ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles.

PubMed

Shibata, Satoshi; Kawanai, Tsubasa; Hara, Takayuki; Yamamoto, Asuka; Chaya, Taro; Tokuhara, Yasunori; Tsuji, Chinami; Sakai, Manabu; Tachibana, Taro; Inagaki, Shinobu

2016-10-01

The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner. © 2016. Published by The Company of Biologists Ltd.

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.

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)

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.

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)

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.

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)

Characterisation of Ga-coated and Ga-brazed aluminium

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

Ferchaud, E.; Christien, F., E-mail: frederic.christien@univ-nantes.fr; Barnier, V.

This work is devoted to the brazing of aluminium using liquid gallium. Gallium was deposited on aluminium samples at {approx} 50 Degree-Sign C using a liquid gallium 'polishing' technique. Brazing was undertaken for 30 min at 500 Degree-Sign C in air. EDS (Energy Dispersive X-ray Spectroscopy) and AES (Auger Electron Spectroscopy) characterisation of Ga-coated samples has shown that the Ga surface layer thickness is of ten (or a few tens of) nanometres. Furthermore, aluminium oxide layer (Al{sub 2}O{sub 3}) was shown to be 'descaled' during Ga deposition, which ensures good conditions for further brazing. Cross-section examination of Ga-coated samples showsmore » that liquid gallium penetrates into the aluminium grain boundaries during deposition. The thickness of the grain boundary gallium film was measured using an original EDS technique and is found to be of a few tens of nanometres. The depth of gallium grain boundary penetration is about 300 {mu}m at the deposition temperature. The fracture stress of the brazed joints was measured from tensile tests and was determined to be 33 MPa. Cross-section examination of brazed joints shows that gallium has fully dissolved into the bulk and that the joint is really autogenous. - Highlights: Black-Right-Pointing-Pointer Aluminium can be brazed using liquid gallium deposited by a 'polishing' technique. Black-Right-Pointing-Pointer The aluminium oxide layer is 'descaled' during liquid Ga 'polishing' deposition. Black-Right-Pointing-Pointer EDS can be used for determination of surface and grain boundary Ga film thickness. Black-Right-Pointing-Pointer The surface and grain boundary Ga film thickness is of a few tens of nm. Black-Right-Pointing-Pointer Surface and grain boundary gallium dissolves in the bulk during brazing.« less

Weld braze technique

DOEpatents

Kanne, Jr., William R.; Kelker, Jr., John W.; Alexander, Robert J.

1982-01-01

High-strength metal joints are formed by a combined weld-braze technique. A hollow cylindrical metal member is forced into an undersized counterbore in another metal member with a suitable braze metal disposed along the bottom of the counterbore. Force and current applied to the members in an evacuated chamber results in the concurrent formation of the weld along the sides of the counterbore and a braze along the bottom of the counterbore in one continuous operation.

The identification of protein domains that mediate functional interactions between Rab-GTPases and RabGAPs using 3D protein modeling.

PubMed

Davie, Jeremiah J; Faitar, Silviu L

2017-01-01

Currently, time-consuming serial in vitro experimentation involving immunocytochemistry or radiolabeled materials is required to identify which of the numerous Rab-GTPases (Rab) and Rab-GTPase activating proteins (RabGAP) are capable of functional interactions. These interactions are essential for numerous cellular functions, and in silico methods of reducing in vitro trial and error would accelerate the pace of research in cell biology. We have utilized a combination of three-dimensional protein modeling and protein bioinformatics to identify domains present in Rab proteins that are predictive of their functional interaction with a specific RabGAP. The RabF2 and RabSF1 domains appear to play functional roles in mediating the interaction between Rabs and RabGAPs. Moreover, the RabSF1 domain can be used to make in silico predictions of functional Rab/RabGAP pairs. This method is expected to be a broadly applicable tool for predicting protein-protein interactions where existing crystal structures for homologs of the proteins of interest are available.

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.

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.

How Do Rab Proteins Determine Golgi Structure?

PubMed Central

Liu, Shijie; Storrie, Brian

2015-01-01

Rab proteins, small GTPases, are key regulators of mammalian Golgi apparatus organization. Based on the effect of Rab activation state, Rab proteins fall into two functional classes. In Class1, inactivation induces Golgi ribbon fragmentation and/or redistribution of Golgi enzymes to the ER, while overexpression of wild type or activation has little, if any, effect on Golgi ribbon organization. In Class 2, the reverse is true. We give emphasis to Rab6, the most abundant Golgi-associated Rab protein. Rab6 depletion in HeLa cells causes an increase in Golgi cisternal number, longer, more continuous cisternae, and a pronounced accumulation of vesicles; the effect of Rab6 on Golgi ribbon organization is probably through regulation of vesicle transport. In effector studies, motor proteins and their regulators are found to be key Rab6 effectors. A related Rab, Rab41, affects Golgi ribbon organization in a contrasting manner. The balance between minus- and plus-end directed motor recruitment may well be the major Rab-dependent factor in Golgi ribbon organization. PMID:25708460

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

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

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.

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.

32 CFR 202.10 - RAB adjournment and dissolution.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 32 National Defense 2 2013-07-01 2013-07-01 false RAB adjournment and dissolution. 202.10 Section... dissolution. (a) RAB adjournment—(1) Requirements for RAB adjournment. An Installation Commander may adjourn a... Commander decides to adjourn the RAB. (b) RAB dissolution—(1) Requirements for RAB dissolution. An...

32 CFR 202.10 - RAB adjournment and dissolution.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 2 2011-07-01 2011-07-01 false RAB adjournment and dissolution. 202.10 Section... dissolution. (a) RAB adjournment—(1) Requirements for RAB adjournment. An Installation Commander may adjourn a... Commander decides to adjourn the RAB. (b) RAB dissolution—(1) Requirements for RAB dissolution. An...

32 CFR 202.10 - RAB adjournment and dissolution.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 2 2012-07-01 2012-07-01 false RAB adjournment and dissolution. 202.10 Section... dissolution. (a) RAB adjournment—(1) Requirements for RAB adjournment. An Installation Commander may adjourn a... Commander decides to adjourn the RAB. (b) RAB dissolution—(1) Requirements for RAB dissolution. An...

32 CFR 202.10 - RAB adjournment and dissolution.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 2 2014-07-01 2014-07-01 false RAB adjournment and dissolution. 202.10 Section... dissolution. (a) RAB adjournment—(1) Requirements for RAB adjournment. An Installation Commander may adjourn a... Commander decides to adjourn the RAB. (b) RAB dissolution—(1) Requirements for RAB dissolution. An...

Development of the weld-braze joining process

NASA Technical Reports Server (NTRS)

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

1973-01-01

A joining process, designated weld-brazing, was developed which combines resistance spot welding and brazing. Resistance spot welding is used to position and aline the parts, as well as to establish a suitable faying-surface gap for brazing. Fabrication is then completed at elevated temperature by capillary flow of the braze alloy into the joint. The process was used successfully to fabricate Ti-6Al-4V alloy joints by using 3003 aluminum braze alloy and should be applicable to other metal-braze systems. Test results obtained on single-overlap and hat-stiffened panel specimens show that weld-brazed joints were superior in tensile shear, stress rupture, fatigue, and buckling compared with joints fabricated by conventional means. Another attractive feature of the process is that the brazed joint is hermetically sealed by the braze material, which may eliminate many of the sealing problems encountered with riveted or spot welded structures. The relative ease of fabrication associated with the weld-brazing process may make it cost effective over conventional joining techniques.

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

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.

Braze Process Optimization Involving Conventional Metal/Ceramic Brazing with 50Au-50Cu Alloy

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

MALIZIA JR.,LOUIS A.; MEREDITH,KEITH W.; APPEL,DANIEL B.

1999-12-15

Numerous process variables can influence the robustness of conventional metal/ceramic brazing processes. Experience with brazing of hermetic vacuum components has identified the following parameters as influencing the outcome of hydrogen furnace brazed Kovar{trademark} to metallized alumina braze joints: (a) Mo-Mn metallization thickness, sinter fire temperature and porosity (b) Nil plate purity, thickness, and sinter firing conditions (c) peak process temperature, time above liquidus and (d) braze alloy washer thickness. ASTM F19 tensile buttons are being used to investigate the above parameters. The F19 geometry permits determination of both joint hermeticity and tensile strength. This presentation will focus on important lessonsmore » learned from the tensile button study: (A) the position of the Kovar{trademark} interlayer can influence the joint tensile strength achieved--namely, off-center interlayers can lead to residual stress development in the ceramic and degrade tensile strength values. Finite element analysis has been used to demonstrate the expected magnitude in strength degradation as a function of misalignment. (B) Time above liquidus (TAL) and peak temperature can influence the strength and alloying level of the resulting braze joint. Excessive TAL or peak temperatures can lead to overbraze conditions where all of the Ni plate is dissolved. (C) Metallize sinter fire processes can influence the morphology and strength obtained from the braze joints.« less

Rab11-family of interacting protein 2 associates with chlamydial inclusions through its Rab-binding domain and promotes bacterial multiplication.

PubMed

Leiva, Natalia; Capmany, Anahí; Damiani, María Teresa

2013-01-01

Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named 'inclusion' and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11-Family of Interacting Proteins, presents at the C-terminus a Rab-binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP-tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab-binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab-binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion. © 2012 Blackwell Publishing Ltd.

Myosin vc interacts with Rab32 and Rab38 proteins and works in the biogenesis and secretion of melanosomes.

PubMed

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

2014-11-28

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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae.

PubMed

Chen, Ming-Chyuan; Cheng, Ying-Min; Sung, Ping-Jyun; Kuo, Cham-En; Fang, Lee-Shing

2003-08-29

The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. In light of the critical roles of the small GTPase, Rab7, as a key regulator of vesicular trafficking, we cloned and characterized the Rab7 protein in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. The Aiptasia homologue of Rab7 proteins, ApRab7 is 88% identical to human Rab7 protein and contains all Rab-specific signature motifs. Results of EGFP reporter analysis, protein fractionation, and immunocytochemistry support that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Significantly, the majority of phagosomes containing live symbionts that either have taken long residency in, or were newly internalized by Aiptasia digestive cells did not contain detectable levels of ApRab7, while most phagosomes containing either heat-killed or photosynthesis-impaired symbionts were positive for ApRab7 staining. Overall, our data suggest that live algal symbionts persist inside their host cells by actively excluding ApRab7 from their phagosomes, and thereby, establish and/or maintain an endosymbiotic relationship with their cnidarian hosts.

Investigation on the influence of nitrogen in process atmospheres on the corrosion behavior of brazed stainless steel joints

NASA Astrophysics Data System (ADS)

Fedorov, V.; Uhlig, T.; Wagner, G.; Langohr, A.; Holländer, U.

2018-06-01

Brazing of stainless steels is commonly carried out using nickel-based brazing fillers, which provide a high corrosion and oxidation resistance of the resulting joints. These brazed stainless steel joints are mostly used for manufacturing of heat exchangers for energy and air conditioning technologies. The joints of the study were produced at temperatures of 1000 °C, 1125 °C and 1150 °C in vacuum furnaces or continuous furnaces. In both cases, the parts interact with process gases like nitrogen within the brazing process, especially during cooling. The amount of nitrogen in the braze metal as well as in the base material was determined by the carrier gas hot extraction technique. The occurring diffusion of nitrogen into the braze metal and the base material causes a shift in the corrosion potentials. In this work, the influence of the nitrogen enrichment on the corrosion behavior was investigated using a capillary microcell. The corrosion measurements were carried out on the braze metal and the base material. The results of samples, brazed with and without the influence of nitrogen, were compared.

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.

Rab3a-Bound CD63 Is Degraded and Rab3a-Free CD63 Is Incorporated into HIV-1 Particles

PubMed Central

Kubo, Yoshinao; Masumoto, Hiroshi; Izumida, Mai; Kakoki, Katsura; Hayashi, Hideki; Matsuyama, Toshifumi

2017-01-01

CD63, a member of the tetraspanin family, is involved in virion production by human immunodeficiency virus type 1 (HIV-1), but its mechanism is unknown. In this study, we showed that a small GTP-binding protein, Rab3a, interacts with CD63. When Rab3a was exogenously expressed, the amounts of CD63 decreased in cells. The Rab3a-mediated reduction of CD63 was suppressed by lysosomal and proteasomal inhibitors. The amount of CD63 was increased by reducing the endogenous Rab3a level using a specific shRNA. These results indicate that Rab3a binds to CD63 to induce the degradation of CD63. Rab3a is thought to be involved in exocytosis, but we found that another function of Rab3a affects the fate of CD63 in lysosomes. CD63 interacted with Rab3a and was incorporated into HIV-1 particles. However, Rab3a was not detected in HIV-1 virions, thereby indicating that Rab3a-free CD63, but not Rab3a-bound CD63, is incorporated into HIV-1 particles. Overexpression or silencing of Rab3a moderately reduced HIV-1 virion formation. Overexpression of Rab3a decreased CD63 levels, but did not affect the incorporation of CD63 into HIV-1 particles. This study showed that Rab3a binds to CD63 to induce the degradation of CD63, and only Rab3a-free CD63 is incorporated into HIV-1 particles. PMID:28900422

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)

Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells.

PubMed

Liu, Chun-Chun; Zhang, Yun-Na; Li, Zhao-Yao; Hou, Jin-Xiu; Zhou, Jing; Kan, Lin; Zhou, Bin; Chen, Pu-Yan

2017-10-01

During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses. IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that

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.

Rab-NANOPS: FRET biosensors for Rab membrane nanoclustering and prenylation detection in mammalian cells.

PubMed

Najumudeen, Arafath Kaja; Guzmán, Camilo; Posada, Itziar M D; Abankwa, Daniel

2015-01-01

Rab proteins constitute the largest subfamily of Ras-like small GTPases. They are central to vesicular transport and organelle definition in eukaryotic cells. Unlike their Ras counterparts, they are not a hallmark of cancer. However, a number of diseases, including cancer, show a misregulation of Rab protein activity. As for all membrane-anchored signaling proteins, correct membrane organization is critical for Rabs to operate. In this chapter, we provide a detailed protocol for the use of a flow cytometry-based Fluorescence Resonance Energy Transfer (FRET)-biosensors assay, which allows to detect changes in membrane anchorage, subcellular distribution, and of the nanoscale organization of Rab-GTPases in mammalian cell lines. This assay is high-throughput amenable and can therefore be utilized in chemical-genomic and drug discovery efforts.

Brazing Inconel 625 Using the Copper Foil

NASA Astrophysics Data System (ADS)

Chen, Wen-Shiang; Wang, Cheng-Yen; Shiue, Ren-Kae

2013-12-01

Brazing Inconel 625 (IN-625) using the copper foil has been investigated in this research. The brazed joint is composed of nanosized CrNi3 precipitates and Cr/Mo/Nb/Ni quaternary compound in the Cu/Ni-rich matrix. The copper filler 50 μm in thickness is enough for the joint filling. However, the application of Cu foil 100 μm in thickness has little effect on the shear strength of the brazed joint. The specimen brazed at 1433 K (1160 °C) for 1800 seconds demonstrates the best shear strength of 470 MPa, and its fractograph is dominated by ductile dimple fracture with sliding marks. Decreasing the brazing temperature slightly decreases the shear strength of the brazed joint due to the presence of a few isolated solidification shrinkage voids smaller than 15 μm. Increasing the brazing temperature, especially for the specimen brazed at 1473 K (1200 °C), significantly deteriorates the shear strength of the joint below 260 MPa because of coalescence of isothermal solidification shrinkage voids in the joint. The Cu foil demonstrates potential in brazing IN-625 for industrial application.

Chem-Braze Abradable Seal Attachment

DTIC Science & Technology

1980-05-01

bonding system for attaching sintered abradable seals such as FELTMETAL® to titanium -, steel- and nickel-base compressor blade tip-shrouds has been... blade tip-shrouds was developed. The improved Chem-Braze system incorporates glycerin as an inhibitor to prevent premature evaporation which prolongs...compressor blade tip-shrouds using the improved Chem-Braze system compared to attachment with gold-nickel braze. p. p. FORM . . yn

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.

Influence of brazing conditions on the strength of brazed joints of alumina dispersion-strengthened copper to 316 stainless steel

NASA Astrophysics Data System (ADS)

Nishi, H.; Kikuchi, K.

1998-10-01

Brazing of alumina dispersion-strengthened copper (DS Cu) to 316 stainless steel were conducted in order to investigate the influence of filler metals and brazing conditions on the joint strength. The brazing were performed with a silver-base (BAg-8) and three kinds of gold-base (BAu-2,4,11) filler metals with varying brazing joint clearance and brazing time. The filler metal had a greater effect on the joint strength than the brazing joint clearance and brazing time. The joint with BAu-2 was superior to the joint with other filler metals. The tensile strength of the joint with BAu-2 was as large as that of DS Cu, however, the Charpy and low cycle fatigue strength were lower than those of DS Cu. The DS Cu melted near the brazed zone, consequently recrystallization and agglomeration of alumina occurred in the diffusion layer for all filler metals. The grain size after the recrystallization was small in order of BAu-2, BAu-4 and BAu-11, that was in accordance with the order of the brazing temperature. The excellent fracture strength for the joint with BAu-2 was attributed to the smallest grain size.

46 CFR 56.30-30 - Brazed joints.

Code of Federal Regulations, 2010 CFR

2010-10-01

....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 or shall be provided in the form of a preinserted ring in a groove in the socket. The brazing alloy shall...

46 CFR 56.30-30 - Brazed joints.

Code of Federal Regulations, 2013 CFR

2013-10-01

....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 or shall be provided in the form of a preinserted ring in a groove in the socket. The brazing alloy shall...

46 CFR 56.30-30 - Brazed joints.

Code of Federal Regulations, 2011 CFR

2011-10-01

....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 or shall be provided in the form of a preinserted ring in a groove in the socket. The brazing alloy shall...

46 CFR 56.30-30 - Brazed joints.

Code of Federal Regulations, 2014 CFR

2014-10-01

....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 or shall be provided in the form of a preinserted ring in a groove in the socket. The brazing alloy shall...

46 CFR 56.30-30 - Brazed joints.

Code of Federal Regulations, 2012 CFR

2012-10-01

....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 or shall be provided in the form of a preinserted ring in a groove in the socket. The brazing alloy shall...

46 CFR 56.75-20 - Brazing qualification.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 2 2014-10-01 2014-10-01 false Brazing qualification. 56.75-20 Section 56.75-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING PIPING SYSTEMS AND APPURTENANCES Brazing § 56.75-20 Brazing qualification. (a) The qualification of the performance of brazers and brazing operators shall be in accordance...

46 CFR 56.75-20 - Brazing qualification.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 2 2012-10-01 2012-10-01 false Brazing qualification. 56.75-20 Section 56.75-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING PIPING SYSTEMS AND APPURTENANCES Brazing § 56.75-20 Brazing qualification. (a) The qualification of the performance of brazers and brazing operators shall be in accordance...

46 CFR 56.75-20 - Brazing qualification.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 2 2013-10-01 2013-10-01 false Brazing qualification. 56.75-20 Section 56.75-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING PIPING SYSTEMS AND APPURTENANCES Brazing § 56.75-20 Brazing qualification. (a) The qualification of the performance of brazers and brazing operators shall be in accordance...

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 APPURTENANCES Brazing § 56.75-20 Brazing qualification. (a) The qualification of the performance of brazers and brazing operators shall be in accordance...

46 CFR 56.75-20 - Brazing qualification.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 2 2011-10-01 2011-10-01 false Brazing qualification. 56.75-20 Section 56.75-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING PIPING SYSTEMS AND APPURTENANCES Brazing § 56.75-20 Brazing qualification. (a) The qualification of the performance of brazers and brazing operators shall be in accordance...

Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4 or Rab11 pathway.

PubMed

Wang, Feifei; Chen, Xiaoqing; Zhang, Xiaoqing; Ma, Lan

2008-08-01

Agonist-induced phosphorylation, internalization, and intracellular trafficking of G protein-coupled receptors are critical in regulating both cellular responsiveness and signal transduction. The current study investigated the role of receptor phosphorylation state in regulation of agonist-induced internalization and intracellular trafficking of mu-opioid receptor (MOR). Our results showed that after agonist stimulation, the recycle of a mutant MOR that lacks the C-terminal residues after Asn(362) (MOR362T) was greatly decreased, whereas a C-terminal phosphorylation sites-mutated MOR (MOR3A), which is deficient in agonist-induced phosphorylation recycled back to the membrane at a level comparable to that of the wild-type receptor, however, interestingly at a slower rate. Inhibition of functions of either Rab4 or Rab11 by dominant-negative mutants and small interfering RNA both significantly impaired the recycling of the wild-type MOR, whereas the recycling of the phosphorylation-deficient mutant was only inhibited by the dominant-negative mutant and small interfering RNA of Rab11, suggesting that the recycling of nonphosphorylated MOR is exclusively via Rab11-mediated pathway. Furthermore, phosphorylated MOR was observed accumulated in Rab5- and Rab4-, but not Rab11-positive vesicles. Our data indicate that both phosphorylated and nonphosphorylated MOR internalize via Rab5-dependent pathway after agonist stimulation, and the phosphorylated and nonphosphorylated MORs recycle through distinct vesicular trafficking pathways mediated by Rab4 and Rab11, respectively, which may ultimately lead to differential cellular responsiveness or downstream signaling.

Interorganellar Regulation of Lysosome Positioning by the Golgi Apparatus through Rab34 Interaction with Rab-interacting Lysosomal Protein

PubMed Central

Wang, Tuanlao; Hong, Wanjin

2002-01-01

We present evidence to suggest the existence of a regulatory pathway for the Golgi apparatus to modulate the spatial positioning of otherwise distantly located lysosomes. Rab34, a new member of the Rab GTPase family, is associated primarily with the Golgi apparatus. Expression of wild-type or GTP-restricted but not GDP-restricted versions of Rab34 causes spatial redistribution of lysosomes from the periphery to the peri-Golgi region. The regulation of lysosomal positioning by Rab34 depends on its association with the membrane mediated by prenylation and its direct interaction with Rab-interacting lysosomal protein (RILP). This biological activity, mediated by Rab34-RILP interaction, is dependent on Lys82 in the switch I region. Our results have uncovered a novel mechanism for the Golgi apparatus to regulate the spatial distribution of another organelle. PMID:12475955

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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

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 ofmore » 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

Automatic-Control System for Safer Brazing

NASA Technical Reports Server (NTRS)

Stein, J. A.; Vanasse, M. A.

1986-01-01

Automatic-control system for radio-frequency (RF) induction brazing of metal tubing reduces probability of operator errors, increases safety, and ensures high-quality brazed joints. Unit combines functions of gas control and electric-power control. Minimizes unnecessary flow of argon gas into work area and prevents electrical shocks from RF terminals. Controller will not allow power to flow from RF generator to brazing head unless work has been firmly attached to head and has actuated micro-switch. Potential shock hazard eliminated. Flow of argon for purging and cooling must be turned on and adjusted before brazing power applied. Provision ensures power not applied prematurely, causing damaged work or poor-quality joints. Controller automatically turns off argon flow at conclusion of brazing so potentially suffocating gas does not accumulate in confined areas.

Microstructure and Mechanical Properties of Stainless Steel/Brass Joints Brazed by Sn-Electroplated Ag Brazing Filler Metals

NASA Astrophysics Data System (ADS)

Wang, Xingxing; Peng, Jin; Cui, Datian

2018-05-01

To develop a high-Sn-content AgCuZnSn brazing filler metal, the BAg50CuZn was used as the base filler metal and a Sn layer was electroplated upon it. Then, the 304 stainless steel and the H62 brass were induction-brazed with the Sn-plated brazing filler metals. The microstructures of the joints were examined with an optical microscope, a scanning electron microscope and an x-ray diffractometer. The corresponding mechanical properties were obtained with a universal tensile testing machine. The results indicated that the induction brazed joints consisted of the Ag phase, the Cu phase and the CuZn phase. When the content of Sn in the Sn-plated Ag brazing filler metal was 6.0 or 7.2 wt.%, the Cu5Zn8, the Cu41Sn11 and the Ag3Sn phases appeared in the brazed joint. The tensile strength of the joints brazed with the Sn-plated filler metal was higher compared to the joints with the base filler metal. When the content of Sn was 6.0 wt.%, the highest tensile strength of the joint reached to 395 MPa. The joint fractures presented a brittle mode, mixed with a low amount of ductile fracture, when the content of Sn exceeded 6.0 wt.%.

Rab2 promotes autophagic and endocytic lysosomal degradation

PubMed Central

Boda, Attila; Glatz, Gábor; Zobel, Martina; Bisi, Sara; Hegedűs, Krisztina; Scita, Giorgio

2017-01-01

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster. We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes. PMID:28483915

Rab proteins in the brain and corpus allatum of Bombyx mori.

PubMed

Uno, Tomohide; Furutani, Masayuki; Watanabe, Chihiro; Sakamoto, Katsuhiko; Uno, Yuichi; Kanamaru, Kengo; Yamagata, Hiroshi; Mizoguchi, Akira; Takeda, Makio

2016-07-01

In eukaryotic cells, Rab guanosine triphosphate-ases serve as key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab3, Rab6, and Rab27 control the regulatory secretory pathway of neuropeptides and neurotransmitters. The cDNAs of Rab3, Rab6, and Rab27 from B. mori were inserted into a plasmid, transformed into Escherichia coli, and then subsequently purified. We then produced antibodies against Rab3, Rab6, and Rab27 of Bombyx mori in rabbits and rats for use in western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue revealed a single band at approximately 26 kDa. Immunohistochemistry results revealed that Rab3, Rab6, and Rab27 expression was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. Rab3 and Rab6 co-localized with bombyxin, an insect neuropeptide. However, there was no Rab that co-localized with prothoracicotropic hormone. The corpus allatum secretes neuropeptides synthesized in the brain into the hemolymph. Results showed that Rab3 and Rab6 co-localized with bombyxin in the corpus allatum. These findings suggest that Rab3 and Rab6 are involved in neurosecretion in B. mori. This study is the first to report a possible relationship between Rab and neurosecretion in the insect corpus allatum.

Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's Syndrome

PubMed Central

Meng, Zhen; Edman, Maria C.; Hsueh, Pang-Yu; Chen, Chiao-Yu; Klinngam, Wannita; Tolmachova, Tanya; Okamoto, Curtis T.

2016-01-01

The mechanism responsible for the altered spectrum of tear proteins secreted by lacrimal gland acinar cells (LGAC) in patients with Sjögren's Syndrome (SS) remains unknown. We have previously identified increased cathepsin S (CTSS) activity as a unique characteristic of tears of patients with SS. Here, we investigated the role of Rab3D, Rab27a, and Rab27b proteins in the enhanced release of CTSS from LGAC. Similar to patients with SS and to the male nonobese diabetic (NOD) mouse model of SS, CTSS activity was elevated in tears of mice lacking Rab3D. Findings of lower gene expression and altered localization of Rab3D in NOD LGAC reinforce a role for Rab3D in suppressing excess CTSS release under physiological conditions. However, CTSS activity was significantly reduced in tears of mice lacking Rab27 isoforms. The reliance of CTSS secretion on Rab27 activity was supported by in vitro findings that newly synthesized CTSS was detected in and secreted from Rab27-enriched secretory vesicles and that expression of dominant negative Rab27b reduced carbachol-stimulated secretion of CTSS in cultured LGAC. High-resolution 3D-structured illumination microscopy revealed microdomains of Rab3D and Rab27 isoforms on the same secretory vesicles but present in different proportions on different vesicles, suggesting that changes in their relative association with secretory vesicles may tailor the vesicle contents. We propose that a loss of Rab3D from secretory vesicles, leading to disproportionate Rab27-to-Rab3D activity, may contribute to the enhanced release of CTSS in tears of patients with SS. PMID:27076615

Reactive eutectic brazing of nitinol

NASA Astrophysics Data System (ADS)

Low, Ke-Bin

Although NiTiNb alloys are well known as wide-hysteresis shape-memory alloys with important applications as coupling materials, the significance of one aspect of the Ni-Ti-Nb ternary system has not been fully appreciated. Based on the existence of a quasibinary NiTi-Nb eutectic isopleth in this ternary system, a novel braze method has been devised to fabricate metallurgical bonds between functional nitinol (NiTi) sections. When NiTi and pure Nb are brought into contact at temperatures above 1170°C, spontaneous melting occurs, forming a liquid that is extremely reactive and not only wets NiTi surfaces, but also apparently dissolves oxide scales, obviating the need for fluxes and providing for efficient capillary flow into joint crevices. The melting process is diffusion-controlled and rate-limited by the diffusivity of Nb in the liquid. The braze liquid will subsequently solidify into microstructures containing predominantly ordered NiTi and disordered bcc-Nb. Mechanical tests revealed that the braze joints are strong, ductile, and biocompatible. With appropriate post-braze aging, the functional performance of the parent NiTi alloy can be restored. Micro-alloying the Nb fluer metal with Zr or tungsten showed great potential for solution-strengthening of the braze joints. For applications where biocompatibility is not an issue, Nb metal can be substituted by pure vanadium as the braze filler, which is demonstrated to possess tensile strengths that can be potentially superior to the Nb counterparts.

Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania.

PubMed

Rastogi, Ruchir; Verma, Jitender Kumar; Kapoor, Anjali; Langsley, Gordon; Mukhopadhyay, Amitabha

2016-07-08

Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania. © 2016 by The American Society

Thin-film diffusion brazing of titanium alloys

NASA Technical Reports Server (NTRS)

Mikus, E. B.

1972-01-01

A thin film diffusion brazing technique for joining titanium alloys by use of a Cu intermediate is described. The method has been characterized in terms of static and dynamic mechanical properties on Ti-6Al-4V alloy. These include tensile, fracture toughness, stress corrosion, shear, corrosion fatigue, mechanical fatigue and acoustic fatigue. Most of the properties of titanium joints formed by thin film diffusion brazing are equal or exceed base metal properties. The advantages of thin film diffusion brazing over solid state diffusion bonding and brazing with conventional braze alloys are discussed. The producibility advantages of this process over others provide the potential for producing high efficiency joints in structural components of titanium alloys for the minimum cost.

Thermal fatigue and oxidation data for alloy/braze combinations

NASA Technical Reports Server (NTRS)

Hill, V. L.; Humphreys, V. E.

1977-01-01

Thermal fatigue and oxidation data were obtained for 62 brazed specimens of 3 iron-, 3 nickel-, and 1 cobalt-base alloy. Fluidized bed thermal cycling was conducted over the range 740/25 C employing 10 cm long single-edge wedge specimens. Immersion time was always 4 minutes in each bed. Types of test specimens employed in the program include those with brazed overlays on the specimen radius, those butt brazed at midspan and those with a brazed foil overlay on the specimen radius. Of the 18 braze overlay specimens, 5 generated fatigue cracks by 7000 cycles. Thermal cracking of butt brazed specimens occurred exclusively through the butt braze. Of the 23 butt brazed specimens, 7 survived 11,000 thermal cycles without cracking. Only 2 of the 21 foil overlaid specimens exhibiting cracking in 7,000 cycles. Blistering of the foil did occur for 2 alloys by 500 cycles. Oxidation of the alloy/braze combination was limited at the test maximum test temperature of 740 C.

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.

New concept in brazing metallic honeycomb panels

NASA Technical Reports Server (NTRS)

Carter, P. D.; Layton, R. E.; Stratton, F. W.

1973-01-01

Aluminum oxide coating provides surface which will not be wetted by brazing alloy and which stops metallic diffusion welding of tooling materials to part being produced. This method eliminates loss of tooling materials and parts from braze wetting and allows fall-apart disassembly of tooling after brazing.

Rab1a regulates sorting of early endocytic vesicles

PubMed Central

Mukhopadhyay, Aparna; Quiroz, Jose A.

2014-01-01

We previously reported that Rab1a is associated with asialoorosomucoid (ASOR)-containing early endocytic vesicles, where it is required for their microtubule-based motility. In Rab1a knockdown (KD) cell lines, ASOR failed to segregate from its receptor and, consequently, did not reach lysosomes for degradation, indicating a defect in early endosome sorting. Although Rab1 is required for Golgi/endoplasmic reticulum trafficking, this process was unaffected, likely due to retained expression of Rab1b in these cells. The present study shows that Rab1a has a more general role in endocytic vesicle processing that extends to EGF and transferrin (Tfn) trafficking. Compared with results in control Huh7 cells, EGF accumulated in aggregates within Rab1a KD cells, failing to reach lysosomal compartments. Tfn, a prototypical example of recycling cargo, accumulated in a Rab11-mediated slow-recycling compartment in Rab1a KD cells, in contrast to control cells, which sort Tfn into a fast-recycling Rab4 compartment. These data indicate that Rab1a is an important regulator of early endosome sorting for multiple cargo species. The effectors and accessory proteins recruited by Rab1a to early endocytic vesicles include the minus-end-directed kinesin motor KifC1, while others remain to be discovered. PMID:24407591

Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6.

PubMed

Cayouette, Sylvie; Bousquet, Simon M; Francoeur, Nancy; Dupré, Emilie; Monet, Michaël; Gagnon, Hugo; Guedri, Youssef B; Lavoie, Christine; Boulay, Guylain

2010-07-01

TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9(S21N), a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11(S25N) abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface. 2010 Elsevier B.V. All rights reserved.

Rab7: roles in membrane trafficking and disease.

PubMed

Zhang, Ming; Chen, Li; Wang, Shicong; Wang, Tuanlao

2009-06-01

The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.

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.

Machining and brazing of accelerating RF cavity

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

Ghodke, S.R.; Barnwal, Rajesh; Mondal, Jayant, E-mail: ghodke_barc@yahoo.co.in

2014-07-01

BARC has developed 2856 MHz accelerating cavities for 6 MeV, 9 MeV and 10 MeV RF Linac. New vendors are developed for mass production of accelerating cavity for future projects. New vendors are developing for diamond turning machining, cleaning and brazing processes. Fabrication involved material testing, CNC diamond turning of cavity, cavity cleaning and brazing. Before and after brazing resonance frequency (RF) of cavity was checked with vector network analyser (VNA). A power feed test setup is also fabricated to test power feed cavity before brazing. This test setup will be used to find out assembly performance of power feedmore » cavity and its coupler. This paper discusses about nano machining, cleaning and brazing processes of RF cavities. (author)« less

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.

Thermal resilient multiple jaw braze fixture

DOEpatents

Ney, R.; Perrone, A.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. 9 figs.

32 CFR 202.9 - Conducting RAB meetings.

Code of Federal Regulations, 2013 CFR

2013-07-01

... public will have a designated time on the agenda to speak to the RAB committee as a whole. (b) Nature of...) 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 publish...

Rab8 Regulates the Actin-based Movement of MelanosomesV⃞

PubMed Central

Chabrillat, Marion L.; Wilhelm, Claire; Wasmeier, Christina; Sviderskaya, Elena V.; Louvard, Daniel; Coudrier, Evelyne

2005-01-01

Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes. PMID:15673612

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.

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.

Restoration Advisory Board (RAB) Implementation Guidelines

EPA Pesticide Factsheets

This document provides recommended procedures for establishing and operating Restoration Advisory Boards (RABs). It is intended as a resource for installation, EPA, and State personnel and citizens who participate in RABs.

Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells.

PubMed

Ahmad, Waqas; Li, Yingying; Guo, Yidi; Wang, Xinyu; Duan, Ming; Guan, Zhenhong; Liu, Zengshan; Zhang, Maolin

2017-06-01

Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID 50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

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.

Rab7a modulates ER stress and ER morphology.

PubMed

Mateus, Duarte; Marini, Elettra Sara; Progida, Cinzia; Bakke, Oddmund

2018-05-01

The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called "ER matrices" together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22 N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Titanium Brazing for Structures and Survivability

DTIC Science & Technology

2007-05-01

materials, such as ceramics. This work focuses on vacuum brazing of titanium (both Ti- 6Al - 4V and commercially pure titanium ) and the effect of...such as ceramics. This work focuses on vacuum brazing of titanium (both Ti- 6Al - 4V and commercially pure titanium ) and the effect of processing...Suzumura, and Onzawa, reported the joining of Ti- 6Al - 4V and CP titanium alloys with zirconium-rich braze alloys.5 They found that these alloys could

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

PubMed

Fu, Yulong; Zhu, Jun-Yi; Zhang, Fujian; Richman, Adam; Zhao, Zhanzheng; Han, Zhe

2017-06-01

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Investigations of nephrocyte cell biological processes are fundamental to understanding the insect renal system. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. We analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. Our results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease. The Drosophila nephrocyte is emerging as a useful in vivo model system for molecular target identification and initial testing of therapeutic approaches in humans.

The role of the small GTPase Rab31 in cancer

PubMed Central

Chua, Christelle En Lin; Tang, Bor Luen

2015-01-01

Members of the small GTPase family Rab are emerging as potentially important factors in cancer development and progression. A good number of Rabs have been implicated or associated with various human cancers, and much recent excitement has been associated with the roles of the Rab11 subfamily member Rab25 and its effector, the Rab coupling protein (RCP), in tumourigenesis and metastasis. In this review, we focus on a Rab5 subfamily member, Rab31, and its implicated role in cancer. Well recognized as a breast cancer marker with good prognostic value, recent findings have provided some insights as to the mechanism underlying Rab31's influence on oncogenesis. Levels of Oestrogen Receptor α (ERα)- responsive Rab31 could be elevated through stabilization of its transcript by the RNA binding protein HuR, or though activation by the oncoprotein mucin1-C (MUC1-C), which forms a transcriptional complex with ERα. Elevated Rab31 stabilizes MUC1-C levels in an auto-inductive loop that could lead to aberrant signalling and gene expression associated with cancer progression. Rab31 and its guanine nucleotide exchange factor GAPex-5 have, however, also been shown to enhance early endosome-late endosome transport and degradation of the epidermal growth factor receptor (EGFR). The multifaceted action and influences of Rab31 in cancer is discussed in the light of its new interacting partners and pathways. PMID:25472813

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.

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.

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.

Integration of two RAB5 groups during endosomal transport in plants

PubMed Central

Ebine, Kazuo; Choi, Seung-won; Ichinose, Sakura; Uemura, Tomohiro; Nakano, Akihiko

2018-01-01

RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells. PMID:29749929

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.

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).

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

Are Rab Proteins the Link Between Golgi Organization and Membrane Trafficking?

PubMed Central

Liu, Shijie; Storrie, Brian

2014-01-01

The fundamental separation of Golgi function between subcompartments termed cisternae is conserved across all eukaryotes. Likewise, Rab proteins, small GTPases of the Ras superfamily, are putative common coordinators of Golgi organization and protein transport. However, despite sequence conservation, e.g., Rab6 and Ypt6 are conserved proteins between humans and yeast, the fundamental organization of the organelle can vary profoundly. In the yeast Sacchromyces cerevisiae, the Golgi cisternae are physically separated from one another while, in mammalian cells, the cisternae are stacked one upon the other. Moreover, in mammalian cells many Golgi stacks are typically linked together to generate a ribbon structure. Do evolutionarily conserved Rab proteins regulate secretory membrane trafficking and diverse Golgi organization in a common manner? In mammalian cells, some Golgi associated Rab proteins function in coordination of protein transport and maintenance of Golgi organization. These include Rab6, Rab33B, Rab1, Rab2, Rab18 and Rab43. In yeast, these include Ypt1, Ypt32 and Ypt6. Here, based on evidence from both yeast and mammalian cells, we speculate on the essential role of Rab proteins in Golgi organization and protein transport. PMID:22581368

BLOC-2, AP-3, and AP-1 Proteins Function in Concert with Rab38 and Rab32 Proteins to Mediate Protein Trafficking to Lysosome-related Organelles*

PubMed Central

Bultema, Jarred J.; Ambrosio, Andrea L.; Burek, Carolyn L.; Di Pietro, Santiago M.

2012-01-01

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis. PMID:22511774

BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.

PubMed

Bultema, Jarred J; Ambrosio, Andrea L; Burek, Carolyn L; Di Pietro, Santiago M

2012-06-01

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling.

PubMed

Chen, Christine Y; Balch, William E

2006-08-01

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.

Rab proteins: The key regulators of intracellular vesicle transport

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

Bhuin, Tanmay; Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in

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 bymore » 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.« less

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

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.

Liquid Film Migration in Warm Formed Aluminum Brazing Sheet

NASA Astrophysics Data System (ADS)

Benoit, M. J.; Whitney, M. A.; Wells, M. A.; Jin, H.; Winkler, S.

2017-10-01

Warm forming has previously proven to be a promising manufacturing route to improve formability of Al brazing sheets used in automotive heat exchanger production; however, the impact of warm forming on subsequent brazing has not previously been studied. In particular, the interaction between liquid clad and solid core alloys during brazing through the process of liquid film migration (LFM) requires further understanding. Al brazing sheet comprised of an AA3003 core and AA4045 clad alloy, supplied in O and H24 tempers, was stretched between 0 and 12 pct strain, at room temperature and 523K (250 °C), to simulate warm forming. Brazeability was predicted through thermal and microstructure analysis. The rate of solid-liquid interactions was quantified using thermal analysis, while microstructure analysis was used to investigate the opposing processes of LFM and core alloy recrystallization during brazing. In general, liquid clad was consumed relatively rapidly and LFM occurred in forming conditions where the core alloy did not recrystallize during brazing. The results showed that warm forming could potentially impair brazeability of O temper sheet by extending the regime over which LFM occurs during brazing. No change in microstructure or thermal data was found for H24 sheet when the forming temperature was increased, and thus warm forming was not predicted to adversely affect the brazing performance of H24 sheet.

Integrin trafficking regulated by Rab21 is necessary for cytokinesis.

PubMed

Pellinen, Teijo; Tuomi, Saara; Arjonen, Antti; Wolf, Maija; Edgren, Henrik; Meyer, Hannelore; Grosse, Robert; Kitzing, Thomas; Rantala, Juha K; Kallioniemi, Olli; Fässler, Reinhard; Kallio, Marko; Ivaska, Johanna

2008-09-01

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

Allosteric binding sites in Rab11 for potential drug candidates

PubMed Central

2018-01-01

Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can preferentially bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may allosterically regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously. PMID:29874286

Rab GTPases and Membrane Trafficking in Neurodegeneration

PubMed Central

Kiral, Ferdi Ridvan; Kohrs, Friederike Elisabeth; Jin, Eugene Jennifer; Hiesinger, Peter Robin

2018-01-01

Defects in membrane trafficking are hallmarks of neurodegeneration. Rab GTPases are key regulators of membrane trafficking. Alterations of Rab GTPases, or the membrane compartments they regulate, are associated with virtually all neuronal activities in health and disease. The observation that many Rab GTPases are associated with neurodegeneration has proven a challenge in the quest for cause and effect. Neurodegeneration can be a direct consequence of a defect in membrane trafficking. Alternatively, changes in membrane trafficking may be secondary consequences or cellular responses. The secondary consequences and cellular responses, in turn, may protect, represent inconsequential correlates or function as drivers of pathology. Here, we attempt to disentangle the different roles of membrane trafficking in neurodegeneration by focusing on selected associations with Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and selected neuropathies. We provide an overview of current knowledge on Rab GTPase functions in neurons and review the associations of Rab GTPases with neurodegeneration with respect to the following classifications: primary cause, secondary cause driving pathology or secondary correlate. This analysis is devised to aid the interpretation of frequently observed membrane trafficking defects in neurodegeneration and facilitate the identification of true causes of pathology. PMID:29689231

Plasma Spray for Difficult-To-Braze Alloys

NASA Technical Reports Server (NTRS)

Brennan, A.

1982-01-01

Nickel plating on surfaces makes brazing easier for some alloys. Sometimes nickel plating may not be feasible because of manufacturing sequence, size of hardware, or lack of suitable source for nickel plating. Alternative surface preparation in such cases is to grit-blast surface lightly and then plasma-spray 1 1/2 to 2 mils of fine nickel powder or braze-alloy material directly on surface. Powder is sprayed from plasma gun, using argon as carrier gas to prevent oxidation of nickel or braze alloy.

Soldering and brazing safety guide: A handbook on space practice for those involved in soldering and brazing

NASA Astrophysics Data System (ADS)

This manual provides those involved in welding and brazing with effective safety procedures for use in performance of their jobs. Hazards exist in four types of general soldering and brazing processes: (1) cleaning; (2) application of flux; (3) application of heat and filler metal; and (4) residue cleaning. Most hazards during those operations can be avoided by using care, proper ventilation, protective clothing and equipment. Specific process hazards for various methods of brazing and soldering are treated. Methods to check ventilation are presented as well as a check of personal hygiene and good maintenance practices are stressed. Several emergency first aid treatments are described.

Expression and localization of exocytic and recycling Rabs from Magnaporthe oryzae in mammalian cells

PubMed Central

Qi, Yaoyao; Marlin, M. Caleb; Liang, Zhimin; Zhang, Dongmei; Zhou, Jie; Wang, Zonghua; Lu, Guodong; Li, Guangpu

2018-01-01

Rab GTPases are master regulators of intracellular membrane trafficking along endocytic and exocytic pathways. In this chapter, we began to characterize the exocytic and recycling Rabs from the filamentous fungus Magnaporthe oryzae (M. oryzae) that causes the rice blast disease. Among the 11 putative Rabs identified from the M. oryzae genome database (MoRabs), MoRab1, MoRab8, and MoRab11 appear orthologs of mammalian Rab1, Rab8, and Rab11 and likely function in exocytosis and endosomal recycling. To test this contention, we cloned, expressed, and determined intracellular localization of the three MoRabs in mammalian cells, in comparison to their human counterparts (hRabs). The MoRabs were well expressed as GFP fusion proteins and colocalized with the tdTomato-labeled hRabs on exocytic and recycling organelles, as determined by immunoblot analysis and confocal fluorescence microscopy. The colocalization supports the contention that the MoRabs are indeed Rab orthologs and may play important roles in the development and pathogenicity of M. oryzae. PMID:26360026

Structural plasticity mediates distinct GAP-dependent GTP hydrolysis mechanisms in Rab33 and Rab5.

PubMed

Majumdar, Soneya; Acharya, Abhishek; Prakash, Balaji

2017-12-01

The classical GTP hydrolysis mechanism, as seen in Ras, employs a catalytic glutamine provided in cis by the GTPase and an arginine supplied in trans by a GTPase activating protein (GAP). The key idea emergent from a large body of research on small GTPases is that GTPases employ a variety of different hydrolysis mechanisms; evidently, these variations permit diverse rates of GTPase inactivation, crucial for temporal regulation of different biological processes. Recently, we unified these variations and argued that a steric clash between active site residues (corresponding to positions 12 and 61 of Ras) governs whether a GTPase utilizes the cis-Gln or the trans-Gln (from the GAP) for catalysis. As the cis-Gln encounters a steric clash, the Rab GTPases employ the so-called dual finger mechanism where the interacting GAP supplies a trans-Gln for catalysis. Using experimental and computational methods, we demonstrate how the cis-Gln of Rab33 overcomes the steric clash when it is stabilized by a residue in the vicinity. In effect, this demonstrates how both cis-Gln- and trans-Gln-mediated mechanisms could operate in the same GTPase in different contexts, i.e. depending on the GAP that regulates its action. Interestingly, in the case of Rab5, which possesses a higher intrinsic GTP hydrolysis rate, a similar stabilization of the cis-Gln appears to overcome the steric clash. Taken together with the mechanisms seen for Rab1, it is evident that the observed variations in Rab and their GAP partners allow structural plasticity, or in other words, the choice of different catalytic mechanisms. © 2017 Federation of European Biochemical Societies.

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania.

PubMed

Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

2015-12-11

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania*

PubMed Central

Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

2015-01-01

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. PMID:26499792

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. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes

PubMed Central

Oda, Seiichiro; Nozawa, Takashi; Nozawa-Minowa, Atsuko; Tanaka, Misako; Aikawa, Chihiro; Harada, Hiroyuki; Nakagawa, Ichiro

2016-01-01

Autophagy acts as a host-defense system against pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we identified Rab30 as a novel regulator of GAS-containing autophagosome-like vacuoles (GcAVs). We found that Rab30, a Golgi-resident Rab, was recruited to GcAVs in response to autophagy induction by GAS infection in epithelial cells. Rab30 recruitment was dependent upon its GTPase activity. In addition, the knockdown of Rab30 expression significantly reduced GcAV formation efficiency and impaired intracellular GAS degradation. Rab30 normally functions to maintain the structural integrity of the Golgi complex, but GcAV formation occurred even when the Golgi apparatus was disrupted. Although Rab30 also colocalized with a starvation-induced autophagosome, Rab30 was not required for autophagosome formation during starvation. These results suggest that Rab30 mediates autophagy against GAS independently of its normal cellular role in the structural maintenance of the Golgi apparatus, and autophagosome biogenesis during bacterial infection involves specific Rab GTPases. PMID:26771875

Rab10 Disruption Results in Delayed OPC Maturation.

PubMed

Zhang, Zhao-Huan; Zhao, Wei-Qian; Ma, Fan-Fei; Zhang, Hui; Xu, Xiao-Hui

2017-10-01

Oligodendrocyte precursor cell (OPC) maturation requires membrane addition for myelin sheath formation. Since the Rab system has been shown to contribute to membrane addition in other cell types, in this study, we explored the role of Rab in OPC maturation. SiRNA and shRNA techniques and conditional knockout mice provided in vitro and in vivo evidence that Rab10 is involved in OPC maturation and may affect myelination during OPC development.

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor.

PubMed

Oesterlin, Lena K; Goody, Roger S; Itzen, Aymelt

2012-04-10

Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

High Rab27A expression indicates favorable prognosis in CRC.

PubMed

Shi, Chuanbing; Yang, Xiaojun; Ni, Yijiang; Hou, Ning; Xu, Li; Zhan, Feng; Zhu, Huijun; Xiong, Lin; Chen, Pingsheng

2015-06-13

Rab27A is a peculiar member in Rab family and has been suggested to play essential roles in the development of human cancers. However, the association between Rab27A expression and clinicopathological characteristics of colorectal cancer (CRC) has not been elucidated yet. One-step quantitative real-time polymerase chain reaction (qPCR) test with 18 fresh-frozen CRC samples and immunohistochemistry (IHC) analysis in 112 CRC cases were executed to evaluate the relationship between Rab27A expression and the clinicopathological features of CRC. Cox regression and Kaplan-Meier survival analyses were performed to identify the prognostic factors for 112 CRC patients. The results specified that the expression levels of Rab27A mRNA and protein were significantly higher in CRC tissues than that in matched non-cancerous tissues, in both qPCR test (p = 0.029) and IHC analysis (p = 0.020). The IHC data indicated that the Rab27A protein expression in CRC was statistically correlated with lymph node metastasis (p = 0.022) and TNM stage (p = 0.026). Cox multi-factor analysis and Kaplan-Meier method suggested Rab27A protein expression (p = 0.012) and tumor differentiation (p = 0.004) were significantly associated with the overall survival of CRC patients. The data indicated the differentiate expression of Rab27A in CRC tissues and matched non-cancerous tissues. Rab27A may be used as a valuable prognostic biomarker for CRC patients.

Retort braze bonding of borsic/aluminum composite sheet to titanium

NASA Technical Reports Server (NTRS)

Webb, B. A.; Dolowy, J. F., Jr.

1975-01-01

Braze bonding studies between Borsic/aluminum composite and titanium sheet were conducted to establish acceptable brazing techniques and to assess potential joint efficiencies. Excellent braze joints were produced which exhibited joint strengths exceeding 117 MPa (17,000 psi) and which retained up to 2/3 of this strength at 589 K (600 F). Noticeable composite strength degradation resulting from the required high temperature braze cycle was found to be a problem.

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

DOEpatents

Howard, Stanley R [Windsor, SC; Korinko, Paul S [Aiken, SC

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.

Role of Rab5 in the formation of macrophage-derived foam cell.

PubMed

Chan, Lokwern; Hong, Jin; Pan, Junjie; Li, Jian; Wen, Zhichao; Shi, Haiming; Ding, Jianping; Luo, Xinping

2017-09-12

Foam cells play a key role in the occurrence and pathogenesis of atherosclerosis. Its formation starts with the ingestion of oxidized low-density lipoprotein (oxLDL). The process is associated with Ras related protein in brain 5 (Rab5) which plays a critical role in regulating endocytosis and early endosomal trafficking. Base on this, we presumed that Rab5 might participate in the maturation of foam cell. The aim of this study is to investigate the effect of Rab5 on macrophage cholesterol during the evolvement of macrophage when induced by oxLDL to the formation of foam cell. Immunohistochemistry was performed to analyze the distribution of macrophages and Rab5 in atherosclerotic plaque. RNA inteference study and transfection of inactive mutant (GFP-Rab5-S34N) and active mutant (GFP-Rab5-Q79L) in U937-derived macrophage were utilized to investigate the impact of Rab5 on the process of macrophage cholesterol, which could be detected by oil red O staining, determination of intracellular lipid content, filipin staining, nile red staining and the costaining of early endosome antigen-1 (EEA-1) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylin dicarbocyanine (Dil)-labelled oxLDL (Dil-oxLDL). Rab5 was found abundantly localized in macrophage rich areas of human atherosclerotic lesions. On the foam cell study, the expression of Rab5 was increased after the incubation of oxLDL. The inteference study indicated the depletion of Rab5 led to the decreases of oil red O staining areas, total cholesterol and cholesterol esters in U937-derived marophages. Moreover, the fluorescence intensity of filipin and nile red staining were lower in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. The confocal study demonstrated less Dil-oxLDL was internalized in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L; the result showed also the decrease in colocalization of internalized Dil-oxLDL and EEA-1 for GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. Rab5 plays an important role in modulating the

Control of vacuum induction brazing system for sealing of instrumentation feed-through

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

Sung Ho Ahn; Jintae Hong; Chang Young Joung

2015-07-01

The integrity of instrumentation cables is an important performance parameter in addition to the sealing performance in the brazing process. An accurate brazing control was developed for the brazing of the instrumentation feed-through in the vacuum induction brazing system in this paper. The experimental results show that the accurate brazing temperature control performance is achieved by the developed control scheme. Consequently, the sealing performances of the instrumentation feed-through and the integrities of the instrumentation cables were satisfied after brazing. (authors)

Control of Vacuum Induction Brazing System for Sealing of Instrumentation Feedthrough

NASA Astrophysics Data System (ADS)

Ahn, Sung Ho; Hong, Jintae; Joung, Chang Young; Heo, Sung Ho

2017-04-01

The integrity of instrumentation cables is an important performance parameter in the brazing process, along with the sealing performance. In this paper, an accurate control scheme for brazing of the instrumentation feedthrough in a vacuum induction brazing system was developed. The experimental results show that the accurate brazing temperature control performance is achieved by the developed control scheme. It is demonstrated that the sealing performances of the instrumentation feedthrough and the integrity of the instrumentation cables are to be acceptable after brazing.

Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly.

PubMed

Pham, Tu M; Tran, Si C; Lim, Yun-Sook; Hwang, Soon B

2017-02-01

Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly. Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP- and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential

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

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).

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..

The Rab GTPase RabG3b Positively Regulates Autophagy and Immunity-Associated Hypersensitive Cell Death in Arabidopsis1[W

PubMed Central

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

2013-01-01

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. PMID:23404918

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin.

PubMed

Los, Ferdinand C O; Kao, Cheng-Yuan; Smitham, Jane; McDonald, Kent L; Ha, Christine; Peixoto, Christina A; Aroian, Raffi V

2011-02-17

Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics. Copyright © 2011 Elsevier Inc. All rights reserved.

Rab GTPases in Immunity and Inflammation.

PubMed

Prashar, Akriti; Schnettger, Laura; Bernard, Elliott M; Gutierrez, Maximiliano G

2017-01-01

Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

Rab7b at the intersection of intracellular trafficking and cell migration.

PubMed

Distefano, Marita Borg; Kjos, Ingrid; Bakke, Oddmund; Progida, Cinzia

2015-01-01

Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).

32 CFR 202.8 - Training RAB members.

Code of Federal Regulations, 2010 CFR

2010-07-01

... provide in-house assistance to discuss technical issues. Funding for training activities must be within... 32 National Defense 2 2010-07-01 2010-07-01 false Training RAB members. 202.8 Section 202.8...) MISCELLANEOUS RESTORATION ADVISORY BOARDS Operating Requirements § 202.8 Training RAB members. Training is not...

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)

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis

PubMed Central

2012-01-01

Background Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. Results Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. Conclusions Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types. PMID:22873208

Role of Rab family GTPases and their effectors in melanosomal logistics.

PubMed

Ohbayashi, Norihiko; Fukuda, Mitsunori

2012-04-01

Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g. Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e. defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.

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.

Simulation model of Al-Ti dissimilar laser welding-brazing and its experimental verification

NASA Astrophysics Data System (ADS)

Behúlová, M.; Babalová, E.; Nagy, M.

2017-02-01

Formation of dissimilar weld joints of light metals and alloys including Al-Ti joints is interesting mainly due to demands on the weight reduction and corrosion resistance of components and structures in automotive, aircraft, aeronautic and other industries. Joining of Al-Ti alloys represents quite difficult problem. Generally, the fusion welding of these materials can lead to the development of different metastable phases and formation of brittle intermetallic compounds. The paper deals with numerical simulation of the laser welding-brazing process of titanium Grade 2 and EN AW 5083 aluminum alloy sheets using the 5087 aluminum filler wire. Simulation model for welding-brazing of testing samples with the dimensions of 50 × 100 × 2 mm was developed in order to perform numerical experiments applying variable welding parameters and to design proper combination of these parameters for formation of sound Al-Ti welded-brazed joints. Thermal properties of welded materials in the dependence on temperature were computed using JMatPro software. The conical model of the heat source was exploited for description of the heat input to the weld due to the moving laser beam source. The sample cooling by convection and radiation to the surrounding air and shielding argon gas was taken into account. Developed simulation model was verified by comparison of obtained results of numerical simulation with the temperatures measured during real experiments of laser welding-brazing by the TruDisk 4002 disk laser.

32 CFR 202.11 - Documenting RAB activities.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 2 2010-07-01 2010-07-01 false Documenting RAB activities. 202.11 Section 202.11 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS RESTORATION ADVISORY BOARDS Operating Requirements § 202.11 Documenting RAB activities. (a) The installation shall document information...

Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A.

PubMed

Zhang, Jinzhong; Johnson, Jennifer L; He, Jing; Napolitano, Gennaro; Ramadass, Mahalakshmi; Rocca, Celine; Kiosses, William B; Bucci, Cecilia; Xin, Qisheng; Gavathiotis, Evripidis; Cuervo, Ana María; Cherqui, Stephanie; Catz, Sergio D

2017-06-23

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns -/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures.

PubMed

Patrussi, Laura; Baldari, Cosima T

2016-01-01

Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Among them, Rab8 is an essential participant in primary cilium formation. In a report recently published in the Journal of Cell Science, Finetti and colleagues identify Rab8 as a novel player in vesicular traffic in the non-ciliated T lymphocytes, which contributes to the assembly of the specialized signaling platform known as the immune synapse. By interacting with the v-SNARE VAMP-3, Rab8 is indeed responsible for the final docking/fusion step in T cell receptor (TCR) recycling to the immune synapse. A second important take-home message which comes to light from this work is that VAMP-3 also interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of Smoothened at the plasma membrane. Hence the data presented in this report, in addition to identifying Rab8 as a novel player in vesicular traffic to the immune synapse, reveal how both ciliated and non-ciliated cells take advantage of a conserved pathway to build highly specific cellular structures.

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

Anterograde Trafficking of KCa3.1 in Polarized Epithelia Is Rab1- and Rab8-Dependent and Recycling Endosome-Independent

PubMed Central

Bertuccio, Claudia A.; Lee, Shih-Liang; Wu, Guangyu; Butterworth, Michael B.; Hamilton, Kirk L.; Devor, Daniel C.

2014-01-01

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized

Rab11 family expression in the human placenta: Localization at the maternal-fetal interface

PubMed Central

Artemiuk, Patrycja A.; Hanscom, Sara R.; Lindsay, Andrew J.; Wuebbolt, Danielle; Breathnach, Fionnuala M.; Tully, Elizabeth C.; Khan, Amir R.; McCaffrey, Mary W.

2017-01-01

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human

Dissimilar Brazed Joints Between Steel and Tungsten Carbide

NASA Astrophysics Data System (ADS)

Voiculescu, I.; Geanta, V.; Binchiciu, H.; Iovanas, D.; Stefanoiu, R.

2017-06-01

Brazing is a joining process used to obtain heterogeneous assemblies between different materials, such as steels, irons, non-ferrous metals, ceramics etc. Some application, like asphalt cutters, require quick solutions to obtain dissimilar joints at acceptable costs, given the very short period of operation of these parts. This paper presents some results obtained during the brazing of dissimilar joints between steel and tungsten carbide by using different types of Ag-Cu system filler materials alloyed with P and Sn. The brazing techniques used were oxygen-gas flame and induction joining. The brazing behaviour was analysed in cross sections by optical and electron microscopy. The metallographic analysis enhanced the adhesion features and the length of penetration in the joining gap. The melting range of the filler materials was measured using thermal analysis.

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.

The breast cancer antigen 5T4 interacts with Rab11, and is a target and regulator of Rab11 mediated trafficking.

PubMed

Harris, Janelle L; Dave, Keyur; Gorman, Jeffrey; Khanna, Kum Kum

2018-06-01

5T4 is a transmembrane glycoprotein with limited expression in normal adult tissues and expression in some solid tumours. It is unclear whether 5T4 is preferentially expressed by stem or differentiated cell types. Modes of 5T4 regulation are unknown despite its ongoing development as a cancer immunotherapy target. Our aims were to clarify the differentiation status of 5T4 expressing cells in breast cancer and to understand the mechanism underlying 5T4 membrane presentation. We analysed 5T4 expression in breast cancer cell populations by flow cytometery and found that 5T4 is highly expressed on differentiated cells, where it localizes to focal adhesions. Using immunoprecipitation and mass spectrometry, we identified interactions between 5T4 and the membrane trafficking proteins Rab11, Rab18 and ARF6. Mechanistically we found that Rab11 and Rab18 have oppositional roles in controlling expression and surface presentation of 5T4. 5T4 depletion stabilizes Rab11 protein expression with a consequent stimulation transferrin surface labelling, indicating that 5T4 represses endocytic activity. Successful immunotherapeutic targeting of 5T4 requires surface presentation and different immunotherapy strategies require surface presentation versus endocytosis. While breast cancer cells with high 5T4 surface expression and rapid cell surface turnover would be susceptible to antibody-drug conjugates that rely on intracellular release, 5T4 positive cells with lower expression or lower turnover may still be responsive to T-cell mediated approaches. We find that endocytosis of 5T4 is strongly Rab11 dependent and as such Rab11 activity could affect the success or failure of 5T4-targetted immunotherapy, particularly for antibody-drug conjugate approaches. In fact, 5T4 itself represses Rab11 expression. This newly uncovered relationship between Rab11 and 5T4 suggests that breast tumours with high 5T4 expression may not have efficient endocytic uptake of 5T4-targetted immunotherapeutics

Brazing Inconel 625 Using Two Ni/(Fe)-Based Amorphous Filler Foils

NASA Astrophysics Data System (ADS)

Chen, Wen-Shiang; Shiue, Ren-Kae

2012-07-01

For MBF-51 filler, the brazed joint consists of interfacial grain boundary borides, coarse Nb6Ni16Si7, and Ni/Cr-rich matrix. In contrast, the VZ-2106 brazed joint is composed of interfacial Nb6Ni16Si7 precipitates as well as grain boundary borides, coarse Nb6Ni16Si7, and Ni/Cr/Fe-rich matrix. The maximum tensile strength of 443 MPa is obtained from the MBF-51 brazed specimen. The tensile strengths of VZ-2106 brazed joints are approximately 300 MPa. Both amorphous filler foils demonstrate potential in brazing IN-625 substrate.

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

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

Silva, Patricio; Soto, Nicolás; Díaz, Jorge

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: • Rab5more » 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.« less

RAB-7 Antagonizes LET-23 EGFR Signaling during Vulva Development in Caenorhabditis elegans

PubMed Central

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. PMID:22558469

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.

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.

Tungsten wire and tubing joined by nickel brazing

NASA Technical Reports Server (NTRS)

1965-01-01

Thin tungsten wire and tungsten tubing are brazed together using a contacting coil of nickel wire heated to its melting point in an inert-gas atmosphere. This method is also effective for brazing tungsten to tungsten-rhenium parts.

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)

Characterization of the human RAB38 and RAB7 genes: exclusion of new major pathological loci for Japanese OCA.

PubMed

Suzuki, Tamio; Miyamura, Yoshinori; Inagaki, Katsuhiko; Tomita, Yasushi

2003-08-01

Oculocutaneous albinisms (OCAs) are due to various gene mutations that cause a disruption of melanogenesis in the melanocyte. Four different genes associated with human OCA have been reported, however, not all of OCA patients can be classified according to these four genes. We have sought to find a new major locus for Japanese OCA. Recently two genes, RAB38 and RAB7, were reported to play an important role in melanogenesis in the melanocyte, suggesting that these two genes could be good candidates for new OCA loci. To determine the structures of the human RAB38 and RAB7 genes, and examine if the two genes are new major loci for Japanese OCA. We screened mutations in these genes of 25 Japanese OCA patients who lacked mutations in the OCA1 and OCA2 genes with SSCP/heteroduplexes method. We determined the both genes, and their genomic organizations to design the primers for SSCP/heteroduplexes method. And then we screened mutations, but no mutation was detected. Neither of the genes is a new major locus for Japanese OCA.

Determination of Rab5 activity in the cell by effector pull-down assay.

PubMed

Qi, Yaoyao; Liang, Zhimin; Wang, Zonghua; Lu, Guodong; Li, Guangpu

2015-01-01

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

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.

Preliminary Process Design of ITER ELM Coil Bracket Brazing

NASA Astrophysics Data System (ADS)

LI, Xiangbin; SHI, Yi

2015-03-01

With the technical requirement of the International Thermonuclear Experimental Reactor (ITER) project, the manufacture and assembly technology of the mid Edge Localized Modes (ELM) coil was developed by the Institute of Plasma Physics, Chinese Academy of Science (ASIPP). As the gap between the bracket and the Stainless Steel jacketed and Mineral Insulated Conductor (SSMIC) can be larger than 0.5 mm instead of 0.01 mm to 0.1 mm as in normal industrial cases, the process of mid ELM coil bracket brazing to the SSMICT becomes quiet challenging, from a technical viewpoint. This paper described the preliminary design of ELM coil bracket brazing to the SSMIC process, the optimal bracket brazing curve and the thermal simulation of the bracket furnace brazing method developed by ANSYS. BAg-6 foil (Bag50Cu34Zn16) plus BAg-1a paste (Bag45CuZnCd) solders were chosen as the brazing filler. By testing an SSMICT prototype, it is shown that the average gap between the bracket and the SSMIC could be controlled to 0.2-0.3 mm, and that there were few voids in the brazing surface. The results also verified that the preliminary design had a favorable heat conducting performance in the bracket.

Influence of cyclic thermal loading on brazed composites for fusion applications

NASA Astrophysics Data System (ADS)

Šmid, I.; Kny, E.; Kneringer, G.; Reheis, N.

1990-04-01

Reactor grade graphite and molybdenum (TZM) were brazed with different high temperature brazes. The resulting tiles had a size of 50 × 50 mm2 with a graphite thickness of 10 mm and a TZM thickness of 5 mm. The brazed composites have been tested in electron beam simulation for their thermal fatigue properties. The parameters of these tests were chosen to match NET design specifications for normal operation and "slow" peak energy deposition. The resulting damage and microstructural changes on the graphites and the brazes are discussed. Additional information is supplied on X-ray diffraction data proving the presence of different phases in the brazes.

Method for controlling brazing

DOEpatents

Hosking, F Michael [Albuquerque, NM; Hall, Aaron C [Albuquerque, NM; Givler, Richard C [Albuquerque, NM; Walker, Charles A [Albuquerque, NM

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.

Chem-Braze Abradable Seal Attachment to Aircraft Gas Turbine Compressor Components.

DTIC Science & Technology

1982-01-01

seals to compressor blade tip-shrouds using the im- proved Chem-Braze system compared to attachment with gold-nickel braze. The Chem-Braze system has been...used successfully to bond abradable seals to titanium ’ cobalt, nickel and iron base alloys; however, attempts to use Chem-Braze to bond seals to...attaching FELTMETALO seals to steel, titanium , and nickel-based alloys, and ICB bonding procedures were investigated for attaching seals to selected

The Joint Strength and Fracture Mechanisms of TC4/TC4 and TA0/TA0 Brazed with Ti-25Cu-15Ni Braze Alloy

NASA Astrophysics Data System (ADS)

Zou, Zhihuan; Zeng, Fanhao; Wu, Haobo; Liu, Jian; Li, Yi; Gu, Yi; Yuan, Tiechui; Zhang, Fuqin

2017-05-01

In this paper, Ti-25Cu-15Ni (mass ratio) braze alloys were prepared by vacuum arc melting. Additionally, the TA0 pure titanium and TC4 titanium alloy were brazed with the Ti-25Cu-15Ni braze alloy at 960, 980, 1000, 1020, and 1040 °C. The effects of the braze temperature on the tensile strength of the TA0 and TC4 joints and their fracture mechanisms were studied. The maximum tensile strength of the TA0 joints of 219.9 ± 0.1 MPa was achieved at a brazing temperature of 980 °C, and the maximum tensile strength of the TC4 joints of 832.9 ± 0.1 MPa was achieved at the same brazing temperature. These results indicate that their ideal joint strength is comparable. According to the fractography results of the TA0 joints, a mixed fracture morphology is indicated. The TA0 fracture surface is dominated by cleavage fracture with a small contribution from ductile fracture. The TC4 joint fracture arises from cleavage.

Development of a Cu-Sn based brazing system with a low brazing and a high remelting temperature

NASA Astrophysics Data System (ADS)

Schmieding, M.; Holländer, U.; Möhwald, K.

2017-03-01

Objective of the project presented is the development of a joining process for hot working steel components at low brazing temperatures leading to a bond with a much higher remelting temperature. This basically is achieved by the use of a Cu-Sn melt spinning foil combined with a pure Cu foil. During brazing, the Sn content of the foil is decreased by diffusion of Sn into the additional Cu resulting in a homogenious joint with a increased remelting temperature of the filler metal. Within this project specimens were brazed and diffusion annealed in a vacuum furnace at 850 °C varying the processing times (0 - 10 h). The samples prepared were studied metallographically and diffusion profiles of Sn were recorded using EDX line scans. The results are discussed in view of further investigations and envisaged applications.

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

Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

PubMed Central

Hsu, FoSheng; Spannl, Stephanie; Ferguson, Charles; Hyman, Anthony A; Parton, Robert G

2018-01-01

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS. PMID:29469808

Wetting and spreading behavior of molten brazing filler metallic alloys on metallic substrate

NASA Astrophysics Data System (ADS)

Kogi, Satoshi; Kajiura, Tetsurou; Hanada, Yukiakira; Miyazawa, Yasuyuki

2014-08-01

Wetting and spreading of molten brazing filler material are important factors that influence the brazing ability of a joint to be brazed. Several investigations into the wetting ability of a brazing filler alloy and its surface tension in molten state, in addition to effects of brazing time and temperature on the contact angle, have been carried out. In general, dissimilar-metals brazing technology and high-performance brazed joint are necessities for the manufacturing field in the near future. Therefore, to address this requirement, more such studies on wetting and spreading of filler material are required for a deeper understanding. Generally, surface roughness and surface conditions affect spreading of molten brazing filler material during brazing. Wetting by and interfacial reactions of the molten brazing filler material with the metallic substrate, especially, affect strongly the spreading of the filler material. In this study, the effects of surface roughness and surface conditions on the spreading of molten brazing filler metallic alloys were investigated. Ag-(40-x)Cu-xIn and Ag- (40-x)Cu-xSn (x=5, 10, 15, 20, 25) alloys were used as brazing filler materials. A mild-steel square plate (S45C (JIS); side: 30 mm; thickness: 3mm) was employed as the substrate. A few surfaces with varying roughness were prepared using emery paper. Brazing filler material and metallic base plate were first washed with acetone, and then a flux was applied to them. The filler, 50 mg, was placed on the center of the metallic base with the flux. A spreading test was performed under Ar gas using an electrically heated furnace, after which, the original spreading area, defined as the sessile drop area, and the apparent spreading area, produced by the capillary grooves, were both evaluated. It was observed that the spreading area decreased with increasing In and Sn content.

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.

Rab1A is required for assembly of classical swine fever virus particle.

PubMed

Lin, Jihui; Wang, Chengbao; Liang, Wulong; Zhang, Jing; Zhang, Longxiang; Lv, Huifang; Dong, Wang; Zhang, Yanming

2018-01-15

Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Brazed graphite/refractory metal composites for first-wall protection elements

NASA Astrophysics Data System (ADS)

Šmid, I.; Croessmann, C. D.; Salmonson, J. C.; Whitley, J. B.; Kny, E.; Reheis, N.; Kneringer, G.; Nickel, H.

1991-03-01

The peak surface heat flux deposition on divertor elements of near term fusion devices is expected to exceed 10 MW/m 2. The needed reliability of brazed plasma interactive components, particularly under abnormal operating conditions with peak surface temperatures well beyond 1000°C, makes refractory metallic substrates and brazes with a high melting point very attractive. TZM, a high temperature alloy of molybdenum, and isotropic graphite, materials very closely matched in their thermal expansion, were brazed with four high-temperature brazes. The brazes used were Zr, 90Ni/10Ti, 90Cu/10Ti and 70Ag/27Cu/3Ti (nominal composition prior to brazing, wt%). The resulting composite tiles of 50 × 50 mm2 with a TZM thickness of 5 mm and a graphite thickness of 10 mm have been tested in high heat flux simulation for their thermal fatigue properties. Up to 600 loading cycles were carried out with an average heat flux of 10 MW/m 2 for 0.5 s pulses. The maximum surface temperature was 1100°C. In support of the experiment, the thermal response and temperature gradients of the samples were investigated using a finite element model.

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.

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.

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.

Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

PubMed Central

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.

2014-01-01

Summary 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 new pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

rab3 mediates cortical granule exocytosis in the sea urchin egg.

PubMed

Conner, S; Wessel, G M

1998-11-15

Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking. Copyright 1998 Academic

Fabrication of large aperture SiC brazing mirror

NASA Astrophysics Data System (ADS)

Li, Ang; Wang, Peipei; Dong, Huiwen; Wang, Peng

2016-10-01

The SiC brazing mirror is the mirror whose blank is made by assembling together smaller SiC pieces with brazing technique. Using such kinds of joining techniques, people can manufacture large and complex SiC assemblies. The key technologies of fabricating and testing SiC brazing flat mirror especially for large aperture were studied. The SiC brazing flat mirror was ground by smart ultrasonic-milling machine, and then it was lapped by the lapping smart robot and measured by Coordinate Measuring Machine (CMM). After the PV of the surface below 4um, we did classic coarse polishing to the surface and studied the shape of the polishing tool which directly effects removal amount distribution. Finally, it was figured by the polishing smart robot and measured by Fizeau interferometer. We also studied the influence of machining path and removal functions of smart robots on the manufacturing results and discussed the use of abrasive in this process. At last, an example for fabricating and measuring a similar SiC brazing flat mirror with the aperture of 600 mm made by Shanghai Institute of Ceramics was given. The mirror blank consists of 6 SiC sectors and the surface was finally processed to a result of the Peak-to-Valley (PV) 150nm and Root Mean Square (RMS) 12nm.

Infrared Brazing of Ti50Ni50 Shape Memory Alloy and Inconel 600 Alloy with Two Ag-Cu-Ti Active Braze Alloys

NASA Astrophysics Data System (ADS)

Shiue, Ren-Kae; Wu, Shyi-Kaan; Yang, Sheng-Hao

2017-02-01

Infrared brazing of Ti50Ni50 SMA and Inconel 600 alloy using Cusil-ABA and Ticusil filler metals has been investigated. The joints were dominated by Ag-Cu eutectic with proeutectic Cu in the Cusil-ABA brazed joint and with proeutectic Ag in the Ticusil one. A continuous curved belt composed of a Ni3Ti layer and a (Cu x Ni1- x )2Ti layer formed in the brazed Ti50Ni50/Ticusil/Inconel 600 joint. On the Ti50Ni50 SMA side, an intermetallic layer of (Cu x Ni1- x )2Ti formed in all joints, with x values around 0.81 and 0.47. Layers of (Cu x Ni1- x )2Ti, Ni3Ti, and mixed Ni3Ti and Ni2Cr intermetallics were observed next to the Inconel 600 substrate in the brazed Ti50Ni50/Cusil-ABA/Inconel 600 joint. The maximum shear strengths of the joints using the Cusil-ABA filler metal and the Ticusil filler metal were 324 and 300 MPa, respectively. In the Cusil-ABA brazed joint, cracks with cleavage-dominated fracture propagated along the (Cu x Ni1- x )2Ti interfacial layer next to the Ti50Ni50 SMA substrate. In the Ticusil brazed joint, ductile dimple fracture occurred in the Ag-rich matrix near the Inconel 600 alloy substrate. The absence of a detrimental Ti-Fe-(Cu) layer on the Inconel 600 substrate side can effectively improve the shear strength of the joint.

A plasmid library of full-length zebrafish rab proteins for in vivo cell biology.

PubMed

Hall, Thomas E; Martel, Nick; Lo, Harriet P; Xiong, Zherui; Parton, Robert G

2017-01-01

The zebrafish is an emerging model for highly sophisticated medium-throughput experiments such as genetic and chemical screens. However, studies of entire protein families within this context are often hampered by poor genetic resources such as clone libraries. Here we describe a complete collection of 76 full-length open reading frame clones for the zebrafish rab protein family. While the mouse genome contains 60 rab genes and the human genome 63, we find that 18 zebrafish rab genes have 2, and in the case of rab38, 3 paralogues. In contrast, we were unable to identify zebrafish orthologues of the mammalian Rab2b, Rab17 or Rab29. We make this resource available through the Addgene repository to facilitate cell biologic approaches using this model.

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.

Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion

PubMed Central

Gundry, Christine; Marco, Sergi; Rainero, Elena; Miller, Bryan; Dornier, Emmanuel; Mitchell, Louise; Caswell, Patrick T.; Campbell, Andrew D.; Hogeweg, Anna; Sansom, Owen J.; Morton, Jennifer P.; Norman, Jim C.

2017-01-01

The Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser435 by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser897 by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinoma—whereas conditional knockout of another RCP cargo, α5 integrin, does not suppress pancreatic cancer metastasis—indicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo. PMID:28294115

Structural Performance of Inconel 625 Superalloy Brazed Joints

NASA Astrophysics Data System (ADS)

Chen, Jianqiang; Demers, Vincent; Cadotte, Eve-Line; Turner, Daniel; Bocher, Philippe

2017-02-01

The purpose of this work was to investigate tensile and fatigue behaviors of Inconel 625 superalloy brazed joints after transient liquid-phase bonding process. Brazing was performed in a vacuum furnace using a nickel-based filler metal in a form of paste to join wrought Inconel 625 plates. Mechanical tests were carried out on single-lap joints under various lap distance-to-thickness ratios. The fatigue crack initiation and crack growth modes were examined via metallographic analysis, and the effect of local stress on fatigue life was assessed by finite element simulations. The fatigue results show that fatigue strength and endurance limit increase with overlap distance, leading to a relatively large scatter of results. Fatigue cracks nucleated in the high-stressed region of the weld fillets from brittle eutectic phases or from internal brazing cavities. The present work proposes to rationalize the results by using the local stress at the brazing fillet. When using this local stress, all fatigue-obtained results find themselves on a single S- N curve, providing a design curve for any joint configuration in fatigue solicitation.

The emerging role of Rab GTPases in the pathogenesis of Parkinson's disease.

PubMed

Gao, Yujing; Wilson, Gabrielle R; Stephenson, Sarah E M; Bozaoglu, Kiymet; Farrer, Matthew J; Lockhart, Paul J

2018-02-01

The identification of pathogenic mutations in Ras analog in brain 39B (RAB39B) and Ras analog in brain 32 (RAB32) that cause Parkinson's disease (PD) has highlighted the emerging role of protein trafficking in disease pathogenesis. Ras analog in brain (Rab) Guanosine triphosphatase (GTPase) function as master regulators of membrane trafficking, including vesicle formation, movement along cytoskeletal networks, and membrane fusion. Recent studies have linked Rab GTPases with α-synuclein, Leucine-rich repeat kinase 2, and Vacuolar protein sorting 35, 3 key proteins in PD pathogenesis. In this review, we discuss the various RAB GTPases associated with PD, current progress in the research, and potential future directions. Investigations into the function of RAB GTPases will likely provide significant insight into the etiology of PD and identify novel therapeutic targets for a currently incurable disease. © 2018 International Parkinson and Movement Disorder Society. © 2018 International Parkinson and Movement Disorder Society.

Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases.

PubMed

Roosterman, Dirk; Cottrell, Graeme S; Schmidlin, Fabien; Steinhoff, Martin; Bunnett, Nigel W

2004-07-16

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.

Analysis of Brazing Effect on Hot Corrosion Behavior of a Nickel-Based Aerospace Superalloy

NASA Astrophysics Data System (ADS)

Esmaeili, N.; Ojo, O. A.

2018-06-01

The effects of brazing and use of composite powder mixture as interlayer material on hot corrosion resistance of brazed IN738 superalloy were studied. Brazing was observed to result in significant reduction in the hot corrosion resistance of the superalloy. However, application of composite powder mixture, which consists of additive superalloy powder, enhanced the hot corrosion resistance of brazed samples. It is also found that although the use of composite powder mixture increased hot corrosion resistance of brazed alloy, if the additive powder completely melts, which is possible during brazing, it can significantly reduce the hot corrosion resistance of the brazed joint. Elemental micro-segregation during solidification of the joint with completely melted powder mixture produces chromium-depleted zones and consequently reduces hot corrosion resistance, since a uniform distribution and adequate chromium concentration are necessary to combat hot corrosion. This has not been previously reported in the literature and it is crucial to the use of composite powder mixture for enhancing the properties of brazed superalloys.

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.

Effect of Microstructure on Diffusional Solidification of 4343/3005/4343 Multi-Layer Aluminum Brazing Sheet

NASA Astrophysics Data System (ADS)

Tu, Yiyou; Tong, Zhen; Jiang, Jianqing

2013-04-01

The effect of microstructure on clad/core interactions during the brazing of 4343/3005/4343 multi-layer aluminum brazing sheet was investigated employing differential scanning calorimetry (DSC) and electron back-scattering diffraction (EBSD). The thickness of the melted clad layer gradually decreased during the brazing operation. It could be completely removed isothermally as a result of diffusional solidification at the brazing temperature. During the brazing cycle, the rate of loss of the melt in the brazing sheet, with small equiaxed grains' core layer, was higher than that with the core layer consisting of elongated large grains. The difference in microstructure affected the amount of liquid formed during brazing.

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.

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.

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

2016-12-01

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.

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus.

PubMed

Zhi, Hui; Wang, Wei; Li, Lingyan; Chai, Baofeng; Sun, Yonghua; Liang, Aihua

2005-08-01

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6-99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.

High heat flux performance of W-Eurofer brazed joints

NASA Astrophysics Data System (ADS)

de Prado, J.; Sánchez, M.; Wirtz, M.; Pintsuk, G.; Du, J.; Linke, J.; Ureña, A.

2018-02-01

The qualification process of the materials and components for the next generation of fusion reactors makes it necessary to expose them to similar service conditions as expected during the service life of the reactor. In the present work, W-Eurofer brazed joints (tungsten block: 8 × 8 × 4 mm; steel block: 8 × 8 × 4 mm; joined to an actively cooled copper heat sink) were exposed to steady state heat loads to study the effect of the thermal fatigue on their microstructure and mechanical integrity. Three different W surface temperatures were tested (400, 500 and 600 °C) varying the number of applied cycles (100 and 1000). The results allowed identifying a braze temperature of 359 °C as threshold condition under which the brazed joints could be used without deterioration. The increase of the surface temperature deteriorated the mechanical integrity of the joints in comparison to those analyzed after the brazing process and accordingly reduced the refrigeration capabilities.

Rab7-a novel redox target that modulates inflammatory pain processing.

PubMed

Kallenborn-Gerhardt, Wiebke; Möser, Christine V; Lorenz, Jana E; Steger, Mirco; Heidler, Juliana; Scheving, Reynir; Petersen, Jonas; Kennel, Lea; Flauaus, Cathrin; Lu, Ruirui; Edinger, Aimee L; Tegeder, Irmgard; Geisslinger, Gerd; Heide, Heinrich; Wittig, Ilka; Schmidtko, Achim

2017-07-01

Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.

Arabidopsis RabF1 (ARA6) Is Involved in Salt Stress and Dark-Induced Senescence (DIS)

PubMed Central

Yin, Congfei; Karim, Sazzad; Zhang, Hongsheng; Aronsson, Henrik

2017-01-01

Arabidopsis small GTPase RabF1 (ARA6) functions in endosomal vesicle transport and may play a crucial role in recycling and degradation of molecules, thus involved in stress responses. Here we have reported that complementary overexpression lines RabF1OE (overexpression), GTPase mutants RabF1Q93L (constitutively active) and RabF1S47N (dominant negative) lines show longer root growth than wild-type, rabF1 knockout and N-myristoylation deletion (Δ1−29, N-terminus) complementary overexpression mutant plants under salt induced stress, which indicates that N-myristoylation of RabF1 is indispensable for salt tolerance. Moreover, RabF1 is highly expressed during senescence and RabF1OE lines were more tolerant of dark-induced senescence (DIS) than wild-type and rabF1. PMID:28157156

Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI).

PubMed

Edler, Eileen; Stein, Matthias

2017-10-25

The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

Reduction of liquid metal embrittlement in copper-brazed stainless steel joints

NASA Astrophysics Data System (ADS)

Uhlig, T.; Fedorov, V.; Elßner, M.; Wagner, G.; Weis, S.

2017-03-01

Due to its very good formability and the low raw material cost, pure copper in form of foils is commonly used to braze plate heat exchangers made of stainless steel. The difference in the electrochemical potentials of brazing filler and base material leads to corrosion effects in contact with electrolytes. This may lead to leakages, which decrease the reliability of the heat exchanger during service in potable water. The dissolution of the emerging corrosion products of brazing filler and base material induces the migration of heavy metal ions, such as Cu2+ and Ni2+, into the potable water. The so-called liquid metal embrittlement, which takes place during the brazing process, may intensify the corrosion. The brazing filler infiltrates the stainless steel along the grain boundaries and causes an embrittlement. This paper deals with the determination of the grain boundary erosion dependent on the degree of deformation and heat treatment of the stainless steel AISI 316L.

Molecular characterization and expression of Rab7 from Clonorchis sinensis and its potential role in autophagy.

PubMed

Jia, Feifei; Li, Ye; Huang, Yan; Chen, Tingjin; Li, Shan; Xu, Yanquan; Wu, Zhongdao; Li, Xuerong; Yu, Xinbing

2013-07-01

Accumulating evidences suggest that Rab7 GTPase is important for the normal progression of autophagy. However, the role of Rab7 GTPase in regulation of autophagy in Clonorchis sinensis is not known. In this study, a gene encoding Rab7 was isolated from C. sinensis adult cDNA. Recombinant CsRab7 was expressed and purified from Escherichia coli. CsRab7 transcripts were detected in the cDNA of adult worm, metacercaria, cercaria, and egg of C. sinensis, and were highly expressed in the metacercaria. Immunohistochemical localization results revealed that CsRab7 was specifically deposited on the vitellarium and eggs of adult worm. Furthermore, EGFP signal of CsRab7WT and the active mutant CsRab7Q67L were associated with autophagic vesicles in transiently transfected 293T cells. It is concluded from the present study that CsRab7 GTPase possibly contributes to the development of C. sinensis and that the autophagy pathway could be an important site of action with respect to the developmental role of CsRab7 in C. sinensis.

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.

Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

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

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. Throughmore » 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.« less

Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish

PubMed Central

Clark, Brian S.; Winter, Mark; Cohen, Andrew R.; Link, Brian A.

2011-01-01

The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions. PMID:21976318

Two Rab5 Homologs Are Essential for the Development and Pathogenicity of the Rice Blast Fungus Magnaporthe oryzae

PubMed Central

Yang, Cheng D.; Dang, Xie; Zheng, Hua W.; Chen, Xiao F.; Lin, Xiao L.; Zhang, Dong M.; Abubakar, Yakubu S.; Chen, Xin; Lu, Guodong; Wang, Zonghua; Li, Guangpu; Zhou, Jie

2017-01-01

The rice blast fungus, Magnaporthe oryzae, infects many economically important cereal crops, particularly rice. It has emerged as an important model organism for studying the growth, development, and pathogenesis of filamentous fungi. RabGTPases are important molecular switches in regulation of intracellular membrane trafficking in all eukaryotes. MoRab5A and MoRab5B are Rab5 homologs in M. oryzae, but their functions in the fungal development and pathogenicity are unknown. In this study, we have employed a genetic approach and demonstrated that both MoRab5A and MoRab5B are crucial for vegetative growth and development, conidiogenesis, melanin synthesis, vacuole fusion, endocytosis, sexual reproduction, and plant pathogenesis in M. oryzae. Moreover, both MoRab5A and MoRab5B show similar localization in hyphae and conidia. To further investigate possible functional redundancy between MoRab5A and MoRab5B, we overexpressed MoRAB5A and MoRAB5B, respectively, in MoRab5B:RNAi and MoRab5A:RNAi strains, but neither could rescue each other’s defects caused by the RNAi. Taken together, we conclude that both MoRab5A and MoRab5B are necessary for the development and pathogenesis of the rice blast fungus, while they may function independently. PMID:28529514

Disruption of endocytic trafficking protein Rab7 impairs invasiveness of cholangiocarcinoma cells.

PubMed

Suwandittakul, Nantana; Reamtong, Onrapak; Molee, Pattamaporn; Maneewatchararangsri, Santi; Sutherat, Maleerat; Chaisri, Urai; Wongkham, Sopit; Adisakwattana, Poom

2017-09-07

Alterations and mutations of endo-lysosomal trafficking proteins have been associated with cancer progression. Identification and characterization of endo-lysosomal trafficking proteins in invasive cholangiocarcinoma (CCA) cells may benefit prognosis and drug design for CCA. To identify and characterize endo-lysosomal trafficking proteins in invasive CCA. A lysosomal-enriched fraction was isolated from a TNF-α induced invasive CCA cell line (KKU-100) and uninduced control cells and protein identification was performed with nano-LC MS/MS. Novel lysosomal proteins that were upregulated in invasive CCA cells were validated by real-time RT-PCR. We selected Rab7 for further studies of protein level using western blotting and subcellular localization using immunofluorescence. The role of Rab7 in CCA invasion was determined by siRNA gene knockdown and matrigel transwell assay. Rab7 mRNA and protein were upregulated in invasive CCA cells compared with non-treated controls. Immunofluorescence studies demonstrated that Rab7 was expressed predominantly in invasive CCA cells and was localized in the cytoplasm and lysosomes. Suppression of Rab7 translation significantly inhibited TNF-α-induced cell invasion compared to non-treated control (p= 0.044). Overexpression of Rab7 in CCA cells was associated with cell invasion, supporting Rab7 as a novel candidate for the development of diagnostic and therapeutic strategies for CCA.

Select Rab GTPases Regulate the Pulmonary Endothelium via Endosomal Trafficking of Vascular Endothelial-Cadherin.

PubMed

Chichger, Havovi; Braza, Julie; Duong, Huetran; Boni, Geraldine; Harrington, Elizabeth O

2016-06-01

Pulmonary edema occurs in settings of acute lung injury, in diseases, such as pneumonia, and in acute respiratory distress syndrome. The lung interendothelial junctions are maintained in part by vascular endothelial (VE)-cadherin, an adherens junction protein, and its surface expression is regulated by endocytic trafficking. The Rab family of small GTPases are regulators of endocytic trafficking. The key trafficking pathways are regulated by Rab4, -7, and -9. Rab4 regulates the recycling of endosomes to the cell surface through a rapid-shuttle process, whereas Rab7 and -9 regulate trafficking to the late endosome/lysosome for degradation or from the trans-Golgi network to the late endosome, respectively. We recently demonstrated a role for the endosomal adaptor protein, p18, in regulation of the pulmonary endothelium through enhanced recycling of VE-cadherin to adherens junction. Thus, we hypothesized that Rab4, -7, and -9 regulate pulmonary endothelial barrier function through modulating trafficking of VE-cadherin-positive endosomes. We used Rab mutants with varying activities and associations to the endosome to study endothelial barrier function in vitro and in vivo. Our study demonstrates a key role for Rab4 activation and Rab9 inhibition in regulation of vascular permeability through enhanced VE-cadherin expression at the interendothelial junction. We further showed that endothelial barrier function mediated through Rab4 is dependent on extracellular signal-regulated kinase phosphorylation and activity. Thus, we demonstrate that Rab4 and -9 regulate VE-cadherin levels at the cell surface to modulate the pulmonary endothelium through extracellular signal-regulated kinase-dependent and -independent pathways, respectively. We propose that regulating select Rab GTPases represents novel therapeutic strategies for patients suffering with acute respiratory distress syndrome.

Microstructure and Mechanical Properties of C/C Composite/TC17 Joints with Ag-Cu-Ti Brazing Alloy

NASA Astrophysics Data System (ADS)

Cao, Xiujie; Zhu, Ying; Guo, Wei; Peng, Peng; Ma, Kaituo

2017-12-01

Carbon/Carbon composite(C/C) was vacuum brazed to titanium alloy (TC17) using Ag-Cu-Ti brazing alloy. The effects of brazing temperature on the interfacial microstructure and joint properties were investigated by energy dispersive spectrometer (EDS), a scanning electron microscope (SEM), X-ray diffraction (XRD) and Gleeble1500D testing machine. Results show that C/C composite and TC17 were successfully brazed using AgCuTi brazing alloy. Various phases including TiC, Ag(s, s), Cu(s, s), Ti3Cu4, TiCu, and Ti2Cu were formed in the brazed joint. The maximum shear strength of the brazed joints with AgCuTi brazing alloy was 24±1 MPa when brazed at 860°C for 15 min.

RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

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

Bello, Oscar D.; Zanetti, M. Natalia; Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM

2012-03-10

Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggestedmore » as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black

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.

Localization and functional analysis of the insect-specific RabX4 in the brain of Bombyx mori.

PubMed

Uno, Tomohide; Furutani, Masayuki; Sakamoto, Katsuhiko; Uno, Yuichi; Kanamaru, Kengo; Mizoguchi, Akira; Hiragaki, Susumu; Takeda, Makio

2017-09-01

Rab proteins are small monomeric GTPases/GTP-binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect-specific Rab protein that has no close homolog in vertebrates. There is little information about insect-specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4-like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori. © 2017 Wiley Periodicals, Inc.

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.

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).

The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network

PubMed Central

2017-01-01

Many intracellular pathogens subvert host membrane trafficking pathways to promote their replication. Toxoplasma multiplies in a membrane-bound parasitophorous vacuole (PV) that interacts with mammalian host organelles and intercepts Golgi Rab vesicles to acquire sphingolipids. The mechanisms of host vesicle internalization and processing within the PV remain undefined. We demonstrate that Toxoplasma sequesters a broad range of Rab vesicles into the PV. Correlative light and electron microscopy analysis of infected cells illustrates that intravacuolar Rab1A vesicles are surrounded by the PV membrane, suggesting a phagocytic-like process for vesicle engulfment. Rab11A vesicles concentrate to an intravacuolar network (IVN), but this is reduced in Δgra2 and Δgra2Δgra6 parasites, suggesting that tubules stabilized by the TgGRA2 and TgGRA6 proteins secreted by the parasite within the PV contribute to host vesicle sequestration. Overexpression of a phospholipase TgLCAT, which is localized to the IVN, results in a decrease in the number of intravacuolar GFP-Rab11A vesicles, suggesting that TgLCAT controls lipolytic degradation of Rab vesicles for cargo release. PMID:29070609

Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs

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

Bello, Oscar Daniel; Cappa, Andrea Isabel; Paola, Matilde de

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 blotmore » 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. - Highlights: • Rab3A has a similar migration pattern to cortical granules in mouse oocytes. • Rab3A can be a

Welding, brazing, and soldering handbook

NASA Technical Reports Server (NTRS)

Kilgore, A. B.; Koehler, M. L.; Metzler, J. W.; Sturges, S. R.

1969-01-01

Handbook gives information on the selection and application of welding, brazing, and soldering techniques for joining various metals. Summary descriptions of processes, criteria for process selection, and advantages of different methods are given.

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.

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

Rab6a/a' are important Golgi regulators of pro-inflammatory TNF secretion in macrophages.

PubMed

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.

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.

Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways.

PubMed

Fuchs, Evelyn; Haas, Alexander K; Spooner, Robert A; Yoshimura, Shin-ichiro; Lord, J Michael; Barr, Francis A

2007-06-18

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.

Rab14 and Its Exchange Factor FAM116 Link Endocytic Recycling and Adherens Junction Stability in Migrating Cells

PubMed Central

Linford, Andrea; Yoshimura, Shin-ichiro; Bastos, Ricardo Nunes; Langemeyer, Lars; Gerondopoulos, Andreas; Rigden, Daniel J.; Barr, Francis A.

2012-01-01

Summary Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions. PMID:22595670

Cell type-specific Rab32 and Rab38 cooperate with the ubiquitous lysosome biogenesis machinery to synthesize specialized lysosome-related organelles

PubMed Central

Bultema, Jarred J.; Di Pietro, Santiago M.

2013-01-01

Lysosome-related organelles (LROs) exist in specialized cells to serve specific functions and typically co-exist with conventional lysosomes. The biogenesis of LROs is known to utilize much of the common protein machinery used in the transport of integral membrane proteins to lysosomes. Consequently, an outstanding question in the field has been how specific cargoes are trafficked to LROs instead of lysosomes, particularly in cells that simultaneously produce both organelles. One LRO, the melanosome, is responsible for the production of the pigment melanin and has long been used as a model system to study the formation of specialized LROs. Importantly, melanocytes, where melanosomes are synthesized, are a cell type that also produces lysosomes and must therefore segregate traffic to each organelle. Two small GTPases, Rab32 and Rab38, are key proteins in the biogenesis of melanosomes and were recently shown to redirect the ubiquitous machinery—BLOC-2, AP-1 and AP-3—to traffic specialized cargoes to melanosomes in melanocytes. In addition, the study revealed Rab32 and Rab38 have both redundant and unique roles in the trafficking of melanin-producing enzymes and overall melanosome biogenesis. Here we review these findings, integrate them with previous knowledge on melanosome biogenesis and discuss their implications for biogenesis of other LROs. PMID:23247405

Rab15 Effector Protein: A Novel Protein for Receptor Recycling from the Endocytic Recycling CompartmentD⃞

PubMed Central

Strick, David J.; Elferink, Lisa A.

2005-01-01

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways. PMID:16195351

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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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

Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.

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 bemore » 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.« less

High-temperature brazing for reliable tungsten CFC joints

NASA Astrophysics Data System (ADS)

Koppitz, Th; Pintsuk, G.; Reisgen, U.; Remmel, J.; Hirai, T.; Sievering, R.; Rojas, Y.; Casalegno, V.

2007-03-01

The joining of tungsten and carbon-based materials is demanding due to the incompatibility of their chemical and thermophysical properties. Direct joining is unfeasible by the reason of brittle tungsten carbide formation. High-temperature brazing has been investigated in order to find a suitable brazing filler metal (BFM) which successfully acts as an intermediary between the incompatible properties of the base materials. So far only low Cr-alloyed Cu-based BFMs provide the preferential combination of good wetting action on both materials, tolerable interface reactions, and a precipitation free braze joint. Attempts to implement a higher melting metal (e.g. Pd, Ti, Zr) as a BFM have failed up to now, because the formation of brittle precipitations and pores in the seam were inevitable. But the wide metallurgical complexity of this issue is regarded to offer further joining potential.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

PubMed

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

Preliminary study on pressure brazing and diffusion welding of Nb-1Zr to Inconel 718

NASA Technical Reports Server (NTRS)

Moore, T. J.

1990-01-01

Future space power systems may include Nb-1Zr/Inconel 718 dissimilar metal joints for operation at 1000 K for 60,000 h. The serviceability of pressure-brazed and diffusion-welded joints was investigated. Ni-based metallic glass foil filler metals were used for brazing. Ni and Fe foils were used as diffusion welding inter-layers. Joint soundness was determined by metallographic examination in the as-brazed and as-welded condition, after aging at 1000 K, and after thermal cycling. Brazed joints thermally cycled in the as-brazed condition and diffusion-welded joints were unsatisfactory because of cracking problems. Brazed joints may meet the service requirements if the joints are aged at 1000 K prior to thermal cycling.

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

PubMed Central

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. PMID:23555015

Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models

PubMed Central

Ramalho, José S; Anders, Ross; Jaissle, Gesine B; Seeliger, Mathias W; Huxley, Clare; Seabra, Miguel C

2002-01-01

Background Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process. Results To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal. Conclusions We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases. PMID:12401133

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)

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. © 2015 Wiley Periodicals, Inc.

A phorbol ester-binding protein is required downstream of Rab5 in endosome fusion.

PubMed

Aballay, A; Barbieri, M A; Colombo, M I; Arenas, G N; Stahl, P D; Mayorga, L S

1998-12-28

Previous observations indicate that a zinc and phorbol ester binding factor is necessary for endosome fusion. To further characterize the role of this factor in the process, we used an in vitro endosome fusion assay supplemented with recombinant Rab5 proteins. Both zinc depletion and addition of calphostin C, an inhibitor of protein kinase C, inhibited endosome fusion in the presence of active Rab5. Addition of the phorbol ester PMA (phorbol 12-myristate 13-acetate) reversed the inhibition of endosome fusion caused by a Rab5 negative mutant. Moreover, PMA stimulated fusion in the presence of Rab5 immunodepleted cytosol. These results suggest that the phorbol ester binding protein is acting downstream of Rab5 in endosome fusion.

Endocytosis in the Shiitake Mushroom Lentinula edodes and Involvement of GTPase LeRAB7▿

PubMed Central

Lee, Ming Tsung; Szeto, Carol Ying Ying; Ng, Tak Pan; Kwan, Hoi Shan

2007-01-01

Endocytosis is the process by which substrates enter a cell without passing through the plasma membrane but rather invaginate the cell membrane and form intracellular vesicles. Rab7 regulates endocytic trafficking between early and late endosomes and between late endosomes and lysosomes. LeRab7 in Lentinula edodes is strongly homologous to Rab7 in Homo sapiens. Receptors for activated C kinase-1 (LeRACK1) and Rab5 GTPase (LeRAB5) were isolated as interacting partners of LeRab7, and the interactions were confirmed by in vivo and in vitro protein interaction assays. The three genes showed differential expression in the various developmental stages of the mushroom. In situ hybridization showed that the three transcripts were localized in regions of active growth, such as the outer region of trama cells, and the subhymenium of the hymenophore of mature fruiting bodies and the prehymenophore of young fruiting bodies. The existence of endocytosis in the mycelium and hymenophores was confirmed by the internalization of FM4-64. LeRAB7 was partially colocalized with the AM4-64 and was located in the late endocytic pathway. This is the first report of the presence of endocytosis in homobasidiomycetes. LeRAB7, LeRAB5, and LeRACK1 may contribute to the growth of L. edodes and cell differentiation in hymenophores. PMID:17921351

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. © 2015. Published by The Company of Biologists Ltd.

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. (c) 2005 Wiley Periodicals, Inc.

TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans

PubMed Central

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M.; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. PMID:22654674

Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

PubMed Central

Zacchi, Paola; Stenmark, Harald; Parton, Robert G.; Orioli, Donata; Lim, Filip; Giner, Angelika; Mellman, Ira; Zerial, Marino; Murphy, Carol

1998-01-01

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells. PMID:9490718

Rab5a‑mediated autophagy regulates the phenotype and behavior of vascular smooth muscle cells.

PubMed

Tan, Jin-Yun; Jia, Luo-Qi; Shi, Wei-Hao; He, Qing; Zhu, Lei; Yu, Bo

2016-11-01

Rab5a, a key member of the Rab family of GTPases, was determined to be a regulator of vascular smooth muscle cell (VSMC) proliferation and migration. However, the exact regulatory mechanism remains unclear. As Rab5a has been shown to be associated with autophagy, which is essential for the conversion of VSMCs from a contractile to a synthetic phenotype in order to prevent cell death due to oxidative stress. The present study hypothesized that autophagy may be responsible for the proliferation and migration of VSMCs via the Rab5a protein. The aim of the present study was to evaluate the effect of Rab5a on autophagy in VSMCs. The human aorta vascular smooth muscle cell line, T/G HA‑VSMCs, was treated with small interfering (si)RNA against Rab5a and/or platelet‑derived growth factor (PDGF). Following treatment, the phenotype transition of the VSMCs was evaluated by detecting the mRNA and protien expression levels of VSMC molecular markers using reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. In addition, autophagy in VSMCs was evaluated by western blotting for autophagy‑associated proteins, flow cytometry of acidic vesicular organelles, punctate fluorescence of microtubule associated protein light chain 3 and transmission electron microscopy of typical scattered double‑membrane vacuolar structures. Additionally, the proliferation, migration, cell cycle and apoptotic response of VSMCs were detected by sulforhodamine B assay, transwell assay and flow cytometry, respectively. The results revealed that transfection with siRNA against Rab5a led to a significant decrease in Rab5a protein expression, while the reduced expression trend of Rab5a was rescued by intervention with PDGF. Furthermore, cells transfected with siRNA against Rab5a inhibited the autophagy of VSMCs. Downregulated Rab5a inhibited the phenotype transition of VSMCs. Additionally, downregulated Rab5a led to slowed cell growth, decreased numbers of

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

PubMed Central

Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

2013-01-01

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

Microstructure characteristics of vacuum glazing brazing joints using laser sealing technique

NASA Astrophysics Data System (ADS)

Liu, Sixing; Yang, Zheng; Zhang, Jianfeng; Zhang, Shanwen; Miao, Hong; Zhang, Yanjun; Zhang, Qi

2018-05-01

Two pieces of plate glass were brazed into a composite of glazing with a vacuum chamber using PbO-TiO2-SiO2-RxOy powder filler alloys to develop a new type of vacuum glazing. The brazing process was carried out by laser technology. The interface characteristics of laser brazed joints formed between plate glass and solder were investigated using optical microscope, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) techniques. The results show that the inter-diffusion of Pb/Ti/Si/O elements from the sealing solder toward the glass and O/Al/Si elements from the glass toward the solder, resulting in a reaction layer in the brazed joints. The microstructure phases of PbTiO3, AlSiO, SiO2 and PbO in the glass/solder interface were confirmed by XRD analysis. The joining of the sealing solder to the glass was realized by the reaction products like fibrous structures on interface, where the wetting layer can help improve the bonding performance and strength between the sealing solder and the plate glass during the laser brazing process.

Brazing of refractory, superalloy, and composite materials for Space Shuttle applications.

NASA Technical Reports Server (NTRS)

Beuyukian, C. S.

1972-01-01

Research work concerning the metallic portion of the shuttle-orbiter heat shield (expected to experience temperatures up to 2500 F) is described. The five metals being evaluated are TD-Ni-Cr, Cb-C129Y, Cb752, Hayes 188, and Inconel 625. Brazing techniques whereby pairs of these materials are joined into thin-membered assemblies for heat shield applications are described. Results obtained with the vacuum-furnace brazing process are examined. In addition, the use of brazed aluminum-boron metal-matrix-contoured composite structures for heat shield applications is evaluated.

Targeting eukaryotic Rab proteins: a smart strategy for chlamydial survival and replication.

PubMed

Damiani, María Teresa; Gambarte Tudela, Julián; Capmany, Anahí

2014-09-01

Chlamydia, an obligate intracellular bacterium which passes its entire lifecycle within a membrane-bound vacuole called the inclusion, has evolved a variety of unique strategies to establish an advantageous intracellular niche for survival. This review highlights the mechanisms by which Chlamydia subverts vesicular transport in host cells, particularly by hijacking the master controllers of eukaryotic trafficking, the Rab proteins. A subset of Rabs and Rab interacting proteins that control the recycling pathway or the biosynthetic route are selectively recruited to the chlamydial inclusion membrane. By interfering with Rab-controlled transport steps, this intracellular pathogen not only prevents its own degradation in the phagocytic pathway, but also creates a favourable intracellular environment for growth and replication. Chlamydia, a highly adapted and successful intracellular pathogen, has several redundant strategies to re-direct vesicles emerging from biosynthetic compartments that carry host molecules essential for bacterial development. Although current knowledge is limited, the latest findings have shed light on the role of Rab proteins in the course of chlamydial infections and could open novel opportunities for anti-chlamydial therapy. © 2014 John Wiley & Sons Ltd.

Novel Regulation of Integrin Trafficking by Rab11-FIP5 in Aggressive Prostate Cancer.

PubMed

Das, Lipsa; Gard, Jaime M C; Prekeris, Rytis; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Miranti, Cindy K; Cress, Anne E

2018-05-14

The laminin-binding integrins, α3β1 and α6β1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. β1 integrins share the Rab11 recycling machinery but the trafficking of α3β1 and α6β1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6β1 trafficking. Rab11-FIP5 and its membrane binding domain were required for α6β1 recycling, without affecting the other laminin-binding integrin (i.e., α3β1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6β1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6β1 recycling in cell-cell locations. Taken together, these data demonstrate that α6β1 is distinct from α3β1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location. Rab11-FIP5 dependent a6b1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Copyright ©2018, American Association for Cancer Research.

Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing.

PubMed

Bartusch, Christina; Döring, Tatjana; Prange, Reinhild

2017-06-21

Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.

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

PubMed

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

2012-02-01

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

Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor.

PubMed

Schmidlin, F; Dery, O; DeFea, K O; Slice, L; Patierno, S; Sternini, C; Grady, E F; Bunnett, N W

2001-07-06

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

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.

Experimental Investigation on High-Cycle Fatigue of Inconel 625 Superalloy Brazed Joints

NASA Astrophysics Data System (ADS)

Chen, Jianqiang; Demers, Vincent; Turner, Daniel P.; Bocher, Philippe

2018-04-01

The high-cycle fatigue performance and crack growth pattern of transient liquid phase-brazed joints in a nickel-based superalloy Inconel 625 were studied. Assemblies with different geometries and types of overlaps were vacuum-brazed using the brazing paste Palnicro-36M in conditions such as to generate eutectic-free joints. This optimal microstructure provides the brazed assemblies with static mechanical strength corresponding to that of the base metal. However, eutectic micro-constituents were observed in the fillet region of the brazed assembly due to an incomplete isothermal solidification within this large volume of filler metal. The fatigue performance increased significantly with the overlap distance for single-lap joints, and the best performance was found for double-lap joints. It was demonstrated that these apparent changes in fatigue properties according to the specimen geometry can be rationalized when looking at the fatigue data as a function of the local stress state at the fillet radii. Fatigue cracks were nucleated from brittle eutectic phases located at the surface of the fillet region. Their propagation occurred through the bimodal microstructure of fillet and the diffusion region to reach the base metal. High levels of crack path tortuosity were observed, suggesting that the ductile phases found in the microstructure may act as a potential crack stopper. The fillet region must be considered as the critical region of a brazed assembly for fatigue applications.

Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes

PubMed Central

Vonderheit, Andreas

2005-01-01

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles. PMID:15954801

Regulation of podocalyxin trafficking by Rab small GTPases in epithelial cells

PubMed Central

Mrozowska, Paulina S.; Fukuda, Mitsunori

2016-01-01

ABSTRACT The characteristic feature of polarity establishment in MDCK II cells is transcytosis of apical glycoprotein podocalyxin (PCX) from the outer plasma membrane to the newly formed apical domain. This transcytotic event consists of multiple steps, including internalization from the plasma membrane, transport through early endosomes and Rab11-positive recycling endosomes, and delivery to the apical membrane. These steps are known to be tightly coordinated by Rab small GTPases, which act as molecular switches cycling between active GTP-bound and inactive GDP-bound states. However, our knowledge regarding which sets of Rabs regulate particular steps of PCX trafficking was rather limited. Recently, we have performed a comprehensive analysis of Rab GTPase engagement in the transcytotic pathway of PCX during polarity establishment in 2-dimensional (2D) and 3-dimensional (3D) MDCK II cell cultures. In this Commentary we summarize our findings and set them in the context of previous reports. PMID:27463697

Quantitative analysis of the interactions between prenyl Rab9, GDP dissociation inhibitor-alpha, and guanine nucleotides.

PubMed

Shapiro, A D; Pfeffer, S R

1995-05-12

Rab9 is a Ras-like GTPase required for the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Rab9 occurs in the cytosol as a complex with GDP dissociation inhibitor (GDI), which we have shown delivers prenyl Rab9 to late endosomes in a functional form. We report here basal rate constants for guanine nucleotide dissociation and GTP hydrolysis for prenyl Rab9. Both rate constants were influenced in part by the hydrophobic environment of the prenyl group. Guanine nucleotide dissociation and GTP hydrolysis rates were lower in the presence of lipid; detergent stimulated intrinsic nucleotide exchange. GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 2.4-fold. GDI-alpha associated with prenyl Rab9 with a KD of 60 nM in 0.1% Lubrol and 23 nM in 0.02% Lubrol. In 0.1% Lubrol, GDI-alpha inhibited GDP dissociation half maximally at 72 +/- 18 nM, consistent with the KD determinations. These data suggest that GDI-alpha associates with prenyl Rab9 with a KD of < or = 23 nM under physiological conditions. Finally, a previously uncharacterized minor form of GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 1.9-fold and bound prenyl Rab9 with a KD of 67 nM in 0.1% Lubrol.

BLOC-3 Mutated in Hermansky-Pudlak Syndrome Is a Rab32/38 Guanine Nucleotide Exchange Factor

PubMed Central

Gerondopoulos, Andreas; Langemeyer, Lars; Liang, Jin-Rui; Linford, Andrea; Barr, Francis A.

2012-01-01

Summary Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting [1–3]. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles [2]. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes [4–6]. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious [7–9]. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38 [10–12]. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles. PMID:23084991

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes*

PubMed Central

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-01

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

Dispersed metal-toughened ceramics and ceramic brazing

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

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 measurementmore » 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.« less

The Protease Activated Receptor2 Promotes Rab5a Mediated Generation of Pro-metastatic Microvesicles.

PubMed

Das, Kaushik; Prasad, Ramesh; Roy, Sreetama; Mukherjee, Ashis; Sen, Prosenjit

2018-05-09

Metastasis, the hallmark of cancer propagation is attributed by the modification of phenotypic/functional behavior of cells to break attachment and migrate to distant body parts. Cancer cell-secreted microvesicles (MVs) contribute immensely in disease propagation. These nano-vesicles, generated from plasma membrane outward budding are taken up by nearby healthy cells thereby inducing phenotypic alterations in those recipient cells. Protease activated receptor 2 (PAR2), activated by trypsin, also contributes to cancer progression by increasing metastasis, angiogenesis etc. Here, we report that PAR2 activation promotes pro-metastatic MVs generation from human breast cancer cell line, MDA-MB-231. Rab5a, located at the plasma membrane plays vital roles in MVs biogenesis. We show that PAR2 stimulation promotes AKT phosphorylation which activates Rab5a by converting inactive Rab5a-GDP to active Rab5a-GTP. Active Rab5a polymerizes actin which critically regulates MVs shedding. Not only MVs generation, has this Rab5a activation also promoted cell migration and invasion. We reveal that Rab5a is over-expressed in human breast tumor specimen and contributes MVs generation in those patients. The involvement of p38 MAPK in MVs-induced cell metastasis has also been highlighted in the present study. Blockade of Rab5a activation can be a potential therapeutic approach to restrict MVs shedding and associated breast cancer metastasis.

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica.

PubMed

Liu, Zhao-liang; Luo, Cong; Dong, Long; Van Toan, Can; Wei, Peng-xiao; He, Xin-hua

2014-04-25

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984bp and contained an open reading frame of 600bp, which encoded a 200 amino acid protein with a molecular weight of 21.83kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant. Copyright © 2014 Elsevier B.V. All rights reserved.

Rab11a and its binding partners regulate the recycling of the β1-adrenergic receptor

PubMed Central

Gardner, Lidia A.; Hajjhussein, Hassan; Frederick, Katherine C.; Bahouth, Suleiman W.

2010-01-01

β1-adrenergic receptors (β1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the β1-AR (S312A) is internalized but does not recycle. We determined that WT β1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by >70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT β1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT β1-AR were colocalized by >70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT β1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the β1-AR. Next, we determined the effect of each of the rab11-intercating proteins on trafficking of the WT β1-AR. The recycling of the β1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role recycling of the human β1-AR. PMID:20727405

Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

PubMed Central

Sadacca, L. Amanda; Bruno, Joanne; Wen, Jennifer; Xiong, Wenyong; McGraw, Timothy E.

2013-01-01

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation. PMID:23804653

Leishmania major large RAB GTPase is highly immunogenic in individuals immune to cutaneous and visceral leishmaniasis.

PubMed

Chamakh-Ayari, Rym; Chenik, Mehdi; Chakroun, Ahmed Sahbi; Bahi-Jaber, Narges; Aoun, Karim; Meddeb-Garnaoui, Amel

2017-04-17

We previously identified a Leishmania (L.) major large RAB GTPase (LmlRAB), a new atypical RAB GTPase protein. It is highly conserved in Leishmania species while displaying low level of homology with mammalian homologues. Leishmania small RAB GTPases proteins have been involved in regulation of exocytic and endocytic pathways whereas the role of large RAB GTPases proteins has not been characterized yet. We report here the immunogenicity of both recombinant rLmlRAB and rLmlRABC, in individuals with immunity against L. major or L. infantum. PBMC were isolated from individuals cured of L. major (CCLm) or from healthy individuals. The latter were subdivided into high or low IFN-γ responders. Healthy high IFN-γ responders, considered as asymptomatics, were living in an endemic area for L. major (HHRLm) or L. infantum (HHRLi). Healthy low IFN-γ responders (HLR) were considered as naïve controls. Cells from all volunteers were stimulated with rLmlRAB or rLmlRABC. Cytokines were analysed by CBA and ELISA and phenotypes of IFN-γ-producing cells were analysed by flow cytometry. Both rLmlRAB and rLmlRABC induced high significant levels of IFN-γ in CCLm, HHRLm and HHRLi groups. Phenotype analysis of rLmlRAB and rLmlRABC-stimulated T cells in CCLm individuals showed a significant increase in the percentage of specific IFN-γ-producing CD4+ and CD8+ T cells. rLmlRAB induced significant granzyme B levels in CCLm and HHRLm. Low but significant granzyme B levels were detected in naïve group. IL-10 was detected in immune and naïve individuals. We showed that rLmlRAB protein and its divergent carboxy-terminal part induced a predominant Th1 response in individuals immune to L. major or L. infantum. Our results suggest that rLmlRAB and rLmlRABC proteins are potential cross-species vaccine candidates against cutaneous and visceral leishmaniasis.

Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

PubMed Central

Catz, Sergio Daniel

2013-01-01

The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. PMID:23378593

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport

PubMed Central

Guichard, Annabel; Jain, Prashant; Moayeri, Mahtab; Cruz-Moreno, Beatriz; Leppla, Stephen H.; Nizet, Victor

2017-01-01

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies. PMID:28945820

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.

PubMed

Guichard, Annabel; Jain, Prashant; Moayeri, Mahtab; Schwartz, Ruth; Chin, Stephen; Zhu, Lin; Cruz-Moreno, Beatriz; Liu, Janet Z; Aguilar, Bernice; Hollands, Andrew; Leppla, Stephen H; Nizet, Victor; Bier, Ethan

2017-09-01

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.

Differential expression and activation of Rab27A in human eosinophils: relationship to blood eosinophilia.

PubMed

Coughlin, Jason J; Odemuyiwa, Solomon O; Davidson, Courtney E; Moqbel, Redwan

2008-08-29

Eosinophil degranulation is thought to play a pathophysiological role in asthma. Rab27A is a GTP-binding protein that is known to be essential for the degranulation of several leukocyte subsets and thus may be essential for eosinophil granule exocytosis. Here, we show that Rab27A mRNA and protein are expressed in human eosinophils. We have developed a novel assay to assess Rab27A activation and have found a similar activation pattern of this protein upon stimulation of eosinophils, neutrophils and NK cells suggesting a similar function in these cell types. Interestingly, Rab27A expression was elevated in eosinophils from asthmatic donors. Furthermore, eosinophils from eosinophilic donors displayed more rapid Rab27A activation kinetics than those from donors with lower eosinophil counts. Given that elevated blood eosinophil numbers correlate with increased priming of eosinophils, this pattern of Rab27A activation suggests differential protein expression in activated cells may allow eosinophils to degranulate more rapidly upon stimulation.

Multi-Scale Computational Modeling of Ni-Base Superalloy Brazed Joints for Gas Turbine Applications

NASA Astrophysics Data System (ADS)

Riggs, Bryan

Brazed joints are commonly used in the manufacture and repair of aerospace components including high temperature gas turbine components made of Ni-base superalloys. For such critical applications, it is becoming increasingly important to account for the mechanical strength and reliability of the brazed joint. However, material properties of brazed joints are not readily available and methods for evaluating joint strength such as those listed in AWS C3.2 have inherent challenges compared with testing bulk materials. In addition, joint strength can be strongly influenced by the degree of interaction between the filler metal (FM) and the base metal (BM), the joint design, and presence of flaws or defects. As a result, there is interest in the development of a multi-scale computational model to predict the overall mechanical behavior and fitness-for-service of brazed joints. Therefore, the aim of this investigation was to generate data and methodology to support such a model for Ni-base superalloy brazed joints with conventional Ni-Cr-B based FMs. Based on a review of the technical literature a multi-scale modeling approach was proposed to predict the overall performance of brazed joints by relating mechanical properties to the brazed joint microstructure. This approach incorporates metallurgical characterization, thermodynamic/kinetic simulations, mechanical testing, fracture mechanics and finite element analysis (FEA) modeling to estimate joint properties based on the initial BM/FM composition and brazing process parameters. Experimental work was carried out in each of these areas to validate the multi-scale approach and develop improved techniques for quantifying brazed joint properties. Two Ni-base superalloys often used in gas turbine applications, Inconel 718 and CMSX-4, were selected for study and vacuum furnace brazed using two common FMs, BNi-2 and BNi-9. Metallurgical characterization of these brazed joints showed two primary microstructural regions; a soft

A Review of Research Progress on Dissimilar Laser Weld-Brazing of Automotive Applications

NASA Astrophysics Data System (ADS)

Krishnaja, Devireddy; Cheepu, Muralimohan; Venkateswarlu, D.

2018-03-01

In recent years, a rapidly growing demand for laser brazing in the transportation industry for automotive parts joining to improve the productivity, quality of the joints and cost efficiency reasons. Due to this, laser brazing technology is extensively used in the major manufacturing companies such as Volkswagen group, General Motors Europe, BMW and Ford manufacturing groups as their openingbulk production solicitation on various parts of vehicles. Laser brazing is different from the welding processes and it will block upanopeningamongst two substrates by mixture of a filler wire on condition that by a concentrated laser beam or any other heat source. Among the all joining processes, laser brazing technique is an alternative and in effect method for welding of dissimilar metals which have large difference in their melting points. It is important to understand therelationsof these phenomena of the fillers of brazing with the substrate surfaces to obtain a high quality joints. The aim of this study is to address the contemporaryenquiriesand its progress on laser-brazing, its importance to the industrial applications and to bring more awareness to the manufacturers about the research results of this technique from various research groups to enhance the research progress and developing new things from this review report.

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.

A role for the Rab6A′ GTPase in the inactivation of the Mad2-spindle checkpoint

PubMed Central

Miserey-Lenkei, Stéphanie; Couëdel-Courteille, Anne; Del Nery, Elaine; Bardin, Sabine; Piel, Matthieu; Racine, Victor; Sibarita, Jean-Baptiste; Perez, Franck; Bornens, Michel; Goud, Bruno

2006-01-01

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A′, regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A′ function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2-spindle checkpoint activated. Furthermore, the Rab6 effector p150Glued, a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase-activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A′ likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2-spindle checkpoint. Rab6A′, through its interaction with p150Glued and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition. PMID:16395330

The Rab-binding Profiles of Bacterial Virulence Factors during Infection*

PubMed Central

So, Ernest C.; Schroeder, Gunnar N.; Carson, Danielle; Mattheis, Corinna; Mousnier, Aurélie; Broncel, Malgorzata; Tate, Edward W.; Frankel, Gad

2016-01-01

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection. PMID:26755725

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

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. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Electron Beam Brazing of Titanium for Construction in Space

NASA Technical Reports Server (NTRS)

Flom, Yury

2006-01-01

An extended presence of humans in space requires an in-situ capability to construct various permanent structures to support scientific research, power generation, communication, radiation shielding and other functions. Electron Beam (EB) vacuum brazing has been identified as one of the best joining processes for in-space joining, particular for making a large quantity of permanent joints as required for construction of the sizeable truss structures. Thin wall titanium tubes are perhaps the best choice because of their high stiffness, excellent strength-to-weight ratio and great metal forming and joining ability. An innovative EB vacuum spot brazing process is being developed at Goddard Space Flight Center to be used for robotic as well as human-assisted construction in space. This paper describes experimental results obtained during the initial effort of EB brazing of titanium tubes with the special emphasis on low temperature aluminum filler metals.

APPLICATION OF ELECTROLESS-NICKEL BRAZING TO TUBULAR FUEL ELEMENTS FOR THE N.S. SAVANNAH. Status Report

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

Lamartine, J T; Thurber, W C

1959-06-01

The feasibility of using electroless nickel, a chemical deposit containing about 10 wt.% phosphorous in nickel, as the brazing alloy for assembling tubular stainless steel fuel elements of the type specified in Core I of the N. S. Savannah was investigated. This material was selected primarily because of the ease of braze-metal preplacement by chemical deposition of the alloy on type 304 stainiess steel ferrule spacers, prior to fuelbundle assembly. Brazed joints produced by this method were generally characterized by a relatively ductile solid-solution region at the thinnest portions of the fillet. This ductile zone should minimize the possibility ofmore » complete propagation of hairline cracks, which form in the brittle, eutectic regions of fillet. The microstructural appearance of the electroless-nickel joints was not appreciably affected by variations in the brazing temperature from 1750 to 1900 deg F or the brazing time from 15 to 60 min. Several plating solutions were evaluated and all were found to be capable of producing deposits suitable for brazing applications. Corrosion tests conducted in static 525 deg F water indicated that no significant attack of joints brazed with electroless nickel had occurred after 300-hr exposure. A small fuel bundle was successfully assembled by brazing with electroless nickel. (auth)« less

Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic.

PubMed

Yamane, Junko; Kubo, Akiharu; Nakayama, Kazuhisa; Yuba-Kubo, Akiko; Katsuno, Tatsuya; Tsukita, Shoichiro; Tsukita, Sachiko

2007-10-01

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.

Extensive in silico analysis of Mimivirus coded Rab GTPase homolog suggests a possible role in virion membrane biogenesis.

PubMed

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.

The Rab11 Effector Protein FIP1 Regulates Adiponectin Trafficking and Secretion

PubMed Central

Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia

2013-01-01

Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release. PMID:24040321

The role of the oncogenic Rab35 in cancer invasion, metastasis, and immune evasion, especially in leukemia.

PubMed

Villagomez, Fabian R; Medina-Contreras, Oscar; Cerna-Cortes, Jorge Francisco; Patino-Lopez, Genaro

2018-05-28

The study of cancer has allowed researchers to describe some biological characteristics that tumor cells acquire during their development, known as the "hallmarks of cancer" but more research is needed to expand our knowledge about cancer biology and to generate new strategies of treatment. The role that RabGTPases might play in some hallmarks of cancer represents interesting areas of study since these proteins are frequently altered in cancer. However, their participation is not well known. Recently, Rab35was recognized as an oncogenic RabGTPase and and because of its association with different cellular functions, distinctly important in immune cells, a possible role of Rab35 in leukemia can be suggested. Nevertheless, the involvement of Rab35 in cancer remains poorly understood and its possible specific role in leukemia remains unknown. In this review, we analyze general aspects of the participation of RabGTPases in cancer, and especially, the plausible role of Rab35 in leukemia.

Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein

PubMed Central

Sato, Miyuki; Sato, Ken; Liou, Willisa; Pant, Saumya; Harada, Akihiro; Grant, Barth D

2008-01-01

Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP–RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP–RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling. PMID:18354496

Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer | Office of Cancer Genomics

Cancer.gov

Recent evidence has established a role for the small GTPase RAB25, as well as related effector proteins, in enacting both pro-oncogenic and anti-oncogenic phenotypes in specific cellular contexts. Here we report the development of all-hydrocarbon stabilized peptides derived from the RAB-binding FIP-family of proteins to target RAB25. Relative to unmodified peptides, optimized stapled peptides exhibit increased structural stability, binding affinity, cell permeability, and inhibition of RAB25:FIP complex formation.

Rab25 acts as an oncogene in luminal B breast cancer and is causally associated with Snail driven EMT

PubMed Central

Mitra, Shreya; Federico, Lorenzo; Zhao, Wei; Dennison, Jennifer; Sarkar, Tapasree Roy; Zhang, Fan; Takiar, Vinita; Cheng, Kwai W.; Mani, Sendurai; Lee, Ju Seog; Mills, Gordon B.

2016-01-01

The Rab GTPases regulate vesicular trafficking machinery that transports and delivers a diverse pool of cargo, including growth factor receptors, integrins, nutrient receptors and junction proteins to specific intracellular sites. The trafficking machinery is indeed a major posttranslational modifier and is critical for cellular homeostasis. Deregulation of this stringently controlled system leads to a wide spectrum of disorders including cancer. Herein we demonstrate that Rab25, a key GTPase, mostly decorating the apical recycling endosome, is a dichotomous variable in breast cancer cell lines with higher mRNA and protein expression in Estrogen Receptor positive (ER+ve) lines. Rab25 and its effector, Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER+ve breast cancers. In contrast, Rab25 levels are decreased in basal-like and almost completely lost in claudin-low tumors. This dichotomy exists despite the presence of the 1q amplicon that hosts Rab25 across breast cancer subtypes and is likely due to differential methylation of the Rab25 promoter. Functionally, elevated levels of Rab25 drive major hallmarks of cancer including indefinite growth and metastasis but in case of luminal B breast cancer only. Importantly, in such ER+ve tumors, coexpression of Rab25 and its effector, RCP is significantly associated with a markedly worsened clinical outcome. Importantly, in claudin-low cell lines, exogenous Rab25 markedly inhibits cell migration. Similarly, during Snail-induced epithelial to mesenchymal transition (EMT) exogenous Rab25 potently reverses Snail-driven invasion. Overall, this study substantiates a striking context dependent role of Rab25 in breast cancer where Rab25 is amplified and enhances aggressiveness in luminal B cancers while in claudin-low tumors, Rab25 is lost indicating possible anti-tumor functions. PMID:27259233

Rab25 acts as an oncogene in luminal B breast cancer and is causally associated with Snail driven EMT.

PubMed

Mitra, Shreya; Federico, Lorenzo; Zhao, Wei; Dennison, Jennifer; Sarkar, Tapasree Roy; Zhang, Fan; Takiar, Vinita; Cheng, Kwai W; Mani, Sendurai; Lee, Ju Seog; Mills, Gordon B

2016-06-28

The Rab GTPases regulate vesicular trafficking machinery that transports and delivers a diverse pool of cargo, including growth factor receptors, integrins, nutrient receptors and junction proteins to specific intracellular sites. The trafficking machinery is indeed a major posttranslational modifier and is critical for cellular homeostasis. Deregulation of this stringently controlled system leads to a wide spectrum of disorders including cancer. Herein we demonstrate that Rab25, a key GTPase, mostly decorating the apical recycling endosome, is a dichotomous variable in breast cancer cell lines with higher mRNA and protein expression in Estrogen Receptor positive (ER+ve) lines. Rab25 and its effector, Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER+ve breast cancers. In contrast, Rab25 levels are decreased in basal-like and almost completely lost in claudin-low tumors. This dichotomy exists despite the presence of the 1q amplicon that hosts Rab25 across breast cancer subtypes and is likely due to differential methylation of the Rab25 promoter. Functionally, elevated levels of Rab25 drive major hallmarks of cancer including indefinite growth and metastasis but in case of luminal B breast cancer only. Importantly, in such ER+ve tumors, coexpression of Rab25 and its effector, RCP is significantly associated with a markedly worsened clinical outcome. Importantly, in claudin-low cell lines, exogenous Rab25 markedly inhibits cell migration. Similarly, during Snail-induced epithelial to mesenchymal transition (EMT) exogenous Rab25 potently reverses Snail-driven invasion. Overall, this study substantiates a striking context dependent role of Rab25 in breast cancer where Rab25 is amplified and enhances aggressiveness in luminal B cancers while in claudin-low tumors, Rab25 is lost indicating possible anti-tumor functions.

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%.

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

PubMed

Miserey-Lenkei, Stéphanie; Bousquet, Hugo; Pylypenko, Olena; Bardin, Sabine; Dimitrov, Ariane; Bressanelli, Gaëlle; Bonifay, Raja; Fraisier, Vincent; Guillou, Catherine; Bougeret, Cécile; Houdusse, Anne; Echard, Arnaud; Goud, Bruno

2017-11-01

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6-Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.

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.

Chlamydia trachomatis Intercepts Golgi-Derived Sphingolipids through a Rab14-Mediated Transport Required for Bacterial Development and Replication

PubMed Central

Capmany, Anahí; Damiani, María Teresa

2010-01-01

Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation. PMID:21124879

Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication.

PubMed

Capmany, Anahí; Damiani, María Teresa

2010-11-22

Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.

Expression profile of Rab5, Rab7, tryptophan aspartate-containing coat protein, leprae lipoarabinomannan, and phenolic glycolipid-1 on the failure of the phagolysosome process in macrophages of leprosy patients as a viability marker of Mycobacterium leprae.

PubMed

Prakoeswa, Cita Rosita Sigit; Wahyuni, Ratna; Iswahyudi; Adriaty, Dinar; Yusuf, Irawan; Sutjipto; Agusni, Indropo; Izumi, Shinzo

2016-06-01

Phagolysosome process in macrophage of leprosy patients' is important in the early phase of eliminating Mycobacterium leprae invasion. This study was to clarify the involvement of Rab5, Rab7, and trytophan aspartate-containing coat protein (TACO) from host macrophage and leprae lipoarabinomannan (Lep-LAM) and phenolic glycolipid-1 (PGL-1) from M. leprae cell wall as the reflection of phagolysosome process in relation to 16 subunit ribosomal RNA (16S rRNA) M. leprae as a marker of viability of M. leprae. Using a cross sectional design study, skin biopsies were obtained from 47 newly diagnosed, untreated leprosy at Dr Soetomo Hospital, Surabaya, Indonesia. RNA isolation and complementary DNA synthesis were performed. Samples were divided into two groups: 16S rRNA M. leprae-positive and 16S rRNA M. leprae-negative. The expressions of Rab5, Rab7, TACO, Lep-LAM, and PGL-1 were assessed with an immunohistochemistry technique. Using Mann-Whitney U analysis, a significant difference in the expression profile of Rab5, Rab7, Lep-LAM, and PGL-1 was found (p<.05), but there was no significant difference of TACO between the two groups (p>.05). Spearman analysis revealed that there was a significant correlation between the score of Rab5, Rab7, Lep-LAM, and PGL-1 and the score of 16S rRNA M. leprae (p<.05). In M. leprae infection, Rab5, Rab7, and Lep-LAM play important roles in the failure of phagolysosome process via a membrane trafficking pathway, while PGL-1 plays a role via blocking lysosomal activities. These inventions might be used for the development of an early diagnostic device in the future. Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Reconstitution of recycling from the phagosomal compartment in streptolysin O-permeabilized macrophages: role of Rab11.

PubMed

Leiva, Natalia; Pavarotti, Martín; Colombo, María I; Damiani, María T

2006-06-10

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells in vesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasma membrane following phagosome biogenesis. Here, we report a new approach to the study of recycling from the phagosomal compartment: streptolysin O- (SLO) permeabilized macrophages. In this semi-intact cell system, energy and cytosol are required to efficiently reconstitute recycling transport. Addition of GDPbetaS strongly inhibits this transport step, suggesting that a GTP-binding protein modulates the dynamics of cargo exit from the phagosomal compartment. GTPases of the Rab family control vesicular trafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role of Rab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilized macrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycled proteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, we confirm that overexpression of Rab11:S25N substantially decreases recycling from phagosomes in intact cells. These findings show the requirement of a functional Rab11 for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilized macrophages likely constitute a useful tool to identify new molecules involved in regulating transport along the phagocytic pathway.

Characterization of a small GTP-binding protein gene TaRab18 from wheat involved in the stripe rust resistance.

PubMed

Jiang, Zhengning; Wang, Hui; Zhang, Guoqin; Zhao, Renhui; Bie, Tongde; Zhang, Ruiqi; Gao, Derong; Xing, Liping; Cao, Aizhong

2017-04-01

The stripe rust resistance gene, Yr26, is commonly used in wheat production. Identification of Yr26 resistance related genes is important for better understanding of the resistance mechanism. TaRab18, a putative small GTP-binding protein, was screened as a resistance regulated gene as it showed differential expression between the Yr26-containing resistant wheat and the susceptible wheat at different time points after Pst inoculation. TaRab18 contains four typical domains (GI to GIV) of the small GTP-binding proteins superfamily and five domains (RabF1 to RabF5) specific to the Rab subfamily. From the phylogenetic tree that TaRab18 was identified as belonging to the RABC1 subfamily. Chromosome location analysis indicated that TaRab18 and its homeoalles were on the homeologous group 7 chromosomes, and the Pst induced TaRab18 was on the 7 B chromosome. Functional analysis by virus induced gene silencing (VIGS) indicated that TaRab18 was positively involved in the stripe rust resistance through regulating the hypersensitive response, and Pst can develop on the leaves of TaRab18 silenced 92R137. However, over-expression of TaRab18 in susceptible Yangmai158 did not enhance its resistance dramatically, only from 9 grade in Yangmai158 to 8 grade in the transgenic plant. However, histological observation indicated that the transgenic plants with over-expressed TaRab18 showed a strong hypersensitive response at the early infection stage. The research herein, will improve our understanding of the roles of Rab in wheat resistance. Copyright © 2017. Published by Elsevier Masson SAS.

A Rab5 GTPase module is important for autophagosome closure

PubMed Central

Lipatova, Zhanna; Sun, Dan; Zhu, Xiaolong; Li, Rui; Wu, Zulin; You, Weiming; Cong, Xiaoxia; Zhou, Yiting; Gyurkovska, Valeriya; Liu, Yutao; Li, Qunli; Li, Wenjing; Cheng, Jie; Segev, Nava

2017-01-01

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure. PMID:28934205

The Rab-binding Profiles of Bacterial Virulence Factors during Infection.

PubMed

So, Ernest C; Schroeder, Gunnar N; Carson, Danielle; Mattheis, Corinna; Mousnier, Aurélie; Broncel, Malgorzata; Tate, Edward W; Frankel, Gad

2016-03-11

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Microstructural Evolution of Vacuum Brazed 1Cr18Ni9Ti Using Various Filler Metals

PubMed Central

Chen, Yunxia; Cui, Haichao; Lu, Binfeng; Lu, Fenggui

2017-01-01

The microstructures and weldability of a brazed joint of 1Cr18Ni9Ti austenitic stainless steel with BNi-2, BNi82CrSiBFe and BMn50NiCuCrCo filler metals in vacuum were investigated. It can be observed that an interdiffusion region existed between the filler metal and the base metal for the brazed joint of Ni-based filler metals. The width of the interdiffusion region was about 10 μm, and the microstructure of the brazed joint of BNi-2 filler metal was dense and free of obvious defects. In the case of the brazed joint of BMn50NiCuCrCo filler metal, there were pits, pores and crack defects in the brazing joint due to insufficient wettability of the filler metal. Crack defects can also be observed in the brazed joint of BNi82CrSiBFe filler metal. Compared with BMn50NiCuCrCo and BNi82CrSiBFe filler metals, BNi-2 filler metal is the best material for 1Cr18Ni9Ti austenitic stainless steel vacuum brazing because of its distinct weldability. PMID:28772745

Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells.

PubMed

Saitoh, Sayaka; Maruyama, Takeshi; Yako, Yuta; Kajita, Mihoko; Fujioka, Yoichiro; Ohba, Yusuke; Kasai, Nobuhiro; Sugama, Natsu; Kon, Shunsuke; Ishikawa, Susumu; Hayashi, Takashi; Yamazaki, Tomohiro; Tada, Masazumi; Fujita, Yasuyuki

2017-03-21

Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.

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.

RabGAP22 Is Required for Defense to the Vascular Pathogen Verticillium longisporum and Contributes to Stomata Immunity

PubMed Central

Roos, Jonas; Bejai, Sarosh; Oide, Shinichi; Dixelius, Christina

2014-01-01

Verticillium longisporum is a soil-borne pathogen with a preference for plants within the family Brassicaceae. Following invasion of the roots, the fungus proliferates in the plant vascular system leading to stunted plant growth, chlorosis and premature senescence. RabGTPases have been demonstrated to play a crucial role in regulating multiple responses in plants. Here, we report on the identification and characterization of the Rab GTPase-activating protein RabGAP22 gene from Arabidopsis, as an activator of multiple components in the immune responses to V. longisporum. RabGAP22Pro:GUS transgenic lines showed GUS expression predominantly in root meristems, vascular tissues and stomata, whereas the RabGAP22 protein localized in the nucleus. Reduced RabGAP22 transcript levels in mutants of the brassinolide (BL) signaling gene BRI1-ASSOCIATED RECEPTOR KINASE 1, together with a reduction of fungal proliferation following BL pretreatment, suggested RabGAP22 to be involved in BL-mediated responses. Pull-down assays revealed SERINE:GLYOXYLATE AMINOTRANSFERASE (AGT1) as an interacting partner during V. longisporum infection and bimolecular fluorescence complementation (BiFC) showed the RabGAP22-AGT1 protein complex to be localized in the peroxisomes. Further, fungal-induced RabGAP22 expression was found to be associated with elevated endogenous levels of the plant hormones jasmonic acid (JA) and abscisic acid (ABA). An inadequate ABA response in rabgap22-1 mutants, coupled with a stomata-localized expression of RabGAP22 and impairment of guard cell closure in response to V. longisporum and Pseudomonas syringae, suggest that RabGAP22 has multiple roles in innate immunity. PMID:24505423

Antiviral effect of PmRab7 knock-down on inhibition of Laem-Singh virus replication in black tiger shrimp.

PubMed

Ongvarrasopone, Chalermporn; Chomchay, Ekapol; Panyim, Sakol

2010-10-01

PmRab7 is a Penaeus monodon small GTPase protein possibly involved in replication of several shrimp viruses. In this study RNA interference (RNAi) using double-stranded RNA (dsRNA) targeting PmRab7 gene (dsRNA-PmRab7) was employed to silence the expression of PmRab7 to investigate the inhibitory effect on Laem-Singh virus (LSNV) replication. Injection of dsRNA-PmRab7 24h before challenge with the virus resulted in a drastic decrease of PmRab7 mRNA and complete inhibition of LSNV replication at 3 days post-challenge. In a therapeutic mode, shrimp injected with dsRNA-PmRab7 1 day but not at 3 or 5 days post-LSNV challenge resulted in inhibition of LSNV replication. These results pave the way to use dsRNA-PmRab7 to prevent or cure LSNV infection in shrimp. Copyright © 2010 Elsevier B.V. All rights reserved.

Wide gap active brazing of ceramic-to-metal-joints for high temperature applications

NASA Astrophysics Data System (ADS)

Bobzin, K.; Zhao, L.; Kopp, N.; Samadian Anavar, S.

2014-03-01

Applications like solid oxide fuel cells and sensors increasingly demand the possibility to braze ceramics to metals with a good resistance to high temperatures and oxidative atmospheres. Commonly used silver based active filler metals cannot fulfill these requirements, if application temperatures higher than 600°C occur. Au and Pd based active fillers are too expensive for many fields of use. As one possible solution nickel based active fillers were developed. Due to the high brazing temperatures and the low ductility of nickel based filler metals, the modification of standard nickel based filler metals were necessary to meet the requirements of above mentioned applications. To reduce thermally induced stresses wide brazing gaps and the addition of Al2O3 and WC particles to the filler metal were applied. In this study, the microstructure of the brazed joints and the thermo-chemical reactions between filler metal, active elements and WC particles were analyzed to understand the mechanism of the so called wide gap active brazing process. With regard to the behavior in typical application oxidation and thermal cycle tests were conducted as well as tensile tests.

FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

PubMed

Saridaki, Theodora; Nippold, Markus; Dinter, Elisabeth; Roos, Andreas; Diederichs, Leonie; Fensky, Luisa; Schulz, Jörg B; Falkenburger, Björn H

2018-05-10

Parkinson disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that overexpression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson disease we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 overexpression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy, and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7 induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7 induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by the dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates

Interfacial microstructure and mechanical properties of brazed aluminum / stainless steel - joints

NASA Astrophysics Data System (ADS)

Fedorov, V.; Elßner, M.; Uhlig, T.; Wagner, G.

2017-03-01

Due to the demand of mass and cost reduction, joints based on dissimilar metals become more and more interesting. Especially there is a high interest for joints between stainless steel and aluminum, often necessary for example for automotive heat exchangers. Brazing offers the possibilities to manufacture several joints in one step at, in comparison to fusion welding, lower temperatures. In the recent work, aluminum / stainless steel - joints are produced by induction brazing using an AlSi10 filler and a non-corrosive flux. The mechanical properties are determined by tensile shear tests as well as fatigue tests at ambient and elevated temperatures. The microstructure of the brazed joints and the fracture surfaces of the tested samples are investigated by SEM.

Rab32 modulates apoptosis onset and mitochondria-associated membrane (MAM) properties.

PubMed

Bui, Michael; Gilady, Susanna Y; Fitzsimmons, Ross E B; Benson, Matthew D; Lynes, Emily M; Gesson, Kevin; Alto, Neal M; Strack, Stefan; Scott, John D; Simmen, Thomas

2010-10-08

The mitochondria-associated membrane (MAM) has emerged as an endoplasmic reticulum (ER) signaling hub that accommodates ER chaperones, including the lectin calnexin. At the MAM, these chaperones control ER homeostasis but also play a role in the onset of ER stress-mediated apoptosis, likely through the modulation of ER calcium signaling. These opposing roles of MAM-localized chaperones suggest the existence of mechanisms that regulate the composition and the properties of ER membrane domains. Our results now show that the GTPase Rab32 localizes to the ER and mitochondria, and we identify this protein as a regulator of MAM properties. Consistent with such a role, Rab32 modulates ER calcium handling and disrupts the specific enrichment of calnexin on the MAM, while not affecting the ER distribution of protein-disulfide isomerase and mitofusin-2. Furthermore, Rab32 determines the targeting of PKA to mitochondrial and ER membranes and through its overexpression or inactivation increases the phosphorylation of Bad and of Drp1. Through a combination of its functions as a PKA-anchoring protein and a regulator of MAM properties, the activity and expression level of Rab32 determine the speed of apoptosis onset.

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.

Rab22a enhances CD147 recycling and is required for lung cancer cell migration and invasion.

PubMed

Zhou, Yang; Wu, Bo; Li, Jiang-Hua; Nan, Gang; Jiang, Jian-Li; Chen, Zhi-Nan

2017-08-01

Rab22a is a member of the Ras-related small GTPase family, which plays a key role in regulating the recycling of cargo proteins entering cells through clathrin-independent endocytosis (CIE). Rab22a is overexpressed in different cancer types, including liver cancer, malignant melanoma, ovarian cancer and osteosarcoma. However, its oncogenic role remains unknown. In this study, we found that silencing of Rab22a suppressed the migration and invasion of lung cancer cells. Furthermore, Rab22a interacts with CD147, and knockdown of Rab22a blocks CD147 recycling and promotes CD147 degradation. Taken together, our findings indicate that Rab22a enhances recycling of CD147, which is required for lung cancer cell migration and invasion,and targeting CD147 recycling may be a rational strategy for lung cancer therapy. Copyright © 2017. Published by Elsevier Inc.

RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis.

PubMed

Lin, Ying-Hung; Ke, Chih-Chun; Wang, Ya-Yun; Chen, Mei-Feng; Chen, Tsung-Ming; Ku, Wei-Chi; Chiang, Han-Sun; Yeh, Chung-Hsin

2017-01-05

According to recent estimates, 2%-15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins ( MGCRABGAPs ) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP-RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release.

PubMed

Müller, Martin; Pym, Edward C G; Tong, Amy; Davis, Graeme W

2011-02-24

Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density, and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen, we have tested 162 new mutations for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. We show that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. We then provide evidence that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define roles for Rab3-GAP and Rab3 in synaptic homeostasis and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity. Copyright © 2011 Elsevier Inc. All rights reserved.

Development of a Brazing Alloy for the Mechanically Alloyed High Temperature Sheet Material INCOLOY Alloy MA 956.

DTIC Science & Technology

1981-09-01

OF A BRAZING ALLOY FOR THE MECHANICALLY ALLOYED HIGH TEMPERATURE SHEET MATERIAL INCOLOY ALLOY MA 956 W. E. Morgan and Dr. P. J. Bridges N. Wiggin...PERIOD COVERED DEVELOPMENT OF A BRAZING ALLOY FOR THE Final Report MECHANICALLY ALLOYED HIGH TEMPERATURE Dec 1978 - March 1981 SHEET MATERIAL INCOLOY...block nomber) High temperature ODS alloys, Braze development, Braze alloys, INCOLOY MA 956, Ni-Cr-Pd, Fe-Cr-Pd, Ni-Cr-Ge, Fe-Cr-Ge, Fe-Cr-B, Fe-Cr-Si

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.

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.

Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

PubMed Central

Hoogenraad, Casper C.; Popa, Ioana; Futai, Kensuke; Sanchez-Martinez, Emma; Wulf, Phebe S.; van Vlijmen, Thijs; Dortland, Bjorn R.; Oorschot, Viola; Govers, Roland; Monti, Maria; Heck, Albert J. R.; Sheng, Morgan; Klumperman, Judith; Rehmann, Holger; Jaarsma, Dick; Kapitein, Lukas C.; van der Sluijs, Peter

2010-01-01

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking. PMID:20098723

Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation.

PubMed

Madero-Pérez, Jesús; Fdez, Elena; Fernández, Belén; Lara Ordóñez, Antonio J; Blanca Ramírez, Marian; Gómez-Suaga, Patricia; Waschbüsch, Dieter; Lobbestael, Evy; Baekelandt, Veerle; Nairn, Angus C; Ruiz-Martínez, Javier; Aiastui, Ana; López de Munain, Adolfo; Lis, Pawel; Comptdaer, Thomas; Taymans, Jean-Marc; Chartier-Harlin, Marie-Christine; Beilina, Alexandria; Gonnelli, Adriano; Cookson, Mark R; Greggio, Elisa; Hilfiker, Sabine

2018-01-23

Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2. Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a. Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.

Trans-Golgi network localized small GTPase RabA1d is involved in cell plate formation and oscillatory root hair growth.

PubMed

Berson, Tobias; von Wangenheim, Daniel; Takáč, Tomáš; Šamajová, Olga; Rosero, Amparo; Ovečka, Miroslav; Komis, George; Stelzer, Ernst H K; Šamaj, Jozef

2014-09-27

Small Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described. The transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal compartments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking. RabA1d is located in early endosomes/TGN and is involved in vesicle

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.

The Interaction Properties of the Human Rab GTPase Family – A Comparative Analysis Reveals Determinants of Molecular Binding Selectivity

PubMed Central

Stein, Matthias; Pilli, Manohar; Bernauer, Sabine; Habermann, Bianca H.; Zerial, Marino; Wade, Rebecca C.

2012-01-01

Background Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood. Methodology/Principal Findings Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics. Conclusions/Significance We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity. PMID:22523562

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

...] Welding, Cutting and Brazing Standard; Extension of the Office of Management and Budget's (OMB) Approval... requirements contained in the Welding, Cutting and Brazing Standard (29 CFR part 1910, subpart Q). The information collected is used by employers and workers whenever welding, cutting and brazing are performed...

Microstructure Evolution During Stainless Steel-Copper Vacuum Brazing with a Ag/Cu/Pd Filler Alloy: Effect of Nickel Plating

NASA Astrophysics Data System (ADS)

Choudhary, R. K.; Laik, A.; Mishra, P.

2017-03-01

Vacuum brazing of stainless steel and copper plates was done using a silver-based filler alloy. In one set of experiments, around 30-µm-thick nickel coatings were electrochemically applied on stainless steel plates before carrying out the brazing runs and its effect in making changes in the braze-zone microstructure was studied. For brazing temperature of 830 °C, scanning electron microscopy examination of the braze-zone revealed that relatively sound joints were obtained when brazing was done with nickel-coated stainless steel than with uncoated one. However, when brazing of nickel-coated stainless steel and copper plates was done at 860 °C, a wide crack appeared in the braze-zone adjacent to copper side. Energy-dispersive x-ray analysis and electron microprobe analysis confirmed that at higher temperature, the diffusion of Cu atoms from copper plate towards the braze-zone was faster than that of Ni atoms from nickel coating. Helium leak rate of the order 10-11 Pa m3/s was obtained for the crack-free joint, whereas this value was higher than 10-4 Pa m3/s for the joint having crack. The shear strength of the joint was found to decrease considerably due to the presence of crack.

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.

PKC and Rab13 mediate Ca2+ signal-regulated GLUT4 traffic.

PubMed

Deng, Bangli; Zhu, Xiaocui; Zhao, Yihe; Zhang, Da; Pannu, Alisha; Chen, Liming; Niu, Wenyan

2018-01-08

Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca 2+ mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca 2+ level to explore the underlying mechanism. We showed that in L6 myoblast muscle cells stably expressing GLUT4myc, ionomycin increased cell surface GLUT4myc levels and the phosphorylation of AS160, TBC1D1. siPKCα and siPKCθ but not siPKCδ and siPKCε inhibited the ionomycin-increased cell surface GLUT4myc level. siPKCα, siPKCθ inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKCα and siPKCθ prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKCθ, but not siPKCα inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRab13 but not siRab8a, siRab10 and siRab14 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycin-promoted exocytosis of GLUT4 is partly reversed by siPKCθ, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKCα and siPKCθ. PKCα and PKCθ contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis. Copyright © 2017 Elsevier Inc. All rights reserved.

The Role of the Rab Coupling Protein in ErbB2-Driven Mammary Tumorigenesis and Metastasis

DTIC Science & Technology

2014-10-01

Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation, Senescence. 16. SECURITY CLASSIFICATION OF: 17...Tyrosine Kinase, Her/ErbB2 signaling, Rab Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation...lines included RCP condition to internalization and detection of E-cadherin, a well-known adherent junction and epithelial mesenchymal transition

Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: Involvement of the effector proteins in the regulatory mechanism

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

Saxena, Sunil K.; Kaur, Simarna

Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) andmore » Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.« less

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

MiR-20a-5p promotes radio-resistance by targeting Rab27B in nasopharyngeal cancer cells.

PubMed

Huang, Dabing; Bian, Geng; Pan, Yueyin; Han, Xinghua; Sun, Yubei; Wang, Yong; Shen, Guodong; Cheng, Min; Fang, Xiang; Hu, Shilian

2017-01-01

MicroRNAs (miRNAs) was reported to be involved in cancer radio-resistance, which remains a major obstacle for effective cancer therapy. The differently expressed miRNAs were detected by RNA-seq experiment in nasopharyngeal cancer (NPC) cells. MiR-20a-5p was selected as our target, which was subject to finding its target gene Rab27B via bioinformatics analysis. The qRT-PCR, western blot and the luciferase reporter assays were performed to confirm Rab27B as the target of miR-20a-5p. In addition, the roles of miR-20a-5p in NPC radio-resistance were detected by transfection of either miR-20a-5p-mimic or miR-20a-5p-antagomiR. The involvement of Rab27B with NPC radio-resistance was also detected by the experiments with siRNA-mediated repression of Rab27B or over-expression of GFP-Rab27B. Wound healing and invasion assays were performed to detect the roles of both miR-20a-5p and Rab27B. MiR-20a-5p promotes NPC radio-resistance. We identified that its target gene Rab27B negatively correlates with miR-20a-5p-mediated NPC radio-resistance by systematic studies of a radio-sensitive (CNE-2) and resistant (CNE-1) NPC cell lines. Repression of Rab27B by siRNA suppresses cell apoptosis and passivates CNE-2 cells, whereas over-expression of Rab27B triggered cell apoptosis and sensitizes CNE-1 cells. MiR-20a-5p and its target gene Rab27B might be involved in the NPC radio-resistance. Thus the key players and regulators involved in this pathway might be the potential targets for developing effective therapeutic strategies against NPC.

Overexpression of Rab11-FIP2 in colorectal cancer cells promotes tumor migration and angiogenesis through increasing secretion of PAI-1.

PubMed

Dong, Wenjie; Wu, Xinai

2018-01-01

Rab11 family-interacting protein 2 (Rab11-FIP2) can interact with MYO5B and plays an important role in regulating plasma membrane recycling. However, little is known about the clinical significance of DUSP2 in colorectal cancer (CRC). In this study, we investigated Rab11-FIP2 expression by immunohistochemistry in 125 patients with colorectal cancer. Conditioned media containing all secreted factors was harvested. Chemokine secretion and expression were analyzed by Chemi-array. We found that the expression level of Rab11-FIP2 was significantly increased in colorectal cancer tissues and high expression of Rab11-FIP2 was closely correlated with nodal metastasis in colorectal cancer patients. Rab11-FIP2 overexpression promoted colorectal cancer metastasis in vitro and in vivo. Finally, we demonstrated that Rab11-FIP2 overexpression may contribute to increased secretion of PAI-1 in human colorectal cancer cells. Our findings reveal a novel mechanism underlying the role of Rab11-FIP2 in colorectal cancer dissemination, suggesting that targeting Rab11-FIP2 might be a promising therapeutic strategy for CRC.

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp.

PubMed

Thagun, Chonprakun; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Narangajavana, Jarunya; Sojikul, Punchapat

2012-09-15

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Classifying the molecular functions of Rab GTPases in membrane trafficking using deep convolutional neural networks.

PubMed

Le, Nguyen-Quoc-Khanh; Ho, Quang-Thai; Ou, Yu-Yen

2018-06-13

Deep learning has been increasingly used to solve a number of problems with state-of-the-art performance in a wide variety of fields. In biology, deep learning can be applied to reduce feature extraction time and achieve high levels of performance. In our present work, we apply deep learning via two-dimensional convolutional neural networks and position-specific scoring matrices to classify Rab protein molecules, which are main regulators in membrane trafficking for transferring proteins and other macromolecules throughout the cell. The functional loss of specific Rab molecular functions has been implicated in a variety of human diseases, e.g., choroideremia, intellectual disabilities, cancer. Therefore, creating a precise model for classifying Rabs is crucial in helping biologists understand the molecular functions of Rabs and design drug targets according to such specific human disease information. We constructed a robust deep neural network for classifying Rabs that achieved an accuracy of 99%, 99.5%, 96.3%, and 97.6% for each of four specific molecular functions. Our approach demonstrates superior performance to traditional artificial neural networks. Therefore, from our proposed study, we provide both an effective tool for classifying Rab proteins and a basis for further research that can improve the performance of biological modeling using deep neural networks. Copyright © 2018 Elsevier Inc. All rights reserved.

The causes of high power diode laser brazed seams fractures of dissimilar materials

NASA Astrophysics Data System (ADS)

Adamiak, Marcin; Czupryński, Artur; Janicki, Damian; Górka, Jacek

2016-12-01

Presented in this article are the results of experiments carried out to determine the causes of braze cracking of dissimilar materials brazed with a ROFIN DL 020 high power diode laser with the use of additional powdered EN AW-1070A aluminium alloy to bond thin aluminium sheets with soft, low alloy DC04+ZE75/75 steel plate which was electrolytically coated with zinc on both sides. Presented are the results of metallographic, macroscopic, microscopic, diffractometric phase analyses of the weld joints. Metallurgical problems arising during processing as well as suggestions regarding technical aspects of laser brazing dissimilar materials in regards to their physical characteristics and chemical composition are explored.

AA6082 to DX56-Steel Laser Brazing: Process Parameter-Intermetallic Formation Correlation

NASA Astrophysics Data System (ADS)

Narsimhachary, D.; Pal, S.; Shariff, S. M.; Padmanabham, G.; Basu, A.

2017-09-01

In the present study, laser-brazed AA6082 to DX56-galvanized steel joints were investigated to understand the influence of process parameters on joint strength in terms of intermetallic layer formation. 1.5-mm-thick sheet of aluminum alloy (AA6082-T6) and galvanized steel (DX56) sheet of 0.7 mm thickness were laser-brazed with 1.5-mm-diameter Al-12% Si solid filler wire. During laser brazing, laser power (4.6 kW) and wire feed rate (3.4 m/min) were kept constant with a varying laser scan speed of 3.5, 3, 2.5, 2, 1.5, and 1 m/min. Microstructure of brazed joint reveals epitaxial growth at the aluminum side and intermetallic layer formation at steel interface. Intermetallic layer formation was confirmed by EDS analysis and XRD study. Hardness profile showed hardness drop in filler region, and failure during tensile testing was initiated through the filler region near the steel interface. As per both experimental study and numerical analysis, it was observed that intermetallic layer thickness decreases with increasing brazing speed. Zn vaporization from galvanized steel interface also affected the joint strength. It was found that high laser scan speed or faster cooling rate can be chosen for suppressing intermetallic layer formation or at least decreasing the layer thickness which results in improved mechanical properties.

Electroplating eliminates gas leakage in brazed areas

NASA Technical Reports Server (NTRS)

Leigh, J. D.

1966-01-01

Electroplating method seals brazed or welded joints against gas leakage under high pressure. Any conventional electroplating process with many different metal anodes can be used, as well as the build up of layers of different metals to any required thickness.

Rab3A Inhibition of Ca2+ -Dependent Dopamine Release From PC12 Cells Involves Interaction With Synaptotagmin I.

PubMed

Dai, Zhipan; Tang, Xia; Chen, Jia; Tang, Xiaochao; Wang, Xianchun

2017-11-01

Rab3 and synaptotagmin have been suggested to play important roles in the regulation of neurotransmitter release and, however, the molecular mechanism has not been completely clear. Here, we studied the effects of Rab3A and synaptotagmin I (Syt I) on dopamine release using PC12 cells as a model system. Rab3A was demonstrated to have effects on both Ca 2+ -independent and Ca 2+ -dependent dopamine releases from the PC12 cells. Application of Rab3A (up to 2500 nM) gradually decreased the amount of Ca 2+ -dependently released dopamine, indicating that Rab3A is a negative modulator that was further supported by the increase in dopamine release caused by Rab3A knockdown. Syt I knockdown weakened the Ca 2+ -dependent dopamine release, suggesting that Syt I plays a positive regulatory role in the cellular process. Treatment of the Syt I-knocked down PC12 cells with Rab3A further decreased Ca 2+ -dependent dopamine release and, however, the decrease magnitude was significantly reduced compared with that before Syt I knockdown, thus for the first time demonstrating that the inhibitory effect of Rab3A on Ca 2+ -dependent dopamine release involves the interaction with Syt I. This work has shed new light on the molecular mechanism for Rab3 and synaptotamin regulation of neurotransmitter release. J. Cell. Biochem. 118: 3696-3705, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Essential Role of RAB27A in Determining Constitutive Human Skin Color

PubMed Central

Yoshida-Amano, Yasuko; Hachiya, Akira; Ohuchi, Atsushi; Kobinger, Gary P.; Kitahara, Takashi; Takema, Yoshinori; Fukuda, Mitsunori

2012-01-01

Human skin color is predominantly determined by melanin produced in melanosomes within melanocytes and subsequently distributed to keratinocytes. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes involved from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. Apart from the activities in the melanogenic rate-limiting enzyme, tyrosinase, in melanocytes and the amounts and distribution patterns of melanosomes in keratinocytes, the abilities of the actin-associated factors in charge of melanosome transport within melanocytes also regulate pigmentation. Mutations in genes encoding melanosome transport-related molecules, such as MYO5A, RAB27A and SLAC-2A, have been reported to cause a human pigmentary disease known as Griscelli syndrome, which is associated with diluted skin and hair color. Thus we hypothesized that process might play a role in modulating skin color variations. To address that hypothesis, the correlations of expression of RAB27A and its specific effector, SLAC2-A, to melanogenic ability were evaluated in comparison with tyrosinase, using human melanocytes derived from 19 individuals of varying skin types. Following the finding of the highest correlation in RAB27A expression to the melanogenic ability, darkly-pigmented melanocytes with significantly higher RAB27A expression were found to transfer significantly more melanosomes to keratinocytes than lightly-pigmented melanocytes in co-culture and in human skin substitutes (HSSs) in vivo, resulting in darker skin color in concert with the difference observed in African-descent and Caucasian skins. Additionally, RAB27A knockdown by a lentivirus-derived shRNA in melanocytes concomitantly demonstrated a significantly reduced number of transferred melanosomes to keratinocytes in co-culture and a significantly diminished epidermal melanin content skin color intensity (ΔL* = 4.4) in the

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.

Rab3a Is Critical for Trapping Alpha-MSH Granules in the High Ca2+-Affinity Pool by Preventing Constitutive Exocytosis

PubMed Central

Sedej, Simon; Klemen, Maša Skelin; Schlüter, Oliver M.; Rupnik, Marjan Slak

2013-01-01

Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of α-MSH. PMID:24205339

Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p†

PubMed Central

Buvelot Frei, Stéphanie; Rahl, Peter B.; Nussbaum, Maria; Briggs, Benjamin J.; Calero, Monica; Janeczko, Stephanie; Regan, Andrew D.; Chen, Catherine Z.; Barral, Yves; Whittaker, Gary R.; Collins, Ruth N.

2006-01-01

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance. PMID:16980630

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

PubMed Central

Patwardhan, Anand; Bardin, Sabine; Miserey-Lenkei, Stéphanie; Larue, Lionel; Goud, Bruno; Raposo, Graça; Delevoye, Cédric

2017-01-01

Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function. PMID:28607494

Disruption of RAB40AL function leads to Martin--Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder.

PubMed

Bedoyan, Jirair Krikor; Schaibley, Valerie M; Peng, Weiping; Bai, Yongsheng; Mondal, Kajari; Shetty, Amol C; Durham, Mark; Micucci, Joseph A; Dhiraaj, Arti; Skidmore, Jennifer M; Kaplan, Julie B; Skinner, Cindy; Schwartz, Charles E; Antonellis, Anthony; Zwick, Michael E; Cavalcoli, James D; Li, Jun Z; Martin, Donna M

2012-05-01

Martin--Probst syndrome (MPS) is a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other features. The authors sought to identify the causative mutation for MPS. Massively parallel sequencing in two affected, related male subjects with MPS identified a RAB40AL (also called RLGP) missense mutation (chrX:102,079,078-102,079,079AC→GA p.D59G; hg18). RAB40AL encodes a small Ras-like GTPase protein with one suppressor of cytokine signalling box. The p.D59G variant is located in a highly conserved region of the GTPase domain between β-2 and β-3 strands. Using RT-PCR, the authors show that RAB40AL is expressed in human fetal and adult brain and kidney, and adult lung, heart, liver and skeletal muscle. RAB40AL appears to be a primate innovation, with no orthologues found in mouse, Xenopus or zebrafish. Western analysis and fluorescence microscopy of GFP-tagged RAB40AL constructs from transiently transfected COS7 cells show that the D59G missense change renders RAB40AL unstable and disrupts its cytoplasmic localisation. This is the first study to show that mutation of RAB40AL is associated with a human disorder. Identification of RAB40AL as the gene mutated in MPS allows for further investigations into the molecular mechanism(s) of RAB40AL and its roles in diverse processes such as cognition, hearing and skeletal development.

Disruption of RAB40AL function leads to Martin–Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder

PubMed Central

Bedoyan, Jirair Krikor; Schaibley, Valerie M; Peng, Weiping; Bai, Yongsheng; Mondal, Kajari; Shetty, Amol C; Durham, Mark; Micucci, Joseph A; Dhiraaj, Arti; Skidmore, Jennifer M; Kaplan, Julie B; Skinner, Cindy; Schwartz, Charles E; Antonellis, Anthony; Zwick, Michael E; Cavalcoli, James D; Li, Jun Z

2012-01-01

Background and aim Martin–Probst syndrome (MPS) is a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other features. The authors sought to identify the causative mutation for MPS. Methods and results Massively parallel sequencing in two affected, related male subjects with MPS identified a RAB40AL (also called RLGP) missense mutation (chrX:102,079,078-102,079,079AC→GA p.D59G; hg18). RAB40AL encodes a small Ras-like GTPase protein with one suppressor of cytokine signalling box. The p.D59G variant is located in a highly conserved region of the GTPase domain between β-2 and β-3 strands. Using RT-PCR, the authors show that RAB40AL is expressed in human fetal and adult brain and kidney, and adult lung, heart, liver and skeletal muscle. RAB40AL appears to be a primate innovation, with no orthologues found in mouse, Xenopus or zebrafish. Western analysis and fluorescence microscopy of GFP-tagged RAB40AL constructs from transiently transfected COS7 cells show that the D59G missense change renders RAB40AL unstable and disrupts its cytoplasmic localisation. Conclusions This is the first study to show that mutation of RAB40AL is associated with a human disorder. Identification of RAB40AL as the gene mutated in MPS allows for further investigations into the molecular mechanism(s) of RAB40AL and its roles in diverse processes such as cognition, hearing and skeletal development. PMID:22581972

Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes.

PubMed

Starling, Georgina P; Yip, Yan Y; Sanger, Anneri; Morton, Penny E; Eden, Emily R; Dodding, Mark P

2016-06-01

The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt-Hoge-Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri-nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi-associated small GTPase Rab34. Rab34-positive peri-nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34-induced peri-nuclear lysosome clustering. FLCN interacts directly via its C-terminal DENN domain with the Rab34 effector RILP Using purified recombinant proteins, we show that the FLCN-DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP We propose a model whereby starvation-induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34-positive peri-nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

32 CFR 202.9 - Conducting RAB meetings.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 2 2014-07-01 2014-07-01 false Conducting RAB meetings. 202.9 Section 202.9 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED... Administrative Record. (2) The records, reports, minutes, appendixes, working papers, drafts, studies, agenda, or...

32 CFR 202.9 - Conducting RAB meetings.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 2 2012-07-01 2012-07-01 false Conducting RAB meetings. 202.9 Section 202.9 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED... Administrative Record. (2) The records, reports, minutes, appendixes, working papers, drafts, studies, agenda, or...

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...

Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack

PubMed Central

1994-01-01

The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (t1/2 < 30 s) extracted Rab1 from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractionates with a molecular mass of approximately 80 kD in the form of a GDI-Rab1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rab1 complex prepared in vitro from recombinant forms of Rab1 and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rab1 complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway. PMID:8089173

RAB GTPASES ASSOCIATE WITH ISOLATED LIPID DROPLETS (LDS) AND SHOW ALTERED CONTENT AFTER ETHANOL ADMINISTRATION: POTENTIAL ROLE IN ALCOHOL-IMPAIRED LD METABOLISM

PubMed Central

Rasineni, Karuna; McVicker, Benita L.; Tuma, Dean J.; McNiven, Mark A.; Casey, Carol A.

2013-01-01

Background Alcoholic liver disease is manifested by the presence of fatty liver, primarily due to accumulation of hepatocellular lipid droplets (LDs). The presence of membrane-trafficking proteins (e.g. Rab GTPases) with LDs indicates that LDs may be involved in trafficking pathways known to be altered in ethanol damaged hepatocytes. Since these Rab GTPases are crucial regulators of protein trafficking, we examined the effect ethanol administration has on hepatic Rab protein content and association with LDs. Methods Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Whole liver and isolated LD fractions were analyzed. Identification of LDs and associated Rab proteins was performed in frozen liver or paraffin-embedded sections followed by immunohistochemical analysis. Results Lipid accumulation was characterized by larger LD vacuoles and increased total triglyceride content in ethanol-fed rats. Rabs 1, 2, 3d, 5, 7 and 18 were analyzed in post-nuclear supernatant (PNS) as well as LDs. All of the Rabs were found in the PNS, and Rabs 1, 2, 5 and 7 did not show alcohol-altered content, while Rab 3d content was reduced by over 80%, and Rab 18 also showed ethanol-induced reduction in content. Rab 3d was not found to associate with LDs, while all other Rabs were found in the LD fractions, and several showed an ethanol-related decrease (Rabs 2, 5, 7, 18). Immunohistochemical analysis revealed the enhanced content of a LD-associated protein, perilipin 2 (PLIN2) that was paralleled with an associated decrease of Rab 18 in ethanol-fed rat sections. Conclusion Chronic ethanol feeding was associated with increased PLIN2 and altered Rab GTPase content in enriched LD fractions. Although mechanisms driving these changes are not established, further studies on intracellular protein trafficking and LD biology after alcohol administration will likely contribute to our understanding of fatty liver disease. PMID:24117505

Feasibility study of fluxless brazing cemented carbides to steel

NASA Astrophysics Data System (ADS)

Tillmann, W.; Sievers, N.

2017-03-01

One of the most important brazing processes is the joints between cemented carbides and steel for the tool industry such as in rotary drill hammers or saw blades. Even though this technique has already been used for several decades, defects in the joint can still occur and lead to quality loss. Mostly, the joining process is facilitated by induction heating and the use of a flux to enhance the wetting of the filler alloy on the surface of the steel and cemented carbide in an ambient atmosphere. However, although the use of flux enables successful joining, it also generates voids within the joint, which reduces the strength of the connection while the chemicals within the flux are toxic and polluting. In this feasibility study, a fluxless brazing process is used to examine the joint between cemented carbides and steel for the first time. For this, ultrasound is applied during induction heating to enable the wetting between the liquid filler metal and the surfaces of the cemented carbide and steel. The ultrasound generates cavitations within the liquid filler metal, which remove the oxides from the surface. Several filler metals such as a silver based alloy Ag449, pure Zn, and an AlSi-alloy were used to reduce the brazing temperature and to lower the thermal residual stresses within the joint. As a result, every filler metal successfully wetted both materials and led to a dense connection. The ultrasound has to be applied carefully to prevent a damage of the cemented carbide. In this regard, it was observed that single grains of the cemented carbide broke out and remained in the joint. This positive result of brazing cemented carbides to steel without a flux but using ultrasound, allows future studies to focus on the shear strength of these joints as well as the behavior of the thermally induced residual stresses.

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

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

Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki

Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our datamore » strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.« less

Some problems of brazing technology for the divertor plate manufacturing

NASA Astrophysics Data System (ADS)

Prokofiev, Yu. G.; Barabash, V. R.; Khorunov, V. F.; Maksimova, S. V.; Gervash, A. A.; Fabritsiev, S. A.; Vinokurov, V. F.

1992-09-01

Among the different design options of the ITER reactor divertor, the joints of the carbon-based materials and molybdenum alloys and joints of tungsten and copper alloys are considered. High-temperature brazing is one of the most promising joining methods for the plasma facing and heat sink materials. The use of brazing for creation of W-Cu and graphite-Mo joints are given here. In addition, the investigation results of microstructure, microhardness and mechanical properties of the joints are presented. For W-Cu samples an influence of the neutron irradiation on the joining strength was studied.

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.

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

2016-10-01

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.

Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

PubMed

Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2002-10-01

Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

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.

Evaluation of mechanically alloyed Cu-based powders as filler alloy for brazing tungsten to a reduced activation ferritic-martensitic steel

NASA Astrophysics Data System (ADS)

de Prado, J.; Sánchez, M.; Ureña, A.

2017-07-01

80Cu-20Ti powders were evaluated for their use as filler alloy for high temperature brazing of tungsten to a reduced activation ferritic/martensitic steel (Eurofer), and its application for the first wall of the DEMO fusion reactor. The use of alloyed powders has not been widely considered for brazing purposes and could improve the operational brazeability of the studied system due to its narrower melting range, determined by DTA analysis, which enhances the spreading capabilities of the filler. Ti contained in the filler composition acts as an activator element, reacting and forming several interfacial layers at the Eurofer-braze, which enhances the wettability properties and chemical interaction at the brazing interface. Brazing thermal cycle also activated the diffusion phenomena, which mainly affected to the Eurofer alloying elements causing in it a softening band of approximately 400 μm of thickness. However, this softening effect did not degrade the shear strength of the brazed joints (94 ± 23 MPa), because failure during testing was always located at the tungsten-braze interface.

Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning.

PubMed

Dodson, Mark W; Zhang, Ting; Jiang, Changan; Chen, Shengdi; Guo, Ming

2012-03-15

LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2(G2019S) allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD.

Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning

PubMed Central

Dodson, Mark W.; Zhang, Ting; Jiang, Changan; Chen, Shengdi; Guo, Ming

2012-01-01

LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2G2019S allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD. PMID:22171073

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

PubMed Central

Gerber, Pehuén Pereyra; Cabrini, Mercedes; Jancic, Carolina; Paoletti, Luciana; Banchio, Claudia; von Bilderling, Catalina; Sigaut, Lorena; Pietrasanta, Lía I.; Duette, Gabriel; Freed, Eric O.; de Saint Basile, Genevieve; Moita, Catarina Ferreira; Moita, Luis Ferreira; Amigorena, Sebastian; Benaroch, Philippe; Geffner, Jorge

2015-01-01

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication. PMID:25940347

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

PubMed Central

Lucas, María; Gaspar, Andrew H.; Pallara, Chiara; Rojas, Adriana Lucely; Fernández-Recio, Juan; Machner, Matthias P.; Hierro, Aitor

2014-01-01

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization. PMID:25114243

Sorting of Clathrin-Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin-Mediated Endocytosis.

PubMed

Dutta, Dipannita; Donaldson, Julie G

2015-09-01

Clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) co-exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6-associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6-GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6-GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin-coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6-GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Rab4b controls an early endosome sorting event by interacting with the γ-subunit of the clathrin adaptor complex 1.

PubMed

Perrin, Laura; Laura, Perrin; Lacas-Gervais, Sandra; Sandra, Lacas-Gervais; Gilleron, Jérôme; Jérôme, Gilleron; Ceppo, Franck; Franck, Ceppo; Prodon, François; François, Prodon; Benmerah, Alexandre; Alexandre, Benmerah; Tanti, Jean-François; Jean-François, Tanti; Cormont, Mireille; Mireille, Cormont

2013-11-01

The endocytic pathway is essential for cell homeostasis and numerous small Rab GTPases are involved in its control. The endocytic trafficking step controlled by Rab4b has not been elucidated, although recent data suggested it could be important for glucose homeostasis, synaptic homeostasis or adaptive immunity. Here, we show that Rab4b is required for early endosome sorting of transferrin receptors (TfRs) to the recycling endosomes, and we identified the AP1γ subunit of the clathrin adaptor AP-1 as a Rab4b effector and key component of the machinery of early endosome sorting. We show that internalised transferrin (Tf) does not reach Vamp3/Rab11 recycling endosomes in the absence of Rab4b, whereas it is rapidly recycled back to the plasma membrane. By contrast, overexpression of Rab4b leads to the accumulation of internalised Tf within AP-1- and clathrin-coated vesicles. These vesicles are poor in early and recycling endocytic markers except for TfR and require AP1γ for their formation. Furthermore, the targeted overexpression of the Rab4b-binding domain of AP1γ to early endosome upon its fusion with FYVE domains inhibited the interaction between Rab4b and endogenous AP1γ, and perturbed Tf traffic. We thus proposed that the interaction between early endocytic Rab4b and AP1γ could allow the budding of clathrin-coated vesicles for subsequent traffic to recycling endosomes. The data also uncover a novel type of endosomes, characterised by low abundance of either early or recycling endocytic markers, which could potentially be generated in cell types that naturally express high level of Rab4b.

Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize.

PubMed

Busk, P K; Jensen, A B; Pagès, M

1997-06-01

The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis-elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis-acting sequences can be divided in embryo-specific, ABA-specific and leaf-specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non-ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought-inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo-specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.

Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

PubMed Central

Lippé, Roger; Miaczynska, Marta; Rybin, Vladimir; Runge, Anja; Zerial, Marino

2001-01-01

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases. PMID:11452015

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.

32 CFR 202.9 - Conducting RAB meetings.

Code of Federal Regulations, 2011 CFR

2011-07-01

... participation. RAB meetings shall be open to the public. (1) The installation co-chair shall prepare and publish... regulations as may be prescribed. Open solicitation of public comments shall be permitted and members of the... under the installation's Community Relations Plan, a public library, or in the offices of the...

32 CFR 202.9 - Conducting RAB meetings.

Code of Federal Regulations, 2010 CFR

2010-07-01

... participation. RAB meetings shall be open to the public. (1) The installation co-chair shall prepare and publish... regulations as may be prescribed. Open solicitation of public comments shall be permitted and members of the... under the installation's Community Relations Plan, a public library, or in the offices of the...

Increasing Ti-6Al-4V brazed joint strength equal to the base metal by Ti and Zr amorphous filler alloys

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

Ganjeh, E., E-mail: navidganjehie@sina.kntu.ac.ir; Sarkhosh, H.; Bajgholi, M.E.

Microstructural features developed along with mechanical properties in furnace brazing of Ti-6Al-4V alloy using STEMET 1228 (Ti-26.8Zr-13Ni-13.9Cu, wt.%) and STEMET 1406 (Zr-9.7Ti-12.4Ni-11.2Cu, wt.%) amorphous filler alloys. Brazing temperatures employed were 900-950 Degree-Sign C for the titanium-based filler and 900-990 Degree-Sign C for the zirconium-based filler alloys, respectively. The brazing time durations were 600, 1200 and 1800 s. The brazed joints were evaluated by ultrasonic test, and their microstructures and phase constitutions analyzed by metallography, scanning electron microscopy and X-ray diffraction analysis. Since microstructural evolution across the furnace brazed joints primarily depends on their alloying elements such as Cu, Ni andmore » Zr along the joint. Accordingly, existence of Zr{sub 2}Cu, Ti{sub 2}Cu and (Ti,Zr){sub 2}Ni intermetallic compounds was identified in the brazed joints. The chemical composition of segregation region in the center of brazed joints was identical to virgin filler alloy content which greatly deteriorated the shear strength of the joints. Adequate brazing time (1800 s) and/or temperature (950 Degree-Sign C for Ti-based and 990 Degree-Sign C for Zr-based) resulted in an acicular Widmanstaetten microstructure throughout the entire joint section due to eutectoid reaction. This microstructure increased the shear strength of the brazed joints up to the Ti-6Al-4V tensile strength level. Consequently, Ti-6Al-4V can be furnace brazed by Ti and Zr base foils produced excellent joint strengths. - Highlights: Black-Right-Pointing-Pointer Temperature or time was the main factors of controlling braze joint strength. Black-Right-Pointing-Pointer Developing a Widmanstaetten microstructure generates equal strength to base metal. Black-Right-Pointing-Pointer Brittle intermetallic compounds like (Ti,Zr){sub 2}Ni/Cu deteriorate shear strength. Black-Right-Pointing-Pointer Ti and Zr base filler alloys were the best choice for

Development of rapidly quenched nickel-based non-boron filler metals for brazing corrosion resistant steels

NASA Astrophysics Data System (ADS)

Ivannikov, A.; Kalin, B.; Suchkov, A.; Penyaz, M.; Yurlova, M.

2016-04-01

Corrosion-resistant steels are stably applied in modern rocket and nuclear technology. Creating of permanent joints of these steels is a difficult task that can be solved by means of welding or brazing. Recently, the use rapidly quenched boron-containing filler metals is perspective. However, the use of such alloys leads to the formation of brittle borides in brazing zone, which degrades the corrosion resistance and mechanical properties of the compounds. Therefore, the development of non-boron alloys for brazing stainless steels is important task. The study of binary systems Ni-Be and Ni-Si revealed the perspective of replacing boron in Ni-based filler metals by beryllium, so there was the objective of studying of phase equilibrium in the system Ni-Be-Si. The alloys of the Ni-Si-Be with different contents of Si and Be are considered in this paper. The presence of two low-melting components is revealed during of their studying by methods of metallography analysis and DTA. Microhardness is measured and X-ray diffraction analysis is conducted for a number of alloys of Ni-Si-Be. The compositions are developed on the basis of these data. Rapidly quenched brazing alloys can be prepared from these compositions, and they are suitable for high temperature brazing of steels.

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.

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.

An Impact of Zirconium Doping of Zn-Al Braze on the Aluminum-Stainless Steel Joints Integrity During Aging

NASA Astrophysics Data System (ADS)

Yang, Jinlong; Xue, Songbai; Sekulic, Dusan P.

2017-01-01

This work offers an analysis of the microstructure and the growth rate of an intermetallic compound within the aged AA 6061 aluminum alloy-304 stainless steel joint brazed with Zn-15Al and Zn-15Al-0.2Zr filler metals. The effect of zirconium addition on mechanical integrity of the brazed joint was studied. The experimental results confirm that the thickness of the Fe-Al intermetallic layer formed at the brazed seam/stainless steel interface increases with the increase of the aging time. Furthermore, it is established that the growth rate of the intermetallic layer for the Zn-15Al-0.2Zr brazed joint was lower than that for Zn-15Al. The results also indicate that the shear strength of both Zn-15Al and Zn-15Al-0.2Zr brazed joints decreases monotonously during aging. The value of the strength after aging lasting for 800 h for Zn-15Al and Zn-15Al-0.2Zr has decreased by 20 and 17%, respectively. The fracture of joints occurred at the interface between the brazed seam and the Fe4Al13 intermetallic layer. The morphology of the surfaces exhibits a cleavage fracture.

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.

Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin

PubMed Central

Chia, Pei Zhi Cheryl; Gasnereau, Isabelle; Lieu, Zi Zhao; Gleeson, Paul A.

2011-01-01

The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin–TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail. PMID:21693586

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.

Brazing copper to dispersion-strengthened copper

NASA Astrophysics Data System (ADS)

Ryding, David G.; Allen, Douglas; Lee, Richard H.

1996-11-01

The advanced photon source 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 than has ben possible to date. The beam is produced by using third- generation insertion devices in a 7-GeV electron/positron storage ring that is 1,104 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$_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, a dispersion-strengthened copper, is the desired design 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.

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.

Corrosion in artificial saliva of a Ni-Cr-based dental alloy joined by TIG welding and conventional brazing.

PubMed

Matos, Irma C; Bastos, Ivan N; Diniz, Marília G; de Miranda, Mauro S

2015-08-01

Fixed prosthesis and partial dental prosthesis frameworks are usually made from welded Ni-Cr-based alloys. These structures can corrode in saliva and have to be investigated to establish their safety. The purpose of this study was to evaluate the corrosion behavior of joints joined by tungsten inert gas (TIG) welding and conventional brazing in specimens made of commercial Ni-Cr alloy in Fusayama artificial saliva at 37°C (pH 2.5 and 5.5). Eighteen Ni-Cr base metal specimens were cast and welded by brazing or tungsten inert gas methods. The specimens were divided into 3 groups (base metal, 2 welded specimens), and the composition and microstructure were qualitatively evaluated. The results of potential corrosion and corrosion current density were analyzed with a 1-way analysis of variance and the Tukey test for pairwise comparisons (α=.05). Base metal and tungsten inert gas welded material showed equivalent results in electrochemical corrosion tests, while the air-torched specimens exhibited low corrosion resistance. The performance was worst at pH 2.5. These results suggest that tungsten inert gas is a suitable welding process for use in dentistry, because the final microstructure does not reduce the corrosion resistance in artificial saliva at 37°C, even in a corrosion-testing medium that facilitates galvanic corrosion processes. Moreover, the corrosion current density of brazed Ni-Cr alloy joints was significantly higher (P<.001) than the base metal and tungsten inert gas welded joints. Copyright © 2015 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Rab14 Regulates Maturation of Macrophage Phagosomes Containing the Fungal Pathogen Candida albicans and Outcome of the Host-Pathogen Interaction

PubMed Central

Okai, Blessing; Lyall, Natalie; Gow, Neil A. R.; Erwig, Lars-Peter

2015-01-01

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogen Candida albicans undergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated by C. albicans hyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment of C. albicans cells. Phagosomes containing live C. albicans cells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing by C. albicans. Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape. PMID:25644001

Golgi-associated Rab14, a new regulator for Chlamydia trachomatis infection outcome.

PubMed

Capmany, Anahí; Leiva, Natalia; Damiani, María Teresa

2011-09-01

Chlamydia trachomatis is the causing agent of the most frequent bacterial sexually-transmitted diseases worldwide and is an underlying cause of chronic pelvic inflammatory diseases and cervical cancer. It is an obligate intracellular bacterium that establishes a close relationship with the Golgi complex and parasites the biosynthetic machinery of host cells. In a recent study, we have demonstrated that Rab14, a newly-described Golgi-associated Rab, is involved in the delivery of sphingolipids to the growing bacteria-containing vacuole. The interference with Rab14-controlled trafficking pathways delays chlamydial inclusion enlargement, decreases bacterial lipid uptake, negatively impact on bacterial differentiation, and reduces bacterial progeny and infectivity. C. trachomatis manipulation of host trafficking pathways for the acquisition of endogenously-biosynthesized nutrients arises as one of the characteristics of this highly evolved pathogen. The development of therapeutic strategies targeted to interfere with bacterium-host cell interaction is a new challenge for pharmacological approaches to control chlamydial infections.

Golgi-associated Rab14, a new regulator for Chlamydia trachomatis infection outcome

PubMed Central

Capmany, Anahí; Leiva, Natalia

2011-01-01

Chlamydia trachomatis is the causing agent of the most frequent bacterial sexually-transmitted diseases worldwide and is an underlying cause of chronic pelvic inflammatory diseases and cervical cancer. It is an obligate intracellular bacterium that establishes a close relationship with the Golgi complex and parasites the biosynthetic machinery of host cells. In a recent study, we have demonstrated that Rab14, a newly-described Golgi-associated Rab, is involved in the delivery of sphingolipids to the growing bacteria-containing vacuole. The interference with Rab14-controlled trafficking pathways delays chlamydial inclusion enlargement, decreases bacterial lipid uptake, negatively impact on bacterial differentiation, and reduces bacterial progeny and infectivity. C. trachomatis manipulation of host trafficking pathways for the acquisition of endogenously-biosynthesized nutrients arises as one of the characteristics of this highly evolved pathogen. The development of therapeutic strategies targeted to interfere with bacterium-host cell interaction is a new challenge for pharmacological approaches to control chlamydial infections. PMID:22046472

The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

PubMed Central

Finetti, Francesca; Patrussi, Laura; Galgano, Donatella; Cassioli, Chiara; Perinetti, Giuseppe; Pazour, Gregory J.; Baldari, Cosima T.

2015-01-01

ABSTRACT IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11+ endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR+ endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis. PMID:26034069

Rab GTPases: The Key Players in the Molecular Pathway of Parkinson’s Disease

PubMed Central

Shi, Meng-meng; Shi, Chang-he; Xu, Yu-ming

2017-01-01

Parkinson’s disease (PD) is a progressive movement disorder with multiple non-motor symptoms. Although family genetic mutations only account for a small proportion of the cases, these mutations have provided several lines of evidence for the pathogenesis of PD, such as mitochondrial dysfunction, protein misfolding and aggregation, and the impaired autophagy-lysosome system. Recently, vesicle trafficking defect has emerged as a potential pathogenesis underlying this disease. Rab GTPases, serving as the core regulators of cellular membrane dynamics, may play an important role in the molecular pathway of PD through the complex interplay with numerous factors and PD-related genes. This might shed new light on the potential therapeutic strategies. In this review, we emphasize the important role of Rab GTPases in vesicle trafficking and summarize the interactions between Rab GTPases and different PD-related genes. PMID:28400718

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

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

Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays withmore » two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.« less

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.

Vacuum Brazing TC4 Titanium Alloy to 304 Stainless Steel with Cu-Ti-Ni-Zr-V Amorphous Alloy Foil

NASA Astrophysics Data System (ADS)

Dong, Honggang; Yang, Zhonglin; Wang, Zengrui; Deng, Dewei; Dong, Chuang

2014-10-01

Dissimilar metal vacuum brazing between TC4 titanium alloy and 304 stainless steel was conducted with newly designed Cu-Ti-Ni-Zr-V amorphous alloy foils as filler metals. Solid joints were obtained due to excellent compatibility between the filler metal and stainless steel substrate. Partial dissolution of stainless steel substrate occurred during brazing. The shear strength of the joint brazed with Cu43.75Ti37.5Ni6.25Zr6.25V6.25 foil was 105 MPa and that with Cu37.5Ti25Ni12.5Zr12.5V12.5 was 116 MPa. All the joints fractured through the gray layer in the brazed seam, revealing brittle fracture features. Cr4Ti, Cu0.8FeTi, Fe8TiZr3 and Al2NiTi3C compounds were found in the fractured joint brazed with Cu43.75Ti37.5Ni6.25Zr6.25V6.25 foil, and Fe2Ti, TiCu, Fe8TiZr3 and NiTi0.8Zr0.3 compounds were detected in the joint brazed with Cu37.5Ti25Ni12.5Zr12.5V12.5 foil. The existence of Cr-Ti, Fe-Ti, Cu-Fe-Ti, and Fe-Ti-V intermetallic compounds in the brazed seam caused fracture of the resultant joints.

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.

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.

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.

An influence of a Glass Braze Composition on the Properties of Li-Ti Ferrite Joints

NASA Astrophysics Data System (ADS)

Lin, Panpan; Lin, Tiesong; He, Peng; Sekulic, Dusan P.; Zhao, Mengyuan; Wang, Shulei

2017-04-01

The influence of the chemical composition of Bi2O3-B2O3-SiO2-ZnO glass brazes on (i) the microstructure, (ii) the mechanical and (iii) the dielectric properties of Li-Ti ferrite joints was systematically investigated. The Bi5(Ti3Fe)O15 whisker and a white block phase consisting of Bi12SiO2 and Bi24B2O39 were observed in the joints of Li-Ti ferrite/Bi25-Ba and Li-Ti ferrite/glass brazes, respectively, containing a higher content of Bi2O3. No crystalline phase was detected in the Li-Ti ferrite/Bi25 and Li-Ti ferrite/Bi20 joints. The joint strength reached the maximum of 48 MPa in the Li-Ti ferrite/Bi25-Ba couples. It is assumed that this is mainly due to the strengthening effect of Bi5(Ti3Fe)O15 whiskers. The bonding temperature (700°C) had little effect on the dielectric properties of Li-Ti ferrite. Moreover, compared to the Bi25-Ba glass brazes, the Bi25 and Bi20 glass brazes had a less pronounced influence on the dielectric properties of joints. Different glass brazes can be tailored to different requirements depending on specific application and joint property requirements.

Vacuum brazing of high volume fraction SiC particles reinforced aluminum matrix composites

NASA Astrophysics Data System (ADS)

Cheng, Dongfeng; Niu, Jitai; Gao, Zeng; Wang, Peng

2015-03-01

This experiment chooses A356 aluminum matrix composites containing 55% SiC particle reinforcing phase as the parent metal and Al-Si-Cu-Zn-Ni alloy metal as the filler metal. The brazing process is carried out in vacuum brazing furnace at the temperature of 550°C and 560°C for 3 min, respectively. The interfacial microstructures and fracture surfaces are investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy spectrum analysis (EDS). The result shows that adequacy of element diffusion are superior when brazing at 560°C, because of higher activity and liquidity. Dislocations and twins are observed at the interface between filler and composite due to the different expansion coefficient of the aluminum alloy matrix and SiC particles. The fracture analysis shows that the brittle fracture mainly located at interface of filler and composites.

CED-10/Rac1 Regulates Endocytic Recycling through the RAB-5 GAP TBC-2

PubMed Central

Sun, Lin; Liu, Ou; Desai, Jigar; Karbassi, Farhad; Sylvain, Marc-André; Shi, Anbing; Zhou, Zheng; Rocheleau, Christian E.; Grant, Barth D.

2012-01-01

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane. PMID:22807685

Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology.

PubMed

Wilson, Gabrielle R; Sim, Joe C H; McLean, Catriona; Giannandrea, Maila; Galea, Charles A; Riseley, Jessica R; Stephenson, Sarah E M; Fitzpatrick, Elizabeth; Haas, Stefan A; Pope, Kate; Hogan, Kirk J; Gregg, Ronald G; Bromhead, Catherine J; Wargowski, David S; Lawrence, Christopher H; James, Paul A; Churchyard, Andrew; Gao, Yujing; Phelan, Dean G; Gillies, Greta; Salce, Nicholas; Stanford, Lynn; Marsh, Ashley P L; Mignogna, Maria L; Hayflick, Susan J; Leventer, Richard J; Delatycki, Martin B; Mellick, George D; Kalscheuer, Vera M; D'Adamo, Patrizia; Bahlo, Melanie; Amor, David J; Lockhart, Paul J

2014-12-04

Advances in understanding the etiology of Parkinson disease have been driven by the identification of causative mutations in families. Genetic analysis of an Australian family with three males displaying clinical features of early-onset parkinsonism and intellectual disability identified a ∼45 kb deletion resulting in the complete loss of RAB39B. We subsequently identified a missense mutation (c.503C>A [p.Thr168Lys]) in RAB39B in an unrelated Wisconsin kindred affected by a similar clinical phenotype. In silico and in vitro studies demonstrated that the mutation destabilized the protein, consistent with loss of function. In vitro small-hairpin-RNA-mediated knockdown of Rab39b resulted in a reduction in the density of α-synuclein immunoreactive puncta in dendritic processes of cultured neurons. In addition, in multiple cell models, we demonstrated that knockdown of Rab39b was associated with reduced steady-state levels of α-synuclein. Post mortem studies demonstrated that loss of RAB39B resulted in pathologically confirmed Parkinson disease. There was extensive dopaminergic neuron loss in the substantia nigra and widespread classic Lewy body pathology. Additional pathological features included cortical Lewy bodies, brain iron accumulation, tau immunoreactivity, and axonal spheroids. Overall, we have shown that loss-of-function mutations in RAB39B cause intellectual disability and pathologically confirmed early-onset Parkinson disease. The loss of RAB39B results in dysregulation of α-synuclein homeostasis and a spectrum of neuropathological features that implicate RAB39B in the pathogenesis of Parkinson disease and potentially other neurodegenerative disorders. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

MicroRNA-16 Alleviates Inflammatory Pain by Targeting Ras-Related Protein 23 (RAB23) and Inhibiting p38 MAPK Activation.

PubMed

Chen, Wenjin; Guo, Shengdong; Wang, Shenggang

2016-10-22

BACKGROUND The purpose of our study was to determine the functional role of microRNA (miR)-16 in chronic inflammatory pain and to disclose its underlying molecular mechanism. MATERIAL AND METHODS Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) to Wistar rats. The pWPXL-miR-16, PcDNA3.1- Ras-related protein (RAB23), and/or SB203580 were delivered intrathecally to the rats. Behavioral tests were detected at 0 h, 4 h, 1 d, 4 d, 7 d, and 14 d after CFA injection. After behavioral tests, L4-L6 dorsal spinal cord were obtained and the levels of miR-16, RAB23, and phosphorylation of p38 (p-p38) were evaluated by quantitative real-time PCR (qRT-PCR). In addition, luciferase reporter assay was performed to explore whether RAB23 was a target of miR-16, and qRT-PCR and Western blotting were used to confirm the regulation between RAB23 and miR-16. RESULTS The level of miR-16 was significantly decreased in the CFA-induced inflammatory pain. Intrathecal injection of miR-16 alleviates pain response and raised pain threshold. The level of RAB23 was significantly increased in the pain model, and intrathecal injection of RAB23 aggravated pain response. Luciferase reporter assay confirmed that RAB23 was a direct target of miR-16, and RAB23 was negatively regulated by miR-16. In addition, we found that simultaneous administration of SB203580 and miR-16 further alleviates pain response compared to only administration of miR-16. CONCLUSIONS Our findings suggest that miR-16 relieves chronic inflammatory pain by targeting RAB23 and inhibiting p38 MAPK activation.

Functional Characterization of Rare RAB12 Variants and Their Role in Musician's and Other Dystonias.

PubMed

Hebert, Eva; Borngräber, Friederike; Schmidt, Alexander; Rakovic, Aleksandar; Brænne, Ingrid; Weissbach, Anne; Hampf, Jennie; Vollstedt, Eva-Juliane; Größer, Leopold; Schaake, Susen; Müller, Michaela; Manzoor, Humera; Jabusch, Hans-Christian; Alvarez-Fischer, Daniel; Kasten, Meike; Kostic, Vladimir S; Gasser, Thomas; Zeuner, Kirsten E; Kim, Han-Joon; Jeon, Beomseok; Bauer, Peter; Altenmüller, Eckart; Klein, Christine; Lohmann, Katja

2017-10-18

Mutations in RAB (member of the Ras superfamily) genes are increasingly recognized as cause of a variety of disorders including neurological conditions. While musician's dystonia (MD) and writer's dystonia (WD) are task-specific movement disorders, other dystonias persistently affect postures as in cervical dystonia. Little is known about the underlying etiology. Next-generation sequencing revealed a rare missense variant (c.586A>G; p.Ile196Val) in RAB12 in two of three MD/WD families. Next, we tested 916 additional dystonia patients; 512 Parkinson's disease patients; and 461 healthy controls for RAB12 variants and identified 10 additional carriers of rare missense changes among dystonia patients (1.1%) but only one carrier in non-dystonic individuals (0.1%; p = 0.005). The detected variants among index patients comprised p.Ile196Val ( n = 6); p.Ala174Thr ( n = 3); p.Gly13Asp; p.Ala148Thr; and p.Arg181Gln in patients with MD; cervical dystonia; or WD. Two relatives of MD patients with WD also carried p.Ile196Val. The two variants identified in MD patients (p.Ile196Val; p.Gly13Asp) were characterized on endogenous levels in patient-derived fibroblasts and in two RAB12-overexpressing cell models. The ability to hydrolyze guanosine triphosphate (GTP), so called GTPase activity, was increased in mutants compared to wildtype. Furthermore, subcellular distribution of RAB12 in mutants was altered in fibroblasts. Soluble Transferrin receptor 1 levels were reduced in the blood of all three tested p.Ile196Val carriers. In conclusion, we demonstrate an enrichment of missense changes among dystonia patients. Functional characterization revealed altered enzyme activity and lysosomal distribution in mutants suggesting a contribution of RAB12 variants to MD and other dystonias.

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.

The interaction of reaction-bonded silicon carbide and inconel 600 with a nickel-based brazing alloy

NASA Astrophysics Data System (ADS)

McDermid, J. R.; Pugh, M. D.; Drew, R. A. L.

1989-09-01

The objective of the present research was to join reaction-bonded silicon carbide (RBSC) to INCONEL 600 (a nickel-based superalloy) for use in advanced heat engine applications using either direct brazing or composite interlayer joining. Direct brazing experiments employed American Welding Society (AWS) BNi-5, a commercial nickel-based brazing alloy, as a filler material; composite interlayers consisted of intimate mixtures of α-SiC and BNi-5 powders. Both methods resulted in the liquid filler metal forming a Ni-Si liquid with the free Si in the RBSC, which, in turn, reacted vigorously with the SiC component of the RBSC to form low melting point constituents in both starting materials and Cr carbides at the metal-ceramic interface. Using solution thermodynamics, it was shown that a Ni-Si liquid of greater than 60 at. pct Ni will decompose a-SiC at the experimental brazing temperature of 1200 ‡C; these calculations are consistent with the experimentally observed composition profiles and reaction morphology within the ceramic. It was concluded that the joining of RBSC to INCONEL 600 using a nickel-based brazing alloy is not feasible due to the inevitability of the filler metal reacting with the ceramic, degrading the high-temperature properties of the base materials.

SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

PubMed Central

Bruno, Joanne; Chaudhary, Natasha; Iaea, David

2016-01-01

RAB10 is a regulator of insulin-stimulated translocation of the GLUT4 glucose transporter to the plasma membrane (PM) of adipocytes, which is essential for whole-body glucose homeostasis. We establish SEC16A as a novel RAB10 effector in this process. Colocalization of SEC16A with RAB10 is augmented by insulin stimulation, and SEC16A knockdown attenuates insulin-induced GLUT4 translocation, phenocopying RAB10 knockdown. We show that SEC16A and RAB10 promote insulin-stimulated mobilization of GLUT4 from a perinuclear recycling endosome/TGN compartment. We propose RAB10–SEC16A functions to accelerate formation of the vesicles that ferry GLUT4 to the PM during insulin stimulation. Because GLUT4 continually cycles between the PM and intracellular compartments, the maintenance of elevated cell-surface GLUT4 in the presence of insulin requires accelerated biogenesis of the specialized GLUT4 transport vesicles. The function of SEC16A in GLUT4 trafficking is independent of its previously characterized activity in ER exit site formation and therefore independent of canonical COPII-coated vesicle function. However, our data support a role for SEC23A, but not the other COPII components SEC13, SEC23B, and SEC31, in the insulin stimulation of GLUT4 trafficking, suggesting that vesicles derived from subcomplexes of COPII coat proteins have a role in the specialized trafficking of GLUT4. PMID:27354378

Investigation on localized corrosion of 304 stainless steel joints brazed using Sn-plated Ag alloy filler in NaCl aqueous solution

NASA Astrophysics Data System (ADS)

Wang, Xingxing; Li, Shuai; Peng, Jin

2018-03-01

Novel AgCuZnSn filler metal with high Sn contents was prepared from BAg50CuZn filler metal by a process of electroplating and thermal diffusion, and the prepared filler metal was applied to induction brazing of 304 stainless steel. The corrosion behavior of the brazed joints was evaluated based on localized corrosion analysis, the morphology of the joints were analyzed by SEM after immersion in a 3.5 vol% NaCl aqueous solution. The results indicated that corrosion groove occurred near the interface between the stainless steel base metal and the brazing seam. A wide range of defects such as holes and cracks appeared on the surface of the base metal, while the brazing seam zone almost no corrosion defects occur. With the increase of corrosion time, the corrosion rates of both the brazing seam and the base metal first exhibited an increasing trend, followed by a decreasing trend, and the corrosion rate of the base metal was slightly greater than that of the brazing seam. The corrosion potential of the brazing seam and 304 stainless steel were -0.7758 V and -0.7863 V, respectively.

Tensile strength and corrosion resistance of brazed and laser-welded cobalt-chromium alloy joints.

PubMed

Zupancic, Rok; Legat, Andraz; Funduk, Nenad

2006-10-01

The longevity of prosthodontic restorations is often limited due to the mechanical or corrosive failure occurring at the sites where segments of a metal framework are joined together. The purpose of this study was to determine which joining method offers the best properties to cobalt-chromium alloy frameworks. Brazed and 2 types of laser-welded joints were compared for their mechanical and corrosion characteristics. Sixty-eight cylindrical cobalt-chromium dental alloy specimens, 35 mm long and 2 mm in diameter, were cast. Sixteen specimens were selected for electrochemical measurements in an artificial saliva solution and divided into 4 groups (n=4). In the intact group, the specimens were left as cast. The specimens of the remaining 3 groups were sectioned at the center, perpendicular to the long-axis, and were subsequently rejoined by brazing (brazing group) or laser welding using an X- or I-shaped joint design (X laser and I laser groups, respectively). Another 16 specimens were selected for electrochemical measurements in a more acidic artificial saliva solution. These specimens were also divided into 4 groups (n=4) as described above. Electrochemical impedance spectroscopy and potentiodynamic polarization were used to assess corrosion potentials, breakdown potentials, corrosion current densities, total impedances at lowest frequency, and polarization charge-transfer resistances. The remaining 36 specimens were used for tensile testing. They were divided into 3 groups in which specimen pairs (n=6) were joined by brazing or laser welding to form 70-mm-long cylindrical rods. The tensile strength (MPa) was measured using a universal testing machine. Differences between groups were analyzed using 1-way analysis of variance (alpha=.05). The fracture surfaces and corrosion defects were examined with a scanning electron microscope. The average tensile strength of brazed joints was 792 MPa and was significantly greater (P<.05) than the tensile strength of both types of

Crystal structure of TBC1D15 GTPase‐activating protein (GAP) domain and its activity on Rab GTPases

PubMed Central

Chen, Yan‐Na; Gu, Xin; Zhou, X. Edward; Wang, Weidong; Cheng, Dandan; Ge, Yinghua; Ye, Fei

2017-01-01

Abstract TBC1D15 belongs to the TBC (Tre‐2/Bub2/Cdc16) domain family and functions as a GTPase‐activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark‐TBC1D15 and Sus‐TBC1D15 belong to the same subfamily of TBC domain‐containing proteins, and their GAP‐domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost. PMID:28168758

Crystal structure of TBC1D15 GTPase-activating protein (GAP) domain and its activity on Rab GTPases.

PubMed

Chen, Yan-Na; Gu, Xin; Zhou, X Edward; Wang, Weidong; Cheng, Dandan; Ge, Yinghua; Ye, Fei; Xu, H Eric; Lv, Zhengbing

2017-04-01

TBC1D15 belongs to the TBC (Tre-2/Bub2/Cdc16) domain family and functions as a GTPase-activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark-TBC1D15 and Sus-TBC1D15 belong to the same subfamily of TBC domain-containing proteins, and their GAP-domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost. © 2017 The Protein Society.

Liver X Receptors Balance Lipid Stores in Hepatic Stellate Cells via Rab18, a Retinoid Responsive Lipid Droplet Protein

PubMed Central

O’Mahony, Fiona; Wroblewski, Kevin; O’Byrne, Sheila M.; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S.; Beaven, Simon W.

2014-01-01

Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ−/− mice have increased lipid droplet (LD) size but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ−/− and wild-type (WT) mice were profiled by gene array during in vitro activation. Lipid content was quantified by HPLC and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with siRNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ−/− HSCs have increased cholesterol and retinyl esters (CEs & REs). The retinoid increase drives intrinsic retinoic acid receptor (RAR) signaling and activation occurs more rapidly in Lxrαβ−/− HSCs. We identify Rab18 as a novel retinoic acid responsive, lipid droplet associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 GTPase activity and isoprenylation are required for stellate cell lipid droplet loss and induction of activation markers. These phenomena are accelerated in the Lxrαβ−/− HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards lipid droplet loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Conclusion Retinoid and cholesterol metabolism are linked in stellate cells by the LD associated protein, Rab18. Retinoid overload helps explain the pro-fibrotic phenotype of Lxrαβ−/− mice and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. PMID:25482505

Liver X receptors balance lipid stores in hepatic stellate cells through Rab18, a retinoid responsive lipid droplet protein.

PubMed

O'Mahony, Fiona; Wroblewski, Kevin; O'Byrne, Sheila M; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S; Beaven, Simon W

2015-08-01

Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ(-/-) mice have increased lipid droplet (LD) size, but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ(-/-) and wild-type mice were profiled by gene array during in vitro activation. Lipid content was quantified by high-performance liquid chromatography and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with small interfering RNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ(-/-) HSCs have increased cholesterol and retinyl esters. The retinoid increase drives intrinsic retinoic acid receptor signaling, and activation occurs more rapidly in Lxrαβ(-/-) HSCs. We identify Rab18 as a novel retinoic acid-responsive, LD-associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 guanosine triphosphatase activity and isoprenylation are required for stellate cell LD loss and induction of activation markers. These phenomena are accelerated in Lxrαβ(-/-) HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards LD loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Retinoid and cholesterol metabolism are linked in stellate cells by the LD-associated protein Rab18. Retinoid overload helps explain the profibrotic phenotype of Lxrαβ(-/-) mice, and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. © 2015 by the American Association for the Study of Liver Diseases.

Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

2010-09-01

The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

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.

Structural Basis for Rab1 De-AMPylation by the Legionella pneumophila Effector SidD

PubMed Central

Neunuebel, M. Ramona; Pallara, Chiara; Brady, Jacqueline; Kinch, Lisa N.; Fernández-Recio, Juan; Rojas, Adriana L.; Machner, Matthias P.; Hierro, Aitor

2013-01-01

The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses. PMID:23696742

The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria.

PubMed

Lizundia, Regina; Chaussepied, Marie; Naissant, Bernina; Masse, Guillemette X; Quevillon, Emmanuel; Michel, Fréderique; Monier, Solange; Weitzman, Jonathan B; Langsley, Gordon

2007-08-01

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

Rab5 GTPase controls chromosome alignment through Lamin disassembly and relocation of the NuMA-like protein Mud to the poles during mitosis

PubMed Central