for neuronal uptake of glucose. It is known that Rab11 activity is diminished in HD{sup 140Q/140Q} neurons. Expression of dominant active Rab11 to enhance the activity of Rab11 normalized glucose uptake in HD{sup 140Q/140Q} neurons. These results suggest that deficient activity of Rab11 is a novel mechanism for glucose hypometabolism in HD.

  12. Charcot-Marie-Tooth disease due to a de novo mutation of the RAB7 gene.

    PubMed

    Meggouh, F; Bienfait, H M E; Weterman, M A J; de Visser, M; Baas, F

    2006-10-24

    We report a 32-year-old patient with Charcot-Marie-Tooth (CMT2B) including foot ulcerations. Genetic analysis identified a de novo mutation in the small GTP-ase late endosomal RAB7 gene, consisting of a c.471G>C, p.Lys157Asn missense mutation. This observation strongly supports the hypothesis that RAB7 mutations are responsible for CMT2B. PMID:17060578

  13. A Heterozygous RAB27A Mutation Associated with Delayed Cytolytic Granule Polarization and Hemophagocytic Lymphohistiocytosis.

    PubMed

    Zhang, Mingce; Bracaglia, Claudia; Prencipe, Giusi; Bemrich-Stolz, Christina J; Beukelman, Timothy; Dimmitt, Reed A; Chatham, W Winn; Zhang, Kejian; Li, Hao; Walter, Mark R; De Benedetti, Fabrizio; Grom, Alexei A; Cron, Randy Q

    2016-03-15

    Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in NK and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report two unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (c.259G→C). We explore the contribution of this Rab27A missense (p.A87P) mutation on NK cell cytolytic function by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant- and wild-type-transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (p.A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay, respectively. Overexpression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein decreased binding to Munc13-4 and delayed granzyme B polarization toward the immunologic synapse. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect. PMID:26880764

  14. Rab7 Mutants Associated with Charcot-Marie-Tooth Disease Exhibit Enhanced NGF-Stimulated Signaling

    PubMed Central

    Romero, Elsa; Wilson, Michael C.; Wandinger-Ness, Angela

    2010-01-01

    Missense mutants in the late endosomal Rab7 GTPase cause the autosomal dominant peripheral neuropathy Charcot-Marie-Tooth disease type 2B (CMT2B). As yet, the pathological mechanisms connecting mutant Rab7 protein expression to altered neuronal function are undefined. Here, we analyze the effects Rab7 CMT2B mutants on nerve growth factor (NGF) dependent intracellular signaling in PC12 cells. The nerve growth factor receptor TrkA interacted similarly with Rab7 wild-type and CMT2B mutant proteins, but the mutant proteins significantly enhanced TrkA phosphorylation in response to brief NGF stimulation. Two downstream signaling pathways (Erk1/2 and Akt) that are directly activated in response to phospho-TrkA were differentially affected. Akt signaling, arising in response to activated TrkA at the plasma membrane was unaffected. However Erk1/2 phosphorylation, triggered on signaling endosomes, was increased. Cytoplasmic phospho-Erk1/2 persisted at elevated levels relative to control samples for up to 24 h following NGF stimulation. Nuclear shuttling of phospho Erk1/2, which is required to induce MAPK phosphatase expression and down regulate signaling, was greatly reduced by the Rab7 CMT2B mutants and explains the previously reported inhibition in PC12 neurite outgrowth. In conclusion, the data demonstrate a mechanistic link between Rab7 CMT2B mutants and altered TrkA and Erk1/2 signaling from endosomes. PMID:21151572

  15. Rab-3 and unc-18 interactions in alcohol sensitivity are distinct from synaptic transmission.

    PubMed

    Johnson, James R; Kashyap, Sudhanva; Rankin, Kim; Barclay, Jeff W

    2013-01-01

    The molecular mechanisms underlying sensitivity to alcohol are incompletely understood. Recent research has highlighted the involvement of two presynaptic proteins, Munc18 and Rab3. We have previously characterised biochemically a number of specific Munc18 point mutations including an E466K mutation that augments a direct Rab3 interaction. Here the phenotypes of this and other Munc18 mutations were assessed in alcohol sensitivity and exocytosis using Caenorhabditis elegans. We found that expressing the orthologous E466K mutation (unc-18 E465K) enhanced alcohol sensitivity. This enhancement in sensitivity was surprisingly independent of rab-3. In contrast unc-18 R39C, which decreases syntaxin binding, enhanced sensitivity to alcohol in a manner requiring rab-3. Finally, overexpression of R39C could suppress partially the reduction in neurotransmitter release in rab-3 mutant worms, whereas wild-type or E465K mutants showed no rescue. These data indicate that the epistatic interactions between unc-18 and rab-3 in modulating sensitivity to alcohol are distinct from interactions affecting neurotransmitter release.

  16. A Heterozygous RAB27A Mutation Associated with Delayed Cytolytic Granule Polarization and Hemophagocytic Lymphohistiocytosis.

    PubMed

    Zhang, Mingce; Bracaglia, Claudia; Prencipe, Giusi; Bemrich-Stolz, Christina J; Beukelman, Timothy; Dimmitt, Reed A; Chatham, W Winn; Zhang, Kejian; Li, Hao; Walter, Mark R; De Benedetti, Fabrizio; Grom, Alexei A; Cron, Randy Q

    2016-03-15

    Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in NK and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report two unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (c.259G→C). We explore the contribution of this Rab27A missense (p.A87P) mutation on NK cell cytolytic function by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant- and wild-type-transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (p.A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay, respectively. Overexpression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein decreased binding to Munc13-4 and delayed granzyme B polarization toward the immunologic synapse. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.

  17. Functional analysis of small Rab GTPases in cytokinesis in Arabidopsis thaliana.

    PubMed

    Qi, Xingyun; Zheng, Huanquan

    2013-01-01

    Rab proteins are key regulators of membrane transport in eukaryotes. Recent evidence from different species supports the notion that some Rab proteins are crucial for cytokinesis, a pivotal procedure for successful cell division. As a family of monomeric small GTPases of the Ras superfamily, the function of Rab proteins is modulated by guanine nucleotide binding and hydrolysis. To investigate the function of Rab proteins, creating dominant negative or constitutively active mutant forms of a Rab protein is a widely used approach. To study cytokinesis in plant cells, using fluorescent dye to highlight the cell shape and the nuclei, and to monitor the formation of the newly formed cell plate in mitotic cells, is easy and useful. In this chapter, we describe detailed methods for (1) generating transgenic plants expressing dominant negative or constitutively active form of RAB-A1c; (2) fluorescent staining of cell shape, cell wall, and nuclei of mitotic root tip cells; (3) fluorescent staining of newly formed cell plate; and (4) detecting fluorescent signals using Confocal Laser Scanning Microscopy in the genetic model plant species Arabidopsis thaliana.

  18. Syntaxin, VAMP, and Rab3 are selectively expressed during sea urchin embryogenesis.

    PubMed

    Conner, S D; Wessel, G M

    2001-01-01

    SNARE and rab protein family members were originally identified in terminally differentiated cell types. These proteins are phylogenetically conserved and while compelling evidence demonstrates their involvement in the secretory pathway, their exact function is debated. We recently identified SNARE protein family members in the sea urchin egg and provided evidence that rab3 functions in the exocytosis of cortical granules. Here we tested the hypothesis that these same proteins might also be present throughout embryogenesis to mediate membrane fusion events. We provide evidence that the sea urchin possesses a low complexity of gene family members of syntaxin, VAMP, and rab3 and that these proteins are not only present during development, but are enriched in regions of the embryo with active secretory roles. We found accumulation of each family member in the apical and basal aspects of cleaving blastomeres, indicative of bidirectional secretion into the extraembryonic environment and blastocoel. Elevated levels of syntaxin, VAMP, and rab3 were also found in the mesodermally derived pigment cells that invade and move within the ectoderm. These cells likely rely on SNARE and rab proteins to enable mobility by mediating the secretion of enzymes that break adhesion to neighboring cells and the extracellular matrix. In addition, these secretory proteins are enriched in the gut following gastrulation. Thus, we conclude that VAMP, syntaxin, and rab3 mediate a variety of secretory events that is important for development.

  19. LMTK1 regulates dendritic formation by regulating movement of Rab11A-positive endosomes

    PubMed Central

    Takano, Tetsuya; Urushibara, Tomoki; Yoshioka, Nozomu; Saito, Taro; Fukuda, Mitsunori; Tomomura, Mineko; Hisanaga, Shin-ichi

    2014-01-01

    Neurons extend two types of neurites—axons and dendrites—that differ in structure and function. Although it is well understood that the cytoskeleton plays a pivotal role in neurite differentiation and extension, the mechanisms by which membrane components are supplied to growing axons or dendrites is largely unknown. We previously reported that the membrane supply to axons is regulated by lemur kinase 1 (LMTK1) through Rab11A-positive endosomes. Here we investigate the role of LMTK1 in dendrite formation. Down-regulation of LMTK1 increases dendrite growth and branching of cerebral cortical neurons in vitro and in vivo. LMTK1 knockout significantly enhances the prevalence, velocity, and run length of anterograde movement of Rab11A-positive endosomes to levels similar to those expressing constitutively active Rab11A-Q70L. Rab11A-positive endosome dynamics also increases in the cell body and growth cone of LMTK1-deficient neurons. Moreover, a nonphosphorylatable LMTK1 mutant (Ser34Ala, a Cdk5 phosphorylation site) dramatically promotes dendrite growth. Thus LMTK1 negatively controls dendritic formation by regulating Rab11A-positive endosomal trafficking in a Cdk5-dependent manner, indicating the Cdk5-LMTK1-Rab11A pathway as a regulatory mechanism of dendrite development as well as axon outgrowth. PMID:24672056

  20. Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour

    PubMed Central

    Breda, Carlo; Nugent, Marie L.; Estranero, Jasper G.; Kyriacou, Charalambos P.; Outeiro, Tiago F.; Steinert, Joern R.; Giorgini, Flaviano

    2015-01-01

    A central pathological hallmark of Parkinson's disease (PD) is the presence of proteinaceous depositions known as Lewy bodies, which consist largely of the protein α-synuclein (aSyn). Mutations, multiplications and polymorphisms in the gene encoding aSyn are associated with familial forms of PD and susceptibility to idiopathic PD. Alterations in aSyn impair neuronal vesicle formation/transport, and likely contribute to PD pathogenesis by neuronal dysfunction and degeneration. aSyn is functionally associated with several Rab family GTPases, which perform various roles in vesicle trafficking. Here, we explore the role of the endosomal recycling factor Rab11 in the pathogenesis of PD using Drosophila models of aSyn toxicity. We find that aSyn induces synaptic potentiation at the larval neuromuscular junction by increasing synaptic vesicle (SV) size, and that these alterations are reversed by Rab11 overexpression. Furthermore, Rab11 decreases aSyn aggregation and ameliorates several aSyn-dependent phenotypes in both larvae and adult fruit flies, including locomotor activity, degeneration of dopaminergic neurons and shortened lifespan. This work emphasizes the importance of Rab11 in the modulation of SV size and consequent enhancement of synaptic function. Our results suggest that targeting Rab11 activity could have a therapeutic value in PD. PMID:25305083

  1. Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole

    NASA Astrophysics Data System (ADS)

    Toshima, Junko Y.; Nishinoaki, Show; Sato, Yoshifumi; Yamamoto, Wataru; Furukawa, Daiki; Siekhaus, Daria Elisabeth; Sawaguchi, Akira; Toshima, Jiro

    2014-03-01

    The yeast Rab5 homologue, Vps21p, is known to be involved both in the vacuolar protein sorting (VPS) pathway from the trans-Golgi network to the vacuole, and in the endocytic pathway from the plasma membrane to the vacuole. However, the intracellular location at which these two pathways converge remains unclear. In addition, the endocytic pathway is not completely blocked in yeast cells lacking all Rab5 genes, suggesting the existence of an unidentified route that bypasses the Rab5-dependent endocytic pathway. Here we show that convergence of the endocytic and VPS pathways occurs upstream of the requirement for Vps21p in these pathways. We also identify a previously unidentified endocytic pathway mediated by the AP-3 complex. Importantly, the AP-3-mediated pathway appears mostly intact in Rab5-disrupted cells, and thus works as an alternative route to the vacuole/lysosome. We propose that the endocytic traffic branches into two routes to reach the vacuole: a Rab5-dependent VPS pathway and a Rab5-independent AP-3-mediated pathway.

  2. Braze Development of Graphite Fiber for Use in Phase Change Material Heat Sinks

    NASA Technical Reports Server (NTRS)

    Quinn, Gregory; Gleason, Brian; Beringer, Woody; Stephen, Ryan

    2010-01-01

    Hamilton Sundstrand (HS), together with NASA Johnson Space Center, developed methods to metallurgically join graphite fiber to aluminum. The goal of the effort was to demonstrate improved thermal conductance, tensile strength and manufacturability compared to existing epoxy bonded techniques. These improvements have the potential to increase the performance and robustness of phase change material heat sinks that use graphite fibers as an interstitial material. Initial work focused on evaluating joining techniques from 4 suppliers, each consisting of a metallization step followed by brazing or soldering of one inch square blocks of Fibercore graphite fiber material to aluminum end sheets. Results matched the strength and thermal conductance of the epoxy bonded control samples, so two suppliers were down-selected for a second round of braze development. The second round of braze samples had up to a 300% increase in strength and up to a 132% increase in thermal conductance over the bonded samples. However, scalability and repeatability proved to be significant hurdles with the metallization approach. An alternative approach was pursued which used nickel and active braze allows to prepare the carbon fibers for joining with aluminum. This approach was repeatable and scalable with improved strength and thermal conductance when compared with epoxy bonding.

  3. Method for braze-joining spirally wound tapes to inner walls of heat exchanger tubes

    DOEpatents

    Garrison, Melton E.

    1984-01-01

    The present invention is directed to a method of fabricating heat exchanger tubes in which twisted tapes are utilized for promoting turbulence and heat transfer. The method of the present invention provides for the brazing of the tapes to the inner walls of the tubes for enhancing heat transfer between the fluid within the conduit and a fluid medium outside of the conduit by conduction through the tape. The braze joint of the present invention is coextensive with the tape over the entire length thereof within the conduit. The practice of the present invention is achieved by placing a filler wire of brazing metal along the tape at a location removed from the side walls and then heating the conduit and tape sufficiently to effect the displacement of the filler metal by wicking to the contact point between the tape and the conduit wall to form a braze joint coextensive with the length of the tape within the conduit. This arrangement provides maximum heat transfer and assures that the tape is in contact with the conduit over the entire common length thereof.

  4. Method for braze-joining spirally wound tapes to inner walls of heat exchanger tubes

    DOEpatents

    Garrison, M.E.

    1982-09-03

    The present invention is directed to a method of fabricating heat exchanger tubes in which twisted tapes are utilized for promoting turbulence and heat transfer. The method of the present invention provides for the brazing of the tapes to the inner walls of the tubes for enhancing heat transfer between the fluid within the conduit and a fluid medium outside of the conduit by conduction through the tape. The braze joint of the present invention is coextensive with the tape over the entire length thereof within the conduit. The practice of the present invention is achieved by placing a filler wire of brazing metal along the tape at a location removed from the side walls and then heating the conduit and tape sufficiently to effect the displacement of the filler metal by wicking to the contact point between the tape and the conduit wall to form a braze joint coextensive with the length of the tape within the conduit. This arrangement provides maximum heat transfer and assures that the tape is in contact with the conduit over the entire common length thereof.

  5. Skylab experiment performance evaluation manual. Appendix G: Experiment M552 exothermic brazing (MSFC)

    NASA Technical Reports Server (NTRS)

    Thomas, O. H., Jr.

    1973-01-01

    Analyses for Experiment M552, Exothermic Brazing (MSFC), to be used for evaluating the performance of the Skylab corollary experiments under preflight, inflight, and post-flight conditions are presented. Experiment contingency plan workaround procedure and malfunction analyses are presented in order to assist in making the experiment operationally successful.

  6. Active vacuum brazing of CNT films to metal substrates for superior electron field emission performance

    NASA Astrophysics Data System (ADS)

    Longtin, Rémi; Sanchez-Valencia, Juan Ramon; Shorubalko, Ivan; Furrer, Roman; Hack, Erwin; Elsener, Hansrudolf; Gröning, Oliver; Greenwood, Paul; Rupesinghe, Nalin; Teo, Kenneth; Leinenbach, Christian; Gröning, Pierangelo

    2015-02-01

    The joining of macroscopic films of vertically aligned multiwalled carbon nanotubes (CNTs) to titanium substrates is demonstrated by active vacuum brazing at 820 °C with a Ag-Cu-Ti alloy and at 880 °C with a Cu-Sn-Ti-Zr alloy. The brazing methodology was elaborated in order to enable the production of highly electrically and thermally conductive CNT/metal substrate contacts. The interfacial electrical resistances of the joints were measured to be as low as 0.35 Ω. The improved interfacial transport properties in the brazed films lead to superior electron field-emission properties when compared to the as-grown films. An emission current of 150 μA was drawn from the brazed nanotubes at an applied electric field of 0.6 V μm-1. The improvement in electron field-emission is mainly attributed to the reduction of the contact resistance between the nanotubes and the substrate. The joints have high re-melting temperatures up to the solidus temperatures of the alloys; far greater than what is achievable with standard solders, thus expanding the application potential of CNT films to high-current and high-power applications where substantial frictional or resistive heating is expected.

  7. Copper-silver-titanium-tin filler metal for direct brazing of structural ceramics

    DOEpatents

    Moorhead, Arthur J.

    1988-04-05

    A method of joining ceramics and metals to themselves and to one another at about 800.degree. C. is described using a brazing filler metal consisting essentially of 35 to 50 at. % copper, 40 to 50 at. % silver, 1 to 15 at. % titanium, and 2 to 8 at. % tin. This method produces strong joints that can withstand high service temperatures and oxidizing environments.

  8. Copper-silver-titanium filler metal for direct brazing of structural ceramics

    DOEpatents

    Moorhead, Arthur J.

    1987-01-01

    A method of joining ceramics and metals to themselves and to one another is described using a brazing filler metal consisting essentially of 35 to 50 atomic percent copper, 15 to 50 atomic percent silver and 10 to 45 atomic percent titanium. This method produces strong joints that can withstand high service temperatures and oxidizing environments.

  9. Training Materials Sourcebook. Welding, Brazing, and Flame-Cutting. Voc./Tec. Resources Series Number 2.

    ERIC Educational Resources Information Center

    Canadian Commission of Employment and Immigration, Ottawa (Ontario).

    Second in a resource series (see note), this annotated bibliography provides detailed information on training curriculum and instructional materials for welding, brazing, and flame-cutting. The materials are divided into thirty-fie sections by topic and type. Specific topic areas include gas and arc welding; arc welding; oxyacetylene welding and…

  10. 49 CFR 178.50 - Specification 4B welded or brazed steel cylinders.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... seams that are forged lap-welded or brazed and with water capacity (nominal) not over 1,000 pounds and a.... Content percent may not exceed the following: Carbon, 0.25; phosphorus, 0.045; sulphur, 0.050. (c...) Where: S = wall stress in psi; P = minimum test pressure prescribed for water jacket test or 450...

  11. 49 CFR 178.50 - Specification 4B welded or brazed steel cylinders.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... seams that are forged lap-welded or brazed and with water capacity (nominal) not over 1,000 pounds and a.... Content percent may not exceed the following: Carbon, 0.25; phosphorus, 0.045; sulphur, 0.050. (c...) Where: S = wall stress in psi; P = minimum test pressure prescribed for water jacket test or 450...

  12. 49 CFR 178.50 - Specification 4B welded or brazed steel cylinders.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... seams that are forged lap-welded or brazed and with water capacity (nominal) not over 1,000 pounds and a.... Content percent may not exceed the following: Carbon, 0.25; phosphorus, 0.045; sulphur, 0.050. (c...) Where: S = wall stress in psi; P = minimum test pressure prescribed for water jacket test or 450...

  13. Micro-nano filler metal foil on vacuum brazing of SiCp/Al composites

    NASA Astrophysics Data System (ADS)

    Wang, Peng; Gao, Zeng; Niu, Jitai

    2016-06-01

    Using micro-nano (Al-5.25Si-26.7Cu)- xTi (wt%, x = 1.0, 1.5, 2.0, 2.5 and 3.0) foils as filler metal, the research obtained high-performance joints of aluminum matrix composites with high SiC particle content (60 vol%, SiCp/Al-MMCs). The effect of brazing process and Ti content on joint properties was investigated, respectively. The experimental results indicate that void free dense interface between SiC particle and metallic brazed seam with C-Al-Si-Ti product was readily obtained, and the joint shear strength enhanced with increasing brazing temperature from 560 to 580 °C or prolonging soaking time from 10 to 90 min. Sound joints with maximum shear strength of 112.5 MPa was achieved at 580 °C for soaking time of 90 min with (Al-5.25Si-26.7Cu)-2Ti filler, where Ti(AlSi)3 intermetallic is in situ strengthening phase dispersed in the joint and fracture occured in the filler metal layer. In this research, the beneficial effect of Ti addition into filler metal on improving wettability between SiC particle and metallic brazed seam was demonstrated, and capable welding parameters were broadened for SiCp/Al-MMCs with high SiC particle content.

  14. Active Brazing of C/C Composite to Copper by AgCuTi Filler Metal

    NASA Astrophysics Data System (ADS)

    Zhang, Kexiang; Xia, Lihong; Zhang, Fuqin; He, Lianlong

    2016-05-01

    Brazing between the carbon-fiber-reinforced carbon composite (C/C composite) and copper has gained increasing interest because of its important application in thermal management systems in nuclear fusion reactors and in the aerospace industry. In order to examine the "interfacial shape effect" on the mechanical properties of the joint, straight and conical interfacial configurations were designed and machined on the surface of C/C composites before joining to copper using an Ag-68.8Cu-4.5Ti (wt pct) alloy. The microstructure and interfacial microchemistry of C/C composite/AgCuTi/Cu brazed joints were comprehensively investigated by using high-resolution transmission electron microscopy. The results indicate that the joint region of both straight and conical joints can be described as a bilayer. Reaction products of Cu3Ti3O and γ-TiO were formed near the copper side in a conical interface joint, while no reaction products were found in the straight case. The effect of Ag on the interfacial reaction was discussed, and the formation mechanism of the joints during brazing was proposed. On the basis of the detailed microstructure presented, the mechanical performance of the brazed joints was discussed in terms of reaction and morphology across the joint.

  15. Braze Development of Graphite Fiber for Use in Phase Change Material Heat Sinks

    NASA Technical Reports Server (NTRS)

    Quinn, Gregory; Beringer, Woody; Gleason, Brian; Stephan, Ryan

    2011-01-01

    Hamilton Sundstrand (HS), together with NASA Johnson Space Center, developed methods to metallurgically join graphite fiber to aluminum. The goal of the effort was to demonstrate improved thermal conductance, tensile strength and manufacturability compared to existing epoxy bonded techniques. These improvements have the potential to increase the performance and robustness of phase change material heat sinks that use graphite fibers as an interstitial material. Initial work focused on evaluating joining techniques from four suppliers, each consisting of a metallization step followed by brazing or soldering of one inch square blocks of Fibercore graphite fiber material to aluminum end sheets. Results matched the strength and thermal conductance of the epoxy bonded control samples, so two suppliers were down-selected for a second round of braze development. The second round of braze samples had up to a 300% increase in strength and up to a 132% increase in thermal conductance over the bonded samples. However, scalability and repeatability proved to be significant hurdles with the metallization approach. An alternative approach was pursued which used a nickel braze allow to prepare the carbon fibers for joining with aluminum. Initial results on sample blocks indicate that this approach should be repeatable and scalable with good strength and thermal conductance when compared with epoxy bonding.

  16. AtRAB-H1b and AtRAB-H1c GTPases, homologues of the yeast Ypt6, target reporter proteins to the Golgi when expressed in Nicotiana tabacum and Arabidopsis thaliana.

    PubMed

    Johansen, Jorunn Nergaard; Chow, Cheung-Ming; Moore, Ian; Hawes, Chris

    2009-01-01

    Ypt/Rab GTPases act as key regulators of intracellular traffic through the conformational differences exhibited by their GTP or GDP-bound forms. In this paper, two Arabidopsis Ypt6 homologues, AtRAB-H1(b) and AtRAB-H1(c) were characterized and compared. Using a live cell imaging approach, it is shown that yellow fluorescent protein-fusions (YFP) of AtRAB-H1(b) and AtRAB-H1(c) locate to the Golgi and to the cytosol in both Nicotiana tabacum and in Arabidopsis thaliana. In addition, YFP-AtRAB-H1(b) targets an as yet unknown compartment not labelled by YFP-AtRAB-H1(c) or Golgi markers. It is also shown that the subcellular location of YFP-AtRAB-H1(b) and YFP-AtRAB-H1(c) is affected by the state of GTP-binding and that expression of a GTP-deficient mutant results in increased apoplastic fluorescence of a secretory form of YFP.

  17. Rab6a/a’ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages

    PubMed Central

    Micaroni, Massimo; Stanley, Amanda C.; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X. F.; Lim, Jet P.; Marsh, Brad J.; Storrie, Brian; Gleeson, Paul A.; Stow, Jennifer L.

    2013-01-01

    Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6–GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages. PMID:23437303

  18. Extensive in silico analysis of Mimivirus coded Rab GTPase homolog suggests a possible role in virion membrane biogenesis

    PubMed Central

    Zade, Amrutraj; Sengupta, Malavi; Kondabagil, Kiran

    2015-01-01

    Rab GTPases are the key regulators of intracellular membrane trafficking in eukaryotes. Many viruses and intracellular bacterial pathogens have evolved to hijack the host Rab GTPase functions, mainly through activators and effector proteins, for their benefit. Acanthamoeba polyphaga mimivirus (APMV) is one of the largest viruses and belongs to the monophyletic clade of nucleo-cytoplasmic large DNA viruses (NCLDV). The inner membrane lining is integral to the APMV virion structure. APMV assembly involves extensive host membrane modifications, like vesicle budding and fusion, leading to the formation of a membrane sheet that is incorporated into the virion. Intriguingly, APMV and all group I members of the Mimiviridae family code for a putative Rab GTPase protein. APMV is the first reported virus to code for a Rab GTPase (encoded by R214 gene). Our thorough in silico analysis of the subfamily specific (SF) region of Mimiviridae Rab GTPase sequences suggests that they are related to Rab5, a member of the group II Rab GTPases, of lower eukaryotes. Because of their high divergence from the existing three isoforms, A, B, and C of the Rab5-family, we suggest that Mimiviridae Rabs constitute a new isoform, Rab5D. Phylogenetic analysis indicated probable horizontal acquisition from a lower eukaryotic ancestor followed by selection and divergence. Furthermore, interaction network analysis suggests that vps34 (a Class III PI3K homolog, coded by APMV L615), Atg-8 and dynamin (host proteins) are recruited by APMV Rab GTPase during capsid assembly. Based on these observations, we hypothesize that APMV Rab plays a role in the acquisition of inner membrane during virion assembly. PMID:26441866

  19. Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction.

    PubMed

    Chen, Shirui; Gendelman, Hannah K; Roche, John P; Alsharif, Peter; Graf, Ethan R

    2015-01-01

    At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.

  20. Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction

    PubMed Central

    Roche, John P.; Alsharif, Peter; Graf, Ethan R.

    2015-01-01

    At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function. PMID:26317909

  1. α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner.

    PubMed

    Yin, Guowei; Lopes da Fonseca, Tomas; Eisbach, Sibylle E; Anduaga, Ane Martín; Breda, Carlo; Orcellet, Maria L; Szegő, Éva M; Guerreiro, Patricia; Lázaro, Diana F; Braus, Gerhard H; Fernandez, Claudio O; Griesinger, Christian; Becker, Stefan; Goody, Roger S; Itzen, Aymelt; Giorgini, Flaviano; Outeiro, Tiago F; Zweckstetter, Markus

    2014-10-01

    Alpha-synuclein (αS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS-Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.

  2. DENND2B activates Rab13 at the leading edge of migrating cells and promotes metastatic behavior

    PubMed Central

    Ioannou, Maria S.; Bell, Emily S.; Girard, Martine; Chaineau, Mathilde; Hamlin, Jason N.R.; Daubaras, Mark; Monast, Anie; Park, Morag; Hodgson, Louis

    2015-01-01

    The small guanosine triphosphatase Rab13 functions in exocytic vesicle trafficking in epithelial cells. Alterations in Rab13 activity have been observed in human cancers, yet the mechanism of Rab13 activation and its role in cancer progression remain unclear. In this paper, we identify the DENN domain protein DENND2B as the guanine nucleotide exchange factor for Rab13 and develop a novel Förster resonance energy transfer–based Rab biosensor to reveal activation of Rab13 by DENND2B at the leading edge of migrating cells. DENND2B interacts with the Rab13 effector MICAL-L2 at the cell periphery, and this interaction is required for the dynamic remodeling of the cell’s leading edge. Disruption of Rab13-mediated trafficking dramatically limits the invasive behavior of epithelial cells in vitro and the growth and migration of highly invasive cancer cells in vivo. Thus, blocking Rab13 activation by DENND2B may provide a novel target to limit the spread of epithelial cancers. PMID:25713415

  3. Tctex-1, a Novel Interaction Partner of Rab3D, Is Required for Osteoclastic Bone Resorption ▿

    PubMed Central

    Pavlos, Nathan J.; Cheng, Tak Sum; Qin, An; Ng, Pei Ying; Feng, Hao-Tian; Ang, Estabelle S. M.; Carrello, Amerigo; Sung, Ching-Hwa; Jahn, Reinhard; Zheng, Ming-Hao; Xu, Jiake

    2011-01-01

    Vesicular transport along microtubules must be strictly regulated to sustain the unique structural and functional polarization of bone-resorbing osteoclasts. However, the molecular mechanisms bridging these vesicle-microtubule interactions remain largely obscure. Rab3D, a member of the Rab3 subfamily (Rab3A/B/C/D) of small exocytotic GTPases, represents a core component of the osteoclastic vesicle transport machinery. Here, we identify a new Rab3D-interacting partner, Tctex-1, a light chain of the cytoplasmic dynein microtubule motor complex, by a yeast two-hybrid screen. We demonstrate that Tctex-1 binds specifically to Rab3D in a GTP-dependent manner and co-occupies Rab3D-bearing vesicles in bone-resorbing osteoclasts. Furthermore, we provide evidence that Tctex-1 and Rab3D intimately associate with the dynein motor complex and microtubules in osteoclasts. Finally, targeted disruption of Tctex-1 by RNA interference significantly impairs bone resorption capacity and mislocalizes Rab3D vesicles in osteoclasts, attesting to the notion that components of the Rab3D-trafficking pathway contribute to the maintenance of osteoclastic resorptive function. PMID:21262767

  4. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

    PubMed

    Lai, Yu-Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B; Dill, Brian D; Woodroof, Helen I; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J; Corti, Olga; Corvol, Jean-Christophe; Campbell, David G; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul Mk

    2015-11-12

    Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism

  5. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

    PubMed

    Lai, Yu-Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B; Dill, Brian D; Woodroof, Helen I; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J; Corti, Olga; Corvol, Jean-Christophe; Campbell, David G; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul Mk

    2015-11-12

    Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism

  6. Structure-function analyses of the small GTPase Rab35 and its effector protein centaurin-β2/ACAP2 during neurite outgrowth of PC12 cells.

    PubMed

    Etoh, Kan; Fukuda, Mitsunori

    2015-04-01

    The small GTPase Rab35 is a molecular switch for membrane trafficking that regulates a variety of cellular events. We previously showed that Rab35 promotes neurite outgrowth of nerve growth factor-stimulated PC12 cells through interaction with centaurin-β2 (also called ACAP2). Centaurin-β2 is the only Rab35-binding protein reported thus far that exclusively recognizes Rab35 and does not recognize any of the other 59 Rabs identified in mammals, but the molecular basis for the exclusive specificity of centaurin-β2 for Rab35 has remained completely unknown. In this study, we performed deletion and mutation analyses and succeeded in identifying the residues of Rab35 and centaurin-β2 that are crucial for formation of a Rab35·centaurin-β2 complex. We found that two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are responsible for binding centaurin-β2 and that the same residues are dispensable for Rab35 recognition by other Rab35-binding proteins. We also determined the minimal Rab35-binding site of centaurin-β2 and identified two asparagine residues (Asn-610 and Asn-691) in the Rab35-binding site as key residues for its specific Rab35 recognition. We further showed by knockdown-rescue approaches that neither a centaurin-β2 binding-deficient Rab35(T76S/T81A) mutant nor a Rab35 binding-deficient centaurin-β2(N610A/N691A) mutant supported neurite outgrowth of PC12 cells, thereby demonstrating the functional significance of the Rab35/centaurin-β2 interaction during neurite outgrowth of PC12 cells.

  7. Roles of plant homologs of Rab1p and Rab7p in the biogenesis of the peribacteroid membrane, a subcellular compartment formed de novo during root nodule symbiosis.

    PubMed

    Cheon, C I; Lee, N G; Siddique, A B; Bal, A K; Verma, D P

    1993-11-01

    The peribacteroid membrane (PBM) in legume root nodules is derived from plasma membrane following endocytosis of Rhizobium by fusion of newly synthesized vesicles. We studied the roles of plant Rab1p and Rab7p homologs, the small GTP-binding proteins involved in vesicular transport, in the biogenesis of the PBM. Three cDNAs encoding legume homologs of mammalian Rab1p and Rab7p were isolated from soybean (sRab1p, sRab7p) and Vigna aconitifolia (vRab7p). sRab1p was confirmed to be a functional counterpart of yeast Ypt1p (Rab1p) by complementation of a yeast ypt1-1 mutant. Both srab1 and vrab7 genes are induced during nodulation with the level of vrab7 mRNA being 12 times higher than that in root meristem and leaves. This induction directly correlates with membrane proliferation in nodules. Antisense constructs of srab1 and vrab7, under a nodule-specific promoter (leghemoglobin, Lbc3), were made in a binary vector and transgenic nodules were developed on soybean hairy roots obtained through Agrobacterium rhizogenes-mediated transformation. Both antisense srab1 and vrab7 nodules were smaller in size and showed lower nitrogenase activity than controls. The antisense srab1 nodules showed lack of expansion of infected cells, fewer bacteroids per cell and their frequent release into vacuoles. In contrast, antisense vrab7 expressing nodules showed accumulation of late endosomal structure and multivesicular bodies in the perinuclear region. These data suggest that both Rab1p and Rab7p are essential for the development of the PBM compartment in effective symbiosis.

  8. A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition.

    PubMed

    Agola, Jacob O; Hong, Lin; Surviladze, Zurab; Ursu, Oleg; Waller, Anna; Strouse, J Jacob; Simpson, Denise S; Schroeder, Chad E; Oprea, Tudor I; Golden, Jennifer E; Aubé, Jeffrey; Buranda, Tione; Sklar, Larry A; Wandinger-Ness, Angela

    2012-06-15

    Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.

  9. Nanoparticle-Assisted Diffusion Brazing of Metal Microchannel Arrays: Nanoparticle Synthesis, Deposition, and Characterization

    NASA Astrophysics Data System (ADS)

    Eluri, Ravindranadh T.

    Microchannel process technology (MPT) offers several advantages to the field of nanomanufacturing: 1) improved process control over very short time intervals owing to shorter diffusional distances; and 2) reduced reactor size due to high surface area to volume ratios and enhanced heat and mass transfer. The objective of this thesis was to consider how nanomaterials, produced in part using MPT, could be used to solve problems associated with the fabrication of MPT devices. Specifically, many MPT devices are produced using transient liquid-phase brazing involving an electroplated interlayer consisting of a brazing alloy designed for melting temperature suppression. Unfortunately, these alloys can form brittle secondary phases which significantly reduce bond strength. In contrast, prior efforts have shown that it is possible to leverage the size-dependent properties of nanomaterials to suppress brazing temperatures. In this prior work, thin films of off-the-shelf elemental nanoparticles were used as interlayers yielding joints with improved mechanical properties. In the present investigation, efforts have been made to characterize the synthesis and deposition of various elemental nanoparticle suspensions for use in the transient liquid-phase brazing of aluminum and stainless steel. Advances were used to demonstrate the nanoparticle-assisted diffusion brazing of a microchannel array. In the first section, a silver nanoparticle (AgNP) interlayer was produced for the diffusion brazing of heat exchanger aluminum. Efforts are made to examine the effect of braze filler particle size (˜5 nm and ˜50 nm) and processing parameters (heating rate: 5ºC/min and 25ºC/min; brazing temperature: 550ºC and 570ºC) on thin coupons of diffusion-brazed 3003 Al. A tensile strength of 69.7 MPa was achieved for a sample brazed at 570°C for 30 min under 1 MPa with an interlayer thickness of approximately 7 microm. Further suppression of the brazing temperature to 500ºC was achieved by

  10. Fatigue FEM analysis in the case of brazed aluminium alloy 3L59 used in aeronautical industry

    NASA Astrophysics Data System (ADS)

    Dimitrescu, A.; Amza, Gh; Niţoi, D. F.; Amza, C. Gh; Apostolescu, Z.

    2016-08-01

    The use, on a larger scale, of brazed aluminum alloys in the aerospace industry led to the need for a detailed study of the assemblies behavior. These are built from 6061 aluminum aloy (3L59) brazed with aluminum aloy A103. Therefore, a finit element simulation (FEM) of durability is necessary, that consists in the observation of gradual deterioration until failure. These studies are required and are previous to the stage of the producing the assembly and test it by traditional methods.

  11. Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation, Guiding the Specific Vesicles to the Cyst Wall

    PubMed Central

    Castillo-Romero, Araceli; Leon-Avila, Gloria; Wang, Ching C.; Perez Rangel, Armando; Camacho Nuez, Minerva; Garcia Tovar, Carlos; Ayala-Sumuano, Jorge Tonatiuh; Luna-Arias, Juan Pedro; Hernandez, Jose Manuel

    2010-01-01

    Background Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. Methodology and Principal Findings In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. Conclusions and Significance Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and

  12. Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.

    PubMed

    Ito, Genta; Katsemonova, Kristina; Tonelli, Francesca; Lis, Pawel; Baptista, Marco A S; Shpiro, Natalia; Duddy, Graham; Wilson, Steve; Ho, Philip Wing-Lok; Ho, Shu-Leong; Reith, Alastair D; Alessi, Dario R

    2016-09-01

    Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1-2 min, markedly more rapidly than the Ser(935) and Ser(1292) biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser(910) and Ser(935) and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

  13. Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

    PubMed Central

    Ito, Genta; Katsemonova, Kristina; Tonelli, Francesca; Lis, Pawel; Baptista, Marco A.S.; Shpiro, Natalia; Duddy, Graham; Wilson, Steve; Ho, Philip Wing-Lok; Ho, Shu-Leong; Reith, Alastair D.; Alessi, Dario R.

    2016-01-01

    Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway. PMID:27474410

  14. Molecular Dynamics of Rab7::REP1::GGTase-II Ternary Complex and Identification of Their Putative Drug Binding Sites.

    PubMed

    Sindhu, Meenakshi; Saini, Vandana; Piplani, Sakshi; Kumar, A

    2013-01-01

    The structure-function correlation of membrane proteins have been a difficult task, particularly in context to transient protein complexes. The molecular simulation of ternary complex of Rab7::REP1::GGTase-II was carried out to understand the basic structural events occurring during the prenylation event of Rab proteins, using the software YASARA. The study suggested that the C-terminus of Rab7 has to be in completely extended conformation during prenylation to reach the active site of RabGGTase-II. Also, attempt was made to find putative drug binding sites on the ternary complex of Rab7::REP1::GGTase-II using Q-SiteFinder programme. The comprehensive consensus probe generated by the program revealed a total of 10 major pockets as putative drug binding sites on Rab7::REP:: GGTase-II ternary complex. These pockets were found on REP protein and GGTase protein subunits. The Rab7 was found to be devoid of any putative drug binding sites in the ternary complex. The phylogenetic analysis of 60 Rab proteins of human was carried out using PHYLIP and study indicated the close phylogenetic relationship between Rab7 and Rab9 proteins of human and hence with further in silico study, the present observations can be extrapolated to Rab9 proteins. The study paves a good platform for further experimental verifications of the findings and other in silico studies like identifying the potential drug targets by searching the putative drug binding sites, generating pharmacophoric pattern, searching or constructing suitable ligand and docking studies. PMID:23901157

  15. RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

    PubMed

    Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

    2016-01-15

    Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. PMID:26620560

  16. Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis.

    PubMed

    Cui, Yong; Zhao, Qiong; Gao, Caiji; Ding, Yu; Zeng, Yonglun; Ueda, Takashi; Nakano, Akihiko; Jiang, Liwen

    2014-05-13

    Rab GTPases serve as multifaceted organizers during vesicle trafficking. Rab7, a member of the Rab GTPase family, has been shown to perform various essential functions in endosome trafficking and in endosome-to-lysosome trafficking in mammalian systems. The Arabidopsis thaliana genome encodes eight putative Rab7 homologs; however, the detailed function and activation mechanism of Rab7 in plants remain unknown. Here, we demonstrate that Arabidopsis RABG3f, a member of the plant Rab7 small GTPase family, localizes to prevacuolar compartments (PVCs) and the tonoplast. The proper activation of Rab7 is essential for both PVC-to-vacuole trafficking and vacuole biogenesis. Expression of a dominant-negative Rab7 mutant (RABG3f(T22N)) induces the formation of enlarged PVCs and affects vacuole morphology in plant cells. We also identify Arabidopsis MON1 (MONENSIN SENSITIVITY1) and CCZ1 (CALCIUM CAFFEINE ZINC SENSITIVITY1) proteins as a dimeric complex that functions as the Rab7 guanine nucleotide exchange factor. The MON1-CCZ1 complex also serves as the Rab5 effector to mediate Rab5-to-Rab7 conversion on PVCs. Loss of functional MON1 causes the formation of enlarged Rab5-positive PVCs that are separated from Rab7-positive endosomes. Similar to the dominant-negative Rab7 mutant, the mon1 mutants show pleiotropic growth defects, fragmented vacuoles, and altered vacuolar trafficking. Thus, Rab7 activation by the MON1-CCZ1 complex is critical for vacuolar trafficking, vacuole biogenesis, and plant growth.

  17. RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

    PubMed

    Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

    2016-01-15

    Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes.

  18. Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

    PubMed Central

    Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

    2015-01-01

    ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex

  19. The effect of a homogenizing optic on residual stresses and shear strength of laser brazed ceramic/steel-joints

    NASA Astrophysics Data System (ADS)

    Südmeyer, I.; Rohde, M.; Besser, H.; Grein, M.; Liesching, B.; Schneider, J.

    2011-03-01

    Oxide and non oxide ceramics (Al2O3, SiC) were brazed to commercial steel with active filler alloys using a CO2-laser (l = 10.64 μm). Two different laser intensity profiles were used for heating up the compound: A laser output beam presenting a Gaussian profile and a homogenized, nearly top head profile were applied for joining the compounds in an Argon stream. The temperature distribution with and without the homogenizing optic was measured during the process and compared to the results of a finite element model simulating the brazing process with the different laser intensity profiles. Polished microsections were prepared for characterization of the different joints by scanning electron micrographs and EDXanalysis. In order to evaluate the effects of the different laser intensity profiles on the compound, the shear strengths of the braze-joints were determined. Additionally residual stresses which were caused by the gradient of thermal expansion between ceramic and metal were determined by finite element modeling. The microsections did not exhibit differences between the joints, which were brazed with different laser profiles. However the shear tests proved, that an explicit increase of compound strength up to 34 MPa of the ceramic/metal joints can be achieved with the top head profile, whereas the joints brazed with the Gaussian profile achieved only shear strength values of 24 MPa. Finally tribological pin-on-disc tests proved the capability of the laser brazed joints with regard to the application conditions.

  20. Effect of Processing Parameters on Thermal Cycling Behavior of Al2O3-Al2O3 Brazed Joints

    NASA Astrophysics Data System (ADS)

    Dandapat, Nandadulal; Ghosh, Sumana; Guha, Bichitra Kumar; Datta, Someswar; Balla, Vamsi Krishna

    2016-06-01

    In the present study, alumina ceramics were active metal brazed at different temperatures ranging from 1163 K to 1183 K (890 °C to 910 °C) using TICUSIL (68.8Ag-26.7Cu-4.5Ti in wt pct) foil as filler alloy of different thicknesses. The brazed joints were subjected to thermal cycling for 100 cycles between 323 K and 873 K (50 °C and 600 °C). The microstructural and elemental composition analysis of the brazed joints were performed by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) before and after thermal cycling. Helium (He) leak test and brazing strength measurement were also conducted after thermal cycling for 100 cycles. The joint could withstand up to 1 × 10-9 Torr pressure and brazing strength was higher than 20 MPa. The experimental results demonstrated that joints brazed at the higher temperature with thinner filler alloy produced strong Al2O3-Al2O3 joints.