Control of phospholipid flip-flop by transmembrane peptides

International Nuclear Information System (INIS)

Kaihara, Masanori; Nakao, Hiroyuki; Yokoyama, Hirokazu; Endo, Hitoshi; Ishihama, Yasushi; Handa, Tetsurou; Nakano, Minoru

2013-01-01

Highlights: ► Phospholipid flip-flop in transmembrane peptide-containing vesicles was investigated. ► Peptides that contained polar residues in the center of the transmembrane region promoted phospholipid flip-flop. ► A bioinformatics approach revealed the presence of polar residues in the transmembrane region of ER membrane proteins. ► Polar residues in ER membrane proteins possibly provide flippase-like activity. - Abstract: We designed three types of transmembrane model peptides whose sequence originates from a frequently used model peptide KALP23, and we investigated their effects on phospholipid flip-flop. Time-resolved small-angle neutron scattering and a dithionite fluorescent quenching assay demonstrated that TMP-L, which has a fully hydrophobic transmembrane region, did not enhance phospholipid flip-flop, whereas TMP-K and TMP-E, which have Lys and Glu, respectively, in the center of their transmembrane regions, enhanced phospholipid flip-flop. Introduction of polar residues in the membrane-spanning helices is considered to produce a locally polar region and enable the lipid head group to interact with the polar side-chain inside the bilayers, thereby reducing the activation energy for the flip-flop. A bioinformatics approach revealed that acidic and basic residues account for 4.5% of the central region of the transmembrane domain in human ER membrane proteins. Therefore, polar residues in ER membrane proteins are considered to provide flippase-like activity

Molecular characterization of the gerbil C5a receptor and identification of a transmembrane domain V amino acid that is crucial for small molecule antagonist interaction.

Science.gov (United States)

Waters, Stephen M; Brodbeck, Robbin M; Steflik, Jeremy; Yu, Jianying; Baltazar, Carolyn; Peck, Amy E; Severance, Daniel; Zhang, Lu Yan; Currie, Kevin; Chenard, Bertrand L; Hutchison, Alan J; Maynard, George; Krause, James E

2005-12-09

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.

[Research advances in CKLF-like MARVEL transmembrane domain containing member 5].

Science.gov (United States)

Yuan, Ye-qing; Xiao, Yun-bei; Liu, Zhen-hua; Zhang, Xiao-wei; Xu, Tao; Wang, Xiao-feng

2012-12-01

CKLF-like MARVEL transmembrane domain containing member(CMTM)is a novel generic family firstly reported by Peking University Center for Human Disease Genomics. CMTM5 belongs to this family and has exhibited tumor-inhibiting activities. It can encode proteins approaching to the transmembrane 4 superfamily(TM4SF). CMTM5 is broadly expressed in normal adult and fetal human tissues, but is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM5 may inhibit the proliferation, migration, and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear, CMTM5 may be involved in various signaling pathways governing the occurrence and development of tumors. CMTM5 may be a new target in the gene therapies for tumors, while further studies on CMTM5 and its anti-tumor mechanisms are warranted.

Characterization of the GXXXG motif in the first transmembrane segment of Japanese encephalitis virus precursor membrane (prM protein

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Wu Suh-Chin

2010-05-01

Full Text Available Abstract The interaction between prM and E proteins in flavivirus-infected cells is a major driving force for the assembly of flavivirus particles. We used site-directed mutagenesis to study the potential role of the transmembrane domains of the prM proteins of Japanese encephalitis virus (JEV in prM-E heterodimerization as well as subviral particle formation. Alanine insertion scanning mutagenesis within the GXXXG motif in the first transmembrane segment of JEV prM protein affected the prM-E heterodimerization; its specificity was confirmed by replacing the two glycines of the GXXXG motif with alanine, leucine and valine. The GXXXG motif was found to be conserved in the JEV serocomplex viruses but not other flavivirus groups. These mutants with alanine inserted in the two prM transmembrane segments all impaired subviral particle formation in cell cultures. The prM transmembrane domains of JEV may play importation roles in prM-E heterodimerization and viral particle assembly.

Activation gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains.

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Sadja, Rona; Reuveny, Eitan

2009-01-01

G-protein-coupled inwardly rectifying potassium channels (GIRK/Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K(+) ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein betagamma subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast activation gating. This result suggests that there is a cooperative movement of either one of these domains to allow fast and efficient activation gating of GIRK channels.

Delineation of the peptide binding site of the human galanin receptor.

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Kask, K; Berthold, M; Kahl, U; Nordvall, G; Bartfai, T

1996-01-01

Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th Images PMID:8617199

Hidden markov model for the prediction of transmembrane proteins using MATLAB.

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Chaturvedi, Navaneet; Shanker, Sudhanshu; Singh, Vinay Kumar; Sinha, Dhiraj; Pandey, Paras Nath

2011-01-01

Since membranous proteins play a key role in drug targeting therefore transmembrane proteins prediction is active and challenging area of biological sciences. Location based prediction of transmembrane proteins are significant for functional annotation of protein sequences. Hidden markov model based method was widely applied for transmembrane topology prediction. Here we have presented a revised and a better understanding model than an existing one for transmembrane protein prediction. Scripting on MATLAB was built and compiled for parameter estimation of model and applied this model on amino acid sequence to know the transmembrane and its adjacent locations. Estimated model of transmembrane topology was based on TMHMM model architecture. Only 7 super states are defined in the given dataset, which were converted to 96 states on the basis of their length in sequence. Accuracy of the prediction of model was observed about 74 %, is a good enough in the area of transmembrane topology prediction. Therefore we have concluded the hidden markov model plays crucial role in transmembrane helices prediction on MATLAB platform and it could also be useful for drug discovery strategy. The database is available for free at bioinfonavneet@gmail.comvinaysingh@bhu.ac.in.

Topology of transmembrane channel-like gene 1 protein.

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Labay, Valentina; Weichert, Rachel M; Makishima, Tomoko; Griffith, Andrew J

2010-10-05

Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N- and C-termini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with that of the shaker-TRP superfamily of ion channels.

MutHTP: Mutations in Human Transmembrane Proteins.

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A, Kulandaisamy; S, Binny Priya; R, Sakthivel; Tarnovskaya, Svetlana; Bizin, Ilya; Hönigschmid, Peter; Frishman, Dmitrij; Gromiha, M Michael

2018-02-01

We have developed a novel database, MutHTP, which contains information on 183395 disease-associated and 17827 neutral mutations in human transmembrane proteins. For each mutation site MutHTP provides a description of its location with respect to the membrane protein topology, structural environment (if available) and functional features. Comprehensive visualization, search, display and download options are available. The database is publicly available at http://www.iitm.ac.in/bioinfo/MutHTP/. The website is implemented using HTML, PHP and javascript and supports recent versions of all major browsers, such as Firefox, Chrome and Opera. gromiha@iitm.ac.in. Supplementary data are available at Bioinformatics online. © The Author (2018). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Substrate-modulated unwinding of transmembrane helices in the NSS transporter LeuT.

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Merkle, Patrick S; Gotfryd, Kamil; Cuendet, Michel A; Leth-Espensen, Katrine Z; Gether, Ulrik; Loland, Claus J; Rand, Kasper D

2018-05-01

LeuT, a prokaryotic member of the neurotransmitter:sodium symporter (NSS) family, is an established structural model for mammalian NSS counterparts. We investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the dynamics associated with transition of the transporter between outward- and inward-facing configurations in a Na + - and K + -dependent manner. The results suggest that partial unwinding of transmembrane helices 1/5/6/7 drives LeuT from a substrate-bound, outward-facing occluded conformation toward an inward-facing open state. These hitherto unknown, large-scale conformational changes in functionally important transmembrane segments, observed for LeuT in detergent-solubilized form and when embedded in a native-like phospholipid bilayer, could be of physiological relevance for the translocation process.

The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

Science.gov (United States)

Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

2012-01-01

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

Possible involvement of membrane lipids peroxidation and oxidation of catalytically essential thiols of the cerebral transmembrane sodium pump as component mechanisms of iron-mediated oxidative stress-linked dysfunction of the pump's activity

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T.I. Omotayo

2015-04-01

Full Text Available The precise molecular events defining the complex role of oxidative stress in the inactivation of the cerebral sodium pump in radical-induced neurodegenerative diseases is yet to be fully clarified and thus still open. Herein we investigated the modulation of the activity of the cerebral transmembrane electrogenic enzyme in Fe2+-mediated in vitro oxidative stress model. The results show that Fe2+ inhibited the transmembrane enzyme in a concentration dependent manner and this effect was accompanied by a biphasic generation of aldehydic product of lipid peroxidation. While dithiothreitol prevented both Fe2+ inhibitory effect on the pump and lipid peroxidation, vitamin E prevented only lipid peroxidation but not inhibition of the pump. Besides, malondialdehyde (MDA inhibited the pump by a mechanism not related to oxidation of its critical thiols. Apparently, the low activity of the pump in degenerative diseases mediated by Fe2+ may involve complex multi-component mechanisms which may partly involve an initial oxidation of the critical thiols of the enzyme directly mediated by Fe2+ and during severe progression of such diseases; aldehydic products of lipid peroxidation such as MDA may further exacerbate this inhibitory effect by a mechanism that is likely not related to the oxidation of the catalytically essential thiols of the ouabain-sensitive cerebral electrogenic pump.

Localization of the transmembrane proteoglycan syndecan-4 and its regulatory kinases in costameres of rat cardiomyocytes: a deconvolution microscopic study

DEFF Research Database (Denmark)

VanWinkle, W Barry; Snuggs, Mark B; De Hostos, Eugenio L

2002-01-01

Syndecan-4 (syn-4), a transmembrane heparan sulfate-containing proteoglycan, is unique among the four members of the syndecan family in its specific cellular localization to complex cytoskeletal adhesion sites, i.e., focal adhesions. During early phenotypic redifferentiation of neonatal cardiomyo...

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

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Balda Maria S

2009-12-01

Full Text Available Abstract Background Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction. Results Here we identify a new transmembrane component of tight junctions: MarvelD3, a four-span transmembrane protein. Its predicted transmembrane helices form a Marvel (MAL and related proteins for vesicle traffic and membrane link domain, a structural motif originally discovered in proteins involved in membrane apposition and fusion events, such as the tight junction proteins occludin and tricellulin. In mammals, MarvelD3 is expressed as two alternatively spliced isoforms. Both isoforms exhibit a broad tissue distribution and are expressed by different types of epithelial as well as endothelial cells. MarvelD3 co-localises with occludin at tight junctions in intestinal and corneal epithelial cells. RNA interference experiments in Caco-2 cells indicate that normal MarvelD3 expression is not required for the formation of functional tight junctions but depletion results in monolayers with increased transepithelial electrical resistance. Conclusions Our data indicate that MarvelD3 is a third member of the tight junction-associated occludin family of transmembrane proteins. Similar to occludin, normal expression of MarvelD3 is not essential for the formation of functional tight junctions. However, MarvelD3 functions as a determinant of epithelial paracellular permeability properties.

PDBTM: Protein Data Bank of transmembrane proteins after 8 years.

Science.gov (United States)

Kozma, Dániel; Simon, István; Tusnády, Gábor E

2013-01-01

The PDBTM database (available at http://pdbtm.enzim.hu), the first comprehensive and up-to-date transmembrane protein selection of the Protein Data Bank, was launched in 2004. The database was created and has been continuously updated by the TMDET algorithm that is able to distinguish between transmembrane and non-transmembrane proteins using their 3D atomic coordinates only. The TMDET algorithm can locate the spatial positions of transmembrane proteins in lipid bilayer as well. During the last 8 years not only the size of the PDBTM database has been steadily growing from ∼400 to 1700 entries but also new structural elements have been identified, in addition to the well-known α-helical bundle and β-barrel structures. Numerous 'exotic' transmembrane protein structures have been solved since the first release, which has made it necessary to define these new structural elements, such as membrane loops or interfacial helices in the database. This article reports the new features of the PDBTM database that have been added since its first release, and our current efforts to keep the database up-to-date and easy to use so that it may continue to serve as a fundamental resource for the scientific community.

A transmembrane polar interaction is involved in the functional regulation of integrin alpha L beta 2.

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Vararattanavech, Ardcharaporn; Chng, Choon-Peng; Parthasarathy, Krupakar; Tang, Xiao-Yan; Torres, Jaume; Tan, Suet-Mien

2010-05-14

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar

U.S. EPA Superfund Program's Policy for Community Involvement at Radioactively Contaminated Sites

International Nuclear Information System (INIS)

Carey, Pat; Walker, Stuart

2008-01-01

This paper describes the Superfund program's statutory requirements for community involvement. It also discusses the efforts the Superfund program has made that go beyond these statutory requirements to involve communities. The Environmental Protection Agency (EPA) implements the Superfund program under the authority of the Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA), as amended by the Superfund Amendments and Reauthorization Act of 1986 (SARA). From the beginning of the Superfund program, Congress envisioned a role for communities. This role has evolved and expanded during the implementation of the Superfund program. Initially, the CERCLA statute had community involvement requirements designed to inform surrounding communities of the work being done at a site. CERCLA's provisions required 1) development of a community relations plan for each site, 2) establishment of information repositories near each site where all publicly available materials related to the site would be accessible for public inspection, 3) opportunities for the public to comment on the proposed remedy for each site and 4) development of a responsiveness summary responding to all significant comments received on the proposed remedy. In recognition of the need for people living near Superfund sites to be well-informed and involved with decisions concerning sites in their communities, SARA expanded Superfund's community involvement activities in 1986. SARA provided the authority to award Technical Assistance Grants (TAGs) to local communities enabling them to hire independent technical advisors to assist them in understanding technical issues and data about the site. The Superfund Community Involvement Program has sought to effectively implement the statutory community involvement requirements, and to go beyond those requirements to find meaningful ways to involve citizens in the cleanup of sites in their communities. We've structured our program around

Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore–quencher pairing

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Pantazis, Antonios

2012-01-01

Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1–S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K+ (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a “decentralized” distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD. PMID:22802360

Impact of axial velocity and transmembrane pressure (TMP) on ARP filter performance

Energy Technology Data Exchange (ETDEWEB)

Poirier, M. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Burket, P. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

2016-02-29

The Savannah River Site (SRS) is currently treating radioactive liquid waste with the Actinide Removal Process (ARP) and the Modular Caustic Side Solvent Extraction Unit (MCU). Recently, the low filter flux through the ARP of approximately 5 gallons per minute has limited the rate at which radioactive liquid waste can be treated. Salt Batch 6 had a lower processing rate and required frequent filter cleaning. Savannah River Remediation (SRR) has a desire to understand the causes of the low filter flux and to increase ARP/MCU throughput. One potential method for increasing filter flux is to adjust the axial velocity and transmembrane pressure (TMP). SRR requested SRNL to conduct bench-scale filter tests to evaluate the effects of axial velocity and transmembrane pressure on crossflow filter flux. The objective of the testing was to determine whether increasing the axial velocity at the ARP could produce a significant increase in filter flux. The authors conducted the tests by preparing slurries containing 6.6 M sodium Salt Batch 6 supernate and 2.5 g MST/L, processing the slurry through a bench-scale crossflow filter unit at varying axial velocity and TMP, and measuring filter flux as a function of time.

U.S. EPA Superfund Program's Policy for Community Involvement at Radioactively Contaminated Sites

International Nuclear Information System (INIS)

Martin, K.; Walker, St.

2009-01-01

This paper describes the EPA Superfund program's statutory requirements for community involvement. It also discusses the efforts the Superfund program has made that go beyond these statutory requirements to involve communities, and what lessons have been learned by EPA when trying to conduct meaningful community involvement at sites. In addition, it discusses tools that EPA has designed to specifically enhance community involvement at radioactively contaminated Superfund sites. In summary, the Superfund program devotes substantial resources to involving the local community in the site cleanup decision making process. We believe community involvement provides us with highly valuable information that must be available to carefully consider remedial alternatives at a site. We also find our employees enjoy their jobs more. Rather than fighting with an angry public they can work collaboratively to solve the problems created by the hazardous waste sites. We have learned the time and resources we devote at the beginning of a project to developing relationships with the local community, and learning about their issues and concerns is time and resources well spent. We believe the evidence shows this up-front investment helps us make better cleanup decisions, and avoids last minute efforts to work with a hostile community who feels left out of the decision-making process. (authors)

Glycine Perturbs Local and Global Conformational Flexibility of a Transmembrane Helix

DEFF Research Database (Denmark)

Högel, Philipp; Götz, Alexander; Kuhne, Felix

2018-01-01

Flexible transmembrane helices frequently support the conformational transitions between different functional states of membrane proteins. While proline is well known to distort and destabilize transmembrane helices, the role of glycine is still debated. Here, we systematically investigated the e...

Expression of transmembrane carbonic anhydrases, CAIX and CAXII, in human development

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Lerman Michael I

2009-03-01

Full Text Available Abstract Background Transmembrane CAIX and CAXII are members of the alpha carbonic anhydrase (CA family. They play a crucial role in differentiation, proliferation, and pH regulation. Expression of CAIX and CAXII proteins in tumor tissues is primarily induced by hypoxia and this is particularly true for CAIX, which is regulated by the transcription factor, hypoxia inducible factor-1 (HIF-1. Their distributions in normal adult human tissues are restricted to highly specialized cells that are not always hypoxic. The human fetus exists in a relatively hypoxic environment. We examined expression of CAIX, CAXII and HIF-1α in the developing human fetus and postnatal tissues to determine whether expression of CAIX and CAXII is exclusively regulated by HIF-1. Results The co-localization of CAIX and HIF-1α was limited to certain cell types in embryonic and early fetal tissues. Those cells comprised the primitive mesenchyma or involved chondrogenesis and skin development. Transient CAIX expression was limited to immature tissues of mesodermal origin and the skin and ependymal cells. The only tissues that persistently expressed CAIX protein were coelomic epithelium (mesothelium and its remnants, the epithelium of the stomach and biliary tree, glands and crypt cells of duodenum and small intestine, and the cells located at those sites previously identified as harboring adult stem cells in, for example, the skin and large intestine. In many instances co-localization of CAIX and HIF-1α was not evident. CAXII expression is restricted to cells involved in secretion and water absorption such as parietal cells of the stomach, acinar cells of the salivary glands and pancreas, epithelium of the large intestine, and renal tubules. Co-localization of CAXII with CAIX or HIF-1α was not observed. Conclusion The study has showed that: 1 HIF-1α and CAIX expression co- localized in many, but not all, of the embryonic and early fetal tissues; 2 There is no evidence of

Sll0528, a Site-2-Protease, Is Critically Involved in Cold, Salt and Hyperosmotic Stress Acclimation of Cyanobacterium Synechocystis sp. PCC 6803

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Haijin Lei

2014-12-01

Full Text Available Site-2-proteases (S2Ps mediated proteolysis of transmembrane transcriptional regulators is a conserved mechanism to regulate transmembrane signaling. The universal presence of S2P homologs in different cyanobacterial genomes suggest conserved and fundamental functions, though limited data has been available. Here we provide the first evidence that Sll0528, a site-2-protease in Synechocystis sp. PCC 6803 is crucial for salt, cold and hyperosmotic stress acclimation. Remarkable induction of sll0528 gene expression was observed under salt, cold and hyperosmotic stress, much higher than induction of the other three S2Ps. Knock-out of sll0528 gene in wild type Synechocystis sp. PCC 6803 increased their sensitivity to salt, cold and hyperosmotic stress, as revealed by retarded growth, reduced pigments and disrupted photosystems. The sll0528 gene was induced to a much smaller extent by high light and mixotrophic growth with glucose. Similar growth responses of the sll0528 knockout mutant and wild type under high light and mixotrophic growth indicated that sll0528 was dispensable for these conditions. Recombinant Sll0528 protein could cleave beta-casein into smaller fragments. These results together suggest that the Sll0528 metalloprotease plays a role in the stress response and lays the foundation for further investigation of its mechanism, as well as providing hints for the functional analysis of other S2Ps in cyanobacteria.

Generation and Nuclear Translocation of Sumoylated Transmembrane Fragment of Cell Adhesion Molecule L1

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Lutz, David; Wolters-Eisfeld, Gerrit; Joshi, Gunjan; Djogo, Nevena; Jakovcevski, Igor; Schachner, Melitta; Kleene, Ralf

2012-01-01

The functions of the cell adhesion molecule L1 in the developing and adult nervous system are triggered by homophilic and heterophilic interactions that stimulate signal transductions that activate cellular responses. Here, we show that stimulation of signaling by function-triggering L1 antibodies or L1-Fc leads to serine protease-dependent cleavage of full-length L1 at the plasma membrane and generation of a sumoylated transmembrane 70-kDa fragment comprising the intracellular and transmembrane domains and part of the extracellular domain. The 70-kDa transmembrane fragment is transported from the plasma membrane to a late endosomal compartment, released from endosomal membranes into the cytoplasm, and transferred from there into the nucleus by a pathway that depends on importin and chromatin-modifying protein 1. Mutation of the sumoylation site at Lys1172 or of the nuclear localization signal at Lys1147 abolished L1-stimulated generation or nuclear import of the 70-kDa fragment, respectively. Nuclear import of the 70-kDa fragment may activate cellular responses in parallel or in association with phosphorylation-dependent signaling pathways. Alterations in the levels of the 70-kDa fragment during development and in the adult after spinal cord injury or in a mouse model of Alzheimer disease suggest that this fragment is functionally implicated in development, regeneration, neurodegeneration, tumorigenesis, and possibly synaptic plasticity in the mature nervous system. PMID:22431726

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

Science.gov (United States)

Johnson, R G; Pfister, D; Carty, S E; Scarpa, A

1979-11-10

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential

A successful effort to involve stakeholders in a facility siting decision using LIPS with stakeholder involvement

International Nuclear Information System (INIS)

Merkhofer, L.; Conway, R.; Anderson, B.

1995-01-01

Local public opposition to federal bureaucratic decisions has resulted in public agencies rethinking the role of stakeholders in decision making. Efforts to include stakeholders directly in the decision-making process are on the increase. Unfortunately, many attempts to involve members of the public in decisions involving complex technical issues have failed. A key problem has been defining a meaningful role for the public in the process of arriving at a technical decision. This paper describes a successful effort by Sandia National Laboratories (SNL) in New Mexico to involve stakeholders in an important technical decision associated with its Environmental Restoration (ER) Project. The decision was where to locate a Corrective Action Management Unit (CAMU), a facility intended to consolidate and store wastes generated from the cleanup of hazardous waste sites. A formal priority setting process known as the Laboratory Integration Prioritization System (LIPS) was adapted to provide an approach for involving the public. Although rarely applied to stakeholder participation, the LIPS process proved surprisingly effective. It produced a consensus over a selected site and enhanced public trust and understanding of Project activities

Stakeholder involvement and public outreach at two Department of Energy sites

International Nuclear Information System (INIS)

Gray, R.H.

1996-01-01

Outreach efforts at two U.S. Department of Energy sites (i.e., the Hanford Site in southeastern Washington and Pantex Plant in the Texas panhandle) have involved a broad spectrum of communications media, including technical presentations and articles, information brochures and fact sheets, video productions; interactive exhibits; presentations at civic and other public meetings; and proactive interactions with the news media, regulators and concerned citizens. In addition, representatives of local communities operate offsite environmental monitoring stations and Native Americans are involved in studying cultural resources, fisheries, and other issues at Hanford, and a program to obtain environmental samples from neighbors property is underway at Pantex. All major environmental programs (e.g., the reconstruction of past radiological doses to offsite human populations at Hanford) are conducted with open public participation, the Pantex Plant has opened an Information office in Amarillo, Texas, and both sites now have Citizen's Advisory Boards. This presentation describes Hanford and Pantex public outreach and involvement efforts, our successes and failures, and the lessons learned

Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae.

Science.gov (United States)

Schuster, M; Wasserbauer, E; Aversa, G; Jungbauer, A

2001-02-01

Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secretion of the transmembrane glycoprotein signaling lymphocytic activation molecule (SLAM) was studied. SLAM belongs to the immunoglobulin superfamily and its engagement results in T-cell expansion and INF-gamma production. The molecule is composed of an extracellular, a single-span transmembrane and a cytoplasmatic domain. The extracellular part may be relevant for stimulation studies in vitro since SLAM is a high-affinity self-ligand. Therefore several fragments of this region have been expressed as Flag-fusions in S. cerevisiae: a full-length fragment containing the transmembrane region and the autologous signal sequence, another without the transmembrane region, and two fragments without the autologous signal sequence with and without the transmembrane region. By molecular cloning, the different deletion mutants of the cDNA encoding the full-length construct have been inserted in a yeast episomal plasmid. Upstream of the cDNA, the alpha-leader sequence of a yeast mating pheromone has been cloned to direct the fusion proteins into the secretory protein maturation pathway. All four fragments were expressed but yield, location, and maturation were highly influenced by the transmembrane domain and the autologous signal sequence. Only the fragment without autologous signal sequence and transmembrane domain could be efficiently secreted. High-mannose glycosylation was analyzed by lectin mapping and digestion with specific glycosidases. After enzyme treatment, a single band product with the theoretical size could be detected and identified as SLAM by a specific monoclonal antibody. The fusion protein concentration in the supernatant was 30 microg/ml. The

Regulation of natural competence by the orphan two-component system sensor kinase ChiS involves a non-canonical transmembrane regulator in Vibrio cholerae.

Science.gov (United States)

Yamamoto, Shouji; Mitobe, Jiro; Ishikawa, Takahiko; Wai, Sun Nyunt; Ohnishi, Makoto; Watanabe, Haruo; Izumiya, Hidemasa

2014-01-01

In Vibrio cholerae, 41 chitin-inducible genes, including the genes involved in natural competence for DNA uptake, are governed by the orphan two-component system (TCS) sensor kinase ChiS. However, the mechanism by which ChiS controls the expression of these genes is currently unknown. Here, we report the involvement of a novel transcription factor termed 'TfoS' in this process. TfoS is a transmembrane protein that contains a large periplasmic domain and a cytoplasmic AraC-type DNA-binding domain, but lacks TCS signature domains. Inactivation of tfoS abolished natural competence as well as transcription of the tfoR gene encoding a chitin-induced small RNA essential for competence gene expression. A TfoS fragment containing the DNA-binding domain specifically bound to and activated transcription from the tfoR promoter. Intracellular TfoS levels were unaffected by disruption of chiS and coexpression of TfoS and ChiS in Escherichia coli recovered transcription of the chromosomally integrated tfoR::lacZ gene, suggesting that TfoS is post-translationally modulated by ChiS during transcriptional activation; however, this regulation persisted when the canonical phosphorelay residues of ChiS were mutated. The results presented here suggest that ChiS operates a chitin-induced non-canonical signal transduction cascade through TfoS, leading to transcriptional activation of tfoR. © 2013 John Wiley & Sons Ltd.

Zn(2+) site engineering at the oligomeric interface of the dopamine transporter.

Science.gov (United States)

Norgaard-Nielsen, Kristine; Norregaard, Lene; Hastrup, Hanne; Javitch, Jonathan A; Gether, Ulrik

2002-07-31

Increasing evidence suggests that Na(+)/Cl(-)-dependent neurotransmitter transporters exist as homo-oligomeric proteins. However, the functional implication of this oligomerization remains unclear. Here we demonstrate the engineering of a Zn(2+) binding site at the predicted dimeric interface of the dopamine transporter (DAT) corresponding to the external end of transmembrane segment 6. Upon binding to this site, which involves a histidine inserted in position 310 (V310H) and the endogenous Cys306 within the same DAT molecule, Zn(2+) potently inhibits [(3)H]dopamine uptake. These data provide indirect evidence that conformational changes critical for the translocation process may occur at the interface between two transporter molecules in the oligomeric structure.

The nectin-1α transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

International Nuclear Information System (INIS)

Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

2005-01-01

Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion

Membrane shape modulates transmembrane protein distribution.

Science.gov (United States)

Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E S; Bassereau, Patricia

2014-01-27

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of cisplatin-binding sites on the large cytoplasmic loop of the Na+/K+-ATPase.

Science.gov (United States)

Šeflová, Jaroslava; Čechová, Petra; Štenclová, Tereza; Šebela, Marek; Kubala, Martin

2018-12-01

Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na + /K + -ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism.

Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.).

Science.gov (United States)

Satheesh, Viswanathan; Chidambaranathan, Parameswaran; Jagannadham, Prasanth Tejkumar; Kumar, Vajinder; Jain, Pradeep K; Chinnusamy, Viswanathan; Bhat, Shripad R; Srinivasan, R

2016-01-01

Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

Management of industrial sites contaminated with radionuclides and stake-holders involvement

International Nuclear Information System (INIS)

Oudiz, A.; Cessac, B.; Brenot, J.; Maigne, J.P.; Santucci, P.

2001-01-01

The method to be used for the assessment and management of the radiation risks associated with sites contaminated with radionuclides was recently developed in France at the request of the authorities. The aim is to provide all the stakeholders (administrations, elected representatives, engineering companies, operators, residents' associations and environmental protection organizations) with a guide describing how to proceed. There are six stages: the removal of doubt, the pre-diagnosis, the initial diagnosis, the simplified risk study, the detailed risk study and the assistance in the selection of the remediation strategy. Each stage of risk assessment involves the stakeholders to a greater or lesser degree depending on the complexity of the site in question. The guide outlines the criteria which enable the assessment sequence to be interrupted and the appropriate decisions to be taken. For example, one can stop at the stage of the simplified risk study when the site is small and if it is relatively easy to remove and store the contaminated soil. However, in many cases a detailed risk study will be needed. The selection of the appropriate strategy presupposes the identification of several alternate strategies which must be characterized in terms of reduction of dosimetric impact, reduction of contamination, costs and associated nuisances. The choice of strategy requires the involvement of the stakeholders. The degree of involvement depends of the sites specific context. The radiological aspect is generally only one of the elements of the choice, and the conditions have to be created to enable the stakeholders to discuss all the relevant aspects in the site's specific context. (authors)

Cholesterol-Binding Sites in GIRK Channels: The Devil is in the Details.

Science.gov (United States)

Rosenhouse-Dantsker, Avia

2018-01-01

In recent years, it has become evident that cholesterol plays a direct role in the modulation of a variety of ion channels. In most cases, cholesterol downregulates channel activity. In contrast, our earlier studies have demonstrated that atrial G protein inwardly rectifying potassium (GIRK) channels are upregulated by cholesterol. Recently, we have shown that hippocampal GIRK currents are also upregulated by cholesterol. A combined computational-experimental approach pointed to putative cholesterol-binding sites in the transmembrane domain of the GIRK2 channel, the primary subunit in hippocampal GIRK channels. In particular, the principal cholesterol-binding site was located in the center of the transmembrane domain in between the inner and outer α-helices of 2 adjacent subunits. Further studies pointed to a similar cholesterol-binding site in GIRK4, a major subunit in atrial GIRK channels. However, a close look at a sequence alignment of the transmembrane helices of the 2 channels reveals surprising differences among the residues that interact with the cholesterol molecule in these 2 channels. Here, we compare the residues that form putative cholesterol-binding sites in GIRK2 and GIRK4 and discuss the similarities and differences among them.

Transmembrane amyloid-related proteins in CSF as potential biomarkers for Alzheimer’s disease

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Inmaculada eLopez-Font

2015-06-01

Full Text Available In the continuing search for new cerebrospinal fluid (CSF biomarkers for Alzheimer’s disease (AD, reasonable candidates are the secretase enzymes involved in the processing of the amyloid precursor protein (APP, as well as the large proteolytic cleavage fragments sAPPα and sAPPβ. The enzymatic activities of some of these secretases, such as BACE1 and TACE, have been investigated as potential AD biomarkers, and it has been assumed that these activities present in human CSF result from the soluble truncated forms of the membrane-bound enzymes. However, we and others recently identified soluble forms of BACE1 and APP in CSF containing the intracellular domains, as well as the multi-pass transmembrane presenilin-1 (PS1 and other subunits of γ-secretase. We also review recent findings that suggest that most of these soluble transmembrane proteins could display self-association properties based on hydrophobic and/or ionic interactions leading to the formation of heteromeric complexes. The oligomerization state of these potential new biomarkers needs to be taken into consideration for assessing their real potential as CSF biomarkers for AD by adequate molecular tools.

Specificity of transmembrane protein palmitoylation in yeast.

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Ayelén González Montoro

Full Text Available Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs, characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD. There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Science.gov (United States)

Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

2008-01-01

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

Identification of an Arabidopsis transmembrane bZIP transcription factor involved in the endoplasmic reticulum stress response

International Nuclear Information System (INIS)

Tajima, Hiromi; Iwata, Yuji; Iwano, Megumi; Takayama, Seiji; Koizumi, Nozomu

2008-01-01

Among 75 bZIP transcription factors identified in Arabidopsis, 3 (AtbZIP17, AtbZIP28, and AtbZIP49) possess a putative transmembrane domain (TMD) in addition to AtbZIP60, which was characterized previously. In the present study, cDNAs of AtbZIP17 and AtbZIP28 were isolated. Truncated forms of AtbZIP17 and AtbZIP28 lacking the C-terminal domain including TMD were examined as putative active forms. One of them, AtbZIP28ΔC, activated BiP1 and BiP3 promoters through the cis-elements P-UPRE and ERSE responsible for the ER stress response. Subsequently, a fusion protein of green fluorescent protein (GFP) and AtbZIP28 was expressed in Arabidopsis cultured cells. Under non-stress conditions, GFP fluorescence localization almost overlapped with an ER marker; however, tunicamycin and dithiothreitol treatment clearly increased GFP fluorescence in the nucleus suggesting that the N-terminal fragment of AtbZIP28 translocates to the nucleus in response to ER stress

The cytoplasmic domain close to the transmembrane region of the glucagon-like peptide-1 receptor contains sequence elements that regulate agonist-dependent internalisation.

Science.gov (United States)

Vázquez, Patricia; Roncero, Isabel; Blázquez, Enrique; Alvarez, Elvira

2005-07-01

In order to gain better insight into the molecular events involved in the signal transduction generated through glucagon-like peptide-1 (GLP-1) receptors, we tested the effect of deletions and point mutations within the cytoplasmic tail of this receptor with a view to establishing relationships between signal transduction desensitisation and receptor internalisation. Wild-type and truncated (deletion of the last 27 amino acids (GLPR 435R) and deletion of 44 amino acids (GLPR 418R)) GLP-1 receptors bound the agonist with similar affinity. Deletion of the last 27 amino acids decreased the internalisation rate by 78%, while deletion of 44 amino acids containing all the phosphorylation sites hitherto described in this receptor decreased the internalisation rate by only 47%. Binding of the ligand to both receptors stimulated adenylyl cyclase. In contrast, deletion of the region containing amino acids 419 to 435 (GLPR 419delta435) increased the internalisation rate by 268%, and the replacement of EVQ(408-410) by alanine (GLPR A(408-410)) increased this process to 296%. In both receptors, the efficacy in stimulating adenylate cyclase was decreased. All the receptors studied were internalised by coated pits, except for the receptor with a deletion of the last 44 amino acids, which also had a faster resensitisation rate. Our findings indicate that the neighbouring trans-membrane domain of the carboxyl-terminal tail of the GLP-1 receptor contains sequence elements that regulate agonist-dependent internalisation and transmembrane signalling.

Coarse-Grained Model for Water Involving a Virtual Site.

Science.gov (United States)

Deng, Mingsen; Shen, Hujun

2016-02-04

In this work, we propose a new coarse-grained (CG) model for water by combining the features of two popular CG water models (BMW and MARTINI models) as well as by adopting a topology similar to that of the TIP4P water model. In this CG model, a CG unit, representing four real water molecules, consists of a virtual site, two positively charged particles, and a van der Waals (vdW) interaction center. Distance constraint is applied to the bonds formed between the vdW interaction center and the positively charged particles. The virtual site, which carries a negative charge, is determined by the locations of the two positively charged particles and the vdW interaction center. For the new CG model of water, we coined the name "CAVS" (charge is attached to a virtual site) due to the involvment of the virtual site. After being tested in molecular dynamic (MD) simulations of bulk water at various time steps, under different temperatures and in different salt (NaCl) concentrations, the CAVS model offers encouraging predictions for some bulk properties of water (such as density, dielectric constant, etc.) when compared to experimental ones.

Fat storage-inducing transmembrane (FIT or FITM proteins are related to lipid phosphatase/phosphotransferase enzymes

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Matthew J Hayes

2017-12-01

Full Text Available Fat storage-inducing transmembrane (FIT or FITM proteins have been implicated in the partitioning of triacylglycerol to lipid droplets and the budding of lipid droplets from the ER. At the molecular level, the sole relevant interaction is that FITMs directly bind to triacyglycerol and diacylglycerol, but how they function at the molecular level is not known. Saccharomyces cerevisiae has two FITM homologues: Scs3p and Yft2p. Scs3p was initially identified because deletion leads to inositol auxotrophy, with an unusual sensitivity to addition of choline. This strongly suggests a role for Scs3p in phospholipid biosynthesis. Looking at the FITM family as widely as possible, we found that FITMs are widespread throughout eukaryotes, indicating presence in the last eukaryotic common ancestor. Protein alignments also showed that FITM sequences contain the active site of lipid phosphatase/phosphotransferase (LPT enzymes. This large family transfers phosphate-containing headgroups either between lipids or in exchange for water. We confirmed the prediction that FITMs are related to LPTs by showing that single amino-acid substitutions in the presumptive catalytic site prevented their ability to rescue growth of the mutants on low inositol/high choline media when over-expressed. The substitutions also prevented rescue of other phenotypes associated with loss of FITM in yeast, including mistargeting of Opi1p, defective ER morphology, and aberrant lipid droplet budding. These results suggest that Scs3p, Yft2p and FITMs in general are LPT enzymes involved in an as yet unknown critical step in phospholipid metabolism.

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

Science.gov (United States)

Goncharuk, M V; Shul'ga, A A; Ermoliuk, Ia S; Tkach, E N; Goncharuk, S A; Pustovalova, Iu E; Mineev, K S; Bocharov, É V; Maslennikov, I V; Arsen'ev, A S; Kirpichnikov, M P

2011-01-01

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

Correlation of Aquaporins and Transmembrane Solute Transporters Revealed by Genome-Wide Analysis in Developing Maize Leaf

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Xun Yue

2012-01-01

Full Text Available Aquaporins are multifunctional membrane channels that facilitate the transmembrane transport of water and solutes. When transmembrane mineral nutrient transporters exhibit the same expression patterns as aquaporins under diverse temporal and physiological conditions, there is a greater probability that they interact. In this study, genome-wide temporal profiling of transcripts analysis and coexpression network-based approaches are used to examine the significant specificity correlation of aquaporins and transmembrane solute transporters in developing maize leaf. The results indicate that specific maize aquaporins are related to specific transmembrane solute transporters. The analysis demonstrates a systems-level correlation between aquaporins, nutrient transporters, and the homeostasis of mineral nutrients in developing maize leaf. Our results provide a resource for further studies into the physiological function of these aquaporins.

A voice from the high wire: Public involvement in a co-operative siting process

International Nuclear Information System (INIS)

Oates, D.J.L.

1995-01-01

The author is a public consultation and communications consultant to the Siting Task Force (STF), Low level Radioactive Waste Management. The STF is a Canadian government-appointed yet independent body implementing a voluntary, co-operative siting process for a long term storage or disposal facility for 1 million cubic metres of LLRW. The presentation will document the experiences of and lessons learned by the author during her role developing and implementing a public involvement program for the process. The Co-operative Siting Process is a new approach to siting controversial facilities. It is based on the belief that communities should accept such a facility in their backyard and not be forced against their will on technical or political grounds. A formal 'ground rules-up-front' process was developed and is now being carried out, with completion slated for April, 1995. Putting these rules and theories into practice has resulted in significant changes being made to the work plan for technical activities, and in a sober second look at the intricacies involved in planning and carrying out a thorough and efficient public involvement program that remain practical and cost-effective. There is a delicate balancing act between meaningful public participation that lays the foundation for trust, confidence and consensus, and public involvement that can result in the process being side-tracked and legitimate solutions and technical activities becoming mired in political and personal agendas

Molecular dynamics simulation studies of transmembrane transport of chemical components in Chinese herbs and the function of platycodin D in a biological membrane

Directory of Open Access Journals (Sweden)

Shufang Yang

2017-04-01

Conclusion: The Martini force field was successfully applied to the study of the interaction between herbal compounds and a biological membrane. By combining the dynamics equilibrium morphology, the distribution of drugs inside and outside the biomembrane, and the interaction sites of drugs on the DPPC bilayer, factors influencing transmembrane transport of drugs were elucidated and the function of platycodin D in a biological membrane was reproduced.

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

Science.gov (United States)

El Hiani, Yassine; Linsdell, Paul

2015-06-19

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

Directory of Open Access Journals (Sweden)

Bryan D Moyer

Full Text Available BACKGROUND: Using fungiform (FG and circumvallate (CV taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive, sour cells (PKD2L1-positive, as well as other taste cell populations. Transmembrane protein 44 (TMEM44, a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1, a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1, a calcium-binding transmembrane protein; and anoctamin 7 (ANO7, a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B, a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

Science.gov (United States)

Marshall, Karen E; Hughson, Andrew; Vascellari, Sarah; Priola, Suzette A; Sakudo, Akikazu; Onodera, Takashi; Baron, Gerald S

2017-01-15

Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP C ) influences PrP C misfolding into the disease-associated isoform, PrP res , as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP C away from rafts. Previous studies showed that nonraft transmembrane PrP C variants resist conversion to PrP res when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP C and GPI-anchored PrP res in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP C might convert to PrP res if seeded by an exogenous source of PrP res not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP C To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP C supported persistent PrP res propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP C is required for persistent PrP res propagation, implicating raft microdomains as a location for conversion. Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP C

Can membrane-bound carotenoid pigment zeaxanthin carry out a transmembrane proton transfer?

Science.gov (United States)

Kupisz, Kamila; Sujak, Agnieszka; Patyra, Magdalena; Trebacz, Kazimierz; Gruszecki, Wiesław I

2008-10-01

Polar carotenoid pigment zeaxanthin (beta,beta-carotene-3,3'-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13 x 10(7) Omega cm2 (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H+-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV-Vis linear dichroism analysis of multibilayer formed with the same lipid-carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21 degrees with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of beta-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane.

Public/stakeholder involvement at two Department of Energy sites: Case studies

International Nuclear Information System (INIS)

Gray, R.H.

1995-01-01

Efforts to communicate the results of environmental studies and involve the public in environmental decisions have increased nationwide. Outreach efforts at two US Department of Energy sites (i.e., the Hanford Site in southeastern Washington State and the Pantex Plant in the Texas Panhandle) have used a broad spectrum of communications media, including technical articles (open literature and symposium publications, annual and topical reports); information brochures and fact sheets; video productions; interactive exhibits, presentations at scientific, technical, civic, and other public meetings; and proactive interactions with the news media and with local, state, federal, and other agencies. In addition, representatives of local communities now operate offsite environmental monitoring stations and Native Americans are involved in studying cultural resources, fisheries, and other issues at Hanford and a program to obtain environmental samples from neighbor's property is underway at the Pantex Plant. All major environmental programs, such as the multi-year effort to reconstruct past radiological doses to offsite human populations at Hanford, are now conducted with open public participation

Public/stakeholder involvement at two Department of Energy sites: Case studies

Energy Technology Data Exchange (ETDEWEB)

Gray, R.H. [Battelle Pantex, Amarillo, TX (United States)

1995-12-31

Efforts to communicate the results of environmental studies and involve the public in environmental decisions have increased nationwide. Outreach efforts at two US Department of Energy sites (i.e., the Hanford Site in southeastern Washington State and the Pantex Plant in the Texas Panhandle) have used a broad spectrum of communications media, including technical articles (open literature and symposium publications, annual and topical reports); information brochures and fact sheets; video productions; interactive exhibits, presentations at scientific, technical, civic, and other public meetings; and proactive interactions with the news media and with local, state, federal, and other agencies. In addition, representatives of local communities now operate offsite environmental monitoring stations and Native Americans are involved in studying cultural resources, fisheries, and other issues at Hanford and a program to obtain environmental samples from neighbor`s property is underway at the Pantex Plant. All major environmental programs, such as the multi-year effort to reconstruct past radiological doses to offsite human populations at Hanford, are now conducted with open public participation.

Who and What Does Involvement Involve?

DEFF Research Database (Denmark)

Hansen, Jeppe Oute; Petersen, Anders; Huniche, Lotte

2015-01-01

This article gives an account of aspects of a multi-sited field study of involvement of relatives in Danish psychiatry. By following metaphors of involvement across three sites of the psychiatric systema family site, a clinical site and a policy sitethe first author (J.O.) investigated how...... theoretical perspective laid out by Ernesto Laclau and Chantal Mouffe, the aim of this study is to show how the dominant discourse about involvement at the political and clinical sites is constituted by understandings of mentally ill individuals and by political objectives of involvement. The analysis...... the responsibility toward the mental health of the ill individual as well as toward the psychological milieu of the family....

The vacuum-assisted closure (V.A.C®) system for surgical site infection with involved vascular grafts.

Science.gov (United States)

Saziye, Karaca; Afksendiyos, Kalangos

2015-04-01

In vascular surgery, surgical site infection is the most common postoperative morbidity, occurring in 5-10% of vascular patients. The optimal management of surgical site infection with involved lower limb vascular grafts remains controversial. We present our 6-year results of using the V.A.C.® system in surgical site infection with involved vascular grafts. A retrospective 6-year review of patient who underwent a VAC® therapy for postoperative surgical site infection in lower limb with involved vascular grafts in our department between January 2006 and December 2011. V.A.C therapy was used in 40 patients. All patients underwent surgical wound revision with VAC® therapy and antibiotics. The mean time of use of the V.A.C. system was 14.2 days. After mean of 12 days in 34 of 40 patients, in whom the use of VAC® therapy resulted in delayed primary closure or healing by secondary intention. The mean postoperative follow-up time was 61.67 months, during which 3 patients died. We showed that the V.A.C.® system is valuable for managing specifically surgical site infection with involved vascular grafts. Using the V.A.C.® system, reoperation rates are reduced; 85% of patients avoided graft replacement. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Cloning and characterization of SCART1, a novel scavenger receptor cysteine-rich type I transmembrane molecule

DEFF Research Database (Denmark)

Holm, Dorte; Fink, Dorte Rosenbek; Grønlund, Jørn

2009-01-01

We have cloned and characterized a novel murine transmembrane molecule, mSCART1 belonging to the scavenger receptor cysteine-rich superfamily. The cDNA encodes a polypeptide chain of 989 amino acids, organized as a type I transmembrane protein that contains eight extracellular SRCR domains followed...

Program Director as Webmaster? Analysis of 131 Anesthesiology Department Web Sites and Program Director Web Site Involvement and Opinion Survey.

Science.gov (United States)

Daneshpayeh, Negin; Lee, Howard; Berger, Jeffrey

2013-01-01

The last formal review of academic anesthesiology department Web sites (ADWs) for content was conducted in 2009. ADWs have been rated as very important by medical students in researching residency training programs; however, the rapid evolution of sites require that descriptive statistics must be more current to be considered reliable. We set out to provide an updated overview of ADW content and to better understand residency program directors' (PD) role and comfort with ADWs. Two independent reviewers (ND and HL) analyzed all 131 Accreditation Council for Graduate Medical Education (ACGME) accredited ADWs. A binary system (Yes/No) was used to determine which features were present. Reviewer reliability was confirmed with inter-rater reliability and percentage agreement calculation. Additionally, a blinded electronic survey (Survey Monkey, Portland, OR) was sent to anesthesiology residency PDs via electronic mail investigating the audiences for ADWs, the frequency of updates and the degree of PD involvement. 13% of anesthesiology departments still lack a Web site with a homepage with links to the residency program and educational offerings (18% in 2009). Only half (55%) of Web sites contain information for medical students, including clerkship information. Furthermore, programs rarely contain up-to-date calendars (13%), accreditation cycle lengths (11%), accreditation dates (7%) or board pass rates (6%). The PD survey, completed by 42 of 131 PDs, noted a correlation (r = 0.36) between the number of years as PD and the frequency of Web site updates - less experienced PDs appear to update their sites more frequently (p = 0.03). Although 86% of PDs regarded a Web site as "very" important in recruitment, only 9% felt "very" comfortable with the skills required to advertise and market a Web site. Despite the overall increase in ADW content since 2009, privacy concerns, limited resources and time constraints may prevent PDs from providing the most up-to-date Web sites for

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

Energy Technology Data Exchange (ETDEWEB)

Ghoshal, Kankana [University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada V6T 1Z4 (Canada); Theilmann, Jane; Reade, Ron; Sanfacon, Helene [Agriculture and Agri-Food Canada Pacific Agri-Food Research Centre, 4200 Hwy 97, Summerland, British Columbia, Canada V0H 1Z0 (Canada); Rochon, D’Ann, E-mail: dann.rochon@agr.gc.ca [University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada V6T 1Z4 (Canada); Agriculture and Agri-Food Canada Pacific Agri-Food Research Centre, 4200 Hwy 97, Summerland, British Columbia, Canada V0H 1Z0 (Canada)

2014-11-15

Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication. - Highlights: • The CLSV p25 auxiliary replicase targets the endoplasmic reticulum (ER). • Targeting of CLSV p25 is associated with ER restructuring. • Restructuring of the ER occurs during CLSV infection. • CLSV p25 contains 3 predicted transmembrane domains 2 of which are required for ER targeting. • Vesicles derived from the ER may be sites of CLSV replication.

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

International Nuclear Information System (INIS)

Ghoshal, Kankana; Theilmann, Jane; Reade, Ron; Sanfacon, Helene; Rochon, D’Ann

2014-01-01

Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication. - Highlights: • The CLSV p25 auxiliary replicase targets the endoplasmic reticulum (ER). • Targeting of CLSV p25 is associated with ER restructuring. • Restructuring of the ER occurs during CLSV infection. • CLSV p25 contains 3 predicted transmembrane domains 2 of which are required for ER targeting. • Vesicles derived from the ER may be sites of CLSV replication

First principles design of a core bioenergetic transmembrane electron-transfer protein

Energy Technology Data Exchange (ETDEWEB)

Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.; Wiedman, Gregory; Moser, Christopher C.; Leslie Dutton, P.; Discher, Bohdana M.

2016-05-01

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Point mutations in the transmembrane region of the clic1 ion channel selectively modify its biophysical properties.

Directory of Open Access Journals (Sweden)

Stefania Averaimo

Full Text Available Chloride intracellular Channel 1 (CLIC1 is a metamorphic protein that changes from a soluble cytoplasmic protein into a transmembrane protein. Once inserted into membranes, CLIC1 multimerises and is able to form chloride selective ion channels. Whilst CLIC1 behaves as an ion channel both in cells and in artificial lipid bilayers, its structure in the soluble form has led to some uncertainty as to whether it really is an ion channel protein. CLIC1 has a single putative transmembrane region that contains only two charged residues: arginine 29 (Arg29 and lysine 37 (Lys37. As charged residues are likely to have a key role in ion channel function, we hypothesized that mutating them to neutral alanine to generate K37A and R29A CLIC1 would alter the electrophysiological characteristics of CLIC1. By using three different electrophysiological approaches: i single channel Tip-Dip in artificial bilayers using soluble recombinant CLIC1, ii cell-attached and iii whole-cell patch clamp recordings in transiently transfected HEK cells, we determined that the K37A mutation altered the single-channel conductance while the R29A mutation affected the single-channel open probability in response to variation in membrane potential. Our results show that mutation of the two charged amino acids (K37 and R29 in the putative transmembrane region of CLIC1 alters the biophysical properties of the ion channel in both artificial bilayers and cells. Hence these charged residues are directly involved in regulating its ion channel activity. This strongly suggests that, despite its unusual structure, CLIC1 itself is able to form a chloride ion channel.

Insight of Transmembrane Processes of Self-Assembling Nanotubes Based on a Cyclic Peptide Using Coarse Grained Molecular Dynamics Simulation.

Science.gov (United States)

Fu, Yankai; Yan, Tingxuan; Xu, Xia

2017-09-28

Transmembrane self-assembling cyclic peptide (SCP) nanotubes are promising candidates for delivering specific molecules through cell membranes. The detailed mechanisms behind the transmembrane processes, as well as stabilization factors of transmembrane structures, are difficult to elucidate through experiments. In this study, the effects of peptide sequence and oligomeric state on the transmembrane capabilities of SCP nanotubes and the perturbation of embedded SCP nanotubes acting on the membrane were investigated based on coarse grained molecular dynamics simulation. The simulation results reveal that hydrophilic SCP oligomers result in the elevation of the energy barrier while the oligomerization of hydrophobic SCPs causes the reduction of the energy barrier, further leading to membrane insertion. Once SCP nanotubes are embedded, membrane properties such as density, thickness, ordering state and lateral mobility are adjusted along the radial direction. This study provides insight into the transmembrane strategy of SCP nanotubes and sheds light on designing novel transport systems.

Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

Science.gov (United States)

Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

2014-07-18

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server

DEFF Research Database (Denmark)

Käll, Lukas; Krogh, Anders; Sonnhammer, Erik L L

2007-01-01

. The method makes an optimal choice between transmembrane segments and signal peptides, and also allows constrained and homology-enriched predictions. We here present a web interface (http://phobius.cgb.ki.se and http://phobius.binf.ku.dk) to access Phobius. Udgivelsesdato: 2007-Jul......When using conventional transmembrane topology and signal peptide predictors, such as TMHMM and SignalP, there is a substantial overlap between these two types of predictions. Applying these methods to five complete proteomes, we found that 30-65% of all predicted signal peptides and 25-35% of all...

Structure of FGFR3 transmembrane domain dimer: implications for signaling and human pathologies.

Science.gov (United States)

Bocharov, Eduard V; Lesovoy, Dmitry M; Goncharuk, Sergey A; Goncharuk, Marina V; Hristova, Kalina; Arseniev, Alexander S

2013-11-05

Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data. Copyright © 2013 Elsevier Ltd. All rights reserved.

Modelling of a transmembrane evaporation module for desalination of seawater

NARCIS (Netherlands)

Guijt, C.M.; Racz, I.G.; van Heuven, Jan Willem; Reith, T.; de Haan, A.B.

1999-01-01

Transmembrane evaporation (often called membrane distillation) carried out in a countercurrent flow module, in which incoming cold seawater is heated by the condensing product water flow, is a promising technology for low-cost seawater desalination. This paper presents a model for preliminary design

Multi-objective decisions in land-use planning involving chemical sites

DEFF Research Database (Denmark)

Rasmussen, B.; Bertelsen, I.; Burchard, V.

1999-01-01

A methodology for land-use planning involving chemical sites has been developed for making decisions in local and regional administrations. The methodology structures the planning process in seven steps, where one can loop through the steps several times.Essential parts of the methodology...... are the specification of objectives and the development of alternatives where the objectives sets the frame in which the alternatives are assessed and compared. The list of objectives includes the following items: safetyand accidents, public distortion and health, environmental impact, cultural and natural heritage...

The concept and evolution of involved site radiation therapy for lymphoma

DEFF Research Database (Denmark)

Specht, Lena; Yahalom, Joachim

2015-01-01

We describe the development of radiation therapy for lymphoma from extended field radiotherapy of the past to modern conformal treatment with involved site radiation therapy based on advanced imaging, three-dimensional treatment planning and advanced treatment delivery techniques. Today, radiation...... therapy is part of the multimodality treatment of lymphoma, and the irradiated tissue volume is much smaller than before, leading to highly significant reductions in the risks of long-term complications....

Transmembrane adaptor molecules: a new category of lymphoid-cell markers

Czech Academy of Sciences Publication Activity Database

Tedoldi, S.; Paterson, J.C.; Hansmann, M.-L.; Natkunam, Y.; Rüdiger, T.; Angelisová, Pavla; Du, M.Q.; Roberton, H.; Roncador, G.; Sanchez, L.; Pozzobon, M.; Masir, N.; Barry, R.; Pileri, S.; Mason, D.Y.; Marafioti, T.; Hořejší, Václav

2006-01-01

Roč. 107, č. 1 (2006), s. 213-221 ISSN 0006-4971 R&D Projects: GA MŠk(CZ) 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : transmembrane adaptors * PAG * LIME Subject RIV: EC - Immunology Impact factor: 10.370, year: 2006

Community Involvement in Tourism Development: A Case Study of Lenggong Valley World Heritage Site

Directory of Open Access Journals (Sweden)

A. Khadar Nur Zafirah

2014-01-01

Full Text Available This paper examines the empirical relationship between the economic impact and community involvement in the Lenggong Valley. Recommendations for improvement in development effectiveness through the development of a community centre for economic and social activities, with specific attention given to types of activity and community involvement stimulating the economic development in the Lenggong Valley. Heritage tourism development is a tourism in which arts, culture and heritage form a key attraction for visitors and it can be represented as an area of significant economic benefit to heritage sites. The tourism industry in Hulu Perak became more widespread after Lenggong Valley is recognized as a World Heritage Site. There is shown a positive effect on the development and economic prosperity.

The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling

Directory of Open Access Journals (Sweden)

Anna Oszmiana

2016-05-01

Full Text Available Super-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR, KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling.

Two novel mutations in the sixth transmembrane segment of the thyrotropin receptor gene causing hyperfunctioning thyroid nodules.

Science.gov (United States)

Gozu, Hulya; Avsar, Melike; Bircan, Rifat; Claus, Maren; Sahin, Serap; Sezgin, Ozlem; Deyneli, Oguzhan; Paschke, Ralf; Cirakoglu, Beyazit; Akalin, Sema

2005-04-01

Autonomously functioning thyroid nodules (AFTNs) can present as hyperfunctioning adenomas or toxic multinodular goiters. In the last decade, a large number of activating mutations have been identified in the thyrotropin receptor (TSHR) gene in autonomously functioning thyroid nodules. Most have been situated close to, or within the sixth transmembrane segment and third intracellular loop of the TSHR where the receptor interacts with the Gs protein. In this study we describe two novel mutations in the sixth transmembrane segment of the TSHR causing hyperfunctioning thyroid nodules. Genomic DNAs were isolated from four hyperfunctioning thyroid nodules, normal tissues and peripheral leukocytes of two patients with toxic multinodular goiter. After amplifying the related regions, TSHR and G(s)alpha genes were analyzed by single-strand conformation polymorphism (SSCP) analysis. The precise localization of the mutations was identified by automatic DNA sequence analysis. Functional studies were done by site-directed mutagenesis and transfection of a mutant construct into COS-7 cells. We identified two novel TSHR mutations in two hyperfunctioning thyroid nodules: Phe631Val in the first patient and Iso630Met in the second patient. Both mutant receptors display an increase in constitutive stimulation of basal cyclic adenosine monophosphate (cAMP) levels compared to the wild-type receptor. This confirms that these mutant receptors cause hyperfunctioning thyroid nodules.

Transmembrane transporter expression regulated by the glucosylceramide pathway in Cryptococcus neoformans.

Science.gov (United States)

Singh, Arpita; Rella, Antonella; Schwacke, John; Vacchi-Suzzi, Caterina; Luberto, Chiara; Del Poeta, Maurizio

2015-11-16

disrupt transmembrane signaling complex, which in turn contributes to cryptococcal osmotic, pH, ion homeostasis and its pathobiology. Six genes identified from gene expression microarrays by gene set enrichment analysis and validated by RT-PCR, are membrane located and associated with the growth defect at neutral-alkaline pH due to the absence and or presence of a structurally modified GlcCer. They may be involved in the transmembrane signaling network in Cryptococcus neoformans, and therefore the pathobiology of the fungus in these conditions.

Role of protein dynamics in transmembrane receptor signalling

DEFF Research Database (Denmark)

Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

2018-01-01

Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function....

Decreasing Risky Behavior on Social Network Sites: The Impact of Parental Involvement in Secondary Education Interventions.

Science.gov (United States)

Vanderhoven, Ellen; Schellens, Tammy; Valcke, Martin

2016-06-01

Teenagers face significant risks when using increasingly popular social network sites. Prevention and intervention efforts to raise awareness about these risks and to change risky behavior (so-called "e-safety" interventions) are essential for the wellbeing of these minors. However, several studies have revealed that while school interventions often affect awareness, they have only a limited impact on pupils' unsafe behavior. Utilizing the Theory of Planned Behavior and theories about parental involvement, we hypothesized that involving parents in an e-safety intervention would positively influence pupils' intentions and behavior. In a quasi-experimental study with pre- and post-test measures involving 207 pupils in secondary education, we compared the impact of an intervention without parental involvement with one that included active parental involvement by means of a homework task. We found that whereas parental involvement was not necessary to improve the intervention's impact on risk awareness, it did change intentions to engage in certain unsafe behavior, such as posting personal and sexual information on the profile page of a social network site, and in reducing existing problematic behavior. This beneficial impact was particularly evident for boys. These findings suggest that developing prevention campaigns with active parental involvement is well worth the effort. Researchers and developers should therefore focus on other efficient strategies to involve parents.

Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor.

Directory of Open Access Journals (Sweden)

Vanessa Chantreau

Full Text Available The thyrotropin receptor (TSHR is a G protein-coupled receptor (GPCR that is member of the leucine-rich repeat subfamily (LGR. In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors.

Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

Science.gov (United States)

Herbst, R; Munemitsu, S; Ullrich, A

1995-01-19

The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

Science.gov (United States)

Vancsok, Catherine; Peñaranda, M Michelle D; Raj, V Stalin; Leroy, Baptiste; Jazowiecka-Rakus, Joanna; Boutier, Maxime; Gao, Yuan; Wilkie, Gavin S; Suárez, Nicolás M; Wattiez, Ruddy; Gillet, Laurent; Davison, Andrew J; Vanderplasschen, Alain F C

2017-11-01

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro , and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs. IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the

Transmembrane protein diffusion in gel-supported dual-leaflet membranes.

Science.gov (United States)

Wang, Chih-Ying; Hill, Reghan J

2014-11-18

Tools to measure transmembrane-protein diffusion in lipid bilayer membranes have advanced in recent decades, providing a need for predictive theoretical models that account for interleaflet leaflet friction on tracer mobility. Here we address the fully three-dimensional flows driven by a (nonprotruding) transmembrane protein embedded in a dual-leaflet membrane that is supported above and below by soft porous supports (e.g., hydrogel or extracellular matrix), each of which has a prescribed permeability and solvent viscosity. For asymmetric configurations, i.e., supports with contrasting permeability, as realized for cells in contact with hydrogel scaffolds or culture media, the diffusion coefficient can reflect interleaflet friction. Reasonable approximations, for sufficiently large tracers on low-permeability supports, are furnished by a recent phenomenological theory from the literature. Interpreting literature data, albeit for hard-supported membranes, provides a theoretical basis for the phenomenological Stokes drag law as well as strengthening assertions that nonhydrodynamic interactions are important in supported bilayer systems, possibly leading to overestimates of the membrane/leaflet viscosity. Our theory provides a theoretical foundation for future experimental studies of tracer diffusion in gel-supported membranes.

Effect of flow rate and temperature on transmembrane blood pressure drop in an extracorporeal artificial lung.

Science.gov (United States)

Park, M; Costa, E L V; Maciel, A T; Barbosa, E V S; Hirota, A S; Schettino, G de P; Azevedo, L C P

2014-11-01

Transmembrane pressure drop reflects the resistance of an artificial lung system to blood transit. Decreased resistance (low transmembrane pressure drop) enhances blood flow through the oxygenator, thereby, enhancing gas exchange efficiency. This study is part of a previous one where we observed the behaviour and the modulation of blood pressure drop during the passage of blood through artificial lung membranes. Before and after the induction of multi-organ dysfunction, the animals were instrumented and analysed for venous-venous extracorporeal membrane oxygenation, using a pre-defined sequence of blood flows. Blood flow and revolutions per minute (RPM) of the centrifugal pump varied in a linear fashion. At a blood flow of 5.5 L/min, pre- and post-pump blood pressures reached -120 and 450 mmHg, respectively. Transmembrane pressures showed a significant spread, particularly at blood flows above 2 L/min; over the entire range of blood flow rates, there was a positive association of pressure drop with blood flow (0.005 mmHg/mL/minute of blood flow) and a negative association of pressure drop with temperature (-4.828 mmHg/(°Celsius). These associations were similar when blood flows of below and above 2000 mL/minute were examined. During its passage through the extracorporeal system, blood is exposed to pressure variations from -120 to 450 mmHg. At high blood flows (above 2 L/min), the drop in transmembrane pressure becomes unpredictable and highly variable. Over the entire range of blood flows investigated (0-5500 mL/min), the drop in transmembrane pressure was positively associated with blood flow and negatively associated with body temperature. © The Author(s) 2014.

Evolution of vertebrate interferon inducible transmembrane proteins

Directory of Open Access Journals (Sweden)

Hickford Danielle

2012-04-01

Full Text Available Abstract Background Interferon inducible transmembrane proteins (IFITMs have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. Results Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. Conclusions Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution.

TMFoldWeb: a web server for predicting transmembrane protein fold class.

Science.gov (United States)

Kozma, Dániel; Tusnády, Gábor E

2015-09-17

Here we present TMFoldWeb, the web server implementation of TMFoldRec, a transmembrane protein fold recognition algorithm. TMFoldRec uses statistical potentials and utilizes topology filtering and a gapless threading algorithm. It ranks template structures and selects the most likely candidates and estimates the reliability of the obtained lowest energy model. The statistical potential was developed in a maximum likelihood framework on a representative set of the PDBTM database. According to the benchmark test the performance of TMFoldRec is about 77 % in correctly predicting fold class for a given transmembrane protein sequence. An intuitive web interface has been developed for the recently published TMFoldRec algorithm. The query sequence goes through a pipeline of topology prediction and a systematic sequence to structure alignment (threading). Resulting templates are ordered by energy and reliability values and are colored according to their significance level. Besides the graphical interface, a programmatic access is available as well, via a direct interface for developers or for submitting genome-wide data sets. The TMFoldWeb web server is unique and currently the only web server that is able to predict the fold class of transmembrane proteins while assigning reliability scores for the prediction. This method is prepared for genome-wide analysis with its easy-to-use interface, informative result page and programmatic access. Considering the info-communication evolution in the last few years, the developed web server, as well as the molecule viewer, is responsive and fully compatible with the prevalent tablets and mobile devices.

Structure and function of the cystic fibrosis transmembrane conductance regulator

Directory of Open Access Journals (Sweden)

M.M. Morales

1999-08-01

Full Text Available Cystic fibrosis (CF is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR. Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs, and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs.

Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

Energy Technology Data Exchange (ETDEWEB)

Satheshkumar, P.S.; Chavre, James; Moss, Bernard, E-mail: bmoss@nih.gov

2013-09-15

The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. - Highlights: • The 35 amino acid O3 protein is required for efficient vaccinia virus entry. • The transmembrane domain of O3 is necessary and sufficient for entry. • Mutagenesis demonstrated extreme sequence flexibility compatible with function.

A negative charge in transmembrane segment 1 of domain II of the cockroach sodium channel is critical for channel gating and action of pyrethroid insecticides

International Nuclear Information System (INIS)

Du Yuzhe; Song Weizhong; Groome, James R.; Nomura, Yoshiko; Luo Ningguang; Dong Ke

2010-01-01

Voltage-gated sodium channels are the primary target of pyrethroids, an important class of synthetic insecticides. Pyrethroids bind to a distinct receptor site on sodium channels and prolong the open state by inhibiting channel deactivation and inactivation. Recent studies have begun to reveal sodium channel residues important for pyrethroid binding. However, how pyrethroid binding leads to inhibition of sodium channel deactivation and inactivation remains elusive. In this study, we show that a negatively charged aspartic acid residue at position 802 (D802) located in the extracellular end of transmembrane segment 1 of domain II (IIS1) is critical for both the action of pyrethroids and the voltage dependence of channel activation. Charge-reversing or -neutralizing substitutions (K, G, or A) of D802 shifted the voltage dependence of activation in the depolarizing direction and reduced channel sensitivity to deltamethrin, a pyrethroid insecticide. The charge-reversing mutation D802K also accelerated open-state deactivation, which may have counteracted the inhibition of sodium channel deactivation by deltamethrin. In contrast, the D802G substitution slowed open-state deactivation, suggesting an additional mechanism for neutralizing the action of deltamethrin. Importantly, Schild analysis showed that D802 is not involved in pyrethroid binding. Thus, we have identified a sodium channel residue that is critical for regulating the action of pyrethroids on the sodium channel without affecting the receptor site of pyrethroids.

System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

Science.gov (United States)

Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

2013-02-05

The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

The allosteric site regulates the voltage sensitivity of muscarinic receptors.

Science.gov (United States)

Hoppe, Anika; Marti-Solano, Maria; Drabek, Matthäus; Bünemann, Moritz; Kolb, Peter; Rinne, Andreas

2018-01-01

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M 1 -Rs and M 3 -Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G q protein cycle. In the presence of ACh, M 1 -R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M 3 -R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M 3 /M 1 -R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M 3 -Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects. Copyright © 2017 Elsevier Inc. All rights reserved.

Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

DEFF Research Database (Denmark)

Sikder, K. U.; Stone, K. A.; Kumar, P. B. S.

2014-01-01

We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that mic......We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find...... that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. (C) 2014 AIP Publishing LLC....

Modeling the Structure of SARS 3a Transmembrane Protein Using a ...

Indian Academy of Sciences (India)

Modeling the structure of SARS 3a Transmembrane protein using a ... for the implicit membrane molecular dynamics (MD) simulations. ... The coordinates during the simulation were saved every 500 steps, and were used for analysis. ... the pair list for calculation of nonbonded interactions being updated after every 10 steps.

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

International Nuclear Information System (INIS)

Watanabe, Yoshihisa; Okui, Akira; Mitsui, Shinichi; Kawarabuki, Kentaro; Yamaguchi, Tatsuyuki; Uemura, Hidetoshi; Yamaguchi, Nozomi

2004-01-01

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression

The Oskarshamn model for public involvement in the siting of nuclear facilities

Energy Technology Data Exchange (ETDEWEB)

Aahagen, H. [Ahagen and Co (Sweden); CarIsson, Torsten [Mayor, Oskarshamn (Sweden); Hallberg, K. [Local Competence Building, Oskarshamn (Sweden); Andersson, Kjell [Karinta-Konsult, Taeby(Sweden)

1999-12-01

The Oskarshamn model has so far worked extremely well as a tool to achieve openness and public participation. The municipality involvement has been successful in several aspects, e.g.: It has been possible to influence the program, to a large extent, to meet certain municipality conditions and to ensure the local perspective. The local competence has increased to a considerable degree. The activities generated by the six working groups with a total of 40 members have generated a large number of contacts with various organisations, schools, mass media, individuals in the general public and interest groups. For the future, clarification of the disposal method and site selection criteria as well as the site selection process as such is crucial. The municipality has also emphasised the importance of SKB having shown the integration between site selection criteria, the feasibility study and the safety assessment. Furthermore, the programs for the encapsulation facility and the repository must be co-ordinated. For Oskarshamn it will be of utmost importance that the repository is well under way to be realised before the encapsulation facility can be built.

The Oskarshamn model for public involvement in the siting of nuclear facilities

International Nuclear Information System (INIS)

Aahagen, H.; CarIsson, Torsten; Hallberg, K.; Andersson, Kjell

1999-01-01

The Oskarshamn model has so far worked extremely well as a tool to achieve openness and public participation. The municipality involvement has been successful in several aspects, e.g.: It has been possible to influence the program, to a large extent, to meet certain municipality conditions and to ensure the local perspective. The local competence has increased to a considerable degree. The activities generated by the six working groups with a total of 40 members have generated a large number of contacts with various organisations, schools, mass media, individuals in the general public and interest groups. For the future, clarification of the disposal method and site selection criteria as well as the site selection process as such is crucial. The municipality has also emphasised the importance of SKB having shown the integration between site selection criteria, the feasibility study and the safety assessment. Furthermore, the programs for the encapsulation facility and the repository must be co-ordinated. For Oskarshamn it will be of utmost importance that the repository is well under way to be realised before the encapsulation facility can be built

The putative Notch ligand HyJagged is a transmembrane protein present in all cell types of adult Hydra and upregulated at the boundary between bud and parent

Directory of Open Access Journals (Sweden)

Tischer Susanne

2011-09-01

Full Text Available Abstract Background The Notch signalling pathway is conserved in pre-bilaterian animals. In the Cnidarian Hydra it is involved in interstitial stem cell differentiation and in boundary formation during budding. Experimental evidence suggests that in Hydra Notch is activated by presenilin through proteolytic cleavage at the S3 site as in all animals. However, the endogenous ligand for HvNotch has not been described yet. Results We have cloned a cDNA from Hydra, which encodes a bona-fide Notch ligand with a conserved domain structure similar to that of Jagged-like Notch ligands from other animals. Hyjagged mRNA is undetectable in adult Hydra by in situ hybridisation but is strongly upregulated and easily visible at the border between bud and parent shortly before bud detachment. In contrast, HyJagged protein is found in all cell types of an adult hydra, where it localises to membranes and endosomes. Co-localisation experiments showed that it is present in the same cells as HvNotch, however not always in the same membrane structures. Conclusions The putative Notch ligand HyJagged is conserved in Cnidarians. Together with HvNotch it may be involved in the formation of the parent-bud boundary in Hydra. Moreover, protein distribution of both, HvNotch receptor and HyJagged indicate a more widespread function for these two transmembrane proteins in the adult hydra, which may be regulated by additional factors, possibly involving endocytic pathways.

Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors

Directory of Open Access Journals (Sweden)

Dongjun Lee

2014-11-01

Full Text Available Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1 or are upstream and regulatory of it (mTORC2. Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2 negatively regulates mTORC2 enzymatic activity, reducing AKTS473, PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway.

Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

Science.gov (United States)

Denys, A; Allain, F; Carpentier, M; Spik, G

1998-12-15

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.

TMDIM: an improved algorithm for the structure prediction of transmembrane domains of bitopic dimers

Science.gov (United States)

Cao, Han; Ng, Marcus C. K.; Jusoh, Siti Azma; Tai, Hio Kuan; Siu, Shirley W. I.

2017-09-01

α-Helical transmembrane proteins are the most important drug targets in rational drug development. However, solving the experimental structures of these proteins remains difficult, therefore computational methods to accurately and efficiently predict the structures are in great demand. We present an improved structure prediction method TMDIM based on Park et al. (Proteins 57:577-585, 2004) for predicting bitopic transmembrane protein dimers. Three major algorithmic improvements are introduction of the packing type classification, the multiple-condition decoy filtering, and the cluster-based candidate selection. In a test of predicting nine known bitopic dimers, approximately 78% of our predictions achieved a successful fit (RMSD PHP, MySQL and Apache, with all major browsers supported.

Biophysical Aspects of Transmembrane Signaling

CERN Document Server

Damjanovich, Sandor

2005-01-01

Transmembrane signaling is one of the most significant cell biological events in the life and death of cells in general and lymphocytes in particular. Until recently biochemists and biophysicists were not accustomed to thinking of these processes from the side of a high number of complex biochemical events and an equally high number of physical changes at molecular and cellular levels at the same time. Both types of researchers were convinced that their findings are the most decisive, having higher importance than the findings of the other scientist population. Both casts were wrong. Life, even at cellular level, has a number of interacting physical and biochemical mechanisms, which finally build up the creation of an "excited" cell that will respond to particular signals from the outer or inner world. This book handles both aspects of the signalling events, and in some cases tries to unify our concepts and help understand the signals that govern the life and death of our cells. Not only the understanding, bu...

Cancer Research Advance in CKLF-like MARVEL Transmembrane Domain Containing Member Family (Review).

Science.gov (United States)

Lu, Jia; Wu, Qian-Qian; Zhou, Ya-Bo; Zhang, Kai-Hua; Pang, Bing-Xin; Li, Liang; Sun, Nan; Wang, Heng-Shu; Zhang, Song; Li, Wen-Jian; Zheng, Wei; Liu, Wei

2016-01-01

CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of genes first reported at international level by Peking University Human Disease Gene Research Center. The gene products are between chemokines and the transmembrane-4 superfamily. Loaceted in several human chromosomes, CMTMs, which are unregulated in kinds of tumors, are potential tumor suppressor genes consisting of CKLF and CMTM1 to CMTM8. CMTMs play important roles in immune, male reproductive and hematopoietic systems. Also, it has been approved that CMTM family has strong connection with diseases of autoimmunity, haematopoietic system and haematopoietic system. The in-depth study in recent years found the close relation between CMTMs and umorigenesis, tumor development and metastasis. CMTM family has a significant clinical value in diagnosis and treatment to the diseases linking to tumor and immune system.

Identification of Putative Transmembrane Proteins Involved in Salinity Tolerance in Chenopodium quinoa by Integrating Physiological Data, RNAseq, and SNP Analyses

KAUST Repository

Schmö ckel, Sandra M.; Lightfoot, Damien; Razali, Rozaimi; Tester, Mark A.; Jarvis, David Erwin

2017-01-01

Chenopodium quinoa (quinoa) is an emerging crop that produces nutritious grains with the potential to contribute to global food security. Quinoa can also grow on marginal lands, such as soils affected by high salinity. To identify candidate salt tolerance genes in the recently sequenced quinoa genome, we used a multifaceted approach integrating RNAseq analyses with comparative genomics and topology prediction. We identified 219 candidate genes by selecting those that were differentially expressed in response to salinity, were specific to or overrepresented in quinoa relative to other Amaranthaceae species, and had more than one predicted transmembrane domain. To determine whether these genes might underlie variation in salinity tolerance in quinoa and its close relatives, we compared the response to salinity stress in a panel of 21 Chenopodium accessions (14 C. quinoa, 5 C. berlandieri, and 2 C. hircinum). We found large variation in salinity tolerance, with one C. hircinum displaying the highest salinity tolerance. Using genome re-sequencing data from these accessions, we investigated single nucleotide polymorphisms and copy number variation (CNV) in the 219 candidate genes in accessions of contrasting salinity tolerance, and identified 15 genes that could contribute to the differences in salinity tolerance of these Chenopodium accessions.

Identification of Putative Transmembrane Proteins Involved in Salinity Tolerance in Chenopodium quinoa by Integrating Physiological Data, RNAseq, and SNP Analyses

KAUST Repository

Schmöckel, Sandra M.

2017-06-21

Chenopodium quinoa (quinoa) is an emerging crop that produces nutritious grains with the potential to contribute to global food security. Quinoa can also grow on marginal lands, such as soils affected by high salinity. To identify candidate salt tolerance genes in the recently sequenced quinoa genome, we used a multifaceted approach integrating RNAseq analyses with comparative genomics and topology prediction. We identified 219 candidate genes by selecting those that were differentially expressed in response to salinity, were specific to or overrepresented in quinoa relative to other Amaranthaceae species, and had more than one predicted transmembrane domain. To determine whether these genes might underlie variation in salinity tolerance in quinoa and its close relatives, we compared the response to salinity stress in a panel of 21 Chenopodium accessions (14 C. quinoa, 5 C. berlandieri, and 2 C. hircinum). We found large variation in salinity tolerance, with one C. hircinum displaying the highest salinity tolerance. Using genome re-sequencing data from these accessions, we investigated single nucleotide polymorphisms and copy number variation (CNV) in the 219 candidate genes in accessions of contrasting salinity tolerance, and identified 15 genes that could contribute to the differences in salinity tolerance of these Chenopodium accessions.

Identification of Putative Transmembrane Proteins Involved in Salinity Tolerance in Chenopodium quinoa by Integrating Physiological Data, RNAseq, and SNP Analyses.

Science.gov (United States)

Schmöckel, Sandra M; Lightfoot, Damien J; Razali, Rozaimi; Tester, Mark; Jarvis, David E

2017-01-01

Chenopodium quinoa (quinoa) is an emerging crop that produces nutritious grains with the potential to contribute to global food security. Quinoa can also grow on marginal lands, such as soils affected by high salinity. To identify candidate salt tolerance genes in the recently sequenced quinoa genome, we used a multifaceted approach integrating RNAseq analyses with comparative genomics and topology prediction. We identified 219 candidate genes by selecting those that were differentially expressed in response to salinity, were specific to or overrepresented in quinoa relative to other Amaranthaceae species, and had more than one predicted transmembrane domain. To determine whether these genes might underlie variation in salinity tolerance in quinoa and its close relatives, we compared the response to salinity stress in a panel of 21 Chenopodium accessions (14 C. quinoa , 5 C. berlandieri , and 2 C. hircinum ). We found large variation in salinity tolerance, with one C. hircinum displaying the highest salinity tolerance. Using genome re-sequencing data from these accessions, we investigated single nucleotide polymorphisms and copy number variation (CNV) in the 219 candidate genes in accessions of contrasting salinity tolerance, and identified 15 genes that could contribute to the differences in salinity tolerance of these Chenopodium accessions.

Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

Science.gov (United States)

Olsen, Richard W

2015-01-01

GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation. © 2015 Elsevier Inc. All rights reserved.

Nanosecond pulsed electric fields depolarize transmembrane potential via voltage-gated K+, Ca2+ and TRPM8 channels in U87 glioblastoma cells.

Science.gov (United States)

Burke, Ryan C; Bardet, Sylvia M; Carr, Lynn; Romanenko, Sergii; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

2017-10-01

Nanosecond pulsed electric fields (nsPEFs) have a variety of applications in the biomedical and biotechnology industries. Cancer treatment has been at the forefront of investigations thus far as nsPEFs permeabilize cellular and intracellular membranes leading to apoptosis and necrosis. nsPEFs may also influence ion channel gating and have the potential to modulate cell physiology without poration of the membrane. This phenomenon was explored using live cell imaging and a sensitive fluorescent probe of transmembrane voltage in the human glioblastoma cell line, U87 MG, known to express a number of voltage-gated ion channels. The specific ion channels involved in the nsPEF response were screened using a membrane potential imaging approach and a combination of pharmacological antagonists and ion substitutions. It was found that a single 10ns pulsed electric field of 34kV/cm depolarizes the transmembrane potential of cells by acting on specific voltage-sensitive ion channels; namely the voltage and Ca2 + gated BK potassium channel, L- and T-type calcium channels, and the TRPM8 transient receptor potential channel. Copyright © 2017 Elsevier B.V. All rights reserved.

Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

DEFF Research Database (Denmark)

Desai, D M; Sap, J; Schlessinger, J

1993-01-01

CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...... that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases....

Electrochemical platform for the detection of transmembrane proteins reconstituted into liposomes

Czech Academy of Sciences Publication Activity Database

Vacek, J.; Zatloukalová, M.; Geletičová, J.; Kubala, M.; Modriansky, M.; Fekete, Ladislav; Mašek, J.; Hubatka, F.; Turánek, J.

2016-01-01

Roč. 88, č. 8 (2016), s. 4548-4556 ISSN 0003-2700 R&D Projects: GA MŠk LO1409; GA MŠk LM2015088 Institutional support: RVO:68378271 Keywords : detection * transmembrane proteins * liposomes * electrochemistry Subject RIV: BM - Solid Matter Physics ; Magnetism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.) Impact factor: 6.320, year: 2016

Cystic Fibrosis Transmembrane Conductance Regulator Potentiation as a Therapeutic Strategy for Pulmonary Edema: A Proof-of-Concept Study in Pigs.

Science.gov (United States)

Li, Xiaopeng; Vargas Buonfiglio, Luis G; Adam, Ryan J; Stoltz, David A; Zabner, Joseph; Comellas, Alejandro P

2017-12-01

To determine the feasibility of using a cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX-770/Kalydeco, Vertex Pharmaceuticals, Boston, MA), as a therapeutic strategy for treating pulmonary edema. Prospective laboratory animal investigation. Animal research laboratory. Newborn and 3 days to 1 week old pigs. Hydrostatic pulmonary edema was induced in pigs by acute volume overload. Ivacaftor was nebulized into the lung immediately after volume overload. Grams of water per grams of dry lung tissue were determined in the lungs harvested 1 hour after volume overload. Ivacaftor significantly improved alveolar liquid clearance in isolated pig lung lobes ex vivo and reduced edema in a volume overload in vivo pig model of hydrostatic pulmonary edema. To model hydrostatic pressure-induced edema in vitro, we developed a method of applied pressure to the basolateral surface of alveolar epithelia. Elevated hydrostatic pressure resulted in decreased cystic fibrosis transmembrane conductance regulator activity and liquid absorption, an effect which was partially reversed by cystic fibrosis transmembrane conductance regulator potentiation with ivacaftor. Cystic fibrosis transmembrane conductance regulator potentiation by ivacaftor is a novel therapeutic approach for pulmonary edema.

Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations.

Directory of Open Access Journals (Sweden)

Heidi O Nousiainen

Full Text Available Prostatic acid phosphatase (PAP, the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG, but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP in the brain by utilizing mice deficient in TMPAP (PAP-/- mice. Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.

Science.gov (United States)

Pirkmajer, Sergej; Kirchner, Henriette; Lundell, Leonidas S; Zelenin, Pavel V; Zierath, Juleen R; Makarova, Kira S; Wolf, Yuri I; Chibalin, Alexander V

2017-07-15

Small transmembrane proteins such as FXYDs, which interact with Na + ,K + -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca 2+ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na + ,K + -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

Science.gov (United States)

Vernon, Robert M; Chong, P Andrew; Lin, Hong; Yang, Zhengrong; Zhou, Qingxian; Aleksandrov, Andrei A; Dawson, Jennifer E; Riordan, John R; Brouillette, Christie G; Thibodeau, Patrick H; Forman-Kay, Julie D

2017-08-25

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A successful effort to involve stakeholders in the selection of a site for a corrective action management unit

International Nuclear Information System (INIS)

Conway, R.; Merkhofer, M.W.; Oms, E.

1995-01-01

As part of the effort to clean up hazardous waste sites, Sandia National Laboratories in New Mexico (SNL/NM) adopted a novel approach to involving stakeholders in a key decision associated with its Environmental Restoration (ER) Project. The decision was where to locate a Corrective Action Management Unit (CAMU), an area designed to consolidate, store, and treat wastes generated from cleanup activities. The decision-making approach was a variation of a technique known as multiattribute utility analysis (MUA). Although MUA has rarely been undertaken during normal Project activities, it proved to be a surprisingly effective means for involving stakeholders in the decision process, generating consensus over a selected site, and enhancing public trust and understanding of Project activities. Requirements and criteria for selecting CAMU sites are provided by the Environmental Protection Agency's (EPA's) CAMU Final Rule (EPA 1993). Recognizing the lack of experience with the Rule and the importance of community understanding and support, the ER Project sought an approach that would allow stakeholders to participate in the site-selection process

Identification of Putative Transmembrane Proteins Involved in Salinity Tolerance in Chenopodium quinoa by Integrating Physiological Data, RNAseq, and SNP Analyses

Directory of Open Access Journals (Sweden)

Sandra M. Schmöckel

2017-06-01

Full Text Available Chenopodium quinoa (quinoa is an emerging crop that produces nutritious grains with the potential to contribute to global food security. Quinoa can also grow on marginal lands, such as soils affected by high salinity. To identify candidate salt tolerance genes in the recently sequenced quinoa genome, we used a multifaceted approach integrating RNAseq analyses with comparative genomics and topology prediction. We identified 219 candidate genes by selecting those that were differentially expressed in response to salinity, were specific to or overrepresented in quinoa relative to other Amaranthaceae species, and had more than one predicted transmembrane domain. To determine whether these genes might underlie variation in salinity tolerance in quinoa and its close relatives, we compared the response to salinity stress in a panel of 21 Chenopodium accessions (14 C. quinoa, 5 C. berlandieri, and 2 C. hircinum. We found large variation in salinity tolerance, with one C. hircinum displaying the highest salinity tolerance. Using genome re-sequencing data from these accessions, we investigated single nucleotide polymorphisms and copy number variation (CNV in the 219 candidate genes in accessions of contrasting salinity tolerance, and identified 15 genes that could contribute to the differences in salinity tolerance of these Chenopodium accessions.

Promiscuous Seven Transmembrane Receptors Sensing L-α-amino Acids

DEFF Research Database (Denmark)

Smajilovic, Sanela; Wellendorph, Petrine; Bräuner-Osborne, Hans

2014-01-01

A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight....... The present review describes the current status of promiscuous L-α-amino acid sensors, the calcium sensing receptor (CaSR), the GPRC6A receptor, the T1R1/T1R3 receptor and also their molecular pharmacology, expression pattern and physiological significance....

Amino acid residues involved in ligand preference of the Snf3 transporter-like sensor in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Dietvorst, J.; Karhumaa, Kaisa; Kielland-Brandt, Morten

2010-01-01

/preferences of Snf3. The ability of cells to sense sugars in vivo was monitored by following the degradation of the Mth1 protein, :ill earl., event ill the signal pathway. Our study reveals that Snf3. ill addition to glucose. also senses fructose and mannose, as well as the glucose analogues 2-deoxyglucose, 3-O......-methylglucoside and 6-deoxyglucose. The signalling proficiency of a non-phosphorylatable analogue strongly supports the notion that sensing through Snf3 does not require sugar phosphorylation. Sequence comparisons of Snf3 to glucose transporters indicated amino acid residues possibly involved in sensing of sugars other...... than glucose. By site-specific mutagenesis of the structural gene, roles of specific residues in Snf3 could he established. Change of isoleucine-374 to valine ill transmembrane segment 7 of Snf3 partially abolished sensing of fructose mannose. while mutagenesis causing it change of phenylalanine-462 (4...

Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

Directory of Open Access Journals (Sweden)

Olivier Barré

Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

Mutational analysis of the antagonist-binding site of the histamine H(1) receptor.

Science.gov (United States)

Wieland, K; Laak, A M; Smit, M J; Kühne, R; Timmerman, H; Leurs, R

1999-10-15

We combined in a previously derived three-dimensional model of the histamine H(1) receptor (Ter Laak, A. M., Timmerman, H., Leurs, H., Nederkoorn, P. H. J., Smit, M. J., and Donne-Op den Kelder, G. M. (1995) J. Comp. Aid. Mol. Design. 9, 319-330) a pharmacophore for the H(1) antagonist binding site (Ter Laak, A. M., Venhorst, J., Timmerman, H., and Donné-Op de Kelder, G. M. (1994) J. Med. Chem. 38, 3351-3360) with the known interacting amino acid residue Asp(116) (in transmembrane domain III) of the H(1) receptor and verified the predicted receptor-ligand interactions by site-directed mutagenesis. This resulted in the identification of the aromatic amino acids Trp(167), Phe(433), and Phe(436) in transmembrane domains IV and VI of the H(1) receptor as probable interaction points for the trans-aromatic ring of the H(1) antagonists. Subsequently, a specific interaction of carboxylate moieties of two therapeutically important, zwitterionic H(1) antagonists with Lys(200) in transmembrane domain V was predicted. A Lys(200) --> Ala mutation results in a 50- (acrivastine) to 8-fold (d-cetirizine) loss of affinity of these zwitterionic antagonists. In contrast, the affinities of structural analogs of acrivastine and cetirizine lacking the carboxylate group, triprolidine and meclozine, respectively, are unaffected by the Lys(200) --> Ala mutation. These data strongly suggest that Lys(200), unique for the H(1) receptor, acts as a specific anchor point for these "second generation" H(1) antagonists.

Role of the transmembrane domain of the VanT serine racemase in resistance to vancomycin in Enterococcus gallinarum BM4174.

Science.gov (United States)

Arias, C A; Peña, J; Panesso, D; Reynolds, P

2003-03-01

Enterococcus gallinarum BM4175 (a vancomycin-susceptible derivative of BM4174 obtained by insertional inactivation of vanC-1) was transformed with plasmid constructs pCA10 (containing the genes necessary for resistance, vanC-1-XYc-T), pJP1 (with a fragment lacking the DNA encoding the transmembrane region of VanT, -vanC-1-XYc-T((Delta))(2-322)-) and with plasmids containing fragments encoding either the transmembrane (mvanT(1-322)) or racemase (svanT(323-698)) domains of VanT under the control of a constitutive promoter. Accumulated peptidoglycan precursors were measured in all strains in the presence of L-Ser, D-Ser (50 mM) or in the absence of any growth supplement. Uptake of 0.1 mM L-[(14)C]serine was also determined in BM4174, BM4175 and BM4175/pCA10. Vancomycin resistance was restored in BM4175 transformed with pCA10(C-1-XYc-T), and the profile of peptidoglycan precursors was similar to wild-type E. gallinarum BM4174. Transformation of E. gallinarum BM4175 with plasmid pJP1(vanC-1-XYc-T((Delta))(2-322)) resulted in: (i) vancomycin MICs remaining within susceptible levels (VanT is likely to be involved in the transport of L-Ser, and that in its absence the resistance phenotype is compromised.

A conserved aromatic lock for the tryptophan rotameric switch in TM-VI of seven-transmembrane receptors

DEFF Research Database (Denmark)

Holst, Birgitte; Nygaard, Rie; Hansen, Louise Valentin

2010-01-01

simulations in rhodopsin demonstrated that rotation around the chi1 torsion angle of Trp-VI:13 brings its side chain close to the equally highly conserved Phe-V:13 (Phe-5.47) in TM-V. In the ghrelin receptor, engineering of high affinity metal-ion sites between these positions confirmed their close spatial...... degree as observed in the constructs where Trp-VI:13 itself was mutated, but again without affecting agonist potency. In a proposed active receptor conformation generated by molecular simulations, where the extracellular segment of TM-VI is tilted inwards in the main ligand-binding pocket, Trp-VI:13......The conserved tryptophan in position 13 of TM-VI (Trp-VI:13 or Trp-6.48) of the CWXP motif located at the bottom of the main ligand-binding pocket in TM-VI is believed to function as a rotameric microswitch in the activation process of seven-transmembrane (7TM) receptors. Molecular dynamics...

Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

Science.gov (United States)

Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

2015-06-01

Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Role of α and β Transmembrane Domains in Integrin Clustering

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Amir Shamloo

2015-11-01

Full Text Available Integrins are transmembrane proteins playing a crucial role in the mechanical signal transduction from the outside to the inside of a cell, and vice versa. Nevertheless, this signal transduction could not be implemented by a single protein. Rather, in order for integrins to be able to participate in signal transduction, they need to be activated and produce clusters first. As integrins consist of α- and β-subunits that are separate in the active state, studying both subunits separately is of a great importance, for, in the active state, the distance between α- and β-subunits is long enough that they do not influence one another significantly. Thus, this study aims to investigate the tendency of transmembrane domains of integrins to form homodimers. We used both Steered and MARTINI Coarse-grained molecular dynamics method to perform our simulations, mainly because of a better resolution and computational feasibility that each of these methods could provide to us. Using the Steered molecular dynamics method for α- and β-subunits, we found that the localized lipid packing prevented them from clustering. Nonetheless, the lipid packing phenomenon was found to be an artifact after investigating this process using a coarse grained (CG model. Exploiting the coarse-grained molecular dynamics simulations, we found that α- and β-subunits tend to form a stable homo-dimer.

Self-Assembling Organic Nanopores as Synthetic Transmembrane Channels with Tunable Functions

Science.gov (United States)

Wei, Xiaoxi

A long-standing goal in the area of supramolecular self-assembly involves the development of synthetic ion/water channels capable of mimicking the mass-transport characteristics of biological channels and pores. Few examples of artificial transmembrane channels with large lumen, high conductivity and selectivity are known. A review of pronounced biological transmembrane protein channels and some representative synthetic models have been provided in Chapter 1, followed by our discovery and initial investigation of shape-persistent oligoamide and phenylene ethynylene macrocycles as synthetic ion/water channels. In Chapter 2, the systematic structural modification of oligoamide macrocycles 1, the so-called first-generation of these shape-persistent macrocycles, has led to third-generation macrocycles 3. The third generation was found to exhibit unprecedented, strong intermolecular association in both the solid state and solution via multiple techniques including X-ray diffraction (XRD), SEM, and 1H NMR. Fluorescence spectroscopy paired with dynamic light scattering (DLS) revealed that macrocycles 3 can assemble into a singly dispersed nanotubular structure in solution. The resultant self-assembling pores consisting of 3 were examined by HPTS-LUVs assays and BLM studies (Chapter 3) and found to form cation-selective (PK+/PCl- = 69:1) transmembrane ion channels with large conductance (200 ˜ 2000 pS for alkali cations) and high stability with open times reaching to 103 seconds. Tuning the aggregation state of macrocycles by choosing an appropriate polar solvent mixture (i.e., 3:1, THF:DMF, v/v) and concentration led to the formation of ion channels with well-defined square top behavior. A parallel study using DLS to examine the size of aggregates was used in conjunction with channel activity assays (LUVs/BLM) to reveal the effects of the aggregation state on channel activity. Empirical evidence now clearly indicates that a preassembled state, perhaps that of a

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

Science.gov (United States)

Furutani, Shogo; Okuhara, Daiki; Hashimoto, Anju; Ihara, Makoto; Kai, Kenji; Hayashi, Hideo; Sattelle, David B; Matsuda, Kazuhiko

2017-10-01

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

Comparative characteristic of transmembrane currents and caffeine-induced responses of intact and irradiated small intestine smooth muscle cells

International Nuclear Information System (INIS)

Stepanov, Yu.V.; Gordienko, D.V.; Preobrazhenskaya, T.D.; Stepanova, L.I.; Vojtsitskij, V.M.

1994-01-01

A comparative investigation of transmembrane ion currents and caffeine-induced responses of single smooth muscle cells isolated from the circular layer of rat small intestine was curried out by the method of 'patch-clamp'. No reliable difference in potential-dependent and amplitude-kinetic characteristics of transmembrane ion currents in cells of intact and irradiated with dose of 3 Gy rats was revealed. In cells of irradiated animals external application of caffeine (4 mM) was not accompanied by strong quick-inactivated transient Ca 2+ -dependent potassium current as in control

Transmembrane helical interactions in the CFTR channel pore.

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Jhuma Das

2017-06-01

Full Text Available Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR gene affect CFTR protein biogenesis or its function as a chloride channel, resulting in dysregulation of epithelial fluid transport in the lung, pancreas and other organs in cystic fibrosis (CF. Development of pharmaceutical strategies to treat CF requires understanding of the mechanisms underlying channel function. However, incomplete 3D structural information on the unique ABC ion channel, CFTR, hinders elucidation of its functional mechanism and correction of cystic fibrosis causing mutants. Several CFTR homology models have been developed using bacterial ABC transporters as templates but these have low sequence similarity to CFTR and are not ion channels. Here, we refine an earlier model in an outward (OWF and develop an inward (IWF facing model employing an integrated experimental-molecular dynamics simulation (200 ns approach. Our IWF structure agrees well with a recently solved cryo-EM structure of a CFTR IWF state. We utilize cysteine cross-linking to verify positions and orientations of residues within trans-membrane helices (TMHs of the OWF conformation and to reconstruct a physiologically relevant pore structure. Comparison of pore profiles of the two conformations reveal a radius sufficient to permit passage of hydrated Cl- ions in the OWF but not the IWF model. To identify structural determinants that distinguish the two conformations and possible rearrangements of TMHs within them responsible for channel gating, we perform cross-linking by bifunctional reagents of multiple predicted pairs of cysteines in TMH 6 and 12 and 6 and 9. To determine whether the effects of cross-linking on gating observed are the result of switching of the channel from open to close state, we also treat the same residue pairs with monofunctional reagents in separate experiments. Both types of reagents prevent ion currents indicating that pore blockage is primarily responsible.

Three tapasin docking sites in TAP cooperate to facilitate transporter stabilization and heterodimerization

Science.gov (United States)

Leonhardt, Ralf M.; Abrahimi, Parwiz; Mitchell, Susan M.; Cresswell, Peter

2014-01-01

The transporter associated with antigen processing (TAP) translocates peptide antigens into the lumen of the endoplasmic reticulum (ER) for loading onto major histocompatibility complex (MHC) class I molecules. MHC class I acquires its peptide cargo in the peptide loading complex (PLC), an oligomeric complex that the chaperone tapasin organizes by bridging TAP to MHC class I and recruiting accessory molecules such as ERp57 and calreticulin. Three tapasin binding sites on TAP have been described, two of which are located in the N-terminal domains (N domains) of TAP1 and TAP2. The third binding site is present in the core transmembrane domain (coreTMD) of TAP1 and is only used by the unassembled subunits. Tapasin is required to promote TAP stability, but through which binding site(s) it is acting is unknown. In particular the role of tapasin binding to the coreTMD of TAP1 single chains is mysterious as this interaction is lost upon TAP2 association. In this study, we map the respective binding site in TAP1 to the polar face of the amphipathic transmembrane helix TM9 and identify key residues that are essential to establish the interaction. We find that this interaction is dispensable for the peptide transport function but essential to achieve full stability of human TAP1. The interaction is also required for proper heterodimerization of the transporter. Based on similar results obtained using TAP mutants lacking tapasin binding to either N domain, we conclude that all three tapasin-binding sites in TAP cooperate to achieve high transporter stability and efficient heterodimerization. PMID:24501197

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

Directory of Open Access Journals (Sweden)

Koji Mizuhashi

Full Text Available Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119, encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS and osteoid maturation time (Omt, and significantly decreased mineral apposition rate (MAR and bone formation rate per bone surface (BFR/BS. In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Resolving the biophysics of axon transmembrane polarization in a single closed-form description

Energy Technology Data Exchange (ETDEWEB)

Melendy, Robert F., E-mail: rfmelendy@liberty.edu [School of Engineering and Computational Sciences, Liberty University, Lynchburg, Virginia 24515 (United States)

2015-12-28

When a depolarizing event occurs across a cell membrane there is a remarkable change in its electrical properties. A complete depolarization event produces a considerably rapid increase in voltage that propagates longitudinally along the axon and is accompanied by changes in axial conductance. A dynamically changing magnetic field is associated with the passage of the action potential down the axon. Over 75 years of research has gone into the quantification of this phenomenon. To date, no unified model exist that resolves transmembrane polarization in a closed-form description. Here, a simple but formative description of propagated signaling phenomena in the membrane of an axon is presented in closed-form. The focus is on using both biophysics and mathematical methods for elucidating the fundamental mechanisms governing transmembrane polarization. The results presented demonstrate how to resolve electromagnetic and thermodynamic factors that govern transmembrane potential. Computational results are supported by well-established quantitative descriptions of propagated signaling phenomena in the membrane of an axon. The findings demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation function together in generating an action potential in a unified closed-form description. The work presented in this paper provides compelling evidence that three basic factors contribute to the propagated signaling in the membrane of an axon. It is anticipated this work will compel those in biophysics, physical biology, and in the computational neurosciences to probe deeper into the classical and quantum features of membrane magnetization and signaling. It is hoped that subsequent investigations of this sort will be advanced by the computational features of this model without having to resort to numerical methods of analysis.

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

Science.gov (United States)

Mizuhashi, Koji; Chaya, Taro; Kanamoto, Takashi; Omori, Yoshihiro; Furukawa, Takahisa

2015-01-01

Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice. First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant. Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Structure and Mechanism of Proton Transport Through the Transmembrane Tetrameric M2 Protein Bundle of the Influenza A Virus

Energy Technology Data Exchange (ETDEWEB)

R Acharya; V Carnevale; G Fiorin; B Levine; A Polishchuk; V Balannick; I Samish; R Lamb; L Pinto; et al.

2011-12-31

The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2{sup +} and 3{sup +} with a pK{sub a} near 6. A 1.65 {angstrom} resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

The cystic fibrosis transmembrane recruiter the alter ego of CFTR as a multi-kinase anchor.

Science.gov (United States)

Mehta, Anil

2007-11-01

This review focuses on a newly discovered interaction between protein kinases involved in cellular energetics, a process that may be disturbed in cystic fibrosis for unknown reasons. I propose a new model where kinase-mediated cellular transmission of energy provides mechanistic insight to a latent role of the cystic fibrosis transmembrane conductance regulator (CFTR). I suggest that CFTR acts as a multi-kinase recruiter to the apical epithelial membrane. My group finds that, in the cytosol, two protein kinases involved in cell energy homeostasis, nucleoside diphosphate kinase (NDPK) and AMP-activated kinase (AMPK), bind one another. Preliminary data suggest that both can also bind CFTR (function unclear). The disrupted role of this CFTR-kinase complex as 'membrane transmitter to the cell' is proposed as an alternative paradigm to the conventional ion transport mediated and CFTR/chloride-centric view of cystic fibrosis pathogenesis. Chloride remains important, but instead, chloride-induced control of the phosphohistidine content of one kinase component (NDPK, via a multi-kinase complex that also includes a third kinase, CK2; formerly casein kinase 2). I suggest that this complex provides the necessary near-equilibrium conditions needed for efficient transmission of phosphate energy to proteins controlling cellular energetics. Crucially, a new role for CFTR as a kinase controller is proposed with ionic concentration acting as a signal. The model posits a regulatory control relay for energy sensing involving a cascade of protein kinases bound to CFTR.

Nanoparticles for Site Specific Genome Editing

Science.gov (United States)

McNeer, Nicole Ali

Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to

Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.

Science.gov (United States)

Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C; Schülein, Ralf; Krause, Gerd

2010-07-01

The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

Structural insight to mutation effects uncover a common allosteric site in class C GPCRs

DEFF Research Database (Denmark)

Harpsøe, Kasper; Boesgaard, Michael W; Munk, Christian

2017-01-01

MOTIVATION: Class C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry....... Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics. RESULTS: We uncover one common site for both positive and negative modulators with different amino acid layouts that can...

CUP-1 Is a Novel Protein Involved in Dietary Cholesterol Uptake in Caenorhabditis elegans

Science.gov (United States)

Valdes, Victor J.; Athie, Alejandro; Salinas, Laura S.; Navarro, Rosa E.; Vaca, Luis

2012-01-01

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane “cholesterol recognition/interaction amino acid consensus” (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals. PMID:22479487

Community Involvement as an Effective Institutional Control at the Weldon Spring Site, a U.S. Department of Energy Office of Legacy Management Site

International Nuclear Information System (INIS)

Deyo, Y.E.; Pauling, T.

2006-01-01

The U.S. Department of Energy (DOE) Weldon Spring Site Remedial Action Project (WSSRAP) was conducted for the purpose of remediating a portion of a former trinitrotoluene and dinitrotoluene production plant that was operational from 1941 to 1945 and a former uranium refinery that was operational from 1957 to 1966. Surface remediation activities concluded in 2001 with the completion of a 45-acre (.18 square kilometer) on-site engineered disposal facility. Long-term surveillance and maintenance activities at the site were officially transferred to the DOE Office of Legacy Management in 2003. The Weldon Spring Site is located within the St. Louis, Missouri, metropolitan area (population 3 million). DOE's close relationship with surrounding land owners created a need for innovative solutions to long-term surveillance and maintenance issues at the site. Through a Secretarial proclamation, a plan was established for development of a comprehensive public involvement and education program. This program would act as an institutional control to communicate the historical legacy of the site and would make information available about contamination present at the site to guide people in making decisions about appropriate site activities. In August 2002, the Weldon Spring Site Interpretive Center opened to the public with exhibits about the history of the area, the remediation work that was completed, and a site information repository that is available to visitors. In addition, the Hamburg Trail for hiking and biking was constructed as a joint DOE/MDC effort. The 8-mile trail travels through both DOE and MDC property; a series of historical markers posted along its length to communicate the history of the area and the remediation work that was done as part of WSSRAP activities. A ramp and viewing platform with informational plaques were constructed on the disposal cell to provide an additional mechanism for public education. With a basic marketing program, site visitor-ship has

Identification of residues of FpvA involved in the different steps of Pvd-Fe uptake in Pseudomonas aeruginosa.

Science.gov (United States)

Nader, Mirella; Dobbelaere, Wim; Vincent, Michel; Journet, Laure; Adams, Hendrik; Cobessi, David; Gallay, Jacques; Schalk, Isabelle J

2007-10-23

FpvA is an outer membrane transporter involved in iron uptake by the siderophore pyoverdine (Pvd) in Pseudomonas aeruginosa. This transporter, like all other proteins of the same family, consists of a transmembrane 22 beta-stranded barrel occluded by a plug domain. The beta-strands of the barrel are connected by large extracellular loops and short periplasmic turns. Site-directed mutagenesis was carried out on FpvA to identify the extracellular loops or parts of these loops involved in the various stages of Pvd-Fe uptake. The G286C, W362C, and W434C mutations in loops L1, L3, and L4, respectively, disturbed the binding of the apo siderophore, as shown by time-resolved fluorescence spectroscopy. Iron uptake experiments followed by fluorescence resonance energy transfer (FRET) or using 55Fe indicated that residues W434 and G701 and, therefore, loops L4 and L9 must be involved in Pvd-Fe uptake by FpvA. The two corresponding mutants incorporated smaller than normal amounts of 55Fe into cells, and no Pvd recycling on FpvA was observed after iron release. Surprisingly, the S603C mutation in loop L7 increased the amount of Pvd-Fe transported. Our results suggest that W434 (L4), S603 (L7), and G701 (L9) are involved in the mechanism of Pvd-Fe uptake.

Are Adolescents Engaged in the Problematic Use of Social Networking Sites More Involved in Peer Aggression and Victimization?

Science.gov (United States)

Martínez-Ferrer, Belén; Moreno, David; Musitu, Gonzalo

2018-01-01

The problematic use of social networking sites is becoming a major public health concern. Previous research has found that adolescents who engage in a problematic use of social networking sites are likely to show maladjustment problems. However, little is known about its links with peer aggression and victimization. The main goal of this study was to analyze the relationship between problematic use of online social networking sites, peer aggression -overt vs. relational and reactive vs. instrumental-, and peer victimization -overt physical and verbal, and relational-, taking into account gender and age (in early and mid-adolescence). Participants were selected using randomized cluster sampling considering school and class as clusters. A battery of instruments was applied to 1,952 adolescents' secondary students from Spain (Andalusia) (50.4% boys), aged 11 to 16 ( M = 14.07, SD = 1.39). Results showed that girls and 14-16 adolescents were more involved in a problematic use of online social networking sites. Furthermore, adolescents with high problematic use of online social networking sites were more involved in overt-reactive and instrumental-and relational-reactive and instrumental-aggressive behaviors, and self-reported higher levels of overt-physical and verbal-and relational victimization. Even though boys indicated higher levels of all types of victimization, girls with high problematic use of online social networking sites scored the highest on relational victimization. Relating to age, early adolescents (aged 11-14) with higher problematic use of online social networking sites reported the highest levels of overt verbal and relational victimization. Overall, results suggested the co-occurrence of problematic use of online social networking sites, peer aggression and victimization. In addition, results showed the influence that gender and age had on peer victimization. This study highlights the continuity between offline and online domains with regard to

Are Adolescents Engaged in the Problematic Use of Social Networking Sites More Involved in Peer Aggression and Victimization?

Directory of Open Access Journals (Sweden)

Belén Martínez-Ferrer

2018-05-01

Full Text Available The problematic use of social networking sites is becoming a major public health concern. Previous research has found that adolescents who engage in a problematic use of social networking sites are likely to show maladjustment problems. However, little is known about its links with peer aggression and victimization. The main goal of this study was to analyze the relationship between problematic use of online social networking sites, peer aggression –overt vs. relational and reactive vs. instrumental–, and peer victimization –overt physical and verbal, and relational–, taking into account gender and age (in early and mid-adolescence. Participants were selected using randomized cluster sampling considering school and class as clusters. A battery of instruments was applied to 1,952 adolescents' secondary students from Spain (Andalusia (50.4% boys, aged 11 to 16 (M = 14.07, SD = 1.39. Results showed that girls and 14–16 adolescents were more involved in a problematic use of online social networking sites. Furthermore, adolescents with high problematic use of online social networking sites were more involved in overt—reactive and instrumental—and relational—reactive and instrumental—aggressive behaviors, and self-reported higher levels of overt—physical and verbal—and relational victimization. Even though boys indicated higher levels of all types of victimization, girls with high problematic use of online social networking sites scored the highest on relational victimization. Relating to age, early adolescents (aged 11–14 with higher problematic use of online social networking sites reported the highest levels of overt verbal and relational victimization. Overall, results suggested the co-occurrence of problematic use of online social networking sites, peer aggression and victimization. In addition, results showed the influence that gender and age had on peer victimization. This study highlights the continuity between offline

Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments

Czech Academy of Sciences Publication Activity Database

Vít, O.; Man, Petr; Kádek, Alan; Hausner, Jiří; Sklenář, A.; Harant, K.; Novák, Petr; Scigelová, M.; Wofferndin, G.; Petrák, J.

2016-01-01

Roč. 146, SI (2016), s. 15-22 ISSN 1874-3919 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Integral membrane proteins * CNBr * Transmembrane Subject RIV: CE - Biochemistry Impact factor: 3.914, year: 2016

The binding sites for cocaine and dopamine in the dopamine transporter overlap

DEFF Research Database (Denmark)

Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L

2008-01-01

Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog Leu......T. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed...... inhibition of dopamine transport by cocaine....

Public involvement in the siting of contentious facilities; Lessons from the radioactive waste repository siting programmes in Canada and the United States, with special reference to the Swedish repository siting process

International Nuclear Information System (INIS)

Richardson, P.J.

1997-08-01

This report describes the conclusion of a two-part programme, begun in 1994 with the overall aim of assisting in the development of an acceptable public participation strategy for use in Sweden for the siting of contentious facilities, with particular reference to the ongoing siting programme for a deep repository for spent nuclear fuel. The first part of the work programme, a global review of siting practice, was reported as SSI Rapport 94-15, in November 1994. This recommended further detailed studies of at least two individual programmes, which have now been carried out in Canada and the United States, and are reported on here. They involved face to face meetings with many of the main stake holders in the two programmes and enabled valuable insight to be gained into the potential problems associated with increased public participation, as well as identifying good practice where it exists. The lessons learned have then been applied to the evolving repository siting process in Sweden. 35 refs

Public involvement in the siting of contentious facilities; Lessons from the radioactive waste repository siting programmes in Canada and the United States, with special reference to the Swedish repository siting process

Energy Technology Data Exchange (ETDEWEB)

Richardson, P J [Geosciences for Development and the Environment (United Kingdom)

1997-08-01

This report describes the conclusion of a two-part programme, begun in 1994 with the overall aim of assisting in the development of an acceptable public participation strategy for use in Sweden for the siting of contentious facilities, with particular reference to the ongoing siting programme for a deep repository for spent nuclear fuel. The first part of the work programme, a global review of siting practice, was reported as SSI Rapport 94-15, in November 1994. This recommended further detailed studies of at least two individual programmes, which have now been carried out in Canada and the United States, and are reported on here. They involved face to face meetings with many of the main stake holders in the two programmes and enabled valuable insight to be gained into the potential problems associated with increased public participation, as well as identifying good practice where it exists. The lessons learned have then been applied to the evolving repository siting process in Sweden. 35 refs.

The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

DEFF Research Database (Denmark)

Yatsenko, A S; Kucherenko, M M; Pantoja, M

2009-01-01

BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C...

Expression and regulation of transmembrane transporters in healthy intestine and gastrointestinal diseases

OpenAIRE

Hruz, Petr

2006-01-01

Transmembrane transporters mediate energy dependent or independent translocation of drugs, potentially toxic compounds, and of various endogenous substrates such as bile acids and bilirubin across membranes. In this thesis the focus is on two classes of transporters, the ATPbinding cassette (ABC) transporters, which mediate ATP dependent transport and the solute carriers (SLC) which use electrochemical gradients for their transport. The transporters are expressed on membranes o...

US DoE's Environmental Management Site-Specific Advisory Board 15 Years of Community Involvement

International Nuclear Information System (INIS)

Nielson, M.; Brennan, C.A.

2009-01-01

This paper provides an overview of the U.S. Department of Energy's Environmental Management Site-Specific Advisory Board (EM SSAB) from its roots in the early 1990's at the Keystone Center to its current activities. The EM SSAB has a unique mandate to provide input regarding the cleanup of nuclear legacy sites in the United States. Chartered under the Federal Advisory Committee Act, the EM SSAB today comprises eight local boards. The Office of Environmental Management has made public participation a fundamental component of its cleanup mission and has found that the EM SSAB has contributed greatly to bringing community values and priorities to the cleanup decision-making processes. Public participation that involves ongoing community engagement has inherent challenges; the EM SSAB has additional challenges that reflect the political and technical nature of the Agency's work. (authors)

Molecular dynamics study of the solvation of an alpha-helical transmembrane peptide by DMSO

NARCIS (Netherlands)

Duarte, A.M.; Mierlo, van C.P.M.; Hemminga, M.A.

2008-01-01

10-ns molecular dynamics study of the solvation of a hydrophobic transmembrane helical peptide in dimethyl sulfoxide (DMSO) is presented. The objective is to analyze how this aprotic polar solvent is able to solvate three groups of amino acid residues (i.e., polar, apolar, and charged) that are

Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity

DEFF Research Database (Denmark)

Rosenkilde, Mette M; Benned-Jensen, Tau; Holst, Peter J

2006-01-01

Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor...

The Potential Mechanism of ZFX Involvement in the Cell Growth

Directory of Open Access Journals (Sweden)

Mahboube Ganji arjenaki

2016-04-01

Full Text Available Background:The zinc-finger X linked (ZFX gene encodes a transcription factor that acts as a regulator of self-renewal of stem cells. Due to the role of ZFX in cell growth, understanding ZFX protein-protein interactions helps to clarify its proper biological functions in signaling pathways. The aim of this study is to define ZFX protein-protein interactions and the role of ZFX in cell growth. Materials and Methods: The PIPs output includes three interacting proteins with ZFX: eukaryotic translation initiation factor 3 subunit I(EIF3I, eukaryotic translation initiation factor 3 subunit G(EIF3G and protein nuclear pore and COPII coat complex component homolog isoform 3 (SEC13L1. Results: As a cargo and transmembrane protein interacting with Sec13,eIF3I and eIF3G, ZFX mediates cargo sorting in COPII vesicles at ER exit sites. While traveling to cis-Golgi, eIF3I is phosphorylated by the mechanistic target of rapamycin (mTOR. Proteins transport by COPI vesicles to the nucleusouter site layer containing SEC13 via the contribution of microtubules. EIF3G and eIF3I interact with coatomer protein complex subunit beta 2 (COPB2 that helps to enclose ZFX in COPI vesicle. ZFX and eIF3G enter nucleolus where activation of transcription from pre rDNA genes occurs. Conclusion:We proposed a model in which ZFX is involved in cell growth by promoting the transcription of rDNA genes.

Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

Directory of Open Access Journals (Sweden)

Adam Bajinting

2017-06-01

Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

Science.gov (United States)

Saadipour, Khalil; MacLean, Michael; Pirkle, Sean; Ali, Solav; Lopez-Redondo, Maria-Luisa; Stokes, David L; Chao, Moses V

2017-10-06

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Site-specific distribution of claudin-based paracellular channels with roles in biological fluid flow and metabolism.

Science.gov (United States)

Tanaka, Hiroo; Tamura, Atsushi; Suzuki, Koya; Tsukita, Sachiko

2017-10-01

The claudins are a family of membrane proteins with at least 27 members in humans and mice. The extracellular regions of claudin proteins play essential roles in cell-cell adhesion and the paracellular barrier functions of tight junctions (TJs) in epithelial cell sheets. Furthermore, the extracellular regions of some claudins function as paracellular channels in the paracellular barrier that allow the selective passage of water, ions, and/or small organic solutes across the TJ in the extracellular space. Structural analyses have revealed a common framework of transmembrane, cytoplasmic, and extracellular regions among the claudin-based paracellular barriers and paracellular channels; however, differences in the claudins' extracellular regions, such as their charges and conformations, determine their properties. Among the biological systems that involve fluid flow and metabolism, it is noted that hepatic bile flow, renal Na + reabsorption, and intestinal nutrient absorption are dynamically regulated via site-specific distributions of paracellular channel-forming claudins in tissue. Here, we focus on how site-specific distributions of claudin-2- and claudin-15-based paracellular channels drive their organ-specific functions in the liver, kidney, and intestine. © 2017 New York Academy of Sciences.

Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.

Science.gov (United States)

Laverty, Duncan; Thomas, Philip; Field, Martin; Andersen, Ole J; Gold, Matthew G; Biggin, Philip C; Gielen, Marc; Smart, Trevor G

2017-11-01

γ-Aminobutyric acid receptors (GABA A Rs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result from receptor dysfunction. A critical component of most native GABA A Rs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact anxiety, stress and depression, and for therapeutic drugs, such as general anesthetics. Understanding the basis for the modulation of GABA A R function requires high-resolution structures. Here we present the first atomic structures of a GABA A R chimera at 2.8-Å resolution, including those bound with potentiating and inhibitory neurosteroids. These structures define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable the exploitation of neurosteroids for therapeutic drug design to regulate GABA A Rs in neurological disorders.

Over half of breakpoints in gene pairs involved in cancer-specific recurrent translocations are mapped to human chromosomal fragile sites

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Pierce Levi CT

2009-01-01

Full Text Available Abstract Background Gene rearrangements such as chromosomal translocations have been shown to contribute to cancer development. Human chromosomal fragile sites are regions of the genome especially prone to breakage, and have been implicated in various chromosome abnormalities found in cancer. However, there has been no comprehensive and quantitative examination of the location of fragile sites in relation to all chromosomal aberrations. Results Using up-to-date databases containing all cancer-specific recurrent translocations, we have examined 444 unique pairs of genes involved in these translocations to determine the correlation of translocation breakpoints and fragile sites in the gene pairs. We found that over half (52% of translocation breakpoints in at least one gene of these gene pairs are mapped to fragile sites. Among these, we examined the DNA sequences within and flanking three randomly selected pairs of translocation-prone genes, and found that they exhibit characteristic features of fragile DNA, with frequent AT-rich flexibility islands and the potential of forming highly stable secondary structures. Conclusion Our study is the first to examine gene pairs involved in all recurrent chromosomal translocations observed in tumor cells, and to correlate the location of more than half of breakpoints to positions of known fragile sites. These results provide strong evidence to support a causative role for fragile sites in the generation of cancer-specific chromosomal rearrangements.

Interprofessional collaboration and family member involvement in intensive care units: emerging themes from a multi-sited ethnography.

Science.gov (United States)

Reeves, Scott; McMillan, Sarah E; Kachan, Natasha; Paradis, Elise; Leslie, Myles; Kitto, Simon

2015-05-01

This article presents emerging findings from the first year of a two-year study, which employed ethnographic methods to explore the culture of interprofessional collaboration (IPC) and family member involvement in eight North American intensive care units (ICUs). The study utilized a comparative ethnographic approach - gathering observation, interview and documentary data relating to the behaviors and attitudes of healthcare providers and family members across several sites. In total, 504 hours of ICU-based observational data were gathered over a 12-month period in four ICUs based in two US cities. In addition, 56 semi-structured interviews were undertaken with a range of ICU staff (e.g. nurses, doctors and pharmacists) and family members. Documentary data (e.g. clinical guidelines and unit policies) were also collected to help develop an insight into how the different sites engaged organizationally with IPC and family member involvement. Directed content analysis enabled the identification and categorization of major themes within the data. An interprofessional conceptual framework was utilized to help frame the coding for the analysis. The preliminary findings presented in this paper illuminate a number of issues related to the nature of IPC and family member involvement within an ICU context. These findings are discussed in relation to the wider interprofessional and health services literature.

The transmembrane region is responsible for targeting of adaptor protein LAX into "heavy rafts''

Czech Academy of Sciences Publication Activity Database

Hrdinka, Matouš; Otáhal, Pavel; Hořejší, Václav

2012-01-01

Roč. 7, č. 5 (2012), e36330 E-ISSN 1932-6203 R&D Projects: GA ČR GEMEM/09/E011; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : LAX * transmembrane domain * DRM Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.730, year: 2012

Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

OpenAIRE

Denys, A; Allain, F; Carpentier, M; Spik, G

1998-01-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes...

Artificial Diels–Alderase based on the transmembrane protein FhuA

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Hassan Osseili

2016-06-01

Full Text Available Copper(I and copper(II complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVFtev. Copper(I was incorporated using an N-heterocyclic carbene (NHC ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels–Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product.

Facilitating Site Specific and Citizens Advisory Boards: Running Effective Meetings that Involve Complex Technical Issues

International Nuclear Information System (INIS)

Freeman, J.

2009-01-01

Environmental cleanup issues at federal sites are more often than not on the agendas of meetings of the Site Specific Advisory Boards (SSABs), also called Citizens Advisory Boards (CABs), that exist at most U.S. Department of Energy (DOE) sites with an Environmental Management (EM) mission. In 1994, when Congress established these committees comprised of local citizens, it enabled community stakeholders to become more directly involved in DOE EM cleanup decisions. This involvement is to help the agency make cost-effective and environmentally sound decisions which lead to faster, safer cleanups. Eight local Boards that fall under the Federal Advisory Committee Act-chartered EM SSAB charter are found in Hanford, Washington; Idaho; Northern New Mexico; Nevada; Oak Ridge, Tennessee; Paducah, Kentucky; Portsmouth, Ohio; and at the Savannah River Site in South Carolina. These boards provide advice and recommendations about EM site-specific issues such as cleanup standards (how clean is clean?), environmental restoration, waste management, the stabilization and disposal of non-stockpile nuclear materials, future land use and long-term stewardship, risk assessment and management, and cleanup science and technology activities. These issues are, by their very nature, loaded with complicated technical terms and strategies, scientific data and interpretations, and long histories of studies and reports. The members of SSABs and CABs rotate on and off the Boards according to defined terms of office, thereby routinely opening the Boards' ranks to new members, many of whom are new to the issues. In addition, members of the public who have access to public comment periods at each Board meeting run up against the same daunting menu of obscure acronyms, scientific terms and notations, and an historical trail which is not always evident except to those involved with the issues over many years. How does a facilitator effectively guide such a group of citizens, each of whom arrives to

Transmembrane adaptor proteins in the high-affinity IgE receptor signaling

Czech Academy of Sciences Publication Activity Database

Dráber, Petr; Hálová, Ivana; Levi-Schaffer, F.; Dráberová, Lubica

2012-01-01

Roč. 2, 11.1. (2012), s. 95 ISSN 1664-3224 R&D Projects: GA MŠk 1M0506; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA AV ČR KAN200520701 Grant - others:AV ČR(CZ) M200520901 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : IgE receptor * LAT/LAT1 * LAX * NTAL/Lab/LAT2 * PAG/Cbp * mast cells * plasma membrane * transmembrane adaptor proteins Subject RIV: EB - Genetics ; Molecular Biology

Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

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Kelly J Culhane

2015-11-01

Full Text Available Although family B G protein-coupled receptors (GPCRs contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

Science.gov (United States)

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Intrinsic potential of cell membranes: opposite effects of lipid transmembrane asymmetry and asymmetric salt ion distribution

DEFF Research Database (Denmark)

Gurtovenko, Andrey A; Vattulainen, Ilpo

2009-01-01

Using atomic-scale molecular dynamics simulations, we consider the intrinsic cell membrane potential that is found to originate from a subtle interplay between lipid transmembrane asymmetry and the asymmetric distribution of monovalent salt ions on the two sides of the cell membrane. It turns out......Cl saline solution and the PE leaflet is exposed to KCl, the outcome is that the effects of asymmetric lipid and salt ion distributions essentially cancel one another almost completely. Overall, our study highlights the complex nature of the intrinsic potential of cell membranes under physiological...... that both the asymmetric distribution of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids across a membrane and the asymmetric distribution of NaCl and KCl induce nonzero drops in the transmembrane potential. However, these potential drops are opposite in sign. As the PC leaflet faces a Na...

Analysis of Light-Induced Transmembrane Ion Gradients and Membrane Potential in Photosystem I Proteoliposomes

International Nuclear Information System (INIS)

Pennisi, Cristian P.; Greenbaum, Elias; Yoshida, Ken

2010-01-01

Photosystem I (PSI) complexes can support a light-driven electrochemical gradient for protons, which is the driving force for energy-conserving reactions across biological membranes. In this work, a computational model that enables a quantitative description of the light-induced proton gradients across the membrane of PSI proteoliposomes is presented. Using a set of electrodiffusion equations, a compartmental model of a vesicle suspended in aqueous medium was studied. The light-mediated proton movement was modeled as a single proton pumping step with backpressure of the electric potential. The model fits determinations of pH obtained from PSI proteoliposomes illuminated in the presence of mediators of cyclic electron transport. The model also allows analysis of the proton gradients in relation to the transmembrane ion fluxes and electric potential. Sensitivity analysis enabled a determination of the parameters that have greater influence on steady-state levels and onset/decay rates of transmembrane pH and electric potential. This model could be used as a tool for optimizing PSI proteoliposomes for photo-electrochemical applications.

Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating.

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Montserrat Samsó

2009-04-01

Full Text Available Ryanodine receptor type 1 (RyR1 produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices". Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right

Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations

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Johanna Wanngren

2014-01-01

Full Text Available The enzyme complex γ-secretase generates amyloid β-peptide (Aβ, a 37–43-residue peptide associated with Alzheimer disease (AD. Mutations in presenilin 1 (PS1, the catalytical subunit of γ-secretase, result in familial AD (FAD. A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio, but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity.

Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites.

Science.gov (United States)

Mariller, C; Haendler, B; Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.

Intact transmembrane isoforms of the neural cell adhesion molecule are released from the plasma membrane

DEFF Research Database (Denmark)

Olsen, M; Krog, L; Edvardsen, K

1993-01-01

. By density-gradient centrifugation it was shown that shed transmembrane NCAM-B was present in fractions of high, as well as low, density, indicating that a fraction of the shed NCAM is associated with minor plasma membrane fragments. Finally, it was shown that isolated soluble NCAM inhibited cell binding...

Structural fragment clustering reveals novel structural and functional motifs in α-helical transmembrane proteins

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Vassilev Boris

2010-04-01

Full Text Available Abstract Background A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology. Results We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to α-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94% appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1 a dimer interface motif found in voltage-gated chloride channels, (2 a proton transfer motif found in heme-copper oxidases, and (3 a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b. Conclusions Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes.

Contribution of Kunitz protease inhibitor and transmembrane domains to amyloid precursor protein homodimerization.

Science.gov (United States)

Ben Khalifa, N; Tyteca, D; Courtoy, P J; Renauld, J C; Constantinescu, S N; Octave, J N; Kienlen-Campard, P

2012-01-01

The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP. Copyright © 2012 S. Karger AG, Basel.

Point mutations in the extracytosolic loop between transmembrane segments M5 and M6 of the yeast Pma1 H+-ATPase: alanine-scanning mutagenesis.

Science.gov (United States)

Petrov, Valery V

2015-01-01

Membrane-spanning segments M4, M5, M6, and M8 of the H(+)-, Ca(2+)-, and K(+), Na(+)-ATPases, which belong to the P2-type pumps are the core through which cations are transported. M5 and M6 loop is a short extracytoplasmic stretch of the seven amino acid residues (714-DNSLDID) connecting two of these segments, M5 and M6, where residues involved in the formation of the proton-binding site(s) are located. In the present study, we have used alanine-scanning mutagenesis to explore the structural and functional relationships within this loop of the yeast plasma membrane Pma1 H(+)-ATPase. Of the 7 Ala mutants made, substitution for the most conserved residue (Leu-717) has led to a severe misfolding and complete block in biogenesis of the mutant enzyme. The replacement of Asp-714 has also caused misfolding leading to significant decrease in the expression of the mutant and loss of activity. The remaining mutants were expressed in secretory vesicles at 21-119% of the wild-type level and were active enough to be analyzed in detail. One of these mutants (I719A) showed five- to threefold decrease in both expression and ATP hydrolyzing and H(+) pumping activities and also threefold reduction in the coupling ratio between ATP hydrolysis and H(+) transport. Thus, Ala substitutions at three positions of the seven seriously affected biogenesis, folding, stability and/or functioning of the enzyme. Taken together, these results lead to suggestion that M5 and M6 loop play an important role in the protein stability and function and is responsible for proper arrangement of transmembrane segments M5 and M6 and probably other domains of the enzyme. Results for additional conserved substitutions (Asn and Glu) at Asp-714 and Asp-720 confirmed this suggestion.

Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

Science.gov (United States)

Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

2007-05-01

Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

Simulations of Skin Barrier Function: Free Energies of Hydrophobic and Hydrophilic Transmembrane Pores in Ceramide Bilayers

NARCIS (Netherlands)

Notman, Rebecca; Anwar, Jamshed; Briels, Willem J.; Noro, Massimo G.; den Otter, Wouter K.

2008-01-01

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO)

Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers-Mast Cell Case.

Science.gov (United States)

Halova, Ivana; Draber, Petr

2016-01-01

The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella.

Science.gov (United States)

Wang, Jing; Wang, Xingliang; Lansdell, Stuart J; Zhang, Jianheng; Millar, Neil S; Wu, Yidong

2016-04-01

Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effect of ionizing radiation on transmembrane potential of Streptococcus

International Nuclear Information System (INIS)

Fomenko, B.S.; Akoev, I.G.

1979-01-01

Treatment of Streptococcus faecalis with ionizing radiation at doses of 5 to 100 krad is shown to reduce the energy-dependent accumulation of dibenzyldimethylammonium (DDA + ) by the cell. Since transmembrane potential is the moving force of DDA + transport across the membrane, the decrease in DDA + accumulation is suggested to be due to potential reduction. This radiation effect was not due to inactivation of the potential-generating mechanism; thus, the ATPase activity and glycolytic activity of the irradiated cells were higher than in the control. At the same time, the membranes exhibited an increased permeability for K + and protons, which is probably due to structural rearrangements in the membranes after irradiation. It is suggested that the potential reduction results from the increase in proton permeability of membranes

What Can We Learn about Cholesterol's Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential?

Czech Academy of Sciences Publication Activity Database

Falkovich, S. G.; Martinez-Seara, Hector; Nesterenko, A. M.; Vattulainen, I.; Gurtovenko, A. A.

2016-01-01

Roč. 7, č. 22 (2016), s. 4585-4590 ISSN 1948-7185 Institutional support: RVO:61388963 Keywords : membrane * cholesterol * membrane asymmetry * membrane dipole potential * transmembrane distribution Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 9.353, year: 2016

Involved-Site Image-Guided Intensity Modulated Versus 3D Conformal Radiation Therapy in Early Stage Supradiaphragmatic Hodgkin Lymphoma

Energy Technology Data Exchange (ETDEWEB)

Filippi, Andrea Riccardo, E-mail: andreariccardo.filippi@unito.it [Department of Oncology, University of Torino, Torino (Italy); Ciammella, Patrizia [Radiation Therapy Unit, Department of Oncology and Advanced Technology, ASMN Hospital IRCCS, Reggio Emilia (Italy); Piva, Cristina; Ragona, Riccardo [Department of Oncology, University of Torino, Torino (Italy); Botto, Barbara [Hematology, Città della Salute e della Scienza, Torino (Italy); Gavarotti, Paolo [Hematology, University of Torino and Città della Salute e della Scienza, Torino (Italy); Merli, Francesco [Hematology Unit, ASMN Hospital IRCCS, Reggio Emilia (Italy); Vitolo, Umberto [Hematology, Città della Salute e della Scienza, Torino (Italy); Iotti, Cinzia [Radiation Therapy Unit, Department of Oncology and Advanced Technology, ASMN Hospital IRCCS, Reggio Emilia (Italy); Ricardi, Umberto [Department of Oncology, University of Torino, Torino (Italy)

2014-06-01

Purpose: Image-guided intensity modulated radiation therapy (IG-IMRT) allows for margin reduction and highly conformal dose distribution, with consistent advantages in sparing of normal tissues. The purpose of this retrospective study was to compare involved-site IG-IMRT with involved-site 3D conformal RT (3D-CRT) in the treatment of early stage Hodgkin lymphoma (HL) involving the mediastinum, with efficacy and toxicity as primary clinical endpoints. Methods and Materials: We analyzed 90 stage IIA HL patients treated with either involved-site 3D-CRT or IG-IMRT between 2005 and 2012 in 2 different institutions. Inclusion criteria were favorable or unfavorable disease (according to European Organization for Research and Treatment of Cancer criteria), complete response after 3 to 4 cycles of an adriamycin- bleomycin-vinblastine-dacarbazine (ABVD) regimen plus 30 Gy as total radiation dose. Exclusion criteria were chemotherapy other than ABVD, partial response after ABVD, total radiation dose other than 30 Gy. Clinical endpoints were relapse-free survival (RFS) and acute toxicity. Results: Forty-nine patients were treated with 3D-CRT (54.4%) and 41 with IG-IMRT (45.6%). Median follow-up time was 54.2 months for 3D-CRT and 24.1 months for IG-IMRT. No differences in RFS were observed between the 2 groups, with 1 relapse each. Three-year RFS was 98.7% for 3D-CRT and 100% for IG-IMRT. Grade 2 toxicity events, mainly mucositis, were recorded in 32.7% of 3D-CRT patients (16 of 49) and in 9.8% of IG-IMRT patients (4 of 41). IG-IMRT was significantly associated with a lower incidence of grade 2 acute toxicity (P=.043). Conclusions: RFS rates at 3 years were extremely high in both groups, albeit the median follow-up time is different. Acute tolerance profiles were better for IG-IMRT than for 3D-CRT. Our preliminary results support the clinical safety and efficacy of advanced RT planning and delivery techniques in patients affected with early stage HL, achieving complete

Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

International Nuclear Information System (INIS)

Perez-Ramirez, B.; Martinez-Carrion, M.

1989-01-01

A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [ 3 H]pyridoxine 5-phosphate ([ 3 H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [ 3 H]PNP at 37 degree C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the α-subunit, internal surface labeling by PLP was greatest for the highest molecular weight (δ) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles

Nonpolar interactions between trans-membrane helical EGF peptide and phosphatidylcholines, sphingomyelins and cholesterol. Molecular dynamics simulation studies

NARCIS (Netherlands)

Róg, T.; Murzyn, K.; Karttunen, M.E.J.; Pasenkiewicz-Gierula, M.

2008-01-01

A molecular dynamics simulation study of four lipid bilayers with inserted trans-membrane helical fragment of epithelial growth factor (EGF) receptor (EGF peptide) was performed. The lipid bilayers differ in their lipid composition and consist of (i) unsaturated phosphatidylcholine

Structural basis of typhod: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

Energy Technology Data Exchange (ETDEWEB)

Balakrishna, A.; Saxena, A; Mok, H; Swaminathan, K

2009-01-01

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

Structural basis of typhoid: Salmonella typhi type IVb pilin (PiLS) and cystic fibrosis transmembrane conductance regulator interaction

Energy Technology Data Exchange (ETDEWEB)

Balakrishna, A.M.; Saxena, A.; Mok, H. Y.-K.; Swaminathan, K.

2009-11-01

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein ({Delta}PilS), which makes the pilus, was determined at 1.9 {angstrom} resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of {Delta}PilS and a target CFTR peptide, determined at 1.8 {angstrom}, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

Structural Basis of Typhoid: Salmonella typhi Type IVb pilin (PilS) and Cystic Fibrosis Transmembrane Conductance Regulatory Interaction

Energy Technology Data Exchange (ETDEWEB)

Balakrishna, A.; Saxena, A; Mok, H; Swaminathan, K

2009-01-01

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

Low rate doses effects of gamma radiation on glycoproteins of transmembrane junctions in fibroblasts

International Nuclear Information System (INIS)

Bringas, J.E.; Caceres, J.L.

1996-01-01

Glycoproteins of trans-membrane junctions are molecules that help to bind cells with the extracellular matrix. Integrins are the most important trans-membrane molecules among others. The damage of gamma radiation on those proteins could be an important early event that causes membrane abnormalities which may lead to cell malfunction and cancer induced by radiation due to cell dissociation. Randomized blocks with 3 repetitions of mouse embryo fibroblast cultures, were irradiated with Cobalt-60 gamma rays, during 20 days. Biological damage to glycoproteins and integrins was evaluated by cellular growth and fibroblast proliferative capacity. Integrins damage was studied by isolation by column immunoaffinity chromatography migrated on SDS-Page under reducing and non reducing conditions, and inhibition of integrins extracellular matrix adhesion by monoclonal antibodies effect. The dose/rate (0.05 Gy/day-0.2 Gy/day) of gamma given to cells did not show damage evidence on glycoproteins and integrins. If damage happened, it was repaired by cells very soon, was delayed by continuous cellular division or by glycoproteins characteristic of being multiple extracellular ligatures. Bio effects became more evident with an irradiation time greater than 20 days or a high dose/rate. (authors). 6 refs

Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

Science.gov (United States)

Sikder, Md. Kabir Uddin; Stone, Kyle A.; Kumar, P. B. Sunil; Laradji, Mohamed

2014-01-01

We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. PMID:25106608

Role of ATP binding and hydrolysis in the gating of the cystic fibrosis transmembrane conductance regulator

Directory of Open Access Journals (Sweden)

Taras Gout

2012-01-01

Full Text Available The CFTR gene is unique within the ATP-binding cassette (ABC protein family, predominantly of transporters, by coding a chloride channel. The gating mechanism of ABC proteins has been characterized by the ATP Switch model in terms cycles of dimer formation and dissociation linked to ATP binding and hydrolysis, respectively. It would be of interest to assess the extent that Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, a functional channel, fits the ATP Switch model for ABC transporters. Additional transporter mechanisms, namely those of Pgp and HlyB, are discussed for perspective. Literature search of databases selected key references in comparing and contrasting the gating mechanism. CFTR is a functional chloride channel facilitating transmembrane anion flow down electrochemical gradients. A dysfunctional CFTR protein results in cystic fibrosis, a fatal pleiotropic disease currently managed symptomatically. Understanding the gating mechanism will help target drug development aimed at alleviating and curing the disease.

The rapid-onset dystonia parkinsonism mutation D923N of the Na+, K+-ATPase alpha3 isoform disrupts Na+ interaction at the third Na+ site.

Science.gov (United States)

Einholm, Anja Pernille; Toustrup-Jensen, Mads S; Holm, Rikke; Andersen, Jens Peter; Vilsen, Bente

2010-08-20

Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific alpha3-isoform of Na(+), K(+)-ATPase. Here, we present the functional consequences of RDP mutation D923N. Relative to the wild type, the mutant exhibits a remarkable approximately 200-fold reduction of Na(+) affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E(1) form with intracellular Na(+). This is the largest effect on Na(+) affinity reported so far for any Na(+), K(+)-ATPase mutant. D923N also affects the interaction with extracellular Na(+) normally driving the E(1)P to E(2)P conformational transition backward. However, no impairment of K(+) binding was observed for D923N, leading to the conclusion that Asp(923) is specifically associated with the third Na(+) site that is selective toward Na(+). The crystal structure of the Na(+), K(+)-ATPase in E(2) form shows that Asp(923) is located in the cytoplasmic half of transmembrane helix M8 inside a putative transport channel, which is lined by residues from the transmembrane helices M5, M7, M8, and M10 and capped by the C terminus, recently found involved in recognition of the third Na(+) ion. Structural modeling of the E(1) form of Na(+), K(+)-ATPase based on the Ca(2+)-ATPase crystal structure is consistent with the hypothesis that Asp(923) contributes to a site binding the third Na(+) ion. These results in conjunction with our previous findings with other RDP mutants suggest that a selective defect in the handling of Na(+) may be a general feature of the RDP disorder.

Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation

DEFF Research Database (Denmark)

Elling, Christian E; Frimurer, Thomas M; Gerlach, Lars-Ole

2006-01-01

for monoamine binding in TM-III, was used as the starting point to engineer activating metal ion sites between the extracellular segments of the beta2-adrenergic receptor. Cu(II) and Zn(II) alone and in complex with aromatic chelators acted as potent (EC50 decreased to 0.5 microm) and efficacious agonists...

Identification of residues involved in nucleotidyltransferase activity of JHP933 from helicobacter pyloriby site-directed mutagenesis

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Ye Xianren

2016-01-01

Full Text Available Helicobacter pylori is a well-known bacterial pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Evidence has suggested that certain strain-specific genes in the plasticity region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. Therefore there is considerable interest in the strain-specific genes located in the plasticity regions of H. pylori. JHP933 is encoded by the gene in the plasticity region of H. pylori strain J99. Recently, the crystal structure of JHP933 has confirmed it as a nucleotidyltransferase (NTase superfamily protein and a putative active site has been proposed. However, no evidence from direct functional assay has been presented to confirm the active site and little is known about the functional mechanism of JHP933. Here, through superimposition with Cid1/NTP complex structures, we modelled the complex structures of JHP933 with different NTPs. Based on the models and using rational site-directed mutagenesis combined with enzymatic activity assays, we confirm the active site and identify several residues important for the nucleotidyl transferring function of JHP933. Furthermore, mutations of these active site residues result in the abolishment of the nucleotidyltransferase activity of JHP933. This work provides preliminary insight into the molecular mechanism underlying the pathophysiological role in H. pylori infection of JHP933 as a novel NTase superfamily protein.

Patterns of failure of diffuse large B-cell lymphoma patients after involved-site radiotherapy

Energy Technology Data Exchange (ETDEWEB)

Holzhaeuser, Eva; Berlin, Maximilian; Bezold, Thomas; Mayer, Arnulf; Schmidberger, Heinz [University Medical Center Mainz, Department of Radiation Oncology and Radiotherapy, Mainz (Germany); Wollschlaeger, Daniel [University Medical Center Mainz, Institute for Medical Biostatistics, Epidemiology and Informatics, Mainz (Germany); Hess, Georg [University Medical Center Mainz, Department of Internal Medicine, Mainz (Germany)

2017-12-15

Radiotherapy (RT) in combination with chemoimmunotherapy is highly efficient in the treatment of diffuse large B-cell lymphoma (DLBCL). This retrospective analysis evaluated the efficacy of the treatment volume and the dose concept of involved-site RT (ISRT). We identified 60 histologically confirmed stage I-IV DLBCL patients treated with multimodal cytotoxic chemoimmunotherapy and followed by consolidative ISRT from 2005-2015. Progression-free survival (PFS) and overall survival (OS) were estimated by Kaplan-Meier method. Univariate analyses were performed by log-rank test and Mann-Whitney U-test. After initial chemoimmunotherapy (mostly R-CHOP; rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), 19 (36%) patients achieved complete response (CR), 34 (64%) partial response (PR) or less. Excluded were 7 (12%) patients with progressive disease after chemoimmunotherapy. All patients underwent ISRT with a dose of 40 Gy. After a median follow-up of 44 months, 79% of the patients remained disease free, while 21% presented with failure, progressive systemic disease, or death. All patients who achieved CR after chemoimmunotherapy remained in CR. Of the patients achieving PR after chemotherapy only 2 failed at the initial site within the ISRT volume. No marginal relapse was observed. Ann Arbor clinical stage I/II showed significantly improved PFS compared to stage III/IV (93% vs 65%; p ≤ 0.021). International Prognostic Index (IPI) score of 0 or 1 compared to 2-5 has been associated with significantly increased PFS (100% vs 70%; p ≤ 0.031). Postchemoimmunotherapy status of CR compared to PR was associated with significantly increased PFS (100% vs 68%; p ≤ 0.004) and OS (100% vs 82%; p ≤ 0.026). Only 3 of 53 patients developed grade II late side effects, whereas grade III or IV side effects have not been observed. These data suggest that a reduction of the RT treatment volume from involved-field (IF) to involved-site (IS) is sufficient because

Transmembrane potential of GlyCl-expressing instructor cells induces a neoplastic-like conversion of melanocytes via a serotonergic pathway

Directory of Open Access Journals (Sweden)

Douglas Blackiston

2011-01-01

Understanding the mechanisms that coordinate stem cell behavior within the host is a high priority for developmental biology, regenerative medicine and oncology. Endogenous ion currents and voltage gradients function alongside biochemical cues during pattern formation and tumor suppression, but it is not known whether bioelectrical signals are involved in the control of stem cell progeny in vivo. We studied Xenopus laevis neural crest, an embryonic stem cell population that gives rise to many cell types, including melanocytes, and contributes to the morphogenesis of the face, heart and other complex structures. To investigate how depolarization of transmembrane potential of cells in the neural crest’s environment influences its function in vivo, we manipulated the activity of the native glycine receptor chloride channel (GlyCl. Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion. A similar effect was observed in human melanocytes in culture. Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT. These data reveal GlyCl as a molecular marker of a sparse and heretofore unknown cell population with the ability to specifically instruct neural crest derivatives, suggest transmembrane potential as a tractable signaling modality by which somatic cells can control stem cell behavior at considerable distance, identify a new biophysical aspect of the environment that confers a neoplastic-like phenotype upon stem cell progeny, reveal a pre-neural role for serotonin and its transporter, and suggest a novel strategy for manipulating stem cell behavior.

Familial CJD Associated PrP Mutants within Transmembrane Region Induced Ctm-PrP Retention in ER and Triggered Apoptosis by ER Stress in SH-SY5Y Cells

Science.gov (United States)

Wang, Xin; Shi, Qi; Xu, Kun; Gao, Chen; Chen, Cao; Li, Xiao-Li; Wang, Gui-Rong; Tian, Chan; Han, Jun; Dong, Xiao-Ping

2011-01-01

Background Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrPC) to the pathogenic one (PrPSc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. Methodology/Principal Findings To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed. Conclusions/Significance The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants

Familial CJD associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells.

Directory of Open Access Journals (Sweden)

Xin Wang

Full Text Available BACKGROUND: Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP gene that are presumed to favor conversion of the cellular isoform of PrP (PrP(C to the pathogenic one (PrP(Sc. The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. METHODOLOGY/PRINCIPAL FINDINGS: To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER, the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL and PrP with three amino acids exchange in transmembrane region (PrP-3AV were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and caspase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K failed. CONCLUSIONS/SIGNIFICANCE: The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them

The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper.

Science.gov (United States)

Inda, Maria E; Oliveira, Rafael G; de Mendoza, Diego; Cybulski, Larisa E

2016-11-01

Thermosensors detect temperature changes and trigger cellular responses crucial for survival at different temperatures. The thermosensor DesK is a transmembrane (TM) histidine kinase which detects a decrease in temperature through its TM segments (TMS). Here, we address a key issue: how a physical stimulus such as temperature can be converted into a cellular response. We show that the thickness of Bacillus lipid membranes varies with temperature and that such variations can be detected by DesK with great precision. On the basis of genetic studies and measurements of in vitro activity of a DesK construct with a single TMS (minimal sensor DesK [MS-DesK]), reconstituted in liposomes, we propose an interplay mechanism directed by a conserved dyad, phenylalanine 8-lysine 10. This dyad is critical to anchor the only transmembrane segment of the MS-DesK construct to the extracellular water-lipid interphase and is required for the transmembrane segment of MS-DesK to function as a caliper for precise measurement of membrane thickness. The data suggest that positively charged lysine 10, which is located in the hydrophobic core of the membrane but is close to the water-lipid interface, pulls the transmembrane region toward the water phase to localize its charge at the interface. Nevertheless, the hydrophobic residue phenylalanine 8, located at the N-terminal extreme of the TMS, has a strong tendency to remain in the lipid phase, impairing access of lysine 10 to the water phase. The outcome of this interplay is a fine-tuned sensitivity to membrane thickness that elicits conformational changes that favor different signaling states of the protein. The ability to sense and respond to extracellular signals is essential for cell survival. One example is the cellular response to temperature variation. How do cells "sense" temperature changes? It has been proposed that the bacterial thermosensor DesK acts as a molecular caliper measuring membrane thickness variations that would occur

Transparency and Public Involvement in Siting a Nuclear Waste Repository in Germany

International Nuclear Information System (INIS)

Lennartz, Hans-Albert; Mussel, Christine; Nies, Alexander

2001-01-01

. In order to receive recommendations for an appropriate answer to this question regarding the German situation, the Federal Minister for the Environment has appointed 15 experts to build the 'Committee on a Selection Procedure for Disposal Sites', called the 'Committee' in this paper. The Committee's mandate is to develop a comprehensible procedure for the selection of sites for radioactive waste disposal in Germany. The procedure shall be built upon well founded criteria and derived in a sound and unprejudiced way. The Committee works independent of any ministerial or political instructions and comprises members of widely different views on nuclear energy in general and radioactive waste management in particular. Funding is available so that the Committee can initiate research projects and be supported by external expertise. It is scheduled that the Committee's final recommendations will be available by 2002. Experience in various countries has made increasingly clear during the recent years, that a scientifically sound and technically satisfying disposal concept is not sufficient in order to gain public acceptance for the selection of a disposal site. In this respect, the Committee considers public participation from the very beginning and in all phases of the selection procedure as an indispensable prerequisite. Only by transparency of the decision process, active public involvement and integration of their proposals chances increase to build up credibility of the responsible decision makers and confidence in their later decisions during the site selection procedure. The paper presents the present status of deliberations regarding public participation before site selection is actually started. It is stressed explicitly that the Committee's development process is not finalised and therefore all thoughts presented in this paper are of a preliminary nature and may be discarded in a later stage of development. In particular, the statements in this paper must not be

Transparency and Public Involvement in Siting a Nuclear Waste Repository in Germany

Energy Technology Data Exchange (ETDEWEB)

Lennartz, Hans-Albert; Mussel, Christine [WIBERA/PWIBERA/PriceWaterhouseCoopers Deutsche Rev., Hannover (Germany); Nies, Alexander [Federal Ministry for the Environment, Bonn (Germany)

2001-07-01

. In order to receive recommendations for an appropriate answer to this question regarding the German situation, the Federal Minister for the Environment has appointed 15 experts to build the 'Committee on a Selection Procedure for Disposal Sites', called the 'Committee' in this paper. The Committee's mandate is to develop a comprehensible procedure for the selection of sites for radioactive waste disposal in Germany. The procedure shall be built upon well founded criteria and derived in a sound and unprejudiced way. The Committee works independent of any ministerial or political instructions and comprises members of widely different views on nuclear energy in general and radioactive waste management in particular. Funding is available so that the Committee can initiate research projects and be supported by external expertise. It is scheduled that the Committee's final recommendations will be available by 2002. Experience in various countries has made increasingly clear during the recent years, that a scientifically sound and technically satisfying disposal concept is not sufficient in order to gain public acceptance for the selection of a disposal site. In this respect, the Committee considers public participation from the very beginning and in all phases of the selection procedure as an indispensable prerequisite. Only by transparency of the decision process, active public involvement and integration of their proposals chances increase to build up credibility of the responsible decision makers and confidence in their later decisions during the site selection procedure. The paper presents the present status of deliberations regarding public participation before site selection is actually started. It is stressed explicitly that the Committee's development process is not finalised and therefore all thoughts presented in this paper are of a preliminary nature and may be discarded in a later stage of development. In particular, the

A portable lipid bilayer system for environmental sensing with a transmembrane protein.

Directory of Open Access Journals (Sweden)

Ryuji Kawano

Full Text Available This paper describes a portable measurement system for current signals of an ion channel that is composed of a planar lipid bilayer. A stable and reproducible lipid bilayer is formed in outdoor environments by using a droplet contact method with a micropipette. Using this system, we demonstrated that the single-channel recording of a transmembrane protein (alpha-hemolysin was achieved in the field at a high-altitude (∼3623 m. This system would be broadly applicable for obtaining environmental measurements using membrane proteins as a highly sensitive sensor.

Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

DEFF Research Database (Denmark)

Rosenkilde, M M; Smit, M J; Waldhoer, M

2008-01-01

A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting...... pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we...

The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor

Directory of Open Access Journals (Sweden)

Buchmeier Michael J

2001-02-01

Full Text Available Abstract Background Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1, and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein. Results Structural models of the transmembrane glycoproteins (GP-2 of the Arenaviruses, lymphochoriomeningitis virus (LCMV and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue. Conclusions These findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.

The Role of Water Distribution Controlled by Transmembrane Potentials in the Cytochrome c-Cardiolipin Interaction: Revealing from Surface-Enhanced Infrared Absorption Spectroscopy.

Science.gov (United States)

Zeng, Li; Wu, Lie; Liu, Li; Jiang, Xiue

2017-11-02

The interaction of cytochrome c (cyt c) with cardiolipin (CL) plays a crucial role in apoptotic functions, however, the changes of the transmembrane potential in governing the protein behavior at the membrane-water interface have not been studied due to the difficulties in simultaneously monitoring the interaction and regulating the electric field. Herein, surface-enhanced infrared absorption (SEIRA) spectroelectrochemistry is employed to study the mechanism of how the transmembrane potentials control the interaction of cyt c with CL membranes by regulating the electrode potentials of an Au film. When the transmembrane potential decreases, the water content at the interface of the membranes can be increased to slow down protein adsorption through decreasing the hydrogen-bond and hydrophobic interactions, but regulates the redox behavior of CL-bound cyt c through a possible water-facilitated proton-coupled electron transfer process. Our results suggest that the potential drop-induced restructure of the CL conformation and the hydration state could modify the structure and function of CL-bound cyt c on the lipid membrane. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase.

Science.gov (United States)

Holm, Rikke; Khandelwal, Jaanki; Einholm, Anja P; Andersen, Jens P; Artigas, Pablo; Vilsen, Bente

2017-01-10

Na + ,K + -ATPase and H + ,K + -ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H + ,K + -ATPase avoids binding of Na + at the site corresponding to the Na + -specific site of the Na + ,K + -ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na + ,K + -ATPase with arginine, present in the H + ,K + -ATPase at the corresponding position, converted the normal 3Na + :2K + :1ATP stoichiometry of the Na + ,K + -ATPase to electroneutral 2Na + :2K + :1ATP stoichiometry similar to the electroneutral transport mode of the H + ,K + -ATPase. The electroneutral C932R mutant of the Na + ,K + -ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K + uptake. Only a relatively minor reduction of apparent Na + affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na + affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine + group of the M8 arginine replaces Na + at the third site, thus preventing Na + binding there, although allowing Na + to bind at the two other sites and become transported. Hence, in the H + ,K + -ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

What Can We Learn about Cholesterol's Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential?

DEFF Research Database (Denmark)

Falkovich, Stanislav G.; Martinez-Seara, Hector; Nesterenko, Alexey M.

2016-01-01

Cholesterol is abundant in the plasma membranes of animal cells and is known to regulate a variety of membrane properties. Despite decades of research, the transmembrane distribution of cholesterol is still a matter of debate. Here we consider this outstanding issue through atomistic simulations ...

Tetraspanins and Transmembrane Adaptor Proteins as Plasma Membrane Organizers – Mast Cell Case

Directory of Open Access Journals (Sweden)

Ivana eHalova

2016-05-01

Full Text Available The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs and transmembrane adaptor protein (TRAP-enriched domains. Recent biophysical, microscopic and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD9, CD53, CD63, CD81, CD151] or TRAPs [linker for activation of T cells (LAT, non-T cell activation linker (NTAL, and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

Energy Technology Data Exchange (ETDEWEB)

Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M. (University of New Mexico, Albuquerque, NM); Lopez, Gabriel P. (University of New Mexico, Albuquerque, NM); Barrick, Todd A. (University of New Mexico, Albuquerque, NM); Flores, Adrean (University of New Mexico, Albuquerque, NM)

2007-03-01

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

Transmembrane Domain Single-Nucleotide Polymorphisms Impair Expression and Transport Activity of ABC Transporter ABCG2

NARCIS (Netherlands)

Sjostedt, N.; Heuvel, J.J.M.W. van den; Koenderink, J.B.; Kidron, H.

2017-01-01

PURPOSE: To study the function and expression of nine naturally occurring single-nucleotide polymorphisms (G406R, F431L, S441N, P480L, F489L, M515R, L525R, A528T and T542A) that are predicted to reside in the transmembrane regions of the ABC transporter ABCG2. METHODS: The transport activity of the

Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding.

Science.gov (United States)

Shi, Jiye; Anderson, Dina; Lynch, Berkley A; Castaigne, Jean-Gabriel; Foerch, Patrik; Lebon, Florence

2011-10-01

LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.

Agonists and inverse agonists for the herpesvirus 8-encoded constitutively active seven-transmembrane oncogene product, ORF-74

DEFF Research Database (Denmark)

Rosenkilde, M M; Kledal, T N; Bräuner-Osborne, Hans

1999-01-01

A number of CXC chemokines competed with similar, nanomolar affinity against 125I-interleukin-8 (IL-8) binding to ORF-74, a constitutively active seven-transmembrane receptor encoded by human herpesvirus 8. However, in competition against 125I-labeled growth-related oncogene (GRO)-alpha, the ORF-74...

Different transport behaviors of NH4 (+) and NH3 in transmembrane cyclic peptide nanotubes.

Science.gov (United States)

Zhang, Mingming; Fan, Jianfen; Xu, Jian; Weng, Peipei; Lin, Huifang

2016-10-01

Two water-filled transmembrane cyclic peptide nanotubes (CPNTs) of 8×cyclo-(WL)n=4,5/POPE were chosen to investigate the dependences of the transport properties of the positive NH4 (+) and neutral NH3 on the channel radius. Molecular dynamic simulations revealed that molecular charge, size, ability to form H-bonds and channel radius all significantly influence the behaviors of NH4 (+) and NH3 in a CPNT. Higher electrostatic interactions, more H-bonds, and water-bridges were found in the NH4 (+) system, resulting in NH4 (+) meeting higher energy barriers, while NH3 can enter, exit and permeate the channels effortlessly. This work sheds a first light on the differences between the mechanisms of NH4 (+) and NH3 moving in a CPNT at an atomic level. Graphical Abstract Snapshot of the simulation system of NH4 (+)_octa-CPNT with an NH4 (+) initially positioned at one mouth of the tube, PMF profiles for single NH4 (+) ion and NH3 molecule moving through water-filled transmembrane CPNTs of 8×cyclo-(WL)n=4,5/POPE and sketch graphs of the possible H-bond forms of NH3 and NH4 (+) with the neighboring water.

Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

Science.gov (United States)

Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

2017-11-09

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

Induction of IL-1 during hemodialysis: Transmembrane passage of intact endotoxins (LPS)

Energy Technology Data Exchange (ETDEWEB)

Laude-Sharp, M.; Caroff, M.; Simard, L.; Pusineri, C.; Kazatchkine, M.D.; Haeffner-Cavaillon, N. (INSERM U 28, Hopital Broussais, Paris (France))

1990-12-01

Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes.

Induction of IL-1 during hemodialysis: Transmembrane passage of intact endotoxins (LPS)

International Nuclear Information System (INIS)

Laude-Sharp, M.; Caroff, M.; Simard, L.; Pusineri, C.; Kazatchkine, M.D.; Haeffner-Cavaillon, N.

1990-01-01

Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes

Stability of transmembrane amyloid β-peptide and membrane integrity tested by molecular modeling of site-specific Aβ42 mutations.

Directory of Open Access Journals (Sweden)

Chetan Poojari

Full Text Available Interactions of the amyloid β-protein (Aβ with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer's disease. Natural mutations in Aβ42, such as the Arctic mutation (E22G have been shown to increase Aβ42 aggregation and neurotoxicity, leading to the early-onset of Alzheimer's disease. A correlation between the propensity of Aβ42 to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ42 peptide it was further revealed that the aggregation of different Aβ42 mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ42 mutation and peptide-membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ42 variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ42 stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ42 peptide and a decreased stability for D23G compared to wild-type Aβ42, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.

Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients.

Science.gov (United States)

Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

2009-09-01

A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium-sensing receptor, the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3, which are sensing L-alpha-amino acids, the carbohydrate-sensing T1R2/T1R3 receptor, the proteolytic degradation product sensor GPR93 (also termed GPR92), and the free fatty acid (FFA) sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the individual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential drug targets, to treat, for example, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance.

Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors

Czech Academy of Sciences Publication Activity Database

Jindřichová, Marie; Vávra, Vojtěch; Obšil, Tomáš; Stojilkovic, S. S.; Zemková, Hana

2009-01-01

Roč. 109, č. 3 (2009), s. 923-934 ISSN 0022-3042 R&D Projects: GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA5011408; GA AV ČR(CZ) IAA500110702; GA AV ČR(CZ) IAA500110910 Institutional research plan: CEZ:AV0Z50110509 Keywords : purinergic receptors * gating * transmembrane domain Subject RIV: FH - Neuro logy Impact factor: 3.999, year: 2009

Differential expression of a novel seven transmembrane domain protein in epididymal fat from aged and diabetic mice.

Science.gov (United States)

Yang, H; Egan, J M; Rodgers, B D; Bernier, M; Montrose-Rafizadeh, C

1999-06-01

To identify novel seven transmembrane domain proteins from 3T3-L1 adipocytes, we used PCR to amplify 3T3-L1 adipocyte complementary DNA (cDNA) with primers homologous to the N- and C-termini of pancreatic glucagon-like peptide-1 (GLP-1) receptor. We screened a cDNA library prepared from fully differentiated 3T3-L1 adipocytes using a 500-bp cDNA PCR product probe. Herein describes the isolation and characterization of a 1.6-kb cDNA clone that encodes a novel 298-amino acid protein that we termed TPRA40 (transmembrane domain protein of 40 kDa regulated in adipocytes). TPRA40 has seven putative transmembrane domains and shows little homology with the known GLP-1 receptor or with other G protein-coupled receptors. The levels of TPRA40 mRNA and protein were higher in 3T3-L1 adipocytes than in 3T3-L1 fibroblasts. TPRA40 is present in a number of mouse and human tissues. Interestingly, TPRA40 mRNA levels were significantly increased by 2- to 3-fold in epididymal fat of 24-month-old mice vs. young controls as well as in db/db and ob/ob mice vs. nondiabetic control littermates. No difference in TPRA40 mRNA levels was observed in brain, heart, skeletal muscle, liver, or kidney. Furthermore, no difference in TPRA40 expression was detected in brown fat of ob/ob mice when compared with age-matched controls. Taken together, these data suggest that TPRA40 represents a novel membrane-associated protein whose expression in white adipose tissue is altered with aging and type 2 diabetes.

Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

International Nuclear Information System (INIS)

Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W.

2007-01-01

P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 123.27, c = 134.43 Å

Transmembrane molecular transport during versus after extremely large, nanosecond electric pulses.

Science.gov (United States)

Smith, Kyle C; Weaver, James C

2011-08-19

Recently there has been intense and growing interest in the non-thermal biological effects of nanosecond electric pulses, particularly apoptosis induction. These effects have been hypothesized to result from the widespread creation of small, lipidic pores in the plasma and organelle membranes of cells (supra-electroporation) and, more specifically, ionic and molecular transport through these pores. Here we show that transport occurs overwhelmingly after pulsing. First, we show that the electrical drift distance for typical charged solutes during nanosecond pulses (up to 100 ns), even those with very large magnitudes (up to 10 MV/m), ranges from only a fraction of the membrane thickness (5 nm) to several membrane thicknesses. This is much smaller than the diameter of a typical cell (∼16 μm), which implies that molecular drift transport during nanosecond pulses is necessarily minimal. This implication is not dependent on assumptions about pore density or the molecular flux through pores. Second, we show that molecular transport resulting from post-pulse diffusion through minimum-size pores is orders of magnitude larger than electrical drift-driven transport during nanosecond pulses. While field-assisted charge entry and the magnitude of flux favor transport during nanosecond pulses, these effects are too small to overcome the orders of magnitude more time available for post-pulse transport. Therefore, the basic conclusion that essentially all transmembrane molecular transport occurs post-pulse holds across the plausible range of relevant parameters. Our analysis shows that a primary direct consequence of nanosecond electric pulses is the creation (or maintenance) of large populations of small pores in cell membranes that govern post-pulse transmembrane transport of small ions and molecules. Copyright © 2011 Elsevier Inc. All rights reserved.

Summary of Epidemiology Studies or Activities Involving Workers at the Savannah River Site or the Surrounding Public: An Update

Energy Technology Data Exchange (ETDEWEB)

Brown, K.T.

2002-10-18

There have been numerous health studies or related activities over time that have involved workers at the Savannah River Site (SRS) or the surrounding public. While most of these epidemiology studies or activities have been performed by external agencies, it has proved useful to provide interested parties an overall summary of such activities. The first such summary was provided in an October 1998 report. The 1998 summary was updated in a February 2000 report. This report provides an update on the status or findings of epidemiology studies or activities involving SRS workers or the surrounding public, as an update to the previous summaries.

Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding.

Science.gov (United States)

Rosenberg, Mark F; Kamis, Alhaji Bukar; Callaghan, Richard; Higgins, Christopher F; Ford, Robert C

2003-03-07

P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.

[Application of Brownian dynamics to the description of transmembrane ion flow as exemplified by the chloride channel of glycine receptor].

Science.gov (United States)

Boronovskiĭ, S E; Nartsissov, Ia R

2009-01-01

Using the Brownian dynamics of the movement of hydrated ion in a viscous water solution, a mathematical model has been built, which describes the transport of charged particles through a single protein pore in a lipid membrane. The dependences of transmembrane ion currents on ion concentrations in solution have been obtained. It was shown that, if the geometry of a membrane pore is identical to that of the inner part of the glycine receptor channel and there is no ion selectivity, then the values of both chloride and sodium currents are not greater than 0.5 pA at the physiological concentrations of these ions. If local charge heterogeneity caused by charged amino acid residues of transmembrane protein segments is included into the model calculations, the chloride current increases to about 3.7 pA, which exceeds more than seven times the value for sodium ions under the conditions of the complex channel geometry in the range of physiological concentrations of ions in the solution. The model takes changes in the density of charge distribution both inside the channel and near the protein surface into account. The alteration of pore geometry can be also considered as a parameter at the researcher's option. Thus, the model appears as an effective tool for the description of transmembrane currents for other types of membrane channels.

Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

DEFF Research Database (Denmark)

Benned-Jensen, Tau; Smethurst, Christopher; Holst, Peter Johannes

2011-01-01

The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we...... with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases...

Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A

International Nuclear Information System (INIS)

Fisher, K.A.

1982-01-01

The technique of monolayer freeze-fracture autoradiography (MONOFARG) has been developed and the principles, quantitation, and application of the method are described. Cell monolayers attached to polylysine-treated glass were freeze-fractured, shadowed, and coated with dry, Parlodion-supported Ilford L4 photographic emulsion at room temperature. Quantitative aspects of MONOFARG were examined using radioiodinated test systems. Background was routinely -4 grains/μm 2 /day, the highest overall efficiency was between 25% and 45%, and grain density and efficiency were dependent on radiation dose for iodine-125 and D-19 development. Corrected grain densities were linearly proportional to iodine-125 concentration. The method was applied to an examination of the transmembrane distribution of radioiodinated and fluoresceinated concanavalin A ( 125 I-FITC-Con-A). Human erythrocytes were labeled, column-purified, freeze-dried or freeze-fractured, autoradiographed, and examined by electron microscopy. The number of silver grains per square micrometer of unsplit single membrane was essentially identical to that of split extracellular membrane halves. These data demonstrate that 125 I-FITC-Con-A partitions exclusively with the extracellular half of the membrane upon freeze-fracturing and can be used as a quantitative marker for the fraction of extracellular split membrane halves. This method should be able to provide new information about certain transmembrane properties of biological membrane molecules and probes, as well as about the process of freeze-fracture per se

Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

Energy Technology Data Exchange (ETDEWEB)

Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W., E-mail: n.isaacs@chem.gla.ac.uk [Department of Chemistry and WestChem, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

2007-07-01

P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6{sub 1}22, with unit-cell parameters a = b = 123.27, c = 134.43 Å.

Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella.

Science.gov (United States)

Steinbach, Denise; Gutbrod, Oliver; Lümmen, Peter; Matthiesen, Svend; Schorn, Corinna; Nauen, Ralf

2015-08-01

Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [(3)H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4-S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where

Full-length cellular β-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization.

Science.gov (United States)

Liebsch, Filip; Aurousseau, Mark R P; Bethge, Tobias; McGuire, Hugo; Scolari, Silvia; Herrmann, Andreas; Blunck, Rikard; Bowie, Derek; Multhaup, Gerd

2017-08-11

The β-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala 463 and Cys 466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Deorphanizing the human transmembrane genome: A landscape of uncharacterized membrane proteins.

Science.gov (United States)

Babcock, Joseph J; Li, Min

2014-01-01

The sequencing of the human genome has fueled the last decade of work to functionally characterize genome content. An important subset of genes encodes membrane proteins, which are the targets of many drugs. They reside in lipid bilayers, restricting their endogenous activity to a relatively specialized biochemical environment. Without a reference phenotype, the application of systematic screens to profile candidate membrane proteins is not immediately possible. Bioinformatics has begun to show its effectiveness in focusing the functional characterization of orphan proteins of a particular functional class, such as channels or receptors. Here we discuss integration of experimental and bioinformatics approaches for characterizing the orphan membrane proteome. By analyzing the human genome, a landscape reference for the human transmembrane genome is provided.

Elastic strain and twist analysis of protein structural data and allostery of the transmembrane channel KcsA

Science.gov (United States)

Mitchell, Michael R.; Leibler, Stanislas

2018-05-01

The abundance of available static protein structural data makes the more effective analysis and interpretation of this data a valuable tool to supplement the experimental study of protein mechanics. Structural displacements can be difficult to analyze and interpret. Previously, we showed that strains provide a more natural and interpretable representation of protein deformations, revealing mechanical coupling between spatially distinct sites of allosteric proteins. Here, we demonstrate that other transformations of displacements yield additional insights. We calculate the divergence and curl of deformations of the transmembrane channel KcsA. Additionally, we introduce quantities analogous to bend, splay, and twist deformation energies of nematic liquid crystals. These transformations enable the decomposition of displacements into different modes of deformation, helping to characterize the type of deformation a protein undergoes. We apply these calculations to study the filter and gating regions of KcsA. We observe a continuous path of rotational deformations physically coupling these two regions, and, we propose, underlying the allosteric interaction between these regions. Bend, splay, and twist distinguish KcsA gate opening, filter opening, and filter-gate coupling, respectively. In general, physically meaningful representations of deformations (like strain, curl, bend, splay, and twist) can make testable predictions and yield insights into protein mechanics, augmenting experimental methods and more fully exploiting available structural data.

The regional issues involved in the siting of power stations

International Nuclear Information System (INIS)

Livingstone, R.

1980-01-01

This paper deals with the regional implications of power station siting in England and Wales and refers to the procedures used by the CEGB to find and develop sites. The resources required for a power station are outlined both for conventional and nuclear stations and the possible development of uses for the rejected heat from power stations as a result of the energy crisis is discussed. (U.K.)

Importance of Leu99 in transmembrane segment M1 of the Na+, K+ -ATPase in the binding and occlusion of K+

DEFF Research Database (Denmark)

Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente

2007-01-01

Twenty-six point mutations were introduced into the N-terminal and middle parts of transmembrane segment M1 of the Na+, K+ -ATPase and its cytosolic extension. None of the alterations to charged and polar residues in the N-terminal part of M1 and its cytosolic extension had any major effect...... on the cation binding properties, thus rejecting the hypothesis that these residues are involved in cation selectivity. By contrast, specific residues in the middle part of M1, particularly Leu(99), were found critical to K+ interaction of the enzyme. Hence, mutation L99A reduced the affinity for K+ activation...... of E2P dephosphorylation 17-fold, and L99F reduced the equilibrium level of the K+-occluded intermediate [K2]E2 and increased the rate of K+ deocclusion 39-fold, i.e. more than seen for mutation E329Q of the cation-binding glutamate in M4. L99Q affected K+ interaction in yet another way...

Defining the transmembrane helix of M2 protein from influenza A by molecular dynamics simulations in a lipid bilayer

NARCIS (Netherlands)

Forrest, LR; Tieleman, DP; Sansom, MSP

Integral membrane proteins containing at least one transmembrane (TM) alpha-helix are believed to account for between 20% and 30% of most genomes. There are several algorithms that accurately predict the number and position of TM helices within,a membrane protein sequence. However, these methods

PheVI:09 (Phe6.44) as a sliding microswitch in seven-transmembrane (7TM) G protein-coupled receptor activation

DEFF Research Database (Denmark)

Valentin-Hansen, Louise; Holst, Birgitte; Frimurer, Thomas M

2012-01-01

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational......-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09...... an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition....

Synthesis and characterization of 18F-labeled active site inhibited factor VII (ASIS)

DEFF Research Database (Denmark)

Erlandsson, Maria; Nielsen, Carsten Haagen; Jeppesen, Troels Elmer

2015-01-01

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example......, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an 18F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[18F]fluorobenzoate, and the [18F]ASIS was purified on a PD-10 desalting...... column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [18F]ASIS to TF and to a specific anti-factor VII...

Identification and functional comparison of seven-transmembrane G-protein-coupled BILF1 receptors in recently discovered nonhuman primate lymphocryptoviruses

DEFF Research Database (Denmark)

Spiess, Katja; Fares, Suzan; Sparre-Ulrich, Alexander H

2015-01-01

Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immuno...

Pattern of extranodal involvement in non hodgkin's lymphoma

International Nuclear Information System (INIS)

Bangash, M.H.; Hussain, I.; Zakaria, M.; Piracha, M.N.

2014-01-01

To study the anatomical and histomorphological pattern of extranodal non Hodgkins lymphoma (NHL). Study Design: Descriptive study Place and Duration of Study: The study was carried out at Oncology department Combined Military Hospital Rawalpindi during July 2012 to April 2013. Materials and Methods: All newly diagnosed patients of NHL with extranodal involvement were included in the study. They were categorized as primary extranodal or secondary extranodal lymphomas. Histological pattern and site of involvement were studied. Results: The male to female ratio was 2.2:1 (Male 31, Female 14), and the mean age was 48.16 years (SD=13.40). Primary extranodal involvement was seen in 55.6% of patients. Secondary extranodal involvement was seen in 44.4% of patients. Diffuse large B-cell lymphoma (DLBCL) was the most common type of NHL observed in both primary and secondary extranodal involvement. Gastrointestinal tract was the most common site for primary extranodal involvement and bone marrow was the most common site for secondary extranodal involvement. Conclusion: High frequency of primary extranodal involvement was noted in our study. DLBCL was the most common morphological type observed. Gastrointestinal tract and secondary bone marrow involvement were the most common anatomical sites for primary and secondary extranodal involvement respectively. (author)

Molecular insights into the m-AAA protease-mediated dislocation of transmembrane helices in the mitochondrial inner membrane.

Science.gov (United States)

Lee, Seoeun; Lee, Hunsang; Yoo, Suji; Kim, Hyun

2017-12-08

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m -AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m -AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m -AAA protease subunit. Furthermore, m -AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m -AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m -AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Exploiting hydrophobicity for efficient production of transmembrane helices for structure determination by NMR spectroscopy

DEFF Research Database (Denmark)

Bugge, Katrine Østergaard; Steinocher, Helena; Brooks, Andrew J.

2015-01-01

-labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations...... of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement and has the potential to facilitate an increase in the number of structures...

Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function.

Science.gov (United States)

Therien, J P Daniel; Baenziger, John E

2017-03-27

Although transmembrane helix-helix interactions must be strong enough to drive folding, they must still permit the inter-helix movements associated with conformational change. Interactions between the outermost M4 and adjacent M1 and M3 α-helices of pentameric ligand-gated ion channels have been implicated in folding and function. Here, we evaluate the role of different physical interactions at this interface in the function of two prokaryotic homologs, GLIC and ELIC. Strikingly, disruption of most interactions in GLIC lead to either a reduction or a complete loss of expression and/or function, while analogous disruptions in ELIC often lead to gains in function. Structural comparisons suggest that GLIC and ELIC represent distinct transmembrane domain archetypes. One archetype, exemplified by GLIC, the glycine and GABA receptors and the glutamate activated chloride channel, has extensive aromatic contacts that govern M4-M1/M3 interactions and that are essential for expression and function. The other archetype, exemplified by ELIC and both the nicotinic acetylcholine and serotonin receptors, has relatively few aromatic contacts that are detrimental to function. These archetypes likely have evolved different mechanisms to balance the need for strong M4 "binding" to M1/M3 to promote folding/expression, and the need for weaker interactions that allow for greater conformational flexibility.

Impact of biofilm accumulation on transmembrane and feed channel pressure drop: Effects of crossflow velocity, feed spacer and biodegradable nutrient

KAUST Repository

Dreszer, C.; Flemming, H. C.; Zwijnenburg, A.; Kruithof, J. C.; Vrouwenvelder, Johannes S.

2014-01-01

. As biodegradable nutrient, acetate was dosed to the feed water (1.0 and 0.25mgL-1 carbon) to enhance biofilm accumulation in the monitors. The studies showed that biofilm formation caused an increased transmembrane resistance and feed channel pressure drop

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER

Directory of Open Access Journals (Sweden)

Haga Christopher L

2007-09-01

Full Text Available Abstract Background In mouse the cytokine interleukin-7 (IL-7 is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER. The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR, a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules.

Transmembrane prostatic acid phosphatase (TMPAP interacts with snapin and deficient mice develop prostate adenocarcinoma.

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Ileana B Quintero

Full Text Available The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP, a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP and a transmembrane type-I (TMPAP. The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/- with C57BL/6J background. The PAP(-/- mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

Transmembrane Prostatic Acid Phosphatase (TMPAP) Interacts with Snapin and Deficient Mice Develop Prostate Adenocarcinoma

Science.gov (United States)

Quintero, Ileana B.; Herrala, Annakaisa M.; Araujo, César L.; Pulkka, Anitta E.; Hautaniemi, Sampsa; Ovaska, Kristian; Pryazhnikov, Evgeny; Kulesskiy, Evgeny; Ruuth, Maija K.; Soini, Ylermi; Sormunen, Raija T.; Khirug, Leonard; Vihko, Pirkko T.

2013-01-01

The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP−/−) with C57BL/6J background. The PAP−/− mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma. PMID:24039861

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

Directory of Open Access Journals (Sweden)

Zhenyong Wu

2017-10-01

Full Text Available Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs. Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores

Public involvement: the critical path in siting controversial facilities. Proceedings of the Nuclear Energy Low-Level Waste Mangement Program conference

International Nuclear Information System (INIS)

1986-01-01

The purpose of the conference was to: exchange information among those responsible for, or interested in, the development of new low-level waste disposal facilities; acquaint participants with past experiences of states and organizations in enfranchising the public in the siting of controversial facilities; and discuss various mechanisms and techniques for effectively involving the public in decision-making processes. The conference addressed four major topics: lessons from past experiences; mechanisms and techniques for public involvement, conflict resolution, and working constructively with the media. A series of presentations on each topic was followed by questions and discussion among presenters and conference participants. Several key points emerged as the conference proceeded

TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

Directory of Open Access Journals (Sweden)

Kwang-Hyung Kim

2009-11-01

Full Text Available The regulation of intracellular levels of reactive oxygen species (ROS is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we

Identification of eight novel mutations in a collaborative analysis of a part of the second transmembrane domain of the CFTR gene

Energy Technology Data Exchange (ETDEWEB)

Mercier, B.; Audrezet, M.P.; Guillermit, H.; Quere, I.; Verlingue, C.; Ferec, C. (CDTS, Brest (France)); Lissens, W.; Bonduelle, M.; Liebaers, I. (University Hospital VUB, Brussels (Belgium)); Novelli, G.; Sangiuolo, F.; Dallapiccola, B. (IRCCS, Rotondo (Italy)); Kalaydjieva, L. (Inst. of Obstetrics, Sofia (Bulgaria)); Arce, M. De; Cashman, S. (Trinity College, Dublin (Ireland)); Kapranov, N. (NRC of medical Genetics, Moscow (Russian Federation)); Canki Klain, N. (Tozd Univerzitetna Ginekoloska Klinika, Ljubljana (Yugoslavia)); Lenoir, G. (Hopital des Enfants Malades Necker, Paris (France)); Chauveau, P. (Centre Hospitalier General, Le Havre (France)); Lanaerts, C. (Centre Hospitalier Regional et Universitaire, Amiens (France)); Rault, G. (Centre Helio-Marin, Roscoff (France))

1993-04-01

Cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible, when mutated, for cystic fibrosis (CF), spans over 230 kb on the long arm of chromosome 7 and is composed of 27 exons. The most common mutation responsible for CF worldwide is the deletion of a phenylalanine amino acid at codon 508 in the first nucleotide-binding fold and accounts for approximately 70% of CF chromosomes studied. More than 250 other mutations have been reported through the CF Genetic Analysis Consortium. The majority of the mutations previously described lie in the two nucleotide-binding folds. To explore exhaustively other regions of the gene, particularly exons coding for transmembrane domains, the authors have initiated a collaborative study between different laboratories to screen 369 non-[Delta]F508 CF chromosomes of seven ethnic European populations (Belgian, French, Breton, Irish, Italian, Yugoslavian, Russian). Among these chromosomes carrying an unidentified mutation, 63 were from Brittany, 50 of various French origin, 45 of Irish origin, 56 of Italian origin, 41 of Belgian origin, 2 of Turkish origin, 38 of Yugoslavian origin, 22 of Russian origin, and 52 of Bulgarian origin. Diagnostic criteria for CF included at least one positive sweat test and pulmonary disease with or without pancreatic disease. Using a denaturing gradient gel electrophoresis (DGGE) assay, they have identified eight novel mutations in exon 17b coding for part of the second transmembrane domain of the CFTR and they describe them in this report. 8 refs., 1 fig., 1 tab.

Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

Science.gov (United States)

Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

2018-05-01

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

Incorporation of transmembrane hydroxide transport into the chemiosmotic theory.

Science.gov (United States)

de Grey, A D

1999-10-01

A cornerstone of textbook bioenergetics is that oxidative ATP synthesis in mitochondria requires, in normal conditions of internal and external pH, a potential difference (delta psi) of well over 100 mV between the aqueous compartments that the energy-transducing membrane separates. Measurements of delta psi inferred from diffusion of membrane-permeant ions confirm this, but those using microelectrodes consistently find no such delta psi--a result ostensibly irreconcilable with the chemiosmotic theory. Transmembrane hydroxide transport necessarily accompanies mitochondrial ATP synthesis, due to the action of several carrier proteins; this nullifies some of the proton transport by the respiratory chain. Here, it is proposed that these carriers' structure causes the path of this "lost" proton flow to include a component perpendicular to the membrane but within the aqueous phases, so maintaining a steady-state proton-motive force between the water at each membrane surface and in the adjacent bulk medium. The conflicting measurements of delta psi are shown to be consistent with the response of this system to its chemical environment.

Transmembrane peptides as sensors of the membrane physical state

Science.gov (United States)

Piotto, Stefano; Di Biasi, Luigi; Sessa, Lucia; Concilio, Simona

2018-05-01

Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state suggested a new approach for the discovery of antimicrobial peptides.

A monocyte chemotaxis inhibiting factor in serum of HIV infected men shares epitopes with the HIV transmembrane protein gp41

NARCIS (Netherlands)

Tas, M.; Drexhage, H. A.; Goudsmit, J.

1988-01-01

This report describes that gp41, the transmembranous envelope protein of HIV, is able to inhibit monocyte chemotaxis (measured as FMLP-induced polarization). To study the presence of such immunosuppressive HIV env proteins in the circulation of HIV-infected men, fractions were prepared from serum

Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74

DEFF Research Database (Denmark)

Rosenkilde, M M; Kledal, T N; Holst, Peter Johannes

2000-01-01

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice...

Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

Energy Technology Data Exchange (ETDEWEB)

Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

2010-07-19

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

Aspartic acid 397 in subunit B of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae forms part of a sodium-binding site, is involved in cation selectivity, and affects cation-binding site cooperativity.

Science.gov (United States)

Shea, Michael E; Juárez, Oscar; Cho, Jonathan; Barquera, Blanca

2013-10-25

The Na(+)-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC).

Coarse Grained Molecular Dynamics Simulations of Transmembrane Protein-Lipid Systems

Directory of Open Access Journals (Sweden)

Peter Spijker

2010-06-01

Full Text Available Many biological cellular processes occur at the micro- or millisecond time scale. With traditional all-atom molecular modeling techniques it is difficult to investigate the dynamics of long time scales or large systems, such as protein aggregation or activation. Coarse graining (CG can be used to reduce the number of degrees of freedom in such a system, and reduce the computational complexity. In this paper the first version of a coarse grained model for transmembrane proteins is presented. This model differs from other coarse grained protein models due to the introduction of a novel angle potential as well as a hydrogen bonding potential. These new potentials are used to stabilize the backbone. The model has been validated by investigating the adaptation of the hydrophobic mismatch induced by the insertion of WALP-peptides into a lipid membrane, showing that the first step in the adaptation is an increase in the membrane thickness, followed by a tilting of the peptide.

Potential Involvement of Draxin in the Axonal Projection of Cranial Nerves, Especially Cranial Nerve X, in the Chick Hindbrain.

Science.gov (United States)

Zhang, Sanbing; Cui, Huixian; Wang, Lei; Kang, Lin; Huang, Guannan; Du, Juan; Li, Sha; Tanaka, Hideaki; Su, Yuhong

2016-07-01

The appropriate projection of axons within the nervous system is a crucial component of the establishment of neural circuitry. Draxin is a repulsive axon guidance protein. Draxin has important functions in the guidance of three commissures in the central nervous system and in the migration of neural crest cells and dI3 interneurons in the chick spinal cord. Here, we report that the distribution of the draxin protein and the location of 23C10-positive areas have a strong temporal and spatial correlation. The overexpression of draxin, especially transmembrane draxin, caused 23C10-positive axon bundles to misproject in the dorsal hindbrain. In addition, the overexpression of transmembrane draxin caused abnormal formation of the ganglion crest of the IX and X cranial nerves, misprojection of some anti-human natural killer-1 (HNK-1)-stained structures in the dorsal roof of the hindbrain, and a simultaneous reduction in the efferent nerves of some motoneuron axons inside the hindbrain. Our data reveal that draxin might be involved in the fascicular projection of cranial nerves in the hindbrain. © 2016 The Histochemical Society.

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

DEFF Research Database (Denmark)

Thiele, Stefanie; Steen, Anne; Jensen, Pia C

2011-01-01

-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5...... preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units....

Transition from site selection to site confirmation phase: New challenge to Posiva's communication and public involvement

International Nuclear Information System (INIS)

Seppaelae, Timo

2001-01-01

In Finland, Posiva Oy is responsible for the final disposal of spent nuclear fuel. In the 1990's investigations have been carried out on four intended sites for final disposal. Posiva implemented Environmental Impact Assessment (EIA) on the investigation sites in 1997-1999. The results of the EIA procedure were published in the EIA report which was attached to the application filed to the Government in May 1999 for a policy decision on the construction of the final disposal facility in Olkiluoto in the municipality of Eurajoki. In December 2000, the Finnish Government made a favourable policy decision ( D ecision in Principle ) on the construction of final disposal facility for spent nuclear fuel in Olkiluoto. According to the decision, the facility is in line with the overall good of the society. According to the Government's decision the prerequisites of the policy decision had been met. The municipality of Eurajoki supported the construction of the facility in Olkiluoto and STUK, the Radiation and Nuclear Safety Authority, also approved of the advancement of the project. The positive policy decision of the Government will still need to be ratified by the Parliament. The parliamentary proceedings of the policy decision will probably start in February 2001. The policy decision will make it possible for Posiva to concentrate the future site confirmation studies in Olkiluoto, Eurajoki, and construct an underground research facility there. The excavation of the research facility in the bedrock is planned to start in a few years' time. The construction of the final disposal facility is scheduled to start after the year 2010. The transition from the site selection to the site confirmation phase in Eurajoki means a new challenge not only to Posiva's research work but also to Posiva's communication. In addition to meeting the safety criteria, Posiva's activities shall in the future also meet all the criteria specified by the surrounding society

Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR).

Science.gov (United States)

Rosenberg, Mark F; Kamis, Alhaji Bukar; Aleksandrov, Luba A; Ford, Robert C; Riordan, John R

2004-09-10

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered.

Simulations of Skin Barrier Function: Free Energies of Hydrophobic and Hydrophilic Transmembrane Pores in Ceramide Bilayers

OpenAIRE

Notman, Rebecca; Anwar, Jamshed; Briels, W. J.; Noro, Massimo G.; den Otter, Wouter K.

2008-01-01

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel pha...

Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor.

Science.gov (United States)

Haas, Ann-Karin; Kleinau, Gunnar; Hoyer, Inna; Neumann, Susanne; Furkert, Jens; Rutz, Claudia; Schülein, Ralf; Gershengorn, Marvin C; Krause, Gerd

2011-01-01

The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.

Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System.

Science.gov (United States)

Chojnacka, Magdalena; Gornicka, Agnieszka; Oeljeklaus, Silke; Warscheid, Bettina; Chacinska, Agnieszka

2015-06-12

The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Requirement of transmembrane domain for CD154 association to lipid rafts and subsequent biological events.

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Nadir Benslimane

Full Text Available Interaction of CD40 with CD154 leads to recruitment of both molecules into lipid rafts, resulting in bi-directional cell activation. The precise mechanism by which CD154 is translocated into lipid rafts and its impact on CD154 signaling remain largely unknown. Our aim is to identify the domain of CD154 facilitating its association to lipid rafts and the impact of such association on signaling events and cytokine production. Thus, we generated Jurkat cell lines expressing truncated CD154 lacking the cytoplasmic domain or chimeric CD154 in which the transmembrane domain was replaced by that of transferrin receptor I, known to be excluded from lipid rafts. Our results show that cell stimulation with soluble CD40 leads to the association of CD154 wild-type and CD154-truncated, but not CD154-chimera, with lipid rafts. This is correlated with failure of CD154-chimera to activate Akt and p38 MAP kinases, known effectors of CD154 signaling. We also found that CD154-chimera lost the ability to promote IL-2 production upon T cell stimulation with anti-CD3/CD28 and soluble CD40. These results demonstrate the implication of the transmembrane domain of CD154 in lipid raft association, and that this association is necessary for CD154-mediated Akt and p38 activation with consequent enhancement of IL-2 production.

Dynamic monitoring of transmembrane potential changes: a study of ion channels using an electrical double layer-gated FET biosensor.

Science.gov (United States)

Pulikkathodi, Anil Kumar; Sarangadharan, Indu; Chen, Yi-Hong; Lee, Geng-Yen; Chyi, Jen-Inn; Lee, Gwo-Bin; Wang, Yu-Lin

2018-03-27

In this research, we have designed, fabricated and characterized an electrical double layer (EDL)-gated AlGaN/GaN high electron mobility transistor (HEMT) biosensor array to study the transmembrane potential changes of cells. The sensor array platform is designed to detect and count circulating tumor cells (CTCs) of colorectal cancer (CRC) and investigate cellular bioelectric signals. Using the EDL FET biosensor platform, cellular responses can be studied in physiological salt concentrations, thereby eliminating complex automation. Upon investigation, we discovered that our sensor response follows the transmembrane potential changes of captured cells. Our whole cell sensor platform can be used to monitor the dynamic changes in the membrane potential of cells. The effects of continuously changing electrolyte ion concentrations and ion channel blocking using cadmium are investigated. This methodology has the potential to be used as an electrophysiological probe for studying ion channel gating and the interaction of biomolecules in cells. The sensor can also be a point-of-care diagnostic tool for rapid screening of diseases.

Social Network Sites, Friends, and Celebrities: The Roles of Social Comparison and Celebrity Involvement in Adolescents’ Body Image Dissatisfaction

Directory of Open Access Journals (Sweden)

Shirley S. Ho

2016-08-01

Full Text Available This study applies the social comparison theory to examine the effects of adolescents’ engagement in comparison with friends and celebrities on social network sites (SNSs on (a their body image dissatisfaction (BID and (b their drive to be thin (DT or muscular (DM. The study also examines celebrity involvement as an antecedent of the outcome variables. Data were collected through a survey of 1,059 adolescents in Singapore. Regression analyses indicate that SNSs use was related to adolescents’ BID. Specifically, social comparison with friends on SNSs was significantly associated with adolescents’ BID, DT, and DM. Gender differences were also observed—social comparison with celebrities was significantly associated with BID and DT among female adolescents. Celebrity involvement was significantly associated with male BID. Theoretical and practical implications were discussed.

The role of public involvement in managing political conflict: A comparative case study of public involvement activities in siting low-level radioactive waste disposal facilities

International Nuclear Information System (INIS)

Tzoumis, K.A.

1992-01-01

A model of political conflict based on the components of scope, intensity and visibility is used to rank the degree of conflict for states hosting a low-level radioactive waste facility in the US. Data were gathered from sixty-five telephone interviews and comparisons made with ten national experts. Public involvement and compensation packages (reviewed in Chapter IV) to be potential conflict management tools for practitioners. The role of the governor and the complexity of the siting process are two additional factors that are important in escalating a conflict. Technical factors of complexity and source of the waste along with the past performance of the company handling the facility have no link to conflict. Conflict escalation is proposed as a means towards understanding the agenda-building process

Proposed training program for construction personnel involved in remedial action work at sites contaminated by naturally occurring radionuclides

International Nuclear Information System (INIS)

Berven, B.A.; Goldsmith, W.A.; Haywood, F.F.; Schiager, K.J.

1979-01-01

Many sites used during the early days of the US atomic energy program are contaminated with radionuclides of the primordial decay chains (uranium, thorium, and actinium series). This contamination consists of residues resulting from refining and processing uranium and thorium. Preparation of these sites for release to unrestricted private use will involve the assistance of construction workers, many of whom have limited knowledge of the hazards associated with radioactive materials. Therefore, there is a need to educate these workers in the fundamentals of radioactive material handling to minimize exposures and possible spread of contamination. This training should disseminate relevant information at an appropriate educational level and should instill a cautious, common-sense attitude toward the handling of radioactive materials. The training should emphasize basic information concerning environmental radiation within a context of relative risk. A multi-media format, including colorful visual aids, demonstration, and discussion, should be used to maximize motivation and retention. A detailed, proposed training program design is presented

Metal transport across biomembranes: emerging models for a distinct chemistry.

Science.gov (United States)

Argüello, José M; Raimunda, Daniel; González-Guerrero, Manuel

2012-04-20

Transition metals are essential components of important biomolecules, and their homeostasis is central to many life processes. Transmembrane transporters are key elements controlling the distribution of metals in various compartments. However, due to their chemical properties, transition elements require transporters with different structural-functional characteristics from those of alkali and alkali earth ions. Emerging structural information and functional studies have revealed distinctive features of metal transport. Among these are the relevance of multifaceted events involving metal transfer among participating proteins, the importance of coordination geometry at transmembrane transport sites, and the presence of the largely irreversible steps associated with vectorial transport. Here, we discuss how these characteristics shape novel transition metal ion transport models.

Ligand-Receptor Interaction-Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics.

Science.gov (United States)

Liang, Junshi; Chen, Pengyu; Dong, Bojun; Huang, Zihan; Zhao, Kongyin; Yan, Li-Tang

2016-05-09

Nearly all nanomedical applications of dendrimer-like soft nanoparticles rely on the functionality of attached ligands. Understanding how the ligands interact with the receptors in cell membrane and its further effect on the cellular uptake of dendrimer-like soft nanoparticles is thereby a key issue for their better application in nanomedicine. However, the essential mechanism and detailed kinetics for the ligand-receptor interaction-mediated transmembrane transport of such unconventional nanoparticles remain poorly elucidated. Here, using coarse-grained simulations, we present the very first study of molecular mechanism and kinetics behaviors for the transmembrane transport of dendrimer-like soft nanoparticles conjugated with ligands. A phase diagram of interaction states is constructed through examining ligand densities and membrane tensions that allows us to identify novel endocytosis mechanisms featured by the direct wrapping and the penetration-extraction vesiculation. The results provide an in-depth insight into the diffusivity of receptors and dendrimer in the membrane plane and demonstrate how the ligand density influences receptor diffusion and uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. The findings facilitate our understanding of some recent experimental observations and could establish fundamental principles for the future development of such important nanomaterials for widespread nanomedical applications.

Implementing Site-based Budgeting.

Science.gov (United States)

Sielke, Catherine C.

2001-01-01

Discusses five questions that must be answered before implementing site-based budgeting: Why are we doing this? What budgeting decisions will be devolved to the school site? How do dollars flow from the central office to the site? Who will be involved at the site? How will accountability be achieved? (Author/PKP)

Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling.

Directory of Open Access Journals (Sweden)

Stefanie Löffek

Full Text Available The hemidesmosomal transmembrane component collagen XVII (ColXVII plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.

Corruption of host seven-transmembrane proteins by pathogenic microbes: a common theme in animals and plants?

Science.gov (United States)

Panstruga, Ralph; Schulze-Lefert, Paul

2003-04-01

Human diseases like AIDS, malaria, and pneumonia are caused by pathogens that corrupt host chemokine G-protein coupled receptors for molecular docking. Comparatively, little is known about plant host factors that are required for pathogenesis and that may serve as receptors for the entry of pathogenic microbes. Here, we review potential analogies between human chemokine receptors and the plant seven-transmembrane MLO protein, a candidate serving a dual role as docking molecule and defence modulator for the phytopathogenic powdery mildew fungus.

SAXS analysis of a soluble cytosolic NgBR construct including extracellular and transmembrane domains.

Directory of Open Access Journals (Sweden)

Joshua Holcomb

Full Text Available The Nogo-B receptor (NgBR is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs. Small Angle X-ray Scattering (SAXS analysis reveals the radius of gyration (Rg of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

NARCIS (Netherlands)

A. Luciani (Alessandro); V.R. Villella (Valeria Rachela); S. Esposito (Susanna); M. Gavina (Manuela); I. Russo (Ilaria); M. Silano (Marco); S. Guido (Stefano); M. Pettoello-Mantovani (Massimo); R. Carnuccio (Rosa); B.J. Scholte (Bob); A. de Matteis (Antonella); M.C. Maiuri (Maria Chiara); V. Raia (Valeria); A. Luini (Alberto); H.K. Kroemer (Heyo); L. Maiuri (Luigi)

2012-01-01

textabstractChannel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded

Transmembrane Peptides as Sensors of the Membrane Physical State

Directory of Open Access Journals (Sweden)

Stefano Piotto

2018-05-01

Full Text Available Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state (MPS suggested a new approach for the discovery of antimicrobial peptides.

Research Advances in CKLFSF-like MARVEL Transmembrane Domain Containing Member 3.

Science.gov (United States)

Hu, Feng-zhan; Sheng, Zheng-zuo; Qin, Cai-peng; Xu, Tao

2016-06-10

CKLF-like MARVEL transmembrane domain containing member/chemokine-like factor super family member (CKLFSF/CMTM) is a novel tumor suppressor gene. CMTM3 is broadly expressed in normal human tissues and evolutionary conserved,especially in testis,spleen,and some cells of peripheral blood mononuclear cells. However,its expression is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM3 may inhibit the proliferation,migration,and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear,CKLFSF3/CMTM3 is closely connected with immune system and associated with sex during tumorigenesis. The study advances of CKLFSF3/CMTM3 are elaborated in this review as CMTM3 may be a new target in the gene therapies for tumors,especially genitourinary tumors,while further studies on CMTM3 and its anti-tumor mechanisms are warranted.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

International Nuclear Information System (INIS)

Kalra, Rajkumar S.; Wadhwa, Renu

2015-01-01

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

Energy Technology Data Exchange (ETDEWEB)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp [Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST Central 4), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

Monitoring of the Proton Electrochemical Gradient in Reconstituted Vesicles: Quantitative Measurements of Both Transmembrane Potential and Intravesicular pH by Ratiometric Fluorescent Probes

Czech Academy of Sciences Publication Activity Database

Holoubek, A.; Večeř, J.; Sigler, Karel

2007-01-01

Roč. 17, - (2007), s. 201-213 ISSN 1053-0509 Institutional research plan: CEZ:AV0Z50200510 Keywords : transmembrane potential * intracellular ph * oxonol dyes Subject RIV: EE - Microbiology, Virology Impact factor: 2.101, year: 2007

Impact of biofilm accumulation on transmembrane and feed channel pressure drop: Effects of crossflow velocity, feed spacer and biodegradable nutrient

KAUST Repository

Dreszer, C.

2014-03-01

Biofilm formation causes performance loss in spiral-wound membrane systems. In this study a microfiltration membrane was used in experiments to simulate fouling in spiral-wound reverse osmosis (RO) and nanofiltration (NF) membrane modules without the influence of concentration polarization. The resistance of a microfiltration membrane is much lower than the intrinsic biofilm resistance, enabling the detection of biofilm accumulation in an early stage. The impact of biofilm accumulation on the transmembrane (biofilm) resistance and feed channel pressure drop as a function of the crossflow velocity (0.05 and 0.20ms-1) and feed spacer presence was studied in transparent membrane biofouling monitors operated at a permeate flux of 20Lm-2h-1. As biodegradable nutrient, acetate was dosed to the feed water (1.0 and 0.25mgL-1 carbon) to enhance biofilm accumulation in the monitors. The studies showed that biofilm formation caused an increased transmembrane resistance and feed channel pressure drop. The effect was strongest at the highest crossflow velocity (0.2ms-1) and in the presence of a feed spacer. Simulating conditions as currently applied in nanofiltration and reverse osmosis installations (crossflow velocity 0.2ms-1 and standard feed spacer) showed that the impact of biofilm formation on performance, in terms of transmembrane and feed channel pressure drop, was strong. This emphasized the importance of hydrodynamics and feed spacer design. Biomass accumulation was related to the nutrient load (nutrient concentration and linear flow velocity). Reducing the nutrient concentration of the feed water enabled the application of higher crossflow velocities. Pretreatment to remove biodegradable nutrient and removal of biomass from the membrane elements played an important part to prevent or restrict biofouling. © 2013 Elsevier Ltd.

In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis.

Science.gov (United States)

Küsters, S; Tiegs, G; Alexopoulou, L; Pasparakis, M; Douni, E; Künstle, G; Bluethmann, H; Wendel, A; Pfizenmaier, K; Kollias, G; Grell, M

1997-11-01

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4+ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.

Expert Radiation Oncologist Interpretations of Involved-Site Radiation Therapy Guidelines in the Management of Hodgkin Lymphoma

Energy Technology Data Exchange (ETDEWEB)

Hoppe, Bradford S. [University of Florida Health Proton Therapy Institute, Jacksonville, Florida (United States); Hoppe, Richard T., E-mail: rhoppe@stanford.edu [Stanford Cancer Institute, Stanford, California (United States)

2015-05-01

Purpose: Recently, involved-site radiation therapy (ISRT) guidelines have been developed and published to replace the previous concept of involved-field radiation therapy for patients with lymphoma. However, these ISRT guidelines may be interpreted in different ways, posing difficulties for prospective clinical trials. This study reports survey results regarding interpretation of the ISRT guidelines. Methods and Materials: Forty-four expert lymphoma radiation oncologists were asked to participate in a survey that included 7 different cases associated with 9 questions. The questions pertained to ISRT contouring and asked respondents to choose between 2 different answers (no “correct” answer) and a third write-in option allowed. Results: Fifty-two percent of those surveyed responded to the questionnaire. Among those who responded, 72% have practiced for >10 years, 46% have treated >20 Hodgkin lymphoma cases annually, and 100% were familiar with the ISRT concept. Among the 9 questions associated with the 7 cases, 3 had concordance among the expert radiation oncologists of greater than 70%. Six of the questions had less than 70% concordance (range, 56%-67%). Conclusions: Even among expert radiation oncologists, interpretation of ISRT guidelines is variable. Further guidance for ISRT field design will be needed to reduce variability among practicing physicians.

A Novel Splice-Site Mutation in Angiotensin I-Converting Enzyme (ACE) Gene, c.3691+1G>A (IVS25+1G>A), Causes a Dramatic Increase in Circulating ACE through Deletion of the Transmembrane Anchor

Science.gov (United States)

Persu, Alexandre; Lambert, Michel; Deinum, Jaap; Cossu, Marta; de Visscher, Nathalie; Irenge, Leonid; Ambroise, Jerôme; Minon, Jean-Marc; Nesterovitch, Andrew B.; Churbanov, Alexander; Popova, Isolda A.; Danilov, Sergei M.; Danser, A. H. Jan; Gala, Jean-Luc

2013-01-01

Background Angiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases. Methods and Results Two Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G>A (c.3691+1G>A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells. Conclusions We describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences. PMID:23560051

Stakeholder Involvement in Swedish Nuclear Waste Management

International Nuclear Information System (INIS)

Elam, Mark; Sundqvist, Goeran

2006-01-01

The focus in this paper is on past, current and emerging patterns of stakeholder involvement in the siting of a deep repository for final disposal of Sweden's spent nuclear fuel. In particular, we concentrate on how the two municipalities of Oskarshamn and Oesthammar have acted as engaged stakeholders, and have gained recognition as such, in the siting process. In general: How has stakeholder involvement gained acceptance as an activity of value in the siting of major waste facilities? What are the issues currently subject to stakeholder involvement and how have these been decided upon? An effect of the history of nuclear activity in Oskarshamn and Oesthammar is that stakeholder involvement over a final repository can be divided into social and technical issues. Both municipalities have out of tradition, as part of their social acceptance of a new repository, been prepared to surrender extended involvement in key safety issues. They have been prepared to do this because they also see themselves being able to delegate these safety issues to the government authorities SSI and SKI. These two authorities have been acceptable to the two municipalities as their legitimate 'technological guardians'. As physical geology re-enters the siting process for a deep repository, Oskarshamn appear more prepared to break with tradition than Oesthammar. Oskarshamn are currently demanding transparency from SKB in relation to the exact technical and geological criteria they will use to choose between them and Oesthammar as a repository site. In contrast to Oesthammar, Oskarshamn are preparing with the expected help of SKI and SSI to dispute their geology and its relation to nuclear safety with SKB if they consider it necessary. If Oskarshamn act to draw safety issues in relation to alternative methods and sitings into the EIA process where might this lead? As environmental groups now enter the process (three groups were granted funding in the first round - 2005) the character of site

Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells.

Science.gov (United States)

Moguilevsky, N; Varsalona, F; Guillaume, J P; Noyer, M; Gillard, M; Daliers, J; Henichart, J P; Bollen, A

1995-01-01

A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

Solid state deuterium nuclear magnetic resonance detection of transmembrane-potential-driven tetraphenylphosphonium redistribution across Giant Unilamellar Vesicle bilayers

International Nuclear Information System (INIS)

Franzin, Carla Maria Mirella

1995-01-01

It has been demonstrated that deuterium nuclear magnetic resonance ( 2 H NMR) of Giant Unilamellar Vesicles (GUVs) consisting of specifically choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), plus 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and cholesterol can be used to monitor the transbilayer redistribution of tetraphenylphosphonium (TPP + ) in response to a transmembrane potential (δψ tm ). The 2 H quadrupolar splittings (δν Q 's) measured reflect the level of TPP + bound at the membrane surface due to the latter's effect on the membrane surface electrostatic potential, ψ s . Results reveal the appearance of two distinct δν Q 's, due to differences in bound TPP + at the inner versus the outer monolayer in response to a δψ tm . The observed values of the δν Q 's agree with theoretical predictions based on a derived mathematical model that takes into account δψ tm , plus ψ s , plus the equilibrium binding of TPP + from solution onto the membrane surface, plus the sensitivity of δν Q to the amount of bound TPP + . This model identifies experimental factors that lead to improvements in spectral resolution. Henceforth, 2 H NMR is a valuable tool for quantifying transmembrane asymmetries of ψ s . (author)

[Analysis on on-site rescue and traumatic features of victims involved in gas explosion accident in Hangzhou].

Science.gov (United States)

Wang, X G; Jin, R H; Liu, F P; Han, C M

2017-10-20

Objective: To investigate the situations of on-site rescue and traumatic features of victims involved in gas explosion accident in Hangzhou, so as to provide more data support for emergency medical rescues of the similar incidents of massive casualty. Methods: Two medical workers with a certain clinical experience were sent to Hangzhou 120 emergency medical centers to collect data of the on-site rescue on 21st July, 2017, including ambulance call-outs, on-site command and traffic conditions, and on-site triage and evacuation of the victims. They were then sent to the hospitals receiving the victims to investigate the situations of these victims including the general information (such as gender, age, admitted hospitals, and number of admission, discharge, and transferring in the first two weeks after the accident) and injury assessment [such as injury position and type, injury severity evaluation by New Injury Severity Scoring (NISS), and burn severity evaluation for victims with burns]. Results: (1) A total of 15 ambulances reached the accident site for rescue. The traffic and transportation were jammed and interrupted after this accident. On-site triage and distribution were disorderly conducted. (2) Clinical data of 53 victims were collected, including 24 males and 29 females, with the age of 8 to 70 (34±14) years old. They were sent into 6 hospitals in Hangzhou. Two victims died on the day of accident. Up to two weeks after this accident, 28 (52.8%) victims were discharged from the hospitals and received follow-up in outpatient department. Five victims with severe injuries were transferred to the other hospitals. (3) Based on the results of NISS, the injury severities were mild in 29 (54.7%) cases, moderate in 9 (17.0%) cases, serious in 3 (5.7%) cases, and severe in 12 (22.6%) cases. Those 2 dead victims were classified into the severe category due to the highest NISS score of 75. For all of the victims, skin and soft tissue defects were most common. Six (11

Serum-dependent selective expression of EhTMKB1-9, a member of Entamoeba histolytica B1 family of transmembrane kinases.

Directory of Open Access Journals (Sweden)

Shiteshu Shrimal

Full Text Available Entamoeba histolytica transmembrane kinases (EhTMKs can be grouped into six distinct families on the basis of motifs and sequences. Analysis of the E. histolytica genome revealed the presence of 35 EhTMKB1 members on the basis of sequence identity (>or=95%. Only six homologs were full length containing an extracellular domain, a transmembrane segment and an intracellular kinase domain. Reverse transcription followed by polymerase chain reaction (RT-PCR of the kinase domain was used to generate a library of expressed sequences. Sequencing of randomly picked clones from this library revealed that about 95% of the clones were identical with a single member, EhTMKB1-9, in proliferating cells. On serum starvation, the relative number of EhTMKB1-9 derived sequences decreased with concomitant increase in the sequences derived from another member, EhTMKB1-18. The change in their relative expression was quantified by real time PCR. Northern analysis and RNase protection assay were used to study the temporal nature of EhTMKB1-9 expression after serum replenishment of starved cells. The results showed that the expression of EhTMKB1-9 was sinusoidal. Specific transcriptional induction of EhTMKB1-9 upon serum replenishment was further confirmed by reporter gene (luciferase expression and the upstream sequence responsible for serum responsiveness was identified. EhTMKB1-9 is one of the first examples of an inducible gene in Entamoeba. The protein encoded by this member was functionally characterized. The recombinant kinase domain of EhTMKB1-9 displayed protein kinase activity. It is likely to have dual specificity as judged from its sensitivity to different kinase inhibitors. Immuno-localization showed EhTMKB1-9 to be a surface protein which decreased on serum starvation and got relocalized on serum replenishment. Cell lines expressing either EhTMKB1-9 without kinase domain, or EhTMKB1-9 antisense RNA, showed decreased cellular proliferation and target cell

Catalytic combustion of propane in a membrane reactor with separate feed of reactants—II. Operation in presence of trans-membrane pressure gradients

NARCIS (Netherlands)

Saracco, Guido; Veldsink, Jan Willem; Versteeg, Geert F.; Swaaij, Wim P.M. van

1995-01-01

This is the second communication of a series dealing with an experimental and modelling study on propane catalytic combustion in a membrane reactor with separate feed of reactants. In paper I the behaviour of the reactor in the absence of trans-membrane pressure gradients was presented and

Biased and Constitutive Signaling in the CC-Chemokine Receptor CCR5 by manipulating the Interface between Transmembrane Helix 6 and 7

DEFF Research Database (Denmark)

Steen, Anne; Thiele, Stefanie; Guo, Dong

2013-01-01

The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biase...

A distance measurement between specific sites on the cytoplasmic surface of bovine rhodopsin in rod outer segment disk membranes.

Science.gov (United States)

Albert, A D; Watts, A; Spooner, P; Groebner, G; Young, J; Yeagle, P L

1997-08-14

Structural information on mammalian integral membrane proteins is scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation with rhodopsin kinase. Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label. Magic angle spinning 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.

Human cytomegalovirus gH stability and trafficking are regulated by ER-associated degradation and transmembrane architecture.

Science.gov (United States)

Gardner, Thomas J; Hernandez, Rosmel E; Noriega, Vanessa M; Tortorella, Domenico

2016-03-30

The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.

Public interest group involvement

International Nuclear Information System (INIS)

Shelley, P.

1986-01-01

Including public interest groups in the siting process for nuclear waste disposal facilities is of great importance. Controversial sitings often result in litigation, but involving public interest groups early in the process will lessen the change of this. They act as surrogates for the general public and should be considered as members of the team. It is important to remember though, that all public interest groups are different. In choosing public panels such as public advisory committees, members should not be chosen on the basis of some quota. Opposition groups should not be excluded. Also, it is important to put the right person in charge of the committee. The goal of public involvement is to identify the conflicts. This must be done during the decision process, because conflicts must be known before they can be eliminated. Regarding litigation, it is important to ease through and around legal battles. If the siting process has integrity and a good faith effort has been shown, the court should uphold the effort. In addition, it is important to be negotiable and to eliminate shortcuts

Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

International Nuclear Information System (INIS)

Usami, Katsuaki; Matsuno, Keita; Igarashi, Manabu; Denda-Nagai, Kaori; Takada, Ayato; Irimura, Tatsuro

2011-01-01

Highlights: → Ebola virus infection is mediated by binding to and fusion with the target cells. → Structural feature of the viral glycoprotein determines the infectivity. → Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. → GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. → There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.

Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

Energy Technology Data Exchange (ETDEWEB)

Usami, Katsuaki [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Matsuno, Keita; Igarashi, Manabu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Denda-Nagai, Kaori [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Takada, Ayato [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Irimura, Tatsuro, E-mail: irimura@mol.f.u-tokyo.ac.jp [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan)

2011-04-01

Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.

NMR studies of transmembrane electron transport in human erythrocytes

International Nuclear Information System (INIS)

Kennett, E.C.; Bubb, W.A.; Kuchel, P.W.

2002-01-01

Full text: Electron transport systems exist in the plasma membranes of all cells. These systems appear to play a role in cell growth and proliferation, intracellular signalling, hormone responses, apoptotic events, cell defence and perhaps most importantly they enable the cell to respond to changes in the redox state of both the intra- and extracellular environments. Previously, 13 C NMR has been used to study transmembrane electron transport in human erythrocytes, specifically the reduction of extracellular 13 C-ferricyanide. NMR is a particularly useful tool for studying such systems as changes in the metabolic state of the cell can be observed concomitantly with extracellular reductase activity. We investigated the oxidation of extracellular NADH by human erythrocytes using 1 H and 31 P NMR spectroscopy. Recent results for glucose-starved human erythrocytes indicate that, under these conditions, extracellular NADH can be oxidised at the plasma membrane with the electron transfer across the membrane resulting in reduction of intracellular NAD + . The activity is inhibited by known trans-plasma membrane electron transport inhibitors (capsaicin and atebrin) and is unaffected by inhibition of the erythrocyte Band 3 anion transporter. These results suggest that electron import from extracellular NADH allows the cell to re-establish a reducing environment after the normal redox balance is disturbed

Two distinctive β subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensis.

Science.gov (United States)

Bao, Haibo; Liu, Yang; Zhang, Yixi; Liu, Zewen

2017-08-01

Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one β subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third β subunits (Locβ3) was identified in this study, which reveals at least three β subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with K d values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locβ1, Locβ2 and Locβ3 respectively. Specific immunodepletion of Locβ1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locβ3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locβ2 had no significant effect on the specific [ 3 H]imidacloprid binding. Taken together, these data indicated that Locβ1 and Locβ3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlβ1 was in two binding sites for imidacloprid. The involvement of two β subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in β subunits in L. migratoria. Copyright © 2017 Elsevier B.V. All rights reserved.

Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding.

Science.gov (United States)

Qiu, Li Yan; Koenderink, Jan B; Swarts, Herman G P; Willems, Peter H G M; De Pont, Jan Joep H H M

2003-11-21

Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.

Sites of extranodal involvement are prognostic in patients with stage 1 follicular lymphoma.

Science.gov (United States)

Shastri, Aditi; Janakiram, Murali; Mantzaris, Ioannis; Yu, Yiting; Londono, Jaime S; Verma, Amit K; Barta, Stefan K

2017-10-03

Follicular lymphoma (FL) is the most common indolent B cell lymphoma in the United States and a quarter of patients present with stage I disease. The objective of this study was to examine if primary site of disease influences survival in early stage lymphoma. The most common extranodal primary sites were the integumentary system (8%), followed by the GI tract (6.4%) and head & neck (5.6%). We stratified patients into a pre-rituximab era (1983-1998) and the rituximab era (1999-2011). In multivariable analysis, integumentary disease was associated with better overall survival (Hazard Ratio [HR], 0.77; Confidence Interval [CI], 0.66-0.9) while primary site FL of the nervous system (HR, 2.40; CI, 1.72-3.38) and the musculoskeletal system (HR, 2.14; CI, 1.44-3.18) were associated with worse overall survival when compared to primary nodal FL. Treatment in the pre-rituximab era, male gender and older age at diagnosis were associated with worse survival. We queried the SEER database from 1983 to 2011. We included all adult patients (>18 years) with histologically confirmed stage I FL, active follow-up, and a single primary tumor. A total of 9,865 patients met eligibility criteria, with 2520 (25%) having an extranodal primary site. We classified the primary sites by organ or anatomic location into 11 sites. Primary site of disease is a prognostic factor for patients with early stage FL and may help identify subsets of patients that could benefit from early, aggressive treatment.

Human Cytomegalovirus Exploits Interferon-Induced Transmembrane Proteins To Facilitate Morphogenesis of the Virion Assembly Compartment

Science.gov (United States)

Xie, Maorong; Xuan, Baoqin; Shan, Jiaoyu; Pan, Deng; Sun, Yamei; Shan, Zhao; Zhang, Jinping; Yu, Dong

2014-01-01

ABSTRACT Recently, interferon-induced transmembrane proteins (IFITMs) have been identified to be key effector molecules in the host type I interferon defense system. The invasion of host cells by a large range of RNA viruses is inhibited by IFITMs during the entry step. However, the roles of IFITMs in DNA virus infections have not been studied in detail. In this study, we report that human cytomegalovirus (HCMV), a large human DNA virus, exploits IFITMs to facilitate the formation of the virion assembly compartment (vAC) during infection of human fibroblasts. We found that IFITMs were expressed constitutively in human embryonic lung fibroblasts (MRC5 cells). HCMV infection inhibited IFITM protein accumulation in the later stages of infection. Overexpression of an IFITM protein in MRC5 cells slightly enhanced HCMV production and knockdown of IFITMs by RNA interference reduced the virus titer by about 100-fold on day 8 postinfection, according to the findings of a virus yield assay at a low multiplicity of infection. Virus gene expression and DNA synthesis were not affected, but the typical round structure of the vAC was not formed after the suppression of IFITMs, thereby resulting in defective virion assembly and the production of less infectious virion particles. Interestingly, the replication of herpes simplex virus, a human herpesvirus that is closely related to HCMV, was not affected by the suppression of IFITMs in MRC5 cells. These results indicate that IFITMs are involved in a specific pathway required for HCMV replication. IMPORTANCE HCMV is known to repurpose the interferon-stimulated genes (ISGs) viperin and tetherin to facilitate its replication. Our results expand the range of ISGs that can be exploited by HCMV for its replication. This is also the first report of a proviral function of IFITMs in DNA virus replication. In addition, whereas previous studies showed that IFITMs modulate virus entry, which is a very early stage in the virus life cycle, we

Bald eagle site management plan for the Hanford Site, south-central Washington

International Nuclear Information System (INIS)

Fitzner, R.F.; Weiss, S.G.

1994-12-01

The CERCLA remedial investigations of waste sites on the Hanford Site will involve lands containing or adjacent to a bald eagle nest, winter concentration areas, or communal night roost. Because these CERCLA investigations may affect bald eagles, the DOE has prepared this Bald Eagle Site Management Plan (BESMP). However, it is intended that this BESMP be used or updated so as to be also applicable to future activities that affect bald eagles on the Hanford Site. Bald eagles regularly use the US Department of Energy's (DOE) Hanford Site in south-central Washington State during winter months for roosting, perching, and foraging. Each of these activities requires buffer zones to protect eagles from human disturbances. Buffer zones developed in this plan follow recommended guidelines and are intended to be used in planning. If Hanford Site activities in the vicinity of identified bald eagle use areas are carried out in accordance with this plan, such actions are not likely to adversely affect the eagles or their habitat. Activities that may be exceptions will involve informal or formal (whichever is appropriate) consultation with the US Fish and Wildlife Service as required by the Endangered Species Act

Hanford Site Development Plan

International Nuclear Information System (INIS)

Rinne, C.A.; Curry, R.H.; Hagan, J.W.; Seiler, S.W.; Sommer, D.J.; Yancey, E.F.

1990-01-01

The Hanford Site Development Plan (Site Development Plan) is intended to guide the short- and long-range development and use of the Hanford Site. All acquisition, development, and permanent facility use at the Hanford Site will conform to the approved plan. The Site Development Plan also serves as the base document for all subsequent studies that involve use of facilities at the Site. This revision is an update of a previous plan. The executive summary presents the highlights of the five major topics covered in the Site Development Plan: general site information, existing conditions, planning analysis, Master Plan, and Five-Year Plan. 56 refs., 67 figs., 31 tabs

Hanford Site Development Plan

Energy Technology Data Exchange (ETDEWEB)

Rinne, C.A.; Curry, R.H.; Hagan, J.W.; Seiler, S.W.; Sommer, D.J. (Westinghouse Hanford Co., Richland, WA (USA)); Yancey, E.F. (Pacific Northwest Lab., Richland, WA (USA))

1990-01-01

The Hanford Site Development Plan (Site Development Plan) is intended to guide the short- and long-range development and use of the Hanford Site. All acquisition, development, and permanent facility use at the Hanford Site will conform to the approved plan. The Site Development Plan also serves as the base document for all subsequent studies that involve use of facilities at the Site. This revision is an update of a previous plan. The executive summary presents the highlights of the five major topics covered in the Site Development Plan: general site information, existing conditions, planning analysis, Master Plan, and Five-Year Plan. 56 refs., 67 figs., 31 tabs.

SitesIdentify: a protein functional site prediction tool

Directory of Open Access Journals (Sweden)

Doig Andrew J

2009-11-01

Full Text Available Abstract Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify, based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/

Transmembrane topology of the acetylcholine receptor examined in reconstituted vesicles

International Nuclear Information System (INIS)

McCrea, P.D.

1987-01-01

Each of the five acetylcholine receptor (AChR) subunits, α 2 β-γδ, is believed to have the same number of transmembrane crossing and to share the same general folding pattern. AChR isolated from the electric organ of electric fish is predominantly dimeric. We have used this bridge as a marker for the C-terminus of the δ subunit, and presumably that of the other subunits in addition. The disulfide's accessibility to hydrophilic reductants, principally glutathione (GSH), was tested in a reconstituted vesicle system. The reduction of the δ-δ desulfide, as evidenced by the transition of AChrR dimers to monomers, was quantitatively monitored on velocity sedimentation sucrose gradients. Alternatively, the reduction of δ 2 to δ was followed by employing non-reducing SDS-PAGE. Reductants such as GSH were able to access the bridge in intact right-side-out vesicles. No acceleration of this process was evident when the vesicles were disrupted by freeze-thaw or by detergents. Control experiments which determined the rate of reduction of entrapped diphtheria toxin, or that of 3 H-GSH efflux, demonstrated that intact reconstituted vesicles provide an adequate permeability barrier to GSH access of their intravesicular space

The structure of the CD3 ζζ transmembrane dimer in POPC and raft-like lipid bilayer: a molecular dynamics study.

Science.gov (United States)

Petruk, Ariel Alcides; Varriale, Sonia; Coscia, Maria Rosaria; Mazzarella, Lelio; Merlino, Antonello; Oreste, Umberto

2013-11-01

Plasma membrane lipids significantly affect assembly and activity of many signaling networks. The present work is aimed at analyzing, by molecular dynamics simulations, the structure and dynamics of the CD3 ζζ dimer in palmitoyl-oleoyl-phosphatidylcholine bilayer (POPC) and in POPC/cholesterol/sphingomyelin bilayer, which resembles the raft membrane microdomain supposed to be the site of the signal transducing machinery. Both POPC and raft-like environment produce significant alterations in structure and flexibility of the CD3 ζζ with respect to nuclear magnetic resonance (NMR) model: the dimer is more compact, its secondary structure is slightly less ordered, the arrangement of the Asp6 pair, which is important for binding to the Arg residue in the alpha chain of the T cell receptor (TCR), is stabilized by water molecules. Different interactions of charged residues with lipids at the lipid-cytoplasm boundary occur when the two environments are compared. Furthermore, in contrast to what is observed in POPC, in the raft-like environment correlated motions between transmembrane and cytoplasmic regions are observed. Altogether the data suggest that when the TCR complex resides in the raft domains, the CD3 ζζ dimer assumes a specific conformation probably necessary to the correct signal transduction. © 2013.

Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells

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Quadri Luis EN

2009-04-01

Full Text Available Abstract Background Cystic fibrosis (CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown. Methods This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection. Results CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1, a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP, a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3β-hydroxysterol-Δ7 reductase (Dhcr7 and stearoyl-CoA desaturase 2 (Scd2, two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC. Conclusion These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

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Fumio Yoshikawa

Full Text Available BACKGROUND: Phospholipase D (PLD catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x(4-Asp (HKD motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE: Results showed that PLD4 is a non

Regulation of KV channel voltage-dependent activation by transmembrane β subunits

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Xiaohui eSun

2012-04-01

Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

Stakeholder involvement in Swedish nuclear waste management

International Nuclear Information System (INIS)

Elam, Mark; Sundqvist, Goeran

2006-09-01

This report concerning Swedish nuclear waste management has been produced as part of a cross national research project: CARL - A Social Science Research Project into the Effects of Stakeholder involvement on Decision-Making in Radioactive Waste Management. Besides Sweden, the participating countries are Belgium, Canada, Finland, Slovenia and United Kingdom. A social science research team, working for three years, is in the first phase conducting research in their own countries in order to produce 6 country reports. During the next years the focus will shift to comparisons of stakeholder involvement practices in the participating countries. The report addresses current practices of Swedish nuclear waste management and their historical development. The main focus is on past, current and emerging patterns of stakeholder involvement in the siting of a deep repository for the final disposal of Sweden's spent nuclear fuel. The general questions attended to in the report are: Who are the main stakeholders, and how have they emerged and gained recognition as such? What are the issues currently subject to stakeholder involvement and how have these been decided upon? How is stakeholder involvement organized locally and nationally and how has this changed over time? How has stakeholder involvement gained acceptance as an activity of value in the siting of major waste facilities? The report have attempted to show the development of stakeholder involvement in the siting of a final repository for Sweden's spent nuclear fuel as resembling something other than a straightforward linear process of improvement and refinement. Stakeholder involvement has developed, over the past 15 years or so, into something more like a patchwork of different shapes and forms. Some of the forces that may well contribute to the further elaboration of the patchwork of stakeholder involvement have been pointed out, contingently modifying once more its overall colour and orientation. Questions have been

Stakeholder involvement in Swedish nuclear waste management

Energy Technology Data Exchange (ETDEWEB)

Elam, Mark; Sundqvist, Goeran [Goeteborg Univ. (Sweden). Section for Science and Technology Studies

2006-09-15

This report concerning Swedish nuclear waste management has been produced as part of a cross national research project: CARL - A Social Science Research Project into the Effects of Stakeholder involvement on Decision-Making in Radioactive Waste Management. Besides Sweden, the participating countries are Belgium, Canada, Finland, Slovenia and United Kingdom. A social science research team, working for three years, is in the first phase conducting research in their own countries in order to produce 6 country reports. During the next years the focus will shift to comparisons of stakeholder involvement practices in the participating countries. The report addresses current practices of Swedish nuclear waste management and their historical development. The main focus is on past, current and emerging patterns of stakeholder involvement in the siting of a deep repository for the final disposal of Sweden's spent nuclear fuel. The general questions attended to in the report are: Who are the main stakeholders, and how have they emerged and gained recognition as such? What are the issues currently subject to stakeholder involvement and how have these been decided upon? How is stakeholder involvement organized locally and nationally and how has this changed over time? How has stakeholder involvement gained acceptance as an activity of value in the siting of major waste facilities? The report have attempted to show the development of stakeholder involvement in the siting of a final repository for Sweden's spent nuclear fuel as resembling something other than a straightforward linear process of improvement and refinement. Stakeholder involvement has developed, over the past 15 years or so, into something more like a patchwork of different shapes and forms. Some of the forces that may well contribute to the further elaboration of the patchwork of stakeholder involvement have been pointed out, contingently modifying once more its overall colour and orientation. Questions

Mock Site Licensing Demonstration Project. Final report

International Nuclear Information System (INIS)

Roop, R.D.

1986-06-01

The Mock Site Licensing Demonstration Project developed the Low-Level Radioactive Waste Siting Simulation, a role-playing exercise designed to facilitate the process of siting and licensing disposal facilities for low-level waste (LLW). This report describes the development, contant, and usefulness of the siting simulation. The simulation was designed by Harvard University's Program on Negotiation; it can be conducted at a workshop or conference, involves 14 or more participants, and requires about eight hours to complete. The simulation consists of two sessions. In the first, participants negotiate the selection of siting criteria, and in the second, a preferred site for a facility is chosen from three candidate sites. The project sponsored two workshops (in Boston, Massachusetts and Richmond, Virginia) in which the simulation was conducted for persons involved in planning for LLW. It is concluded that the siting simulation can be useful in three ways: (1) as a tool for information dissemination, (2) as a vehicle that can foste communication among parties in conflict, and (3) as a step toward consensus building and conflict resolution. The DOE National Low-Level Waste Management Program is now making the siting simulation available for use by states, regional compacts, and other organizations involved in development of LLW disposal facilities

Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists

DEFF Research Database (Denmark)

Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina

2010-01-01

(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity......Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR...... and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation...

The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

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Eliran Moshe Reuven

2014-08-01

Full Text Available HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD of the HIV-1 envelope (ENV directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

Synthesis of a designed transmembrane protein by thioether ligation of solubilised segments : Nα-haloacetylated peptides survived resin cleavage using TFA with EDT as scavenger

NARCIS (Netherlands)

Englebretsen, D.R; Choma, C.T.; Robillard, G.T.

1998-01-01

Nα-haloacetylated peptides made by Fmoc solid phase synthesis survived cleavage when EDT was used as a cleavage component. Two segments of a desgned transmembrane protein, one bromoacetylated, the other containing a cysteine, and each bearing a "solubilising tail" peptide, were synthesised by Fmoc

Molecular Simulations of Sequence-Specific Association of Transmembrane Proteins in Lipid Bilayers

Science.gov (United States)

Doxastakis, Manolis; Prakash, Anupam; Janosi, Lorant

2011-03-01

Association of membrane proteins is central in material and information flow across the cellular membranes. Amino-acid sequence and the membrane environment are two critical factors controlling association, however, quantitative knowledge on such contributions is limited. In this work, we study the dimerization of helices in lipid bilayers using extensive parallel Monte Carlo simulations with recently developed algorithms. The dimerization of Glycophorin A is examined employing a coarse-grain model that retains a level of amino-acid specificity, in three different phospholipid bilayers. Association is driven by a balance of protein-protein and lipid-induced interactions with the latter playing a major role at short separations. Following a different approach, the effect of amino-acid sequence is studied using the four transmembrane domains of the epidermal growth factor receptor family in identical lipid environments. Detailed characterization of dimer formation and estimates of the free energy of association reveal that these helices present significant affinity to self-associate with certain dimers forming non-specific interfaces.

Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF.

Science.gov (United States)

Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

2015-10-01

Poxviruses encode up to four different soluble TNF receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific pro-inflammatory and immunological activities. Here, we report that viral TNF receptors bound and inhibited tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus, CrmE, which does not inhibit mouse soluble TNF, could block murine tmTNF-induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes.

Science.gov (United States)

Ytterberg, A Jimmy; Peltier, Jean-Benoit; van Wijk, Klaas J

2006-03-01

Plastoglobules (PGs) are oval or tubular lipid-rich structures present in all plastid types, but their specific functions are unclear. PGs contain quinones, alpha-tocopherol, and lipids and, in chromoplasts, carotenoids as well. It is not known whether PGs contain any enzymes or regulatory proteins. Here, we determined the proteome of PGs from chloroplasts of stressed and unstressed leaves of Arabidopsis (Arabidopsis thaliana) as well as from pepper (Capsicum annuum) fruit chromoplasts using mass spectrometry. Together, this showed that the proteome of chloroplast PGs consists of seven fibrillins, providing a protein coat and preventing coalescence of the PGs, and an additional 25 proteins likely involved in metabolism of isoprenoid-derived molecules (quinines and tocochromanols), lipids, and carotenoid cleavage. Four unknown ABC1 kinases were identified, possibly involved in regulation of quinone monooxygenases. Most proteins have not been observed earlier but have predicted N-terminal chloroplast transit peptides and lack transmembrane domains, consistent with localization in the PG lipid monolayer particles. Quantitative differences in PG composition in response to high light stress and degreening were determined by differential stable-isotope labeling using formaldehyde. More than 20 proteins were identified in the PG proteome of pepper chromoplasts, including four enzymes of carotenoid biosynthesis and several homologs of proteins observed in the chloroplast PGs. Our data strongly suggest that PGs in chloroplasts form a functional metabolic link between the inner envelope and thylakoid membranes and play a role in breakdown of carotenoids and oxidative stress defense, whereas PGs in chromoplasts are also an active site for carotenoid conversions.

Nuclear site selection studies

International Nuclear Information System (INIS)

Gharib, A.; Zohoorian Izadpanah, A.A.; Iranmanesh, H.

2000-01-01

It is of special importance, especially from the nuclear safety viewpoint, to select suitable sites for different nuclear structures with the considered future activities. Site selection sometimes involves high costs not necessarily for merely selecting of site but for some preliminary measures to be taken so as the site may have the necessary characteristics. The more suitable the natural characteristics of the site for the considered project, the more successful and efficient the project, the lower the project costs and the longer the project operation period. If so, the project will cause the growth of public culture and sustainable socioeconomic development. This paper is the result of the conclusion of numerous massive reports of this activity in the preliminary phase based on theories, practices and the related safety principles on this ground as well as the application of data and information of the past and a glance to the future. The conception of need for a site for medium structures and nuclear research projects and how to perform this process are presented step by step here with a scientific approach to its selection during the investigations. In this study, it is practically described how the site is selected, by determining and defining the characteristics of research and nuclear projects with medium structures and also its fitting to the optimum site. The discovered sites typically involve the best advantages in technical and economic aspects and no particular contrast with the concerned structures

Cystic Fibrosis Transmembrane Regulator Modulators: Implications for the Management of Depression and Anxiety in Cystic Fibrosis.

Science.gov (United States)

Talwalkar, Jaideep S; Koff, Jonathan L; Lee, Hochang B; Britto, Clemente J; Mulenos, Arielle M; Georgiopoulos, Anna M

Individuals with cystic fibrosis (CF) are at high risk for depression and anxiety, which are associated with worse medical outcomes. Novel therapies for CF hold great promise for improving physical health, but the effects of these therapies on mental health remain poorly understood. This review aims to familiarize psychiatrists with the potential effect of novel CF therapies on depression and anxiety. We discuss novel therapies that directly target the mutant CF protein, the CF transmembrane regulator (CFTR), which are called CFTR modulators. We summarize depression and anxiety screening and treatment guidelines under implementation in accredited CF centers. Case vignettes highlight the complexities of caring for individuals with CF with comorbid depression and anxiety, including patients experiencing worsening depression and anxiety proximate to initiation of CFTR modulator therapy, and management of drug-drug interactions. Although CFTR modulator therapies provide hope for improving clinical outcomes, worsening depression and anxiety occurs in some patients when starting these novel agents. This phenomenon may be multifactorial, with hypothesized contributions from CFTR modulator-psychotropic medication interactions, direct effects of CFTR modulators on central nervous system function, the psychologic effect of starting a potentially life-altering drug, and typical triggers of depression and anxiety such as stress, pain, and inflammation. The medical and psychiatric complexity of many individuals with CF warrants more direct involvement of mental health specialists on the multidisciplinary CF team. Inclusion of mental health variables in patients with CF registries will facilitate further examination at an epidemiologic level. Copyright © 2017 The Academy of Psychosomatic Medicine. Published by Elsevier Inc. All rights reserved.

Short length transmembrane domains having voluminous exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex

OpenAIRE

Quiroga, Rodrigo; Trenchi, Alejandra; Gonzalez Montoro, Ayelén; Valdez, Javier Esteban; Maccioni, Hugo Jose Fernando

2017-01-01

It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the ...

Overexpression of transmembrane protein 168 in the mouse nucleus accumbens induces anxiety and sensorimotor gating deficit.

Directory of Open Access Journals (Sweden)

Kequan Fu

Full Text Available Transmembrane protein 168 (TMEM168 comprises 697 amino acid residues, including some putative transmembrane domains. It is reported that TMEM168 controls methamphetamine (METH dependence in the nucleus accumbens (NAc of mice. Moreover, a strong link between METH dependence-induced adaptive changes in the brain and mood disorders has been evaluated. In the present study, we investigated the effects of accumbal TMEM168 in a battery of behavioral paradigms. The adeno-associated virus (AAV Tmem168 vector was injected into the NAc of C57BL/6J mice (NAc-TMEM mice. Subsequently, the accumbal TMEM168 mRNA was increased approximately by seven-fold when compared with the NAc-Mock mice (controls. The NAc-TMEM mice reported no change in the locomotor activity, cognitive ability, social interaction, and depression-like behaviors; however, TMEM168 overexpression enhanced anxiety in the elevated-plus maze and light/dark box test. The increased anxiety was reversed by pretreatment with the antianxiety drug diazepam (0.3 mg/kg i.p.. Moreover, the NAc-TMEM mice exhibited decreased prepulse inhibition (PPI in the startle response test, and the induced schizophrenia-like behavior was reversed by pretreatment with the antipsychotic drug risperidone (0.01 mg/kg i.p.. Furthermore, accumbal TMEM168 overexpression decreased the basal levels of extracellular GABA in the NAc and the high K+ (100 mM-stimulated GABA elevation; however, the total contents of GABA in the NAc remained unaffected. These results suggest that the TMEM168-regulated GABAergic neuronal system in the NAc might become a novel target while studying the etiology of anxiety and sensorimotor gating deficits.

The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

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Deng W-M

2009-02-01

Full Text Available Abstract Background Dystroglycan (Dg is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. Results We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. Conclusion Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling.

Science.gov (United States)

Inda, Carolina; Dos Santos Claro, Paula A; Bonfiglio, Juan J; Senin, Sergio A; Maccarrone, Giuseppina; Turck, Christoph W; Silberstein, Susana

2016-07-18

Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein-dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP. © 2016 Inda et al.

Trafficking and function of the cystic fibrosis transmembrane conductance regulator: a complex network of posttranslational modifications

Science.gov (United States)

McClure, Michelle L.; Barnes, Stephen; Brodsky, Jeffrey L.

2016-01-01

Posttranslational modifications add diversity to protein function. Throughout its life cycle, the cystic fibrosis transmembrane conductance regulator (CFTR) undergoes numerous covalent posttranslational modifications (PTMs), including glycosylation, ubiquitination, sumoylation, phosphorylation, and palmitoylation. These modifications regulate key steps during protein biogenesis, such as protein folding, trafficking, stability, function, and association with protein partners and therefore may serve as targets for therapeutic manipulation. More generally, an improved understanding of molecular mechanisms that underlie CFTR PTMs may suggest novel treatment strategies for CF and perhaps other protein conformational diseases. This review provides a comprehensive summary of co- and posttranslational CFTR modifications and their significance with regard to protein biogenesis. PMID:27474090

Public involvement in environmental surveillance at Hanford

International Nuclear Information System (INIS)

Hanf, R.W. Jr.; Patton, G.W.; Woodruff, R.K.; Poston, T.M.

1994-08-01

Environmental surveillance at the Hanford Site began during the mid-1940s following the construction and start-up of the nation's first plutonium production reactor. Over the past approximately 45 years, surveillance operations on and off the Site have continued, with virtually all sampling being conducted by Hanford Site workers. Recently, the US Department of Energy Richland Operations Office directed that public involvement in Hanford environmental surveillance operations be initiated. Accordingly, three special radiological air monitoring stations were constructed offsite, near hanford's perimeter. Each station is managed and operated by two local school teaches. These three stations are the beginning of a community-operated environmental surveillance program that will ultimately involve the public in most surveillance operations around the Site. The program was designed to stimulate interest in Hanford environmental surveillance operations, and to help the public better understand surveillance results. The program has also been used to enhance educational opportunities at local schools

Interactions between charged residues in the transmembrane segments of the voltage-sensing domain in the hERG channel.

Science.gov (United States)

Zhang, M; Liu, J; Jiang, M; Wu, D-M; Sonawane, K; Guy, H R; Tseng, G-N

2005-10-01

Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel's voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4's positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the 'mutant cycle analysis' to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538.

Comparative exploration of hydrogen sulfide and water transmembrane free energy surfaces via orthogonal space tempering free energy sampling.

Science.gov (United States)

Lv, Chao; Aitchison, Erick W; Wu, Dongsheng; Zheng, Lianqing; Cheng, Xiaolin; Yang, Wei

2016-03-05

Hydrogen sulfide (H2 S), a commonly known toxic gas compound, possesses unique chemical features that allow this small solute molecule to quickly diffuse through cell membranes. Taking advantage of the recent orthogonal space tempering (OST) method, we comparatively mapped the transmembrane free energy landscapes of H2 S and its structural analogue, water (H2 O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. As revealed by our OST sampling results, in contrast to the highly polar water solute, hydrogen sulfide is evidently amphipathic, and thus inside membrane is favorably localized at the interfacial region, that is, the interface between the polar head-group and nonpolar acyl chain regions. Because the membrane binding affinity of H2 S is mainly governed by its small hydrophobic moiety and the barrier height inbetween the interfacial region and the membrane center is largely determined by its moderate polarity, the transmembrane free energy barriers to encounter by this toxic molecule are very small. Moreover when H2 S diffuses from the bulk solution to the membrane center, the above two effects nearly cancel each other, so as to lead to a negligible free energy difference. This study not only explains why H2 S can quickly pass through cell membranes but also provides a practical illustration on how to use the OST free energy sampling method to conveniently analyze complex molecular processes. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Advances in characterizing ubiquitylation sites by mass spectrometry

DEFF Research Database (Denmark)

Sylvestersen, K.B.; Young, C.; Nielsen, M.L.

2013-01-01

of ubiquitylation is a two-fold challenge that involves the mapping of ubiquitylation sites and the determination of ubiquitin chain topology. This review focuses on the technical advances in the mass spectrometry-based characterization of ubiquitylation sites, which have recently involved the large...

Role of the transmembrane domain of FXYD7 in structural and functional interactions with Na,K-ATPase.

Science.gov (United States)

Li, Ciming; Crambert, Gilles; Thuillard, Delphine; Roy, Sophie; Schaer, Danièle; Geering, Käthi

2005-12-30

Members of the FXYD family are tissue-specific regulators of the Na,K-ATPase. Here, we have investigated the contribution of amino acids in the transmembrane (TM) domain of FXYD7 to the interaction with Na,K-ATPase. Twenty amino acids of the TM domain were replaced individually by tryptophan, and combined mutations and alanine insertion mutants were constructed. Wild type and mutant FXYD7 were expressed in Xenopus oocytes with Na,K-ATPase. Mutational effects on the stable association with Na,K-ATPase and on the functional regulation of Na,K-ATPase were determined by co-immunoprecipitation and two-electrode voltage clamp techniques, respectively. Most residues important for the structural and functional interaction of FXYD7 are clustered in a face of the TM helix containing the two conserved glycine residues, but others are scattered over two-thirds of the FXYD TM helix. Ile-35, Ile-43, and Ile-44 are only involved in the stable association with Na,K-ATPase. Glu-26, Met-30, and Ile-44 are important for the functional effect and/or the efficient association of FXYD7 with Na,K-ATPase, consistent with the prediction that these amino acids contact TM domain 9 of the alpha subunit (Li, C., Grosdidier, A., Crambert, G., Horisberger, J.-D., Michielin, O., and Geering, K. (2004) J. Biol. Chem. 279, 38895-38902). Several amino acids that are not implicated in the efficient association of FXYD7 with the Na,K-ATPase are specifically involved in the functional effect of FXYD7. Leu-32 and Phe-37 influence the apparent affinity for external K+, whereas Val-28 and Ile-42 are implicated in the apparent affinity for both external K+ and external Na+. These amino acids act in a synergistic way. These results highlight the important structural and functional role of the TM domain of FXYD7 and delineate the determinants that mediate the complex interactions of FXYD7 with Na,K-ATPase.

Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40

Energy Technology Data Exchange (ETDEWEB)

Lu, Jun; Byrne, Noel; Wang, John; Bricogne, Gerard; Brown, Frank K.; Chobanian, Harry R.; Colletti, Steven L.; Di Salvo, Jerry; Thomas-Fowlkes, Brande; Guo, Yan; Hall, Dawn L.; Hadix, Jennifer; Hastings, Nicholas B.; Hermes, Jeffrey D.; Ho, Thu; Howard, Andrew D.; Josien, Hubert; Kornienko, Maria; Lumb, Kevin J.; Miller, Michael W.; Patel, Sangita B.; Pio, Barbara; Plummer, Christopher W.; Sherborne, Bradley S.; Sheth, Payal; Souza, Sarah; Tummala, Srivanya; Vonrhein, Clemens; Webb, Maria; Allen, Samantha J.; Johnston, Jennifer M.; Weinglass, Adam B.; Sharma, Sujata; Soisson, Stephen M. (Merck); (Globel Phasing)

2017-06-05

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Common structural features of cholesterol binding sites in crystallized soluble proteins.

Science.gov (United States)

Bukiya, Anna N; Dopico, Alejandro M

2017-06-01

Cholesterol-protein interactions are essential for the architectural organization of cell membranes and for lipid metabolism. While cholesterol-sensing motifs in transmembrane proteins have been identified, little is known about cholesterol recognition by soluble proteins. We reviewed the structural characteristics of binding sites for cholesterol and cholesterol sulfate from crystallographic structures available in the Protein Data Bank. This analysis unveiled key features of cholesterol-binding sites that are present in either all or the majority of sites: i ) the cholesterol molecule is generally positioned between protein domains that have an organized secondary structure; ii ) the cholesterol hydroxyl/sulfo group is often partnered by Asn, Gln, and/or Tyr, while the hydrophobic part of cholesterol interacts with Leu, Ile, Val, and/or Phe; iii ) cholesterol hydrogen-bonding partners are often found on α-helices, while amino acids that interact with cholesterol's hydrophobic core have a slight preference for β-strands and secondary structure-lacking protein areas; iv ) the steroid's C21 and C26 constitute the "hot spots" most often seen for steroid-protein hydrophobic interactions; v ) common "cold spots" are C8-C10, C13, and C17, at which contacts with the proteins were not detected. Several common features we identified for soluble protein-steroid interaction appear evolutionarily conserved. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant.

Science.gov (United States)

Zhong, Huailing; Hansen, Kasper B; Boyle, Noel J; Han, Kiho; Muske, Galina; Huang, Xinyan; Egebjerg, Jan; Sánchez, Connie

2009-10-25

The human serotonin transporter (hSERT) has primary and allosteric binding sites for escitalopram and R-citalopram. Previous studies have established that the interaction of these two compounds at a low affinity allosteric binding site of hSERT can affect the dissociation of [(3)H]escitalopram from hSERT. The allosteric binding site involves a series of residues in the 10th, 11th, and 12th trans-membrane domains of hSERT. The low affinity allosteric activities of escitalopram and R-citalopram are essentially eliminated in a mutant hSERT with changes in some of these residues, namely A505V, L506F, I507L, S574T, I575T, as measured in dissociation binding studies. We confirm that in association binding experiments, R-citalopram at clinically relevant concentrations reduces the association rate of [(3)H]escitalopram as a ligand to wild type hSERT. We demonstrate that the ability of R-citalopram to reduce the association rate of escitalopram is also abolished in the mutant hSERT (A505V, L506F, I507L, S574T, I575T), along with the expected disruption the low affinity allosteric function on dissociation binding. This suggests that the allosteric binding site mediates both the low affinity and higher affinity interactions between R-citalopram, escitalopram, and hSERT. Our data add an additional structural basis for the different efficacies of escitalopram compared to racemic citalopram reported in animal studies and clinical trials, and substantiate the hypothesis that hSERT has complex allosteric mechanisms underlying the unexplained in vivo activities of its inhibitors.

MRI findings of multiple sclerosis involving the brainstem

International Nuclear Information System (INIS)

Park, Jeong Hoon; Jeong, Hae Woong; Kim, Hyun Jin; Cho, Jae Kwoeng; Kim, Chang Soo

2001-01-01

To describe MRI findings of multiple sclerosis involving the brainstem. Among 35 cases of clinically definite multiple sclerosis, the authors retrospectively analysed 20 in which the brainstem was involved. MR images were analysed with regard to involvement sites in the brainstem or other locations, signal intensity, multiplicity, shape, enhancement pattern, and contiguity of brainstem lesions with cisternal or ventricular CSF space. The brainstem was the only site of involvement in five cases (25%), while simultaneous involvement of the brainstem and other sites was observed in 15 cases (75%). No case involved only the midbrain or medulla oblongata, and simultaneous involvement of the midbrain, pons and medulla oblongata was noted in 12 cases (60%). The most frequently involved region of the brainstem was the medulla oblongata (n=13; 90%), followed by the pons (n=17; 85%) and the midbrain (n=16; 80%). Compared with normal white matter, brainstem lesions showed low signal intensity on T1 weighted images, and high signal intensity on T2 weighted, proton density weighted, and FLAIR images. In 17 cases (85%), multiple intensity was observed, and the shape of lesions varied: oval, round, elliptical, patchy, crescentic, confluent or amorphous were seen on axial MR images, and in 14 cases (82%), coronal or sagittal scanning showed that lesions were long and tubular. Contiguity between brainstem lesions and cisternal or ventricular CSF space was seen in all cases (100%) involving midbrain (16/16) and medulla oblongata (18/18) and in 15 of 17 (88%) involving the pons. Contrast enhancement was apparent in 7 of 12 cases (58%). In the brainstem, MRI demonstrated partial or total contiguity between lesions and cisternal or ventricular CSF space, and coronal or sagittal images showed that lesions were long and tubuler

Prediction of FAD binding sites in electron transport proteins according to efficient radial basis function networks and significant amino acid pairs.

Science.gov (United States)

Le, Nguyen-Quoc-Khanh; Ou, Yu-Yen

2016-07-30

Cellular respiration is a catabolic pathway for producing adenosine triphosphate (ATP) and is the most efficient process through which cells harvest energy from consumed food. When cells undergo cellular respiration, they require a pathway to keep and transfer electrons (i.e., the electron transport chain). Due to oxidation-reduction reactions, the electron transport chain produces a transmembrane proton electrochemical gradient. In case protons flow back through this membrane, this mechanical energy is converted into chemical energy by ATP synthase. The convert process is involved in producing ATP which provides energy in a lot of cellular processes. In the electron transport chain process, flavin adenine dinucleotide (FAD) is one of the most vital molecules for carrying and transferring electrons. Therefore, predicting FAD binding sites in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. We used an independent data set to evaluate the performance of the proposed method, which had an accuracy of 69.84 %. We compared the performance of the proposed method in analyzing two newly discovered electron transport protein sequences with that of the general FAD binding predictor presented by Mishra and Raghava and determined that the accuracy of the proposed method improved by 9-45 % and its Matthew's correlation coefficient was 0.14-0.5. Furthermore, the proposed method enabled reducing the number of false positives significantly and can provide useful information for biologists. We developed a method that is based on PSSM profiles and SAAPs for identifying FAD binding sites in newly discovered electron transport protein sequences. This approach achieved a significant improvement after we added SAAPs to PSSM features to analyze FAD binding proteins in the electron transport chain. The proposed method can serve as an effective tool for predicting FAD binding sites in electron

Structural details (kinks and non-α conformations) in transmembrane helices are intrahelically determined and can be predicted by sequence pattern descriptors

Science.gov (United States)

Rigoutsos, Isidore; Riek, Peter; Graham, Robert M.; Novotny, Jiri

2003-01-01

One of the promising methods of protein structure prediction involves the use of amino acid sequence-derived patterns. Here we report on the creation of non-degenerate motif descriptors derived through data mining of training sets of residues taken from the transmembrane-spanning segments of polytopic proteins. These residues correspond to short regions in which there is a deviation from the regular α-helical character (i.e. π-helices, 310-helices and kinks). A ‘search engine’ derived from these motif descriptors correctly identifies, and discriminates amongst instances of the above ‘non-canonical’ helical motifs contained in the SwissProt/TrEMBL database of protein primary structures. Our results suggest that deviations from α-helicity are encoded locally in sequence patterns only about 7–9 residues long and can be determined in silico directly from the amino acid sequence. Delineation of such variations in helical habit is critical to understanding the complex structure–function relationships of polytopic proteins and for drug discovery. The success of our current methodology foretells development of similar prediction tools capable of identifying other structural motifs from sequence alone. The method described here has been implemented and is available on the World Wide Web at http://cbcsrv.watson.ibm.com/Ttkw.html. PMID:12888523

Structural details (kinks and non-alpha conformations) in transmembrane helices are intrahelically determined and can be predicted by sequence pattern descriptors.

Science.gov (United States)

Rigoutsos, Isidore; Riek, Peter; Graham, Robert M; Novotny, Jiri

2003-08-01

One of the promising methods of protein structure prediction involves the use of amino acid sequence-derived patterns. Here we report on the creation of non-degenerate motif descriptors derived through data mining of training sets of residues taken from the transmembrane-spanning segments of polytopic proteins. These residues correspond to short regions in which there is a deviation from the regular alpha-helical character (i.e. pi-helices, 3(10)-helices and kinks). A 'search engine' derived from these motif descriptors correctly identifies, and discriminates amongst instances of the above 'non-canonical' helical motifs contained in the SwissProt/TrEMBL database of protein primary structures. Our results suggest that deviations from alpha-helicity are encoded locally in sequence patterns only about 7-9 residues long and can be determined in silico directly from the amino acid sequence. Delineation of such variations in helical habit is critical to understanding the complex structure-function relationships of polytopic proteins and for drug discovery. The success of our current methodology foretells development of similar prediction tools capable of identifying other structural motifs from sequence alone. The method described here has been implemented and is available on the World Wide Web at http://cbcsrv.watson.ibm.com/Ttkw.html.

3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

Directory of Open Access Journals (Sweden)

Johanna L Höög

2016-01-01

Full Text Available Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners.

Science.gov (United States)

Muramatsu, Takashi

2016-05-01

Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society.

Effects of transmembrane hydraulic pressure on performance of forward osmosis membranes.

Science.gov (United States)

Coday, Bryan D; Heil, Dean M; Xu, Pei; Cath, Tzahi Y

2013-03-05

Forward osmosis (FO) is an emerging membrane separation process that continues to be tested and implemented in various industrial water and wastewater treatment applications. The growing interests in the technology have prompted laboratories and manufacturers to adopt standard testing methods to ensure accurate comparison of membrane performance under laboratory-controlled conditions; however, standardized methods might not capture specific operating conditions unique to industrial applications. Experiments with cellulose triacetate (CTA) and polyamide thin-film composite (TFC) FO membranes demonstrated that hydraulic transmembrane pressure (TMP), common in industrial operation of FO membrane elements, could affect membrane performance. Experiments were conducted with three FO membranes and with increasing TMP up to a maximum of 50 psi (3.45 bar). The feed solution was a mixture of salts and the draw solution was either a NaCl solution or concentrated seawater at similar osmotic pressure. Results revealed that TMP minimally affected water flux, reverse salt flux (RSF), and solute rejection of the CTA membrane. However, water flux through TFC membranes might slightly increase with increasing TMP, and RSF substantially declines with increasing TMP. It was observed that rejection of feed constituents was influenced by TMP and RSF.

Identification of Loop D Domain Amino Acids in the Human Aquaporin-1 Channel Involved in Activation of the Ionic Conductance and Inhibition by AqB011

Directory of Open Access Journals (Sweden)

Mohamad Kourghi

2018-04-01

Full Text Available Aquaporins are integral proteins that facilitate the transmembrane transport of water and small solutes. In addition to enabling water flux, mammalian Aquaporin-1 (AQP1 channels activated by cyclic GMP can carry non-selective monovalent cation currents, selectively blocked by arylsulfonamide compounds AqB007 (IC50 170 μM and AqB011 (IC50 14 μM. In silico models suggested that ligand docking might involve the cytoplasmic loop D (between AQP1 transmembrane domains 4 and 5, but the predicted site of interaction remained to be tested. Work here shows that mutagenesis of two conserved arginine residues in loop D slowed the activation of the AQP1 ion conductance and impaired the sensitivity of the channel to block by AqB011. Substitution of residues in loop D with proline showed effects on ion conductance amplitude that varied with position, suggesting that the structural conformation of loop D is important for AQP1 channel gating. Human AQP1 wild type, AQP1 mutant channels with alanines substituted for two arginines (R159A+R160A, and mutants with proline substituted for single residues threonine (T157P, aspartate (D158P, arginine (R159P, R160P, or glycine (G165P were expressed in Xenopus laevis oocytes. Conductance responses were analyzed by two-electrode voltage clamp. Optical osmotic swelling assays and confocal microscopy were used to confirm mutant and wild type AQP1-expressing oocytes were expressed in the plasma membrane. After application of membrane-permeable cGMP, R159A+R160A channels had a significantly slower rate of activation as compared with wild type, consistent with impaired gating. AQP1 R159A+R160A channels showed no significant block by AqB011 at 50 μM, in contrast to the wild type channel which was blocked effectively. T157P, D158P, and R160P mutations had impaired activation compared to wild type; R159P showed no significant effect; and G165P appeared to augment the conductance amplitude. These findings provide evidence for the

Design of an environmental site assessment template for open radioactive site contamination : a radioecological risk approach and case study

International Nuclear Information System (INIS)

Nguyen, T.

2004-01-01

To reduce redundancy, cost, and time, while at the same time ultimately increasing the effectiveness of the radioactive risk management process, a logical framework incorporating risk assessments (human cancer and environmental risks) into the environmental site assessment process was designed for radioactive open site contamination. Risk-based corrective action is becoming an increasingly more acceptable approach for the remediation of contaminated sites. In the past, cleanup goals were usually established without any regard to the risk involved, by mandating remediation goals based solely on maximum contamination levels. Now, a multi-stage environmental site assessment template has been developed on a radioecological approach. The template gives a framework for making environmentally sound decisions based on relevant regulations and guidelines. The first stage involves the comparison of the background screening activity level to the regulated activity level, the second stage involves the use of site-specific information to determine the risk involved with the contamination, and the third stage provides a remediation decision matrix based on results from the first two stages. This environmental site assessment template is unique because it incorporates the modified Canadian National Classification System for radioactive contaminated sites and two different types of risk assessments (human cancer risks and the newly designed ecological risk) into the decision making process. The template was used to assess a radiologically contaminated site at the Canadian Forces Base at Suffield (Alberta) as a case study, and it reaffirms the Department of National Defence's action as appropriate. This particular site is a Class 3, has an overall insignificant human cancer risk ( -6 ) and a low environmental risk, and conforms to all regulated guidelines. Currently, it is restricted and should be left as is, provided that the subsurface is not disturbed. (author)

Overlapping binding site for the endogenous agonist, small-molecule agonists, and ago-allosteric modulators on the ghrelin receptor

DEFF Research Database (Denmark)

Holst, Birgitte; Frimurer, Thomas M; Mokrosinski, Jacek

2008-01-01

A library of robust ghrelin receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone......, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing ghrelin's maximal efficacy overlapped with the common....... It is concluded that although each of the ligands in addition exploits other parts of the receptor, a large, common binding site for both small-molecule agonists--including ago-allosteric modulators--and the endogenous agonist is found on the opposing faces of TM-III and -VI of the ghrelin receptor....

Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes1[W

Science.gov (United States)

Ytterberg, A. Jimmy; Peltier, Jean-Benoit; van Wijk, Klaas J.

2006-01-01

Plastoglobules (PGs) are oval or tubular lipid-rich structures present in all plastid types, but their specific functions are unclear. PGs contain quinones, α-tocopherol, and lipids and, in chromoplasts, carotenoids as well. It is not known whether PGs contain any enzymes or regulatory proteins. Here, we determined the proteome of PGs from chloroplasts of stressed and unstressed leaves of Arabidopsis (Arabidopsis thaliana) as well as from pepper (Capsicum annuum) fruit chromoplasts using mass spectrometry. Together, this showed that the proteome of chloroplast PGs consists of seven fibrillins, providing a protein coat and preventing coalescence of the PGs, and an additional 25 proteins likely involved in metabolism of isoprenoid-derived molecules (quinines and tocochromanols), lipids, and carotenoid cleavage. Four unknown ABC1 kinases were identified, possibly involved in regulation of quinone monooxygenases. Most proteins have not been observed earlier but have predicted N-terminal chloroplast transit peptides and lack transmembrane domains, consistent with localization in the PG lipid monolayer particles. Quantitative differences in PG composition in response to high light stress and degreening were determined by differential stable-isotope labeling using formaldehyde. More than 20 proteins were identified in the PG proteome of pepper chromoplasts, including four enzymes of carotenoid biosynthesis and several homologs of proteins observed in the chloroplast PGs. Our data strongly suggest that PGs in chloroplasts form a functional metabolic link between the inner envelope and thylakoid membranes and play a role in breakdown of carotenoids and oxidative stress defense, whereas PGs in chromoplasts are also an active site for carotenoid conversions. PMID:16461379

CERCLA site assessment workbook

International Nuclear Information System (INIS)

1994-08-01

This contains comments for each chapter of exercises (in Vol. 1) which illustrate how to conduct site assessments for CERCLA regulation. A through analysis of the exercises is provided so that work and solutions from Vol 1 can be critiqued and comments are also included on the strategy of site assessment whereas the exercises illustrate the principles involved. Covered exercises include the following: A preliminary assessment of a ground water site; waste characteristics and characterization of sources; documentation of observed releases and actual contamination of targets; the strategy of an SI at a surface water site; the soil exposure pathway; the air pathway

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

International Nuclear Information System (INIS)

Kaperonis, A.A.; Chien, S.

1989-01-01

Plasma 125 I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the 125 I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies

15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.

Science.gov (United States)

Chekmenev, Eduard Y; Hu, Jun; Gor'kov, Peter L; Brey, William W; Cross, Timothy A; Ruuge, Andres; Smirnov, Alex I

2005-04-01

This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.

Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development.

Science.gov (United States)

Sun, Hao; Lagarrigue, Frederic; Gingras, Alexandre R; Fan, Zhichao; Ley, Klaus; Ginsberg, Mark H

2018-04-02

Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development. © 2018 Sun et al.

Non-equilibrium dynamics of 2D liquid crystals driven by transmembrane gas flow.

Science.gov (United States)

Seki, Kazuyoshi; Ueda, Ken; Okumura, Yu-ichi; Tabe, Yuka

2011-07-20

Free-standing films composed of several layers of chiral smectic liquid crystals (SmC*) exhibited unidirectional director precession under various vapor transfers across the films. When the transferred vapors were general organic solvents, the precession speed linearly depended on the momentum of the transmembrane vapors, where the proportional constant was independent of the kind of vapor. In contrast, the same SmC* films under water transfer exhibited precession in the opposite direction. As a possible reason for the rotational inversion, we suggest the competition of two origins for the torques, one of which is microscopic and the other macroscopic. Next, we tried to move an external object by making use of the liquid crystal (LC) motion. When a solid or a liquid particle was set on a film under vapor transfer, the particle was rotated in the same direction as the LC molecules. Using home-made laser tweezers, we measured the force transmitted from the film to the particle, which we found to be several pN.

Site decommissioning management plan

International Nuclear Information System (INIS)

Fauver, D.N.; Austin, J.H.; Johnson, T.C.; Weber, M.F.; Cardile, F.P.; Martin, D.E.; Caniano, R.J.; Kinneman, J.D.

1993-10-01

The Nuclear Regulatory Commission (NRC) staff has identified 48 sites contaminated with radioactive material that require special attention to ensure timely decommissioning. While none of these sites represent an immediate threat to public health and safety they have contamination that exceeds existing NRC criteria for unrestricted use. All of these sites require some degree of remediation, and several involve regulatory issues that must be addressed by the Commission before they can be released for unrestricted use and the applicable licenses terminated. This report contains the NRC staff's strategy for addressing the technical, legal, and policy issues affecting the timely decommissioning of the 48 sites and describes the status of decommissioning activities at the sites

Site decommissioning management plan

Energy Technology Data Exchange (ETDEWEB)

Fauver, D.N.; Austin, J.H.; Johnson, T.C.; Weber, M.F.; Cardile, F.P.; Martin, D.E.; Caniano, R.J.; Kinneman, J.D.

1993-10-01

The Nuclear Regulatory Commission (NRC) staff has identified 48 sites contaminated with radioactive material that require special attention to ensure timely decommissioning. While none of these sites represent an immediate threat to public health and safety they have contamination that exceeds existing NRC criteria for unrestricted use. All of these sites require some degree of remediation, and several involve regulatory issues that must be addressed by the Commission before they can be released for unrestricted use and the applicable licenses terminated. This report contains the NRC staff`s strategy for addressing the technical, legal, and policy issues affecting the timely decommissioning of the 48 sites and describes the status of decommissioning activities at the sites.

Signal peptide discrimination and cleavage site identification using SVM and NN.

Science.gov (United States)

Kazemian, H B; Yusuf, S A; White, K

2014-02-01

About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model. © 2013 Published by Elsevier Ltd.

Effect of transmembrane pressure control on energy efficiency during skim milk concentration by ultrafiltration at 10 and 50°C.

Science.gov (United States)

Méthot-Hains, S; Benoit, S; Bouchard, C; Doyen, A; Bazinet, L; Pouliot, Y

2016-11-01

The efficiency of the ultrafiltration process during skim milk concentration was studied using both dynamic and constant (465 or 672kPa) transmembrane pressure experiments at refrigerated temperature (10°C) and high temperature (50°C). The pilot-scale module was equipped with a 10-kDa polyethersulfone spiral-wound membrane element with a surface area of 2.04m 2 . Permeation flux, resistance-in-series model, mineral and protein rejection, and energy consumption were studied as a function of temperature and transmembrane pressure applied. Higher permeation flux values were systematically obtained at 50°C. Also, a significant temperature effect was found for calcium rejection, which was lower at 10°C compared with 50°C. Total hydraulic resistance and reversible fouling resistance were higher at 50°C than at 10°C. No change in protein rejection was observed, depending on the operating mode studied. Permeation flux, which was higher at 50°C, had lower pumping energy consumption compared with ultrafiltration at the colder temperature. Also, the low ultrafiltration temperature required a higher total energy consumption to reach the 3.6× retentate compared with ultrafiltration at 50°C. Overall, our study shows that the operating parameters and temperature can be optimized using an energy efficiency ratio. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Interactions involved in pH protection of the alphavirus fusion protein

Energy Technology Data Exchange (ETDEWEB)

Fields, Whitney; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

2015-12-15

The alphavirus membrane protein E1 mediates low pH-triggered fusion of the viral and endosome membranes during virus entry. During virus biogenesis E1 associates as a heterodimer with the transmembrane protein p62. Late in the secretory pathway, cellular furin cleaves p62 to the mature E2 protein and a peripheral protein E3. E3 remains bound to E2 at low pH, stabilizing the heterodimer and thus protecting E1 from the acidic pH of the secretory pathway. Release of E3 at neutral pH then primes the virus for fusion during entry. Here we used site-directed mutagenesis and revertant analysis to define residues important for the interactions at the E3–E2 interface. Our data identified a key residue, E2 W235, which was required for E1 pH protection and alphavirus production. Our data also suggest additional residues on E3 and E2 that affect their interacting surfaces and thus influence the pH protection of E1 during alphavirus exit.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

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Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

2009-08-07

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide-Localization of a putative binding site.

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Dayan, Avraham; Babin, Gilad; Ganoth, Assaf; Kayouf, Nivin Samir; Nitoker Eliaz, Neta; Mukkala, Srijana; Tsfadia, Yossi; Fleminger, Gideon

2017-08-01

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO 2 )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO 2 -binding capabilities with TiBP. Intrigued by the unique TiO 2 -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO 2 surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO 2 , indicating that neither the enzyme undergoes major conformational changes nor the TiO 2 binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO 2 through similar regions located far from the active site and the dimerization sites. The putative TiO 2 -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins. Copyright © 2017 John Wiley & Sons, Ltd.

Expression of the fructose receptor BmGr9 and its involvement in the promotion of feeding, suggested by its co-expression with neuropeptide F1 in Bombyx mori.

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Mang, Dingze; Shu, Min; Tanaka, Shiho; Nagata, Shinji; Takada, Tomoyuki; Endo, Haruka; Kikuta, Shingo; Tabunoki, Hiroko; Iwabuchi, Kikuo; Sato, Ryoichi

2016-08-01

Insect gustatory receptors (Grs) are members of a large family of proteins with seven transmembrane domains that provide insects with the ability to detect chemical signals critical for feeding, mating, and oviposition. To date, 69 Bombyx mori Grs (BmGrs) genes have been identified via genome studies. BmGr9 has been shown to respond specifically to fructose and to function as a ligand-gated ion channel selectively activated by fructose. However, the sites where this Gr are expressed remain unclear. We demonstrated using reverse transcription (RT)-PCR that BmGr9 is widely expressed in the central nervous system (CNS), as well as oral sensory organs. Additionally, immunohistochemistry was performed using anti-BmGr9 antiserum to show that BmGr9 is expressed in cells of the oral sensory organs, including the maxillary galea, maxillary palps, labrum, and labium, as well as in putative neurosecretory cells of the CNS. Furthermore, double immunohistochemical analysis showed that most BmGr9-expressing cells co-localized with putative neuropeptide F1-expressing cells in the brain, suggesting that BmGr9 is involved in the promotion of feeding behaviors. In addition, a portion of BmGr9-expressing cells in the brain co-localized with cells expressing BmGr6, a molecule of the sugar receptor clade, suggesting that sugars other than fructose are involved in the regulation of feeding behaviors in B. mori larvae. Copyright © 2016 Elsevier Ltd. All rights reserved.

Emerging role of cystic fibrosis transmembrane conductance regulator- an epithelial chloride channel in gastrointestinal cancers

Institute of Scientific and Technical Information of China (English)

Yuning Hou; Xiaoqing Guan; Zhe Yang; Chunying Li

2016-01-01

Cystic fibrosis transmembrane conductance regulator(CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers.

Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.

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Choi, Sun-Hye; Lee, Byung-Hwan; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyun-Sook; Shin, Ho-Chul; Lee, Jun-Hee; Kim, Hyoung-Chun; Rhim, Hyewhon; Hwang, Sung-Hee; Ha, Tal Soo; Kim, Hyun-Ji; Cho, Hana; Nah, Seung-Yeol

2014-09-01

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca(2+)]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K(+) (I(Ks)) channel is a cardiac K(+) channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I(Ks) channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I(Ks) channel activity by expressing human I(Ks) channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the I(Ks) channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the I(Ks) channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I(Ks) channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca(2+)]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I(Ks) channel. However, gintonin had no effect on hERG K(+) channel activity. These results show that gintonin-mediated enhancement of I(Ks) channel currents is achieved through binding of the [Ca(2+)]i/CaM complex to the C terminus of KCNQ1 subunit.

Sites of extranodal involvement are prognostic in patients with stage 1 follicular lymphoma

OpenAIRE

Shastri, Aditi; Janakiram, Murali; Mantzaris, Ioannis; Yu, Yiting; Londono, Jaime S.; Verma, Amit K.; Barta, Stefan K.

2017-01-01

Objectives Follicular lymphoma (FL) is the most common indolent B cell lymphoma in the United States and a quarter of patients present with stage I disease. The objective of this study was to examine if primary site of disease influences survival in early stage lymphoma. Results The most common extranodal primary sites were the integumentary system (8%), followed by the GI tract (6.4%) and head & neck (5.6%). We stratified patients into a pre-rituximab era (1983-1998) and the rituximab era (1...

Conformational Plasticity of the Influenza A M2 Transmembrane Helix in Lipid Bilayers Under Varying pH, Drug Binding and Membrane Thickness

Science.gov (United States)

Hu, Fanghao; Luo, Wenbin; Cady, Sarah D.; Hong, Mei

2010-01-01

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). 13C and 15N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, non-ideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and non-ideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (ϕ, ψ) torsion angles for three basis states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins. PMID:20883664

Progesterone modulation of transmembrane helix-helix interactions between the α-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane

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Askari Amir

2010-05-01

Full Text Available Abstract Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit. Results Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020 successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME, can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. Conclusion We propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

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Nicole Hofmann

Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

Ethics Review for a Multi-Site Project Involving Tribal Nations in the Northern Plains.

Science.gov (United States)

Angal, Jyoti; Petersen, Julie M; Tobacco, Deborah; Elliott, Amy J

2016-04-01

Increasingly, Tribal Nations are forming ethics review panels, which function separately from institutional review boards (IRBs). The emergence of strong community representation coincides with a widespread effort supported by the U.S. Department of Health & Human Services and other federal agencies to establish a single IRB for all multi-site research. This article underscores the value of a tribal ethics review board and describes the tribal oversight for the Safe Passage Study-a multi-site, community-based project in the Northern Plains. Our experience demonstrates the benefits of tribal ethics review and makes a strong argument for including tribal oversight in future regulatory guidance for multi-site, community-based research. © The Author(s) 2016.

General aspects of siting and safety considerations

International Nuclear Information System (INIS)

Rutgers, E.

1980-01-01

The siting process from site selection to the different stages of review by the regulatory body is described. Special attention is payed to the role and responsibilities of the licensing authority. Next, the basic considerations involved in the siting process are reviewed. They include system planning, engineering, safety, environmental impact (including land use) and economics. Case studies illustrating different aspects of the siting process (e.g. site selection) are presented. (orig.)

Evolutionary and Comparative Genomics to Drive Rational Drug Design, with Particular Focus on Neuropeptide Seven-Transmembrane Receptors.

Science.gov (United States)

Furlong, Michael; Seong, Jae Young

2017-01-01

Seven transmembrane receptors (7TMRs), also known as G protein-coupled receptors, are popular targets of drug development, particularly 7TMR systems that are activated by peptide ligands. Although many pharmaceutical drugs have been discovered via conventional bulk analysis techniques the increasing availability of structural and evolutionary data are facilitating change to rational, targeted drug design. This article discusses the appeal of neuropeptide-7TMR systems as drug targets and provides an overview of concepts in the evolution of vertebrate genomes and gene families. Subsequently, methods that use evolutionary concepts and comparative analysis techniques to aid in gene discovery, gene function identification, and novel drug design are provided along with case study examples.

Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss

International Nuclear Information System (INIS)

Izquierdo-Vega, Jeannett A.; Sanchez-Gutierrez, Manuel; Razo, Luz Maria del

2008-01-01

Fluorosis, caused by drinking water contamination with inorganic fluoride, is a public health problem in many areas around the world. The aim of the study was to evaluate the effect of environmentally relevant doses of fluoride on in vitro fertilization (IVF) capacity of spermatozoa, and its relationship to spermatozoa mitochondrial transmembrane potential (ΔΨ m ). Male Wistar rats were administered at 5 mg fluoride/kg body mass/24 h, or deionized water orally for 8 weeks. We evaluated several spermatozoa parameters in treated and untreated rats: i) standard quality analysis, ii) superoxide dismutase (SOD) activity, iii) the generation of superoxide anion (O 2 ·- ), iv) lipid peroxidation concentration, v) ultrastructural analyses of spermatozoa using transmission electron microscopy, vi) ΔΨ m , vii) acrosome reaction, and viii) IVF capability. Spermatozoa from fluoride-treated rats exhibited a significant decrease in SOD activity (∼ 33%), accompanied with a significant increase in the generation of O 2 · (∼ 40%), a significant decrease in ΔΨ m (∼ 33%), and a significant increase in lipid peroxidation concentration (∼ 50%), relative to spermatozoa from the control group. Consistent with this finding, spermatozoa from fluoride-treated rats exhibited altered plasmatic membrane. In addition, the percentage of fluoride-treated spermatozoa capable of undergoing the acrosome reaction was decreased relative to control spermatozoa (34 vs. 55%), while the percentage fluoride-treated spermatozoa capable of oocyte fertilization was also significantly lower than the control group (13 vs. 71%). These observations suggest that subchronic exposure to fluoride causes oxidative stress damage and loss of mitochondrial transmembrane potential, resulting in reduced fertility

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

Energy Technology Data Exchange (ETDEWEB)

Kaperonis, A.A.; Chien, S.

1989-02-01

Plasma /sup 125/I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the /sup 125/I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies.

The PTK7-related transmembrane proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for male fertility.

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Linnemannstöns, Karen; Ripp, Caroline; Honemann-Capito, Mona; Brechtel-Curth, Katja; Hedderich, Marie; Wodarz, Andreas

2014-07-01

Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk) are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964), which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.

Extrapulmonary involvement in pediatric tuberculosis.

Science.gov (United States)

Kritsaneepaiboon, Supika; Andres, Mariaem M; Tatco, Vincent R; Lim, Cielo Consuelo Q; Concepcion, Nathan David P

2017-09-01

Tuberculosis in childhood is clinically challenging, but it is a preventable and treatable disease. Risk factors depend on age and immunity status. The most common form of pediatric tuberculosis is pulmonary disease, which comprises more than half of the cases. Other forms make up the extrapulmonary tuberculosis that involves infection of the lymph nodes, central nervous system, gastrointestinal system, hepatobiliary tree, and renal and musculoskeletal systems. Knowledge of the imaging characteristics of pediatric tuberculosis provides clues to diagnosis. This article aims to review the imaging characteristics of common sites for extrapulmonary tuberculous involvement in children.

Fluorophores, environments, and quantification techniques in the analysis of transmembrane helix interaction using FRET.

Science.gov (United States)

Khadria, Ambalika S; Senes, Alessandro

2015-07-01

Förster resonance energy transfer (FRET) has been widely used as a spectroscopic tool in vitro to study the interactions between transmembrane (TM) helices in detergent and lipid environments. This technique has been instrumental to many studies that have greatly contributed to quantitative understanding of the physical principles that govern helix-helix interactions in the membrane. These studies have also improved our understanding of the biological role of oligomerization in membrane proteins. In this review, we focus on the combinations of fluorophores used, the membrane mimetic environments, and measurement techniques that have been applied to study model systems as well as biological oligomeric complexes in vitro. We highlight the different formalisms used to calculate FRET efficiency and the challenges associated with accurate quantification. The goal is to provide the reader with a comparative summary of the relevant literature for planning and designing FRET experiments aimed at measuring TM helix-helix associations. © 2015 Wiley Periodicals, Inc.

When a transmembrane channel isn't, or how biophysics and biochemistry (mis)communicate.

Science.gov (United States)

Reviakine, Ilya

2018-02-12

Annexins are a family of soluble proteins that bind to acidic phospholipids such as phosphatidylserine in a calcium-dependent manner. The archetypical member of the annexin family is annexin A5. For many years, its function remained unknown despite the availability of a high-resolution structure. This, combined with the observations of specific ion conductance in annexin-bound membranes, fueled speculations about the possible membrane-spanning forms of annexins that functioned as ion channels. The channel hypothesis remained controversial and did not gather sufficient evidence to become accepted. Yet, it continues to draw attention as a framework for interpreting indirect (e.g., biochemical) data. The goal of the mini-review is to examine the data on annexin-lipid interactions from the last ~30 years from the point of view of the controversy between the two lines of inquiry: the well-characterized peripheral assembly of the annexins at membranes vs. their putative transmembrane insertion. In particular, the potential role of lipid rearrangements induced by annexin binding is highlighted. Copyright © 2018 Elsevier B.V. All rights reserved.

Transmembrane carbonic anhydrase isozymes IX and XII in the female mouse reproductive organs

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Tomas Eija

2004-10-01

Full Text Available Abstract Background Carbonic anhydrase (CA classically catalyses the reversible hydration of dissolved CO2 to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems. Methods The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract. Results The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta. Conclusion The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.

Molecular Dynamics Simulations of Orai Reveal How the Third Transmembrane Segment Contributes to Hydration and Ca2+ Selectivity in Calcium Release-Activated Calcium Channels.

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Alavizargar, Azadeh; Berti, Claudio; Ejtehadi, Mohammad Reza; Furini, Simone

2018-04-26

Calcium release-activated calcium (CRAC) channels open upon depletion of Ca 2+ from the endoplasmic reticulum, and when open, they are permeable to a selective flux of calcium ions. The atomic structure of Orai, the pore domain of CRAC channels, from Drosophila melanogaster has revealed many details about conduction and selectivity in this family of ion channels. However, it is still unclear how residues on the third transmembrane helix can affect the conduction properties of the channel. Here, molecular dynamics and Brownian dynamics simulations were employed to analyze how a conserved glutamate residue on the third transmembrane helix (E262) contributes to selectivity. The comparison between the wild-type and mutated channels revealed a severe impact of the mutation on the hydration pattern of the pore domain and on the dynamics of residues K270, and Brownian dynamics simulations proved that the altered configuration of residues K270 in the mutated channel impairs selectivity to Ca 2+ over Na + . The crevices of water molecules, revealed by molecular dynamics simulations, are perfectly located to contribute to the dynamics of the hydrophobic gate and the basic gate, suggesting a possible role in channel opening and in selectivity function.

Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid secretion in airway epithelia

Science.gov (United States)

Turner, Mark J.; Saint‐Criq, Vinciane; Patel, Waseema; Ibrahim, Salam H.; Verdon, Bernard; Ward, Christopher; Garnett, James P.; Tarran, Robert; Cann, Martin J.

2015-01-01

Key points Raised arterial blood CO2 (hypercapnia) is a feature of many lung diseases.CO2 has been shown to act as a cell signalling molecule in human cells, notably by influencing the levels of cell signalling second messengers: cAMP and Ca2+.Hypercapnia reduced cAMP‐stimulated cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid transport in Calu‐3 cells and primary human airway epithelia but did not affect cAMP‐regulated HCO3 − transport via pendrin or Na+/HCO3 − cotransporters.These results further support the role of CO2 as a cell signalling molecule and suggests CO2‐induced reductions in airway anion and fluid transport may impair innate defence mechanisms of the lungs. Abstract Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist‐stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP‐regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin‐stimulated elevations in intracellular cAMP as well as both adenosine‐ and forskolin‐stimulated increases in CFTR‐dependent transepithelial short‐circuit current, in polarised cultures of Calu‐3 human airway cells. This CO2‐induced reduction in anion secretion was not due to a decrease in HCO3 − transport given that neither a change in CFTR‐dependent HCO3 − efflux nor Na+/HCO3 − cotransporter‐dependent HCO3 − influx were CO2‐sensitive. Hypercapnia also reduced the volume of forskolin‐stimulated fluid

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

Science.gov (United States)

Papa, S; Lorusso, M; Izzo, G; Capuano, F

1981-02-15

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi

Mutations of C19orf12, coding for a transmembrane glycine zipper containing mitochondrial protein, cause mis-localization of the protein, inability to respond to oxidative stress and increased mitochondrial Ca2+.

Directory of Open Access Journals (Sweden)

Paola eVenco

2015-05-01

Full Text Available Mutations in C19orf12 have been identified in patients affected by Neurodegeneration with Brain Iron Accumulation (NBIA, a clinical entity characterized by iron accumulation in the basal ganglia. By using western blot analysis with specific antibody and confocal studies, we showed that wild-type C19orf12 protein was not exclusively present in mitochondria, but also in the Endoplasmic Reticulum (ER and MAM (Mitochondria Associated Membrane, while mutant C19orf12 variants presented a different localization. Moreover, after induction of oxidative stress, a GFP-tagged C19orf12 wild-type protein was able to relocate to the cytosol. On the contrary, mutant isoforms were not able to respond to oxidative stress. High mitochondrial calcium concentration and increased H2O2 induced apoptosis were found in fibroblasts derived from one patient as compared to controls.C19orf12 protein is a 17kDa mitochondrial membrane-associated protein whose function is still unknown. Our in silico investigation suggests that, the glycine zipper motifs of C19orf12 form helical regions spanning the membrane. The N- and C-terminal regions with respect to the transmembrane portion, on the contrary, are predicted to rearrange in a structural domain, which is homologues to the N-terminal regulatory domain of the magnesium transporter MgtE, suggesting that C19orf12 may act as a regulatory protein for human MgtE transporters. The mutations here described affect respectively one glycine residue of the glycine zipper motifs, which are involved in dimerization of transmembrane helices and predicted to impair the correct localization of the protein into the membranes, and one residue present in the regulatory domain, which is important for protein-protein interaction.

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

Science.gov (United States)

Caballero-Rivera, Daniel; Cruz-Nieves, Omar A; Oyola-Cintrón, Jessica; Torres-Núñez, David A; Otero-Cruz, José D

2011-01-01

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the δM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the δM3 transmembrane domain were characterized by two-electrode voltage clamp and 125I-labeled α-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300–301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery. PMID:21785268

Qualified public involvement in the decision making process of siting a waste repository

International Nuclear Information System (INIS)

Rodrigues, Danielle Monegalha; Almeida, Ivan Pedro Salati de

2009-01-01

The main objective of this paper is to identify the most important characteristics required for the qualification of local communities for participating in the process of defining a specific site for a radioactive waste repository. It also compares the strategies used by Hungary, United Kingdom and Belgium to stimulate the public participation in the decision-making process of building and operating a radioactive waste repository, considering both the stepwise process and the spontaneous candidacy. Two main aspects are discussed as prerequisites to constitute a qualified public. The first aspect is how well the person or entity can be considered an effective representative of the community affected by the repository. This means the conditions the representative has to speak on behalf of the community and participate in the decision making process as its voice. The second characteristic is the level and quality of the information that the community and its representatives must have to participate actively in the decision-making process and what can be done to improve this status. Referring to the strategy to public involvement, this paper discusses the importance of transparency in the process, aiming the credibility of the entrepreneur as the first pace to gaining the confidence of the public affected by the project. Implementing an open dialog and listening to the needs and claims of the population are the first steps to being accepted as a true partner of the community. Preliminary discussions and explanations are important to introduce the subject and to reduce beliefs of false threats in the affected community. The constitution of a local committee is suggested, to act as a legal and formal channel to facilitate the partnership between local community, neighbors and the entrepreneur in order to achieve a positive result in the whole process. (author)

The recognition unit of FIBCD1 organizes into a noncovalently linked tetrameric structure and uses a hydrophobic funnel (S1) for acetyl group recognition

DEFF Research Database (Denmark)

Thomsen, Theresa; Moeller, Jesper B; Schlosser, Anders

2010-01-01

We have recently identified FIBCD1 (Fibrinogen C domain containing 1) as a type II transmembrane endocytic receptor located primarily in the intestinal brush border. The ectodomain of FIBCD1 comprises a coiled coil, a polycationic region, and a C-terminal FReD (fibrinogen-related domain) that ass......We have recently identified FIBCD1 (Fibrinogen C domain containing 1) as a type II transmembrane endocytic receptor located primarily in the intestinal brush border. The ectodomain of FIBCD1 comprises a coiled coil, a polycationic region, and a C-terminal FReD (fibrinogen-related domain......) that assembles into disulfide-linked homotetramers. The FIBCD1-FReD binds Ca(2+) dependently to acetylated structures like chitin, N-acetylated carbohydrates, and amino acids. FReDs are present in diverse innate immune pattern recognition proteins including the ficolins and horseshoe crab TL5A. Here, we use...... combined with site-directed mutagenesis to define the binding site involved in the interaction of FIBCD1 with acetylated structures. We show that mutations of central residues (A432V and H415G) in the hydrophobic funnel (S1) abolish the binding of FIBCD1 to acetylated bovine serum albumin and chitin...

Fixed-site physical protection system modeling

International Nuclear Information System (INIS)

Chapman, L.D.

1975-01-01

An evaluation of a fixed-site safeguard security system must consider the interrelationships of barriers, alarms, on-site and off-site guards, and their effectiveness against a forcible adversary attack whose intention is to create an act of sabotage or theft. A computer model has been developed at Sandia Laboratories for the evaluation of alternative fixed-site security systems. Trade-offs involving on-site and off-site response forces and response times, perimeter alarm systems, barrier configurations, and varying levels of threat can be analyzed. The computer model provides a framework for performing inexpensive experiments on fixed-site security systems for testing alternative decisions, and for determining the relative cost effectiveness associated with these decision policies

On multi-site damage identification using single-site training data

Science.gov (United States)

Barthorpe, R. J.; Manson, G.; Worden, K.

2017-11-01

This paper proposes a methodology for developing multi-site damage location systems for engineering structures that can be trained using single-site damaged state data only. The methodology involves training a sequence of binary classifiers based upon single-site damage data and combining the developed classifiers into a robust multi-class damage locator. In this way, the multi-site damage identification problem may be decomposed into a sequence of binary decisions. In this paper Support Vector Classifiers are adopted as the means of making these binary decisions. The proposed methodology represents an advancement on the state of the art in the field of multi-site damage identification which require either: (1) full damaged state data from single- and multi-site damage cases or (2) the development of a physics-based model to make multi-site model predictions. The potential benefit of the proposed methodology is that a significantly reduced number of recorded damage states may be required in order to train a multi-site damage locator without recourse to physics-based model predictions. In this paper it is first demonstrated that Support Vector Classification represents an appropriate approach to the multi-site damage location problem, with methods for combining binary classifiers discussed. Next, the proposed methodology is demonstrated and evaluated through application to a real engineering structure - a Piper Tomahawk trainer aircraft wing - with its performance compared to classifiers trained using the full damaged-state dataset.

Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness.

Science.gov (United States)

Cybulski, Larisa Estefanía; Ballering, Joost; Moussatova, Anastassiia; Inda, Maria Eugenia; Vazquez, Daniela B; Wassenaar, Tsjerk A; de Mendoza, Diego; Tieleman, D Peter; Killian, J Antoinette

2015-05-19

DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called "minimal sensor DesK" (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein-lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.

Measuring site occupancy

DEFF Research Database (Denmark)

Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna; Williamson, James

2014-01-01

occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance. We make use of high resolution mass...... peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise...... and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site occupancy in cellular redox response....

Decommissioning: guiding principles and best practices for involving local stakeholders

International Nuclear Information System (INIS)

Keyes, D.

2005-01-01

Full text: A wide range of nuclear facilities covering the entire nuclear fuel cycle have been constructed and operated for many years worldwide. For communities where the facilities are located, concerns about safety and environmental contamination are paramount. Working together with elected officials, local community leaders and the public at large during the earliest planning stages will help alleviate concerns about facility operation and ultimate disposition, and will result in better decisions about facility design, location, construction, operation and, ultimately, decommissioning. Such comprehensive community involvement has been the exception rather than the norm. Now that older facilities are being considered for decommissioning, efforts to involve local stakeholders and alleviate their concerns face major challenges. This is particularly true where some residual radioactive contamination will remain onsite and future use of the site may need to be restricted. Plans for stakeholder involvement at the decommissioning stage should be carefully designed and provide for honest, authentic and meaningful involvement of all stakeholders affected by decommissioning decisions. A set of principles and best practices is proposed to help guide the design and implementation of effective community involvement programs. The principles and best practices are drawn from the experiences of public involvement practitioners in a variety of environmental contamination applications. Successful community involvement is the result of a carefully crafted set of coordinated activities conducted over the long term. Ideally, facility decommissioning is simply the end stage of the involvement process, or the beginning of a site stewardship process in those cases where decommissioning does not produce an uncontaminated site. In either case, decommissioning will not be a new, unexpected event, and stakeholders could be involved just as they have been over the life of the facility. In

Pilot-Scale Demonstration of In-Situ Chemical Oxidation Involving Chlorinated Volatile Organic Compounds - Design and Deployment Guidelines (Parris Island, SC, U.S. Marine Corp Recruit Depot, Site 45 Pilot Study)

Science.gov (United States)

A pilot-scale in situ chemical oxidation (ISCO) demonstration, involving subsurface injections of sodium permanganate (NaMnO4), was performed at the US Marine Corp Recruit Depot (MCRD), site 45 (Parris Island (PI), SC). The ground water was originally contaminated with perchloroe...

Decision aiding techniques for site remediation

Energy Technology Data Exchange (ETDEWEB)

Van de Walle, B

1996-09-18

Decision making problems in the nuclear domain are known for their complexity since they usually involve a wide range of technical, social, and political considerations. Site restoration is a typical example of a complex nuclear decision problem, and more and more decision makers realize that they need new tools to assist in the decision making process. This paper reports on multi-criteria decision analysis, a powerful tool for handling complex decisions involving multiple criteria. The motivation to use multi-criteria decision analysis in the domain of site restoration is illustrated. New developments and challenges in this field are addressed.

Decision aiding techniques for site remediation

International Nuclear Information System (INIS)

Van de Walle, B.

1996-01-01

Decision making problems in the nuclear domain are known for their complexity since they usually involve a wide range of technical, social, and political considerations. Site restoration is a typical example of a complex nuclear decision problem, and more and more decision makers realize that they need new tools to assist in the decision making process. This paper reports on multi-criteria decision analysis, a powerful tool for handling complex decisions involving multiple criteria. The motivation to use multi-criteria decision analysis in the domain of site restoration is illustrated. New developments and challenges in this field are addressed

Direct evidence that scorpion α-toxins (site-3 modulate sodium channel inactivation by hindrance of voltage-sensor movements.

Directory of Open Access Journals (Sweden)

Zhongming Ma

Full Text Available The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4 and S5-S6 in Domain 1 (D1 and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS. The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH33 (+ trimethylammonium, MTSET and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx, but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage

Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis.

Science.gov (United States)

Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Pagliarani, Alessandra

2011-07-01

Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F(1)F(0)-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F(0), namely oligomycin and N,N'-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F(1), maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F(1) and F(0) domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F(0) proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F(1)F(0)-ATPase oligomycin sensitivity loss. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids.

Directory of Open Access Journals (Sweden)

Gonzalo E Yévenes

Full Text Available Glycine receptors (GlyRs are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA are positive modulators of α(1, α(2 and α(3 GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly potentiate α(1 GlyRs but inhibit α(2 and α(3. This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM region 2 and intracellular lysine 385 determine the positive modulation of α(1 GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in α(2 converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of α(1 GlyRs, without affecting inhibition of α(2 and α(3. Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain.

Hanford Site environmental management specification

Energy Technology Data Exchange (ETDEWEB)

Grygiel, M.L.

1998-06-10

The US Department of Energy, Richland Operations Office (RL) uses this Hanford Site Environmental Management Specification (Specification) to document top-level mission requirements and planning assumptions for the prime contractors involved in Hanford Site cleanup and infrastructure activities under the responsibility of the US Department of Energy, Office of Environmental Management. This Specification describes at a top level the activities, facilities, and infrastructure necessary to accomplish the cleanup of the Hanford Site and assigns this scope to Site contractors and their respective projects. This Specification also references the key National Environmental Policy Act of 1969 (NEPA), Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA), and safety documentation necessary to accurately describe the cleanup at a summary level. The information contained in this document reflects RL`s application of values, priorities, and critical success factors expressed by those involved with and affected by the Hanford Site project. The prime contractors and their projects develop complete baselines and work plans to implement this Specification. These lower-level documents and the data that support them, together with this Specification, represent the full set of requirements applicable to the contractors and their projects. Figure 1-1 shows the relationship of this Specification to the other basic Site documents. Similarly, the documents, orders, and laws referenced in this specification represent only the most salient sources of requirements. Current and contractual reference data contain a complete set of source documents.

Hanford Site environmental management specification

International Nuclear Information System (INIS)

Grygiel, M.L.

1998-01-01

The US Department of Energy, Richland Operations Office (RL) uses this Hanford Site Environmental Management Specification (Specification) to document top-level mission requirements and planning assumptions for the prime contractors involved in Hanford Site cleanup and infrastructure activities under the responsibility of the US Department of Energy, Office of Environmental Management. This Specification describes at a top level the activities, facilities, and infrastructure necessary to accomplish the cleanup of the Hanford Site and assigns this scope to Site contractors and their respective projects. This Specification also references the key National Environmental Policy Act of 1969 (NEPA), Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA), and safety documentation necessary to accurately describe the cleanup at a summary level. The information contained in this document reflects RL's application of values, priorities, and critical success factors expressed by those involved with and affected by the Hanford Site project. The prime contractors and their projects develop complete baselines and work plans to implement this Specification. These lower-level documents and the data that support them, together with this Specification, represent the full set of requirements applicable to the contractors and their projects. Figure 1-1 shows the relationship of this Specification to the other basic Site documents. Similarly, the documents, orders, and laws referenced in this specification represent only the most salient sources of requirements. Current and contractual reference data contain a complete set of source documents

Siting guidelines and their role in repository site selection

International Nuclear Information System (INIS)

Hanlon, C.L.

1985-01-01

The first requirement of the Nuclear Waste Policy Act was for the Secretary of Energy to issue general guidelines for siting repositories. The guidelines were to specify detailed geologic considerations that would be the primary criteria for the selection of sites in various host rocks, as well as factors that would qualify or disqualify any site from development as a repository. These guidelines were clearly intended to provide not only the framework for the siting program but also the stimulus for establishing effective communication and consultation among the parties involved in the program. The Act further required that the guidelines be a factor in the development of all future decision documents of the Office of Civilian Radioactive Waste Management, including the environmental assessments that would accompany the nomination of sites for characterization, the site-characterization plans that are to be prepared before the sinking of exploratory shafts at any candidate site, and the environmental impact statement that is to support the recommendation of a site for development as a repository. More than two years after its passage, the intention of the Act for the guidelines has been realized. Concurred in by the Nuclear Regulatory Commission on June 22, 1984, and issued by the Department in November 1984, the guidelines include postclosure technical guidelines that apply to conditions governing the long-term performance of the repository system; preclosure technical guidelines that apply to conditions governing the siting, construction, operation, and closure of the repository; and system guidelines whose objective is to ensure that the regulatory requirements of the Environmental Protection Agency and the Nuclear Regulatory Commission are met

Multiple regulatory roles of the mouse transmembrane adaptor protein NTAL in gene transcription and mast cell physiology.

Directory of Open Access Journals (Sweden)

Iva Polakovicova

Full Text Available Non-T cell activation linker (NTAL; also called LAB or LAT2 is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.

Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

Directory of Open Access Journals (Sweden)

Blom Nikolaj

2004-06-01

Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

Siting simulation for low-level waste disposal facilities

International Nuclear Information System (INIS)

Roop, R.D.; Rope, R.C.

1985-01-01

The Mock Site Licensing Demonstration Project has developed the Low-Level Radioactive Waste Siting Simulation, a role-playing exercise designed to facilitate the process of siting and licensing disposal facilities for low-level waste (LLW). This paper describes the development, content, and usefulness of the siting simulation. The simulation can be conducted at a workshop or conference, involves 14 or more participants, and requires about eight hours to complete. The simulation consists of two sessions; in the first, participants negotiate the selection of siting criteria, and in the second, a preferred disposal site is chosen from three candidate sites. The project has sponsored two workshops (in Boston, Massachusetts and Richmond, Virginia) in which the simulation has been conducted for persons concerned with LLW management issues. It is concluded that the simulation can be valuable as a tool for disseminating information about LLW management; a vehicle that can foster communication; and a step toward consensus building and conflict resolution. The DOE National Low-Level Waste Management Program is now making the siting simulation available for use by states, regional compacts, and other organizations involved in development of LLW disposal facilities

Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

Science.gov (United States)

Balfanz, Sabine; Ehling, Petra; Wachten, Sebastian; Jordan, Nadine; Erber, Joachim; Mujagic, Samir; Baumann, Arnd

2012-06-01

The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. Copyright © 2012 Elsevier Ltd. All rights reserved.

Siting controversial facilities

International Nuclear Information System (INIS)

Baird, R.D.; Blacker, P.B.

1985-01-01

There is often significant difficulty involved with siting controversial facilities. The social and political problems are frequently far more difficult to resolve than the technical and economic issues. The tendancy for most developing organizations is to address only technical issues in the search for a technically optimal site, to the exclusion of such weighting considerations as the social and political climate associated with potential sites--an approach which often imperils the success of the project. The site selection processes currently suggested is summarized and two contemporary examples of their application are cited. The difference between developers' real objectives and the objectives they have implicitly assumed by adopting the recommended approaches without augmentation are noted. The resulting morass of public opposition is attributed to the failure to consider the needs of individuals and groups who stand to be negatively impacted by the development. A comprehensive implementation strategy which addresses non-technical consideration in parallel with technical ones is presented and evaluated

Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues

DEFF Research Database (Denmark)

Magni, M; Pandiella, A; Helin, K

1991-01-01

a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather...... in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed......, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion...

Protein kinase A (PKA) phosphorylation of Na+/K+-ATPase opens intracellular C-terminal water pathway leading to third Na+-binding site in molecular dynamics simulations

DEFF Research Database (Denmark)

Poulsen, Hanne; Nissen, Poul; Mouritsen, Ole G.

2012-01-01

-atom Molecular Dynamics (MD) simulations to investigate the structural consequences of phosphorylating the Na+/K+- ATPase (NKA) residue S936, which is the best characterized phosphorylation site in NKA, targeted in vivo by Protein Kinase A (PKA) (1-3). The MD simulations suggest that S936 phosphorylation opens......Phosphorylation is one of the major mechanisms for posttranscriptional modification of proteins. The addition of a compact, negatively charged moiety to a protein can significantly change its function and localization by affecting its structure and interaction network. We have used all...... a C-terminal hydrated pathway leading to D926, a transmembrane residue proposed to form part of the third sodium ion-binding site (4). Simulations of a S936E mutant form, for which only subtle effects are observed when expressed in Xenopus oocytes and studied with electrophysiology, does not mimic...

DOE site performance assessment activities

International Nuclear Information System (INIS)

1990-07-01

Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions

GABAA receptor: Positive and negative allosteric modulators.

Science.gov (United States)

Olsen, Richard W

2018-01-31

gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane transport of GABA, the GABA receptors Type A (GABA A R) and Type B (GABA B R) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABA B R is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABA A R pharmacology, the topic of this article. GABA A R are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABA A R the targets of agonist depressants and antagonist convulsants, but most GABA A R drugs act at other (allosteric) binding sites on the GABA A R proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABA A R subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABA A R subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABA A R subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABA A R subtype-dependent extracellular domain sites. Thus GABA A R subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of

The PTK7-Related Transmembrane Proteins Off-track and Off-track 2 Are Co-receptors for Drosophila Wnt2 Required for Male Fertility

OpenAIRE

Linnemannstöns, Karen; Ripp, Caroline; Honemann-Capito, Mona; Brechtel-Curth, Katja; Hedderich, Marie; Wodarz, Andreas

2014-01-01

Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-recept...

Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

Science.gov (United States)

Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

2014-08-22

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Synthesis and characterization of (18)F-labeled active site inhibited factor VII (ASIS).

Science.gov (United States)

Erlandsson, Maria; Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Jesper B; Petersen, Lars C; Madsen, Jacob; Kjaer, Andreas

2015-05-15

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress. Copyright © 2015 John Wiley & Sons, Ltd.

G. Nuclear power plant siting

International Nuclear Information System (INIS)

1976-01-01

The selection of a site for a nuclear power site is a complex process involving considerations of public health and safety, engineering design, economics, and environmental impact. Although policies adopted in various countries differ in some details, a common philosophy usually underlies the criteria employed. The author discusses the basic requirements, as they relate to New Zealand, under the headings: engineering and economics; health and safety; environmental factors

Computer-Based Testing: Test Site Security.

Science.gov (United States)

Rosen, Gerald A.

Computer-based testing places great burdens on all involved parties to ensure test security. A task analysis of test site security might identify the areas of protecting the test, protecting the data, and protecting the environment as essential issues in test security. Protecting the test involves transmission of the examinations, identifying the…

Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3.

Science.gov (United States)

Joensuu, T; Hämäläinen, R; Yuan, B; Johnson, C; Tegelberg, S; Gasparini, P; Zelante, L; Pirvola, U; Pakarinen, L; Lehesjoki, A E; de la Chapelle, A; Sankila, E M

2001-10-01

Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterized by progressive hearing loss, severe retinal degeneration, and variably present vestibular dysfunction, assigned to 3q21-q25. Here, we report on the positional cloning of the USH3 gene. By haplotype and linkage-disequilibrium analyses in Finnish carriers of a putative founder mutation, the critical region was narrowed to 250 kb, of which we sequenced, assembled, and annotated 207 kb. Two novel genes-NOPAR and UCRP-and one previously identified gene-H963-were excluded as USH3, on the basis of mutational analysis. USH3, the candidate gene that we identified, encodes a 120-amino-acid protein. Fifty-two Finnish patients were homozygous for a termination mutation, Y100X; patients in two Finnish families were compound heterozygous for Y100X and for a missense mutation, M44K, whereas patients in an Italian family were homozygous for a 3-bp deletion leading to an amino acid deletion and substitution. USH3 has two predicted transmembrane domains, and it shows no homology to known genes. As revealed by northern blotting and reverse-transcriptase PCR, it is expressed in many tissues, including the retina.

Targeting a genetic defect: cystic fibrosis transmembrane conductance regulator modulators in cystic fibrosis

Directory of Open Access Journals (Sweden)

Nico Derichs

2013-03-01

Full Text Available Cystic fibrosis (CF is caused by genetic mutations that affect the cystic fibrosis transmembrane conductance regulator (CFTR protein. These mutations can impact the synthesis and transfer of the CFTR protein to the apical membrane of epithelial cells, as well as influencing the gating or conductance of chloride and bicarbonate ions through the channel. CFTR dysfunction results in ionic imbalance of epithelial secretions in several organ systems, such as the pancreas, gastrointestinal tract, liver and the respiratory system. Since discovery of the CFTR gene in 1989, research has focussed on targeting the underlying genetic defect to identify a disease-modifying treatment for CF. Investigated management strategies have included gene therapy and the development of small molecules that target CFTR mutations, known as CFTR modulators. CFTR modulators are typically identified by high-throughput screening assays, followed by preclinical validation using cell culture systems. Recently, one such modulator, the CFTR potentiator ivacaftor, was approved as an oral therapy for CF patients with the G551D-CFTR mutation. The clinical development of ivacaftor not only represents a breakthrough in CF care but also serves as a noteworthy example of personalised medicine.

Siting nuclear power plants

International Nuclear Information System (INIS)

Yellin, J.; Joskow, P.L.

1980-01-01

The first edition of this journal is devoted to the policies and problems of siting nuclear power plants and the question of how far commercial reactors should be placed from urban areas. The article is divided into four major siting issues: policies, risk evaluation, accident consequences, and economic and physical constraints. One concern is how to treat currently operating reactors and those under construction that were established under less-stringent criteria if siting is to be used as a way to limit the consequences of accidents. Mehanical cost-benefit analyses are not as appropriate as the systematic use of empirical observations in assessing the values involved. Stricter siting rules are justified because (1) opposition because of safety is growing: (2) remote siting will make the industry more stable; (3) the conflict is eliminated between regulatory policies and the probability basis for nuclear insurance; and (4) joint ownership of utilities and power-pooling are increasing. 227 references, 7 tables

Comparative biology of the ubiquitous Na+/H+ exchanger, NHE1: lessons from erythrocytes

DEFF Research Database (Denmark)

Pedersen, Stine Falsig; Cala, Peter Michael

2004-01-01

of NHE1 in erythrocytes, in their own right and as model systems, but pertinent findings from non-erythroid cells are also discussed. NHE1 plays a key role in the regulation of cell volume and pH, and consequently in the control of such diverse processes as blood O2/CO2 transport, and cell proliferation......, motility, and survival. Disturbances in NHE1 function are involved in important pathological states such as hypoxic cell damage and cancer development. NHE1 has a predicted topology of 12 transmembrane domains, and a hydrophilic C-terminus thought to be the major site for NHE1 regulation. NHE1 is highly...... conserved throughout the vertebrate phylum, particularly in the transmembrane region and the proximal part of the C-terminus. In non-erythroid, and probably also in erythroid cells, this part of the hydrophilic C-terminus interacts with multiple binding partners important for NHE1 function. Erythrocyte NHE1...

Allosteric regulation of rhomboid intramembrane proteolysis.

Science.gov (United States)

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-09-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

The PTK7-related transmembrane proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for male fertility.

Directory of Open Access Journals (Sweden)

Karen Linnemannstöns

2014-07-01

Full Text Available Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7 was identified as a Wnt co-receptor required for control of planar cell polarity (PCP in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964, which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.

Stakeholder involvement in the decommissioning of Dounreay

International Nuclear Information System (INIS)

Harrisson, Norman; LOVE, June; Murray, Marc

2006-01-01

The United Kingdom Atomic Energy Authority (UKAEA) was established in the 1950's to pioneer the development of nuclear energy within the UK. Today its primary mission is to decommission UK's former nuclear research sites and restore its environment in a way that is safe and secure, environmentally friendly, value for money and publicly Acceptable. UKAEA Dounreay celebrated its 50 birthday in 2005, having pioneered the development of fast reactor technology since 1955. Today the site is now leading the way in decommissioning. The Dounreay nuclear site licence covers an area of approximately 140 acres and includes 3 reactors: the Dounreay Material Test Reactor (DMTR), the Dounreay Fast Reactor (DFR), and the Prototype Fast Reactor (PFR). In addition there are 180 facilities on site which have supported the fast reactor programme, including a fuel reprocessing capability, laboratories and administration buildings. The reactors are now all in advanced stages of decommissioning. In October 2000 the Dounreay Site Restoration Plan (DSRP) was published to provide a framework for the site's restoration. The plan's objective was to reduce the site's hazards progressively by decontaminating and dismantling the plant, equipment and facilities, remediating contaminated ground and treating and packaging waste so it is suitable for long term storage or disposal. Whilst hailed as the most detailed plan integrating some 1500 activities and spanning 60 years it was criticised for having no stakeholder involvement. In response to this criticism, UKAEA developed a process for public participation over the following 2 years and launched its stakeholder engagement programme in October 2002. In order to provide a larger platform for the engagement process an advertisement was placed in the Scottish media inviting people to register as stakeholders in the Dounreay Site Restoration Plan. The stakeholder list now total over 1000. In October 2002 UKAEA launched their commitment to public

Environmental Regulatory Compliance Plan for site: Draft characterization of the Yucca Mountain site:Draft

International Nuclear Information System (INIS)

1988-01-01

The objective of the EMMP is to document compliance with the NWPA. To do so, a summary description of site characterization activites is provided, based on the consultation draft of the SCP. Subsequent chpaters identify those technical areas having the potential to be impacted by site characterization activities and the monitoring plans proposed to identify whether those impacts acutally occur. Should monitoring confirm the potential for significant adverse impact, mitigative measures will be developed. In the context of site characterization, mitigation is defined as those changes in site characterization activities that serve to avoid or minimize, to the maximum extent practicle, any significant adverse environmental impacts. Although site characterization activies involve both surface and subsurface activities, it is the surface-based aspect of site characterization that is addressed in detailed by the EMMP. The schedule and duration of these activities is given in the consultation draft of the SCP. A breif summary of all proposed activities is given in the EMMP. 10 refs., 8 figs

The Community Environmental Monitoring Program: Reducing Public Perception of Risk through Stakeholder Involvement

International Nuclear Information System (INIS)

William T. Hartwell

2007-01-01

The Community Environmental Monitoring Program (CEMP) has promoted stakeholder involvement, awareness, and understanding of radiological surveillance in communities surrounding the Nevada Test Site (NTS) since 1981. It involves stakeholders in the operation, data collection, and dissemination of information obtained from a network of 29 stations across a wide area of Nevada, Utah and California. It is sponsored by the U.S. Department of Energy, National Nuclear Security Administration's Nevada Site Office (NNSA/NSO) and administered by the Desert Research Institute (DRI) of the Nevada System of Higher Education. Integration of a near real-time communications system, a public web site, training workshops for involved stakeholders, and educational programs all help to alleviate public perception of risk of health effects from past activities conducted at the NTS

The effect of types of banner ad, Web localization, and customer involvement on Internet users' attitudes.

Science.gov (United States)

Chen, Jengchung Victor; Ross, William H; Yen, David C; Akhapon, Lerdsuwankij

2009-02-01

In this study, three characteristics of Web sites were varied: types of banner ad, Web localization, and involvement in purchasing a product. The dependent variable was attitude toward the site. In laboratory experiments conducted in Thailand and Taiwan, participants browsed versions of a Web site containing different types of banner ads and products. As a within-participants factor, each participant browsed both a standardized English-language Web site and a localized Web site. Results showed that animated (rather than static) banner ads, localized versions (rather than a standardized version) of Web sites, and high (rather than low) product involvement led to favorable attitudes toward the site.

A chimeric prokaryotic-eukaryotic pentameric ligand gated ion channel reveals interactions between the extracellular and transmembrane domains shape neurosteroid modulation.

Science.gov (United States)

Ghosh, Borna; Tsao, Tzu-Wei; Czajkowski, Cynthia

2017-10-01

Pentameric ligand-gated ion channels (pLGICs) are the targets of several clinical and endogenous allosteric modulators including anesthetics and neurosteroids. Molecular mechanisms underlying allosteric drug modulation are poorly understood. Here, we constructed a chimeric pLGIC by fusing the extracellular domain (ECD) of the proton-activated, cation-selective bacterial channel GLIC to the transmembrane domain (TMD) of the human ρ1 chloride-selective GABA A R, and tested the hypothesis that drug actions are regulated locally in the domain that houses its binding site. The chimeric channels were proton-gated and chloride-selective demonstrating the GLIC ECD was functionally coupled to the GABAρ TMD. Channels were blocked by picrotoxin and inhibited by pentobarbital, etomidate and propofol. The point mutation, ρ TMD W328M, conferred positive modulation and direct gating by pentobarbital. The data suggest that the structural machinery mediating general anesthetic modulation resides in the TMD. Proton-activation and neurosteroid modulation of the GLIC-ρ chimeric channels, however, did not simply mimic their respective actions on GLIC and GABAρ revealing that across domain interactions between the ECD and TMD play important roles in determining their actions. Proton-induced current responses were biphasic suggesting that the chimeric channels contain an additional proton sensor. Neurosteroid modulation of the GLIC-ρ chimeric channels by the stereoisomers, 5α-THDOC and 5β-THDOC, were swapped compared to their actions on GABAρ indicating that positive versus negative neurosteroid modulation is not encoded solely in the TMD nor by neurosteroid isomer structure but is dependent on specific interdomain connections between the ECD and TMD. Our data reveal a new mechanism for shaping neurosteroid modulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum

Directory of Open Access Journals (Sweden)

Thea Bismo Strøm

2014-01-01

Full Text Available More than 1700 mutations in the low density lipoprotein receptor (LDLR gene have been found to cause familial hypercholesterolemia (FH. These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

Geology, Bedrock, Tabular data involving the location of design specifics for wells related to the Low-level Radioactive Waste (LLRW) Site Characterization., Published in 1998, North Carolina Department of Environment and Natural Resources (DENR).

Data.gov (United States)

NSGIC State | GIS Inventory — Geology, Bedrock dataset current as of 1998. Tabular data involving the location of design specifics for wells related to the Low-level Radioactive Waste (LLRW) Site...

Maize plasma membrane aquaporin ZmPIP2;5, but not ZmPIP1;2, facilitates transmembrane diffusion of hydrogen peroxide.

Science.gov (United States)

Bienert, Gerd P; Heinen, Robert B; Berny, Marie C; Chaumont, François

2014-01-01

Plant aquaporins play important roles in transmembrane water transport processes, but some also facilitate the diffusion of other small uncharged solutes ranging from gases to metalloids. Recent evidence suggests that the transmembrane movement of hydrogen peroxide, an intra- and intercellular multifunctional signaling and defense compound, can be regulated by aquaporins. We addressed the question whether maize aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily facilitate hydrogen peroxide diffusion using heterologous expression in the yeast Saccharomyces cerevisiae. We showed that ZmPIP proteins belonging to the PIP1 and PIP2 groups were significantly expressed in yeast cells only after codon optimization of their cDNA. In accordance with previous localization studies in oocytes and plants, ZmPIP1;2 was mainly retained in intracellular membranes, while ZmPIP2;5 was localized to the plasma membrane. However, upon co-expression with ZmPIP2;5, ZmPIP1;2 was re-localized to the plasma membrane. Using a non-functional plasma membrane-localized ZmPIP2;5 mutant to deliver ZmPIP1;2 to the plasma membrane, we demonstrated that, in contrast to wild type ZmPIP2;5, ZmPIP1;2 was not permeable to hydrogen peroxide. Our study further highlighted the fact that, when using the yeast system, which is widely employed to study substrates for plant aquaporins and other transporters, although positive transport assay results allow direct conclusions to be drawn regarding solute permeability, negative results require additional control experiments to show that the protein is expressed and localized correctly before concluding on the lack of transport activity. © 2013.

The voluntary siting process: The solution to siting in the Northeast Compact

International Nuclear Information System (INIS)

Deshais, J.B.

1995-01-01

The date: June 10, 1991. The decision: the announcement of three 'candidate sites' for a low-level radioactive waste (LLRW) facility in Connecticut following statewide screening. The debate: the right of the citizens of the state to refuse to have this type of facility in their 'backyard' versus the State's responsibility to provide for the safe management of LLRW generated within its borders. The debacle: vigorous opposition, political involvement, no opportunity for effective dialogue with the candidate towns. The end: a legislative mandate to terminate the siting process. This series of events, familiar to those in the business of attempting to site and develop unwanted and unwelcome facilities, would have thwarted efforts to provide for disposal capacity in the Northeast Compact region. Connecticut's efforts to site a LLRW disposal facility pursuant to a traditional 'decide-announce-defend' approach had apparently failed. New Jersey, its partner in the Compact, was also ready to proceed with a similar process that would lead to the naming of several candidate sites, but suspended its efforts to review other siting alternatives. The problem: a new approach was needed. The answer: both states would pursue voluntary siting for the LLRW facilities. The result: the best chance for successful development of LLRW disposal capacity in the Northeast Compact region

Identification and characterization of a novel Plasmodium falciparum adhesin involved in erythrocyte invasion.

Directory of Open Access Journals (Sweden)

Nidhi Hans

Full Text Available Malaria remains a major health problem worldwide. All clinical symptoms of malaria are attributed to the asexual blood stages of the parasite life cycle. Proteins resident in apical organelles and present on the surface of P. falciparum merozoites are considered promising candidates for the development of blood stage malaria vaccines. In the present study, we have identified and characterized a microneme associated antigen, PfMA [PlasmoDB Gene ID: PF3D7_0316000, PFC0700c]. The gene was selected by applying a set of screening criteria such as transcriptional upregulation at late schizogony, inter-species conservation and the presence of signal sequence or transmembrane domains. The gene sequence of PfMA was found to be conserved amongst various Plasmodium species. We experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion. PfMA was localized by immunofluorescence and immuno-electron microscopy to be in the micronemes, an apical organelle of merozoites. The functional role of the PfMA protein in erythrocyte invasion was identified as a parasite adhesin involved in direct attachment with the target erythrocyte. PfMA was demonstrated to bind erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner and its antibodies inhibited P. falciparum erythrocyte invasion. Invasion of erythrocytes is a complex multistep process that involves a number of redundant ligand-receptor interactions many of which still remain unknown and even uncharacterized. Our work has identified and characterized a novel P. falciparum adhesin involved in erythrocyte invasion.

Site characterization plan overview: Yucca Mountain Site, Nevada Research and Development Area, Nevada: Consultation Draft

International Nuclear Information System (INIS)

1988-01-01

The consultation draft of the site characterization plan is a lengthy document that describes in considerable detail the program that will be conducted to characterize the geologic, hydrologic, and other conditions relevant to the suitability of the site for a repository. The overview presented here consists of brief summaries of important topics covered in the consultation draft of the site-characterization plan; it is not a substitute for the site-characterization plan. The arrangement of the overview is similar to that of the plan itself, with brief descriptions of the disposal system -- the site, the repository, and the waste package -- preceding the discussion of the characterization program to be carried out at the Yucca Mountain site. It is intended primarily for the management staff of organizations involved in the DOE's repository program -- staff who might wish to understand the general scope of the site-characterization program, the activities to be conducted, and the facilities to be constructed rather than the technical details of site characterization. 22 figs., 1 tab

Gains of ubiquitylation sites in highly conserved proteins in the human lineage

Directory of Open Access Journals (Sweden)

Kim Dong Seon

2012-11-01

Full Text Available Abstract Background Post-translational modification of lysine residues of specific proteins by ubiquitin modulates the degradation, localization, and activity of these target proteins. Here, we identified gains of ubiquitylation sites in highly conserved regions of human proteins that occurred during human evolution. Results We analyzed human ubiquitylation site data and multiple alignments of orthologous mammalian proteins including those from humans, primates, other placental mammals, opossum, and platypus. In our analysis, we identified 281 ubiquitylation sites in 252 proteins that first appeared along the human lineage during primate evolution: one protein had four novel sites; four proteins had three sites each; 18 proteins had two sites each; and the remaining 229 proteins had one site each. PML, which is involved in neurodevelopment and neurodegeneration, acquired three sites, two of which have been reported to be involved in the degradation of PML. Thirteen human proteins, including ERCC2 (also known as XPD and NBR1, gained human-specific ubiquitylated lysines after the human-chimpanzee divergence. ERCC2 has a Lys/Gln polymorphism, the derived (major allele of which confers enhanced DNA repair capacity and reduced cancer risk compared with the ancestral (minor allele. NBR1 and eight other proteins that are involved in the human autophagy protein interaction network gained a novel ubiquitylation site. Conclusions The gain of novel ubiquitylation sites could be involved in the evolution of protein degradation and other regulatory networks. Although gains of ubiquitylation sites do not necessarily equate to adaptive evolution, they are useful candidates for molecular functional analyses to identify novel advantageous genetic modifications and innovative phenotypes acquired during human evolution.

G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

Science.gov (United States)

Kratochwil, Nicole A; Gatti-McArthur, Silvia; Hoener, Marius C; Lindemann, Lothar; Christ, Andreas D; Green, Luke G; Guba, Wolfgang; Martin, Rainer E; Malherbe, Pari; Porter, Richard H P; Slack, Jay P; Winnig, Marcel; Dehmlow, Henrietta; Grether, Uwe; Hertel, Cornelia; Narquizian, Robert; Panousis, Constantinos G; Kolczewski, Sabine; Steward, Lucinda

2011-01-01

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor. © 2011 Bentham Science Publishers

Strategic planning model for achieving stakeholder involvement in environmental at DOE weapons complex sites

International Nuclear Information System (INIS)

Weber, G.

1994-01-01

Within today's reality a public manager often needs to develop cooperative relationships among a number of individual, program, and organizational stakeholders to accomplish particular projects, programs, or policies. A DOE site manager charged with accomplishing environmental restoration and conversion at former weapons production sites is no exception. Important reasons for this include the technical and political complexity of the clean-up problem; limits on the funding, authority, and other resources available to DOE; authority, responsibilities, and interests of other stakeholders; and the ever present potential for conflict among stakeholders, and power of any one to hinder, if not halt, the clean-up process if conflicts aren't managed and cooperative relationships established and maintained

Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α2-adrenergic receptor

International Nuclear Information System (INIS)

Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W.

1989-01-01

The human platelet α 2 -adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [ 3 H]SKF 102229 (an antagonist) or p-azido[ 3 H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [ 3 H]SKF 102229 labeled receptor yielded one peptide of M r 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M r 4000, which was further digested to the M r 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[ 3 H]clonidine-labeled receptor, a similar M r 2400 peptide was obtained by lysylendopeptidase cleavage. This M r 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α 2 -adrenergic receptor

Opinion on the radio-ecological monitoring of waters around nuclear installations and on the management of old nuclear waste warehousing sites: 18 recommendations to improve information, transparency and dialogue with involved parties

International Nuclear Information System (INIS)

2008-01-01

After a presentation of the different actors (agencies, institutions, companies) involved in the activities of the various French nuclear installations (base nuclear installations and those concerning the national defence), this report describes the radio-ecological monitoring performed around these nuclear sites: water surveillance on these sites and within their environment, regulatory requirements on effluents and surveillance, information provided by operators and by institutional organisations, assessment of the radio-ecological status of nuclear sites and of potential environmental and health impacts. It describes regulatory obligation in terms of public information, information and communication actions, and gives an assessment of the High committee about public information quality. It discusses ways to improve this quality for a higher transparency, to reinforce the role of local information commissions (CLI), and improve site monitoring. All these aspects are grouped in 18 recommendations

Accelerated SDS depletion from proteins by transmembrane electrophoresis: Impacts of Joule heating.

Science.gov (United States)

Unterlander, Nicole; Doucette, Alan Austin

2018-02-08

SDS plays a key role in proteomics workflows, including protein extraction, solubilization and mass-based separations (e.g. SDS-PAGE, GELFrEE). However, SDS interferes with mass spectrometry and so it must be removed prior to analysis. We recently introduced an electrophoretic platform, termed transmembrane electrophoresis (TME), enabling extensive depletion of SDS from proteins in solution with exceptional protein yields. However, our prior TME runs required 1 h to complete, being limited by Joule heating which causes protein aggregation at higher operating currents. Here, we demonstrate effective strategies to maintain lower TME sample temperatures, permitting accelerated SDS depletion. Among these strategies, the use of a magnetic stir bar to continuously agitate a model protein system (BSA) allows SDS to be depleted below 100 ppm (>98% removal) within 10 min of TME operations, while maintaining exceptional protein recovery (>95%). Moreover, these modifications allow TME to operate without any user intervention, improving throughput and robustness of the approach. Through fits of our time-course SDS depletion curves to an exponential model, we calculate SDS depletion half-lives as low as 1.2 min. This promising electrophoretic platform should provide proteomics researchers with an effective purification strategy to enable MS characterization of SDS-containing proteins. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plant virus cell-to-cell movement is not dependent on the transmembrane disposition of its movement protein.

Science.gov (United States)

Martínez-Gil, Luis; Sánchez-Navarro, Jesús A; Cruz, Antonio; Pallás, Vicente; Pérez-Gil, Jesús; Mingarro, Ismael

2009-06-01

The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.

Site acceptability and power availability: needed institutional changes

International Nuclear Information System (INIS)

Haggard, J.E.

1975-01-01

Timely assurance of power plant site availability is threatened by institutional inabilities to resolve often competing environmental/energy requirements. Institutional changes are needed. The issue of site approval should be separated from that of plant approval. A ''one-stop'' forum for site approval, modeled after Washington State's Thermal Power Plant Siting Act, is needed. The one-stop process utilizes one forum composed of officials drawn from all agencies involved in site related issues. A joint Federal/State Siting Council, with sole jurisdiction over site approval, is recommended. The State Council would have a determinative vote on all issues not otherwise preempted by federal legislation. 21 references. (U.S.)

Bone marrow involvement by lymphoproliferative disorders after renal transplantation: PTLD. Int. Survey

Directory of Open Access Journals (Sweden)

Morteza Izadi

2012-01-01

Conclusions: Renal recipients with BM PTLD represent worse outcome and more unfavorable histopathological phenomenon than in other organ involvements. Moreover, a concomitant PTLD involvement site in liver was found which necessitates full hepatic evaluation for a potential complication by the disease in renal recipients whose BM is involved.

Geothermal Well Site Restoration and Plug and Abandonment of Wells

Energy Technology Data Exchange (ETDEWEB)

Rinehart, Ben N.

1994-08-01

A report is presented on the final phase of an energy research program conducted by the U.S. Department of Energy (DOE) involving two geothermal well sites in the State of Louisiana-the Gladys McCall site and the Willis Hulin site. The research program was intended to improve geothermal technology and to determine the efficacy of producing electricity commercially from geopressured resource sites. The final phase of the program consisted of plug and abandonment (P&A) of the wells and restoration of the well sites. Restoration involved (a) initial soil and water sampling and analysis; (b) removal and disposal of well pads, concrete, utility poles, and trash; (c) plugging of monitor and freshwater wells; and (d) site leveling and general cleanup. Restoration of the McCall site required removal of naturally occurring radioactive material (NORM), which was costly and time-consuming. Exhibits are included that provide copies of work permits and authorizations, P&A reports and procedures, daily workover and current conditions report, and cost and salvage reports. Site locations, grid maps, and photographs are provided.

CUTANEOUS INVOLVEMENT IN MULTIPLE MYELOMA AT AN UNUSUAL SITE: A RARE CASE REPORT

Directory of Open Access Journals (Sweden)

Lohit Kumar

2015-05-01

Full Text Available Multiple myeloma is a rare cancer. According to the most recent data from the Surveillance, Epidemiology, and End Results (SEER program, multiple myeloma is the second most common haematological malignancy in the U.S. (after non - Hodgkin lymphoma, constitutes 1% of all cancers and constitutes 2% of all cancer deaths. Cutaneous involvement of multiple myeloma during treatment period is uncommon with fewer described in literature. Moreover, metastatic cutaneous involvement at the sole of the foot during treatment period of a IgA kappa type multiple myeloma patient followed by death has not encountered in literature. We have reported such a case

The observational approach for site remediation at federal facilities

International Nuclear Information System (INIS)

Myers, R.S.; Gianti, S.J.

1989-11-01

The observational approach, developed by geotechnical engineers to cope with the uncertainty associated with subsurface construction such as tunnels and dams, can be applied to hazardous waste site remediation. During the last year, the observational approach has gained increasing attention as a means of addressing the uncertainties involved in site remediation. In order to evaluate the potential advantages and constraints of applying the observational approach to site restoration at federal facilities, a panel of scientists and engineers from Pacific Northwest Laboratory and CH2M Hill was convened. Their review evaluated potential technical and institutional advantages and constraints that may affect the use of the observational approach for site remediation. This paper summarizes the panel's comments and conclusions about the application of the observational approach to site remediation at federal facilities. Key issues identified by the panel include management of uncertainty, cost and schedule, regulations and guidance, public involvement, and implementation. 5 refs

MemBrain: An Easy-to-Use Online Webserver for Transmembrane Protein Structure Prediction

Science.gov (United States)

Yin, Xi; Yang, Jing; Xiao, Feng; Yang, Yang; Shen, Hong-Bin

2018-03-01

Membrane proteins are an important kind of proteins embedded in the membranes of cells and play crucial roles in living organisms, such as ion channels, transporters, receptors. Because it is difficult to determinate the membrane protein's structure by wet-lab experiments, accurate and fast amino acid sequence-based computational methods are highly desired. In this paper, we report an online prediction tool called MemBrain, whose input is the amino acid sequence. MemBrain consists of specialized modules for predicting transmembrane helices, residue-residue contacts and relative accessible surface area of α-helical membrane proteins. MemBrain achieves a prediction accuracy of 97.9% of A TMH, 87.1% of A P, 3.2 ± 3.0 of N-score, 3.1 ± 2.8 of C-score. MemBrain-Contact obtains 62%/64.1% prediction accuracy on training and independent dataset on top L/5 contact prediction, respectively. And MemBrain-Rasa achieves Pearson correlation coefficient of 0.733 and its mean absolute error of 13.593. These prediction results provide valuable hints for revealing the structure and function of membrane proteins. MemBrain web server is free for academic use and available at www.csbio.sjtu.edu.cn/bioinf/MemBrain/. [Figure not available: see fulltext.

Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

Directory of Open Access Journals (Sweden)

Thomas A Prohaska

Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

Trans-membrane electron transfer in red blood cells immobilized in a chitosan film on a glassy carbon electrode

International Nuclear Information System (INIS)

Yu, Chunmei; Wang, Li; Zhu, Zhenkun; Bao, Ning; Gu, Haiying

2014-01-01

We have studied the trans-membrane electron transfer in human red blood cells (RBCs) immobilized in a chitosan film on a glassy carbon electrode (GCE). Electron transfer results from the presence of hemoglobin (Hb) in the RBCs. The electron transfer rate (k s ) of Hb in RBCs is 0.42 s −1 , and <1.13 s −1 for Hb directly immobilized in the chitosan film. Only Hb molecules in RBCs that are closest to the plasma membrane and the surface of the electrode can undergo electron transfer to the electrode. The immobilized RBCs displayed sensitive electrocatalytic response to oxygen and hydrogen peroxide. It is believed that this cellular biosensor is of potential significance in studies on the physiological status of RBCs based on observing their electron transfer on the modified electrode. (author)

Transmembrane neural cell-adhesion molecule (NCAM), but not glycosyl-phosphatidylinositol-anchored NCAM, down-regulates secretion of matrix metalloproteinases

DEFF Research Database (Denmark)

Edvardsen, K; Chen, W; Rucklidge, G

1993-01-01

proteinases, and proteinase inhibitors all participate in the construction, maintenance, and remodeling of extracellular matrix by cells. The neural cell-adhesion molecule (NCAM)-negative rat glioma cell line BT4Cn secretes substantial amounts of metalloproteinases, as compared with its NCAM-positive mother......During embryogenesis interactions between cells and extracellular matrix play a central role in the modulation of cell motility, growth, and differentiation. Modulation of matrix structure is therefore crucial during development; extracellular matrix ligands, their receptors, extracellular...... cell line BT4C. We have transfected the BT4Cn cell line with cDNAs encoding the human NCAM-B and -C isoforms. We report here that the expression of transmembrane NCAM-B, but not of glycosyl-phosphatidylinositol-linked NCAM-C, induces a down-regulation of 92-kDa gelatinase (matrix metalloproteinase 9...

An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

Science.gov (United States)

Jaquemar, D; Schenker, T; Trueb, B

1999-03-12

We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

Declassification and restoration of nuclear sites; Declassement et restauration des sites nucleaires

Energy Technology Data Exchange (ETDEWEB)

Noynaert, L; Rahier, A; Deboodt, P; Massaut, V

1998-09-01

The report describes the legal and technical aspects of the declassification and restoration of nuclear sites. This involves a number of technical and administrative operations. Different declassification strategies are discussed. The evaluation of the risks and impact on the environment are discussed as well as research and development needs, costs and possible sources for funding.

Cleanup around an old waste site

International Nuclear Information System (INIS)

Vandergaast, G.; Moffett, D.; Lawrence, B.E.

1988-01-01

42,500 m 3 of contaminated soil were removed from off-site areas around an old, low-level radioactive waste site near Port Hope, Ontario. The cleanup was done by means of conventional excavation equipment to criteria developed by Eldorado specific to the land use around the company's waste management facility. These cleanup criteria were based on exposure analyses carried out for critical receptors in two different scenarios. The excavated soils, involving eight different landowners, were placed on the original burial area of the waste management facility. Measures were also undertaken to stabilize the soils brought on-site and to ensure that there would be no subsequent recontamination of the off-site areas

Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and opportunities for mutation-specific treatment.

LENUS (Irish Health Repository)

Rogan, Mark P

2012-02-01

Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?

The disposal of Canada's nuclear fuel waste: site screening and site evaluation technology

International Nuclear Information System (INIS)

Davison, C.C.; Brown, A.; Everitt, R.A.; Gascoyne, M.; Kozak, E.T.; Lodha, G.S.; Martin, C.D.; Soonawala, N.M.; Stevenson, D.R.; Thorne, G.A.; Whitaker, S.H.

1994-06-01

The concept for the disposal of Canada's nuclear fuel waste is to dispose of the waste in an underground vault, nominally at 500 m to 1000 m depth, at a suitable site in plutonic rock of the Canadian Shield. The feasibility of this concept and assessments of its impact on the environment and human health, will be documented by AECL in an Environmental Impact Statement (EIS). This report is one of nine primary references for the EIS. It describes the approach and methods that would be used during the siting stage of the disposal project to identify a preferred candidate disposal site and to confirm its suitability for constructing a disposal facility. The siting stage is divided into two distinct but closely related substages, site screening and site evaluation. Site screening would mainly involve reconnaissance investigations of siting regions of the Shield to identify potential candidate areas where suitable vault locations are likely to exist. Site screening would identify a small number of candidate areas where further detailed investigations were warranted. Site evaluation would involve progressively more detailed surface and subsurface investigations of the candidate areas to first identify potentially suitable vault locations within the candidate areas, and then characterize these potential disposal sites to identify the preferred candidate location for constructing the disposal vault. Site evaluation would conclude with the construction of exploratory shafts and tunnels at the preferred vault location, and underground characterization would be done to confirm the suitability of the preferred candidate site. An integrated program of geological, geophysical, hydrogeological, geochemical and geomechanical investigations would be implemented to obtain the geoscience information needed to assess the suitability of the candidate siting areas and candidate sites for locating a disposal vault. The candidate siting areas and candidate disposal vault sites would be

Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation

Directory of Open Access Journals (Sweden)

Kazutaka Sawada

2016-01-01

Full Text Available Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus. Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation.

Dissection of seroreactivity against the tryptophan-rich motif of the feline immunodeficiency virus transmembrane glycoprotein

International Nuclear Information System (INIS)

Freer, Giulia; Giannecchini, Simone; Tissot, Alain; Bachmann, Martin F.; Rovero, Paolo; Serres, Pierre Francoise; Bendinelli, Mauro

2004-01-01

Immunogenicity of the tryptophan-rich motif (TrpM) in the membrane-proximal ectodomain of the transmembrane (TM) glycoprotein of feline immunodeficiency virus (FIV) was investigated. Peptide 59, a peptide containing the TrpM of the TM of FIV, was covalently coupled to Qβ phage virus-like particles (Qβ-59) in the attempt to induce potent anti-TrpM B cell responses in cats. All Qβ-59 immunized cats, but not cats that received a mixture of uncoupled Qβ and peptide 59, developed antibodies that reacted with a same epitope in extensive binding and binding competition assays. The epitope recognized was composed of three amino acids, two of which are adjacent. However, Qβ-59-immune sera failed to recognize whole FIV in all binding and neutralization assays performed. Furthermore, no reactivity against the TrpM was detected by screening sera from FIV-infected cats that had reacted with TM peptides, confirming that this epitope does not seem to be serologically functional in the FIV virion. The data suggest that TrpM may not be a suitable target for antiviral vaccine design

Site Decommissioning Management Plan. Supplement 1

International Nuclear Information System (INIS)

Fauver, D.N.; Weber, M.F.; Johnson, T.C.; Kinneman, J.D.

1995-11-01

The Nuclear Regulatory Commission (NRC) staff has identified 51 sites contaminated with radioactive material that require special attention to ensure timely decommissioning. While none of these sites represent an immediate threat to public health and safety, they have contamination that exceeds existing NRC criteria for unrestricted use. All of these sites require some degree of remediation, and several involve regulatory issues that must be addressed by the Commission before they can be released for unrestricted use and the applicable licenses terminated. This report contains the NRC stairs strategy for addressing the technical, legal, and policy issues affecting the timely decommissioning of the 51 sites and describes the status of decommissioning activities at the sites. This is supplement number one to NUREG-1444, which was published in October 1993

Roles of transmembrane segment M1 of Na(+),K (+)-ATPase and Ca (2+)-ATPase, the gatekeeper and the pivot

DEFF Research Database (Denmark)

Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente

2007-01-01

In this review we summarize mutagenesis work on the structure-function relationship of transmembrane segment M1 in the Na(+),K(+)-ATPase and the sarco(endo)plasmic reticulum Ca(2+)-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported...... cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca(2+) interaction/occlusion in Ca(2+)-ATPase and K(+) interaction/occlusion in Na(+),K(+)-ATPase. Leu(65) of the Ca(2+)-ATPase and Leu(99) of the Na(+),K(+)-ATPase, located...... of the extracytoplasmic gate in both the Ca(2+)-ATPase and the Na(+),K(+)-ATPase. Udgivelsesdato: 2007-Dec...

Old radioactive waste storage sites

International Nuclear Information System (INIS)

2008-01-01

After a recall of the regulatory context for the management of old sites used for the storage of radioactive wastes with respect with their activity, the concerned products, the disposal or storage type, this document describes AREVA's involvement in the radioactive waste management process in France. Then, for the different kinds of sites (currently operated sites having radioactive waste storage, storage sites for uranium mineral processing residues), it indicates their location and name, their regulatory status and their control authority, the reference documents. It briefly presents the investigation on the long term impact of uranium mineral processing residues on health and environment, evokes some aspects of public information transparency, and presents the activities of an expertise group on old uranium mines. The examples of the sites of Bellezane (uranium mineral processing residues) and COMURHEX Malvesi (assessment of underground and surface water quality at the vicinity of this installation) are given in appendix

Primary cutaneous amyloidosis involving the external ears along with the classical sites

Directory of Open Access Journals (Sweden)

Khaitan Binod

2001-01-01

Full Text Available A 26- year- old woman had multiple itchy persistent gradually progressive papular lesions on the forearms and shins for 10 and 4 years respectively. She also noticed similar lesions on both the ears for 4 years. There were no systemic symptoms. Cutaneous examination revealed multiple 2-3 mm discrete firm hyperpigmented papules on the extensors of forearms, shins and earlobes. Skin biopsy from all sites demonstrated deposits of amyloid in the papillary dermis. The patient was treated with cyclophosphamide 50 mg daily orally. There was more than 50% improvement in her lesions.

Nuclear power plant site selection: a case study

International Nuclear Information System (INIS)

Lugasi, Y.; Mehrez, A.; Sinuany-Stern, Z.

1985-01-01

Selecting the site for a nuclear power plant involves the evaluation of numerous criteria and the professional judgment of various experts. The Israel Atomic Energy Commission has been concerned with the problem of selecting a site for a nuclear power station. Previous studies have been performed by the commission to identify potential sites. There were initial screenings where potential sites were chosen according to various minimal criteria and international standards. Only sites that met all the criteria were chosen. A study was made to find the most preferred site among the potential sites that met all the criteria. Two mathematical approaches were used: Keeney's multiattribute utility function and Saaty's eigenvalue prioritization technique. Both models ranked the same site as the most desirable; however, the models differed in their ranking of the other sites

Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

Directory of Open Access Journals (Sweden)

Metzger Kelsey J

2010-05-01

Full Text Available Abstract Background CC chemokine receptor proteins (CCR1 through CCR10 are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions. In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events. Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR gene family. The results of neutral vs. adaptive evolution (positive selection hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or ω >1. Results Of the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive

MPL-net at ARM Sites

Science.gov (United States)

Spinhirne, J. D.; Welton, E. J.; Campbell, J. R.; Berkoff, T. A.; Starr, David OC. (Technical Monitor)

2002-01-01

The NASA MPL-net project goal is consistent data products of the vertical distribution of clouds and aerosol from globally distributed lidar observation sites. The four ARM micro pulse lidars are a basis of the network to consist of over twelve sites. The science objective is ground truth for global satellite retrievals and accurate vertical distribution information in combination with surface radiation measurements for aerosol and cloud models. The project involves improvement in instruments and data processing and cooperation with ARM and other partners.

Beta2-adrenergic receptor homodimers: Role of transmembrane domain 1 and helix 8 in dimerization and cell surface expression.

Science.gov (United States)

Parmar, Vikas K; Grinde, Ellinor; Mazurkiewicz, Joseph E; Herrick-Davis, Katharine

2017-09-01

Even though there are hundreds of reports in the published literature supporting the hypothesis that G protein-coupled receptors (GPCR) form and function as dimers this remains a highly controversial area of research and mechanisms governing homodimer formation are poorly understood. Crystal structures revealing homodimers have been reported for many different GPCR. For adrenergic receptors, a potential dimer interface involving transmembrane domain 1 (TMD1) and helix 8 (H8) was identified in crystal structures of the beta 1 -adrenergic (β 1 -AR) and β 2 -AR. The purpose of this study was to investigate a potential role for TMD1 and H8 in dimerization and plasma membrane expression of functional β 2 -AR. Charged residues at the base of TMD1 and in the distal portion of H8 were replaced, singly and in combination, with non-polar residues or residues of opposite charge. Wild type and mutant β 2 -AR, tagged with YFP and expressed in HEK293 cells, were evaluated for plasma membrane expression and function. Homodimer formation was evaluated using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and fluorescence correlation spectroscopy. Amino acid substitutions at the base of TMD1 and in the distal portion of H8 disrupted homodimer formation and caused receptors to be retained in the endoplasmic reticulum. Mutations in the proximal region of H8 did not disrupt dimerization but did interfere with plasma membrane expression. This study provides biophysical evidence linking a potential TMD1/H8 interface with ER export and the expression of functional β 2 -AR on the plasma membrane. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

Involved Node, Site, Field and Residual Volume Radiotherapy for Lymphoma: A Comparison of Organ at Risk Dosimetry and Second Malignancy Risks.

Science.gov (United States)

Murray, L; Sethugavalar, B; Robertshaw, H; Bayman, E; Thomas, E; Gilson, D; Prestwich, R J D

2015-07-01

Recent radiotherapy guidelines for lymphoma have included involved site radiotherapy (ISRT), involved node radiotherapy (INRT) and irradiation of residual volume after full-course chemotherapy. In the absence of late toxicity data, we aim to compare organ at risk (OAR) dose-metrics and calculated second malignancy risks. Fifteen consecutive patients who had received mediastinal radiotherapy were included. Four radiotherapy plans were generated for each patient using a parallel pair photon technique: (i) involved field radiotherapy (IFRT), (ii) ISRT, (iii) INRT, (iv) residual post-chemotherapy volume. The radiotherapy dose was 30 Gy in 15 fractions. The OARs evaluated were: breasts, lungs, thyroid, heart, oesophagus. Relative and absolute second malignancy rates were estimated using the concept of organ equivalent dose. Significance was defined as P risks of second cancers were significantly higher with IFRT compared with ISRT for lung, breast and thyroid; INRT and residual volume resulted in significantly lower relative risks compared with ISRT for lung, breast and thyroid. The median excess absolute risks of second cancers were consistently lowest for the residual technique and highest for IFRT in terms of thyroid, lung and breast cancers. The risk of oesophageal cancer was similar for all four techniques. Overall, the absolute risk of second cancers was very similar for ISRT and INRT. Decreasing treatment volumes from IFRT to ISRT, INRT or residual volume reduces radiation exposure to OARs. Second malignancy modelling suggests that this reduction in treatment volumes will lead to a reduction in absolute excess second malignancy. Little difference was observed in second malignancy risks between ISRT and INRT, supporting the use of ISRT in the absence of a pre-chemotherapy positron emission tomography scan in the radiotherapy treatment position. Copyright © 2015 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

Science.gov (United States)

Cvicek, Vaclav; Goddard, William A.; Abrol, Ravinder

2016-01-01

The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs

Nuclear Power Plant project site selection geotechnical considerations

International Nuclear Information System (INIS)

Katti, V.J.; Banerjee, D.C.

1997-01-01

During the selection of a site for Nuclear Power Plant (NPP) and Radioactive Waste Plant (RWP), geotechnical investigations play a significant role in deciding merits and demerits of the sites. Any accidents in these units can play havoc on mankind and may leave bitter imprints on generations to come. Hence proper care has to be taken at the early stage for selecting the sites. Site selection procedure is a complicated one, because it involves experts from various disciplines like geology, geophysics, civil, mechanical electrical engineering, health-physics and other fields

Germline variant FGFR4  p.G388R exposes a membrane-proximal STAT3 binding site.

Science.gov (United States)

Ulaganathan, Vijay K; Sperl, Bianca; Rapp, Ulf R; Ullrich, Axel

2015-12-24

Variant rs351855-G/A is a commonly occurring single-nucleotide polymorphism of coding regions in exon 9 of the fibroblast growth factor receptor FGFR4 (CD334) gene (c.1162G>A). It results in an amino-acid change at codon 388 from glycine to arginine (p.Gly388Arg) in the transmembrane domain of the receptor. Despite compelling genetic evidence for the association of this common variant with cancers of the bone, breast, colon, prostate, skin, lung, head and neck, as well as soft-tissue sarcomas and non-Hodgkin lymphoma, the underlying biological mechanism has remained elusive. Here we show that substitution of the conserved glycine 388 residue to a charged arginine residue alters the transmembrane spanning segment and exposes a membrane-proximal cytoplasmic signal transducer and activator of transcription 3 (STAT3) binding site Y(390)-(P)XXQ(393). We demonstrate that such membrane-proximal STAT3 binding motifs in the germline of type I membrane receptors enhance STAT3 tyrosine phosphorylation by recruiting STAT3 proteins to the inner cell membrane. Remarkably, such germline variants frequently co-localize with somatic mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Using Fgfr4 single nucleotide polymorphism knock-in mice and transgenic mouse models for breast and lung cancers, we validate the enhanced STAT3 signalling induced by the FGFR4 Arg388-variant in vivo. Thus, our findings elucidate the molecular mechanism behind the genetic association of rs351855 with accelerated cancer progression and suggest that germline variants of cell-surface molecules that recruit STAT3 to the inner cell membrane are a significant risk for cancer prognosis and disease progression.

Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

Science.gov (United States)

Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun

2018-04-15

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of

Coordinated site characterization and performance assessment - an iterative approach for the site evaluation

International Nuclear Information System (INIS)

Papp, T.; Ericsson, L.O.; Thegerstroem, C.; Almen, K.E.

1995-01-01

SKB planning for siting a deep repository involves feasibility studies in 5-10 municipalities surface based characterization and drilling on two candidate sites and detailed characterization of one site including a shaft to proposed repository depth. The selection of a site or the detailed layout of the repository defines characteristics that might influence safety in a broad sense. There is a strong ling between the safety, technical (engineering) and functional aspects. The site selection will be based on general geoscientific information, i.e. mechanical stability, ground-water chemistry, slow ground-water movements and complicating factors like high potential for mineralization. The general layout of the repository in the actual geological structure of the site must be done with regard to a number of guidelines, e.g. to hydraulically separate the parts of the repository containing the spent nuclear fuel from those for other types of long lived waste and to separate the two stages of the spent fuel repository so they can be handled separately in the licensing process. When the various parts of the repository have been tentatively located the consequence of the multiple barrier principle is that the layout of the various parts should be made with the aim to utilize the available natural barrier system at the site as well as possible. (authors). 2 refs., 3 figs., 2 tabs

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein▿ †

Science.gov (United States)

Martínez-Gil, Luis; Sánchez-Navarro, Jesús A.; Cruz, Antonio; Pallás, Vicente; Pérez-Gil, Jesús; Mingarro, Ismael

2009-01-01

The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement. PMID:19321624

NMR studies of the fifth transmembrane segment of Na+,K+-ATPase reveals a non-helical ion-binding region

DEFF Research Database (Denmark)

Underhaug, Jarl; Jakobsen, Louise Odgaard; Esmann, Mikael

2006-01-01

The structure of a synthetic peptide corresponding to the fifth membrane-spanning segment (M5) in Na(+),K(+)-ATPase in sodium dodecyl sulfate (SDS) micelles was determined using liquid-state nuclear magnetic resonance (NMR) spectroscopy. The spectra reveal that this peptide is substantially less...... transmembrane element of the Ca(2+)-ATPase. Furthermore, this region spans the residues implicated in Na(+) and K(+) transport, where they are likely to offer the flexibility needed to coordinate Na(+) as well as K(+) during active transport....... alpha-helical than the corresponding M5 peptide of Ca(2+)-ATPase. A well-defined alpha-helix is shown in the C-terminal half of the peptide. Apart from a short helical stretch at the N-terminus, the N-terminal half contains a non-helical region with two proline residues and sequence similarity to a non-structured...

Molecular mechanism of 7TM receptor activation--a global toggle switch model

DEFF Research Database (Denmark)

Schwartz, Thue W; Frimurer, Thomas M; Holst, Birgitte

2006-01-01

the accumulated biophysical data supporting an outward rigid-body movement of the intracellular segments, as well as the recent data derived from activating metal ion sites and tethered ligands, which suggests an opposite, inward movement of the extracellular segments of the transmembrane helices. According...... to this model, a vertical see-saw movement of TM-VI-and to some degree TM-VII-around a pivot corresponding to the highly conserved prolines will occur during receptor activation, which may involve the outer segment of TM-V in an as yet unclear fashion. Small-molecule agonists can stabilize such a proposed...

Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

Science.gov (United States)

Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

2015-08-14

Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4

Science.gov (United States)

Auvray, Frédéric; Coddeville, Michèle; Ordonez, Romy Catoira; Ritzenthaler, Paul

1999-01-01

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3′ end of a tRNASer gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNASer gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model. PMID:10572145

Long-term monitoring for closed special sites

International Nuclear Information System (INIS)

Golchert, N.W.; Sedlet, J.

1987-01-01

A methodology is presented for planning and implementing a long-term environmental monitoring program for closed special radioactive waste disposal sites. The steps in the method involve collection of the available background information on the site history, site and area characteristics, waste inventory, pathway analysis, prior monitoring programs, applicable standards, and the legal/regulatory requirements. This information is coupled with factors such as experience, half-life, radionuclide migration rates, and potential hazard to develop a monitoring program. As an example, a site-specific long-term monitoring program is described for the AMAX site using the available information. Sampling techniques and practices for the monitoring program are discussed and techniques and practices expected to be available in the future are considered. 7 references, 2 tables

DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

Science.gov (United States)

Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

2013-01-01

Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

Posttransplantation lymphoproliferative disease involving the pituitary gland.

Science.gov (United States)

Meriden, Zina; Bullock, Grant C; Bagg, Adam; Bonatti, Hugo; Cousar, John B; Lopes, M Beatriz; Robbins, Mark K; Cathro, Helen P

2010-11-01

Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus-negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD. Copyright © 2010 Elsevier Inc. All rights reserved.

Architecture and Development of DCMP Web Site

Science.gov (United States)

Bariakhtar, Irina

2003-03-01

The multi-tier implementation of DCMP Web site is discussed. It is based upon newly developed PHP technology. The technology allows for creating dynamic content and scalable solutions for Web site capabilities. There are several aspects as to what type of information is to be on the site. First, it should serve the immediate needs of the researchers in the field, namely, conferences, journals, news, funds, etc. This is currently available on the site, but can be extended and improved if needed. Second, the site will reflect the connection between Condensed matter physics and the technological breakthroughs that drive the economy. Third, the site will carry an educational mission helping educate the general public, and on the other hand, help young people to start their careers in the field. The content of the DCMP Web site is under active development. It depends upon wide involvement of DCMP members.

Intramolecular cross-linking in a bacterial homolog of mammalian SLC6 neurotransmitter transporters suggests an evolutionary conserved role of transmembrane segments 7 and 8

DEFF Research Database (Denmark)

Kniazeff, Julie; Loland, Claus Juul; Goldberg, Naomi

2005-01-01

The extracellular concentration of the neurotransmitters dopamine, serotonin, norepinephrine, GABA and glycine is tightly controlled by plasma membrane transporters belonging to the SLC6 gene family. A very large number of putative transport proteins with a remarkable homology to the SLC6...... proximity between TM 7 and 8 in the tertiary structure of TnaT as previously suggested for the mammalian counterparts. Furthermore, the inhibition of uptake upon cross-linking the two cysteines provides indirect support for a conserved conformational role of these transmembrane domains in the transport...

A hybrid peptide PTS that facilitates transmembrane delivery and its application for the rapid in vivo imaging via near-infrared fluorescence imaging

Directory of Open Access Journals (Sweden)

Xuejiao eYan

2016-03-01

Full Text Available Background and purpose: Intravital imaging provides invaluable readouts for clinical diagnoses and therapies and shows great potential in the design of individualized drug dosage regimes. Ts is a mammalian free cell membrane-penetrating peptide. This study aimed to introduce a novel approach to the design of a cancer-selective peptide on the basis of a membrane-penetrating peptide and to explore its potential as a carrier of medical substances. Experimental approach: Ts was linked with a αvβ3-binding peptide P1c to create a hybrid referred to as PTS. The hybrid was labeled with an FITC or Cy5.5 as an imaging indicator to evaluate its in vitro and in vivo bioactivity. Key results: Hemolysis tests proved that in comparison with Ts, PTS caused similar or even less leakage of human erythrocytes at concentrations of up to 1 mmol/L. Flow cytometry assay and confocal microscopy demonstrated the following. 1 P1c alone could target and mostly halt at the cancer cell membrane. 2 Ts alone could not bind to the membrane sufficiently. 3 P1c greatly enhanced the binding affinity of PTS with MDA-MB-231 breast cancer cells that upregulated αvβ3. 4 Ts conferred PTS with the ability to traverse a cell membrane and thus facilitate the transmembrane delivery of imaging probes. In vivo near-infrared fluorescence (NIRF imaging demonstrated that the imaging probes were rapidly concentrated in a MDA-MB-231 tumor tissue within 1 h after intravenous injection. Conclusions and implications: PTS exhibited the capability of targeting specific tumors and greatly facilitating the transmembrane delivery of imaging probes.

Muscarinic acetylcholine receptors: location of the ligand binding site

International Nuclear Information System (INIS)

Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

1987-01-01

The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, 3 H-propylbenzilycholine mustard aziridinium ion ( 3 H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that 3 H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin

Integration of a working site in its regional context

International Nuclear Information System (INIS)

Couture, J.

1986-01-01

This paper shows how a local representative has felt the decision of construction of the Paluel nuclear power plant and the problems involved, and how it has been tried to resolve them: insertion in the site, reception of the population of this big working site [fr

Remediation of a former fuel loading site using phytoremediation

Energy Technology Data Exchange (ETDEWEB)

Kotecha, P [Jacques Whitford Environment Ltd., Mont-Royal, PQ (Canada)

2001-07-01

The degradation and/or removal of pollutants from a contaminated medium is caused, mediated, and/or assisted by vegetation is defined as phytoremediation. It is a method widely used for the degradation, removal, and/or stabilisation of soils, sludges, sediments, or wastewaters. Some of the substances that can be cleaned up using phytoremediation are heavy metals, radionucleotides, petroleum hydrocarbons, energetics, chlorinated hydrocarbons, biocides, metalloids, nutrients, salts, and volatile organic contaminants. A former fuel loading site currently owned by Ultramar was remediated by Jacques Whitford Environment Limited using phytoremediation. A gasoline loading facility, a fuel loading facility, and a berm along an adjacent creek were all located at the site. All buildings and petroleum equipment had been removed in the mid-1980s, and the site is now vacant. The first phase involved the revegetation of the site with a phytoremediation grass cover and hybrid poplars, then the tree roots were allowed to infiltrate the ground to act as intake paths for contaminated water, the tree roots acted as a barrier to the contaminants headed to the river. Some of the advantages of phytoremediation are: low cost technique that can be applied using solar-powered ecotechnology in situ, wide applicability, involves minimum site disruption, has wide public acceptance, produces were few by-products requiring disposal, if any, and the harvested material can be easily disposed of in cases involving plant harvesting. The outcome of the project was also presented.

Direct action of aldosterone on transmembrane 22Na efflux from arterial smooth muscle. Rapid and delayed effects

International Nuclear Information System (INIS)

Moura, A.M.; Worcel, M.

1984-01-01

Acute subcutaneous (s.c.) administration of aldosterone increases ex vivo 22 Na efflux from rat tail artery smooth muscle, which appears to be due to a specific action on mineralocorticoid receptors. Indeed, this effect is blocked by the antimineralocorticoid compounds RU 28318 [17 beta-hydroxy-3-oxo,7 alpha-propyl(17 alpha)-pregn 4-ene, 21 potassium carboxylate] and spironolactone. The specific glucocorticoid receptor agonist RU 26988 does not modify 22 Na efflux. The authors show here that aldosterone has, at physiological concentrations, a mineralocorticoid specific stimulating effect on passive and sodium pump dependent transmembrane movements of sodium from the rat tail artery smooth muscle. Aldosterone exerts two types of action on sodium transport: 1) a delayed stimulation of ouabain-dependent 22 Na efflux and ouabain-independent 22 Na efflux, which are completely blocked by actinomycin D; and 2) a very rapid increase of passive 22 Na efflux, which is insensitive to actinomycin D and therefore does not seem to depend on transcription of genomic information

Influence of economic factor and site milestones on the salience of environmental issues at Department of Energy sites

International Nuclear Information System (INIS)

O'Connor, S.; Yin, J.

1997-01-01

The change in the US Department of Energy's (DOE) mission from nuclear weapons production to remediation and restoration of its installations, in 1989, challenged all citizens around the nation's weapons complex to get involved in DOE's environmental management decision-making process. The purpose of this study is to determine whether, and to what extent, economic factors and site milestones influence the rise and fall of environmental issues and participation. One might believe that citizen participation would be influenced by economic conditions, particularly with the down-sizing that occurred at many DOE sites. Another factor that might influence salience of environmental issues is the occurrence of crucial events, or site milestones. Important events occurring at DOE installations are well publicized by local and national media, and citizens' interest and opinions are influenced by media. In this study, the authors use the number of comments expressed by the public and Indian tribes as a measure of public involvement and salience of issues. Their study, using multiple regression analysis, examined the relationships between the number of comments expressed and the economic conditions as indicated by monthly unemployment rates, and the relationship between the number of comments expressed and the occurrence of crucial site events or milestones

Remaining Sites Verification Package for the 100-B-26 Spillway. Attachment to Waste Site Reclassification Form 2006-052

International Nuclear Information System (INIS)

Dittmer, L.M.

2006-01-01

The 100-B-26 Spillway waste site is a spillway that served as an emergency discharge point for the 132-C-2 outfall in the event that the 100-B-15 river effluent pipelines were blocked, damaged, or undergoing maintenance. The selected action involved demonstrating through confirmatory sampling that cleanup goals have been met and proposing a reclassification of this site to No Action. The results of the confirmatory sampling demonstrate that residual contaminant concentrations remaining in the soil beneath the riprap are more protective of groundwater and the Columbia River than the risk they would pose if the site was remediated

Mutaciones en el gen regulador de la conductancia transmembranal de la fibrosis quística en tres países latinoamericanos

OpenAIRE

Restrepo, CM; Pineda, L.; Gómez, Y.; González, A.; Silva, CT; Villalobos, MC.; Morales, A.; Barrera-Saldaña, H.

2011-01-01

La Fibrosis Quística (FQ) es la enfermedad autosómica recesiva más común en la población caucásica, es causada por la alteración del gen regulador de la conductancia transmembranal de la Fibrosis Quística (CFTR), descrito por primera vez en 1.989, hasta el momento han
sido descritas más de 500 mutaciones, la principal es la mutación DF508, una deleción de tres pares de bases (3 pb), la cual resulta en la pérdida de un residuo de fenilalanina en la posición 508 de la proteína, esta ...

Hypereosinophilic syndrome with hepatic involvement in a young child

Energy Technology Data Exchange (ETDEWEB)

Jung, Mi Ran; Goo, Hyun Woo; Hong, Seong Sook; Yoon, Chong Hyun [Ulsan University College of Medicine, Seoul (Korea, Republic of)

2003-09-01

Hypereosinophilic syndrome, whose etiology in unknown, involves the infiltration of various organs by a large number of eosinophils. The sites of involvement are the heart, skin, lung, liver, nervous system, and gastrointestinal tract. The disorder occurs mostly in middle-aged men and is characterized by striking peripheral eosinophilia. There have been few reports of hypereosinophilic syndrome in patients younger than 15 years and the disease also shows a predilection for males. We report a case of hypereosinophilic syndrome with hepatic involvement in a 17-month-old girl, and correlate the imaging features with the pathologic findings.

Hypereosinophilic syndrome with hepatic involvement in a young child

International Nuclear Information System (INIS)

Jung, Mi Ran; Goo, Hyun Woo; Hong, Seong Sook; Yoon, Chong Hyun

2003-01-01

Hypereosinophilic syndrome, whose etiology in unknown, involves the infiltration of various organs by a large number of eosinophils. The sites of involvement are the heart, skin, lung, liver, nervous system, and gastrointestinal tract. The disorder occurs mostly in middle-aged men and is characterized by striking peripheral eosinophilia. There have been few reports of hypereosinophilic syndrome in patients younger than 15 years and the disease also shows a predilection for males. We report a case of hypereosinophilic syndrome with hepatic involvement in a 17-month-old girl, and correlate the imaging features with the pathologic findings

Consultation draft: Site characterization plan overview, Deaf Smith County Site, Texas: Nuclear Waste Policy Act (Section 113)

International Nuclear Information System (INIS)

1988-01-01

The Department of Energy (DOE) is preparing a site characterization plan for the candidate site in Deaf Smith County, Texas. The DOE has provided, for information and review, a consultation draft of the plan to the State of Texas and the US Nuclear Regulatory Commission. The site characterization plan is a lengthy document that describes in considerable detail the program that will be conducted to characterize the geologic, hydrologic, and other conditions relevant to the suitability of the site for a repository. The overview presented here consists of brief summaries of important topics covered in the consultation draft of the site characterization plan; it is not a substitute for the site characterization plan. The arrangement of the overview is similar to that of the plan itself, with brief descriptions of the repository system - the site, the repository, and the waste package - preceding the discussion of the characterization program to be carried out at the Deaf Smith County site. It is intended primarily for the management staff of organizations involved in the DOE's repository program or other persons who might wish to understand the general scope of the site-characterization program, the activities to be conducted, and the facilities to be constructed rather than the technical details of site characterization. 15 figs., 1 tab

Rocketdyne division annual site environmental report, Santa Susana Field Laboratory and De Soto Sites, 1992

Energy Technology Data Exchange (ETDEWEB)

none,

1993-12-14

This annual report discusses environmental monitoring at two manufacturing and test operations sites operated in the Los Angeles area by the Rocketdyne Division of Rockwell International Corporation. These are identified as the Santa Susana Field Laboratory (SSFL) and the De Soto site. These sites have been used for manufacturing, R&D, engineering, and testing in a broad range of technical fields, primarily rocket engine propulsion and nuclear reactor technology. The De Soto site is essentially light industry with some laboratory-scale R&D and has little potential impact on the environment. The SSFL site, because of its large size (2,668 acres), warranted comprehensive monitoring to assure protection of the environment. The purpose of this report is to present information on environmental and effluent monitoring primarily for the regulatory agencies involved in controlling environmental remediation, i.e., the U.S. DOE, the Nuclear Regulatory Commission (NRC), and the California State Department of Health Services (DHS) Radiologic Health Branch (RHB). For that reason, information concentrates on Area IV at SSFL as this is the site of the former nuclear operations. While the major area of interest is radiological, this report also includes a discussion of nonradiological monitoring at SSFL.