Sample records for t3ss transmembrane regions

  1. 3D reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout

    PubMed Central

    Hodgkinson, Julie L.; Horsley, Ashley; Stabat, David; Simon, Martha; Johnson, Steven; da Fonseca, Paula C. A.; Morris, Edward P.; Wall, Joseph S.; Lea, Susan M.; Blocker, Ariel J.

    2009-01-01

    Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base bridging both bacterial membranes into which a hollow, external needle is embedded. When isolated, the latter two parts are termed ‘needle complex’ (NC). Incomplete understanding of NC structure hampers studies of T3SS function. To estimate the stoichiometry of its components, the mass f its sub-domains was measured by scanning transmission electron microscopy (STEM). Subunit symmetries were determined by analysis of top and side views within negatively stained samples in low dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25Å resolution three-dimensional (3D) reconstruction of the NC base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component. PMID:19396171

  2. YebC controls virulence by activating T3SS gene expression in the pathogen Edwardsiella piscicida.

    PubMed

    Wei, Lifan; Wu, Yanyan; Qiao, Haoxian; Xu, Wensheng; Zhang, Yuanxing; Liu, Xiaohong; Wang, Qiyao

    2018-06-12

    Edwardsiella piscicida is an infectious Gram-negative bacterium that causes great losses to the aquaculture industry worldwide. Based on pattern analysis of conditional essentiality (PACE), a new method for transposon insertion sequencing (Tn-seq) data analysis, we investigated the genome-wide genetic requirements during the dynamic process of infection and colonization in turbot in this study. As a result, disruption of ETAE_1437 was discovered to lead to substantially reduced colonization, which was similar to the in vivo dynamic patterns of the mutants of T3SS or T6SS. Bioinformatics analysis indicated that ETAE_1437 is a YebC/PmpR family regulator. Moreover, we found that ETAE_1437 not only regulated quorum sensing by directly binding to the edwR promoter region but also activated T3SS expression by directly binding to the promoter region of the T3SS gene ETAE_0873. In addition, ETAE_1437 mutants exhibited substantial colonization defects and significantly decreased virulence in turbot. Overall, this study identified ETAE_1437 as a novel virulence regulator in E. piscicida and enriched our understanding of the pathogenesis of E. piscicida in fish. We thus reannotated ETAE_1437 as YebC.

  3. Pseudomonas aeruginosa uses T3SS to inhibit diabetic wound healing

    PubMed Central

    Goldufsky, Josef; Wood, Stephen J.; Jayaraman, Vijayakumar; Majdobeh, Omar; Chen, Lin; Qin, Shanshan; Zhang, Chunxiang; DiPietro, Luisa A.; Shafikhani, Sasha H.

    2015-01-01

    Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram-negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram-negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa–induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS. PMID:25912785

  4. Pseudomonas aeruginosa uses T3SS to inhibit diabetic wound healing.

    PubMed

    Goldufsky, Josef; Wood, Stephen J; Jayaraman, Vijayakumar; Majdobeh, Omar; Chen, Lin; Qin, Shanshan; Zhang, Chunxiang; DiPietro, Luisa A; Shafikhani, Sasha H

    2015-01-01

    Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram-negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram-negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa-induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS. © 2015 by the Wound Healing Society.

  5. Type 3 Secretion System (T3SS) of Bradyrhizobium sp. DOA9 and Its Roles in Legume Symbiosis and Rice Endophytic Association.

    PubMed

    Songwattana, Pongpan; Noisangiam, Rujirek; Teamtisong, Kamonluck; Prakamhang, Janpen; Teulet, Albin; Tittabutr, Panlada; Piromyou, Pongdet; Boonkerd, Nantakorn; Giraud, Eric; Teaumroong, Neung

    2017-01-01

    The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod , nif , and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod -box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (Ω rhcN ) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four ( Arachis hypogea , Vigna radiata , Crotalaria juncea , and Macroptilium atropurpureum ) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one ( Stylosanthes hamata ) responded negatively (fewer nodules and less nitrogen fixation) and four species ( Aeschynomene americana , Aeschynomene afraspera , Indigofera tinctoria , and Desmodium tortuosum ) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice.

  6. Type 3 Secretion System (T3SS) of Bradyrhizobium sp. DOA9 and Its Roles in Legume Symbiosis and Rice Endophytic Association

    PubMed Central

    Songwattana, Pongpan; Noisangiam, Rujirek; Teamtisong, Kamonluck; Prakamhang, Janpen; Teulet, Albin; Tittabutr, Panlada; Piromyou, Pongdet; Boonkerd, Nantakorn; Giraud, Eric; Teaumroong, Neung

    2017-01-01

    The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod, nif, and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod-box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (ΩrhcN) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four (Arachis hypogea, Vigna radiata, Crotalaria juncea, and Macroptilium atropurpureum) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one (Stylosanthes hamata) responded negatively (fewer nodules and less nitrogen fixation) and four species (Aeschynomene americana, Aeschynomene afraspera, Indigofera tinctoria, and Desmodium tortuosum) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice. PMID:28979252

  7. Presence of T3SS2β genes in trh⁺ Vibrio parahaemolyticus isolated from seafood harvested along Mangalore coast, India.

    PubMed

    Kumar, B K; Deekshit, V K; Rai, P; Shekar, M; Karunasagar, I; Karunasagar, I

    2014-05-01

    Vibrio parahaemolyticus is a seafood-borne pathogen autochthonous to the marine and estuarine ecosystem, responsible for gastroenteritis when contaminated raw seafood is consumed. The pathogenicity has been associated with thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH). Of late, the presence of T3SS2α and T3SS2β gene clusters has been well documented in clinical isolates of Vibrio parahaemolyticus and known to play an essential role in pathogenesis. However, reports on the presence of T3SSβ genes in V. parahaemolyticus isolated from the seafood and/or environmental samples are scanty. In this study, we have identified and analysed the distribution of the T3SS2β genes in V. parahaemolyticus isolated from seafood harvested along southwest coast of India. Results showed that T3SS2β genes are solely associated with trh⁺ and tdh⁺ /trh⁺ strains of V. parahaemolyticus. Reverse transcriptase PCR (RT-PCR) showed that the T3SS2β genes identified in trh⁺ V. parahaemolyticus were transcriptionally active. To our knowledge, this study appears to be the first description on the presence of T3SS2β-positive V. parahaemolyticus isolated from seafood in India. The study of T3SS2 along with other virulence factors will help in better understanding of the risk of seafood-borne illness due to V. parahaemolyticus. T3SSs (α or β) are the important virulence factors of Vibrio parahaemolyticus that contribute to their pathogenicity in humans. This study demonstrated the presence of T3SS2β genes in V. parahaemolyticus isolated from the seafood harvested along Mangalore coast. RT-PCR showed that the T3SS2β genes identified in seafood isolates of V. parahaemolyticus were found to be functional. To the best of our knowledge, this is the first description of T3SS2β genes in trh⁺ V. parahaemolyticus isolated from seafood in India. The presence of T3SS2 along with other virulence factors such as TDH and/or TRH highlights a potential health risk for

  8. Identification of the Vibrio parahaemolyticus type III secretion system 2-associated chaperone VocC for the T3SS2-specific effector VopC.

    PubMed

    Akeda, Yukihiro; Kodama, Toshio; Saito, Kazunobu; Iida, Tetsuya; Oishi, Kazunori; Honda, Takeshi

    2011-11-01

    The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V. parahaemolyticus. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  9. Chitin-induced T6SS in Vibrio cholerae is dependent on ChiS activation.

    PubMed

    Chourashi, Rhishita; Das, Suman; Dhar, Debarpan; Okamoto, Keinosuke; Mukhopadhyay, Asish K; Chatterjee, Nabendu Sekhar

    2018-05-01

    Vibrio cholerae regularly colonizes the chitinous exoskeleton of crustacean shells in the aquatic region. The type 6 secretion system (T6SS) in V. cholerae is an interbacterial killing device. This system is thought to provide a competitive advantage to V. cholerae in a polymicrobial community of the aquatic region under nutrient-poor conditions. V. cholerae chitin sensing is known to be initiated by the activation of a two-component sensor histidine kinase ChiS in the presence of GlcNAc2 (N,N'-diacetylchitobiose) residues generated by the action of chitinases on chitin. It is known that T6SS in V. cholerae is generally induced by chitin. However, the effect of ChiS activation on T6SS is unknown. Here, we found that ChiS inactivation resulted in impaired bacterial killing and reduced expression of T6SS genes. Active ChiS positively affected T6SS-mediated natural transformation in V. cholerae. ChiS depletion or inactivation also resulted in reduced colonization on insoluble chitin surfaces. Therefore, we have shown that V. cholerae colonization on chitinous surfaces activates ChiS, which promotes T6SS-dependent bacterial killing and horizontal gene transfer. We also highlight the importance of chitinases in T6SS upregulation.

  10. The Post-transcriptional Regulator rsmA/csrA Activates T3SS by Stabilizing the 5′ UTR of hrpG, the Master Regulator of hrp/hrc Genes, in Xanthomonas

    PubMed Central

    Andrade, Maxuel O.; Farah, Chuck S.; Wang, Nian

    2014-01-01

    The RsmA/CsrA family of the post-transcriptional regulators of bacteria is involved in the regulation of many cellular processes, including pathogenesis. In this study, we demonstrated that rsmA not only is required for the full virulence of the phytopathogenic bacterium Xanthomonas citri subsp. citri (XCC) but also contributes to triggering the hypersensitive response (HR) in non-host plants. Deletion of rsmA resulted in significantly reduced virulence in the host plant sweet orange and a delayed and weakened HR in the non-host plant Nicotiana benthamiana. Microarray, quantitative reverse-transcription PCR, western-blotting, and GUS assays indicated that RsmA regulates the expression of the type 3 secretion system (T3SS) at both transcriptional and post-transcriptional levels. The regulation of T3SS by RsmA is a universal phenomenon in T3SS-containing bacteria, but the specific mechanism seems to depend on the interaction between a particular bacterium and its hosts. For Xanthomonads, the mechanism by which RsmA activates T3SS remains unknown. Here, we show that RsmA activates the expression of T3SS-encoding hrp/hrc genes by directly binding to the 5′ untranslated region (UTR) of hrpG, the master regulator of the hrp/hrc genes in XCC. RsmA stabilizes hrpG mRNA, leading to increased accumulation of HrpG proteins and subsequently, the activation of hrp/hrc genes. The activation of the hrp/hrc genes by RsmA via HrpG was further supported by the observation that ectopic overexpression of hrpG in an rsmA mutant restored its ability to cause disease in host plants and trigger HR in non-host plants. RsmA also stabilizes the transcripts of another T3SS-associated hrpD operon by directly binding to the 5′ UTR region. Taken together, these data revealed that RsmA primarily activates T3SS by acting as a positive regulator of hrpG and that this regulation is critical to the pathogenicity of XCC. PMID:24586158

  11. Role of T3SS-1 SipD Protein in Protecting Mice against Non-typhoidal Salmonella Typhimurium

    PubMed Central

    Jneid, Bakhos; Moreau, Karine; Plaisance, Marc; Rouaix, Audrey; Dano, Julie

    2016-01-01

    Background Salmonella enterica species are enteric pathogens that cause severe diseases ranging from self-limiting gastroenteritis to enteric fever and sepsis in humans. These infectious diseases are still the major cause of morbidity and mortality in low-income countries, especially in children younger than 5 years and immunocompromised adults. Vaccines targeting typhoidal diseases are already marketed, but none protect against non-typhoidal Salmonella. The existence of multiple non-typhoidal Salmonella serotypes as well as emerging antibiotic resistance highlight the need for development of a broad-spectrum protective vaccine. All Salmonella spp. utilize two type III Secretion Systems (T3SS 1 and 2) to initiate infection, allow replication in phagocytic cells and induce systemic disease. T3SS-1, which is essential to invade epithelial cells and cross the barrier, forms an extracellular needle and syringe necessary to inject effector proteins into the host cell. PrgI and SipD form, respectively, the T3SS-1 needle and the tip complex at the top of the needle. Because they are common and highly conserved in all virulent Salmonella spp., they might be ideal candidate antigens for a subunit-based, broad-spectrum vaccine. Principal Findings We investigated the immunogenicity and protective efficacy of PrgI and SipD administered by subcutaneous, intranasal and oral routes, alone or combined, in a mouse model of Salmonella intestinal challenge. Robust IgG (in all immunization routes) and IgA (in intranasal and oral immunization routes) antibody responses were induced against both proteins, particularly SipD. Mice orally immunized with SipD alone or SipD combined with PrgI were protected against lethal intestinal challenge with Salmonella Typhimurium (100 Lethal Dose 50%) depending on antigen, route and adjuvant. Conclusions and Significance Salmonella T3SS SipD is a promising antigen for the development of a protective Salmonella vaccine, and could be developed for

  12. Ferulic Acid, But Not All Hydroxycinnamic Acids, Is a Novel T3SS Inducer of Ralstonia solanacearum and Promotes Its Infection Process in Host Plants under Hydroponic Condition.

    PubMed

    Zhang, Yong; Li, Jing; Zhang, Weiqi; Wang, Rongsheng; Qiu, Qiaoqing; Luo, Feng; Hikichi, Yasufumi; Ohnishi, Kouhei; Ding, Wei

    2017-01-01

    Hydroxycinnamic acids (HCAs) are typical monocyclic phenylpropanoids, including cinnamic acid (Cin), coumaric acid (Cou), caffeic acid (Caf), ferulic acid (FA) and their isomers, and involved in the interactions between pathogens and host plants. Here, we focused on the impact of HCAs on expression of type III secretion system (T3SS) in Ralstonia solanacearum . FA significantly induced the expression of the T3SS and some type III effectors (T3Es) genes in hrp -inducing medium, while did not the other HCAs. However, exogenously supplemented FA did not affect the T3SS expression in planta and the elicitation of the hypersensitive response (HR) in tobacco leaves. Consistent with its central roles in pathogenicity, the FA-induced expression of the T3SS led to significant promotion on infection process of R. solanacearum in tomato plants under hydroponics cultivation. Moreover, the FA-induced expression of the T3SS was specifically mediated by the well-characterized signaling cascade PrhA-prhI/R-PrhJ-HrpG-HrpB, independent of the other known regulatory pathways. In summary, our results demonstrated that FA, a novel inducer of the T3SS in R. solanacearum , was able to promote its infection process in host plants under hydroponics condition.

  13. AtlasT4SS: a curated database for type IV secretion systems.

    PubMed

    Souza, Rangel C; del Rosario Quispe Saji, Guadalupe; Costa, Maiana O C; Netto, Diogo S; Lima, Nicholas C B; Klein, Cecília C; Vasconcelos, Ana Tereza R; Nicolás, Marisa F

    2012-08-09

    The type IV secretion system (T4SS) can be classified as a large family of macromolecule transporter systems, divided into three recognized sub-families, according to the well-known functions. The major sub-family is the conjugation system, which allows transfer of genetic material, such as a nucleoprotein, via cell contact among bacteria. Also, the conjugation system can transfer genetic material from bacteria to eukaryotic cells; such is the case with the T-DNA transfer of Agrobacterium tumefaciens to host plant cells. The system of effector protein transport constitutes the second sub-family, and the third one corresponds to the DNA uptake/release system. Genome analyses have revealed numerous T4SS in Bacteria and Archaea. The purpose of this work was to organize, classify, and integrate the T4SS data into a single database, called AtlasT4SS - the first public database devoted exclusively to this prokaryotic secretion system. The AtlasT4SS is a manual curated database that describes a large number of proteins related to the type IV secretion system reported so far in Gram-negative and Gram-positive bacteria, as well as in Archaea. The database was created using the RDBMS MySQL and the Catalyst Framework based in the Perl programming language and using the Model-View-Controller (MVC) design pattern for Web. The current version holds a comprehensive collection of 1,617 T4SS proteins from 58 Bacteria (49 Gram-negative and 9 Gram-Positive), one Archaea and 11 plasmids. By applying the bi-directional best hit (BBH) relationship in pairwise genome comparison, it was possible to obtain a core set of 134 clusters of orthologous genes encoding T4SS proteins. In our database we present one way of classifying orthologous groups of T4SSs in a hierarchical classification scheme with three levels. The first level comprises four classes that are based on the organization of genetic determinants, shared homologies, and evolutionary relationships: (i) F-T4SS, (ii) P-T4SS, (iii

  14. R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport.

    PubMed

    Suzuki, Satomi; Nanatani, Kei; Abe, Keietsu

    2016-01-01

    The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)-a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport.

  15. T3SS-dependent differential modulations of the jasmonic acid pathway in susceptible and resistant genotypes of Malus spp. challenged with Erwinia amylovora.

    PubMed

    Dugé De Bernonville, Thomas; Gaucher, Matthieu; Flors, Victor; Gaillard, Sylvain; Paulin, Jean-Pierre; Dat, James F; Brisset, Marie-Noëlle

    2012-06-01

    Fire blight is a bacterial disease of Maloideae caused by Erwinia amylovora (Ea). This necrogenic enterobacterium uses a type III secretion system (T3SS) to inject type III effectors into the plant cells to cause disease on its susceptible hosts, including economically important crops like apple and pear. The expressions of marker genes of the salicylic acid (SA) and jasmonic acid (JA) defense regulation pathways were monitored by RT-qPCR in leaves of two apple genotypes, one susceptible and one resistant, challenged with a wild type strain, a T3SS-deficient strain or water. The transcriptional data taken together with hormone level measurements indicated that the SA pathway was similarly induced in both apple genotypes during infection by Ea. On the contrary, the data clearly showed a strong T3SS-dependent down-regulation of the JA pathway in leaves of the susceptible genotype but not in those of the resistant one. Accordingly, methyl-jasmonate treated susceptible plants displayed an increased resistance to Ea. Bacterial mutant analysis indicated that JA manipulation by Ea mainly relies on the type III effector DspA/E. Taken together, our data suggest that the T3SS-dependent down-regulation of the JA pathway is a critical step in the infection process of Malus spp. by Ea. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  16. Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase.

    PubMed

    Harris, Darby M; Corbin, Kendall; Wang, Tuo; Gutierrez, Ryan; Bertolo, Ana L; Petti, Carloalberto; Smilgies, Detlef-M; Estevez, José Manuel; Bonetta, Dario; Urbanowicz, Breeanna R; Ehrhardt, David W; Somerville, Chris R; Rose, Jocelyn K C; Hong, Mei; Debolt, Seth

    2012-03-13

    The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

  17. Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase

    PubMed Central

    Harris, Darby M.; Corbin, Kendall; Wang, Tuo; Gutierrez, Ryan; Bertolo, Ana L.; Petti, Carloalberto; Smilgies, Detlef-M.; Estevez, José Manuel; Bonetta, Dario; Urbanowicz, Breeanna R.; Ehrhardt, David W.; Somerville, Chris R.; Rose, Jocelyn K. C.; Hong, Mei; DeBolt, Seth

    2012-01-01

    The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1A903V and CESA3T942I in Arabidopsis thaliana. Using 13C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1A903V and CESA3T942I displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1A903V and CESA3T942I have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization. PMID:22375033

  18. The Vibrio alginolyticus T3SS effectors, Val1686 and Val1680, induce cell rounding, apoptosis and lysis of fish epithelial cells

    PubMed Central

    Zhao, Zhe; Liu, Jinxin; Deng, Yiqin; Huang, Wen; Ren, Chunhua; Call, Douglas R.; Hu, Chaoqun

    2018-01-01

    ABSTRACT Vibrio alginolyticus is a Gram-negative bacterium that is an opportunistic pathogen of both marine animals and people. Its pathogenesis likely involves type III secretion system (T3SS) mediated induction of rapid apoptosis, cell rounding and osmotic lysis of infected eukaryotic cells. Herein, we report that effector proteins, Val1686 and Val1680 from V. alginolyticus, were responsible for T3SS-mediated death of fish cells. Val1686 is a Fic-domain containing protein that not only contributed to cell rounding by inhibiting Rho guanosine triphosphatases (GTPases), but was requisite for the induction of apoptosis because the deletion mutant (Δval1686) was severely weakened in its ability to induce cell rounding and apoptosis in fish cells. In addition, Val1686 alone was sufficient to induce cell rounding and apoptosis as evidenced by the transfection of Val1686 into fish cells. Importantly, the Fic-domain essential for cell rounding activity was equally important to activation of apoptosis of fish cells, indicating that apoptosis is a downstream event of Val1686-dependent GTPase inhibition. V. alginolyticus infection likely activates JNK and ERK pathways with sequential activation of caspases (caspase-8/-10, -9 and -3) and subsequent apoptosis. Val1680 contributed to T3SS-dependent lysis of fish cells in V. alginolyticus, but did not induce autophagy as has been reported for its homologue (VopQ) in V. parahaemolyticus. Together, Val1686 and Val1680 work together to induce apoptosis, cell rounding and cell lysis of V. alginolyticus-infected fish cells. These findings provide new insights into the mechanism of cell death caused by T3SS of V. alginolyticus. PMID:29252102

  19. Evolutionarily Conserved nodE, nodO, T1SS, and Hydrogenase System in Rhizobia of Astragalus membranaceus and Caragana intermedia.

    PubMed

    Yan, Hui; Xie, Jian Bo; Ji, Zhao Jun; Yuan, Na; Tian, Chang Fu; Ji, Shou Kun; Wu, Zhong Yu; Zhong, Liang; Chen, Wen Xin; Du, Zheng Lin; Wang, En Tao; Chen, Wen Feng

    2017-01-01

    Mesorhizobium species are the main microsymbionts associated with the medicinal or sand-fixation plants Astragalus membranaceus and Caragana intermedia (AC) in temperate regions of China, while all the Mesorhizobium strains isolated from each of these plants could nodulate both of them. However, Rhizobium yanglingense strain CCBAU01603 could nodulate AC plants and it's a high efficiency symbiotic and competitive strain with Caragana . Therefore, the common features shared by these symbiotic rhizobia in genera of Mesorhizobium and Rhizobium still remained undiscovered. In order to study the genomic background influencing the host preference of these AC symbiotic strains, the whole genomes of two ( M. silamurunense CCBAU01550, M. silamurunense CCBAU45272) and five representative strains ( M. septentrionale CCBAU01583, M. amorphae CCBAU01570, M. caraganae CCBAU01502, M. temperatum CCBAU01399, and R. yanglingense CCBAU01603) originally isolated from AC plants were sequenced, respectively. As results, type III secretion systems (T3SS) of AC rhizobia evolved in an irregular pattern, while an evolutionarily specific region including nodE, nodO , T1SS, and a hydrogenase system was detected to be conserved in all these AC rhizobia. Moreover, nodO was verified to be prevalently distributed in other AC rhizobia and was presumed as a factor affecting the nodule formation process. In conclusion, this research interpreted the multifactorial features of the AC rhizobia that may be associated with their host specificity at cross-nodulation group, including nodE, nodZ , T1SS as the possible main determinants; and nodO , hydrogenase system, and T3SS as factors regulating the bacteroid formation or nitrogen fixation efficiency.

  20. Escherichia coli type III secretion system 2: a new kind of T3SS?

    PubMed

    Zhou, Mingxu; Guo, Zhiyan; Duan, Qiangde; Hardwidge, Philip R; Zhu, Guoqiang

    2014-03-19

    Type III secretion systems (T3SSs) are employed by Gram-negative bacteria to deliver effector proteins into the cytoplasm of infected host cells. Enteropathogenic Escherichia coli use a T3SS to deliver effector proteins that result in the creation of the attaching and effacing lesions. The genome sequence of the Escherichia coli pathotype O157:H7 revealed the existence of a gene cluster encoding components of a second type III secretion system, the E. coli type III secretion system 2 (ETT2). Researchers have revealed that, although ETT2 may not be a functional secretion system in most (or all) strains, it still plays an important role in bacterial virulence. This article summarizes current knowledge regarding the E. coli ETT2, including its genetic characteristics, prevalence, function, association with virulence, and prospects for future work.

  1. Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

    PubMed

    Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

    2016-05-01

    Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.

  2. Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling.

    PubMed

    Brdicka, Tomás; Imrich, Martin; Angelisová, Pavla; Brdicková, Nadezda; Horváth, Ondrej; Spicka, Jirí; Hilgert, Ivan; Lusková, Petra; Dráber, Petr; Novák, Petr; Engels, Niklas; Wienands, Jürgen; Simeoni, Luca; Osterreicher, Jan; Aguado, Enrique; Malissen, Marie; Schraven, Burkhart; Horejsí, Václav

    2002-12-16

    A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.

  3. Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane.

    PubMed

    Ji, Hongtao; Dong, Hansong

    2015-09-01

    Many plant- and animal-pathogenic Gram-negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel-like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two-step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two-domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin-rich middle lamella by the bacterial pilus. One-domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin-type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly. © 2014 BSPP AND JOHN WILEY & SONS LTD.

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

    PubMed

    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.

  5. MIX and match: mobile T6SS MIX-effectors enhance bacterial fitness

    PubMed Central

    Salomon, Dor

    2016-01-01

    ABSTRACT Protein secretion systems that mediate interbacterial competition secret a wide repertoire of antibacterial toxins. A major player in these competitions is the newly discovered bacterial type VI secretion system (T6SS). We recently found that a subset of polymorphic MIX-effectors, which are a widespread class of effectors secreted by T6SSs, are horizontally shared between marine bacteria and are used to diversify their T6SS effector repertoires, thus enhancing their environmental fitness. In this commentary, I expand on the ideas that were introduced in the previous report, and further speculate on the possible mobility of other MIX-effectors. In addition, I discuss the possible role of horizontal gene transfer in the dissemination of MIX-effectors through bacterial genomes, as well as its possible role in diversifying the T6SS effector repertoire. PMID:27066305

  6. CalR is required for the expression of T6SS2 and the adhesion of Vibrio parahaemolyticus to HeLa cells.

    PubMed

    Zhang, Lingyu; Osei-Adjei, George; Zhang, Ying; Gao, He; Yang, Wenhui; Zhou, Dongsheng; Huang, Xinxiang; Yang, Huiying; Zhang, Yiquan

    2017-08-01

    Vibrio parahaemolyticus expresses one major virulence determinant T6SS2, which is constituted into three putative operons, i.e., VPA1027-1024, VPA1043-1028, and VPA1044-1046. CalR, a LysR-type transcriptional regulator, was originally identified as a repressor of the swarming motility and T3SS1 gene expression. As shown in this study, CalR binds to the promoter-proximal region of each of the three operons to activate their transcription, and moreover, CalR activates the adhesion of V. parahaemolyticus to HeLa cells. In addition, competitive EMSAs demonstrated that CalR acts as an antagonist of H-NS in V. parahaemolyticus. Collectively, these studies confirmed a new physiological role for CalR in V. parahaemolyticus.

  7. Further Characterization of a Type III Secretion System (T3SS) and of a New Effector Protein from a Clinical Isolate of Aeromonas Hydrophila - Part I

    EPA Science Inventory

    A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular cha...

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

    PubMed

    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.

  9. Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

    PubMed

    Lallemand, Mathilde; Login, Frédéric H; Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E

    2013-01-01

    The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

  10. Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System

    PubMed Central

    Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E.

    2013-01-01

    The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine. PMID:24223969

  11. T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage.

    PubMed

    Stolle, Anne-Sophie; Norkowski, Stefanie; Körner, Britta; Schmitz, Jürgen; Lüken, Lena; Frankenberg, Maj; Rüter, Christian; Schmidt, M Alexander

    2017-01-01

    Effector proteins secreted by the type 3 secretion system (T3SS) of pathogenic bacteria have been shown to precisely modulate important signaling cascades of the host for the benefit of the pathogens. Among others, the non-LEE encoded T3SS effector protein NleC of enteropathogenic Escherichia coli (EPEC) is a Zn-dependent metalloprotease and suppresses innate immune responses by directly targeting the NF-κB signaling pathway. Many pathogenic bacteria release potent bacterial toxins of the A-B type, which-in contrast to the direct cytoplasmic injection of T3SS effector proteins-are released first into the environment. In this study, we found that NleC displays characteristics of bacterial A-B toxins, when applied to eukaryotic cells as a recombinant protein. Although lacking a B subunit, that typically mediates the uptake of toxins, recombinant NleC (rNleC) induces endocytosis via lipid rafts and follows the endosomal-lysosomal pathway. The conformation of rNleC is altered by low pH to facilitate its escape from acidified endosomes. This is reminiscent of the homologous A-B toxin AIP56 of the fish pathogen Photobacterium damselae piscicida ( Phdp ). The recombinant protease NleC is functional inside eukaryotic cells and cleaves p65 of the NF-κB pathway. Here, we describe the endocytic uptake mechanism of rNleC, characterize its intracellular trafficking and demonstrate that its specific activity of cleaving p65 requires activation of host cells e.g., by IL1β. Further, we propose an evolutionary link between some T3SS effector proteins and bacterial toxins from apparently unrelated bacteria. In summary, these properties might suggest rNleC as an interesting candidate for future applications as a potential therapeutic against immune disorders.

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

    PubMed

    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.

  13. A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition

    PubMed Central

    Lin, Jinshui; Zhang, Weipeng; Cheng, Juanli; Yang, Xu; Zhu, Kaixiang; Wang, Yao; Wei, Gehong; Qian, Pei-Yuan; Luo, Zhao-Qing; Shen, Xihui

    2017-01-01

    Iron sequestration by host proteins contributes to the defence against bacterial pathogens, which need iron for their metabolism and virulence. A Pseudomonas aeruginosa mutant lacking all three known iron acquisition systems retains the ability to grow in media containing iron chelators, suggesting the presence of additional pathways involved in iron uptake. Here we screen P. aeruginosa mutants defective in growth in iron-depleted media and find that gene PA2374, proximal to the type VI secretion system H3 (H3-T6SS), functions synergistically with known iron acquisition systems. PA2374 (which we have renamed TseF) appears to be secreted by H3-T6SS and is incorporated into outer membrane vesicles (OMVs) by directly interacting with the iron-binding Pseudomonas quinolone signal (PQS), a cell–cell signalling compound. TseF facilitates the delivery of OMV-associated iron to bacterial cells by engaging the Fe(III)-pyochelin receptor FptA and the porin OprF. Our results reveal links between type VI secretion, cell–cell signalling and classic siderophore receptors for iron acquisition in P. aeruginosa. PMID:28348410

  14. Tn-Seq Analysis of Vibrio cholerae Intestinal Colonization Reveals a Role for T6SS-Mediated Antibacterial Activity in the Host

    PubMed Central

    Fu, Yang; Waldor, Matthew K.; Mekalanos, John J.

    2014-01-01

    SUMMARY Analysis of genes required for host infection will provide clues to the drivers of evolutionary fitness of pathogens like Vibrio cholerae, a mounting threat to global heath. We used transposon insertion site sequencing (Tn-seq) to comprehensively assess the contribution of nearly all V. cholerae genes toward growth in the infant rabbit intestine. Four hundred genes were identified as critical to V. cholerae in vivo fitness. These included most known colonization factors and several new genes affecting the bacterium's metabolic properties, resistance to bile, and ability to synthesize cyclic AMP-GMP. Notably, a mutant carrying an insertion in tsiV3, encoding immunity to a bacteriocidal type VI secretion system (T6SS) effector VgrG3, exhibited a colonization defect. The reduced in vivo fitness of tsiV3 mutants depends on their cocolonization with bacterial cells carrying an intact T6SS locus and VgrG3 gene, suggesting that the V. cholerae T6SS is functional and mediates antagonistic interbacterial interactions during infection. PMID:24331463

  15. APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

    NASA Astrophysics Data System (ADS)

    Shlyakhtenko, Luda S.; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S.; Lyubchenko, Yuri L.

    2015-10-01

    APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.

  16. APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies.

    PubMed

    Shlyakhtenko, Luda S; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S; Lyubchenko, Yuri L

    2015-10-27

    APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.

  17. APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

    PubMed Central

    Shlyakhtenko, Luda S.; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S.; Lyubchenko, Yuri L.

    2015-01-01

    APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA. PMID:26503602

  18. Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region.

    PubMed

    Minato, Yuichi; Suzuki, Shiho; Hara, Tomoaki; Kofuku, Yutaka; Kasuya, Go; Fujiwara, Yuichiro; Igarashi, Shunsuke; Suzuki, Ei-Ichiro; Nureki, Osamu; Hattori, Motoyuki; Ueda, Takumi; Shimada, Ichio

    2016-04-26

    Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s(-1)), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.

  19. Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

    PubMed Central

    Minato, Yuichi; Suzuki, Shiho; Hara, Tomoaki; Kofuku, Yutaka; Kasuya, Go; Fujiwara, Yuichiro; Igarashi, Shunsuke; Suzuki, Ei-ichiro; Nureki, Osamu; Hattori, Motoyuki; Ueda, Takumi; Shimada, Ichio

    2016-01-01

    Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s−1), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region. PMID:27071117

  20. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

    PubMed Central

    Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

    2017-01-01

    Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

  1. Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

    PubMed

    Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

    2017-01-01

    Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

  2. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    PubMed

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  3. The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function

    PubMed Central

    Lasica, Anna M.; Ksiazek, Miroslaw; Madej, Mariusz; Potempa, Jan

    2017-01-01

    Protein secretion systems are vital for prokaryotic life, as they enable bacteria to acquire nutrients, communicate with other species, defend against biological and chemical agents, and facilitate disease through the delivery of virulence factors. In this review, we will focus on the recently discovered type IX secretion system (T9SS), a complex translocon found only in some species of the Bacteroidetes phylum. T9SS plays two roles, depending on the lifestyle of the bacteria. It provides either a means of movement (called gliding motility) for peace-loving environmental bacteria or a weapon for pathogens. The best-studied members of these two groups are Flavobacterium johnsoniae, a commensal microorganism often found in water and soil, and Porphyromonas gingivalis, a human oral pathogen that is a major causative agent of periodontitis. In P. gingivalis and some other periodontopathogens, T9SS translocates proteins, especially virulence factors, across the outer membrane (OM). Proteins destined for secretion bear a conserved C-terminal domain (CTD) that directs the cargo to the OM translocon. At least 18 proteins are involved in this still enigmatic process, with some engaged in the post-translational modification of T9SS cargo proteins. Upon translocation across the OM, the CTD is removed by a protease with sortase-like activity and an anionic LPS is attached to the newly formed C-terminus. As a result, a cargo protein could be secreted into the extracellular milieu or covalently attached to the bacterial surface. T9SS is regulated by a two-component system; however, the precise environmental signal that triggers it has not been identified. Exploring unknown systems contributing to bacterial virulence is exciting, as it may eventually lead to new therapeutic strategies. During the past decade, the major components of T9SS were identified, as well as hints suggesting the possible mechanism of action. In addition, the list of characterized cargo proteins is

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

    PubMed

    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 (< or =4 mg/L) in the absence of any growth supplement, but increasing to 8 mg/L when either L-Ser or D-Ser was added to the medium; and (ii) the relative amounts of accumulated UDP-MurNAc-pentapeptide[D-Ser] and tetrapeptide precursors decreasing substantially compared with BM4175/pCA10 and BM4174. The effect on the appearance of tetrapeptide appeared to be host dependent, since a substantial amount was present when the same plasmid construct pJP1(vanC-1-XYc-T((Delta))(2-322)) was electroporated into Enterococcus faecalis JH2-2. The uptake of L-[(14)C]Ser at 240 s was decreased by approximately 40% in BM4175 compared with BM4174. Plasmid pCA10(C-1-XY(C)-T) restored uptake of L-[(14)C]Ser at 180 and 240 s in BM4175. The results suggest that the transmembrane domain of VanT is likely to be involved in the transport of L-Ser, and that in its absence the resistance phenotype is compromised.

  5. All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences.

    PubMed

    Hayat, Sikander; Sander, Chris; Marks, Debora S; Elofsson, Arne

    2015-04-28

    Transmembrane β-barrels (TMBs) carry out major functions in substrate transport and protein biogenesis but experimental determination of their 3D structure is challenging. Encouraged by successful de novo 3D structure prediction of globular and α-helical membrane proteins from sequence alignments alone, we developed an approach to predict the 3D structure of TMBs. The approach combines the maximum-entropy evolutionary coupling method for predicting residue contacts (EVfold) with a machine-learning approach (boctopus2) for predicting β-strands in the barrel. In a blinded test for 19 TMB proteins of known structure that have a sufficient number of diverse homologous sequences available, this combined method (EVfold_bb) predicts hydrogen-bonded residue pairs between adjacent β-strands at an accuracy of ∼70%. This accuracy is sufficient for the generation of all-atom 3D models. In the transmembrane barrel region, the average 3D structure accuracy [template-modeling (TM) score] of top-ranked models is 0.54 (ranging from 0.36 to 0.85), with a higher (44%) number of residue pairs in correct strand-strand registration than in earlier methods (18%). Although the nonbarrel regions are predicted less accurately overall, the evolutionary couplings identify some highly constrained loop residues and, for FecA protein, the barrel including the structure of a plug domain can be accurately modeled (TM score = 0.68). Lower prediction accuracy tends to be associated with insufficient sequence information and we therefore expect increasing numbers of β-barrel families to become accessible to accurate 3D structure prediction as the number of available sequences increases.

  6. Structural elucidation of transmembrane transporter protein bilitranslocase: conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy.

    PubMed

    Roy Choudhury, Amrita; Perdih, Andrej; Zuperl, Spela; Sikorska, Emilia; Solmajer, Tom; Jurga, Stefan; Zhukov, Igor; Novič, Marjana

    2013-11-01

    Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing projects. They account for up to two thirds of known drugable targets, which emphasizes their critical pharmaceutical importance. Here we present a study on bilitranslocase (BTL) (TCDB 2.A.65), a membrane protein primarily involved in the transport of bilirubin from blood to liver cells. Bilitranslocase has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its 3D structure. However, at present, only a limited knowledge is available beyond the primary structure of BTL. It has been recently confirmed experimentally that one of the four computationally predicted transmembrane segments of bilitranslocase, TM3, has a helical structure with hydrophilic amino acid residues oriented towards one side, which is typical for transmembrane domains of membrane proteins. In this study we confirmed by the use of multidimensional NMR spectroscopy that the second transmembrane segment, TM2, also appears in a form of α-helix. The stability of this polypeptide chain was verified by molecular dynamics (MD) simulation in dipalmitoyl phosphatidyl choline (DPPC) and in sodium dodecyl sulfate (SDS) micelles. The two α-helices, TM2 corroborated in this study, and TM3 confirmed in our previous investigation, provide reasonable building blocks of a potential transmembrane channel for transport of bilirubin and small hydrophilic molecules, including pharmaceutically active compounds. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. XopN-T3SS effector of Xanthomonas axonopodis pv. punicae localizes to the plasma membrane and modulates ROS accumulation events during blight pathogenesis in pomegranate.

    PubMed

    Kumar, Rishikesh; Soni, Madhvi; Mondal, Kalyan K

    2016-12-01

    Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a major disease of pomegranate. Xap secretes effector proteins via type III secretion system (T3SS) to suppress pathogen-associated molecular pattern (PAMP)-triggered plant immunity (PTI). Previously we reported that XopN, a conserved effector of Xap, modulate in planta bacterial growth, and blight disease. In continuation to that here we report the deletion of XopN from Xap caused higher accumulation of reactive oxygen species (ROS) including H 2 O 2 and O 2 - . We quantitatively assessed the higher accumulation of H 2 O 2 in pomegranate leaves infiltrated with Xap ΔxopN compared to Xap wild-type. We analysed that 1.5 to 3.3 fold increase in transcript expression of ROS and flg22-inducible genes, namely FRK1, GST1, WRKY29, PR1, PR2 and PR5 in Arabidopsis when challenged with Xap ΔxopN; contrary, the up-regulation of all the genes were compromised when challenged with either Xap wild-type or Xap ΔxopN+xopN. Further, we demonstrated the plasma-membrane based localization of XopN protein both in its natural and experimental hosts. All together, the present study suggested that XopN-T3SS effector of Xap gets localized in the plasma membrane and suppresses ROS-mediated early defense responses during blight pathogenesis in pomegranate. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor.

    PubMed Central

    Parrish, William; Eilers, Markus; Ying, Weiwen; Konopka, James B

    2002-01-01

    The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors. PMID:11861550

  9. A conserved αβ transmembrane interface forms the core of a compact T-cell receptor–CD3 structure within the membrane

    PubMed Central

    Krshnan, Logesvaran; Park, Soohyung; Im, Wonpil; Call, Melissa J.; Call, Matthew E.

    2016-01-01

    The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αβ and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαβ:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR–CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling. PMID:27791034

  10. A conserved αβ transmembrane interface forms the core of a compact T-cell receptor-CD3 structure within the membrane.

    PubMed

    Krshnan, Logesvaran; Park, Soohyung; Im, Wonpil; Call, Melissa J; Call, Matthew E

    2016-10-25

    The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αβ and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαβ:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR-CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling.

  11. An atlas of H-alpha-emitting regions in M33: A systematic search for SS433 star candidates

    NASA Technical Reports Server (NTRS)

    Calzetti, Daniela; Kinney, Anne L.; Ford, Holland; Doggett, Jesse; Long, Knox S.

    1995-01-01

    We report finding charts and accurate positions for 432 compact H-alpha emitting regions in the Local Group galaxy M 33 (NGC 598), in an effort to isolate candidates for an SS433-like stellar system. The objects were extracted from narrow band images, centered in the rest-frame H-alpha (lambda 6563 A) and in the red continuum at 6100 A. The atlas is complete down to V approximately equal to 20 and includes 279 compact HII regions and 153 line emitting point-like sources. The point-like sources undoubtedly include a variety of objects: very small HII regions, early type stars with intense stellar winds, and Wolf-Rayet stars, but should also contain objects with the characteristics of SS433. This extensive survey of compact H-alpha regions in M 33 is a first step towards the identification of peculiar stellar systems like SS433 in external galaxies.

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

    PubMed

    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.

  13. APOBEC3B cytidine deaminase targets the non-transcribed strand of tRNA genes in yeast.

    PubMed

    Saini, Natalie; Roberts, Steven A; Sterling, Joan F; Malc, Ewa P; Mieczkowski, Piotr A; Gordenin, Dmitry A

    2017-05-01

    Variations in mutation rates across the genome have been demonstrated both in model organisms and in cancers. This phenomenon is largely driven by the damage specificity of diverse mutagens and the differences in DNA repair efficiency in given genomic contexts. Here, we demonstrate that the single-strand DNA-specific cytidine deaminase APOBEC3B (A3B) damages tRNA genes at a 1000-fold higher efficiency than other non-tRNA genomic regions in budding yeast. We found that A3B-induced lesions in tRNA genes were predominantly located on the non-transcribed strand, while no transcriptional strand bias was observed in protein coding genes. Furthermore, tRNA gene mutations were exacerbated in cells where RNaseH expression was completely abolished (Δrnh1Δrnh35). These data suggest a transcription-dependent mechanism for A3B-induced tRNA gene hypermutation. Interestingly, in strains proficient in DNA repair, only 1% of the abasic sites formed upon excision of A3B-deaminated cytosines were not repaired leading to mutations in tRNA genes, while 18% of these lesions failed to be repaired in the remainder of the genome. A3B-induced mutagenesis in tRNA genes was found to be efficiently suppressed by the redundant activities of both base excision repair (BER) and the error-free DNA damage bypass pathway. On the other hand, deficiencies in BER did not have a profound effect on A3B-induced mutations in CAN1, the reporter for protein coding genes. We hypothesize that differences in the mechanisms underlying ssDNA formation at tRNA genes and other genomic loci are the key determinants of the choice of the repair pathways and consequently the efficiency of DNA damage repair in these regions. Overall, our results indicate that tRNA genes are highly susceptible to ssDNA-specific DNA damaging agents. However, increased DNA repair efficacy in tRNA genes can prevent their hypermutation and maintain both genome and proteome homeostasis. Published by Elsevier B.V.

  14. Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri

    PubMed Central

    van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S.; Britton, Robert A.

    2012-01-01

    Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution. PMID:22750793

  15. Comparative proteomic analysis reveals that T3SS, Tfp, and xanthan gum are key factors in initial stages of Citrus sinensis infection by Xanthomonas citri subsp. citri.

    PubMed

    Facincani, Agda P; Moreira, Leandro M; Soares, Márcia R; Ferreira, Cristiano B; Ferreira, Rafael M; Ferro, Maria I T; Ferro, Jesus A; Gozzo, Fabio C; de Oliveira, Julio C F

    2014-03-01

    The bacteria Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. The disease symptoms are characterized by localized host cell hyperplasia followed by tissue necrosis at the infected area. An arsenal of bacterial pathogenicity- and virulence-related proteins is expressed to ensure a successful infection process. At the post-genomic stage of Xac, we used a proteomic approach to analyze the proteins that are displayed differentially over time when the pathogen attacks the host plant. Protein extracts were prepared from infectious Xac grown in inducing medium (XAM1) for 24 h or from host citrus plants for 3 or 5 days after infection, detached times to evaluate the adaptation and virulence of the pathogen. The protein extracts were proteolyzed, and the peptides derived from tryptic digestion were investigated using liquid chromatography and tandem mass spectrometry. Changes in the protein expression profile were compared with the Xac genome and the proteome recently described under non-infectious conditions. An analysis of the proteome of Xac under infectious conditions revealed proteins directly involved in virulence such as the type III secretion system (T3SS) and effector proteins (T3SS-e), the type IV pilus (Tfp), and xanthan gum biosynthesis. Moreover, four new mutants related to proteins detected in the proteome and with different functions exhibited reduced virulence relative to the wild-type proteins. The results of the proteome analysis of infectious Xac define the processes of adaptation to the host and demonstrate the induction of the virulence factors of Xac involved in plant-pathogen interactions.

  16. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    PubMed Central

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

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

    PubMed

    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.

  18. Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae.

    PubMed

    Wawrzycka, Donata; Markowska, Katarzyna; Maciaszczyk-Dziubinska, Ewa; Migocka, Magdalena; Wysocki, Robert

    2017-01-01

    Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H + and Sb(III)/H + antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Predicting the transmembrane secondary structure of ligand-gated ion channels.

    PubMed

    Bertaccini, E; Trudell, J R

    2002-06-01

    Recent mutational analyses of ligand-gated ion channels (LGICs) have demonstrated a plausible site of anesthetic action within their transmembrane domains. Although there is a consensus that the transmembrane domain is formed from four membrane-spanning segments, the secondary structure of these segments is not known. We utilized 10 state-of-the-art bioinformatics techniques to predict the transmembrane topology of the tetrameric regions within six members of the LGIC family that are relevant to anesthetic action. They are the human forms of the GABA alpha 1 receptor, the glycine alpha 1 receptor, the 5HT3 serotonin receptor, the nicotinic AChR alpha 4 and alpha 7 receptors and the Torpedo nAChR alpha 1 receptor. The algorithms utilized were HMMTOP, TMHMM, TMPred, PHDhtm, DAS, TMFinder, SOSUI, TMAP, MEMSAT and TOPPred2. The resulting predictions were superimposed on to a multiple sequence alignment of the six amino acid sequences created using the CLUSTAL W algorithm. There was a clear statistical consensus for the presence of four alpha helices in those regions experimentally thought to span the membrane. The consensus of 10 topology prediction techniques supports the hypothesis that the transmembrane subunits of the LGICs are tetrameric bundles of alpha helices.

  20. The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3.

    PubMed

    Liger, D; Nizard, P; Gaillard, C; vanderSpek, J C; Murphy, J R; Pitard, B; Gillet, D

    1998-11-01

    We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T-(Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine-dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.

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

    PubMed

    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

  2. Structural insights into the T6SS effector protein Tse3 and the Tse3-Tsi3 complex from Pseudomonas aeruginosa reveal a calcium-dependent membrane-binding mechanism.

    PubMed

    Lu, Defen; Shang, Guijun; Zhang, Heqiao; Yu, Qian; Cong, Xiaoyan; Yuan, Jupeng; He, Fengjuan; Zhu, Chunyuan; Zhao, Yanyu; Yin, Kun; Chen, Yuanyuan; Hu, Junqiang; Zhang, Xiaodan; Yuan, Zenglin; Xu, Sujuan; Hu, Wei; Cang, Huaixing; Gu, Lichuan

    2014-06-01

    The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex. © 2014 John Wiley & Sons Ltd.

  3. Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8.

    PubMed

    Shiheido, Hirokazu; Kitagori, Koji; Sasaki, Chiyomi; Kobayashi, Shio; Aoyama, Takane; Urata, Kozue; Oku, Takuma; Hirayama, Yoshitaka; Yoshitomi, Hiroyuki; Hikida, Masaki; Yoshifuji, Hajime; Mimori, Tsuneyo; Watanabe, Takeshi; Shimizu, Jun

    2014-06-01

    BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3(+) T cells). BEND3(+) T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4(+) and CD8(+) T cells, and were also observed in cord blood. The stimulation of BEND3(+) T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3(+) T cells were higher than those by BEND3(-) T cells. The proportion of BEND3(+) T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3(+) T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3(+) T cells may be of importance and useful in understanding human T cell immunology.

  4. [Efficient extraction of transmembrane proteins using ProteoExtract Transmembrane Protein Extraction Kit].

    PubMed

    Błachnio, Karina

    2010-01-01

    Detergents commonly used for solubilization of membrane proteins may be ionic or non-ionic. Exposing membrane proteins to detergents, however, can adversely affect their native structure, which can be a major hindrance for functional studies. This is especially true for proteins with multiple transmembrane domains. The ProteoExtract Transmembrane Protein Extraction Kit (TM-PEK), offered by Merck, provides a detergent-free novel reagents to enable the mild and efficient extraction of proteins containing seven transmembrane domains, such as GPCRs (G-Protein Coupled Receptors) e.g.: Frizzled-4 and CELSR-3, from mammalian cells. The fraction enriched in transmembrane proteins using TM-PEK is directly compatible with enzyme assays, non-denaturing gel electrophoresis, 1- and 2-D SDS-PAGE, MS analysis, Western blotting, immunoprecipitation and ELISA. Unlike many alternatives, TM-PEK extraction procedure does not require sonication, extended rigorous vortexing, ultracentrifugation, or incubation of samples at elevated temperatures--thus minimizing the risk of post-extraction degradation or modifications.

  5. Accurate computational design of multipass transmembrane proteins.

    PubMed

    Lu, Peilong; Min, Duyoung; DiMaio, Frank; Wei, Kathy Y; Vahey, Michael D; Boyken, Scott E; Chen, Zibo; Fallas, Jorge A; Ueda, George; Sheffler, William; Mulligan, Vikram Khipple; Xu, Wenqing; Bowie, James U; Baker, David

    2018-03-02

    The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  6. Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram-Positive Genus Streptococcus

    PubMed Central

    Chen, Chen; Gao, George F.

    2012-01-01

    A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans. PMID:23056296

  7. The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids.

    PubMed

    Ennouri, Habiba; d'Abzac, Paul; Hakil, Florence; Branchu, Priscilla; Naïtali, Murielle; Lomenech, Anne-Marie; Oueslati, Ridha; Desbrières, Jacques; Sivadon, Pierre; Grimaud, Régis

    2017-01-01

    The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n-hexadecane-water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra-cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo-proteins secreted through the type-2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n-hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8

    PubMed Central

    Shiheido, Hirokazu; Kitagori, Koji; Sasaki, Chiyomi; Kobayashi, Shio; Aoyama, Takane; Urata, Kozue; Oku, Takuma; Hirayama, Yoshitaka; Yoshitomi, Hiroyuki; Hikida, Masaki; Yoshifuji, Hajime; Mimori, Tsuneyo; Watanabe, Takeshi; Shimizu, Jun

    2014-01-01

    BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3+ T cells). BEND3+ T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4+ and CD8+ T cells, and were also observed in cord blood. The stimulation of BEND3+ T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3+ T cells were higher than those by BEND3T cells. The proportion of BEND3+ T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3+ T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3+ T cells may be of importance and useful in understanding human T cell immunology. PMID:25400923

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

    PubMed

    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.

  10. Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton.

    PubMed

    Veenendaal, Andreas K J; Sundin, Charlotta; Blocker, Ariel J

    2009-01-01

    Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

  11. Singleshot T1 Mapping using Simultaneous Acquisitions of Spin- and STimulated-Echo Planar Imaging (2D ss-SESTEPI)

    PubMed Central

    Shi, Xianfeng; Kim, Seong-Eun; Jeong, Eun-Kee

    2011-01-01

    The conventional stimulated-echo NMR sequence only measures the longitudinal component, while discarding the transverse component, after tipping up the prepared magnetization. This transverse magnetization can be used to measure a spin-echo, in addition to the stimulated-echo. 2D ss-SESTEPI is an EPI-based singleshot imaging technique that simultaneously acquires a spin-echo-planar image (SEPI) and a stimulated-echo-planar image (STEPI) after a single RF excitation. The magnitudes of SEPI and STEPI differ by T1 decay and diffusion weighting for perfect 90° RF, and thus can be used to rapidly measure T1. However, the spatial variation of B1 amplitude induces un-even splitting of the transverse magnetization for SEPI and STEPI within the imaging FOV. Correction for B1 inhomogeneity is therefore critical for 2D ss-SESTEPI to be used for T1 measurement. We developed a method for B1 inhomogeneity correction by acquiring an additional STEPI with minimal mixing time, calculating the difference between the spin-echo and the stimulated-echo and multiplying the STEPI by the inverse functional map. Diffusion-induced decay is corrected by measuring the average diffusivity during the prescanning. Rapid singleshot T1 mapping may be useful for various applications, such as dynamic T1 mapping for real-time estimation of the concentration of contrast agent in DCE-MRI. PMID:20564579

  12. Human in vitro induced T regulatory cells and memory T cells share common demethylation of specific FOXP3 promoter region.

    PubMed

    Bégin, Philippe; Schulze, Janika; Baron, Udo; Olek, Sven; Bauer, Rebecca N; Passerini, Laura; Baccheta, Rosa; Nadeau, Kari C

    2015-01-01

    The FOXP3 gene is the master regulator for T regulatory cells and is under tight DNA methylation control at the Treg specific demethylated region (TSDR) in its first intron. This said, methylation of its promoter region, the significance of which is unknown, has also been associated with various immune-related disease states such as asthma, food allergy, auto-immunity and cancer. Here, we used induced T regulatory cells (iTreg) as a target cell population to identify candidate hypomethylated CpG sites in the FOXP3 gene promoter to design a DNA methylation quantitative assay for this region. Three CpG sites at the promoter region showed clear demethylation pattern associated with high FOXP3 expression after activation in presence of TGFβ and were selected as primary targets to design methylation-dependent RT-PCR primers and probes. We then examined the methylation of this 'inducible-promoter-demethylated-region' (IPDR) in various FOXP3+ T cell subsets. Both naïve and memory thymic-derived Treg cells were found to be fully demethylated at both the IPDR and TSDR. Interestingly, in addition to iTregs, both CD25- and CD25(lo) conventional memory CD4+CD45RA- T cells displayed a high fraction of IPDR demethylated cells in absence of TSDR demethylation. This implies that the fraction of memory T cells should be taken in account when interpreting FOXP3 promoter methylation results from clinical studies. This approach, which is available for testing in clinical samples could have diagnostic and prognostic value in patients with immune or auto-inflammatory diseases.

  13. Large Multiple Transmembrane Domain Fragments of a G Protein-Coupled Receptor: Biosynthesis, Purification, and Biophysical Studies

    PubMed Central

    Potetinova, Zhanna; Tantry, Subramanyam; Cohen, Leah S.; Caroccia, Katrina E.; Arshava, Boris; Becker, Jeffrey M.; Naider, Fred

    2013-01-01

    To conduct biophyiscal analyses on large domains of GPCRs, multi-milligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of 4 transmembrane and 5 transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G31-A198] (4TMN), four C-terminal Ste2p[T155-L340] (4TMC) or five C-terminal Ste2p[I120-L340] (5TMC) transmembrane segments of Ste2p. 4TMN was expressed as a fusion protein using a modified pMMHa vector in L-arabinose-induced Escherichia coli BL21-AI, and cleaved with cyanogen bromide. 4TMC and 5TMC were obtained by direct expression using a pET21a vector in IPTG-induced Escherichia coli BL21(DE3) cells. 4TMC and 5TMC were biosynthesized on a preparative scale, isolated in multi-milligram amounts, characterized by MS and investigated by biophysical methods. CD spectroscopy indicated the expected highly α-helical content for 4TMC and 5TMC in membrane mimetic environments. Tryptophan fluorescence showed that 5TMC integrated into the nonpolar region of 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles. HSQC-TROSY investigations revealed that [15N]-labeled 5TMC in 50% trifluoroethanol-d2/H2O/0.05% trifluoroacetic acid was stable enough to conduct long multidimensional NMR measurements. The entire Ste2p GPCR was not readily reconstituted from the first two and last five or first three and last four transmembrane domains. PMID:23203693

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

    PubMed Central

    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

  15. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

    PubMed Central

    de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2016-01-01

    In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

  16. Investigation of Regional Influence of Magic-Angle Effect on T2 in Human Articular Cartilage with Osteoarthritis at 3 T

    PubMed Central

    Wang, Ligong; Regatte, Ravinder R.

    2014-01-01

    Rationale and Objectives The objectives of this research study were to determine the magic-angle effect on different subregions of in vivo human femoral cartilage through the quantitative assessment of the effect of static magnetic field orientation (B0) on transverse (T2) relaxation time at 3.0 T. Materials and Methods Healthy volunteers (n = 5l; mean age, 36.4 years) and clinical patients (n = 5; mean age, 64 years) with early osteoarthritis (OA) were scanned at 3.0-T magnetic resonance using an 8-channel phased-array knee coil (transmit-receive). Results The T2 maps revealed significantly greater values in ventral than in dorsal regions. When the cartilage regions were oriented at 55° to B0 (magic angle), the longest T2 values were detected in comparison with the neighboring regions oriented 90° and 180° (0°) to B0. The subregions oriented 180° (0°) to B0 showed the lowest T2 values. Conclusions The differences in T2 values of different subregions suggest that magic-angle effect needs to be considered when interpreting cartilage abnormalities in OA patients. PMID:25481517

  17. Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3.

    PubMed

    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.

  18. Cardiac MOLLI T1 mapping at 3.0 T: comparison of patient-adaptive dual-source RF and conventional RF transmission.

    PubMed

    Rasper, Michael; Nadjiri, Jonathan; Sträter, Alexandra S; Settles, Marcus; Laugwitz, Karl-Ludwig; Rummeny, Ernst J; Huber, Armin M

    2017-06-01

    To prospectively compare image quality and myocardial T 1 relaxation times of modified Look-Locker inversion recovery (MOLLI) imaging at 3.0 T (T) acquired with patient-adaptive dual-source (DS) and conventional single-source (SS) radiofrequency (RF) transmission. Pre- and post-contrast MOLLI T 1 mapping using SS and DS was acquired in 27 patients. Patient wise and segment wise analysis of T 1 times was performed. The correlation of DS MOLLI measurements with a reference spin echo sequence was analysed in phantom experiments. DS MOLLI imaging reduced T 1 standard deviation in 14 out of 16 myocardial segments (87.5%). Significant reduction of T 1 variance could be obtained in 7 segments (43.8%). DS significantly reduced myocardial T 1 variance in 16 out of 25 patients (64.0%). With conventional RF transmission, dielectric shading artefacts occurred in six patients causing diagnostic uncertainty. No according artefacts were found on DS images. DS image findings were in accordance with conventional T 1 mapping and late gadolinium enhancement (LGE) imaging. Phantom experiments demonstrated good correlation of myocardial T 1 time between DS MOLLI and spin echo imaging. Dual-source RF transmission enhances myocardial T 1 homogeneity in MOLLI imaging at 3.0 T. The reduction of signal inhomogeneities and artefacts due to dielectric shading is likely to enhance diagnostic confidence.

  19. The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

    NASA Astrophysics Data System (ADS)

    Wu, Sangwook

    2016-04-01

    The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

  20. Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton▿ †

    PubMed Central

    Veenendaal, Andreas K. J.; Sundin, Charlotta; Blocker, Ariel J.

    2009-01-01

    Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds. PMID:18996990

  1. Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential

    PubMed Central

    Saint Clair, Erica C.; Ogren, John I.; Mamaev, Sergey; Russano, Daniel; Kralj, Joel M.; Rothschild, Kenneth J.

    2013-01-01

    Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3–11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (Oall-trans) is found. At higher pH a second 13-cis retinal N-like species (N13-cis) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N13-cis detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N13-cis to M13-cis levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential

  2. Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

    PubMed Central

    Lee, Wonbae; Gillies, John P.; Jose, Davis; Israels, Brett A.; von Hippel, Peter H.; Marcus, Andrew H.

    2016-01-01

    Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed. PMID:27694621

  3. T3SEdb: data warehousing of virulence effectors secreted by the bacterial Type III Secretion System.

    PubMed

    Tay, Daniel Ming Ming; Govindarajan, Kunde Ramamoorthy; Khan, Asif M; Ong, Terenze Yao Rui; Samad, Hanif M; Soh, Wei Wei; Tong, Minyan; Zhang, Fan; Tan, Tin Wee

    2010-10-15

    Effectors of Type III Secretion System (T3SS) play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE) sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i) comprehensive annotation of experimental status of effectors, ii) submission and curation review of records by users of the database, and iii) the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression) are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%). Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors. The T3SEdb represents a platform for inclusion of

  4. Topology of transmembrane channel-like gene 1 protein.

    PubMed

    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.

  5. The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis

    PubMed Central

    Yamamoto, Yoshiko; Negi, Juntaro; Isogai, Yasuhiro; Schroeder, Julian I.; Iba, Koh

    2016-01-01

    The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response. PMID:26764376

  6. The Symbiotic System SS73 17 seen with Suzaku

    NASA Technical Reports Server (NTRS)

    Smith, Randall K.; Mushotzky, Richard; Kallman, Tim; Tueller, Jack; Mukai, Koji; Markwardt, Craig

    2007-01-01

    We observed with Suzaku the symbiotic star SS73 17, motivated by the discovery by the INTEGRAL satellite and the Swift BAT survey that it emits hard X-rays. Our observations showed a highly-absorbed X-ray spectrum with NH > loz3 emp2, equivalent to Av > 26, although the source has B magnitude 11.3 and is also bright in UV. The source also shows strong, narrow iron lines including fluorescent Fe K as well as Fe xxv and Fe XXVI. The X-ray spectrum can be fit with a thermal model including an absorption component that partially covers the source. Most of the equivalent width of the iron fluorescent line in this model can be explained as a combination of reprocessing in a dense absorber plus reflection off a white dwarf surface, but it is likely that the continuum is partially seen in reflection as well. Unlike other symbiotic systems that show hard X-ray emission (CH Cyg, RT Cru, T CrB, GX1+4), SS73 17 is not known to have shown nova-like optical variability, X-ray flashes, or pulsations, and has always shown faint soft X-ray emission. As a result, although it is likely a white dwarf, the nature of the compact object in SS73 17 is still uncertain. SS73 17 is probably an extreme example of the recently discovered and relatively small class of hard X-ray emitting symbiotic systems.

  7. A substitution in the transmembrane region of the glycoprotein leads to an unstable attenuation of Machupo virus.

    PubMed

    Patterson, Michael; Koma, Takaaki; Seregin, Alexey; Huang, Cheng; Miller, Milagros; Smith, Jennifer; Yun, Nadezhda; Smith, Jeanon; Paessler, Slobodan

    2014-09-01

    Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing a reverse-genetics system recently developed, we report the rescue of a rationally modified recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice, we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild-type genotype. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

    PubMed

    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.

  9. The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis.

    PubMed

    Yamamoto, Yoshiko; Negi, Juntaro; Wang, Cun; Isogai, Yasuhiro; Schroeder, Julian I; Iba, Koh

    2016-02-01

    The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response. © 2016 American Society of Plant Biologists. All rights reserved.

  10. The type III secretion system needle tip complex mediates host cell sensing and translocon insertion.

    PubMed

    Veenendaal, Andreas K J; Hodgkinson, Julie L; Schwarzer, Lynn; Stabat, David; Zenk, Sebastian F; Blocker, Ariel J

    2007-03-01

    Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.

  11. Sub-kT/q Subthreshold-Slope Using Negative Capacitance in Low-Temperature Polycrystalline-Silicon Thin-Film Transistor

    PubMed Central

    Park, Jae Hyo; Jang, Gil Su; Kim, Hyung Yoon; Seok, Ki Hwan; Chae, Hee Jae; Lee, Sol Kyu; Joo, Seung Ki

    2016-01-01

    Realizing a low-temperature polycrystalline-silicon (LTPS) thin-film transistor (TFT) with sub-kT/q subthreshold slope (SS) is significantly important to the development of next generation active-matrix organic-light emitting diode displays. This is the first time a sub-kT/q SS (31.44 mV/dec) incorporated with a LTPS-TFT with polycrystalline-Pb(Zr,Ti)O3 (PZT)/ZrTiO4 (ZTO) gate dielectrics has been demonstrated. The sub-kT/q SS was observed in the weak inversion region at −0.5 V showing ultra-low operating voltage with the highest mobility (250.5 cm2/Vsec) reported so far. In addition, the reliability of DC negative bias stress, hot carrier stress and self-heating stress in LTPS-TFT with negative capacitance was investigated for the first time. It was found that the self-heating stress showed accelerated SS degradation due to the PZT Curie temperature. PMID:27098115

  12. Validity of Automated Choroidal Segmentation in SS-OCT and SD-OCT.

    PubMed

    Zhang, Li; Buitendijk, Gabriëlle H S; Lee, Kyungmoo; Sonka, Milan; Springelkamp, Henriët; Hofman, Albert; Vingerling, Johannes R; Mullins, Robert F; Klaver, Caroline C W; Abràmoff, Michael D

    2015-05-01

    To evaluate the validity of a novel fully automated three-dimensional (3D) method capable of segmenting the choroid from two different optical coherence tomography scanners: swept-source OCT (SS-OCT) and spectral-domain OCT (SD-OCT). One hundred eight subjects were imaged using SS-OCT and SD-OCT. A 3D method was used to segment the choroid and quantify the choroidal thickness along each A-scan. The segmented choroidal posterior boundary was evaluated by comparing to manual segmentation. Differences were assessed to test the agreement between segmentation results of the same subject. Choroidal thickness was defined as the Euclidian distance between Bruch's membrane and the choroidal posterior boundary, and reproducibility was analyzed using automatically and manually determined choroidal thicknesses. For SS-OCT, the average choroidal thickness of the entire 6- by 6-mm2 macular region was 219.5 μm (95% confidence interval [CI], 204.9-234.2 μm), and for SD-OCT it was 209.5 μm (95% CI, 197.9-221.0 μm). The agreement between automated and manual segmentations was high: Average relative difference was less than 5 μm, and average absolute difference was less than 15 μm. Reproducibility of choroidal thickness between repeated SS-OCT scans was high (coefficient of variation [CV] of 3.3%, intraclass correlation coefficient [ICC] of 0.98), and differences between SS-OCT and SD-OCT results were small (CV of 11.0%, ICC of 0.73). We have developed a fully automated 3D method for segmenting the choroid and quantifying choroidal thickness along each A-scan. The method yielded high validity. Our method can be used reliably to study local choroidal changes and may improve the diagnosis and management of patients with ocular diseases in which the choroid is affected.

  13. Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor*

    PubMed Central

    Tong, Yufeng; Hota, Prasanta K.; Penachioni, Junia Y.; Hamaneh, Mehdi B.; Kim, SoonJeung; Alviani, Rebecca S.; Shen, Limin; He, Hao; Tempel, Wolfram; Tamagnone, Luca; Park, Hee-Won; Buck, Matthias

    2009-01-01

    Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 Å crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120GAP·H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation. PMID:19843518

  14. Identification of MICA alleles with a long Leu-repeat in the transmembrane region and no cytoplasmic tail due to a frameshift-deletion in exon 4.

    PubMed

    Obuchi, N; Takahashi, M; Nouchi, T; Satoh, M; Arimura, T; Ueda, K; Akai, J; Ota, M; Naruse, T; Inoko, H; Numano, F; Kimura, A

    2001-06-01

    MHC class I chain-related gene A (MICA) is located close to HLA-B gene and expressed in epithelial cells. The MICA gene is reported to be highly polymorphic as are the classical class I genes. To further assess the polymorphism in the MICA gene, we analyzed a total of 60 HLA-homozygous cells for the sequences spanning exons 2-6. In the analysis, four new MICA alleles were identified and six variations were recognized in exon 6. MICA*017, which was identified in three HLA-B57 homozygous cells (DBB, DEM and WIN), differed from MICA*002 in exon 3 and had a guanine deletion at the 3' end of exon 4. MICA*015 identified in an HLA-B45 homozygous cell (OMW) also had the same deletion that causes a frameshift mutation resulting in complete change of the transmembrane region and premature termination in the cytoplasmic tail; these alleles have a long hydrophobic leucine-rich region instead of the alanine repeat in the transmembrane region and terminate at the second position in the cytoplasmic domain. The frameshift deletion was found only in HLA-B45- or -B57-positive panels tested, suggesting a strong linkage disequilibrium between the deletion and B45 or B57. MICA*048, which was different in exon 5 from MICA*008, was identified in an HLA-B61 homozygous cell (TA21), while MICA*00901 identified in HLA-B51 homozygous cells (LUY and KT2) was distinguished from MICA*009 by exon 6.

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

    PubMed

    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.

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

    PubMed

    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.

  17. Transmembrane Segments Form Tertiary Hairpins in the Folding Vestibule of the Ribosome.

    PubMed Central

    Tu, LiWei; Khanna, Pooja; Deutsch, Carol

    2013-01-01

    Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100 Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a ‘folding vestibule’ that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains, but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved, intramolecular crosslinking assay, that the helical transmembrane S3b-S4 hairpin (‘paddle’) of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane. PMID:24055377

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

    DOEpatents

    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.

  19. Lactic acid bacteria increase antiallergic effect of Artemisia princeps pampanini SS-1.

    PubMed

    Lee, Seung-Hoon; Shin, Yong-Wook; Bae, Eun-Ah; Lee, Bomi; Min, Sungwon; Baek, Nam-In; Chung, Hae-Gon; Kim, Nam-Jae; Kim, Dong-Hyun

    2006-09-01

    Artemisia princeps Pampanini, which is called Ssajuarissuk in Korean (SS-1), was fermented with lactic acid bacteria (LAB) and their passive cutaneous anaphylaxis reaction-inhibitory activity was investigated. Of these fermented agents, SS-1 extract fermented with Bifidobacterium infantis K-525 (F-SS-1) most effectively inhibited the release of P-hexosamindase from RBL-2H3 cells induced IgE. In IgE-induced RBL-2H3 cells, F-SS-1 inhibited proinflammatory cytokines IL-6 and TNF-alpha mRNA expression. Oral administration of SS-1 and F-SS-1 to mice inhibited passive cutaneous anaphylaxis (PCA) reaction induced by IgE and scratching behaviors induced by compound 48/80. The inhibitory activity of F-SS-1 against scratching behaviors was more effective than that of SS-1. These findings suggest that the fermentation of SS-1 with LAB can increase its antiallergic activity.

  20. Resource for structure related information on transmembrane proteins

    NASA Astrophysics Data System (ADS)

    Tusnády, Gábor E.; Simon, István

    Transmembrane proteins are involved in a wide variety of vital biological processes including transport of water-soluble molecules, flow of information and energy production. Despite significant efforts to determine the structures of these proteins, only a few thousand solved structures are known so far. Here, we review the various resources for structure-related information on these types of proteins ranging from the 3D structure to the topology and from the up-to-date databases to the various Internet sites and servers dealing with structure prediction and structure analysis. Abbreviations: 3D, three dimensional; PDB, Protein Data Bank; TMP, transmembrane protein.

  1. Transmembrane Polyproline Helix.

    PubMed

    Kubyshkin, Vladimir; Grage, Stephan L; Bürck, Jochen; Ulrich, Anne S; Budisa, Nediljko

    2018-05-03

    The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold and demonstrate its transmembrane alignment in model lipid bilayers by means of solid-state 19 F NMR. As result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding.

  2. Common T cell receptor clonotype in lacrimal glands and labial salivary glands from patients with Sjögren's syndrome.

    PubMed Central

    Matsumoto, I; Tsubota, K; Satake, Y; Kita, Y; Matsumura, R; Murata, H; Namekawa, T; Nishioka, K; Iwamoto, I; Saitoh, Y; Sumida, T

    1996-01-01

    Sjogren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into lacrimal and salivary glands leading to symptomatic dry eyes and mouth. Immunohistological studies have clarified that the majority of infiltrating lymphocytes around the lacrimal glands and labial salivary glands are CD4 positive alphabeta T cells. To analyze the pathogenesis of T cells infiltrating into lacrimal and labial salivary glands, we examined T cell clonotype of these cells in both glands from four SS patients using PCR-single-strand conformation polymorphism (SSCP) and a sequencing method. SSCP analysis showed that some infiltrating T cells in both glands expand clonally, suggesting that the cells proliferate by antigen-driven stimulation. Intriguingly, six to sixteen identical T cell receptor (TCR) Vbeta genes were commonly found in lacrimal glands and labial salivary glands from individual patients. This indicates that some T cells infiltrating into both glands recognize the shared epitopes on autoantigens. Moreover, highly conserved amino acid sequence motifs were found in the TCR CDR3 region bearing the same TCR Vbeta family gene from four SS patients, supporting the notion that the shared epitopes on antigens are limited. In conclusion, these findings suggest that some autoreactive T cells infiltrating into the lips and eyes recognized restricted epitopes of a common autoantigen in patients with SS. PMID:8621782

  3. On the self-association potential of transmembrane tight junction proteins.

    PubMed

    Blasig, I E; Winkler, L; Lassowski, B; Mueller, S L; Zuleger, N; Krause, E; Krause, G; Gast, K; Kolbe, M; Piontek, J

    2006-02-01

    Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.

  4. A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi.

    PubMed

    Login, Frédéric H; Fries, Markus; Wang, Xiaohui; Pickersgill, Richard W; Shevchik, Vladimir E

    2010-05-01

    The type II secretion system (T2SS) is widely exploited by proteobacteria to secrete enzymes and toxins involved in bacterial survival and pathogenesis. The outer membrane pore formed by the secretin OutD and the inner membrane protein OutC are two key components of the secretion complex, involved in secretion specificity. Here, we show that the periplasmic regions of OutC and OutD interact directly and map the interaction site of OutC to a 20-residue peptide named OutCsip (secretin interacting peptide, residues 139-158). This peptide interacts in vitro with two distinct sites of the periplasmic region of OutD, one located on the N0 subdomain and another overlapping the N2-N3' subdomains. The two interaction sites of OutD have different modes of binding to OutCsip. A single substitution, V143S, located within OutCsip prevents its interaction with one of the two binding sites of OutD and fully inactivates the T2SS. We show that the N0 subdomain of OutD interacts also with a second binding site within OutC located in the region proximal to the transmembrane segment. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machine.

  5. Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

    DOE PAGES

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; ...

    2014-12-22

    Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is notmore » necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Finally, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.« less

  6. CAR T-cells targeting FLT3 have potent activity against FLT3-ITD+ AML and act synergistically with the FLT3-inhibitor crenolanib.

    PubMed

    Jetani, Hardikkumar; Garcia-Cadenas, Irene; Nerreter, Thomas; Thomas, Simone; Rydzek, Julian; Meijide, Javier Briones; Bonig, Halvard; Herr, Wolfgang; Sierra, Jordi; Einsele, Hermann; Hudecek, Michael

    2018-05-01

    FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein expressed on normal hematopoietic stem and progenitor cells (HSC) and retained on malignant blasts in acute myeloid leukemia (AML). We engineered CD8 + and CD4 + T-cells expressing a FLT3-specific chimeric antigen receptor (CAR) and demonstrate they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3 with internal tandem duplication (FLT3-ITD). We also show that treatment with the FLT3-inhibitor crenolanib leads to increased surface expression of FLT3 specifically on FLT3-ITD + AML cells and consecutively, enhanced recognition by FLT3-CAR T-cells in vitro and in vivo. As anticipated, we found that FLT3-CAR T-cells recognize normal HSCs in vitro and in vivo, and disrupt normal hematopoiesis in colony-formation assays, suggesting that adoptive therapy with FLT3-CAR T-cells will require subsequent CAR T-cell depletion and allogeneic HSC transplantation to reconstitute the hematopoietic system. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD + AML. Further, our data provide the first proof-of-concept that CAR T-cell immunotherapy and small molecule inhibition can be used synergistically, as exemplified by our data showing superior antileukemia efficacy of FLT3-CAR T-cells in combination with crenolanib.

  7. Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

    PubMed Central

    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

  8. Succession of splicing regulatory elements determines cryptic 5΄ss functionality

    PubMed Central

    Brillen, Anna-Lena; Schöneweis, Katrin; Walotka, Lara; Hartmann, Linda; Müller, Lisa; Ptok, Johannes; Kaisers, Wolfgang; Poschmann, Gereon; Stühler, Kai; Buratti, Emanuele

    2017-01-01

    Abstract A critical step in exon definition is the recognition of a proper splice donor (5΄ss) by the 5’ end of U1 snRNA. In the selection of appropriate 5΄ss, cis-acting splicing regulatory elements (SREs) are indispensable. As a model for 5΄ss recognition, we investigated cryptic 5΄ss selection within the human fibrinogen Bβ-chain gene (FGB) exon 7, where we identified several exonic SREs that simultaneously acted on up- and downstream cryptic 5΄ss. In the FGB exon 7 model system, 5΄ss selection iteratively proceeded along an alternating sequence of U1 snRNA binding sites and interleaved SREs which in principle supported different 3’ exon ends. Like in a relay race, SREs either suppressed a potential 5΄ss and passed the splicing baton on or splicing actually occurred. From RNA-Seq data, we systematically selected 19 genes containing exons with silent U1 snRNA binding sites competing with nearby highly used 5΄ss. Extensive SRE analysis by different algorithms found authentic 5΄ss significantly more supported by SREs than silent U1 snRNA binding sites, indicating that our concept may permit generalization to a model for 5΄ss selection and 3’ exon end definition. PMID:28039323

  9. Phosphorylation of the Antiviral Protein Interferon-inducible Transmembrane Protein 3 (IFITM3) Dually Regulates Its Endocytosis and Ubiquitination*

    PubMed Central

    Chesarino, Nicholas M.; McMichael, Temet M.; Hach, Jocelyn C.; Yount, Jacob S.

    2014-01-01

    Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr20) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr27. Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr20 caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr27 mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr20 is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXXΦ (where X is any amino acid and Φ is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr20 both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr20 may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3. PMID:24627473

  10. Safe and effective error rate monitors for SS7 signaling links

    NASA Astrophysics Data System (ADS)

    Schmidt, Douglas C.

    1994-04-01

    This paper describes SS7 error monitor characteristics, discusses the existing SUERM (Signal Unit Error Rate Monitor), and develops the recently proposed EIM (Error Interval Monitor) for higher speed SS7 links. A SS7 error monitor is considered safe if it ensures acceptable link quality and is considered effective if it is tolerant to short-term phenomena. Formal criteria for safe and effective error monitors are formulated in this paper. This paper develops models of changeover transients, the unstable component of queue length resulting from errors. These models are in the form of recursive digital filters. Time is divided into sequential intervals. The filter's input is the number of errors which have occurred in each interval. The output is the corresponding change in transmit queue length. Engineered EIM's are constructed by comparing an estimated changeover transient with a threshold T using a transient model modified to enforce SS7 standards. When this estimate exceeds T, a changeover will be initiated and the link will be removed from service. EIM's can be differentiated from SUERM by the fact that EIM's monitor errors over an interval while SUERM's count errored messages. EIM's offer several advantages over SUERM's, including the fact that they are safe and effective, impose uniform standards in link quality, are easily implemented, and make minimal use of real-time resources.

  11. Clinical evaluation of single-shot and readout-segmented diffusion-weighted imaging in stroke patients at 3 T.

    PubMed

    Morelli, John; Porter, David; Ai, Fei; Gerdes, Clint; Saettele, Megan; Feiweier, Thorsten; Padua, Abraham; Dix, James; Marra, Michael; Rangaswamy, Rajesh; Runge, Val

    2013-04-01

    Diffusion-weighted imaging (DWI) magnetic resonance imaging (MRI) is most commonly performed utilizing a single-shot echo-planar imaging technique (ss-EPI). Susceptibility artifact and image blur are severe when this sequence is utilized at 3 T. To evaluate a readout-segmented approach to DWI MR in comparison with single-shot echo planar imaging for brain MRI. Eleven healthy volunteers and 14 patients with acute and early subacute infarctions underwent DWI MR examinations at 1.5 and 3T with ss-EPI and readout-segmented echo-planar (rs-EPI) DWI at equal nominal spatial resolutions. Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) calculations were made, and two blinded readers ranked the scans in terms of high signal intensity bulk susceptibility artifact, spatial distortions, image blur, overall preference, and motion artifact. SNR and CNR were greatest with rs-EPI (8.1 ± 0.2 SNR vs. 6.0 ± 0.2; P <10(-4) at 3T). Spatial distortions were greater with single-shot (0.23 ± 0.03 at 3T; P <0.001) than with rs-EPI (0.12 ± 0.02 at 3T). Combined with blur and artifact reduction, this resulted in a qualitative preference for the readout-segmented scans overall. Substantial image quality improvements are possible with readout-segmented vs. single-shot EPI - the current clinical standard for DWI - regardless of field strength (1.5 or 3 T). This results in improved image quality secondary to greater real spatial resolution and reduced artifacts from susceptibility in MR imaging of the brain.

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

    PubMed

    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.

  13. French validation of the Maslach Burnout Inventory-Student Survey (MBI-SS).

    PubMed

    Faye-Dumanget, Christine; Carré, Julie; Le Borgne, Margaux; Boudoukha, Pr Abdel Halim

    2017-12-01

    Several international studies have been conducted on student burnout. To contribute to the clinical examination as well as research on the mental health of students, the MBI-SS (Maslach Burnout Inventory-Student Survey) has been validated and used in different countries but not in French. The aim of this study is to examine the validity of the 3-dimensional model of the French version of the MBI-SS, which is characterized by emotional "Emotional Exhaustion" (EE); "Cynicism" (CY); and low scores in "Academic Efficacy" (AE). A total of 667 university students were questioned to study the 3-dimensional structure of the French translation of the MBI-SS. The results validate the 3-dimensional structure of the MBI-SS and indicate satisfactory psychometric values. It is concluded that the MBI-SS can be used to assess burnout in French students. © 2017 John Wiley & Sons, Ltd.

  14. Multi-scale virtual view on the precessing jet SS433

    NASA Astrophysics Data System (ADS)

    Monceau-Baroux, R.; Porth, O.; Meliani, Z.; Keppens, R.

    2014-07-01

    Observations of SS433 infer how an X-ray binary gives rise to a corkscrew patterned relativistic jet. XRB SS433 is well known on a large range of scales for wich we realize 3D simulation and radio mappings. For our study we use relativistic hydrodynamic in special relativity using a relativistic effective polytropic index. We use parameters extracted from observations to impose thermodynamical conditions of the ISM and jet. We follow the kinetic and thermal energy content, of the various ISM and jet regions. Our simulation follows simultaneously the evolution of the population of electrons which are accelerated by the jet. The evolving spectrum of these electrons, together with an assumed equipartition between dynamic and magnetic pressure, gives input for estimating the radio emission from our simulation. Ray tracing according to a direction of sight then realizes radio mappings of our data. Single snapshots are realised to compare with VLA observation as in Roberts et al. 2008. A radio movie is realised to compare with the 41 days movie made with the VLBA instrument. Finaly a larger scale simulation explore the discrepancy of opening angle between 10 and 20 degree between the large scale observation of SS433 and its close in observation.

  15. RORγt antagonist suppresses M3 muscarinic acetylcholine receptor-induced Sjögren's syndrome-like sialadenitis.

    PubMed

    Tahara, M; Tsuboi, H; Segawa, S; Asashima, H; Iizuka-Koga, M; Hirota, T; Takahashi, H; Kondo, Y; Matsui, M; Matsumoto, I; Sumida, T

    2017-02-01

    We showed recently that M3 muscarinic acetylcholine receptor (M3R)-reactive CD3 + T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid-related orphan receptor-gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R -/- ) mice immunized with murine M3R peptide mixture were inoculated into recombination-activating gene 1 knockout (Rag-1 -/- ) mice (M3R -/- →Rag-1 -/- ) with MIS. Immunized M3R -/- mice (pretransfer treatment) and M3R -/- →Rag-1 -/- mice (post-transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide-stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme-linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4 + T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)-γ and interleukin (IL)-17 production significantly compared with phosphate-buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4 + T cells rather than CD11c + cells. Post-transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN-γ and IL-17 production (P < 0·05). In vitro, A213 suppressed IFN-γ and IL-17 production from M3R-stimulated splenocytes and CD4 + T cells of immunized M3R -/- mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL-17 production from Th17 differentiated CD4 + T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS-like sialadenitis through suppression of IL-17 and IFN-γ production

  16. PDB_TM: selection and membrane localization of transmembrane proteins in the protein data bank.

    PubMed

    Tusnády, Gábor E; Dosztányi, Zsuzsanna; Simon, István

    2005-01-01

    PDB_TM is a database for transmembrane proteins with known structures. It aims to collect all transmembrane proteins that are deposited in the protein structure database (PDB) and to determine their membrane-spanning regions. These assignments are based on the TMDET algorithm, which uses only structural information to locate the most likely position of the lipid bilayer and to distinguish between transmembrane and globular proteins. This algorithm was applied to all PDB entries and the results were collected in the PDB_TM database. By using TMDET algorithm, the PDB_TM database can be automatically updated every week, keeping it synchronized with the latest PDB updates. The PDB_TM database is available at http://www.enzim.hu/PDB_TM.

  17. Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage.

    PubMed

    Voiniciuc, Catalin; Dean, Gillian H; Griffiths, Jonathan S; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L; Estelle, Mark; Haughn, George W

    2013-03-01

    Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.

  18. Grafting PNIPAAm from β-barrel shaped transmembrane nanopores.

    PubMed

    Charan, Himanshu; Kinzel, Julia; Glebe, Ulrich; Anand, Deepak; Garakani, Tayebeh Mirzaei; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich; Böker, Alexander

    2016-11-01

    The research on protein-polymer conjugates by grafting from the surface of proteins has gained significant interest in the last decade. While there are many studies with globular proteins, membrane proteins have remained untouched to the best of our knowledge. In this study, we established the conjugate formation with a class of transmembrane proteins and grow polymer chains from the ferric hydroxamate uptake protein component A (FhuA; a β-barrel transmembrane protein of Escherichia coli). As the lysine residues of naturally occurring FhuA are distributed over the whole protein, FhuA was reengineered to have up to 11 lysines, distributed symmetrically in a rim on the membrane exposed side (outside) of the protein channel and exclusively above the hydrophobic region. Reengineering of FhuA ensures a polymer growth only on the outside of the β-barrel and prevents blockage of the channel as a result of the polymerization. A water-soluble initiator for controlled radical polymerization (CRP) was consecutively linked to the lysine residues of FhuA and N-isopropylacrylamide (NIPAAm) polymerized under copper-mediated CRP conditions. The conjugate formation was analyzed by using MALDI-ToF mass spectrometry, SDS-PAGE, circular dichroism spectroscopy, analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy and size exclusion chromatography. Such conjugates combine the specific functions of the transmembrane proteins, like maintaining membrane potential gradients or translocation of substrates with the unique properties of synthetic polymers such as temperature and pH stimuli handles. FhuA-PNIPAAm conjugates will serve as functional nanosized building blocks for applications in targeted drug delivery, self-assembly systems, functional membranes and transmembrane protein gated nanoreactors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. [Study and development of a new automatic suction system (SS) of intratracheal sputum in order to improve QOL of many patients with tracheostomy mechanical ventilation (TMV) and their family members by this new autonomic SS].

    PubMed

    Hokezu, Youichi; Yamamoto, Makoto; Tokunaga, Syuichi; Niikura, Makoto; Nagamatsu, Keiji; Kira, Jun-ichi; Fukunaga, Toshihide; Shima, Kouji; Kikuchi, Seiji; Kimura, Itaru; Kondo, Kiyohiko; Mori, Teruhiko; Goto, Katumasa; Takigami, Shigeru; Shioya, Keiichi; Uehara, Minako

    2009-11-01

    In Japan, many patients equipped with TMV are under medical treatment at home after 1990. These patients can't put out sputa in trachea, so that these patient's family members must suck these patient's intratracheal sputa all days. Mr Yamamoto and Mr Tokunaga, main researchers of this study, began the study on the automatic SS of itratracheal sputa from 1999. In first stage, They developed the intermittent SS in detaining the suction tube within tracheal cannula, monitering the intratracheal pressure, but this system takes the ventilation away from the patient. Hypoventilation caused by this SS may cause the serious accident in patient. Therefore, we remodel the SS from intermittent SS to rollerpomp-type SS continuing to suck the itratracheal sputa with low volume from 2004, and thereafter we made up the SS of piston pomp type-SS finally at 2007. We developed the tracheal cannula with double suction holes of inner and lower hole in the lower part of its cannula together with the suction machine. We think that the practical use of this automatic SS will bring these patients with TMV and their family members great benefits. We desire that the practical use of this SS will be realized as soon as possible.

  20. Highly conserved CDR3 region in circulating CD4+Vβ5+ T cells may be associated with cytotoxic activity in Chagas disease

    PubMed Central

    Menezes, C A S; Sullivan, A K; Falta, M T; Mack, D G; Freed, B M; Rocha, M O C; Gollob, K J; Fontenot, A P; Dutra, W O

    2012-01-01

    Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4+ T cells from chagasic patients preferentially express T cell receptor (TCR) β-chain variable region (Vβ) 5. The aim of this work was to determine whether T cells expressing this particular Vβ region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vβ5·1 expressing CD4+ T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jβ region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4+Vβ5·1+ cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy. PMID:22774985

  1. First insights into the genetic diversity of Echinococcus granulosus sensu stricto (s.s.) in Serbia.

    PubMed

    Debeljak, Zoran; Boufana, Belgees; Interisano, Maria; Vidanovic, Dejan; Kulisic, Zoran; Casulli, Adriano

    2016-06-15

    Cystic echinococcosis (CE) caused by Echinococcus granulosus sensu stricto (s.s.) is a cosmopolitan zoonotic infection which is endemic in Serbia where it is subject to mandatory reporting. However, information on the incidence of the disease in humans and prevalence of hydatid infection in livestock remains limited. We used sequenced data of the cytochrome c oxidase subunit 1 (cox 1) mitochondrial gene to examine the genetic diversity and population structure of E. granulosus (s.s.) from intermediate hosts from Serbia. We also compared our generated nucleotide sequences with those reported for neighbouring European countries. Echinococcus canadensis was molecularly confirmed from pig and human hydatid isolates. E. granulosus (G1) was confirmed from sheep and cattle hydatid isolates as well as the first molecular confirmation in Serbia of E. granulosus G2 in sheep and E. granulosus G3 in sheep and cattle hydatid isolates. The Serbian E. granulosus (s.s.) parsimony network displayed 2 main haplotypes (SB02 and SB05) which together with the neutrality indices were suggestive of bottleneck and/or balancing selection. Haplotype analysis showed the presence of the common E. granulosus haplotype described from other worldwide regions. Investigation of the pairwise fixation (Fst) index suggested that Serbian populations of E. granulosus (s.s.) from sheep and cattle hosts showed moderate genetic differentiation. Six of the Serbian haplotypes (SB02-SB07) were shared with haplotypes from Bulgaria, Hungary and/or Romania. Further studies using a larger number of hydatid isolates from various locations across Serbia will provide more information on the genetic structure of E. granulosus (s.s.) within this region. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Structure of a Type-1 Secretion System ABC Transporter.

    PubMed

    Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

    2017-03-07

    Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Internal control regions for transcription of eukaryotic tRNA genes.

    PubMed Central

    Sharp, S; DeFranco, D; Dingermann, T; Farrell, P; Söll, D

    1981-01-01

    We have identified the region within a eukaryotic tRNA gene required for initiation of transcription. These results were obtained by systematically constructing deletions extending from the 5' or the 3' flanking regions into a cloned Drosophila tRNAArg gene by using nuclease BAL 31. The ability of the newly generated deletion clones to direct the in vitro synthesis of tRNA precursors was measured in transcription systems from Xenopus laevis oocytes, Drosophila Kc cells, and HeLa cells. Two control regions within the coding sequence were identified. The first was essential for transcription and was contained between nucleotides 8 and 25 of the mature tRNA sequence. Genes devoid of the second control region, which was contained between nucleotides 50 and 58 of the mature tRNA sequence, could be transcribed but with reduced efficiency. Thus, the promoter regions within a tRNA gene encode the tRNA sequences of the D stem and D loop, the invariant uridine at position 8, and the semi-invariant G-T-psi-C sequence. Images PMID:6947245

  4. Mechanism of the formation of the RecA-ssDNA nucleoprotein filament structure: a coarse-grained approach.

    PubMed

    Mukherjee, Goutam; Pal, Arumay; Levy, Yaakov

    2017-11-21

    In prokaryotes, the RecA protein catalyzes the repair and strand exchange of double-stranded DNA. RecA binds to single-stranded DNA (ssDNA) and forms a presynaptic complex in which the protein polymerizes around the ssDNA to form a right-handed helical nucleoprotein filament structure. In the present work, the mechanism for the formation of the RecA-ssDNA filament structure is modeled using coarse-grained molecular dynamics simulations. Information from the X-ray structure was used to model the protein itself but not its interactions; the interactions between the protein and the ssDNA were modeled solely by electrostatic, aromatic, and repulsive energies. For the present study, the monomeric, dimeric, and trimeric units of RecA and 4, 8, and 11 NT-long ssDNA, respectively, were studied. Our results indicate that monomeric RecA is not sufficient for nucleoprotein filament formation; rather, dimeric RecA is the elementary binding unit, with higher multimeric units of RecA facilitating filament formation. Our results reveal that loop region flexibility at the primary binding site of RecA is essential for it to bind the incoming ssDNA, that the aromatic residues present in the loop region play an important role in ssDNA binding, and that ATP may play a role in guiding the ssDNA by changing the electrostatic potential of the RecA protein.

  5. High T3, Low T4 Serum Levels in Mct8 Deficiency Are Not Caused by Increased Hepatic Conversion through Type I Deiodinase.

    PubMed

    Wirth, Eva K; Rijntjes, Eddy; Meyer, Franziska; Köhrle, Josef; Schweizer, Ulrich

    2015-09-01

    The Allan-Herndon-Dudley syndrome is a severe psychomotor retardation accompanied by specific changes in circulating thyroid hormone levels (high T3, low T4). These are caused by mutations in the thyroid hormone transmembrane transport protein monocarboxylate transporter 8 (MCT8). To test the hypothesis that circulating low T4 and high T3 levels are caused by enhanced conversion of T4 via increased activity of hepatic type I deiodinase (Dio1). We crossed mice deficient in Mct8 with mice lacking Dio1 activity in hepatocytes. Translation of the selenoenzyme Dio1 was abrogated by hepatocyte-specific inactivation of selenoprotein biosynthesis. Inactivation of Dio1 activity in the livers of global Mct8-deficient mice does not restore normal circulating thyroid hormone levels. Our data suggest that although hepatic Dio1 activity is increased in Mct8-deficient mice, it does not cause the observed abnormal circulating thyroid hormone levels. Since global inactivation of Dio1 in Mct8-deficient mice does normalize circulating thyroid hormone levels, the underlying mechanism and relevant tissues involved remain to be elucidated.

  6. Microstructural and Electrochemical Evaluation of Fusion Welded Low-Nickel and 304 SS at Different Heat Input

    NASA Astrophysics Data System (ADS)

    Bansod, Ankur V.; Patil, Awanikumar P.; Moon, Abhijeet P.; Shukla, Sourabh

    2017-12-01

    The present research study investigates the effect of heat input using E 308 electrode (controlled by welding current, i.e., 70, 85 and 100 A) on microstructure, mechanical properties and corrosion behavior of low-nickel and 304 stainless steel (SS) weldments produced by shielded metal arc welding technique. SEM investigation shows that with the higher heat input, δ-ferrite content was reduced. Dendrite and inter-dendritic length is also reduced by lowering the heat input. For all the heat inputs, it is observed that δ-ferrite content was higher in 304 stainless steel (SS) as compared to that of low-nickel austenitic stainless steel (Cr-Mn SS). Considering the heat input for Cr-Mn SS, coarse grains were observed in the heat-affected zone region. For low heat input (LHI), tensile fracture surface has exhibited river-like pattern with dimple appearance. Corrosion studies show better pitting resistance for low heat input (LHI) samples due to higher δ-ferrite present in the weld region. Similarly, higher interphase corrosion resistance is observed in both the SS grades causing more dissolution in the LHI samples.

  7. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

    PubMed Central

    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

  8. Usefulness of free-breathing readout-segmented echo-planar imaging (RESOLVE) for detection of malignant liver tumors: comparison with single-shot echo-planar imaging (SS-EPI).

    PubMed

    Tokoro, Hirokazu; Fujinaga, Yasunari; Ohya, Ayumi; Ueda, Kazuhiko; Shiobara, Aya; Kitou, Yoshihiro; Ueda, Hitoshi; Kadoya, Masumi

    2014-10-01

    We aimed to clarify the usefulness of free-breathing readout-segmented echo-planar imaging (RESOLVE), which is multi-shot echo-planar imaging based on a 2D-navigator-based reacquisition technique, for detecting malignant liver tumor. In 77 patients with malignant liver tumors, free-breathing RESOLVE and respiratory-triggered single-shot echo-planar imaging (SS-EPI) at 3-T MR unit were performed. We set a scan time up to approximately 5 min (300s) before examination, measured actual scan time and assessed (1) susceptibility and (2) motion artifacts in the right and left liver lobes (3, no artifact; 1, marked), and (3) detectability of malignant liver tumors (3, good; 1, poor) using a 3-point scale. The median actual scan time of RESOLVE/SS-EPI was 365/423s. The median scores of each factor in RESOLVE/SS-EPI were as following in this order: (1) 3/2 (right lobe); 3/3 (left lobe), (2) 2/3 (right lobe); 1/2 (left lobe), and (3) 3/3, respectively. Significant differences were noted between RESOLVE and SS-EPI in all evaluated factors (P<0.05) except for susceptibility of left lobe and detectability of the lesions. Despite the effect of motion artifacts, RESOLVE provides a comparable detectability of the lesion and the advantage of reducing scanning time compared with SS-EPI. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. SS-mPMG and SS-GA: tools for finding pathways and dynamic simulation of metabolic networks.

    PubMed

    Katsuragi, Tetsuo; Ono, Naoaki; Yasumoto, Keiichi; Altaf-Ul-Amin, Md; Hirai, Masami Y; Sriyudthsak, Kansuporn; Sawada, Yuji; Yamashita, Yui; Chiba, Yukako; Onouchi, Hitoshi; Fujiwara, Toru; Naito, Satoshi; Shiraishi, Fumihide; Kanaya, Shigehiko

    2013-05-01

    Metabolomics analysis tools can provide quantitative information on the concentration of metabolites in an organism. In this paper, we propose the minimum pathway model generator tool for simulating the dynamics of metabolite concentrations (SS-mPMG) and a tool for parameter estimation by genetic algorithm (SS-GA). SS-mPMG can extract a subsystem of the metabolic network from the genome-scale pathway maps to reduce the complexity of the simulation model and automatically construct a dynamic simulator to evaluate the experimentally observed behavior of metabolites. Using this tool, we show that stochastic simulation can reproduce experimentally observed dynamics of amino acid biosynthesis in Arabidopsis thaliana. In this simulation, SS-mPMG extracts the metabolic network subsystem from published databases. The parameters needed for the simulation are determined using a genetic algorithm to fit the simulation results to the experimental data. We expect that SS-mPMG and SS-GA will help researchers to create relevant metabolic networks and carry out simulations of metabolic reactions derived from metabolomics data.

  10. Leukocyte common antigen-related phosphatase (LRP) gene structure: Conservation of the genomic organization of transmembrane protein tyrosine phosphatases

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

    Wong, E.C.C.; Mullersman, J.E.; Thomas, M.L.

    1993-07-01

    The leukocyte common antigen-related protein tyrosine phosphatase (LRP) is a widely expressed transmembrane glycoprotein thought to be involved in cell growth and differentiation. Similar to most other transmembrane protein tyrosine phosphatases, LRP contains two tandem cytoplasmic phosphatase domains. To understand further the regulation and evolution of LRP, the authors have isolated and characterized mouse [lambda] genomic clones. Thirteen genomic clones could be divided into two non-overlapping clusters. The first cluster contained the transcription initiation site and the exon encoding most of the 5[prime] untranslated region. The second cluster contained the remaining exons encoding the protein and the 3[prime] untranslated region.more » The gene consists of 22 exons spanning over 75 kb. The distance between exon 1 and exon 2 is at least 25 kb. Characterization of the 5[prime] ends of LRP mRNA by S1 nuclease protection identifies putative initiation start sites within a G/C-rich region. The upstream region does not contain a TATA box. Comparison of the LRP gene structure to the mammalian protein tyrosine phosphatase gene, CD45, shows striking similarities in size and genomic organization. 29 refs., 5 figs., 1 tab.« less

  11. The T-cell receptor beta chain CDR3 region of BV8S1/BJ1S5 transcripts in type 1 diabetes.

    PubMed

    Naserke, H E; Durinovic-Bellò, I; Seidel, D; Ziegler, A G

    1996-01-01

    We recently described the T-cell receptor (TCR) beta chain CDR3 motif S-SDRLG-NQPQH (BV8S1-BJ1S5) in an islet-specific T-cell clone (K2.12) from a type 1 diabetic patient (AS). A similar motif (RLGNQ) was also reported in a T-cell clone of non-obese diabetic (NOD) mice by others. In order to determine the frequency of our motif in selected and unselected T-cell populations, we cloned and sequenced the CDR3 region of BV8S1-BJ1S5 transcripts. These transcripts were derived from unstimulated peripheral blood T lymphocytes from two type 1 diabetic patients (AS and FS) and their non-diabetic sibling (WS), as well as from an islet-specific T-cell line of one of the patients. In addition, we compared the structure and composition of the CDR3 region in BV8S1-BJ1S5 transcripts from peripheral blood T cells between the patients and their non-diabetic sibling (>50 sequences each). We found that 30% of the islet-specific T-cell line cDNA clones expressed the entire sequence-motif, whereas it was absent in the clones of unstimulated peripheral blood T cells from both patients and their non-diabetic sibling. The average length of the CDR3 region was shorter in the patients (mean AS 9.9, FS 9.9, versus WS 10.7, p = 0.0037) and the number of inserted nucleotides in N nucleotide addition at the DJ-junction lower (mean AS 3.5, FS 3. 2, versus WS 5.2, P = <10(-4)) as compared with their non-diabetic sibling. Moreover, the pattern of amino acid usage in the CDR3 region was dissimilar at positions 5 and 6, where polar amino acids predominated in both diabetic siblings. In contrast, basic amino acids are preferentially used at position 5 in the clones of the non-diabetic sibling. These data provide information on the general structure of the TCR(BV8S1-BJ1S5) CDR3 region in type 1 diabetes and may indicate differences in the amino and nucleic acid composition of the TCR beta chain CDR3 region between two type 1 diabetic patients and their non-diabetic sibling.

  12. SS 383: A NEW S-TYPE YELLOW SYMBIOTIC STAR?

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

    Baella, N. O.; Pereira, C. B.; Miranda, L. F.

    Symbiotic stars are key objects in understanding the formation and evolution of interacting binary systems, and are probably the progenitors of Type Ia supernovae. However, the number of known symbiotic stars is much lower than predicted. We aim to search for new symbiotic stars, with particular emphasis on the S-type yellow symbiotic stars, in order to determine their total population, evolutionary timescales, and physical properties. The Two Micron All Sky Survey (2MASS) (J – H) versus (H – K {sub s}) color-color diagram has been previously used to identify new symbiotic star candidates and show that yellow symbiotics are locatedmore » in a particular region of that diagram. Candidate symbiotic stars are selected on the basis of their locus in the 2MASS (J – H) versus (H – K {sub s}) diagram and the presence of Hα line emission in the Stephenson and Sanduleak Hα survey. This diagram separates S-type yellow symbiotic stars from the rest of the S-type symbiotic stars, allowing us to select candidate yellow symbiotics. To establish the true nature of the candidates, intermediate-resolution spectroscopy is obtained. We have identified the Hα emission line source SS 383 as an S-type yellow symbiotic candidate by its position in the 2MASS color-color diagram. The optical spectrum of SS 383 shows Balmer, He I, He II, and [O III] emission lines, in combination with TiO absorption bands that confirm its symbiotic nature. The derived electron density (≅10{sup 8-9} cm{sup –3}), He I emission line intensity ratios, and position in the [O III] λ5007/Hβ versus [O III] λ4363/Hγ diagram indicate that SS 383 is an S-type symbiotic star, with a probable spectral type of K7-M0 deduced for its cool component based on TiO indices. The spectral type and the position of SS 383 (corrected for reddening) in the 2MASS color-color diagram strongly suggest that SS 383 is an S-type yellow symbiotic. Our result points out that the 2MASS color-color diagram is a powerful

  13. Experimental measurement of stationary SS 304, SS 316L and 8630 GTA weld pool surface temperatures

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

    Kraus, H.G.

    1989-07-01

    The optical spectral radiometric/laser reflectance experimental method, previously developed by the author, was extended to obtain high-resolution surface temperature maps of stationary GTA molten weld pools using thick-plate SS 304, SS316L, and 8630 steel. Increasing the welding current from 50 to 200 A resulted in peak pool surface temperatures from 1050{sup 0} to 2400{sup 0}C for the SS 304. At a constant welding current of 150 A, the SS 304 and various heats of SS 316L and 8630 resulted in peak weld pool temperatures from 2300{sup 0} to 2700{sup 0}C. Temperature contour plots of all the welds made are given.more » Surface temperature maps are classified into types that are believed to be indicative of the convective circulation patterns present in the weld pools.« less

  14. Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

    PubMed Central

    Garneau, Line; Klein, Hélène; Lavoie, Marie-France; Brochiero, Emmanuelle; Parent, Lucie

    2014-01-01

    The Ca2+-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca2+ concentrations (Pomax) is low, typically 0.1–0.2 for KCa3.1 wild type. This observation argues for the binding of Ca2+ to the calmodulin (CaM)–KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca2+-dependent gating of KCa3.1 originates from the binding of Ca2+ to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic–aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic–aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators. PMID:24470490

  15. Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

    PubMed

    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.

  16. The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB

    PubMed Central

    Yokoyama, Katsushi; Nogami, Hideki; Kabasawa, Mamiko; Ebihara, Sonomi; Shimowasa, Ai; Hashimoto, Keiko; Kawashima, Tsuyoshi; Ishijima, Sanae A.; Suzuki, Masashi

    2009-01-01

    The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship. PMID:19468044

  17. The 3’-Jα Region of the TCRα Locus Bears Gene Regulatory Activity in Thymic and Peripheral T Cells

    PubMed Central

    Kučerová-Levisohn, Martina; Knirr, Stefan; Mejia, Rosa I.; Ortiz, Benjamin D.

    2015-01-01

    Much progress has been made in understanding the important cis-mediated controls on mouse TCRα gene function, including identification of the Eα enhancer and TCRα locus control region (LCR). Nevertheless, previous data have suggested that other cis-regulatory elements may reside in the locus outside of the Eα/LCR. Based on prior findings, we hypothesized the existence of gene regulatory elements in a 3.9-kb region 5’ of the Cα exons. Using DNase hypersensitivity assays and TCRα BAC reporter transgenes in mice, we detected gene regulatory activity within this 3.9-kb region. This region is active in both thymic and peripheral T cells, and selectively affects upstream, but not downstream, gene expression. Together, these data indicate the existence of a novel cis-acting regulatory complex that contributes to TCRα transgene expression in vivo. The active chromatin sites we discovered within this region would remain in the locus after TCRα gene rearrangement, and thus may contribute to endogenous TCRα gene activity, particularly in peripheral T cells, where the Eα element has been found to be inactive. PMID:26177549

  18. Clinical characteristics of RA patients with secondary SS and association with joint damage.

    PubMed

    Brown, Lindsay E; Frits, Michelle L; Iannaccone, Christine K; Weinblatt, Michael E; Shadick, Nancy A; Liao, Katherine P

    2015-05-01

    Secondary SS (sSS) is a common extra-articular manifestation of RA. There are conflicting data regarding the association of sSS with worse joint damage. This study aims to characterize sSS patients in an RA cohort and study the association between sSS and joint damage. We conducted a cross-sectional study of RA patients with ≥1 year of follow-up at a large academic centre. Subjects with co-morbid diseases that can also result in sicca symptoms were excluded from the analysis. Subjects were considered to have sSS if they were reported as having sSS by their rheumatologist at recruitment into the cohort and had the diagnosis confirmed by chart review. The primary outcome was Sharp score using bilateral hand radiographs at recruitment. We constructed a linear regression model to determine the association of sSS status and Sharp score adjusted by age, gender, disease duration and ACPA and RF status. We studied 829 RA subjects, mean age 57 years, 83% female, mean RA duration 13 years, 74% seropositive; 85 subjects (10.3%) had sSS. We observed a female predominance (95.3%), longer mean disease duration (16.9 years) and higher frequency of RF or ACPA positive among patients with sSS and RA. Having sSS at baseline was associated with higher Sharp scores (P = 0.03), independent of age, gender, RA disease duration and seropositive disease. In our RA cohort, RA subjects with sSS had worse joint damage, suggesting that sSS is a marker of more aggressive disease. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins.

    PubMed

    Langó, Tamás; Róna, Gergely; Hunyadi-Gulyás, Éva; Turiák, Lilla; Varga, Julia; Dobson, László; Várady, György; Drahos, László; Vértessy, Beáta G; Medzihradszky, Katalin F; Szakács, Gergely; Tusnády, Gábor E

    2017-02-13

    Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins.

  20. Serotoninergic neuronal systems and ethanol sensitivity in long-sleep (LS) and short-sleep (SS) mice

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

    French, T.A.; Weiner, N.

    LS and SS mice selectively bred for differences in CNS sensitivity to ethanol (EtOH) exhibit markedly different sleep times (LS = 120 min; SS = 13 min) and differ considerably in hypothermic response following EtOH< 4 g/kg i.p. Basal levels of serotonin (5HT), 5-hydroxyindole acetic acid and 5HT turnover (defined as 5-hydroxy-tryptophan accumulation after inhibition of aromatic L-amino acid decarboxylase) in 6 brain regions (hypthalamus (HYP), dorsal raphe (DR), pontine-medullary raphe (PMR), striatum (STR), hippocampus (HIP) and cortex (CTX)) differ minimally between the two lines. 5 HT turnover in STR, HIP, CTX and PMR of either LS or SS micemore » or in HYP or DR of SS mice was not altered at either 20 or 120 min after EtOH, 4 g/kg. However, in LS mice, 5HT turnover was decreased 23% in HYP at 20 min and 30-34% in HYP and DR at 120 min. LS mice become 1.2 and 2.2/sup 0/ more hypothermic than SS mice at 20 and 120 min after EtOH, 4 g/kg. When approximately equi-effective hypothermic doses of EtOH were administered to LS (3.5 g/kg) and SS (5.0 g/kg) mice, differences in 5HT turnover were still apparent. Pentobarbital administration (65 mg/kg) both decreased brain 5HT turnover 30-60% and produced comparable degrees of hypothermia in LS and SS mice. These data suggest that the apparent increase in sensitivity to inhibition by EtOH of 5HT neurons of HYP and DR of LS mice may contribute to the greater degree of hypothermia induced in this line.« less

  1. Depolarization of the Electrogenic Transmembrane Electropotential of Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Toxin, Azide, Cyanide, Dodecyl Succinic Acid, or Cold Temperature 1

    PubMed Central

    Mertz, Stuart M.; Arntzen, Charles J.

    1978-01-01

    The transmembrane electrical potential of root cells of Zea mays L. cv. W64A in a modified 1× Higinbotham solution was partially depolarized by semipurified toxin obtained from Bipolaris (Helminthosporium) maydis race T. At a given toxin concentration depolarization of Texas cytoplasm cells was much greater than for normal cytoplasm cells. This observation correlated directly to the differential host susceptibility to the fungus. The time course and magnitude of depolarization were dependent on toxin concentration; at high concentration the electropotential difference change was rapid. Cortex cells depolarized more slowly than epidermal cells indicating that the toxin slowly permeated intercellular regions. Toxin concentrations which affected electropotential difference were of the same magnitude as those required to inhibit root growth, ion uptake, and mitochondrial processes. Azide, cyanide, and cold temperature (5 C) gave the same partial depolarization as did the toxin. Dodecyl succinic acid caused complete depolarization. These and other data indicate that one of the primary actions of the toxin is to inhibit electrogenic ion pumps in the plasmalemma. PMID:16660605

  2. An improved DNA force field for ssDNA interactions with gold nanoparticles

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

    Jiang, Xiankai; Huai, Ping; Fan, Chunhai

    The widespread applications of single-stranded DNA (ssDNA) conjugated gold nanoparticles (AuNPs) have spurred an increasing interest in the interactions between ssDNA and AuNPs. Despite extensive studies using the most sophisticated experimental techniques, the detailed molecular mechanisms still remain largely unknown. Large scale molecular dynamics (MD) simulations can thus be used to supplement experiments by providing complementary information about ssDNA-AuNP interactions. However, up to now, all modern force fields for DNA were developed based on the properties of double-stranded DNA (dsDNA) molecules, which have hydrophilic outer backbones “protecting” hydrophobic inner nucleobases from water. Without the double-helix structure of dsDNA and thusmore » the “protection” by the outer backbone, the nucleobases of ssDNA are directly exposed to solvent, and their behavior in water is very different from that of dsDNA, especially at the interface with nanoparticles. In this work, we have improved the force field of ssDNA for use with nanoparticles, such as AuNPs, based on recent experimental results and quantum mechanics calculations. With the new improved force field, we demonstrated that a poly(A) sequence adsorbed on a AuNP surface is much more stable than a poly(T) sequence, which is consistent with recent experimental observations. On the contrary, the current standard force fields, including AMBER03, CHARMM27, and OPLSAA, all gave erroneous results as compared to experiments. The current improved force field is expected to have wide applications in the study of ssDNA with nanomaterials including AuNPs, which might help promote the development of ssDNA-based biosensors and other bionano-devices.« less

  3. An improved DNA force field for ssDNA interactions with gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Jiang, Xiankai; Gao, Jun; Huynh, Tien; Huai, Ping; Fan, Chunhai; Zhou, Ruhong; Song, Bo

    2014-06-01

    The widespread applications of single-stranded DNA (ssDNA) conjugated gold nanoparticles (AuNPs) have spurred an increasing interest in the interactions between ssDNA and AuNPs. Despite extensive studies using the most sophisticated experimental techniques, the detailed molecular mechanisms still remain largely unknown. Large scale molecular dynamics (MD) simulations can thus be used to supplement experiments by providing complementary information about ssDNA-AuNP interactions. However, up to now, all modern force fields for DNA were developed based on the properties of double-stranded DNA (dsDNA) molecules, which have hydrophilic outer backbones "protecting" hydrophobic inner nucleobases from water. Without the double-helix structure of dsDNA and thus the "protection" by the outer backbone, the nucleobases of ssDNA are directly exposed to solvent, and their behavior in water is very different from that of dsDNA, especially at the interface with nanoparticles. In this work, we have improved the force field of ssDNA for use with nanoparticles, such as AuNPs, based on recent experimental results and quantum mechanics calculations. With the new improved force field, we demonstrated that a poly(A) sequence adsorbed on a AuNP surface is much more stable than a poly(T) sequence, which is consistent with recent experimental observations. On the contrary, the current standard force fields, including AMBER03, CHARMM27, and OPLSAA, all gave erroneous results as compared to experiments. The current improved force field is expected to have wide applications in the study of ssDNA with nanomaterials including AuNPs, which might help promote the development of ssDNA-based biosensors and other bionano-devices.

  4. An improved DNA force field for ssDNA interactions with gold nanoparticles.

    PubMed

    Jiang, Xiankai; Gao, Jun; Huynh, Tien; Huai, Ping; Fan, Chunhai; Zhou, Ruhong; Song, Bo

    2014-06-21

    The widespread applications of single-stranded DNA (ssDNA) conjugated gold nanoparticles (AuNPs) have spurred an increasing interest in the interactions between ssDNA and AuNPs. Despite extensive studies using the most sophisticated experimental techniques, the detailed molecular mechanisms still remain largely unknown. Large scale molecular dynamics (MD) simulations can thus be used to supplement experiments by providing complementary information about ssDNA-AuNP interactions. However, up to now, all modern force fields for DNA were developed based on the properties of double-stranded DNA (dsDNA) molecules, which have hydrophilic outer backbones "protecting" hydrophobic inner nucleobases from water. Without the double-helix structure of dsDNA and thus the "protection" by the outer backbone, the nucleobases of ssDNA are directly exposed to solvent, and their behavior in water is very different from that of dsDNA, especially at the interface with nanoparticles. In this work, we have improved the force field of ssDNA for use with nanoparticles, such as AuNPs, based on recent experimental results and quantum mechanics calculations. With the new improved force field, we demonstrated that a poly(A) sequence adsorbed on a AuNP surface is much more stable than a poly(T) sequence, which is consistent with recent experimental observations. On the contrary, the current standard force fields, including AMBER03, CHARMM27, and OPLSAA, all gave erroneous results as compared to experiments. The current improved force field is expected to have wide applications in the study of ssDNA with nanomaterials including AuNPs, which might help promote the development of ssDNA-based biosensors and other bionano-devices.

  5. The Colony-Stimulating Factor 3 Receptor T640N Mutation Is Oncogenic, Sensitive to JAK Inhibition, and Mimics T618I.

    PubMed

    Maxson, Julia E; Luty, Samuel B; MacManiman, Jason D; Paik, Jason C; Gotlib, Jason; Greenberg, Peter; Bahamadi, Swaleh; Savage, Samantha L; Abel, Melissa L; Eide, Christopher A; Loriaux, Marc M; Stevens, Emily A; Tyner, Jeffrey W

    2016-02-01

    Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in the majority of chronic neutrophilic leukemia (CNL) and a smaller percentage of atypical chronic myeloid leukemia (aCML) cases. Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. In this study, we assess the importance of the CSF3R T640N mutation as a marker of CNL/aCML and potential therapeutic target. Sanger sequencing of leukemia samples was performed to identify CSF3R mutations in CNL and aCML. The oncogenicity of the CSF3R T640N mutation relative to the T618I mutation was assessed by cytokine independent growth assays and by mouse bone marrow transplant. Receptor dimerization and O-glycosylation of the mutants was assessed by Western blot, and JAK inhibitor sensitivity was assessed by colony assay. Here, we identify a CSF3R T640N mutation in two patients with CNL/aCML, one of whom was originally diagnosed with MDS and acquired the T640N mutation upon evolution of disease to aCML. The T640N mutation is oncogenic in cellular transformation assays and an in vivo mouse bone marrow transplantation model. It exhibits many similar phenotypic features to T618I, including ligand independence and altered patterns of O-glycosylation--despite the transmembrane location of T640 preventing access by GalNAc transferase enzymes. Cells transformed by the T640N mutation are sensitive to JAK kinase inhibition to a similar degree as cells transformed by CSF3R T618I. Because of its similarities to CSF3R T618I, the T640N mutation likely has diagnostic and therapeutic relevance in CNL/aCML. ©2015 American Association for Cancer Research.

  6. FLYING SAUCER1 Is a Transmembrane RING E3 Ubiquitin Ligase That Regulates the Degree of Pectin Methylesterification in Arabidopsis Seed Mucilage[W

    PubMed Central

    Voiniciuc, Cătălin; Dean, Gillian H.; Griffiths, Jonathan S.; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L.; Estelle, Mark; Haughn, George W.

    2013-01-01

    Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca2+ ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca2+ or completely rescued using alkaline Ca2+ chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1–yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells. PMID:23482858

  7. Transmembrane protein CD93 diffuses by a continuous time random walk.

    NASA Astrophysics Data System (ADS)

    Goiko, Maria; de Bruyn, John; Heit, Bryan

    Molecular motion within the cell membrane is a poorly-defined process. In this study, we characterized the diffusion of the transmembrane protein CD93. By careful analysis of the dependence of the ensemble-averaged mean squared displacement (EA-MSD, r2) on time t and the ensemble-averaged, time-averaged MSD (EA-TAMSD, δ2) on lag time τ and total measurement time T, we showed that the motion of CD93 is well-described by a continuous-time random walk (CTRW). CD93 tracks were acquired using single particle tracking. The tracks were classified as confined or free, and the behavior of the MSD analyzed. EA-MSDs of both populations grew non-linearly with t, indicative of anomalous diffusion. Their EA-TAMSDs were found to depend on both τ and T, indicating non-ergodicity. Free molecules had r2 tα and δ2 (τ /T 1 - α) , with α 0 . 5 , consistent with a CTRW. Mean maximal excursion analysis supported this result. Confined CD93 had r2 t0 and δ2 (τ / T) α , with α 0 . 3 , consistent with a confined CTRW. CTRWs are described by a series of random jumps interspersed with power-law distributed waiting times, and may arise due to the interactions of CD93 with the endocytic machinery. NSERC.

  8. ASCA observations of SS Cygni during an anomalous outburst

    NASA Technical Reports Server (NTRS)

    Nousek, John A.; Baluta, Christopher J.; Corbet, Robin H. D.; Mukai, Koji; Osborne, Julian P.; Ishida, Manabu

    1994-01-01

    SS Cygni was observed by the ASCA satellite on 1993 May 27, the first cataclysmic variable studied by ASCA. The observations were conducted while the system was in an outburst of the 'anomalous' variety. The SIS spectrum cannot be explained by two-temperature Raymond-Smith coronal plasma models as invoked in previous studies with lower spectral resolution. Significantly better agreement is found for models with plasma emission at kT = 0.8, 3.5 keV and thermal bremsstrahlung at kT = 18 keV. The gas imaging spectrometer (GIS) data are consistent with the solid state imaging spectrometer (SIS) data, showing evidence for Fe line emission but showing no evidence of pulsation over times ranging from seconds to minutes. These observations seem at variance with standard theory in two regards: we simultaneously see hard X-rays and optically thin soft X-rays while the system is in outburst, and we see a nonsmooth emission measure distribution. We speculate on possible scenarios which might resolve these differences.

  9. Effect of 3,5,3'-Triiodothyronine (T3) administration on dio1 gene expression and T3 metabolism in normal and type 1 deiodinase-deficient mice.

    PubMed

    Maia, A L; Kieffer, J D; Harney, J W; Larsen, P R

    1995-11-01

    The type 1 deiodinase (D1) catalyzes the monodeiodination of T4 to produce T3, the active thyroid hormone. In the C3H mouse, hepatic D1 and the dio1 messenger RNA (mRNA) are only 10% that in the C57 strain, the common phenotype. Low activity cosegregated with a series of five GCT repeats located in the 5'-flanking region of the C3H dio1 gene that impaired C3H promoter potency and provided a partial explanation for the lower D1. The present studies were performed to search for additional explanations for low D1 activity in C3H mice. Previous studies have shown that T3 up-regulates the dio1 gene. Therefore, loss of the capacity to respond to endogenous T3 is a possible additional cause of the lower D1 levels in the C3H mice. The hepatic C3H dio1 mRNA increases 10- to 20 fold after T3 administration. The t3 effect occurs at a transplantation level and T3 does not alter the dio1 mRNA half-life. Despite the transcriptional response to T3, no functional thyroid response elements were identified in the 1.5-kilobase 5'-flanking region of either the C57 or C3H dio1 gene. After the same dose of exogenous T3, both dio1 mRNA and D1 of the C3H mouse respond to a greater extent than those of the C57 strain. This can be explained in part by the reduction in T3 clearance due to the lower D1 levels in C3H mice in which higher concentrations of circulating T3 are maintained. The decrease in serum T3 levels and T3 production observed in fasting and systemic illness in both human and experimental animals has been attributed in part to a decrease in hepatic D1. In contrast, despite markedly lower hepatic and renal D1 levels, serum T3 concentrations remain normal in C3H mice. The present studies suggest that the absence of stress-induced hypothalamic-pituitary suppression that allows T4 production to be maintained together with the reduced clearance of T3 and T4 via inner ring deiodination compensate for the D1 deficiency.

  10. Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

    PubMed Central

    Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J

    1993-01-01

    Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins. Images Fig. 2 Fig. 4 PMID:8265602

  11. Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

    PubMed

    Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J

    1993-12-15

    Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.

  12. Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

    PubMed

    Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya

    2009-08-11

    The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFbeta, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP- cells. Remarkably, CD25+GARP- T cells expanded in culture contained 3-5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25-GARP- cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) -infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation.

  13. Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells

    PubMed Central

    Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya

    2009-01-01

    The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFβ, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP− cells. Remarkably, CD25+GARP− T cells expanded in culture contained 3–5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25−GARP− cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) −infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation. PMID:19666573

  14. Detection of human T lymphotropic virus type-I bZIP factor and tax in the salivary glands of Sjögren's syndrome patients.

    PubMed

    Nakamura, Hideki; Hasegawa, Hiroo; Sasaki, Daisuke; Takatani, Ayuko; Shimizu, Toshimasa; Kurushima, Shota; Horai, Yoshiro; Nakashima, Yoshikazu; Nakamura, Tatsufumi; Fukuoka, Junya; Kawakami, Atsushi

    2018-03-20

    To detect HTLV-I bZIP factor (HBZ), tax and relevant molecules in labial salivary glands (LSGs) from patients with Sjögren's syndrome (SS). The expressions of HBZ and tax in T cell lines and LSGs were analysed by in situ hybridization (ISH) or real time PCR. The expressions of forkhead box P3 (Foxp3) and p65 in immunohistochemistry were quantified. After specificity of ISH probes was determined in 5 T cell lines, in LSGs from an adult T-cell leukemia (ATL) patient and 3 HTLV-I-associated myelopathy (HAM)-SS patients, both HBZ and tax signals were detected in infiltrating mononuclear cells (MNCs) and ducts, and HBZ and tax were dominantly expressed in MNCs of ATL and HAM-SS, respectively. HBZ was dominantly observed in LSGs from 8 HTLV-I asymptomatic carrier (AC)-SS patients; faint expression of HBZ was observed in LSGs from 5 HTLV-I-seronegative SS patients. No cell adhesion molecule 1(CADM1) expressed in LSGs from the ATL patient. Although Foxp3 expression was observed in LSG MNCs of all of the SS patients, the ATL patient's expression was significantly greater than that of the AC-SS (p<0.01) and HTLV-I-seronegative SS (p<0.01) patients. The Foxp3 expression was similar in ATL and HAMSS, but significantly higher in HAM-SS than AC-SS (p<0.05). p65 was expressed in LSG MNC nuclei from all SS patients and co-expressed with Foxp3. The expressions of Foxp3 in ducts differed according to HTLV-I infection. These results suggest that HBZ-mediated Foxp3 expression is partly associated with the pathogenesis of HTLV-I-seropositive SS.

  15. High T3, Low T4 Serum Levels in Mct8 Deficiency Are Not Caused by Increased Hepatic Conversion through Type I Deiodinase

    PubMed Central

    Wirth, Eva K.; Rijntjes, Eddy; Meyer, Franziska; Köhrle, Josef; Schweizer, Ulrich

    2015-01-01

    Background The Allan-Herndon-Dudley syndrome is a severe psychomotor retardation accompanied by specific changes in circulating thyroid hormone levels (high T3, low T4). These are caused by mutations in the thyroid hormone transmembrane transport protein monocarboxylate transporter 8 (MCT8). Objective: To test the hypothesis that circulating low T4 and high T3 levels are caused by enhanced conversion of T4 via increased activity of hepatic type I deiodinase (Dio1). Methods We crossed mice deficient in Mct8 with mice lacking Dio1 activity in hepatocytes. Translation of the selenoenzyme Dio1 was abrogated by hepatocyte-specific inactivation of selenoprotein biosynthesis. Results Inactivation of Dio1 activity in the livers of global Mct8-deficient mice does not restore normal circulating thyroid hormone levels. Conclusions Our data suggest that although hepatic Dio1 activity is increased in Mct8-deficient mice, it does not cause the observed abnormal circulating thyroid hormone levels. Since global inactivation of Dio1 in Mct8-deficient mice does normalize circulating thyroid hormone levels, the underlying mechanism and relevant tissues involved remain to be elucidated. PMID:26601078

  16. Trans-membrane transport of n-octadecane by Pseudomonas sp. DG17.

    PubMed

    Hua, Fei; Wang, Hong Qi; Li, Yi; Zhao, Yi Cun

    2013-12-01

    The trans-membrane transport of hydrocarbons is an important and complex aspect of the process of biodegradation of hydrocarbons by microorganisms. The mechanism of transport of (14)C n-octadecane by Pseudomonas sp. DG17, an alkane-degrading bacterium, was studied by the addition of ATP inhibitors and different substrate concentrations. When the concentration of n-octadecane was higher than 4.54 μmol/L, the transport of (14)C n-octadecane was driven by a facilitated passive mechanism following the intra/extra substrate concentration gradient. However, when the cells were grown with a low concentration of the substrate, the cellular accumulation of n-octadecane, an energy-dependent process, was dramatically decreased by the presence of ATP inhibitors, and n-octadecane accumulation continually increased against its concentration gradient. Furthermore, the presence of non-labeled alkanes blocked (14)C n-octadecane transport only in the induced cells, and the trans-membrane transport of n-octadecane was specific with an apparent dissociation constant K t of 11.27 μmol/L and V max of 0.96 μmol/min/mg protein. The results indicated that the trans-membrane transport of n-octadecane by Pseudomonas sp. DG17 was related to the substrate concentration and ATP.

  17. A novel, highly divergent ssDNA virus identified in Brazil infecting apple, pear and grapevine.

    PubMed

    Basso, Marcos Fernando; da Silva, José Cleydson Ferreira; Fajardo, Thor Vinícius Martins; Fontes, Elizabeth Pacheco Batista; Zerbini, Francisco Murilo

    2015-12-02

    Fruit trees of temperate and tropical climates are of great economical importance worldwide and several viruses have been reported affecting their productivity and longevity. Fruit trees of different Brazilian regions displaying virus-like symptoms were evaluated for infection by circular DNA viruses. Seventy-four fruit trees were sampled and a novel, highly divergent, monopartite circular ssDNA virus was cloned from apple, pear and grapevine trees. Forty-five complete viral genomes were sequenced, with a size of approx. 3.4 kb and organized into five ORFs. Deduced amino acid sequences showed identities in the range of 38% with unclassified circular ssDNA viruses, nanoviruses and alphasatellites (putative Replication-associated protein, Rep), and begomo-, curto- and mastreviruses (putative coat protein, CP, and movement protein, MP). A large intergenic region contains a short palindromic sequence capable of forming a hairpin-like structure with the loop sequence TAGTATTAC, identical to the conserved nonanucleotide of circoviruses, nanoviruses and alphasatellites. Recombination events were not detected and phylogenetic analysis showed a relationship with circo-, nano- and geminiviruses. PCR confirmed the presence of this novel ssDNA virus in field plants. Infectivity tests using the cloned viral genome confirmed its ability to infect apple and pear tree seedlings, but not Nicotiana benthamiana. The name "Temperate fruit decay-associated virus" (TFDaV) is proposed for this novel virus. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Comparative analysis of the Hrp pathogenicity island of Rubus- and Spiraeoideae-infecting Erwinia amylovora strains identifies the IT region as a remnant of an integrative conjugative element.

    PubMed

    Mann, Rachel A; Blom, Jochen; Bühlmann, Andreas; Plummer, Kim M; Beer, Steven V; Luck, Joanne E; Goesmann, Alexander; Frey, Jürg E; Rodoni, Brendan C; Duffy, Brion; Smits, Theo H M

    2012-08-01

    The Hrp pathogenicity island (hrpPAI) of Erwinia amylovora not only encodes a type III secretion system (T3SS) and other genes required for pathogenesis on host plants, but also includes the so-called island transfer (IT) region, a region that originates from an integrative conjugative element (ICE). Comparative genomic analysis of the IT regions of two Spiraeoideae- and three Rubus-infecting strains revealed that the regions in Spiraeoideae-infecting strains were syntenic and highly conserved in length and genetic information, but that the IT regions of the Rubus-infecting strains varied in gene content and length, showing a mosaic structure. None of the ICEs in E. amylovora strains were complete, as conserved ICE genes and the left border were missing, probably due to reductive genome evolution. Comparison of the hrpPAI region of E. amylovora strains to syntenic regions from other Erwinia spp. indicates that the hrpPAI and the IT regions are the result of several insertion and deletion events that have occurred within the ICE. It also suggests that the T3SS was present in a common ancestor of the pathoadapted Erwinia spp. and that insertion and deletion events in the IT region occurred during speciation. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Multiscale simulations on conformational dynamics and membrane interactions of the non-structural 2 (NS2) transmembrane domain.

    PubMed

    Hung, Huynh Minh; Hang, Tran Dieu; Nguyen, Minh Tho

    2016-09-09

    Hepatitis C virus (HCV) is one of the most crucial global health issues, in which the HCV non-structural protein 2 (NS2), particularly its three transmembrane segments, plays a crucial role in HCV assembly. In this context, multiscale MD simulations have been applied to investigate the preferred orientation of transmembrane domain of NS2 protein (TNS2) in a POPC bilayer, structural stability and characteristic of intramembrane protein-lipid and protein-protein interaction. Our study indicates that NS2 protein adopts three trans-membrane segments with highly stable α-helix structure in a POPC bilayer and a short helical luminal segment. While the first and second TM segment involved in continuous helical domain, the third TM segment is however cleaved into two sub-segments with different tilt angles via a kink at L87G88. Salt bridges K81-E45, R32-PO4 and R43-PO4 are determined as the key factor to stabilize the structure of TM2 and TM3 which consist of charged residues located in the hydrophobic region of the membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. ssDNA damage dependence from singlet oxygen concentration at photodynamic interaction

    NASA Astrophysics Data System (ADS)

    Klimenko, V. V.; Kaydanov, N. E.; Emelyanov, A. K.; Bogdanov, A. A.

    2017-11-01

    Single stranded DNA damage at photodynamic treatment with Radachlorin photosensitizer was investigated. Chemical trap method was used to evaluate generation of singlet oxygen in water solution. Interaction of singlet oxygen with ssDNA resulted into decrease of the replication activity of ssDNA. DNA stopped replicating during PCR at irradiation doses greater than 15 J/cm2 and concentration of photosensitizer [PS] = 3.8 μM. The dependence of replication activity of ssDNA on generated singlet oxygen concentration was identified.

  1. Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure*

    PubMed Central

    Dixon, Ann M.; Drake, Lisa; Hughes, Kelly T.; Sargent, Elizabeth; Hunt, Danielle; Harton, Jonathan A.; Drake, James R.

    2014-01-01

    Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2+ class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2+ I-Ak conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2+ versus Ia.2− I-Ak class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response. PMID:24619409

  2. Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents

    PubMed Central

    Dezi, Manuela; Di Cicco, Aurelie; Bassereau, Patricia; Lévy, Daniel

    2013-01-01

    Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs. PMID:23589883

  3. A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins.

    PubMed

    Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia; Navarro-Garcia, Fernando

    2017-03-28

    The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia , require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens. IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an "injectisome" conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia , and EspC, an autotransporter protein secreted by enteropathogenic E. coli , require a

  4. Control of rectification and permeation by two distinct sites after the second transmembrane region in Kir2.1 K+ channel

    PubMed Central

    Kubo, Yoshihiro; Murata, Yoshimichi

    2001-01-01

    The rectification property of the inward rectifier K+ channel is chiefly due to the block of outward current by cytoplasmic Mg2+ and polyamines. In the cloned inward rectifier K+ channel Kir2.1 (IRK1), Asp172 in the second transmembrane region (M2) and Glu224 in the putative cytoplasmic region after M2 are reported to be critical for the sensitivity to these blockers. However, the difference in the inward rectification properties between Kir2.1 and a very weak inward rectifier sWIRK could not be explained by differences at these two sites. Following sequence comparison of Kir2.1 and sWIRK, we focused this study on Glu299 located in the centre of the putative cytoplasmic region after M2. Single-point mutants of Kir2.1 (Glu224Gly and Glu299Ser) and a double-point mutant (Glu224Gly-Glu299Ser) were made and expressed in Xenopus oocytes or in HEK293T cells. Their electrophysiological properties were compared with those of wild-type (WT) Kir2.1 and the following observations were made. (a) Glu299Ser showed a weaker inward rectification, a slower activation upon hyperpolarization, a slower decay of the outward current upon depolarization, a lower sensitivity to block by cytoplasmic spermine and a smaller single-channel conductance than WT. (b) The features of Glu224Gly were similar to those of Glu299Ser. (c) In the double mutant (Glu224Gly-Glu299Ser), the differences from WT described above were more prominent. These results demonstrate that Glu299 as well as Glu224 control rectification and permeation, and suggest the possibility that the two sites contribute to the inner vestibule of the channel pore. The slowing down of the on- and off-blocking processes by mutation of these sites implies that Glu224 and Glu299 function to facilitate the entry (and exit) of spermine to (and from) the blocking site. PMID:11251047

  5. MUFOLD-SS: New deep inception-inside-inception networks for protein secondary structure prediction.

    PubMed

    Fang, Chao; Shang, Yi; Xu, Dong

    2018-05-01

    Protein secondary structure prediction can provide important information for protein 3D structure prediction and protein functions. Deep learning offers a new opportunity to significantly improve prediction accuracy. In this article, a new deep neural network architecture, named the Deep inception-inside-inception (Deep3I) network, is proposed for protein secondary structure prediction and implemented as a software tool MUFOLD-SS. The input to MUFOLD-SS is a carefully designed feature matrix corresponding to the primary amino acid sequence of a protein, which consists of a rich set of information derived from individual amino acid, as well as the context of the protein sequence. Specifically, the feature matrix is a composition of physio-chemical properties of amino acids, PSI-BLAST profile, and HHBlits profile. MUFOLD-SS is composed of a sequence of nested inception modules and maps the input matrix to either eight states or three states of secondary structures. The architecture of MUFOLD-SS enables effective processing of local and global interactions between amino acids in making accurate prediction. In extensive experiments on multiple datasets, MUFOLD-SS outperformed the best existing methods and other deep neural networks significantly. MUFold-SS can be downloaded from http://dslsrv8.cs.missouri.edu/~cf797/MUFoldSS/download.html. © 2018 Wiley Periodicals, Inc.

  6. Rapid detection of ssDNA and RNA using multi-walled carbon nanotubes modified screen-printed carbon electrode.

    PubMed

    Ye, Yongkang; Ju, Huangxian

    2005-11-15

    A method for rapid sensitive detection of DNA or RNA was designed using a composite screen-printed carbon electrode modified with multi-walled carbon nanotubes (MWNTs). MWNTs showed catalytic characteristics for the direct electrochemical oxidation of guanine or adenine residues of signal strand DNA (ssDNA) and adenine residues of RNA, leading to indicator-free detection of ssDNA and RNA concentrations. With an accumulation time of 5 min, the proposed method could be used for detection of calf thymus ssDNA ranging from 17.0 to 345 microg ml(-1) with a detection limit of 2.0 microg ml(-1) at 3 sigma and yeast tRNA ranging from 8.2 microg ml(-1) to 4.1 mg ml(-1). AC impedance was employed to characterize the surface of modified electrodes. The advantages of convenient fabrication, low-cost detection, short analysis time and combination with nanotechnology for increasing the sensitivity made the subject worthy of special emphasis in the research programs and sources of new commercial products.

  7. Immunological Functions of the Membrane Proximal Region of MHC Class II Molecules

    PubMed Central

    Harton, Jonathan; Jin, Lei; Hahn, Amy; Drake, Jim

    2016-01-01

    Major histocompatibility complex (MHC) class II molecules present exogenously derived antigen peptides to CD4 T cells, driving activation of naïve T cells and supporting CD4-driven immune functions. However, MHC class II molecules are not inert protein pedestals that simply bind and present peptides. These molecules also serve as multi-functional signaling molecules delivering activation, differentiation, or death signals (or a combination of these) to B cells, macrophages, as well as MHC class II-expressing T cells and tumor cells. Although multiple proteins are known to associate with MHC class II, interaction with STING (stimulator of interferon genes) and CD79 is essential for signaling. In addition, alternative transmembrane domain pairing between class II α and β chains influences association with membrane lipid sub-domains, impacting both signaling and antigen presentation. In contrast to the membrane-distal region of the class II molecule responsible for peptide binding and T-cell receptor engagement, the membrane-proximal region (composed of the connecting peptide, transmembrane domain, and cytoplasmic tail) mediates these “non-traditional” class II functions. Here, we review the literature on the function of the membrane-proximal region of the MHC class II molecule and discuss the impact of this aspect of class II immunobiology on immune regulation and human disease. PMID:27006762

  8. INFRARED SPECTROSCOPY OF SYMBIOTIC STARS. X. ORBITS FOR THREE S-TYPE SYSTEMS: V1044 CENTAURI, HEN 3-1213, AND SS 73-96

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

    Fekel, Francis C.; Hinkle, Kenneth H.; Joyce, Richard R.

    Employing new infrared radial velocities, we have computed orbits of the cool giants in three southern S-type symbiotic systems. The orbit for V1044 Cen, an M5.5 giant, has a period of 985 days and a modest eccentricity of 0.16. Hen 3-1213 is a K4 giant, yellow symbiotic with an orbital period of 533 days and a similar eccentricity of 0.18. For the M2 giant SS 73-96 the orbital period is 828 days, and this system has a somewhat larger eccentricity of 0.26. Measurement of the H i Paschen δ emission lines, which may at least partially reflect the motion ofmore » the secondary in SS 73-96, results in a mass ratio of 2.4 for the M giant relative to the presumed white dwarf. The estimated orbital inclinations of V1044 Cen and Hen 3-1213 are low, about 40°. However, for SS 73-96 the predicted inclination is 90°, and so an ephemeris for eclipses of the secondary or the hot nebula surrounding it is provided. A search of the orbital velocity residuals of V1044 Cen and SS 73-96 for pulsation periods produced no realistic or convincing period for either star.« less

  9. [The province of East Prussia and "euthanasia" during national socialism: the SS-"Aktion Lange" and "Aktion T4"].

    PubMed

    Topp, Sascha; Fuchs, Petra; Hohendorf, Gerrit; Richter, Paul; Rotzoll, Maike

    2008-01-01

    During World War II, psychiatric patients hospitalized in asylums in Eastern Prussia became victims of two separate killing programmes: first, by the SS-special command Lange, second by the centrally (in Berlin) organized "euthanasia"-"Aktion T4". By an analysis of the patient files of the victims, the present paper shows that the historical actors responsible for the killings were communicating with each other. It is now also possible to reconstruct the exact dynamic in time and space of the killings. A comparative analysis of the selection criteria within the total population of the asylums documents that in both programs, the responsible historical actors included physicians and provincial administrative personnel; it further shows that under the conditions of war, only patients who were able to contribute to the asylum work and economy, and were behaviourally adapted could survive.

  10. Activation of phosphatidylinositol-3-kinase by platelet-derived growth factor and insulin-like growth factor-1 is inhibited by a transmembrane phosphotyrosine phosphatase.

    PubMed

    Way, B A; Mooney, R A

    1993-12-15

    Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit hormone-dependent tyrosine phosphorylation and mitogenesis (Mooney, R. A., Freund, G. G., Way, B. A., and Bordwell, K. L. (1992) J. Biol. Chem. 267, 23443-23446). Here the impact of PTPase expression on insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor- (PDGF) dependent activation of PI-3-K was investigated. In PTPase+ cells, IGF-1 and PDGF-dependent PI-3-K activity in antiphosphotyrosine immunoprecipitates was decreased by 62 +/- 13 and 46 +/- 17%, respectively, compared to control cells. Similar decreases in PI-3-K activity associated with anti-PDGF receptor and anti-insulin receptor substrate-1 (IRS-1) immunoprecipitates were also observed. Association of PI-3-K with the hormone-activated PDGF receptor decreased approximately 55%, paralleling its loss of activation in PTPase+ cells. Tyrosine phosphorylation of the 85-kDa subunit of PI-3-K was also inhibited. Similarly, IGF-1 dependent tyrosine phosphorylation of IRS-1 was decreased by 45%, and its association with PI-3-K was decreased by 65% in PTPase+ cells. Finally, PDGF-dependent tyrosine phosphorylation of phospholipase C-gamma 1 and GTPase-activating protein was reduced by 60-70% in the PTPase+ cells as was tyrosine phosphorylation of the PDGF receptor associated with these proteins. In summary, expression of a transmembrane PTPase decreased hormone-dependent PI-3-K activation, tyrosine phosphorylation of receptor substrates, and their association with signaling complexes. These data support a role for transmembrane PTPases in the regulation of receptor signal transduction pathways.

  11. SWIFT Observations of a Far UV Luminosity Component in SS433

    NASA Technical Reports Server (NTRS)

    Cannizzo, J. K.; Boyd, P. T.; Dolan, J. F.

    2007-01-01

    SS433 is a binary system showing relativistic Doppler shifts in its two sets of emission lines. The origin of its UV continuum is not well established. We observed SS433 to determine the emission mechanism responsible for its far UV spectrum. The source was observed at several different phases of both its 13 d orbital period and 162.5 d precession period using the UVOT and XRT detector systems on Swift. The far UV spectrum down to 1880 Angstrom lies significantly above the spectral flux distribution predicted by extrapolating the reddened blackbody continuum that fits the spectrum above 3500 Angstroms. The intensity of the far UV flux varies over a period of days and the variability is correlated with the variability of the soft X-ray flux from the source. An emission mechanism in addition to those previously detected in the optical and X-ray regions must exist in the far UV spectrum of SS433.

  12. Role of protein kinase D in Golgi exit and lysosomal targeting of the transmembrane protein, Mcoln1

    PubMed Central

    Marks, David L.; Holicky, Eileen L.; Wheatley, Christine L.; Frumkin, Ayala; Bach, Gideon; Pagano, Richard E.

    2012-01-01

    The targeting of lysosomal transmembrane proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of Mcoln1, the transmembrane protein defective in the autosomal recessive disease, Mucolipidosis, type IV, was studied by over-expressing full length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53 amino acid C-terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ~35 kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that co-expression of full length Mcoln1 with kinase-inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident transmembrane proteins from the Golgi to the lysosomes. PMID:22268962

  13. Structural Analysis of a Peptide Fragment of Transmembrane Transporter Protein Bilitranslocase

    PubMed Central

    Župerl, Špela; Sikorska, Emilia; Zhukov, Igor; Solmajer, Tom; Novič, Marjana

    2012-01-01

    Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane. PMID:22745694

  14. Mechanochemical regulations of RPA's binding to ssDNA

    NASA Astrophysics Data System (ADS)

    Chen, Jin; Le, Shimin; Basu, Anindita; Chazin, Walter J.; Yan, Jie

    2015-03-01

    Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. Previous studies of RPA's binding behavior on ssDNA were conducted in the absence of force; therefore the RPA-ssDNA conformations regulated by force remain unclear. Here, using a combination of atomic force microscopy imaging and mechanical manipulation of single ssDNA tethers, we show that force mediates a switch of the RPA bound ssDNA from amorphous aggregation to a much more regular extended conformation. Further, we found an interesting non-monotonic dependence of the binding affinity on monovalent salt concentration in the presence of force. In addition, we discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state. These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA.

  15. Productive interaction between transmembrane mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor beta receptor.

    PubMed

    Lai, Char-Chang; Edwards, Anne P B; DiMaio, Daniel

    2005-02-01

    The bovine papillomavirus E5 protein is a 44-amino-acid transmembrane protein that transforms cells by binding to the transmembrane region of the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in sustained receptor signaling. However, there are published reports that certain mutants with amino acid substitutions in the membrane-spanning segment of the E5 protein transform cells without activating the PDGF beta receptor. We re-examined several of these transmembrane mutants, and here we present five lines of evidence that these mutants do in fact activate the PDGF beta receptor, resulting in cellular signaling and transformation.

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

    PubMed

    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.

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

    PubMed

    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.

  18. alpha(4)beta(7) independent pathway for CD8(+) T cell-mediated intestinal immunity to rotavirus.

    PubMed

    Kuklin, N A; Rott, L; Darling, J; Campbell, J J; Franco, M; Feng, N; Müller, W; Wagner, N; Altman, J; Butcher, E C; Greenberg, H B

    2000-12-01

    Rotavirus (RV), which replicates exclusively in cells of the small intestine, is the most important cause of severe diarrhea in young children worldwide. Using a mouse model, we show that expression of the intestinal homing integrin alpha(4)ss(7) is not essential for CD8(+) T cells to migrate to the intestine or provide immunity to RV. Mice deficient in ss7 expression (ss7(-/-)) and unable to express alpha(4)ss(7) integrin were found to clear RV as quickly as wild-type (wt) animals. Depletion of CD8(+) T cells in ss7(-/-) animals prolonged viral shedding, and transfer of immune ss7(-/-) CD8(+) T cells into chronically infected Rag-2-deficient mice resolved RV infection as efficiently as wt CD8(+) T cells. Paradoxically, alpha(4)ss(7)(hi) memory CD8(+) T cells purified from wt mice that had been orally immunized cleared RV more efficiently than alpha(4)ss(7)(low) CD8(+) T cells. We explained this apparent contradiction by demonstrating that expression of alpha(4)ss(7) on effector CD8(+) T cells depends upon the site of initial antigen exposure: oral immunization generates RV-specific CD8(+) T cells primarily of an alpha(4)ss(7)(hi) phenotype, but subcutaneous immunization yields both alpha(4)ss(7)(hi) and alpha(4)ss(7)(low) immune CD8(+) T cells with anti-RV effector capabilities. Thus, alpha(4)ss(7) facilitates normal intestinal immune trafficking to the gut, but it is not required for effective CD8(+) T cell immunity.

  19. Functions of transmembrane domain 3 of human melanocortin-4 receptor.

    PubMed

    Mo, Xiu-Lei; Yang, Rui; Tao, Ya-Xiong

    2012-12-01

    The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critical for maintaining energy homeostasis. Transmembrane domain 3 (TM3) of MC4R contains residues that were suggested to be essential in ligand binding and signaling. Several MC4R mutations in TM3 are associated with human obesity. To gain a better understanding of the functions of TM3, we analyzed the functions of 26 residues in TM3 using alanine-scanning mutagenesis. We showed that all mutants had normal cell-surface expression. Four mutants were defective in ligand binding and signaling and six mutants had normal ligand binding but impaired cAMP production. L140A had increased basal cAMP level. To further characterize the function of L140, we generated 17 additional L140 mutants. Fifteen L140 mutants had significantly decreased cell-surface expression, with L140R and L140V expressed normally. Ten L140 mutants had increased basal cAMP activities. Four L140 mutants were defective in ligand-stimulated cAMP generation. Interestingly, with the ERK1/2 pathway, we showed that nine constitutively active mutants had similar levels of basal pERK1/2 as that of WT, and two signaling defective mutants had similar levels of pERK1/2 as that of WT upon agonist stimulation, different from their cAMP signaling properties, suggesting biased signaling in these mutant receptors. In summary, we identified 13 residues in TM3 that were essential for ligand binding and/or signaling. Moreover, L140 was critical for locking MC4R in inactive conformation and several mutants showed biased signaling in cAMP and ERK1/2 signaling pathways.

  20. Functional Analysis of Dendritic Cells Generated from T-iPSCs from CD4+ T Cell Clones of Sjögren's Syndrome.

    PubMed

    Iizuka-Koga, Mana; Asashima, Hiromitsu; Ando, Miki; Lai, Chen-Yi; Mochizuki, Shinji; Nakanishi, Mahito; Nishimura, Toshinobu; Tsuboi, Hiroto; Hirota, Tomoya; Takahashi, Hiroyuki; Matsumoto, Isao; Otsu, Makoto; Sumida, Takayuki

    2017-05-09

    Although it is important to clarify the pathogenic functions of T cells in human samples, their examination is often limited due to difficulty in obtaining sufficient numbers of dendritic cells (DCs), used as antigen-presenting cells, especially in autoimmune diseases. We describe the generation of DCs from induced pluripotent stem cells derived from T cells (T-iPSCs). We reprogrammed CD4+ T cell clones from a patient with Sjögren's syndrome (SS) into iPSCs, which were differentiated into DCs (T-iPS-DCs). T-iPS-DCs had dendritic cell-like morphology, and expressed CD11c, HLA-DR, CD80, CD86, and also BDCA-3. Compared with monocyte-derived DCs, the capacity for antigen processing was similar, and T-iPS-DCs induced the proliferative response of autoreactive CD4+ T cells. Moreover, we could evaluate T cell functions of the patient with SS. In conclusion, we obtained adequate numbers of DCs from T-iPSCs, which could be used to characterize pathogenic T cells in autoimmune diseases such as SS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. LoCuSS: Testing hydrostatic equilibrium in galaxy clusters

    NASA Astrophysics Data System (ADS)

    Smith, G. P.; Mazzotta, P.; Okabe, N.; Ziparo, F.; Mulroy, S. L.; Babul, A.; Finoguenov, A.; McCarthy, I. G.; Lieu, M.; Bahé, Y. M.; Bourdin, H.; Evrard, A. E.; Futamase, T.; Haines, C. P.; Jauzac, M.; Marrone, D. P.; Martino, R.; May, P. E.; Taylor, J. E.; Umetsu, K.

    2016-02-01

    We test the assumption of hydrostatic equilibrium in an X-ray luminosity selected sample of 50 galaxy clusters at 0.15 < z < 0.3 from the Local Cluster Substructure Survey (LoCuSS). Our weak-lensing measurements of M500 control systematic biases to sub-4 per cent, and our hydrostatic measurements of the same achieve excellent agreement between XMM-Newton and Chandra. The mean ratio of X-ray to lensing mass for these 50 clusters is β_X= 0.95± 0.05, and for the 44 clusters also detected by Planck, the mean ratio of Planck mass estimate to LoCuSS lensing mass is β_P= 0.95± 0.04. Based on a careful like-for-like analysis, we find that LoCuSS, the Canadian Cluster Comparison Project, and Weighing the Giants agree on β_P ≃ 0.9-0.95 at 0.15 < z < 0.3. This small level of hydrostatic bias disagrees at ˜5σ with the level required to reconcile Planck cosmology results from the cosmic microwave background and galaxy cluster counts.

  2. Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

    PubMed

    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.

  3. Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

    PubMed Central

    Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

    2011-01-01

    The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

  4. Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data.

    PubMed

    Roy Choudhury, Amrita; Sikorska, Emilia; van den Boom, Johannes; Bayer, Peter; Popenda, Łukasz; Szutkowski, Kosma; Jurga, Stefan; Bonomi, Massimiliano; Sali, Andrej; Zhukov, Igor; Passamonti, Sabina; Novič, Marjana

    2015-01-01

    We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery.

  5. Expansion and productive HIV-1 infection of Foxp3 positive CD4 T cells at pleural sites of HIV/TB co-infection

    PubMed Central

    Hirsch, Christina S; Baseke, Joy; Kafuluma, John Lusiba; Nserko, Mary; Mayanja-Kizza, Harriet; Toossi, Zahra

    2016-01-01

    Background CD4 T-cells expressing Foxp3 are expanded systemically during active tuberculosis (TB) regardless of HIV-1 co-infection. Foxp3+ CD4 T cells are targets of HIV-1 infection. However, expansion of HIV-1 infected Foxp3+ CD4 T cells at sites of HIV/TB co-infection, and whether they contribute to promotion of HIV-1 viral activity is not known. Methods Pleural fluid mononuclear cells (PFMC) from HIV/TB co-infected patients with pleural TB were characterized by immune-staining and FACS analysis for surface markers CD4, CD127, CCR5, CXCR4, HLA-DR and intracellular expression of Foxp3, HIVp24, IFN-γ and Bcl-2. Whole PFMC and bead separated CD4+CD25+CD127− T cells were assessed for HIV-1 LTR strong stop (SS) DNA by real-time PCR, which represents viral DNA post cell entry and initiation of reverse transcription. Results High numbers of HIV-1 p24 positive Foxp3+ and Foxp3+CD127− CD4 T cells were identified in PFMC from HIV/TB co-infected subjects. CD4+Foxp3+CD127− T cells displayed high expression of the cellular activation marker, HLA-DR. Further, expression of the HIV-1 co-receptors, CCR5 and CXCR4, were higher on CD4+Foxp3+T cells compared to CD4+Foxp3T cells. Purified CD4+CD25+CD127− T cells isolated from PFMC of HIV/TB co-infected patients, were over 90% CD4+Foxp3+T cells, and exhibited higher HIV-1 SS DNA as compared to whole PFMC, and as compared to CD4+CD25+CD127− T cells from an HIV-infected subject with pleural mesothelioma. HIV-1 p24+ Foxp3+ CD4+T cells from HIV/TB patients higher in Bcl-2 expression as compared to both HIV-1 p24+ Foxp3− CD4 T cells, and Foxp3+ CD4+T cells without HIV-p24 expression. Conclusion Foxp3+ CD4 T cells in PFMC from HIV/TB co-infected subjects are predisposed to productive HIV-1 infection and have survival advantage as compared to Foxp3 negative CD4 T cells. PMID:28124031

  6. Molecular determinants of the DprA−RecA interaction for nucleation on ssDNA

    PubMed Central

    Lisboa, Johnny; Andreani, Jessica; Sanchez, Dyana; Boudes, Marion; Collinet, Bruno; Liger, Dominique; van Tilbeurgh, Herman; Guérois, Raphael; Quevillon-Cheruel, Sophie

    2014-01-01

    Natural transformation is a major mechanism of horizontal gene transfer in bacteria that depends on DNA recombination. RecA is central to the homologous recombination pathway, catalyzing DNA strand invasion and homology search. DprA was shown to be a key binding partner of RecA acting as a specific mediator for its loading on the incoming exogenous ssDNA. Although the 3D structures of both RecA and DprA have been solved, the mechanisms underlying their cross-talk remained elusive. By combining molecular docking simulations and experimental validation, we identified a region on RecA, buried at its self-assembly interface and involving three basic residues that contact an acidic triad of DprA previously shown to be crucial for the interaction. At the core of these patches, DprAM238 and RecAF230 are involved in the interaction. The other DprA binding regions of RecA could involve the N-terminal α-helix and a DNA-binding region. Our data favor a model of DprA acting as a cap of the RecA filament, involving a DprA−RecA interplay at two levels: their own oligomeric states and their respective interaction with DNA. Our model forms the basis for a mechanistic explanation of how DprA can act as a mediator for the loading of RecA on ssDNA. PMID:24782530

  7. Visualization of ligand-induced transmembrane signaling in the full-length human insulin receptor

    PubMed Central

    2018-01-01

    Insulin receptor (IR) signaling plays a critical role in the regulation of metabolism and growth in multicellular organisms. IRs are unique among receptor tyrosine kinases in that they exist exclusively as covalent (αβ)2 homodimers at the cell surface. Transmembrane signaling by the IR can therefore not be based on ligand-induced dimerization as such but must involve structural changes within the existing receptor dimer. In this study, using glycosylated full-length human IR reconstituted into lipid nanodiscs, we show by single-particle electron microscopy that insulin binding to the dimeric receptor converts its ectodomain from an inverted U-shaped conformation to a T-shaped conformation. This structural rearrangement of the ectodomain propagates to the transmembrane domains, which are well separated in the inactive conformation but come close together upon insulin binding, facilitating autophosphorylation of the cytoplasmic kinase domains. PMID:29453311

  8. 3-Helium in Obscure H II Regions

    NASA Astrophysics Data System (ADS)

    Bania, T. M.; Rood, R. T.; Balser, D. S.

    1999-05-01

    The light isotope of helium, (3) He, can serve as a probe of cosmology, the evolution of low mass stars, and the chemical evolution of the Galaxy. Its abundance can be determined via measurements of the 3.46 cm hyperfine transition of (3) He(+) . Potentially observable sources of ionized gas include H ii regions and planetary nebulae. The selection of (3) He targets is counter-intuitive because the (3) He(+) hyperfine line strength is proportional to the source density, while one usually thinks of H ii regions in terms of radio continuum or recombination line strength both of which depend on the square of the density. The (3) He(+) line strength depends on the (3) He(+) abundance ratio and a number of other factors: $ TL(A}({) (3) He(+)) ~ frac {N((3) He(+)}{N() H(+)}) frac {({TC(A}}D)({1/2)) Te(1/4) (theta_obs (2) - theta_beam (2})({3/4}}{Delta {v}({)) (3) He(+)) [ln(5.717 x 10(-3}Te({3/2})]^{1/2)) theta_obs } where T_L^A and Delta v are the antenna temperature and FWHM of the ^3He^+ line, D is the nebular distance, T_C^A and theta_obs are the antenna temperature and observed FWHM angular size of the continuum emission, theta_beam is the telescope's FWHM beam, and Te is the nebular electron temperature. For H {sc ii} regions much larger than the telescope beam we can select targets using the criterion: TLA(^3He^+)\\sim\\sqrt{TCA D\\theta_obs}. This is the case since we can neglect the weak dependence on T_e and because we do not know either ^3He^{+}/H^+ or Delta v. Thus big, distant H ii regions could be potential ^3He^+ targets even if their continuum emission is weak. Armed with this knowledge we included H ii regions like S209 in our early observing list along with more famous sources like W43. Still we did not have the temerity to push this reasoning to the limit. We have now found, however, that this selection criterion is valid for even the wimpiest known H ii regions. Here we report on the detection of ^3He^+$ emission in 8 distant, low density H ii regions.

  9. Kidney Disease among Patients with Sickle Cell Disease, Hemoglobin SS and SC

    PubMed Central

    Drawz, Paul; Ayyappan, Sabarish; Nouraie, Mehdi; Saraf, Santosh; Gordeuk, Victor; Hostetter, Thomas; Gladwin, Mark T.

    2016-01-01

    Background and objectives Sickle cell disease (SCD) is an inherited anemia that afflicts millions worldwide. Kidney disease is a major contributor to its morbidity and mortality. We examined contemporary and historical SCD populations to understand how renal disease behaved in hemoglobin SS (HbSS) compared with HbSC. Design, setting, participants, & measurements Kidney function was examined in the multicentered Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk-PHaSST) Trial (HbSS=463; HbSC=127; years 2007–2009) and historical comparator populations from the Cooperative Study of Sickle Cell Disease (CSSCD; HbSS=708) and the Multicenter Study of Hydroxyurea in Sickle Cell Disease (MSH; HbSS=299). Results In adults with SCD, eGFR was lower among older individuals: −1.78 ml/min per 1.73 m2 per year of age (95% confidence interval [95% CI], −2.06 to −1.50; Walk-PHaSST Trial), −1.75 ml/min per 1.73 m2 per year of age (95% CI, −2.05 to −1.44; MSH), and −1.69 ml/min per 1.73 m2 per year of age (95% CI, −2.00 to −1.38; CSSCD) in HbSS compared with −1.09 ml/min per 1.73 m2 per year of age (95% CI, −1.39 to −0.75) in HbSC (Walk-PHaSST Trial). Macroalbuminuria was seen in 20% of participants with SCD (HbSS or HbSC; P=0.45; Walk-PHaSST Trial), but microalbuminuria was more prevalent in HbSS (44% versus 23% in HbSC; P<0.002). In the Walk-PHaSST Trial, albuminuria was associated with hemolysis (higher lactate dehydrogenase, P<0.001; higher absolute reticulocyte count, P<0.02; and lower Hb, P=0.07) and elevated systolic BP (P<0.001) in HbSS. One half of all participants with HbSS (20 of 39) versus one fifth without (41 of 228) elevated tricuspid regurgitant jet velocity (≥3 m/s; adverse prognostic indicator in SCD) had macroalbuminuria (P<0.001). In the CSSCD, overt proteinuria, detected (less sensitively) by urine dipstick, associated with higher 3-year mortality (odds ratio, 2.48; 95% CI, 1.07 to 5.77). Serum

  10. Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease

    PubMed Central

    Nelson, Tarah

    2017-01-01

    Juvenile CLN3 (Batten) disease, a fatal, childhood neurodegenerative disorder, results from mutations in the CLN3 gene encoding a lysosomal/endosomal transmembrane protein. The exact physiological function of CLN3 is still unknown and it is unclear how CLN3 mutations lead to selective neurodegeneration. To study the tissue expression and subcellular localization of the CLN3 protein, a number of anti-CLN3 antibodies have been generated using either the whole CLN3 protein or short peptides from CLN3 for immunization. The specificity of these antibodies, however, has never been tested properly. Using immunoblot experiments, we show that commercially available or researcher-generated anti-CLN3 antibodies lack specificity: they detect the same protein bands in wild-type (WT) and Cln3−/− mouse brain and kidney extracts prepared with different detergents, in membrane proteins isolated from the cerebellum, cerebral hemisphere and kidney of WT and Cln3−/− mice, in cell extracts of WT and Cln3−/− mouse embryonic fibroblast cultures, and in lysates of BHK cells lacking or overexpressing human CLN3. Protein BLAST searches with sequences from peptides used to generate anti-CLN3 antibodies identified short motifs present in a number of different mouse and human proteins, providing a plausible explanation for the lack of specificity of anti-CLN3 antibodies. Our data provide evidence that immunization against a transmembrane protein with low to medium expression level does not necessarily generate specific antibodies. Because of the possible cross-reactivity to other proteins, the specificity of an antibody should always be checked using tissue samples from an appropriate knock-out animal or using knock-out cells. PMID:29089465

  11. Type 3 secretion system effector genotype and secretion phenotype of longitudinally collected Pseudomonas aeruginosa isolates from young children diagnosed with cystic fibrosis following newborn screening.

    PubMed

    Hu, H; Harmer, C; Anuj, S; Wainwright, C E; Manos, J; Cheney, J; Harbour, C; Zablotska, I; Turnbull, L; Whitchurch, C B; Grimwood, K; Rose, B

    2013-03-01

    Studies of the type 3 secretion system (T3SS) in Pseudomonas aeruginosa isolates from chronically infected older children and adults with cystic fibrosis (CF) show a predominantly exoS+/exoU- (exoS+) genotype and loss of T3SS effector secretion over time. Relatively little is known about the role of the T3SS in the pathogenesis of early P. aeruginosa infection in the CF airway. In this longitudinal study, 168 P. aeruginosa isolates from 58 children diagnosed with CF following newborn screening and 47 isolates from homes of families with or without children with CF were genotyped by pulsed-field gel electrophoresis (PFGE) and T3SS genotype and phenotype determined using multiplex PCR and western blotting. Associations were sought between T3SS data and clinical variables and comparisons made between T3SS data of clinical and environmental PFGE genotypes. Seventy-seven of the 92 clinical strains were exoS+ (71% secretors (ExoS+)) and 15 were exoU+ (93% secretors (ExoU+)). Initial exoS+ strains were five times more likely to secrete ExoS than subsequent exoS+ strains at first isolation. The proportion of ExoS+ strains declined with increasing age at acquisition. No associations were found between T3SS characteristics and gender, site of isolation, exacerbation, a persistent strain or pulmonary outcomes. Fourteen of the 23 environmental strains were exoS+ (79% ExoS+) and nine were exoU+ (33% ExoU+). The exoU+ environmental strains were significantly less likely to secrete ExoU than clinical strains. This study provides new insight into the T3SS characteristics of P. aeruginosa isolated from the CF airway early in life. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

  12. THGS: a web-based database of Transmembrane Helices in Genome Sequences

    PubMed Central

    Fernando, S. A.; Selvarani, P.; Das, Soma; Kumar, Ch. Kiran; Mondal, Sukanta; Ramakumar, S.; Sekar, K.

    2004-01-01

    Transmembrane Helices in Genome Sequences (THGS) is an interactive web-based database, developed to search the transmembrane helices in the user-interested gene sequences available in the Genome Database (GDB). The proposed database has provision to search sequence motifs in transmembrane and globular proteins. In addition, the motif can be searched in the other sequence databases (Swiss-Prot and PIR) or in the macromolecular structure database, Protein Data Bank (PDB). Further, the 3D structure of the corresponding queried motif, if it is available in the solved protein structures deposited in the Protein Data Bank, can also be visualized using the widely used graphics package RASMOL. All the sequence databases used in the present work are updated frequently and hence the results produced are up to date. The database THGS is freely available via the world wide web and can be accessed at http://pranag.physics.iisc.ernet.in/thgs/ or http://144.16.71.10/thgs/. PMID:14681375

  13. Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells

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

    Ozawa, H.; Iwaguchi, T.; Kataoka, T.

    1987-12-01

    The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurredmore » between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the effector phase as well as in the induction phase. The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the bystander effect.« less

  14. Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients.

    PubMed

    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.

  15. 3D-FE Modeling of 316 SS under Strain-Controlled Fatigue Loading and CFD Simulation of PWR Surge Line

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

    Mohanty, Subhasish; Barua, Bipul; Listwan, Joseph

    In financial year 2017, we are focusing on developing a mechanistic fatigue model of surge line pipes for pressurized water reactors (PWRs). To that end, we plan to perform the following tasks: (1) conduct stress- and strain-controlled fatigue testing of surge-line base metal such as 316 stainless steel (SS) under constant, variable, and random fatigue loading, (2) develop cyclic plasticity material models of 316 SS, (3) develop one-dimensional (1D) analytical or closed-form model to validate the material models and to understand the mechanics associated with 316 SS cyclic hardening and/or softening, (4) develop three-dimensional (3D) finite element (FE) models withmore » implementation of evolutionary cyclic plasticity, and (5) develop computational fluid dynamics (CFD) model for thermal stratification, thermal-mechanical stress, and fatigue of example reactor components, such as a PWR surge line under plant heat-up, cool-down, and normal operation with/without grid-load-following. This semi-annual progress report presents the work completed on the above tasks for a 316 SS laboratory-scale specimen subjected to strain-controlled cyclic loading with constant, variable, and random amplitude. This is the first time that the accurate 3D-FE modeling of the specimen for its entire fatigue life, including the hardening and softening behavior, has been achieved. We anticipate that this work will pave the way for the development of a fully mechanistic-computer model that can be used for fatigue evaluation of safety-critical metallic components, which are traditionally evaluated by heavy reliance on time-consuming and costly test-based approaches. This basic research will not only help the nuclear reactor industry for fatigue evaluation of reactor components in a cost effective and less time-consuming way, but will also help other safety-related industries, such as aerospace, which is heavily dependent on test-based approaches, where a single full-scale fatigue test can

  16. X-ray variability of SS 433: Evidence for supercritical accretion

    NASA Astrophysics Data System (ADS)

    Atapin, K. E.; Fabrika, S. N.

    2016-08-01

    We study the X-ray variability of SS 433 based on data from the ASCA observatory and the MAXI and RXTE/ASM monitoring missions. Based on the ASCA data, we have constructed the power spectrum of SS 433 in the frequency range from 10-6 to 0.1 Hz, which confirms the presence of a flat portion in the spectrum at frequencies 3 × 10-5-10-3 Hz. The periodic variability (precession, nutation, eclipses) begins to dominate significantly over the stochastic variability at lower frequencies, which does not allow the stochastic variability to be studied reliably. The model in which the flat portion extends to 9.5 × 10-6 Hz, while a power-law rise with an index of 2.6 occurs below provides the best agreement with the observations. The nutational oscillations of the jets with a period of about three days suggests that the time for the passage of material through the disk is less than this value. At frequencies below 4 × 10-6 Hz, the shape of the power spectrum probably does not reflect the disk structure but is determined by external factors, for example, by a change in the amount of material supplied by the donor. The flat portion can arise from a rapid decrease in the viscous time in the supercritical or radiative disk zones. The flat spectrum is associated with the variability of the X-ray jets that are formed in the supercritical disk region.

  17. Response of GWALP Transmembrane Peptides to Changes in the Tryptophan Anchor Positions†

    PubMed Central

    Vostrikov, Vitaly V.; Koeppe, Roger E.

    2011-01-01

    While the interfacial partitioning of charged or aromatic anchor residues may determine the preferred orientations of transmembrane peptide helices, the dependence of helix orientation on anchor residue position is not well understood. When anchor residue locations are changed systematically, some adaptations of the peptide-lipid interactions may be required to compensate the altered interfacial interactions. Recently we have developed a novel transmembrane peptide, termed GW5,19ALP23 (acetyl-GGALW5LALALALALALALW19LAGA-ethanolamide), which proves to be a well behaved sequence for an orderly investigation of protein-lipid interactions. Its roughly symmetric nature allows for shifting the anchoring Trp residues by one Leu-Ala pair inward (GW7,17ALP23) or outward (GW3,21ALP23), thus providing fine adjustments of the formal distance between the tryptophan residues. With no other obvious anchoring features present, we postulate that the inter-Trp distance may be crucial for aspects of the peptide-lipid interaction. Importantly, the amino acid composition is identical for each of the resulting related GWALP23 sequences, and the radial separation between the pairs of Trp residues on each side of the transmembrane α-helix remains similar. Here we address the adaptation of the aforementioned peptides to the varying Trp locations by means of solid-state 2H NMR experiments in varying lipid bilayer membrane environments. All of the GWx,yALP23 sequence isomers adopt transmembrane orientations in DOPC, DMPC and DLPC environments, even when the Trp residues are quite closely spaced, in GW7,17ALP23. Furthermore, the dynamics for each peptide isomer are less extensive than for peptides possessing additional interfacial Trp residues. The helical secondary structure is maintained more strongly within the Trp-flanked core region than outside of the Trp boundaries. Deuterium labeled tryptophan indole rings in the GWx,yALP23 peptides provide additional insights into the behavior of the

  18. CKLF-Like MARVEL Transmembrane Domain-Containing Member 3 (CMTM3) Inhibits the Proliferation and Tumorigenisis in Hepatocellular Carcinoma Cells.

    PubMed

    Li, Wujun; Zhang, Shaobo

    2017-01-26

    The CKLF-like MARVEL transmembrane domain-containing 3 (CMTM3), a member of the CMTM family, was found in several human tumors and plays an important role in the development and progression of tumors. However, the role of CMTM3 in hepatocellular carcinoma (HCC) remains largely unknown. Thus, in the present study, we explored its expression pattern in human HCC cell lines, as well as its functions in HCC cells. Our results demonstrated that the expression of CMTM3 is lowly expressed in HCC cell lines. In vitro, we found that overexpression of CMTM3 obviously inhibited the proliferation, invasion, and EMT process in HCC cells. Furthermore, overexpression of CMTM3 significantly downregulated the expression levels of phosphorylation of JAK2 and STAT3 in HepG2 cells. In vivo, overexpression of CMTM3 attenuated the tumor growth in Balb/c nude mice. In conclusion, we demonstrated that CMTM3 could play an important role in HCC metastasis by EMT induction via, at least partially, suppressing the JAK2/STAT3 signaling pathway. Therefore, CMTM3 may serve as a potential molecular target in the prevention and/or treatment of HCC invasion and metastasis.

  19. Common regions of deletion in chromosome regions 3p12 and 3p14.2 in primary clear cell renal carcinomas.

    PubMed

    Lubinski, J; Hadaczek, P; Podolski, J; Toloczko, A; Sikorski, A; McCue, P; Druck, T; Huebner, K

    1994-07-15

    Nearly all clear cell renal cell carcinomas (RCCs) exhibit loss of alleles on the short arm of chromosome 3. Loss and mutation at the von Hippel-Lindau (VHL) gene at 3p25 probably occurs in most RCCs and, since the VHL gene was recently cloned, data on VHL involvement in RCCs is accumulating. However, the region 3p14-p12, a region that contains the familial RCC-associated t(3;8)(p14.2;q24) chromosome translocation and the small cell lung carcinoma-associated homozygous deletion at 3p13-12, has also been reported to exhibit allele loss in a large fraction of RCCs. In order to focus future studies on potential suppressor genes in the 3p14-p12 region, we have studied allele loss in 30 RCCs with 9 polymorphic simple sequence repeat markers spanning 3p21.1-p12. Partial losses in the 3p21-p12 region were observed, allowing determination of common regions of loss of heterozygosity overlap in 15 RCCs. Results suggested that most RCCs exhibit loss in a region which brackets the t(3;8) familial chromosome translocation at 3p14.2, and some show additional deletions within the U2020 small cell lung carcinoma deletion at 3p12.

  20. Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex

    PubMed Central

    Witosch, Justine; Wolf, Eva; Mizuno, Naoko

    2014-01-01

    The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork. PMID:25348395

  1. Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro.

    PubMed

    Smith, S E; Gibson, M S; Wash, R S; Ferrara, F; Wright, E; Temperton, N; Kellam, P; Fife, M

    2013-12-01

    Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.

  2. Transmembrane chloride flux in tissue-cultured chick heart cells

    PubMed Central

    1983-01-01

    To evaluate the transmembrane movement of chloride in a preparation of cardiac muscle lacking the extracellular diffusion limitations of natural specimens, intracellular chloride concentration ( [Cl] i) and transmembrane 36Cl efflux have been determined in growth-oriented embryonic chick heart cells in tissue culture. Using the method of isotopic equilibrium, [Cl]i was 25.1 +/- 7.3 mmol x (liter cell water)- 1, comparable to the value of 24.9 +/- 5.4 mmol x (liter cell water)-1 determined by coulometric titration. Two cellular 36Cl compartments were found; one exchanged with a rate constant of 0.67 +/- 0.12 min-1 and was associated with the cardiac muscle cells; the other, attributed to the fibroblasts, exchanged with a rate constant of 0.18 +/- 0.05 min- 1. At 37 degrees C, transmembrane Cl flux of cardiac muscle under steady-state conditions was 30 pmol x cm-2 x s-1. In K-free, normal, or high-Ko solutions, the responses of the membrane potential to changes in external Cl concentration suggested that chloride conductance was low. These results indicate that Cl transport across the myocardial cell membrane is more rapid than K transport and is largely electrically silent. PMID:6864192

  3. Characterization of the Interaction between the Salmonella Type III Secretion System Tip Protein SipD and the Needle Protein PrgI by Paramagnetic Relaxation Enhancement*

    PubMed Central

    Rathinavelan, Thenmalarchelvi; Tang, Chun; De Guzman, Roberto N.

    2011-01-01

    Many Gram-negative bacteria that cause major diseases and mortality worldwide require the type III secretion system (T3SS) to inject virulence proteins into their hosts and cause infections. A structural component of the T3SS is the needle apparatus, which consists of a base, an external needle, and a tip complex. In Salmonella typhimurium, the external needle is assembled by the polymerization of the needle protein PrgI. On top of this needle sits a tip complex, which is partly formed by the tip protein SipD. How SipD interacts with PrgI during the assembly of the T3SS needle apparatus remains unknown. The central region of PrgI forms an α-helical hairpin, whereas SipD has a long central coiled-coil, which is a defining structural feature of other T3SS tip proteins as well. Using NMR paramagnetic relaxation enhancement, we have identified a specific region on the SipD coiled-coil that interacts directly with PrgI. We present a model of how SipD might dock at the tip of the needle based on our paramagnetic relaxation enhancement results, thus offering new insight about the mechanism of assembly of the T3SS needle apparatus. PMID:21138848

  4. TMSEG: Novel prediction of transmembrane helices.

    PubMed

    Bernhofer, Michael; Kloppmann, Edda; Reeb, Jonas; Rost, Burkhard

    2016-11-01

    Transmembrane proteins (TMPs) are important drug targets because they are essential for signaling, regulation, and transport. Despite important breakthroughs, experimental structure determination remains challenging for TMPs. Various methods have bridged the gap by predicting transmembrane helices (TMHs), but room for improvement remains. Here, we present TMSEG, a novel method identifying TMPs and accurately predicting their TMHs and their topology. The method combines machine learning with empirical filters. Testing it on a non-redundant dataset of 41 TMPs and 285 soluble proteins, and applying strict performance measures, TMSEG outperformed the state-of-the-art in our hands. TMSEG correctly distinguished helical TMPs from other proteins with a sensitivity of 98 ± 2% and a false positive rate as low as 3 ± 1%. Individual TMHs were predicted with a precision of 87 ± 3% and recall of 84 ± 3%. Furthermore, in 63 ± 6% of helical TMPs the placement of all TMHs and their inside/outside topology was correctly predicted. There are two main features that distinguish TMSEG from other methods. First, the errors in finding all helical TMPs in an organism are significantly reduced. For example, in human this leads to 200 and 1600 fewer misclassifications compared to the second and third best method available, and 4400 fewer mistakes than by a simple hydrophobicity-based method. Second, TMSEG provides an add-on improvement for any existing method to benefit from. Proteins 2016; 84:1706-1716. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. New Insights into Cytosolic Glucose Levels during Differentiation of 3T3-L1 Fibroblasts into Adipocytes*

    PubMed Central

    Kovacic, Petra Brina; Chowdhury, Helena H.; Velebit, Jelena; Kreft, Marko; Jensen, Jørgen; Zorec, Robert

    2011-01-01

    Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolic free glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600μ. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only 56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with the one recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (ΔR) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p < 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose. PMID:21349852

  6. Molecular determinants of the interactions between proteins and ssDNA.

    PubMed

    Mishra, Garima; Levy, Yaakov

    2015-04-21

    ssDNA binding proteins (SSBs) protect ssDNA from chemical and enzymatic assault that can derail DNA processing machinery. Complexes between SSBs and ssDNA are often highly stable, but predicting their structures is challenging, mostly because of the inherent flexibility of ssDNA and the geometric and energetic complexity of the interfaces that it forms. Here, we report a newly developed coarse-grained model to predict the structure of SSB-ssDNA complexes. The model is successfully applied to predict the binding modes of six SSBs with ssDNA strands of lengths of 6-65 nt. In addition to charge-charge interactions (which are often central to governing protein interactions with nucleic acids by means of electrostatic complementarity), an essential energetic term to predict SSB-ssDNA complexes is the interactions between aromatic residues and DNA bases. For some systems, flexibility is required from not only the ssDNA but also, the SSB to allow it to undergo conformational changes and the penetration of the ssDNA into its binding pocket. The association mechanisms can be quite varied, and in several cases, they involve the ssDNA sliding along the protein surface. The binding mechanism suggests that coarse-grained models are appropriate to study the motion of SSBs along ssDNA, which is expected to be central to the function carried out by the SSBs.

  7. The Sinorhizobium (Ensifer) fredii HH103 Type 3 Secretion System Suppresses Early Defense Responses to Effectively Nodulate Soybean.

    PubMed

    Jiménez-Guerrero, Irene; Pérez-Montaño, Francisco; Monreal, José Antonio; Preston, Gail M; Fones, Helen; Vioque, Blanca; Ollero, Francisco Javier; López-Baena, Francisco Javier

    2015-07-01

    Plants that interact with pathogenic bacteria in their natural environments have developed barriers to block or contain the infection. Phytopathogenic bacteria have evolved mechanisms to subvert these defenses and promote infection. Thus, the type 3 secretion system (T3SS) delivers bacterial effectors directly into the plant cells to alter host signaling and suppress defenses, providing an appropriate environment for bacterial multiplication. Some rhizobial strains possess a symbiotic T3SS that seems to be involved in the suppression of host defenses to promote nodulation and determine the host range. In this work, we show that the inactivation of the Sinorhizobium (Ensifer) fredii HH103 T3SS negatively affects soybean nodulation in the early stages of the symbiotic process, which is associated with a reduction of the expression of early nodulation genes. This symbiotic phenotype could be the consequence of the bacterial triggering of soybean defense responses associated with the production of salicylic acid (SA) and the impairment of the T3SS mutant to suppress these responses. Interestingly, the early induction of the transcription of GmMPK4, which negatively regulates SA accumulation and defense responses in soybean via WRKY33, could be associated with the differential defense responses induced by the parental and the T3SS mutant strain.

  8. Cuscuta chinensis seeds water extraction protecting murine osteoblastic MC3T3-E1 cells against tertiary butyl hydroperoxide induced injury.

    PubMed

    Gao, Jian-mei; Li, Ran; Zhang, Lei; Jia, Li-long; Ying, Xi-xiang; Dou, De-qiang; Li, Jian-chun; Li, Hai-bo

    2013-07-09

    Cuscuta chinensis (C. chinensis) is a well-known traditional Chinese herb that has been used to treat heart disease, diabetes, liver injury, cancer, and aging. Murine osteoblastic MC3T3-E1 cells were treated with various concentrations of C. chinensis water extraction at different time intervals. The antioxidant effect of C. chinensis on MC3T3-E1 cells was evaluated using MTT and TUNEL assays. The effect of C. chinensis on cell cycle was analyzed by flow cytometry with propidium iodide. Lipid peroxidation was measured by the HPLC method. The cellular redox status was determined from the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) and the enzymes involved in glutathione metabolism, including glutathione reductase (GR), Glutathione S-transferase (GST), and Glucose-6-phosphate dehydrogenase (G6PD). The changes in relative mitochondrial transmembrane potential (ΔΨm) in the MC3T3-E1 cells were analyzed with rhodamine 123 staining. Western blot analysis was used to evaluate the levels of cytochrome c (cyto c), Bax, Bcl-2, caspase 3, Sirt3, and IDH2 expressions. The C. chinensis water extraction protects tertiary butyl hydroperoxide (TBHP)-treated MC3T3-E1 cells from death in a dose-dependent manner. C. chinensis treatment significantly inhibited the reactive oxygen species (ROS) generation, malondialdehyde (MDA) production, and increased the activity of superoxide dismutase (SOD), GR, GST, and G6PD. The release of cyto c from mitochondria was reduced by C. chinensis, which increased the expression of antiapoptotic IDH2, Sirt3, and Bcl-2 and decreased the expression of Bax, cyto c, and caspase 3. C. chinensis modulated the oxidative stress-induced apoptosis in MC3T3-E1 cells, probably due to its antioxidant activity and functioning via mitochondria-dependent pathways. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  9. Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

    PubMed

    Liu, Binyan; Gu, Shiling; Liang, Nengsong; Xiong, Mei; Xue, Qizhen; Lu, Shuguang; Hu, Fuquan; Zhang, Huidong

    2016-08-01

    Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa.

  10. Constraints on particle acceleration in SS433/W50 from MAGIC and H.E.S.S. observations

    NASA Astrophysics Data System (ADS)

    MAGIC Collaboration; Ahnen, M. L.; Ansoldi, S.; Antonelli, L. A.; Arcaro, C.; Babić, A.; Banerjee, B.; Bangale, P.; Barres de Almeida, U.; Barrio, J. A.; Becerra González, J.; Bednarek, W.; Bernardini, E.; Berti, A.; Biasuzzi, B.; Biland, A.; Blanch, O.; Bonnefoy, S.; Bonnoli, G.; Borracci, F.; Carosi, R.; Carosi, A.; Chatterjee, A.; Colin, P.; Colombo, E.; Contreras, J. L.; Cortina, J.; Covino, S.; Cumani, P.; da Vela, P.; Dazzi, F.; de Angelis, A.; de Lotto, B.; de Oña Wilhelmi, E.; di Pierro, F.; Doert, M.; Domínguez, A.; Dominis Prester, D.; Dorner, D.; Doro, M.; Einecke, S.; Eisenacher Glawion, D.; Elsaesser, D.; Engelkemeier, M.; Fallah Ramazani, V.; Fernández-Barral, A.; Fidalgo, D.; Fonseca, M. V.; Font, L.; Fruck, C.; Galindo, D.; García López, R. J.; Garczarczyk, M.; Gaug, M.; Giammaria, P.; Godinović, N.; Gora, D.; Griffiths, S.; Guberman, D.; Hadasch, D.; Hahn, A.; Hassan, T.; Hayashida, M.; Herrera, J.; Hose, J.; Hrupec, D.; Hughes, G.; Ishio, K.; Konno, Y.; Kubo, H.; Kushida, J.; Kuveždić, D.; Lelas, D.; Lindfors, E.; Lombardi, S.; Longo, F.; López, M.; López-Oramas, A.; Majumdar, P.; Makariev, M.; Maneva, G.; Manganaro, M.; Mannheim, K.; Maraschi, L.; Mariotti, M.; Martínez, M.; Mazin, D.; Menzel, U.; Minev, M.; Mirzoyan, R.; Moralejo, A.; Moreno, V.; Moretti, E.; Munar-Adrover, P.; Neustroev, V.; Niedzwiecki, A.; Nievas Rosillo, M.; Nilsson, K.; Nishijima, K.; Noda, K.; Nogués, L.; Paiano, S.; Palacio, J.; Paneque, D.; Paoletti, R.; Paredes, J. M.; Paredes-Fortuny, X.; Pedaletti, G.; Peresano, M.; Perri, L.; Persic, M.; Prada Moroni, P. G.; Prandini, E.; Puljak, I.; Garcia, J. R.; Reichardt, I.; Rhode, W.; Ribó, M.; Rico, J.; Saito, T.; Satalecka, K.; Schroeder, S.; Schweizer, T.; Shore, S. N.; Sillanpää, A.; Sitarek, J.; Šnidarić, I.; Sobczynska, D.; Stamerra, A.; Strzys, M.; Surić, T.; Takalo, L.; Tavecchio, F.; Temnikov, P.; Terzić, T.; Tescaro, D.; Teshima, M.; Torres, D. F.; Torres-Albà, N.; Treves, A.; Vanzo, G.; Vazquez Acosta, M.; Vovk, I.; Ward, J. E.; Will, M.; Wu, M. H.; Zarić, D.; H.E.S.S. Collaboration; Abdalla, H.; Abramowski, A.; Aharonian, F.; Ait Benkhali, F.; Akhperjanian, A. G.; Andersson, T.; Angüner, E. O.; Arakawa, M.; Arrieta, M.; Aubert, P.; Backes, M.; Balzer, A.; Barnard, M.; Becherini, Y.; Becker Tjus, J.; Berge, D.; Bernhard, S.; Bernlöhr, K.; Blackwell, R.; Böttcher, M.; Boisson, C.; Bolmont, J.; Bordas, P.; Bregeon, J.; Brun, F.; Brun, P.; Bryan, M.; Büchele, M.; Bulik, T.; Capasso, M.; Carr, J.; Casanova, S.; Cerruti, M.; Chakraborty, N.; Chalme-Calvet, R.; Chaves, R. C. G.; Chen, A.; Chevalier, J.; Chrétien, M.; Coffaro, M.; Colafrancesco, S.; Cologna, G.; Condon, B.; Conrad, J.; Cui, Y.; Davids, I. D.; Decock, J.; Degrange, B.; Deil, C.; Devin, J.; Dewilt, P.; Dirson, L.; Djannati-Ataï, A.; Domainko, W.; Donath, A.; Drury, L. O.'c.; Dutson, K.; Dyks, J.; Edwards, T.; Egberts, K.; Eger, P.; Ernenwein, J.-P.; Eschbach, S.; Farnier, C.; Fegan, S.; Fernandes, M. V.; Fiasson, A.; Fontaine, G.; Förster, A.; Funk, S.; Füßling, M.; Gabici, S.; Gajdus, M.; Gallant, Y. A.; Garrigoux, T.; Giavitto, G.; Giebels, B.; Glicenstein, J. F.; Gottschall, D.; Goyal, A.; Grondin, M.-H.; Hahn, J.; Haupt, M.; Hawkes, J.; Heinzelmann, G.; Henri, G.; Hermann, G.; Hervet, O.; Hinton, J. A.; Hofmann, W.; Hoischen, C.; Holler, M.; Horns, D.; Ivascenko, A.; Iwasaki, H.; Jacholkowska, A.; Jamrozy, M.; Janiak, M.; Jankowsky, D.; Jankowsky, F.; Jingo, M.; Jogler, T.; Jouvin, L.; Jung-Richardt, I.; Kastendieck, M. A.; Katarzyński, K.; Katsuragawa, M.; Katz, U.; Kerszberg, D.; Khangulyan, D.; Khélifi, B.; Kieffer, M.; King, J.; Klepser, S.; Klochkov, D.; Kluźniak, W.; Kolitzus, D.; Komin, Nu.; Kosack, K.; Krakau, S.; Kraus, M.; Krüger, P. P.; Laffon, H.; Lamanna, G.; Lau, J.; Lees, J.-P.; Lefaucheur, J.; Lefranc, V.; Lemière, A.; Lemoine-Goumard, M.; Lenain, J.-P.; Leser, E.; Lohse, T.; Lorentz, M.; Liu, R.; López-Coto, R.; Lypova, I.; Marandon, V.; Marcowith, A.; Mariaud, C.; Marx, R.; Maurin, G.; Maxted, N.; Mayer, M.; Meintjes, P. J.; Meyer, M.; Mitchell, A. M. W.; Moderski, R.; Mohamed, M.; Mohrmann, L.; Morå, K.; Moulin, E.; Murach, T.; Nakashima, S.; de Naurois, M.; Niederwanger, F.; Niemiec, J.; Oakes, L.; O'Brien, P.; Odaka, H.; Öttl, S.; Ohm, S.; Ostrowski, M.; Oya, I.; Padovani, M.; Panter, M.; Parsons, R. D.; Pekeur, N. W.; Pelletier, G.; Perennes, C.; Petrucci, P.-O.; Peyaud, B.; Piel, Q.; Pita, S.; Poon, H.; Prokhorov, D.; Prokoph, H.; Pühlhofer, G.; Punch, M.; Quirrenbach, A.; Raab, S.; Reimer, A.; Reimer, O.; Renaud, M.; de Los Reyes, R.; Richter, S.; Rieger, F.; Romoli, C.; Rowell, G.; Rudak, B.; Rulten, C. B.; Safi-Harb, S.; Sahakian, V.; Saito, S.; Salek, D.; Sanchez, D. A.; Santangelo, A.; Sasaki, M.; Schlickeiser, R.; Schüssler, F.; Schulz, A.; Schwanke, U.; Schwemmer, S.; Seglar-Arroyo, M.; Settimo, M.; Seyffert, A. S.; Shafi, N.; Shilon, I.; Simoni, R.; Sol, H.; Spanier, F.; Spengler, G.; Spies, F.; Stawarz, Ł.; Steenkamp, R.; Stegmann, C.; Stycz, K.; Sushch, I.; Takahashi, T.; Tavernet, J.-P.; Tavernier, T.; Taylor, A. M.; Terrier, R.; Tibaldo, L.; Tiziani, D.; Tluczykont, M.; Trichard, C.; Tsuji, N.; Tuffs, R.; Uchiyama, Y.; van der Walt, D. J.; van Eldik, C.; van Rensburg, C.; van Soelen, B.; Vasileiadis, G.; Veh, J.; Venter, C.; Viana, A.; Vincent, P.; Vink, J.; Voisin, F.; Völk, H. J.; Vuillaume, T.; Wadiasingh, Z.; Wagner, S. J.; Wagner, P.; Wagner, R. M.; White, R.; Wierzcholska, A.; Willmann, P.; Wörnlein, A.; Wouters, D.; Yang, R.; Zabalza, V.; Zaborov, D.; Zacharias, M.; Zanin, R.; Zdziarski, A. A.; Zech, A.; Zefi, F.; Ziegler, A.; Zywucka, N.

    2018-04-01

    Context. The large jet kinetic power and non-thermal processes occurring in the microquasar SS 433 make this source a good candidate for a very high-energy (VHE) gamma-ray emitter. Gamma-ray fluxes above the sensitivity limits of current Cherenkov telescopes have been predicted for both the central X-ray binary system and the interaction regions of SS 433 jets with the surrounding W50 nebula. Non-thermal emission at lower energies has been previously reported, indicating that efficient particle acceleration is taking place in the system. Aim. We explore the capability of SS 433 to emit VHE gamma rays during periods in which the expected flux attenuation due to periodic eclipses (Porb 13.1 days) and precession of the circumstellar disk (Ppre 162 days) periodically covering the central binary system is expected to be at its minimum. The eastern and western SS 433/W50 interaction regions are also examined using the whole data set available. We aim to constrain some theoretical models previously developed for this system with our observations. Methods: We made use of dedicated observations from the Major Atmospheric Gamma Imaging Cherenkov telescopes (MAGIC) and High Energy Spectroscopic System (H.E.S.S.) of SS 433 taken from 2006 to 2011. These observation were combined for the first time and accounted for a total effective observation time of 16.5 h, which were scheduled considering the expected phases of minimum absorption of the putative VHE emission. Gamma-ray attenuation does not affect the jet/medium interaction regions. In this case, the analysis of a larger data set amounting to 40-80 h, depending on the region, was employed. Results: No evidence of VHE gamma-ray emission either from the central binary system or from the eastern/western interaction regions was found. Upper limits were computed for the combined data set. Differential fluxes from the central system are found to be ≲ 10-12-10-13 TeV-1 cm-2 s-1 in an energy interval ranging from few × 100 Ge

  11. Seismic Anisotropy in Mantle Transition Zone: Constraints from Observations and Synthetic Modeling of SS Precursors

    NASA Astrophysics Data System (ADS)

    Huang, Q.; Schmerr, N. C.; Waszek, L.; Beghein, C.; Weidner, E. C.

    2017-12-01

    Mantle transition zone (MTZ) is delineated by the 410 and 660 km discontinuities and plays an important role in mantle convection. Mineral physics experiments predict that wadsleyite and ringwoodite can have 13% and 2% single-crystal anisotropy respectively, indicating that seismic anisotropy is likely to exist in the upper part of the MTZ when MTZ minerals are aligned by mantle flow (e.g. subducting slabs). Here we use the SS precursors to study the topography change and seismic anisotropy in the vicinity of MTZ discontinuities. An up-to-date SS precursor dataset consisting of 45,624 records was collected to investigate MTZ topography and anisotropy. We stacked the whole dataset into 9 geographical caps to obtain the global topography of 410 and 660 km discontinuities. The MTZ is thickened by 15 km beneath subduction zones (e.g. Japan and South America) and also thinned by 15 km beneath mantle plume regions (e.g. Bowie and Iceland hotspots), which is consistent with thermal heterogeneity in the mid-mantle. We identify four locations with sufficient bounce point density and azimuthal coverage of SS precursors to study azimuthal anisotropy in MTZ; the central Pacific, the northwest Pacific, Greenland and the central Atlantic. We stack the data by the azimuth of SS bounce points falling within the range of 2000 km in these four locations. The goal is to detect the azimuthal dependence of travel time and amplitude of SS precursors, thus to constrain azimuthal anisotropy in MTZ. The central Pacific bin has fast direction at 110° for both S410S and S660S azimuthal stacks, which is interpreted as seismic anisotropy in the overlying upper mantle. We also stack data in subduction zones by the relative azimuths of bounce points compared to mantle flow directions to test the hypothesis that subducting slabs can cause azimuthal anisotropy in MTZ. A trench-parallel fast direction is observed for both S410S and S660S travel times and amplitudes, but not for their differential

  12. Molecular determinants of the DprA-RecA interaction for nucleation on ssDNA.

    PubMed

    Lisboa, Johnny; Andreani, Jessica; Sanchez, Dyana; Boudes, Marion; Collinet, Bruno; Liger, Dominique; van Tilbeurgh, Herman; Guérois, Raphael; Quevillon-Cheruel, Sophie

    2014-06-01

    Natural transformation is a major mechanism of horizontal gene transfer in bacteria that depends on DNA recombination. RecA is central to the homologous recombination pathway, catalyzing DNA strand invasion and homology search. DprA was shown to be a key binding partner of RecA acting as a specific mediator for its loading on the incoming exogenous ssDNA. Although the 3D structures of both RecA and DprA have been solved, the mechanisms underlying their cross-talk remained elusive. By combining molecular docking simulations and experimental validation, we identified a region on RecA, buried at its self-assembly interface and involving three basic residues that contact an acidic triad of DprA previously shown to be crucial for the interaction. At the core of these patches, (DprA)M238 and (RecA)F230 are involved in the interaction. The other DprA binding regions of RecA could involve the N-terminal α-helix and a DNA-binding region. Our data favor a model of DprA acting as a cap of the RecA filament, involving a DprA-RecA interplay at two levels: their own oligomeric states and their respective interaction with DNA. Our model forms the basis for a mechanistic explanation of how DprA can act as a mediator for the loading of RecA on ssDNA. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Transmembrane Topology and Signal Peptide Prediction Using Dynamic Bayesian Networks

    PubMed Central

    Reynolds, Sheila M.; Käll, Lukas; Riffle, Michael E.; Bilmes, Jeff A.; Noble, William Stafford

    2008-01-01

    Hidden Markov models (HMMs) have been successfully applied to the tasks of transmembrane protein topology prediction and signal peptide prediction. In this paper we expand upon this work by making use of the more powerful class of dynamic Bayesian networks (DBNs). Our model, Philius, is inspired by a previously published HMM, Phobius, and combines a signal peptide submodel with a transmembrane submodel. We introduce a two-stage DBN decoder that combines the power of posterior decoding with the grammar constraints of Viterbi-style decoding. Philius also provides protein type, segment, and topology confidence metrics to aid in the interpretation of the predictions. We report a relative improvement of 13% over Phobius in full-topology prediction accuracy on transmembrane proteins, and a sensitivity and specificity of 0.96 in detecting signal peptides. We also show that our confidence metrics correlate well with the observed precision. In addition, we have made predictions on all 6.3 million proteins in the Yeast Resource Center (YRC) database. This large-scale study provides an overall picture of the relative numbers of proteins that include a signal-peptide and/or one or more transmembrane segments as well as a valuable resource for the scientific community. All DBNs are implemented using the Graphical Models Toolkit. Source code for the models described here is available at http://noble.gs.washington.edu/proj/philius. A Philius Web server is available at http://www.yeastrc.org/philius, and the predictions on the YRC database are available at http://www.yeastrc.org/pdr. PMID:18989393

  14. The role of transmembrane segment 5 (TM5) in Na2 release and the conformational transition of neurotransmitter:sodium symporters toward the inward-open state

    PubMed Central

    Stolzenberg, Sebastian; Li, Zheng; Quick, Matthias; Malinauskaite, Lina; Nissen, Poul; Weinstein, Harel; Javitch, Jonathan A.; Shi, Lei

    2017-01-01

    Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by the reuptake of released neurotransmitters. This active accumulation of substrate against its concentration gradient is driven by the transmembrane Na+ gradient and requires that the transporter traverses several conformational states. LeuT, a prokaryotic NSS homolog, has been crystallized in outward-open, outward-occluded, and inward-open states. Two crystal structures of another prokaryotic NSS homolog, the multihydrophobic amino acid transporter (MhsT) from Bacillus halodurans, have been resolved in novel inward-occluded states, with the extracellular vestibule closed and the intracellular portion of transmembrane segment 5 (TM5i) in either an unwound or a helical conformation. We have investigated the potential involvement of TM5i in binding and unbinding of Na2, i.e. the Na+ bound in the Na2 site, by carrying out comparative molecular dynamics simulations of the models derived from the two MhsT structures. We find that the helical TM5i conformation is associated with a higher propensity for Na2 release, which leads to the repositioning of the N terminus and transition to an inward-open state. By using comparative interaction network analysis, we also identify allosteric pathways connecting TM5i and the Na2 binding site to the extracellular and intracellular regions. Based on our combined computational and mutagenesis studies of MhsT and LeuT, we propose that TM5i plays a key role in Na2 binding and release associated with the conformational transition toward the inward-open state, a role that is likely to be shared across the NSS family. PMID:28320858

  15. Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex.

    PubMed

    Witosch, Justine; Wolf, Eva; Mizuno, Naoko

    2014-11-10

    The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Transmembrane domain dependent inhibitory function of FcγRIIB.

    PubMed

    Wang, Junyi; Li, Zongyu; Xu, Liling; Yang, Hengwen; Liu, Wanli

    2018-03-01

    FcγRIIB, the only inhibitory IgG Fc receptor, functions to suppress the hyper-activation of immune cells. Numerous studies have illustrated its inhibitory function through the ITIM motif in the cytoplasmic tail of FcγRIIB. However, later studies revealed that in addition to the ITIM, the transmembrane (TM) domain of FcγRIIB is also indispensable for its inhibitory function. Indeed, recent epidemiological studies revealed that a non-synonymous single nucleotide polymorphism (rs1050501) within the TM domain of FcγRIIB, responsible for the I232T substitution, is associated with the susceptibility to systemic lupus erythematosus (SLE). In this review, we will summarize these epidemiological and functional studies of FcγRIIB-I232T in the past few years, and will further discuss the mechanisms accounting for the functional loss of FcγRIIB-I232T. Our review will help the reader gain a deeper understanding of the importance of the TM domain in mediating the inhibitory function of FcγRIIB and may provide insights to a new therapeutic target for the associated diseases.

  17. Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

    PubMed

    Katragadda, M; Chopra, A; Bennett, M; Alderfer, J L; Yeagle, P L; Albert, A D

    2001-07-01

    An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.

  18. Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen

    PubMed Central

    Park, Chin-Ju; Lee, Joon-Hwa; Choi, Byong-Seok

    2005-01-01

    Replication protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA-binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA–T antigen (T-ag) interaction required for Simian virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate the similarities and differences between these two homologous proteins, we determined the solution structure of scRPA70A, which closely resembled the structure of hRPA70A. The structure of ssDNA-bound scRPA70A, as simulated by residual dipolar coupling-based homology modeling, suggested that the positioning of the ssDNA is the same for scRPA70A and hRPA70A, although the conformational changes that occur in the two proteins upon ssDNA binding are not identical. NMR titrations of hRPA70A with T-ag showed that the T-ag binding surface is separate from the ssDNA-binding region and is more neutral than the corresponding part of scRPA70A. These differences might account for the species-specific nature of the hRPA70A–T-ag interaction. Our results provide insight into how these two homologous RPA proteins can exhibit functional differences, but still both retain their ability to bind ssDNA. PMID:16043636

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

    PubMed Central

    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

  20. Transmembrane Domains of Attraction on the TSH Receptor

    PubMed Central

    Ali, M. Rejwan; Mezei, Mihaly; Davies, Terry F.

    2015-01-01

    The TSH receptor (TSHR) has the propensity to form dimers and oligomers. Our data using ectodomain-truncated TSHRs indicated that the predominant interfaces for oligomerization reside in the transmembrane (TM) domain. To map the potentially interacting residues, we first performed in silico studies of the TSHR transmembrane domain using a homology model and using Brownian dynamics (BD). The cluster of dimer conformations obtained from BD analysis indicated that TM1 made contact with TM4 and two residues in TM2 made contact with TM5. To confirm the proximity of these contact residues, we then generated cysteine mutants at all six contact residues predicted by the BD analysis and performed cysteine cross-linking studies. These results showed that the predicted helices in the protomer were indeed involved in proximity interactions. Furthermore, an alternative experimental approach, receptor truncation experiments and LH receptor sequence substitution experiments, identified TM1 harboring a major region involved in TSHR oligomerization, in agreement with the conclusion from the cross-linking studies. Point mutations of the predicted interacting residues did not yield a substantial decrease in oligomerization, unlike the truncation of the TM1, so we concluded that constitutive oligomerization must involve interfaces forming domains of attraction in a cooperative manner that is not dominated by interactions between specific residues. PMID:25406938

  1. A new theoretical model for transmembrane potential and ion currents induced in a spherical cell under low frequency electromagnetic field.

    PubMed

    Zheng, Yu; Gao, Yang; Chen, Ruijuan; Wang, Huiquan; Dong, Lei; Dou, Junrong

    2016-10-01

    Time-varying electromagnetic fields (EMF) can induce some physiological effects in neuronal tissues, which have been explored in many applications such as transcranial magnetic stimulation. Although transmembrane potentials and induced currents have already been the subjects of many theoretical studies, most previous works about this topic are mainly completed by utilizing Maxwell's equations, often by solving a Laplace equation. In previous studies, cells were often considered to be three-compartment models with different electroconductivities in different regions (three compartments are often intracellular regions, membrane, and extracellular regions). However, models like that did not take dynamic ion channels into consideration. Therefore, one cannot obtain concrete ionic current changes such as potassium current change or sodium current change by these models. The aim of the present work is to present a new and more detailed model for calculating transmembrane potentials and ionic currents induced by time-varying EMF. Equations used in the present paper originate from Nernst-Plank equations, which are ionic current-related equations. The main work is to calculate ionic current changes induced by EMF exposure, and then transmembrane potential changes are calculated with Hodgkin-Huxley model. Bioelectromagnetics. 37:481-492, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. VMD-SS: A graphical user interface plug-in to calculate the protein secondary structure in VMD program.

    PubMed

    Yahyavi, Masoumeh; Falsafi-Zadeh, Sajad; Karimi, Zahra; Kalatarian, Giti; Galehdari, Hamid

    2014-01-01

    The investigation on the types of secondary structure (SS) of a protein is important. The evolution of secondary structures during molecular dynamics simulations is a useful parameter to analyze protein structures. Therefore, it is of interest to describe VMD-SS (a software program) for the identification of secondary structure elements and its trajectories during simulation for known structures available at the Protein Data Bank (PDB). The program helps to calculate (1) percentage SS, (2) SS occurrence in each residue, (3) percentage SS during simulation, and (4) percentage residues in all SS types during simulation. The VMD-SS plug-in was designed using TCL script and stride to calculate secondary structure features. The database is available for free at http://science.scu.ac.ir/HomePage.aspx?TabID=13755.

  3. A Mechanical Switch Couples T Cell Receptor Triggering to the Cytoplasmic Juxtamembrane Regions of CD3ζζ.

    PubMed

    Lee, Mark S; Glassman, Caleb R; Deshpande, Neha R; Badgandi, Hemant B; Parrish, Heather L; Uttamapinant, Chayasith; Stawski, Philipp S; Ting, Alice Y; Kuhns, Michael S

    2015-08-18

    The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

    PubMed Central

    Pérez Santángelo, Agustín; Corti Bielsa, Rodrigo M.; Sala, Andrea; Ginart, Santiago; Corach, Daniel

    2017-01-01

    Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). PMID:29099837

  5. Helicobacter pylori modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis

    PubMed Central

    Faber, Eugenia; Bats, Simon H.; Murillo, Tatiana; Speidel, Yvonne; Coombs, Nina

    2017-01-01

    Highly virulent Helicobacter pylori cause proinflammatory signaling inducing the transcriptional activation and secretion of cytokines such as IL-8 in epithelial cells. Responsible in part for this signaling is the cag pathogenicity island (cagPAI) that codetermines the risk for pathological sequelae of an H. pylori infection such as gastric cancer. The Cag type IV secretion system (CagT4SS), encoded on the cagPAI, can translocate various molecules into cells, the effector protein CagA, peptidoglycan metabolites and DNA. Although these transported molecules are known to contribute to cellular responses to some extent, a major part of the cagPAI-induced signaling leading to IL-8 secretion remains unexplained. We report here that biosynthesis of heptose-1,7-bisphosphate (HBP), an important intermediate metabolite of LPS inner heptose core, contributes in a major way to the H. pylori cagPAI-dependent induction of proinflammatory signaling and IL-8 secretion in human epithelial cells. Mutants defective in the genes required for synthesis of HBP exhibited a more than 95% reduction of IL-8 induction and impaired CagT4SS-dependent cellular signaling. The loss of HBP biosynthesis did not abolish the ability to translocate CagA. The human cellular adaptor TIFA, which was described before to mediate HBP-dependent activity in other Gram-negative bacteria, was crucial in the cagPAI- and HBP pathway-induced responses by H. pylori in different cell types. The active metabolite was present in H. pylori lysates but not enriched in bacterial supernatants. These novel results advance our mechanistic understanding of H. pylori cagPAI-dependent signaling mediated by intracellular pattern recognition receptors. They will also allow to better dissect immunomodulatory activities by H. pylori and to improve the possibilities of intervention in cagPAI- and inflammation-driven cancerogenesis. PMID:28715499

  6. The Power of SS 433's Jets

    NASA Technical Reports Server (NTRS)

    Band, David F.

    1999-01-01

    We observed SS 433 for the third time with RXTE, this time simultaneously with a Nobeyama millimeter band monitoring campaign. These observations extended the RXTE coverage of SS 433's precession phases. and once again monitored the source during a binary eclipse. Our AO-2 campaign of a joint RXTE-VLBA-VLA-MERLIN observation was delayed to little more than a month before the AO-3 observations. We also had an AO-1 observation of SS 433. In each case we observed an eclipse of the compact ob'ect by the companion star, and had contemporaneous optical observations. We are analyzing all three sets of observations together. A publication is in preparation; Its completion will be supported by the remaining AO-2 funds. Here I will summarize the general results and point out the relevant features of the AO-3 observations. The spectrum is detected to approx. 50 keV, which is entirely within the PCA energy band. We find that the HEXTE data do not add significantly to the spectrum, and most of our fits are without HEXTE. We find that the continuum can be fit with a power law with an exponential cutoff; the photon index is usually approx. 1.4. A line at approx. 6.4 keV is definitely required, and the fits improve significant, if we add a second line, also with an energy in the iron K(alpha) complex, and an absorption edge. The edge usually has an energy of approx. 5.8 keV, which does not correspond to any known physical feature. and may be an instrumental artifact. This is under further investigation.

  7. Transmembrane S1 mutations in CNGA3 from achromatopsia 2 patients cause loss of function and impaired cellular trafficking of the cone CNG channel.

    PubMed

    Patel, Kirti A; Bartoli, Kristen M; Fandino, Richard A; Ngatchou, Anita N; Woch, Gustaw; Carey, Jannette; Tanaka, Jacqueline C

    2005-07-01

    Achromatopsia 2, an inherited retinal disorder resulting in attenuation or loss of cone function, is caused by mutations in the alpha subunit of the cone cyclic nucleotide-gated (CNG) channel gene CNGA3. Examination of mutations that cluster in the first transmembrane segment of the protein may provide insight into its role in CNG channel structure, function, biogenesis, and pathophysiology. The human CNGA3 gene was tagged at the C terminus with green fluorescent protein. Four mutations, Y181C, N182Y, L186F, and C191Y, were expressed in human embryonic kidney cells. Protein expression was evaluated with immunoblot analysis and cellular localization was determined by immunocytochemistry. Channel function was evaluated by patch-clamp electrophysiology. All the mutations result in loss of channel function, as determined by the failure of cGMP to activate wild-type currents in excised patches. Full-length mutant proteins were synthesized but retained in the endoplasmic reticulum. Glycerol treatment did not rescue channel function nor did coexpression with CNGB3, a subunit of native hetero-tetrameric cone channels. A control mutant, C191S, exhibited cGMP current activation with significantly reduced cooperativity, suggesting that mutations in the first transmembrane domain alter in inter- or intrasubunit communication. The results implicate the first transmembrane segment in both maturation and function of CNG channels. The defects are not reversed with glycerol, a chemical chaperone that rescues channel function in some channelopathies. Molecular analysis of achromatopsia 2 mutations may be useful in evaluating potential therapeutic approaches for treatment of this channelopathy.

  8. Transmembrane proteins in the Protein Data Bank: identification and classification.

    PubMed

    Tusnády, Gábor E; Dosztányi, Zsuzsanna; Simon, István

    2004-11-22

    Integral membrane proteins play important roles in living cells. Although these proteins are estimated to constitute 25% of proteins at a genomic scale, the Protein Data Bank (PDB) contains only a few hundred membrane proteins due to the difficulties with experimental techniques. The presence of transmembrane proteins in the structure data bank, however, is quite invisible, as the annotation of these entries is rather poor. Even if a protein is identified as a transmembrane one, the possible location of the lipid bilayer is not indicated in the PDB because these proteins are crystallized without their natural lipid bilayer, and currently no method is publicly available to detect the possible membrane plane using the atomic coordinates of membrane proteins. Here, we present a new geometrical approach to distinguish between transmembrane and globular proteins using structural information only and to locate the most likely position of the lipid bilayer. An automated algorithm (TMDET) is given to determine the membrane planes relative to the position of atomic coordinates, together with a discrimination function which is able to separate transmembrane and globular proteins even in cases of low resolution or incomplete structures such as fragments or parts of large multi chain complexes. This method can be used for the proper annotation of protein structures containing transmembrane segments and paves the way to an up-to-date database containing the structure of all known transmembrane proteins and fragments (PDB_TM) which can be automatically updated. The algorithm is equally important for the purpose of constructing databases purely of globular proteins.

  9. Application of Quantitative MRI for Brain Tissue Segmentation at 1.5 T and 3.0 T Field Strengths

    PubMed Central

    West, Janne; Blystad, Ida; Engström, Maria; Warntjes, Jan B. M.; Lundberg, Peter

    2013-01-01

    Background Brain tissue segmentation of white matter (WM), grey matter (GM), and cerebrospinal fluid (CSF) are important in neuroradiological applications. Quantitative Mri (qMRI) allows segmentation based on physical tissue properties, and the dependencies on MR scanner settings are removed. Brain tissue groups into clusters in the three dimensional space formed by the qMRI parameters R1, R2 and PD, and partial volume voxels are intermediate in this space. The qMRI parameters, however, depend on the main magnetic field strength. Therefore, longitudinal studies can be seriously limited by system upgrades. The aim of this work was to apply one recently described brain tissue segmentation method, based on qMRI, at both 1.5 T and 3.0 T field strengths, and to investigate similarities and differences. Methods In vivo qMRI measurements were performed on 10 healthy subjects using both 1.5 T and 3.0 T MR scanners. The brain tissue segmentation method was applied for both 1.5 T and 3.0 T and volumes of WM, GM, CSF and brain parenchymal fraction (BPF) were calculated on both field strengths. Repeatability was calculated for each scanner and a General Linear Model was used to examine the effect of field strength. Voxel-wise t-tests were also performed to evaluate regional differences. Results Statistically significant differences were found between 1.5 T and 3.0 T for WM, GM, CSF and BPF (p<0.001). Analyses of main effects showed that WM was underestimated, while GM and CSF were overestimated on 1.5 T compared to 3.0 T. The mean differences between 1.5 T and 3.0 T were -66 mL WM, 40 mL GM, 29 mL CSF and -1.99% BPF. Voxel-wise t-tests revealed regional differences of WM and GM in deep brain structures, cerebellum and brain stem. Conclusions Most of the brain was identically classified at the two field strengths, although some regional differences were observed. PMID:24066153

  10. Pharmacogenomic Strain Differences in Cardiovascular Sensitivity to Propofol

    PubMed Central

    Stekiel, Thomas A.; Contney, Stephen J.; Roman, Richard J.; Weber, Craig A.; Stadnicka, Anna; Bosnjak, Zeljko J.; Greene, Andrew S; Moreno, Carol

    2011-01-01

    Introduction A pharmacogenomic approach was used to further localize the genetic region responsible for previously observed enhanced cardiovascular sensitivity to propofol in Dahl Salt Sensitive (SS) vs. control Brown Norway (BN) rats. Methods Propofol infusion levels that decreased blood pressure by 50% were measured in BN.13SS rats (substitution of SS chromosome 13 into BN) and in 5 congenic (partial substitution) strains of SS.13BN. The effect of superfused 2,6 diisopropylphenol on small mesenteric arterial vascular smooth muscle transmembrane potential was measured in congenic strains before and during superfusion with Rp-cAMPS and Rp-8-pCPT-cGMPS, inhibitors of protein kinase A and G respectively. The genetic locus and potential role of the renin gene in mediating VSM sensitivity to propofol were determined in three selected sub-congenic SS.BN13 strains. Results A 30 – 32% smaller propofol infusion rate reduced blood pressure by 50% in BN.13SS compared to BN and the SS.13BN congenic containing a 80 BN gene substitution. Compared to the latter, SS exhibited greater protein kinase A dependent vascular smooth muscle hyperpolarization in response to propofol. Using sub-congenics, the increased propofol-induced cardiovascular sensitivity and hyperpolarization was further localized to an 8-gene region (containing the BN renin gene). Blockade of angiotensin (AT1) receptors with losartan in this sub-congenic, elevated propofol-induced hyperpolarization by 3 fold, to that observed in SS. Conclusions Enhanced cardiovascular sensitivity to propofol in SS (compared to BN) is caused by an altered renin gene. Through modified second messenger function, this differentially regulates VSM contractile state and reduces vascular tone exacerbating cardiovascular depression by propofol. PMID:22020141

  11. YneA, an SOS-induced inhibitor of cell division in Bacillus subtilis, is regulated posttranslationally and requires the transmembrane region for activity.

    PubMed

    Mo, Allison H; Burkholder, William F

    2010-06-01

    Cell viability depends on the stable transmission of genetic information to each successive generation. Therefore, in the event of intrinsic or extrinsic DNA damage, it is important that cell division be delayed until DNA repair has been completed. In Bacillus subtilis, this is accomplished in part by YneA, an inhibitor of division that is induced as part of the SOS response. We sought to gain insight into the mechanism by which YneA blocks cell division and the processes involved in shutting off YneA activity. Our data suggest that YneA is able to inhibit daughter cell separation as well as septum formation. YneA contains a LysM peptidoglycan binding domain and is predicted to be exported. We established that the YneA signal peptide is rapidly cleaved, resulting in secretion of YneA into the medium. Mutations within YneA affect both the rate of signal sequence cleavage and the activity of YneA. YneA does not stably associate with the cell wall and is rapidly degraded by extracellular proteases. Based on these results, we hypothesize that exported YneA is active prior to signal peptide cleavage and that proteolysis contributes to the inactivation of YneA. Finally, we identified mutations in the transmembrane segment of YneA that abolish the ability of YneA to inhibit cell division, while having little or no effect on YneA export or stability. These data suggest that protein-protein interactions mediated by the transmembrane region may be required for YneA activity.

  12. HOPX1 Ea (Eop3) in Erwinia Amylovora functions as an avirulence gene in apple and is regulated by HRPL

    USDA-ARS?s Scientific Manuscript database

    Fire blight is a devastating disease of rosaceous plants caused by the Gram-negative bacterium E. amylovora. This pathogen delivers virulence proteins into host cells utilizing the Type-Three Secretion System (T3SS). Expression of the T3SS and associated substrates are activated by the alternative s...

  13. Simultaneous quantification of T4, T3, rT3, 3,5-T2 and 3,3'-T2 in larval zebrafish (Danio rerio) as a model to study exposure to polychlorinated biphenyls.

    PubMed

    Chen, Xiaopeng; Walter, Kyla M; Miller, Galen W; Lein, Pamela J; Puschner, Birgit

    2018-06-01

    Environmental toxicants that interfere with thyroid hormone (TH) signaling can impact growth and development in animals and humans. Zebrafish represent a model to study chemically induced TH disruption, prompting the need for sensitive detection of THs. Simultaneous quantification of 3,3',5-triiodo-l-thyronine (T3), thyroxine (T4), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2) and 3,3'-diiodo-l-thyronine (3,3'-T2) in zebrafish larvae was achieved by ultra-performance liquid chromatography-tandem mass spectrometry in positive ion mode. Solid-phase extraction with SampliQ cartridges and derivatization with 3 m hydrochloric acid in n-butanol reduced matrix effects. Derivatized compounds were separated on an Acquity UPLC BEH C 18 column with mobile phases consisting of 0.1% acetic acid in deionized water and 0.1% acetic acid in methanol. The limits of detection ranged from 0.5 to 0.6 pg injected on column. The method was validated by evaluating recovery (77.1-117.2%), accuracy (87.3-123.9%) and precision (0.5-12.4%) using diluted homogenized zebrafish embryos spiked with all target compounds. This method was then applied to zebrafish larvae collected after 114 h of exposure to polychlorinated biphenyls (PCBs), including PCB 28, PCB 66 and PCB 95, or the technical mixture Aroclor 1254. Exposure to PCB 28 and PCB 95 increased the T4:T3 ratio and decreased the T3:rT3 ratio, demonstrating that this method can effectively detect PCB-induced alterations in THs. Copyright © 2018 John Wiley & Sons, Ltd.

  14. Design of High Performance Si/SiGe Heterojunction Tunneling FETs with a T-Shaped Gate

    NASA Astrophysics Data System (ADS)

    Li, Wei; Liu, Hongxia; Wang, Shulong; Chen, Shupeng; Yang, Zhaonian

    2017-03-01

    In this paper, a new Si/SiGe heterojunction tunneling field-effect transistor with a T-shaped gate (HTG-TFET) is proposed and investigated by Silvaco-Atlas simulation. The two source regions of the HTG-TFET are placed on both sides of the gate to increase the tunneling area. The T-shaped gate is designed to overlap with N+ pockets in both the lateral and vertical directions, which increases the electric field and tunneling rate at the top of tunneling junctions. Moreover, using SiGe in the pocket regions leads to the smaller tunneling distance. Therefore, the proposed HTG-TFET can obtain the higher on-state current. The simulation results show that on-state current of HTG-TFET is increased by one order of magnitude compared with that of the silicon-based counterparts. The average subthreshold swing (SS) of HTG-TFET is 44.64 mV/dec when V g is varied from 0.1 to 0.4 V, and the point SS is 36.59 mV/dec at V g = 0.2 V. Besides, this design cannot bring the sever Miller capacitance for the TFET circuit design. By using the T-shaped gate and SiGe pocket regions, the overall performance of the TFET is optimized.

  15. Design of High Performance Si/SiGe Heterojunction Tunneling FETs with a T-Shaped Gate.

    PubMed

    Li, Wei; Liu, Hongxia; Wang, Shulong; Chen, Shupeng; Yang, Zhaonian

    2017-12-01

    In this paper, a new Si/SiGe heterojunction tunneling field-effect transistor with a T-shaped gate (HTG-TFET) is proposed and investigated by Silvaco-Atlas simulation. The two source regions of the HTG-TFET are placed on both sides of the gate to increase the tunneling area. The T-shaped gate is designed to overlap with N + pockets in both the lateral and vertical directions, which increases the electric field and tunneling rate at the top of tunneling junctions. Moreover, using SiGe in the pocket regions leads to the smaller tunneling distance. Therefore, the proposed HTG-TFET can obtain the higher on-state current. The simulation results show that on-state current of HTG-TFET is increased by one order of magnitude compared with that of the silicon-based counterparts. The average subthreshold swing (SS) of HTG-TFET is 44.64 mV/dec when V g is varied from 0.1 to 0.4 V, and the point SS is 36.59 mV/dec at V g  = 0.2 V. Besides, this design cannot bring the sever Miller capacitance for the TFET circuit design. By using the T-shaped gate and SiGe pocket regions, the overall performance of the TFET is optimized.

  16. Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins*

    PubMed Central

    Pinto, Bernardo I.; García, Isaac E.; Pupo, Amaury; Retamal, Mauricio A.; Martínez, Agustín D.; Latorre, Ramón; González, Carlos

    2016-01-01

    Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity. PMID:27143357

  17. Polymorphisms rs12998 and rs5780218 in KiSS1 suppressor metastasis gene in Mexican patients with breast cancer.

    PubMed

    Quevedo, Edhit Guadalupe Cruz; Aguilar, Gabriela Monserrat Mimendi; Aguilar, Luis Anselmo Juárez; Rubio, Susan Andrea Gutierrez; Martínez, Silvia Esperanza Flores; Rodríguez, Ingrid Patricia Dávalos; Corona, José Sánchez; Morán, Martha Isabel Torres; Gómez, Roberto Carlos Rosales; Moguel, María Cristina Morán

    2015-01-01

    KiSS1 is a metastasis suppressor gene associated with inhibition of cellular chemotaxis and invasion attenuating the metastasis in melanoma and breast cancer cell lines. Along the KiSS-1 gene at least 294 SNPs have been described; however the association of these polymorphisms as genetic markers for metastasis in breast cancer studies has not been investigated. Here we describe two simple PCR-RFLPs protocols to identify the rs5780218 (9DelT) and the rs12998 (E20K) KiSS1 polymorphisms and the allelic, genotypic, and haplotypic frequencies in Mexican general population (GP) and patients with benign breast disease (BBD) or breast cancer (BC). The rs5780218 polymorphism was individually associated with breast cancer (P = 0.0332) and the rs12998 polymorphism shows statistically significant differences when GP versus case (BC and BBD) groups were compared (P < 0.0001). The H1 Haplotype (G/-) occurred more frequently in BC group (0.4256) whereas H2 haplotype (G/T) was the most prevalent in BBD group (0.4674). Our data indicated that the rs5780218 polymorphism individually confers susceptibility for development of breast cancer in Mexican population and a possible role as a genetic marker in breast cancer metastasis for H1 haplotype (Wt/variant) in KiSS1 gene must be analyzed in other populations.

  18. Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase.

    PubMed

    Van, Ru C; Pan, Yih J; Hsu, Shen H; Huang, Yun T; Hsiao, Yi Y; Pan, Rong L

    2005-08-15

    Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.

  19. Construction, characterization and evaluation of the protective efficacy of the Streptococcus suis double mutant strain ΔSsPep/ΔSsPspC as a live vaccine candidate in mice.

    PubMed

    Hu, Jin; You, Wujin; Wang, Bin; Hu, Xueying; Tan, Chen; Liu, Jinlin; Chen, Huanchun; Bei, Weicheng

    2015-01-01

    Streptococcus suis serotype 2 (S. suis 2) causes sepsis and meningitis in piglets and humans, and results in one of the most serious bacterial diseases affecting the production of commercial pigs around the world. Due to the failure of the current inactivated vaccine to protect against the disease, development of a new attenuated live vaccine against S. suis 2 by deleting essential virulence factors is urgently needed. We have previously reported the construction and characterization of an SsPep single gene deletion mutant strain ΔSsPep based on S. suis 2. Our previous results have shown that SsPep plays a critical role in the pathogenesis of S. suis 2. In this study, a precisely defined double-deletion mutant ΔSsPep/ΔSsPspC of S. suis 2 without antibiotic-resistance markers was constructed based on ΔSsPep, and the levels of virulence of the wild-type (WT) and ΔSsPep/ΔSsPspC were compared in a mouse experimental infection model. We demonstrated that the double mutant ΔSsPep/ΔSsPspC was less virulent than the WT, and could induce a noticeable antibody response. Analysis of IgG subclasses (IgG1 and IgG2a) indicated that both Th1 and Th2 responses were induced by ΔSsPep/ΔSsPspC, although the IgG2a (Th1) response predominated over the IgG1 (Th2) response. Moreover, ΔSsPep/ΔSsPspC could confer 90% protective efficacy against challenge with a lethal dose of fully virulent S. suis 2. Taken together, these data demonstrate that ΔSsPep/ΔSsPspC can be used as an effective live vaccine and provide a novel strategy against infection of S. suis 2. Copyright © 2014 Elsevier GmbH. All rights reserved.

  20. Interdependence of the kinetics of NTP hydrolysis and the stability of the RecA-ssDNA complex.

    PubMed

    Katz, F S; Bryant, F R

    2001-09-18

    The ssDNA-dependent NTP hydrolysis activity of the RecA protein was examined using a series of dTn oligomers ranging in size from dT10 to dT2000 as the ssDNA effector. There were three distinct manifestations of the dTn-dependent NTP hydrolysis reaction, depending on the length of the dTn effector that was used. With longer dTn oligomers, NTP hydrolysis occurred with a turnover number of 20-25 min(-1) and the observed S0.5 value for the NTP was independent of the concentration of the dTn oligomer (DNA concentration-independent hydrolysis). With dTn oligomers of intermediate length, NTP hydrolysis still occurred with a turnover number of 20-25 min(-1), but the observed S0.5 for the NTP decreased with increasing dTn concentration until reaching a value similar to that obtained with the longer dTn oligomers (DNA concentration-dependent hydrolysis). With shorter dTn oligomers, the NTP hydrolysis activity was effectively eliminated. Although this general progression of kinetic behavior was observed for the three structurally related NTPs (dATP, ATP, and GTP), the dTn oligomer length at which DNA concentration-independent, DNA concentration-dependent, and no NTP hydrolysis was observed depended on the NTP being considered. For example, dATP (S0.5 = 35 microM) was hydrolyzed in the presence of dT20, whereas ATP (S0.5 = 70 microM) and GTP (S0.5 = 1200 microM) required at least dT50 and dT200 for hydrolysis, respectively. These results are discussed in terms of a kinetic model in which the stability of the RecA-ssDNA-NTP complex is dependent on the intrinsic S0.5 value of the NTP being hydrolyzed.

  1. T-tubule disease: Relationship between t-tubule organization and regional contractile performance in human dilated cardiomyopathy.

    PubMed

    Crossman, David J; Young, Alistair A; Ruygrok, Peter N; Nason, Guy P; Baddelely, David; Soeller, Christian; Cannell, Mark B

    2015-07-01

    Evidence from animal models suggest that t-tubule changes may play an important role in the contractile deficit associated with heart failure. However samples are usually taken at random with no regard as to regional variability present in failing hearts which leads to uncertainty in the relationship between contractile performance and possible t-tubule derangement. Regional contraction in human hearts was measured by tagged cine MRI and model fitting. At transplant, failing hearts were biopsy sampled in identified regions and immunocytochemistry was used to label t-tubules and sarcomeric z-lines. Computer image analysis was used to assess 5 different unbiased measures of t-tubule structure/organization. In regions of failing hearts that showed good contractile performance, t-tubule organization was similar to that seen in normal hearts, with worsening structure correlating with the loss of regional contractile performance. Statistical analysis showed that t-tubule direction was most highly correlated with local contractile performance, followed by the amplitude of the sarcomeric peak in the Fourier transform of the t-tubule image. Other area based measures were less well correlated. We conclude that regional contractile performance in failing human hearts is strongly correlated with the local t-tubule organization. Cluster tree analysis with a functional definition of failing contraction strength allowed a pathological definition of 't-tubule disease'. The regional variability in contractile performance and cellular structure is a confounding issue for analysis of samples taken from failing human hearts, although this may be overcome with regional analysis by using tagged cMRI and biopsy mapping. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Expression and Purification of a Matrix Metalloprotease Transmembrane Domain in Escherichia coli.

    PubMed

    Galea, Charles A

    2017-01-01

    Membrane tethered matrix metalloproteases are bound to the plasma membrane by a glycosylphosphatidylinositol-anchor or a transmembrane domain. To date, most studies of membrane-bound matrix metalloprotease have focused on the globular catalytic and protein-protein interaction domains of these enzymes. However, the transmembrane domains have been poorly studied even though they are known to mediate intracellular signaling via interaction with various cellular proteins. The expression and purification of the transmembrane domain of these proteins can be challenging due to their hydrophobic nature. In this chapter we describe the purification of a transmembrane domain for a membrane-bound matrix metalloprotease expressed in E. coli and its initial characterization by NMR spectroscopy.

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

    PubMed

    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.

  4. Comparison of Diffusion-Weighted Imaging in the Human Brain Using Readout-Segmented EPI and PROPELLER Turbo Spin Echo With Single-Shot EPI at 7 T MRI.

    PubMed

    Kida, Ikuhiro; Ueguchi, Takashi; Matsuoka, Yuichiro; Zhou, Kun; Stemmer, Alto; Porter, David

    2016-07-01

    The purpose of the present study was to compare periodically rotated overlapping parallel lines with enhanced reconstruction-type turbo spin echo diffusion-weighted imaging (pTSE-DWI) and readout-segmented echo planar imaging (rsEPI-DWI) with single-shot echo planar imaging (ssEPI-DWI) in a 7 T human MR system. We evaluated the signal-to-noise ratio (SNR), image distortion, and apparent diffusion coefficient values in the human brain. Six healthy volunteers were included in this study. The study protocol was approved by our institutional review board. All measurements were performed at 7 T using pTSE-DWI, rsEPI-DWI, and ssEPI-DWI sequences. The spatial resolution was 1.2 × 1.2 mm in-plane with a 3-mm slice thickness. Signal-to-noise ratio was measured using 2 scans. The ssEPI-DWI sequence showed significant image blurring, whereas pTSE-DWI and rsEPI-DWI sequences demonstrated high image quality with low geometrical distortion compared with reference T2-weighted, turbo spin echo images. Signal loss in ventral regions near the air-filled paranasal sinus/nasal cavity was found in ssEPI-DWI and rsEPI-DWI but not pTSE-DWI. The apparent diffusion coefficient values for ssEPI-DWI were 824 ± 17 × 10 and 749 ± 25 × 10 mm/s in the gray matter and white matter, respectively; the values obtained for pTSE-DWI were 798 ± 21 × 10 and 865 ± 40 × 10 mm/s; and the values obtained for rsEPI-DWI were 730 ± 12 × 10 and 722 ± 25 × 10 mm/s. The pTSE-DWI images showed no additional distortion comparison to the T2-weighted images, but had a lower SNR than ssEPI-DWI and rsEPI-DWI. The rsEPI-DWI sequence provided high-quality images with minor distortion and a similar SNR to ssEPI-DWI. Our results suggest that the benefits of the rsEPI-DWI and pTSE-DWI sequences, in terms of SNR, image quality, and image distortion, appear to outweigh those of ssEPI-DWI. Thus, pTSE-DWI and rsEPI-DWI at 7 T have great potential use for clinical diagnoses. However, it is noteworthy that both

  5. Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

    PubMed Central

    Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

  6. A Triad of Molecular Regions Contribute to the Formation of Two Distinct MHC Class II Conformers

    PubMed Central

    Drake, Lisa A.; Drake, James R.

    2016-01-01

    MHC class II molecules present antigen-derived peptides to CD4 T cells to drive the adaptive immune response. Previous work has established that class II αβ dimers can adopt two distinct conformations, driven by the differential pairing of transmembrane domain GxxxG dimerization motifs. These class II conformers differ in their ability to be loaded with antigen-derived peptide and to effectively engage CD4 T cells. Motif 1 (M1) paired I-Ak class II molecules are efficiently loaded with peptides derived from the processing of B cell receptor-bound antigen, have unique B cell signaling properties and high T cell stimulation activity. The 11-5.2 mAb selectively binds M1 paired I-Ak class II molecules. However, the molecular determinants of 11-5.2 binding are currently unclear. Here, we report the ability of a human class II transmembrane domain to drive both M1 and M2 class II conformer formation. Protease sensitivity analysis further strengthens the idea that there are conformational differences between the extracellular domains of M1 and M2 paired class II. Finally, MHC class II chain alignments and site directed mutagenesis reveals a triad of molecular regions that contributes to 11-5.2 mAb binding. In addition to transmembrane GxxxG motif domain pairing, 11-5.2 binding is influenced directly by α chain residue Glu-71 and indirectly by the region around the inter-chain salt bridge formed by α chain Arg-52 and β chain Glu-86. These findings provide insight into the complexity of 11-5.2 mAb recognition of the M1 paired I-Ak class II conformer and further highlight the molecular heterogeneity of peptide-MHC class II complexes that drive T cell antigen recognition. PMID:27148821

  7. Mechanisms for RNA capture by ssDNA viruses: grand theft RNA.

    PubMed

    Stedman, Kenneth

    2013-06-01

    Viruses contain three common types of packaged genomes; double-stranded DNA (dsDNA), RNA (mostly single and occasionally double stranded) and single-stranded DNA (ssDNA). There are relatively straightforward explanations for the prevalence of viruses with dsDNA and RNA genomes, but the evolutionary basis for the apparent success of ssDNA viruses is less clear. The recent discovery of four ssDNA virus genomes that appear to have been formed by recombination between co-infecting RNA and ssDNA viruses, together with the high mutation rate of ssDNA viruses provide possible explanations. RNA-DNA recombination allows ssDNA viruses to access much broader sequence space than through nucleotide substitution and DNA-DNA recombination alone. Multiple non-exclusive mechanisms, all due to the unique replication of ssDNA viruses, are proposed for this unusual RNA capture. RNA capture provides an explanation for the evolutionary success of the ssDNA viruses and may help elucidate the mystery of integrated RNA viruses in viral and cellular DNA genomes.

  8. Deficiency of slow skeletal muscle troponin T causes atrophy of type I slow fibres and decreases tolerance to fatigue.

    PubMed

    Wei, Bin; Lu, Yingru; Jin, J-P

    2014-03-15

    The total loss of slow skeletal muscle troponin T (ssTnT encoded by TNNT1 gene) due to a nonsense mutation in codon Glu(180) causes a lethal form of recessively inherited nemaline myopathy (Amish nemaline myopathy, ANM). To investigate the pathogenesis and muscle pathophysiology of ANM, we studied the phenotypes of partial and total loss of ssTnT in Tnnt1 gene targeted mice. An insertion of neomycin resistance cassette in intron 10 of Tnnt1 gene caused an approximately 60% decrease in ssTnT protein expression whereas cre-loxP-mediated deletion of exons 11-13 resulted in total loss of ssTnT, as seen in ANM muscles. In diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that ssTnT deficiency resulted in significantly decreased levels of other slow fibre-specific myofilament proteins whereas fast fibre-specific myofilament proteins were increased correspondingly. Immunohistochemical studies revealed that ssTnT deficiency produced significantly smaller type I slow fibres and compensatory growth of type II fast fibres. Along with the slow fibre atrophy and the changes in myofilament protein isoform contents, ssTnT deficiency significantly reduced the tolerance to fatigue in soleus muscle. ssTnT-deficient soleus muscle also contains significant numbers of small-sized central nuclei type I fibres, indicating active regeneration. The data provide strong support for the essential role of ssTnT in skeletal muscle function and the causal effect of its loss in the pathology of ANM. This observation further supports the hypothesis that the function of slow fibres can be restored in ANM patients if a therapeutic supplement of ssTnT is achieved.

  9. Deficiency of slow skeletal muscle troponin T causes atrophy of type I slow fibres and decreases tolerance to fatigue

    PubMed Central

    Wei, Bin; Lu, Yingru; Jin, J-P

    2014-01-01

    The total loss of slow skeletal muscle troponin T (ssTnT encoded by TNNT1 gene) due to a nonsense mutation in codon Glu180 causes a lethal form of recessively inherited nemaline myopathy (Amish nemaline myopathy, ANM). To investigate the pathogenesis and muscle pathophysiology of ANM, we studied the phenotypes of partial and total loss of ssTnT in Tnnt1 gene targeted mice. An insertion of neomycin resistance cassette in intron 10 of Tnnt1 gene caused an approximately 60% decrease in ssTnT protein expression whereas cre-loxP-mediated deletion of exons 11–13 resulted in total loss of ssTnT, as seen in ANM muscles. In diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that ssTnT deficiency resulted in significantly decreased levels of other slow fibre-specific myofilament proteins whereas fast fibre-specific myofilament proteins were increased correspondingly. Immunohistochemical studies revealed that ssTnT deficiency produced significantly smaller type I slow fibres and compensatory growth of type II fast fibres. Along with the slow fibre atrophy and the changes in myofilament protein isoform contents, ssTnT deficiency significantly reduced the tolerance to fatigue in soleus muscle. ssTnT-deficient soleus muscle also contains significant numbers of small-sized central nuclei type I fibres, indicating active regeneration. The data provide strong support for the essential role of ssTnT in skeletal muscle function and the causal effect of its loss in the pathology of ANM. This observation further supports the hypothesis that the function of slow fibres can be restored in ANM patients if a therapeutic supplement of ssTnT is achieved. PMID:24445317

  10. In vivo fate of 125I SS-B (La) injected into mice.

    PubMed Central

    Schrieber, L; Erhardt, C C; Melsom, R D; Venables, P J; McCarthy, D A; Mumford, P A; Horsfall, A C; Maini, R N

    1983-01-01

    The fate of the radiolabelled soluble cellular antigen SS-B (La) was compared with that of other 125I-labelled proteins of known molecular weight (MW) and electrostatic charge, following i.v. injection into BALB/c mice. The plasma half-life of 125I-SS-B was 3 min, while that of 125I-bovine serum albumin (similar MW and electrostatic charge) was 270 min. 125I-heat-aggregated IgG (MW greater than 1 x 10(6)) and 125I-7S human IgG (MW 168,000) had plasma half-lives of 40 min and greater than 300 min, respectively. Liver and kidney showed preferential uptake of 125I-SS-B, followed by a rapid decrease in radioactivity. During this time low MW, trichloroacetic acid (TCA) soluble, material appeared in urine. This suggests a specific uptake mechanism followed by a catabolic phase. These studies demonstrate that normal mice remove 125I-SS-B rapidly from the circulation and then degrade it. This rapid antigen elimination may protect against the induction of potentially harmful autoantibody responses. PMID:6832805

  11. Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs.

    PubMed

    Wang, C; Deber, C M

    2000-05-26

    Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.

  12. Synthesis of the new compound CaFe(CO 3) 2 and experimental constraints on the (Ca,Fe)CO 3 join

    NASA Astrophysics Data System (ADS)

    Davidson, Paula M.; Symmes, Gregory H.; Cohen, Barbara A.; Reeder, Richard J.; Lindsley, Donald H.

    1993-12-01

    Synthesis of the new (disordered) compound CaFe(CO 3) 2 has been achieved with the use of Fe-substituted CaCO 3(Cc ss) + Ca-substituted FeCO 3(Sid ss) as starting materials, and high CO 2 pressures. High pressure (20-30 kbar) is needed to stabilize FeCO 3 to sufficiently high temperatures for disordered CaFe(CO 3) 2 to form. Experiments provide reversed compositions of coexisting disordered phases in the CaFe join and locate the solvus temperature for CaFe(C) 3) 2 between 815 and 845°C at 30 kbars. Calculated phase relations predict that the stability of ordered CaFe(CO 3) 2 is limited to T < ˜450°C by the breakdown to Cc ss + Sid ss. A comparison of the unit-cell volume measured for disordered CaFe(CO 3) 2 vs. that estimated for ordered CaFe(CO 3) 2 suggests that increasing pressure stabilizes the disordered phase.

  13. Remedial Investigation for Cold Regions Research and Engineering Laboratory (CRREL) Hanover, New Hampshire. Phase 2. Volume 3. Appendices. Revision 2

    DTIC Science & Technology

    1994-03-18

    93 6-Aug-943 U1421 STYR tOOL 5.0c :0SS33 MIST044 ;AOI.905 AZ;ýI A -Aug-9 3 R-Auq-93 01121 .S’YR Uý 5.3( 1,:5337 91ST046 JA02194 ADYR Rt 20-Aug-93 25...Zffe-t-s E’ac-ilitY at sam-1v locaticns 378, 379, and 382. i three point azmm1y of learne icai i~taiuity wa also icdertifiaeucd ~ s of the L09st-IcS and...Retrieval operations are dependent of surface comxtiaw and rcetinely (xxsist of the folloiwhg tw metho. 2e first methd applies to grass covered or

  14. Simulation of debris flow events in Sicily by cellular automata model SCIDDICA_SS3

    NASA Astrophysics Data System (ADS)

    Cancelliere, A.; Lupiano, V.; Peres, D. J.; Stancanelli, L.; Avolio, M.; Foti, E.; Di Gregorio, S.

    2013-12-01

    Debris flow models are widely used for hazard mapping or for evaluating the effectiveness of risk mitigation measures. Several models analyze the dynamics of debris flow runout solving Partial Differential Equations. In use of such models, difficulties arise in estimating kinematic geotechnical soil parameters for real phenomena. In order to overcome such difficulties, alternative semi-empirical approaches can be employed, such as macroscopic Cellular Automata (CA). In particular, for CA simulation purposes, the runout of debris flows emerges from local interactions in a dynamical system, subdivided into elementary parts, whose state evolves within a spatial and temporal discretum. The attributes of each cell (substates) describe physical characteristics. For computational reasons, the natural phenomenon is splitted into a number of elementary processes, whose proper composition makes up the CA transition function. By simultaneously applying this function to all the cells, the evolution of the phenomenon can be simulated in terms of modifications of the substates. In this study, we present an application of the macroscopic CA semi-empirical model SCIDDICA_SS3 to the Peloritani Mountains area in Sicily island, Italy. The model was applied using detailed data from the 1 October 2009 debris flow event, which was triggered by a rainfall event of about 250 mm falling in 9 hours, that caused the death of 37 persons. This region is characterized by river valleys with large hillslope angles (30°-60°), catchment basins of small extensions (0.5-12 km2) and soil composed by metamorphic material, which is easy to be eroded. CA usage implies a calibration phase, that identifies an optimal set of parameters capable of adequately play back the considered case, and a validation phase, that tests the model on a sufficient (and different) number of cases similar in terms of physical and geomorphological properties. The performance of the model can be measured in terms of a fitness

  15. Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

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

    Turley, E.A.; Moore, D.; Hayden, L.J.

    1987-06-02

    A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weightmore » range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.« less

  16. Fast T2*-weighted MRI of the prostate at 3 Tesla.

    PubMed

    Hardman, Rulon L; El-Merhi, Fadi; Jung, Adam J; Ware, Steve; Thompson, Ian M; Friel, Harry T; Peng, Qi

    2011-04-01

    To describe a rapid T2*-weighted (T2*W), three-dimensional (3D) echo planar imaging (EPI) sequence and its application in mapping local magnetic susceptibility variations in 3 Tesla (T) prostate MRI. To compare the sensitivity of T2*W EPI with routinely used T1-weighted turbo-spin echo sequence (T1W TSE) in detecting hemorrhage and the implications on sequences sensitive to field inhomogeneities such as MR spectroscopy (MRS). B(0) susceptibility weighted mapping was performed using a 3D EPI sequence featuring a 2D spatial excitation pulse with gradients of spiral k-space trajectory. A series of 11 subjects were imaged using 3T MRI and combination endorectal (ER) and six-channel phased array cardiac coils. T1W TSE and T2*W EPI sequences were analyzed quantitatively for hemorrhage contrast. Point resolved spectroscopy (PRESS MRS) was performed and data quality was analyzed. Two types of susceptibility variation were identified: hemorrhagic and nonhemorrhagic T2*W-positive areas. Post-biopsy hemorrhage lesions showed on average five times greater contrast on the T2*W images than T1W TSE images. Six nonhemorrhage regions of severe susceptibility artifact were apparent on the T2*W images that were not seen on standard T1W or T2W images. All nonhemorrhagic susceptibility artifact regions demonstrated compromised spectral quality on 3D MRS. The fast T2*W EPI sequence identifies hemorrhagic and nonhemorrhagic areas of susceptibility variation that may be helpful in prostate MRI planning at 3.0T. Copyright © 2011 Wiley-Liss, Inc.

  17. Complementarity-Determining Region 3 Size Spectratypes of T Cell Receptor β Chains in CD8+ T Cells following Antiviral Treatment of Chronic Hepatitis B▿

    PubMed Central

    Ma, Shi-Wu; Li, Yong-Yin; Zhang, Guang-Wen; Huang, Xuan; Sun, Jian; Li, Chris; Abbott, William G. H.; Hou, Jin-Lin

    2011-01-01

    An increased CD8+ T cell response to hepatitis B virus (HBV) peptides occurs between 12 and 24 weeks after starting antiviral therapy for chronic hepatitis B. It is not known whether these cells have antiviral function. The aim of this study was to determine whether clonal expansions of CD8+ T cells at these time points predict the virological response to therapy. Peripheral blood CD8+ T cells were obtained from 20 patients treated with lamivudine or telbivudine for chronic hepatitis B at baseline, 12 weeks, and 24 weeks. The CDR3 spectratype of each T cell receptor (TCR) β chain variable region (Vβ) gene family was analyzed, and the changes in the numbers of Vβ families with clonal expansions were compared in subjects with (n = 12) and without (n = 8) a virological response (52 week HBV DNA < 300 copies/ml). The number of CD8+ TCR Vβ families with clonal expansions at 12 weeks relative to baseline (median [10th to 90th percentile], +2.5 [0 to +7] versus +1 [0 to +2], P = 0.03) and at 24 weeks relative to 12 weeks (+1 [0 to +2] versus −1 [−3 to +4], P = 0.006) was higher in subjects with a virological response versus subjects without a virological response, as were interleukin-2 (IL-2) but not IL-21 mRNA levels in peripheral blood mononuclear cells. The duration of new expansions at 12 weeks was higher (P < 0.0001) in responders. Increased numbers of CD8+ T cell expansions after antiviral therapy are associated with a virological response to treatment. These CD8+ T cells are a potential target for a therapeutic vaccine for chronic hepatitis B. PMID:21098256

  18. Synthesis and characterization of PEG-P(MAA-SS-VCL) nanoparticles

    NASA Astrophysics Data System (ADS)

    Yu, L. L.; Yang, K.; Mu, R. H.; Zhang, N.; Su, L.

    2016-07-01

    The PEG-P(MAA-SS-VCL) nanoparticles were obtained using disulfide containing dimethacrylate (SS) as cross-linking agent, using polyethylene glycol methyl acrylate (PEGMA), N-Vinyl-ε-caprolactam (VCL), and methacrylic acid (MAA) as monomers via homogeneous polymerization in aqueous. The PEG-P(MAA-SS-VCL) nanoparticles were characterized by FT-IR and TGA. The particle size and morphology variation in different environments were detected by dynamic light scattering (DLS) and scanning electron microscopy (SEM). It is the very method that PEG-P(MAA-SS-VCL) nanoparticles can be obtained in this study.

  19. The Masses and Evolutionary State of the Stars in the Dwarf Nova SS Cygni

    NASA Astrophysics Data System (ADS)

    Bitner, Martin A.; Robinson, Edward L.; Behr, Bradford B.

    2007-06-01

    The dwarf nova SS Cygni is a close binary star consisting of a K star transferring mass to a white dwarf by way of an accretion disk. We have obtained new spectroscopic observations of SS Cyg. Fits of synthetic spectra for Roche lobe-filling stars to the absorption-line spectrum of the K star yield the amplitude of the K star's radial velocity curve and the mass ratio, KK=162.5+/-1.0 km s-1 and q=MK/MWD=0.685+/-0.015. The fits also show that the accretion disk and white dwarf contribute a fraction f=0.535+/-0.075 of the total flux at 5500 Å. Taking the weighted average of our results with previously published results obtained using similar techniques, we find =163.7+/-0.7 km s-1 and =0.683+/-0.012. The orbital light curve of SS Cyg shows an ellipsoidal variation diluted by light from the disk and white dwarf. From an analysis of the ellipsoidal variations, we limit the orbital inclination to the range 45deg<=i<=56deg. The derived masses of the K star and white dwarf are MK=0.55+/-0.13 Msolar and MWD=0.81+/-0.19 Msolar, where the uncertainties are dominated by systematic errors in the orbital inclination. The K star in SS Cyg is 10%-50% larger than an unevolved star with the same mass and thus does not follow the mass-radius relation for zero-age main-sequence stars, nor does it follow the ZAMS mass-spectral type relation. Its mass and spectral type are, however, consistent with models in which the core hydrogen has been significantly depleted. Based on observations obtained with the Hobby-Eberly Telescope, which is a joint project of the University of Texas at Austin, the Pennsylvania State University, Stanford University, Ludwig-Maximilians-Universität München, and Georg-August-Universität Göttingen.

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

    PubMed

    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

  1. A mutation within the transmembrane domain of melanosomal protein Silver (Pmel17) changes lumenal fragment interactions

    PubMed Central

    Kuliawat, Regina; Santambrogio, Laura

    2009-01-01

    Melanocytes synthesize and store melanin within tissue-specific organelles, the melanosomes. Melanin deposition takes place along fibrils found within these organelles and fibril formation is known to depend on trafficking of the membrane glycoprotein Silver/Pmel17. However, correctly targeted, full-length Silver/Pmel17 cannot form fibers. Proteolytic processing in endosomal compartments and the generation of a lumenal Mα fragment that is incorporated into amyloid-like structures is also essential. Dominant White (DWhite), a mutant form of Silver/Pmel17 first described in chicken, causes disorganized fibers and severe hypopigmentation due to melanocyte death. Surprisingly, the DWhite mutation is an insertion of three amino acids into the transmembrane domain; the DWhite-Mα fragment is unaffected. To determine the functional importance of the transmembrane domain in organized fibril assembly, we investigated membrane trafficking and multimerization of Silver/Pmel17/DWhite proteins. We demonstrate that the DWhite mutation changes lipid interactions and disulfide bond-mediated associations of lumenal domains. Thus, partitioning into membrane microdomains and effects on conformation explain how the transmembrane region may contribute to the structural integrity of Silver/Pmel17 oligomers or influence toxic, amyloidogenic properties. PMID:19679373

  2. YneA, an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity▿ †

    PubMed Central

    Mo, Allison H.; Burkholder, William F.

    2010-01-01

    Cell viability depends on the stable transmission of genetic information to each successive generation. Therefore, in the event of intrinsic or extrinsic DNA damage, it is important that cell division be delayed until DNA repair has been completed. In Bacillus subtilis, this is accomplished in part by YneA, an inhibitor of division that is induced as part of the SOS response. We sought to gain insight into the mechanism by which YneA blocks cell division and the processes involved in shutting off YneA activity. Our data suggest that YneA is able to inhibit daughter cell separation as well as septum formation. YneA contains a LysM peptidoglycan binding domain and is predicted to be exported. We established that the YneA signal peptide is rapidly cleaved, resulting in secretion of YneA into the medium. Mutations within YneA affect both the rate of signal sequence cleavage and the activity of YneA. YneA does not stably associate with the cell wall and is rapidly degraded by extracellular proteases. Based on these results, we hypothesize that exported YneA is active prior to signal peptide cleavage and that proteolysis contributes to the inactivation of YneA. Finally, we identified mutations in the transmembrane segment of YneA that abolish the ability of YneA to inhibit cell division, while having little or no effect on YneA export or stability. These data suggest that protein-protein interactions mediated by the transmembrane region may be required for YneA activity. PMID:20400548

  3. Downregulation of SS18-SSX1 expression in synovial sarcoma by small interfering RNA enhances the focal adhesion pathway and inhibits anchorage-independent growth in vitro and tumor growth in vivo.

    PubMed

    Takenaka, Satoshi; Naka, Norifumi; Araki, Nobuhito; Hashimoto, Nobuyuki; Ueda, Takafumi; Yoshioka, Kiyoko; Yoshikawa, Hideki; Itoh, Kazuyuki

    2010-04-01

    Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by a unique t(X;18) translocation resulting in expression of SS18-SSX fusion protein. In order to investigate the biological function of this fusion protein and to develop a novel therapeutic option, we examined downregulation of SS18-SSX1 expression by small interfering RNA targeting SS18-SSX1 in three human SS cell lines. Microarray analysis comparing SS18-SSX1-silenced cells with control cells in three SS cell lines showed that SS18-SSX1 mainly affected the focal adhesion pathway. In accord with the array data, silencing of SS18-SSX1 enhances adhesion to the extracellular matrix through the induction of expression of myosin light-chain kinase. Furthermore, the silencing of SS18-SSX1 inhibits anchorage-independent growth in vitro and systemic delivery of siRNA against SS18-SSX1 using a nanoparticle system inhibited tumor growth in a nude mouse xenograft model. Our results demonstrate that siRNA targeting of SS18-SSX1 has therapeutic potential for the treatment of SS.

  4. Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide.

    PubMed

    Heiat, Mohammad; Ranjbar, Reza; Latifi, Ali Mohammad; Rasaee, Mohammad Javad

    2016-08-01

    Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52±2.44E-10 and 5.87±1.3E-9M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33±1.15E-9 and 4.11±1.09E-9M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P<0.05). They also significantly reduced the serum sodium level and increased the urine volume (P<0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins.

    PubMed

    Pinto, Bernardo I; García, Isaac E; Pupo, Amaury; Retamal, Mauricio A; Martínez, Agustín D; Latorre, Ramón; González, Carlos

    2016-07-22

    Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Computational approach to analyze isolated ssDNA aptamers against angiotensin II.

    PubMed

    Heiat, Mohammad; Najafi, Ali; Ranjbar, Reza; Latifi, Ali Mohammad; Rasaee, Mohammad Javad

    2016-07-20

    Aptamers are oligonucleotides with highly structured molecules that can bind to their targets through specific 3-D conformation. Commonly, not all the nucleotides such as primer binding fixed region and some other sequences are vital for aptamers folding and interaction. Elimination of unnecessary regions needs trustworthy prediction tools to reduce experimental efforts and errors. Here we introduced a manipulated in-silico approach to predict the 3-D structure of aptamers and their target interactions. To design an approach for computational analysis of isolated ssDNA aptamers (FLC112, FLC125 and their truncated core region including CRC112 and CRC125), their secondary and tertiary structures were modeled by Mfold and RNA composer respectively. Output PDB files were modified from RNA to DNA in the discovery studio visualizer software. Using ZDOCK server, the aptamer-target interactions were predicted. Finally, the interaction scores were compared with the experimental results. In-silico interaction scores and the experimental outcomes were in the same descending arrangement of FLC112>CRC125>CRC112>FLC125 with similar intensity. The consistent results of innovative in-silico method with experimental outputs, affirmed that the present method may be a reliable approach. Also, it showed that the exact in-silico predictions can be utilized as a credible reference to find aptameric fragments binding potency. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Transmembrane Mucins: Signaling Receptors at the Intersection of Inflammation and Cancer

    PubMed Central

    van Putten, Jos P.M.; Strijbis, Karin

    2017-01-01

    Mucosal surfaces line our body cavities and provide the interaction surface between commensal and pathogenic microbiota and the host. The barrier function of the mucosal layer is largely maintained by gel-forming mucin proteins that are secreted by goblet cells. In addition, mucosal epithelial cells express cell-bound mucins that have both barrier and signaling functions. The family of transmembrane mucins consists of diverse members that share a few characteristics. The highly glycosylated extracellular mucin domains inhibit invasion by pathogenic bacteria and can form a tight mesh structure that protects cells in harmful conditions. The intracellular tails of transmembrane mucins can be phosphorylated and connect to signaling pathways that regulate inflammation, cell-cell interactions, differentiation, and apoptosis. Transmembrane mucins play important roles in preventing infection at mucosal surfaces, but are also renowned for their contributions to the development, progression, and metastasis of adenocarcinomas. In general, transmembrane mucins seem to have evolved to monitor and repair damaged epithelia, but these functions can be highjacked by cancer cells to yield a survival advantage. This review presents an overview of the current knowledge of the functions of transmembrane mucins in inflammatory processes and carcinogenesis in order to better understand the diverse functions of these multifunctional proteins. PMID:28052300

  8. Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle.

    PubMed

    Rezaei, Hossein B; Kamato, Danielle; Ansari, Ghazaleh; Osman, Narin; Little, Peter J

    2012-08-01

    The transforming growth factor (TGF)-β superfamily of ligands regulates a diverse set of cellular functions. Transforming growth factor-β induces its biological effects through Type I and Type II transmembrane receptors that have serine/threonine kinase activities and weak tyrosine kinase activity. In vascular smooth muscle, TGF-β binds to the TGF-β Type II receptor (TβRII) at the cell surface, recruiting the Type I receptor (TβRI) to form a heterocomplex. Consequently, after phosphorylation and activation of TβRI, the transcription factors receptor activated (R-) Smad2 and Smad3 are recruited and activated through phosphorylation of C terminal residues. Overall, Smad2/3 and co-Smad4 have similar structures consisting of three regions an N-terminal MH1 domain, a C-terminal MH2 domain and a central linker region. Phosphorylation of the Smad linker region appears to have an important role in the regulation of Smad activity and function. The mitogen-activated protein kinase (MAPK) family, CDK2, CDK4 and calcium-calmodulin dependent kinase are the main kinases that phosphorylate sites in the linker region. The role of the linker region includes enabling the formation of Smad homo-oligomers and provision of phosphorylation sites for MAPK and other kinases. In some instances, linker region phosphorylation regulates the inhibition of the nuclear translocation of Smads. In the present review, we describe TGF-β signalling through Smad2/3 and the importance of the linker region in the regulation and expression of genes induced by TGF-β superfamily ligands in the context of vascular smooth muscle. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

  9. [111In-DOTA]LTT-SS28, a first pansomatostatin radioligand for in vivo targeting of somatostatin receptor-positive tumors.

    PubMed

    Maina, Theodosia; Cescato, Renzo; Waser, Beatrice; Tatsi, Aikaterini; Kaloudi, Aikaterini; Krenning, Eric P; de Jong, Marion; Nock, Berthold A; Reubi, Jean Claude

    2014-08-14

    Radiolabeled pansomatostatin-like analogues are expected to enhance the diagnostic sensitivity and to expand the clinical indications of currently applied sst2-specific radioligands. In this study, we present the somatostatin mimic [DOTA]LTT-SS28 {[(DOTA)Ser1,Leu8,D-Trp22,Tyr25]SS28} and its 111In radioligand. [DOTA]LTT-SS28 exhibited a pansomatostatin-like profile binding with high affinity to all five hsst1-hsst5 subtypes (IC50 values in the lower nanomolar range). Furthermore, [DOTA]LTT-SS28 behaved as an agonist at hsst2, hsst3, and hsst5, efficiently stimulating internalization of the three receptor subtypes. Radioligand [111In-DOTA]LTT-SS28 showed good stability in the mouse bloodstream. It displayed strong and specific uptake in AR42J tumors 4 h postinjection (9.3±1.6% ID/g vs 0.3±0.0% ID/g during sst2 blockade) in mice. Significant and specific uptake was also observed in HEK293-hsst2-, HEK293-hsst3-, and HEK293-hsst5-expressing tumors (4.43±1.5, 4.88±1.1, and <3% ID/g, respectively, with values of <0.5% ID/g during receptor blockade). In conclusion, the somatostatin mimic [111In-DOTA]LTT-SS28 specifically localizes in sst2-, sst3-, and sst5-expressing xenografts in mice showing promise for multi-sst1-sst5 targeted tumor imaging.

  10. Dynamic Cooperation of Hydrogen Binding and π Stacking in ssDNA Adsorption on Graphene Oxide.

    PubMed

    Xu, Zhen; Lei, Xiaoling; Tu, Yusong; Tan, Zhi-Jie; Song, Bo; Fang, Haiping

    2017-09-21

    Functional nanoscale structures consisting of a DNA molecule coupled to graphene or graphene oxide (GO) have great potential for applications in biosensors, biomedicine, nanotechnology, and materials science. Extensive studies using the most sophisticated experimental techniques and theoretical methods have still not clarified the dynamic process of single-stranded DNA (ssDNA) adsorbed on GO surfaces. Based on a molecular dynamics simulation, this work shows that an ssDNA segment could be stably adsorbed on a GO surface through hydrogen bonding and π-π stacking interactions, with preferential binding to the oxidized rather than to the unoxidized region of the GO surface. The adsorption process shows a dynamic cooperation adsorption behavior; the ssDNA segment first captures the oxidized groups of the GO surface by hydrogen bonding interaction, and then the configuration relaxes to maximize the π-π stacking interactions between the aromatic rings of the nucleobases and those of the GO surface. We attributed this behavior to the faster forming hydrogen bonding interaction compared to π-π stacking; the π-π stacking interaction needs more relaxation time to regulate the configuration of the ssDNA segment to fit the aromatic rings on the GO surface. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Excessive Counterion Condensation on Immobilized ssDNA in Solutions of High Ionic Strength

    PubMed Central

    Rant, Ulrich; Arinaga, Kenji; Fujiwara, Tsuyoshi; Fujita, Shozo; Tornow, Marc; Yokoyama, Naoki; Abstreiter, Gerhard

    2003-01-01

    We present experiments on the bias-induced release of immobilized, single-stranded (ss) 24-mer oligonucleotides from Au-surfaces into electrolyte solutions of varying ionic strength. Desorption is evidenced by fluorescence measurements of dye-labeled ssDNA. Electrostatic interactions between adsorbed ssDNA and the Au-surface are investigated with respect to 1), a variation of the bias potential applied to the Au-electrode; and 2), the screening effect of the electrolyte solution. For the latter, the concentration of monovalent salt in solution is varied from 3 to 1600 mM. We find that the strength of electric interaction is predominantly determined by the effective charge of the ssDNA itself and that the release of DNA mainly occurs before the electrochemical double layer has been established at the electrolyte/Au interface. In agreement with Manning's condensation theory, the measured desorption efficiency (ηrel) stays constant over a wide range of salt concentrations; however, as the Debye length is reduced below a value comparable to the axial charge spacing of the DNA, ηrel decreases substantially. We assign this effect to excessive counterion condensation on the DNA in solutions of high ionic strength. In addition, the relative translational diffusion coefficient of ssDNA in solution is evaluated for different salt concentrations. PMID:14645075

  12. Excessive counterion condensation on immobilized ssDNA in solutions of high ionic strength.

    PubMed

    Rant, Ulrich; Arinaga, Kenji; Fujiwara, Tsuyoshi; Fujita, Shozo; Tornow, Marc; Yokoyama, Naoki; Abstreiter, Gerhard

    2003-12-01

    We present experiments on the bias-induced release of immobilized, single-stranded (ss) 24-mer oligonucleotides from Au-surfaces into electrolyte solutions of varying ionic strength. Desorption is evidenced by fluorescence measurements of dye-labeled ssDNA. Electrostatic interactions between adsorbed ssDNA and the Au-surface are investigated with respect to 1), a variation of the bias potential applied to the Au-electrode; and 2), the screening effect of the electrolyte solution. For the latter, the concentration of monovalent salt in solution is varied from 3 to 1600 mM. We find that the strength of electric interaction is predominantly determined by the effective charge of the ssDNA itself and that the release of DNA mainly occurs before the electrochemical double layer has been established at the electrolyte/Au interface. In agreement with Manning's condensation theory, the measured desorption efficiency (etarel) stays constant over a wide range of salt concentrations; however, as the Debye length is reduced below a value comparable to the axial charge spacing of the DNA, etarel decreases substantially. We assign this effect to excessive counterion condensation on the DNA in solutions of high ionic strength. In addition, the relative translational diffusion coefficient of ssDNA in solution is evaluated for different salt concentrations.

  13. Structure elucidation of dimeric transmembrane domains of bitopic proteins.

    PubMed

    Bocharov, Eduard V; Volynsky, Pavel E; Pavlov, Konstantin V; Efremov, Roman G; Arseniev, Alexander S

    2010-01-01

    The interaction between transmembrane helices is of great interest because it directly determines biological activity of a membrane protein. Either destroying or enhancing such interactions can result in many diseases related to dysfunction of different tissues in human body. One much studied form of membrane proteins known as bitopic protein is a dimer containing two membrane-spanning helices associating laterally. Establishing structure-function relationship as well as rational design of new types of drugs targeting membrane proteins requires precise structural information about this class of objects. At present time, to investigate spatial structure and internal dynamics of such transmembrane helical dimers, several strategies were developed based mainly on a combination of NMR spectroscopy, optical spectroscopy, protein engineering and molecular modeling. These approaches were successfully applied to homo- and heterodimeric transmembrane fragments of several bitopic proteins, which play important roles in normal and in pathological conditions of human organism.

  14. A Genome-Wide Association Study Identifies Genomic Regions for Virulence in the Non-Model Organism Heterobasidion annosum s.s

    PubMed Central

    Dalman, Kerstin; Himmelstrand, Kajsa; Olson, Åke; Lind, Mårten; Brandström-Durling, Mikael; Stenlid, Jan

    2013-01-01

    The dense single nucleotide polymorphisms (SNP) panels needed for genome wide association (GWA) studies have hitherto been expensive to establish and use on non-model organisms. To overcome this, we used a next generation sequencing approach to both establish SNPs and to determine genotypes. We conducted a GWA study on a fungal species, analysing the virulence of Heterobasidion annosum s.s., a necrotrophic pathogen, on its hosts Picea abies and Pinus sylvestris. From a set of 33,018 single nucleotide polymorphisms (SNP) in 23 haploid isolates, twelve SNP markers distributed on seven contigs were associated with virulence (P<0.0001). Four of the contigs harbour known virulence genes from other fungal pathogens and the remaining three harbour novel candidate genes. Two contigs link closely to virulence regions recognized previously by QTL mapping in the congeneric hybrid H. irregulare × H. occidentale. Our study demonstrates the efficiency of GWA studies for dissecting important complex traits of small populations of non-model haploid organisms with small genomes. PMID:23341945

  15. Crystal structure of the complete integrin αVβ3 ectodomain plus an α/β transmembrane fragment

    PubMed Central

    Xiong, Jian-Ping; Mahalingham, Bhuvaneshwari; Alonso, Jose Luis; Borrelli, Laura Ann; Rui, Xianliang; Anand, Saurabh; Hyman, Bradley T.; Rysiok, Thomas; Müller-Pompalla, Dirk; Goodman, Simon L.

    2009-01-01

    We determined the crystal structure of 1TM-αVβ3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the αV and β3 subunits. 1TM-αVβ3 is more compact and less active in solution when compared with ΔTM-αVβ3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the α–β interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length αVβ3 on live cells yields a donor–membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2–thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state. PMID:19704023

  16. Novel Immunocytokine IL12-SS1 (Fv) Inhibits Mesothelioma Tumor Growth in Nude Mice

    PubMed Central

    Kim, Heungnam; Gao, Wei; Ho, Mitchell

    2013-01-01

    Mesothelin is a glycosylphosphatidylinositol-anchored glycoprotein that is highly expressed on the cell surface of malignant mesothelioma. Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma. Immunocytokines represent a novel class of armed antibodies. To provide an alternative approach to current mesothelin-targeted antibody therapies, we have developed a novel immunocytokine based on interleukin-12 (IL12) and the SS1 Fv specific for mesothelin. IL12 possesses potent anti-tumor activity in a wide variety of solid tumors. The newly-developed recombinant immunocytokine, IL12-SS1 (Fv), was produced in insect cells using a baculovirus-insect cell expression system. The SS1 single-chain Fv was fused to the C terminus of the p35 subunit of IL12 through a short linker (GSADGG). The single-chain IL12-SS1 (Fv) immunocytokine bound native mesothelin proteins on malignant mesothelioma (NCI-H226) and ovarian (OVCAR-3) cells as well as recombinant mesothelin on A431/H9 cells. The immunocytokine retained sufficient bioactivity of IL12 and significantly inhibited human malignant mesothelioma (NCI-H226) grown in the peritoneal cavity of nude mice and showed comparable anti-tumor activity to that of the SS1P immunotoxin. IL12-SS1 (Fv) is the first reported immunocytokine to mesothelin-positive tumors and may be an attractive addition to mesothelin-targeted cancer therapies. PMID:24260587

  17. Installation Restoration Program. Volume 3. 152nd Tactical Reconnaissance Group, Nevada Air National Guard, Reno Cannon International Airport, Reno, Nevada

    DTIC Science & Technology

    1994-04-01

    N F-67 PROJECTt REINO AZR NATIONAL GUARD DATE:03/25/94 ANALYSIS: BNA - CONTANINATZON REPORT REVIEWER: DENNIS MART27...10SS 0.569 0.7021 -23.4 T 12/14/92 2259 CRIoemmA 3 loSS 0.027 1.011 -22.2 T 12/14/92 2259 DIO 1055 l 0.703 .0.11 -22.5 T 12/14/92 2259 NTUL= CULORIDS...seetma. rule VOL DIo -U-T...z..-- 1064 1055 Perocnt D in the Coatcal ezood limits. wa 1064 10ss The ouaes.. does not met 52/102 no contemin. rule NIT

  18. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  19. Re Effects on Phase Stability and Mechanical Properties of MoSS+Mo3Si+Mo5SiB2 alloys

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

    Yang, Ying; Bei, Hongbin; George, Easo P

    2013-01-01

    Because of their high melting points and good oxidation resistance Mo-Si-B alloys are of interest as potential ultrahigh-temperature structural materials. But their major drawbacks are poor ductility and fracture toughness at room temperature. Since alloying with Re has been suggested as a possible solution, we investigate here the effects of Re additions on the microstructure and mechanical properties of a ternary alloy with the composition Mo-12.5Si-8.5B (at.%). This alloy has a three-phase microstructure consisting of Mo solid-solution (MoSS), Mo3Si, and Mo5SiB2 and our results show that up to 8.4 at.% Re can be added to it without changing its microstructuremore » or forming any brittle phase at 1600 C. Three-point bend tests using chevron-notched specimens showed that Re did not improve fracture toughness of the three-phase alloy. Nanoindentation performed on the MoSS phase in the three-phase alloy showed that Re increases Young s modulus, but does not lower hardness as in some Mo solid solution alloys. Based on our thermodynamic calculations and microstructural analyses, the lack of a Re softening effect is attributed to the increased Si levels in the Re-containing MoSS phase since Si is known to increase its hardness. This lack of softening is possibly why there is no Re-induced improvement in fracture toughness.« less

  20. Light-induced exposure of the cytoplasmic end of transmembrane helix seven in rhodopsin

    PubMed Central

    Abdulaev, Najmoutin G.; Ridge, Kevin D.

    1998-01-01

    A key step in signal transduction in the visual cell is the light-induced conformational change of rhodopsin that triggers the binding and activation of the guanine nucleotide-binding protein. Site-directed mAbs against bovine rhodopsin were produced and used to detect and characterize these conformational changes upon light activation. Among several antibodies that bound exclusively to the light-activated state, an antibody (IgG subclass) with the highest affinity (Ka ≈ 6 × 10−9 M) was further purified and characterized. The epitope of this antibody was mapped to the amino acid sequence 304–311. This epitope extends from the central region to the cytoplasmic end of the seventh transmembrane helix and incorporates a part of a highly conserved NPXXY motif, a critical region for signaling and agonist-induced internalization of several biogenic amine and peptide receptors. In the dark state, no binding of the antibody to rhodopsin was detected. Accessibility of the epitope to the antibody correlated with formation of the metarhodopsin II photointermediate and was reduced significantly at the metarhodopsin III intermediate. Further, incubation of the antigen–antibody complex with 11-cis-retinal failed to regenerate the native rhodopsin chromophore. These results suggest significant and reversible conformational changes in close proximity to the cytoplasmic end of the seventh transmembrane helix of rhodopsin that might be important for folding and signaling. PMID:9789004

  1. Automatic regional analysis of myocardial native T1 values: left ventricle segmentation and AHA parcellations.

    PubMed

    Huang, Hsiao-Hui; Huang, Chun-Yu; Chen, Chiao-Ning; Wang, Yun-Wen; Huang, Teng-Yi

    2018-01-01

    Native T1 value is emerging as a reliable indicator of abnormal heart conditions related to myocardial fibrosis. Investigators have extensively used the standardized myocardial segmentation of the American Heart Association (AHA) to measure regional T1 values of the left ventricular (LV) walls. In this paper, we present a fully automatic system to analyze modified Look-Locker inversion recovery images and to report regional T1 values of AHA segments. Ten healthy individuals participated in the T1 mapping study with a 3.0 T scanner after providing informed consent. First, we obtained masks of an LV blood-pool region and LV walls by using an image synthesis method and a layer-growing method. Subsequently, the LV walls were divided into AHA segments by identifying the boundaries of the septal regions and by using a radial projection method. The layer-growing method significantly enhanced the accuracy of the derived myocardium mask. We compared the T1 values that were obtained using manual region of interest selections and those obtained using the automatic system. The average T1 difference of the calculated segments was 4.6 ± 1.5%. This study demonstrated a practical and robust method of obtaining native T1 values of AHA segments in LV walls.

  2. SynBioSS-aided design of synthetic biological constructs.

    PubMed

    Kaznessis, Yiannis N

    2011-01-01

    We present walkthrough examples of using SynBioSS to design, model, and simulate synthetic gene regulatory networks. SynBioSS stands for Synthetic Biology Software Suite, a platform that is publicly available with Open Licenses at www.synbioss.org. An important aim of computational synthetic biology is the development of a mathematical modeling formalism that is applicable to a wide variety of simple synthetic biological constructs. SynBioSS-based modeling of biomolecular ensembles that interact away from the thermodynamic limit and not necessarily at steady state affords for a theoretical framework that is generally applicable to known synthetic biological systems, such as bistable switches, AND gates, and oscillators. Here, we discuss how SynBioSS creates links between DNA sequences and targeted dynamic phenotypes of these simple systems. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Predicting protein aggregation during storage in lyophilized solids using solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS).

    PubMed

    Moorthy, Balakrishnan S; Schultz, Steven G; Kim, Sherry G; Topp, Elizabeth M

    2014-06-02

    Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products.

  4. Predicting Protein Aggregation during Storage in Lyophilized Solids Using Solid State Amide Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)

    PubMed Central

    2015-01-01

    Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products. PMID:24816133

  5. Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT, a Member of the l-Amino Acid Transporter (LAT) Family*

    PubMed Central

    Bartoccioni, Paola; del Rio, César; Ratera, Merce; Kowalczyk, Lukasz; Baldwin, Jocelyn M.; Zorzano, Antonio; Quick, Matthias; Baldwin, Stephen A.; Vázquez-Ibar, José Luis; Palacín, Manuel

    2010-01-01

    System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC50 similar to the apparent KM of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT. PMID:20610400

  6. Upper Mantle Discontinuity Structure Beneath the Western Atlantic Ocean and Eastern North America from SS Precursors

    NASA Astrophysics Data System (ADS)

    Schmerr, N. C.; Beghein, C.; Kostic, D.; Baldridge, A. M.; West, J. D.; Nittler, L. R.; Bull, A. L.; Montesi, L.; Byrne, P. K.; Hummer, D. R.; Plescia, J. B.; Elkins-Tanton, L. T.; Lekic, V.; Schmidt, B. E.; Elkins, L. J.; Cooper, C. M.; ten Kate, I. L.; Van Hinsbergen, D. J. J.; Parai, R.; Glass, J. B.; Ni, J.; Fuji, N.; McCubbin, F. M.; Michalski, J. R.; Zhao, C.; Arevalo, R. D., Jr.; Koelemeijer, P.; Courtier, A. M.; Dalton, H.; Waszek, L.; Bahamonde, J.; Schmerr, B.; Gilpin, N.; Rosenshein, E.; Mach, K.; Ostrach, L. R.; Caracas, R.; Craddock, R. A.; Moore-Driskell, M. M.; Du Frane, W. L.; Kellogg, L. H.

    2015-12-01

    Seismic discontinuities within the mantle arise from a wide range of mechanisms, including changes in mineralogy, major element composition, melt content, volatile abundance, anisotropy, or a combination of the above. In particular, the depth and sharpness of upper mantle discontinuities at 410 and 660 km depth are attributed to solid-state phase changes sensitive to both mantle temperature and composition, where regions of thermal heterogeneity produce topography and chemical heterogeneity changes the impedance contrast across the discontinuity. Seismic mapping of this topography and sharpness thus provides constraint on the thermal and compositional state of the mantle. The EarthScope USArray is providing unprecedented access to a wide variety of new regions previously undersampled by the SS precursors. This includes the boundary between the oceanic plate in the western Atlantic Ocean and continental margin of eastern North America. Here we use a seismic array approach to image the depth, sharpness, and topography of the upper mantle discontinuities, as well as other possible upper mantle reflectors beneath this region. This array approach utilizes seismic waves that reflect off the underside of a mantle discontinuity and arrive several hundred seconds prior to the SS seismic phase as precursory energy. In this study, we collected high-quality broadband data SS precursors data from shallow focus (< 30 km deep), mid-Atlantic ridge earthquakes recorded by USArray seismometers in Alaska. We generated 4th root vespagrams to enhance the SS precursors and determine how they sample the mantle. Our data show detection of localized structure on the discontinuity boundaries as well as additional horizons, such as the X-discontinuity and a potential reflection from a discontinuity near the depth of the lithosphere-asthenosphere boundary. These structures are related to the transition from predominantly old ocean lithosphere to underlying continental lithosphere, as while

  7. Critical analysis of the discrepancy between V(beta) and V(ss) for drugs exhibiting different two-compartment disposition profiles.

    PubMed

    Sobol, Eyal; Bialer, Meir

    2005-03-01

    It is well known that in the two-compartment open body model the values of apparent volume of distribution (V(beta)) and volume of distribution at steady state (V(ss)) are never identical. There are at least two conditions when V(beta) significantly overestimates V(ss). The first is when most of a drug is eliminated relatively rapidly but a small fraction of the dose persists and gives rise to an extremely long half-life. The second is when a drug is rapidly cleared from the central compartment with a short half-life. The primary purpose of the current paper was to investigate how different two-compartment disposition profiles affect the magnitude of difference between V(beta) and V(ss). Novel equations have been developed that relate the V(beta)/V(ss) ratio to f1 (fraction of drug elimination associated with the distributive phase) and to beta/alpha (ratio of the exponential coefficients). This paper demonstrates mathematically that an increasing value of f1 is associated with a greater divergence between V(beta) and V(ss). A similar relationship was also found for the divergence between the terminal half-life (t(1/2beta)) and the mean residence time (MRT). An increase in the beta/alpha ratio results in a substantial decrease of this discrepancy and provides a maximal possible value, or an upper limit to the V(beta)/V(ss) ratio. The newly derived equations along with their graphical presentation may serve as an excellent predictive tool for checking the accuracy of the experimentally obtained values of V(beta) and V(ss). Copyright 2004 John Wiley & Sons, Ltd.

  8. CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

    PubMed Central

    Zhang, Xiaolong; Chang Li, Xian; Xiao, Xiang; Sun, Rui; Tian, Zhigang; Wei, Haiming

    2013-01-01

    The peripheral Foxp3+ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4+ T cells can be readily converted to Foxp3+ iTreg in vitro, and memory CD4+ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3+ T cells from various CD4+ T-cell subsets in human peripheral blood. Though naive CD4+ T cells were readily converted to Foxp3+ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3+ T cells did not express the memory marker CD45RO as do Foxp3+ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4+ T cells, defined as CD62L+ central memory T cells, could be induced by TGF-β to differentiate into Foxp3+ T cells. It is well known that Foxp3+ T cells derived from human CD4+CD25- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4+CD62L+ central memory T cell-derived Foxp3+ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4+ T cell-derived Foxp3+ T cells. But further research showed that mouse CD4+ central memory T cells also could be induced to differentiate into Foxp3+ T cells, such Foxp3+ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4+CD62L+ central memory T cells as a novel potential source of iTreg. PMID:24155942

  9. Controllable g5p-Protein-Directed Aggregation of ssDNA-Gold Nanoparticles

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

    Lee, S.; Maye, M; Zhang, Y

    We assembled single-stranded DNA (ssDNA) conjugated nanoparticles using the phage M13 gene 5 protein (g5p) as the molecular glue to bind two antiparallel noncomplementary ssDNA strands. The entire process was controlled tightly by the concentration of the g5p protein and the presence of double-stranded DNA. The g5p-ssDNA aggregate was disintegrated by hybridization with complementary ssDNA (C-ssDNA) that triggers the dissociation of the complex. Polyhistidine-tagged g5p was bound to nickel nitrilotriacetic acid (Ni2+-NTA) conjugated nanoparticles and subsequently used to coassemble the ssDNA-conjugated nanoparticles into multiparticle-type aggregates. Our approach offers great promise for designing biologically functional, controllable protein/nanoparticle composites.

  10. Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes

    PubMed Central

    Connell, Jennifer Petsche; Augustini, Emily; Moise, Kenneth J; Johnson, Anthony; Jacot, Jeffrey G

    2013-01-01

    Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC. PMID:23634988

  11. Duck Interferon-Inducible Transmembrane Protein 3 Mediates Restriction of Influenza Viruses.

    PubMed

    Blyth, Graham A D; Chan, Wing Fuk; Webster, Robert G; Magor, Katharine E

    2016-01-01

    Interferon-inducible transmembrane proteins (IFITMs) can restrict the entry of a wide range of viruses. IFITM3 localizes to endosomes and can potently restrict the replication of influenza A viruses (IAV) and several other viruses that also enter host cells through the endocytic pathway. Here, we investigate whether IFITMs are involved in protection in ducks, the natural host of influenza virus. We identify and sequence duck IFITM1, IFITM2, IFITM3, and IFITM5. Using quantitative PCR (qPCR), we demonstrate the upregulation of these genes in lung tissue in response to highly pathogenic IAV infection by 400-fold, 30-fold, 30-fold, and 5-fold, respectively. We express each IFITM in chicken DF-1 cells and show duck IFITM1 localizes to the cell surface, while IFITM3 localizes to LAMP1-containing compartments. DF-1 cells stably expressing duck IFITM3 (but not IFITM1 or IFITM2) show increased restriction of replication of H1N1, H6N2, and H11N9 IAV strains but not vesicular stomatitis virus. Although duck and human IFITM3 share only 38% identity, critical residues for viral restriction are conserved. We generate chimeric and mutant IFITM3 proteins and show duck IFITM3 does not require its N-terminal domain for endosomal localization or antiviral function; however, this N-terminal end confers endosomal localization and antiviral function on IFITM1. In contrast to mammalian IFITM3, the conserved YXXθ endocytosis signal sequence in the N-terminal domain of duck IFITM3 is not essential for correct endosomal localization. Despite significant structural and amino acid divergence, presumably due to host-virus coevolution, duck IFITM3 is functional against IAV. Immune IFITM genes are poorly conserved across species, suggesting that selective pressure from host-specific viruses has driven this divergence. We wondered whether coevolution between viruses and their natural host would result in the evasion of IFITM restriction. Ducks are the natural host of avian influenza A viruses

  12. Administration of antioxidant peptide SS-31 attenuates transverse aortic constriction-induced pulmonary arterial hypertension in mice.

    PubMed

    Lu, Hung-I; Huang, Tien-Hung; Sung, Pei-Hsun; Chen, Yung-Lung; Chua, Sarah; Chai, Han-Yan; Chung, Sheng-Ying; Liu, Chu-Feng; Sun, Cheuk-Kwan; Chang, Hsueh-Wen; Zhen, Yen-Yi; Lee, Fan-Yen; Yip, Hon-Kan

    2016-05-01

    Antioxidant peptide SS-31 is a class of cell-permeable small peptides, which selectively resides on the inner mitochondrial membrane and possesses intrinsic mitochondrial protective capacities. In this study we investigated the therapeutic effects of antioxidant peptide SS-31 on transverse aortic constriction (TAC)-induced pulmonary arterial hypertension (PAH) in a murine model. Adult male mice were divided into 3 groups: sham-operated mice, TAC mice, and TAC+SS-31 mice that underwent TAC surgery and received SS-31 (2 mg/d, ip) for 60 d. The right ventricular systolic blood pressure (RVSBP) was measured on d 60 prior to sacrificing the mice; then their right heart and lung tissues were collected for histological and biochemical examinations. Lung injury scores were defined by the increased crowded area and decreased number of alveolar sacs. TAC mice showed significantly higher RVSBP compared with sham-operated mice, the elevation was substantially suppressed in TAC+SS-31 mice. The same pattern of changes was found in pulmonary levels of oxidative stress proteins (NOX-1/NOX-2/oxidized proteins), cytosolic cytochrome c, biomarkers related to inflammation (MMP-9/TNF-α/iNOS), calcium overload index (TRPC1, 2, 4, 6), apoptosis (mitochondrial BAX, cleaved caspase 3/PARP), fibrosis (Smad3/TGF-β), hypoxic (HIF-1α), DNA damage (γ-H2AX) and endothelial function (eNOS/ET-1R), as well as in lung injury score, number of muscularized vessels in lungs, number of TRPC1(+) and HIF-1α(+) cells in pulmonary artery, and number of γ-H2AX(+) and Ki-67(+) cells in lung parenchyma. An opposite pattern of changes was observed in pulmonary anti-fibrotic markers (Smad1/5, BMP-2), number of small vessels, and number of alveolar sacs. In contrast, the levels of antioxidant proteins (HO-1/NQO-1/GR/GPx) in lung parenchyma were progressively and significantly increased from sham-operated mice, TAC mice to TAC+SS-31 mice. Antioxidant peptide SS-31 administration effectively attenuates TAC

  13. SS Edgar M. Queeny collision with the Liberian S/T Corinthos, Marcus Hook, Pennsylvania, 31 January 1975. Marine casualty report

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

    Not Available

    1977-10-27

    At 0029, on January 31, 1975, the U.S. Registered tankship SS EDGAR M. QUEENY, laden with chemical and petroleum products, was maneuvering into Marcus Hook channel of the Delaware River in Pennsylvania, when it collided with the Liberian tanker S/T CORINTHOS which was moored and discharging a bulk cargo of crude oil at the British Petroleum Company dock. The port anchor of the QUEENY slightly penetrated the port side plating of the CORINTHOS at an angle of about 39 deg. into one or more of the wing cargo tanks, which were being pumped and were approximately half full. Almost immediately,more » a series of increasingly intense explosions began in the CORINTHOS, and the vessel was engulfed in flames. Twenty-six persons were killed or are missing and 11 were injured in this accident. The QUEENY suffered minor damage but the CORINTHOS was destroyed. The Delaware River was polluted by oil about Marcus Hook. Property damage was estimated to be $20 million.« less

  14. When a transmembrane channel isn't, or how biophysics and biochemistry (mis)communicate.

    PubMed

    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.

  15. The meaning behind observed pT regions at the LHC energies

    NASA Astrophysics Data System (ADS)

    Suleymanov, M.

    We argue that pT distribution data from the LHC on the invariant differential yield of the charged primary particles in pp collisions at s = 0.9TeV, 2.76TeV, 7TeV and in Pb-Pb collisions at sNN = 2.76TeV with six centrality bins contains several pT regions with special properties. These distributions were analyzed by fitting the data with exponential functions. We conclude that the regions reflect features of fragmentation and hadronization of partons through the string dynamics. The nuclear transparency results in negligible influence of the medium in the III region (pT > 17-20GeV/c), which has highest pT values. The effects and changes by the medium start to appear weakly in the II region (4-6GeV/c < pT < 17-20GeV/c) and become stronger in the I region (pT < 4-6GeV/c). It seems that the II region has highest number of strings. The increase in string density in this region could lead to fusion of strings, appearance of a new string and collective behavior of the partons in the most central collisions. These phenomena can explain anomalous behavior of the Nuclear Modification Factor in the II region. We propose the II region as a possible area of Quark Gluon Plasma formation through string fusion. The first pT regions are the ones with the maximum number of hadrons and minimum number of strings due to direct hadronization of the low energy strings into two quark systems-mesons.

  16. Membrane shape modulates transmembrane protein distribution.

    PubMed

    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.

  17. Perspectives of the KM3NeT project

    NASA Astrophysics Data System (ADS)

    Margiotta, A.; KM3NeT Collaboration

    2016-10-01

    KM3NeT is a large distributed research infrastructure that comprises a network of deep-sea neutrino telescopes in the Mediterranean Sea with user ports for Earth and Sea sciences. The main objectives of KM3NeT are the discovery and subsequent observation of high-energy neutrino sources in the Universe (ARCA) and the determination of the mass hierarchy of neutrinos (ORCA). Technically, the network of telescopes will consist of building blocks of 115 vertical detection units anchored at the seabed and connected to shore via a deep sea electro-optical cable. Each detection unit carries 18 optical modules equipped with 31 3; photomultipliers. Two configurations for the building blocks are defined to optimally detect neutrinos in different ranges of energy. The modular technical design of the KM3NeT telescope allows for a progressive implementation and for data taking even with an incomplete detector. The first phase of implementation has started. The next phase foresees the installation of three building blocks: two building blocks, for a total instrumented volume of 1 km3 (ARCA), at the KM3NeT-It site, at a depth of 3500 m, about 100 km offshore Capo Passero, Sicily. The main scientific goals of the ARCA detector is the exploration of the neutrino sky with unprecedented resolution, searching for neutrinos coming from defined sources or sky regions, like the Galactic Plane. It will also look for diffuse high energy neutrino fluxes following the indication provided by the IceCube signal. The third building block, with a more compact distribution of the optical modules, will be deployed at the KM3NeT-Fr site, 40 km offshore Toulon at a depth of 2500 m (ORCA). The main objective of ORCA is studying the neutrino mass-hierarchy problem and exploring the low energy region of the spectrum. The status of the first phase of the KM3NeT implementation is described and a survey of the physics potentiality of the telescope is given in this contribution, with particular emphasis on

  18. Comparison of FSE T2 W PROPELLER and 3D-FIESTA of 3 T MR for the internal auditory canal.

    PubMed

    Wu, Hai-Bo; Yuan, Hui-Shu; Ma, Furong; Zhao, Qiang

    The study compared the use of periodically rotated overlapping parallel lines with enhanced reconstruction (PROPELLER) technique fast spin echo (FSE) T2 W and the sequence of three-dimensional fast imaging employing steady-state acquisition (3D-FIESTA) technique in the MRI of the internal auditory canal for overall image quality improvement. One hundred thirty-two patients undergoing FSE T2 W PROPELLER and 3D-FIESTA examinations of the internal auditory canal were included. All examinations were performed at 3.0 T with comparison of a sagittal oblique FSE T2 W sequence with the PROPELLER technique to 3D-FIESTA in the same reconstructed orientation with PROPELLER. Image quality was evaluated by two radiologists using a 4-point scale. The Wilcoxon signed rank test was used to compare the data of the two techniques. The image quality of FSE T2 W PROPELLER was significantly improved compared to the reconstructed images of 3D-FIESTA. Observer 1: median FSE T2 W with PROPELLER, 4 [mean, 3.455] versus median reconstructed 3D-FIESTA, 3 [mean, 3.15], (P<.001); Observer 2: median FSE T2 W with PROPELLER, 4 [mean, 3.47] versus median reconstructed 3D-FIESTA, 3 [mean, 3.25], (P<.001). Interobserver agreement was good (k value, 0.73) for the rating of the overall image quality. The FSE T2 W PROPELLER technique for MRI of internal auditory canal reduced uncertainty caused by motion artifact and improved the quality of the image compared to the reconstructed 3D-FIESTA. It was affected by different parameters including the blade width, echo train length (ETL). This is explained by data oversampling at the center region of k-space, which requires additional imaging time over conventional MRI techniques. Increasing blade was expected to improve motion correction effects but also the signal-to-noise ratio. ETL increases the image sharpness and the overall image quality. Copyright © 2016. Published by Elsevier Inc.

  19. Interfacial Interaction between Transmembrane Ocular Mucins and Adhesive Polymers and Dendrimers Analyzed by Surface Plasmon Resonance

    PubMed Central

    Noiray, M.; Briand, E.; Woodward, A. M.; Argüeso, P.; Molina Martínez, I. T.; Herrero-Vanrell, R.; Ponchel, G.

    2013-01-01

    Purpose Development of the first in vitro method based on biosensor chip technology designed for probing the interfacial interaction phenomena between transmembrane ocular mucins and adhesive polymers and dendrimers intended for ophthalmic administration. Methods The surface plasmon resonance (SPR) technique was used. A transmembrane ocular mucin surface was prepared on the chip surface and characterized by QCM-D (Quartz Crystal Microbalance with Dissipation) and XPS (X-ray photoelectron spectroscopy). The mucoadhesive molecules tested were: hyaluronic acid (HA), carboxymethyl cellulose (CMC), hydroxypropylmethyl cellulose (HPMC), chitosan (Ch) and polyamidoamine dendrimers (PAMAM). Results While Ch originated interfacial interaction with ocular transmembrane mucins, for HA, CMC and HPMC, chain interdiffusion seemed to be mandatory for bioadherence at the concentrations used in ophthalmic clinical practise. Interestingly, PAMAM dendrimers developed permanent interfacial interactions with transmembrane ocular mucins whatever their surface chemical groups, showing a relevant importance of co-operative effect of these multivalent systems. Polymers developed interfacial interactions with ocular membrane-associated mucins in the following order: Ch(1 %) > G4PAMAM-NH2(2 %) = G4PAMAM-OH(2 %) > G3.5PAMAM-COOH(2 %)≫ CMC(0.5 %) = HA(0.2 %) = HPMC(0.3 %). Conclusions The method proposed is useful to discern between the mucin-polymer chemical interactions at molecular scale. Results reinforce the usefulness of chitosan and den-drimers as polymers able to increase the retention time of drugs on the ocular surface and hence their bioavailability. PMID:22565639

  20. Network Management and FDIR for SpaceWire Networks (N-MaSS)

    NASA Astrophysics Data System (ADS)

    Montano, Giuseppe; Jameux, David; Cook, Barry; Peel, Rodger; McCormick, Ecaterina; Walker, Paul; Kollias, Vangelis; Pogkas, Nikos

    2014-08-01

    The SpaceWire network management layer, which manages network topology and routing, is not yet standardised. This paper presents the European Space Agency (ESA) N-MaSS study, which focuses on implementation and standardisation of Fault Detection, Isolation and Recovery (FDIR) functions within the SpaceWire network management layer. N-MaSS provides an autonomous FDIR solution. It is defined at the SpaceWire network layer in order to achieve efficient re-use for heterogeneous missions, allowing for the incorporation of legacy equipment. The N-MaSS FDIR functions identify SpaceWire link and node failures and provide recovery using redundant nodes.This paper provides an overview of the overall N- MaSS study. In particular, the following topics are discussed: (a) how user requirements have been captured from the industry, SpaceWire Working Group and ESA; (b) how the N-MaSS architecture was organically shaped on the basis of the requirements captured; (c) how the N-MaSS concept is currently being implemented in a demonstrator and verified.

  1. Participation of primary motor cortex area 4a in complex sensory processing: 3.0-T fMRI study.

    PubMed

    Terumitsu, Makoto; Ikeda, Kotaro; Kwee, Ingrid L; Nakada, Tsutomu

    2009-05-06

    The precise movement of human fingers requires continuous and reciprocal interaction between motor and sensory systems. Similar to other primates, there is double representation of the digits and wrists within the human primary motor cortex (M1), which are generally referred to as area 4 anterior (M1-4a) and area 4 posterior (M1-4p). In this high-field (3.0 T) functional magnetic resonance imaging (fMRI) study, we hypothesized that M1-4p is more important for initiation of motion, whereas M1-4a is important for execution of a given motion involving more complex sensoriomotor interaction. We investigated M1-4a and M1-4p activation associated with two representative motor tasks, namely, finger tapping (voluntary motion, VM) and passive finger movement accomplished by continuous pressure (passive motor, PM), and two representative sensory stimulations, namely, simple stimulation of flutter vibration (simple sensory, SS), and complex stimulation by a row of pins moving either vertically or horizontally (complex sensory, CS). Both M1-4a and M1-4p were activated in both motor tasks, VM and PM. M1-4p was not activated by either of the two sensory tasks, whereas M1-4a was activated by CS but not by SS. Analysis of the center of gravities (COG) of the activated areas showed that VM and PM moved COG towards M1-4p and 3a. SS moved COG towards somatosensory cortex Brodmann areas 1, 2, and 3b, whereas CS towards M1-4a. The result clearly showed that M1-4a represents the area of secondary motor execution, which actively participates in CS processing.

  2. Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

    PubMed

    Iwamoto, T; Grove, A; Montal, M O; Montal, M; Tomich, J M

    1994-06-01

    A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

  3. Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity.

    PubMed

    Saether, Per C; Hoelsbrekken, Sigurd E; Fossum, Sigbjørn; Dissen, Erik

    2011-12-15

    Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.

  4. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

    2015-05-01

    Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

  5. Mapping and characterization of the major quantitative trait locus qSS7 associated with increased length and decreased width of rice seeds.

    PubMed

    Qiu, Xianjin; Gong, Rong; Tan, Youbin; Yu, Sibin

    2012-12-01

    Seed shape in rice (Oryza sativa) is an important factor that determines grain appearance, cooking quality and grain yield. Here, we report a major quantitative trait locus qSS7 on the long arm of chromosome 7 for seed length, seed width and the ratio of seed length to width, identified using a segregating population derived from a cross between an indica variety Zhenshan97 and a chromosomal segment substitution line of a japonica variety Cypress within the genetic background of Zhenshan97. The Cypress allele at qSS7 contributes to an increase in seed length and the ratio of length to width, but a decrease in seed width, without significantly changing seed weight, plant height, heading date or number of spikelets per panicle. Using a large F(2) population generated from a substitution line that carries only a heterozygous single segment surrounding qSS7, we delimited the QTL to a 23-kb region containing two annotated genes. Progeny testing of the informative recombinants suggested that this qSS7 region is a composite QTL in which at least two genes contribute to seed length and width. Sequence comparison and expression analysis of two probable candidate genes revealed differences between the parental lines. These results will facilitate cloning of the gene(s) underlying qSS7 as well as marker-assisted transfer of desirable genes for seed shape in rice improvement.

  6. Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding

    DTIC Science & Technology

    2008-12-23

    glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain... peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the...terminal signal peptide (SP), which directs the precursor to the endoplasmic retic- ulum (ER) for further processing [11]. The SP, which has been

  7. Discovery of Plant Phenolic Compounds That Act as Type III Secretion System Inhibitors or Inducers of the Fire Blight Pathogen, Erwinia amylovora

    PubMed Central

    Khokhani, Devanshi; Zhang, Chengfang; Li, Yan; Wang, Qi; Zeng, Quan; Yamazaki, Akihiro; Hutchins, William; Zhou, Shan-Shan

    2013-01-01

    Erwinia amylovora causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by E. amylovora in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer, trans-2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the rsmBEa-RsmAEa system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of E. amylovora. In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens. PMID:23770912

  8. Discovery of plant phenolic compounds that act as type III secretion system inhibitors or inducers of the fire blight pathogen, Erwinia amylovora.

    PubMed

    Khokhani, Devanshi; Zhang, Chengfang; Li, Yan; Wang, Qi; Zeng, Quan; Yamazaki, Akihiro; Hutchins, William; Zhou, Shan-Shan; Chen, Xin; Yang, Ching-Hong

    2013-09-01

    Erwinia amylovora causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by E. amylovora in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer, trans-2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the rsmBEa-RsmAEa system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of E. amylovora. In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.

  9. A Mutation in the Start Codon of γ-Crystallin D Leads to Nuclear Cataracts in the Dahl SS/Jr-Ctr Strain

    PubMed Central

    Johnson, Ashley C.; Lee, Jonathan W.; Harmon, Ashlyn C.; Morris, Zaliya; Wang, Xuexiang; Fratkin, Jonathan; Rapp, John P.; Gomez-Sanchez, Elise; Garrett, Michael R.

    2013-01-01

    Cataracts are a major cause of blindness. The most common forms of cataracts are age and UV related and develops mostly in the elderly, while congenital cataracts appear at birth or in early childhood. The Dahl salt-sensitive (SS/Jr) rat is an extensively used model of salt-sensitive hypertension that exhibits concomitant renal disease. In the mid 1980’s, cataracts appeared in a few animals in the Dahl S colony, presumably the result of a spontaneous mutation. The mutation was fixed and bred to establish the SS/Jr-Ctr substrain. The SS/Jr-Ctr substrain has been exclusively used by a single investigator to study the role of steroids and hypertension. Using a classical positional cloning approach, we localized the cataract gene with high-resolution to a less than 1 Mbp region on chromosome 9 using an F1 (SS/Jr-Ctr X SHR) X SHR backcross population. The 1 Mbp region contained only 13 genes, including 4 genes from the γ-crystallins (Cryg) gene family which are known to play a role in cataract formation. All of the γ-crystallins were sequenced and a novel point mutation in the start codon (ATG → GTG) of the Crygd gene was identified which led to the complete absence of CRYGD protein in the eyes of the SS/Jr-Ctr strain. In summary, the identification of the genetic cause in this novel cataract model may provide an opportunity to better understand the development of cataracts, particularly in the context of hypertension. PMID:23404175

  10. Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis *

    PubMed Central

    Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

    2013-01-01

    OBJECTIVE: To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). METHODS: This was a cross-sectional study involving adolescents and adults aged ≥ 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. RESULTS: We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. CONCLUSIONS: The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis. PMID:23670503

  11. Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis.

    PubMed

    Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

    2013-01-01

    To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). This was a cross-sectional study involving adolescents and adults aged > 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis.

  12. Highly Coarse-Grained Representations of Transmembrane Proteins

    PubMed Central

    2017-01-01

    Numerous biomolecules and biomolecular complexes, including transmembrane proteins (TMPs), are symmetric or at least have approximate symmetries. Highly coarse-grained models of such biomolecules, aiming at capturing the essential structural and dynamical properties on resolution levels coarser than the residue scale, must preserve the underlying symmetry. However, making these models obey the correct physics is in general not straightforward, especially at the highly coarse-grained resolution where multiple (∼3–30 in the current study) amino acid residues are represented by a single coarse-grained site. In this paper, we propose a simple and fast method of coarse-graining TMPs obeying this condition. The procedure involves partitioning transmembrane domains into contiguous segments of equal length along the primary sequence. For the coarsest (lowest-resolution) mappings, it turns out to be most important to satisfy the symmetry in a coarse-grained model. As the resolution is increased to capture more detail, however, it becomes gradually more important to match modular repeats in the secondary structure (such as helix-loop repeats) instead. A set of eight TMPs of various complexity, functionality, structural topology, and internal symmetry, representing different classes of TMPs (ion channels, transporters, receptors, adhesion, and invasion proteins), has been examined. The present approach can be generalized to other systems possessing exact or approximate symmetry, allowing for reliable and fast creation of multiscale, highly coarse-grained mappings of large biomolecular assemblies. PMID:28043122

  13. Fatigue Crack Propagation from Notched Specimens of 304 SS in elevated Temperature Aqueous Environment

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

    Wire, G. L.; Mills, W. J.

    2002-08-01

    Fatigue crack propagation (FCP) rates for 304 stainless steel (304SS) were determined in 24 degree C and 288 degree C air and 288 degree C water using double-edged notch (DEN) specimens of 304 stainless steel (304 SS). Test performed at matched loading conditions in air and water at 288 degree C with 20-6- cc h[sub]2/kg h[sub]2O provided a direct comparison of the relative crack growth rates in air and water over a wide range of crack growth rates. The DEN crack extension ranged from short cracks (0.03-0.25 mm) to long cracks up to 4.06 mm, which are consistent with conventionalmore » deep crack tests. Crack growth rates of 304 SS in water were about 12 times the air rate. This 12X environmental enhancement persisted to crack extensions up to 4.06 mm, far outside the range associated with short crack effects. The large environmental degradation for 304 SS crack growth is consistent with the strong reduction of fatigue life in high hydrogen water. Further, very similar environmental effects w ere reported in fatigue crack growth tests in hydrogen water chemistry (HWC). Most literature data in high hydrogen water show only a mild environmental effect for 304 SS, of order 2.5 times air or less, but the tests were predominantly performed at high cyclic stress intensity or equivalently, high air rates. The environmental effect in low oxygen environments at low stress intensity depends strongly on both the stress ratio, R, and the load rise time, T[sub]r, as recently reported for austenitic stainless steel in BWR water. Fractography was performed for both tests in air and water. At 288 degree C in water, the fracture surfaces were crisply faceted with a crystallographic appearance, and showed striations under high magnification. The cleavage-like facets on the fracture surfaces suggest that hydrogen embrittlement is the primary cause of accelerated cracking.« less

  14. Role of Transmembrane Potential and Defects on the Permeabilization of Lipid Bilayers by Alamethicin, an Ion-Channel-Forming Peptide.

    PubMed

    Su, ZhangFei; Shodiev, Muzaffar; Leitch, J Jay; Abbasi, Fatemeh; Lipkowski, Jacek

    2018-05-29

    The insertion and ion-conducting channel properties of alamethicin reconstituted into a 1,2-di- O-phytanyl- sn-glycero-3-phosphocholine bilayer floating on the surface of a gold (111) electrode modified with a 1-thio-β-d-glucose (β-Tg) self-assembled monolayer were investigated using a combination of electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The hydrophilic β-Tg monolayer separated the bilayer from the gold substrate and created a water-rich spacer region, which better represents natural cell membranes. The EIS measurements acquired information about the membrane resistivity (a measure of membrane porosity), and the PM-IRRAS experiments provided insight into the conformation and orientation of the membrane constituents as a function of the transmembrane potential. The results showed that the presence of alamethicin had a small effect on the conformation and orientation of phospholipid molecules within the bilayer for all studied potentials. In contrast, the alamethicin peptides assumed a surface state, where the helical axes adopted a large tilt angle with respect to the surface normal, at small transmembrane potentials, and inserted into the bilayer at sufficiently negative transmembrane potentials forming pores, which behaved as barrel-stave ion channels for ionic transport across the membrane. The results indicated that insertion of alamethincin peptides into the bilayer was driven by the dipole-field interactions and that the transitions between the inserted and surface states were electrochemically reversible. Additionally, the EIS measurements performed on phospholipid bilayers without alamethicin also showed that the application of negative transmembrane potentials introduces defects into the bilayer. The membrane resistances measured in both the absence and presence of alamethicin show similar dependencies on the electrode potential, suggesting that the insertion of

  15. Xylanase production by a newly isolated Aspergillus niger SS7 in submerged culture.

    PubMed

    Bakri, Yasser; Al-Jazairi, Manal; Al-Kayat, Ghassan

    2008-01-01

    Xylanase production by a newly isolated Aspergillus niger SS7 was studied in submerged culture. The optimum initial pH for xylanase production was found to be 7.0. Different agricultural and industrial wastes were evaluated for their ability to induce xylanase production by this isolate. The best xylanase production (293.82 IU/ml) was recorded at 3% (w/v) corn cob hulls after 120 h of incubation. The Aspergillus niger SS7 isolate grown in a simple medium, proved to be a promising microorganism for xylanase production.

  16. Comparison of post-contrast 3D-T1-MPRAGE, 3D-T1-SPACE and 3D-T2-FLAIR MR images in evaluation of meningeal abnormalities at 3-T MRI.

    PubMed

    Jeevanandham, Balaji; Kalyanpur, Tejas; Gupta, Prashant; Cherian, Mathew

    2017-06-01

    This study was to assess the usefulness of newer three-dimensional (3D)-T 1 sampling perfection with application optimized contrast using different flip-angle evolutions (SPACE) and 3D-T 2 fluid-attenuated inversion recovery (FLAIR) sequences in evaluation of meningeal abnormalities. 78 patients who presented with high suspicion of meningeal abnormalities were evaluated using post-contrast 3D-T 2 -FLAIR, 3D-T 1 magnetization-prepared rapid gradient-echo (MPRAGE) and 3D-T 1 -SPACE sequences. The images were evaluated independently by two radiologists for cortical gyral, sulcal space, basal cisterns and dural enhancement. The diagnoses were confirmed by further investigations including histopathology. Post-contrast 3D-T 1 -SPACE and 3D-T 2 -FLAIR images yielded significantly more information than MPRAGE images (p < 0.05 for both SPACE and FLAIR images) in detection of meningeal abnormalities. SPACE images best demonstrated abnormalities in dural and sulcal spaces, whereas FLAIR was useful for basal cisterns enhancement. Both SPACE and FLAIR performed equally well in detection of gyral enhancement. In all 10 patients, where both SPACE and T 2 -FLAIR images failed to demonstrate any abnormality, further analysis was also negative. The 3D-T 1 -SPACE sequence best demonstrated abnormalities in dural and sulcal spaces, whereas FLAIR was useful for abnormalities in basal cisterns. Both SPACE and FLAIR performed holds good for detection of gyral enhancement. Post-contrast SPACE and FLAIR sequences are superior to the MPRAGE sequence for evaluation of meningeal abnormalities and when used in combination have the maximum sensitivity for leptomeningeal abnormalities. The negative-predictive value is nearly 100%, where no leptomeningeal abnormality was detected on these sequences. Advances in knowledge: Post-contrast 3D-T 1 -SPACE and 3D-T 2 -FLAIR images are more useful than 3D-T 1 -MPRAGE images in evaluation of meningeal abnormalities.

  17. CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy

    PubMed Central

    Bridgeman, J S; Ladell, K; Sheard, V E; Miners, K; Hawkins, R E; Price, D A; Gilham, D E

    2014-01-01

    Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions. PMID:24116999

  18. New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome

    PubMed Central

    Tsuboi, H; Matsumoto, I; Wakamatsu, E; Nakamura, Y; Iizuka, M; Hayashi, T; Goto, D; Ito, S; Sumida, T

    2010-01-01

    M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca2+ concentrations [(Ca2+)i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca2+)i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca2+)i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. PMID:20731676

  19. EuroPhenome and EMPReSS: online mouse phenotyping resource

    PubMed Central

    Mallon, Ann-Marie; Hancock, John M.

    2008-01-01

    EuroPhenome (http://www.europhenome.org) and EMPReSS (http://empress.har.mrc.ac.uk/) form an integrated resource to provide access to data and procedures for mouse phenotyping. EMPReSS describes 96 Standard Operating Procedures for mouse phenotyping. EuroPhenome contains data resulting from carrying out EMPReSS protocols on four inbred laboratory mouse strains. As well as web interfaces, both resources support web services to enable integration with other mouse phenotyping and functional genetics resources, and are committed to initiatives to improve integration of mouse phenotype databases. EuroPhenome will be the repository for a recently initiated effort to carry out large-scale phenotyping on a large number of knockout mouse lines (EUMODIC). PMID:17905814

  20. EuroPhenome and EMPReSS: online mouse phenotyping resource.

    PubMed

    Mallon, Ann-Marie; Blake, Andrew; Hancock, John M

    2008-01-01

    EuroPhenome (http://www.europhenome.org) and EMPReSS (http://empress.har.mrc.ac.uk/) form an integrated resource to provide access to data and procedures for mouse phenotyping. EMPReSS describes 96 Standard Operating Procedures for mouse phenotyping. EuroPhenome contains data resulting from carrying out EMPReSS protocols on four inbred laboratory mouse strains. As well as web interfaces, both resources support web services to enable integration with other mouse phenotyping and functional genetics resources, and are committed to initiatives to improve integration of mouse phenotype databases. EuroPhenome will be the repository for a recently initiated effort to carry out large-scale phenotyping on a large number of knockout mouse lines (EUMODIC).

  1. Complementary DNA sequences of the constant regions of T-cell antigen receptors α, β and γ in mandarin fish, Siniperca chuatsi Basilewsky, and their transcriptional changes after stimulation with Flavobacterium columnare.

    PubMed

    Tian, J Y; Qi, Z T; Wu, N; Chang, M X; Nie, P

    2014-02-01

    In this study, the constant-region genes (Cα, Cβ and Cγ) that encode the T-cell antigen receptor (TCR) α, β and γ chains were cloned from mandarin fish, Siniperca chuatsi Basilewsky, an important freshwater fish species in China. The complementary DNA sequences of Cα, Cβ and Cγ were 843, 716 and 906 base pairs (bp) in length and had a 465-, 289- and 360-bp 3' untranslated region, encoding 125, 142 and 182 amino acids, respectively. The amino-acid sequences of the constant regions of mandarin fish TCR α, β and γ chains (encoded by Cα, Cβ and Cγ, respectively) were most similar to those of their teleost counterparts, showing 60% similarity with pufferfish, 48% similarity with Atlantic salmon and 57% similarity with flounder, respectively. The phylogenetic analysis revealed that the mandarin fish Cα, Cβ and Cγ were clustered, respectively, with their vertebrate counterparts. The mandarin fish Cα, Cβ and Cγ could also be separated into four domains: immunoglobulin; connecting peptide (CP); transmembrane (TM); and cytoplasmic tail. Several conserved features in mammalian TCRs were also found in those of mandarin fish, such as a conserved cysteine residue in the CP domain of Cα, necessary for creating an interchain disulphide bond with the TCR β chain, and a conserved antigen receptor TM motif in Cα and Cβ. Meanwhile, transcripts of Cα, Cβ and Cγ were detectable in all examined organs, with a stronger signal observed in lymphoid organs. In addition, the temporal transcriptional changes for Cα and Cγ were investigated, 1, 2, 3, 4, 5, 6 and 8 weeks after stimulation with Flavobacterium columnare, in head kidney, spleen, blood, thymus, gill and intestine, using real-time polymerase chain reaction. The results demonstrated stimulation-dependent up-regulations in almost all tissues examined, which indicates that T cells may play important roles in preventing mandarin fish from bacterial invasion. In particular, apart from thymus, T cells were

  2. Photosensitized electron transport across lipid vesicle walls: Enhancement of quantum yield by ionophores and transmembrane potentials

    PubMed Central

    Laane, Colja; Ford, William E.; Otvos, John W.; Calvin, Melvin

    1981-01-01

    The photosensitized reduction of heptylviologen in the bulk aqueous phase of phosphatidylcholine vesicles containing EDTA inside and a membrane-bound tris(2,2′-bipyridine)ruthenium(2+) derivative is enhanced by a factor of 6.5 by the addition of valinomycin in the presence of K+. A 3-fold stimulation by gramicidin and carbonyl cyanide m-chlorophenylhydrazone is observed. The results suggest that, under these conditions, the rate of photoinduced electron transfer across vesicle walls in the absence of ion carriers is limited by cotransport of cations. The rate of electron transfer across vesicle walls could be influenced further by generating transmembrane potentials with K+ gradients in the presence of valinomycin. When vesicles are made with transmembrane potentials, interior more negative, the quantum yield of heptylviologen reduction is doubled, and, conversely, when vesicles are made with transmembrane potentials, interior more positive, the quantum yield is decreased and approaches the value found in the absence of valinomycin. PMID:16593002

  3. Activation of intestinal Cl- secretion by lubiprostone requires the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Bijvelds, Marcel J C; Bot, Alice G M; Escher, Johanna C; De Jonge, Hugo R

    2009-09-01

    Lubiprostone alleviates constipation by stimulating intestinal fluid secretion, purportedly through activation of ClC-2-type Cl(-) channels. Intestinal obstruction is also a recurrent cause of distress in cystic fibrosis (CF) patients, caused by loss of CF transmembrane conductance regulator (CFTR) Cl(-) channel activity. Because ClC-2 recruitment might be beneficial to CF patients, we investigated lubiprostone's mode of action. Cl(-) transport was measured in an Ussing chamber, in 3 model systems: (1) T84 colonocytes, (2) intestinal epithelium of wild-type and CF mice, and (3) intestinal epithelium of CF patients and controls. In T84 monolayers, lubiprostone induced a robust secretory response. Selective permeabilization of the basolateral plasma membrane revealed that lubiprostone activated an apical Cl(-) conductance. The lubiprostone response was attenuated by H89, an inhibitor of the cAMP-dependent protein kinase, and lubiprostone precluded responsiveness to the cAMP agonist forskolin. CFTR blockage by CFTRinh172, but not ClC-2 blockage by CdCl(2), inhibited the lubiprostone response. Lubiprostone induced a CdCl(2)-insensitive secretory response in mouse intestine, but failed to induce intestinal Cl(-) secretion in Cftr-null mice. Correspondingly, lubiprostone induced a secretory response in human intestinal epithelium, but not in tissue of CF patients. The EP(4)-type prostanoid receptor antagonist L-161,982 blocked the lubiprostone response in all 3 models studied. In T84 cells, lubiprostone induced a rise in cAMP levels that was sensitive to EP(4)-receptor blockage. Lubiprostone enhances intestinal Cl(-) and fluid secretion via prostanoid receptor signaling, triggering activation of CFTR. Therefore, it is of limited use for treatment of CF-related intestinal disease.

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

    PubMed Central

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

    2015-01-01

    Background 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. Results 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. Conclusions 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. PMID:26207632

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

    PubMed

    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.

  6. Adhesion activity of glyceraldehyde-3-phosphate dehydrogenase in a Chinese Streptococcus suis type 2 strain.

    PubMed

    Wang, Kaicheng; Lu, Chengping

    2007-01-01

    A total of 36 streptococcal strains, including seven S. equi ssp.zooepidemicus, two S. suis type 1 (SS1), 24 SS2, two SS9, and one SS7, were tested for glyceraldehyde-3-phosphate dehydrogenase gene (gapdh). Except from non-virulent SS2 strain T1 5, all strains harboured gapdh. The gapdh of Chinese Sichuan SS2 isolate ZY05719 and Jiangsu SS2 isolate HA9801 were sequenced and then compared with published sequences in the GenBank. The comparison revealed a 99.9 % and 99.8 % similarity of ZY05719 and HA9801, respectively, with the published sequence. Adherence assay data demonstrated a significant ((p<0.05)) reduction in adhesion of SS2 in HEp-2 cells pre-incubated with purified GAPDH compared to non pre-incubated controls, suggesting the GAPDH mediates SS2 bacterial adhesion to host cells.

  7. Can T1 w/T2 w ratio be used as a myelin-specific measure in subcortical structures? Comparisons between FSE-based T1 w/T2 w ratios, GRASE-based T1 w/T2 w ratios and multi-echo GRASE-based myelin water fractions.

    PubMed

    Uddin, Md Nasir; Figley, Teresa D; Marrie, Ruth Ann; Figley, Chase R

    2018-03-01

    Given the growing popularity of T 1 -weighted/T 2 -weighted (T 1 w/T 2 w) ratio measurements, the objective of the current study was to evaluate the concordance between T 1 w/T 2 w ratios obtained using conventional fast spin echo (FSE) versus combined gradient and spin echo (GRASE) sequences for T 2 w image acquisition, and to compare the resulting T 1 w/T 2 w ratios with histologically validated myelin water fraction (MWF) measurements in several subcortical brain structures. In order to compare these measurements across a relatively wide range of myelin concentrations, whole-brain T 1 w magnetization prepared rapid acquisition gradient echo (MPRAGE), T 2 w FSE and three-dimensional multi-echo GRASE data were acquired from 10 participants with multiple sclerosis at 3 T. Then, after high-dimensional, non-linear warping, region of interest (ROI) analyses were performed to compare T 1 w/T 2 w ratios and MWF estimates (across participants and brain regions) in 11 bilateral white matter (WM) and four bilateral subcortical grey matter (SGM) structures extracted from the JHU_MNI_SS 'Eve' atlas. Although the GRASE sequence systematically underestimated T 1 w/T 2 w values compared to the FSE sequence (revealed by Bland-Altman and mountain plots), linear regressions across participants and ROIs revealed consistently high correlations between the two methods (r 2 = 0.62 for all ROIs, r 2 = 0.62 for WM structures and r 2 = 0.73 for SGM structures). However, correlations between either FSE-based or GRASE-based T 1 w/T 2 w ratios and MWFs were extremely low in WM structures (FSE-based, r 2 = 0.000020; GRASE-based, r 2 = 0.0014), low across all ROIs (FSE-based, r 2 = 0.053; GRASE-based, r 2 = 0.029) and moderate in SGM structures (FSE-based, r 2 = 0.20; GRASE-based, r 2 = 0.17). Overall, our findings indicated a high degree of correlation (but not equivalence) between FSE-based and GRASE-based T 1 w/T 2 w ratios, and low correlations between T 1 w/T 2 w ratios and MWFs. This

  8. In vivo estimation of transverse relaxation time constant (T2 ) of 17 human brain metabolites at 3T.

    PubMed

    Wyss, Patrik O; Bianchini, Claudio; Scheidegger, Milan; Giapitzakis, Ioannis A; Hock, Andreas; Fuchs, Alexander; Henning, Anke

    2018-08-01

    The transverse relaxation times T 2 of 17 metabolites in vivo at 3T is reported and region specific differences are addressed. An echo-time series protocol was applied to one, two, or three volumes of interest with different fraction of white and gray matter including a total number of 106 healthy volunteers and acquiring a total number of 128 spectra. The data were fitted with the 2D fitting tool ProFit2, which included individual line shape modeling for all metabolites and allowed the T 2 calculation of 28 moieties of 17 metabolites. The T 2 of 10 metabolites and their moieties have been reported for the first time. Region specific T 2 differences in white and gray matter enriched tissue occur in 16 of 17 metabolites examined including single resonance lines and coupled spin systems. The relaxation time T 2 is regions specific and has to be considered when applying tissue composition correction for internal water referencing. Magn Reson Med 80:452-461, 2018. © 2018 International Society for Magnetic Resonance in Medicine. © 2018 International Society for Magnetic Resonance in Medicine.

  9. Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

    PubMed

    Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

    2008-04-01

    Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

  10. Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage

    PubMed Central

    Brok-Volchanskaya, Vera S.; Kadyrov, Farid A.; Sivogrivov, Dmitry E.; Kolosov, Peter M.; Sokolov, Andrey S.; Shlyapnikov, Michael G.; Kryukov, Valentine M.; Granovsky, Igor E.

    2008-01-01

    Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3′ 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TψC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages. PMID:18281701

  11. KM3NeT

    NASA Astrophysics Data System (ADS)

    de Jong, M.

    2015-07-01

    KM3NeT is a large research infrastructure, that will consist of a network of deep-sea neutrino telescopes in the Mediterranean Sea. The main objective of KM3NeT is the discovery and subsequent observation of high-energy neutrino sources in the Universe. A further physics perspective is the measurement of the mass hierarchy of neutrinos. A corresponding study, ORCA, is ongoing within KM3NeT. A cost effective technology for (very) large water Cherenkov detectors has been developed based on a new generation of low price 3-inch photo-multiplier tubes. Following the successful deployment and operation of two prototypes, the construction of the KM3NeT research infrastructure has started. The prospects of the different phases of the implementation of KM3NeT are summarised.

  12. Comparison of T1 and T2 metabolite relaxation times in glioma and normal brain at 3 T

    PubMed Central

    Li, Yan; Srinivasan, Radhika; Ratiney, Helene; Lu, Ying; Chang, Susan M.; Nelson, Sarah J.

    2011-01-01

    Purpose To measure T1 and T2 relaxation times of metabolites in glioma patients at 3T and to investigate how these values influence the observed metabolite levels. Materials and Methods Twenty-three patients with gliomas and ten volunteers were studied with single voxel 2D J-resolved PRESS using a 3T MR scanner. Voxels were chosen in normal appearing white matter and in regions of tumor. The T1 and T2 of choline containing compounds (Cho), creatine (Cr) and N-acetyl aspartate (NAA) were estimated. Results Metabolite T1 relaxation values in gliomas were not significantly different from values in normal white matter. The T2 of Cho and Cr were statistically significantly longer for Grade 4 gliomas than for normal white matter but the T2 of NAA was similar. These differences were large enough to impact the corrections of metabolite levels for relaxation times with tumor grade in terms of metabolite ratios (P<0.001). Conclusion The differential increase in T2 for Cho and Cr relative to NAA means that the ratios of Cho/NAA and Cr/NAA are higher in tumor at longer echo times relative to values in normal appearing brain. Having this information may be useful in defining the acquisition parameters for optimizing contrast between tumor and normal tissue in MRSI data, where limited time is available and only one echo time can be used. PMID:18666155

  13. The Phage Lysin PlySs2 Decolonizes Streptococcus suis from Murine Intranasal Mucosa.

    PubMed

    Gilmer, Daniel B; Schmitz, Jonathan E; Thandar, Mya; Euler, Chad W; Fischetti, Vincent A

    2017-01-01

    Streptococcus suis infects pigs worldwide and may be zoonotically transmitted to humans with a mortality rate of up to 20%. S. suis has been shown to develop in vitro resistance to the two leading drugs of choice, penicillin and gentamicin. Because of this, we have pursued an alternative therapy to treat these pathogens using bacteriophage lysins. The bacteriophage lysin PlySs2 is derived from an S. suis phage and displays potent lytic activity against most strains of that species including serotypes 2 and 9. At 64 μg/ml, PlySs2 reduced multiple serotypes of S. suis by 5 to 6-logs within 1 hour in vitro and exhibited a minimum inhibitory concentration (MIC) of 32 μg/ml for a S. suis serotype 2 strain and 64 μg/ml for a serotype 9 strain. Using a single 0.1-mg dose, the colonizing S. suis serotype 9 strain was reduced from the murine intranasal mucosa by >4 logs; a 0.1-mg dose of gentamicin reduced S. suis by <3-logs. A combination of 0.05 mg PlySs2 + 0.05 mg gentamicin reduced S. suis by >5-logs. While resistance to gentamicin was induced after systematically increasing levels of gentamicin in an S. suis culture, the same protocol resulted in no observable resistance to PlySs2. Thus, PlySs2 has both broad and high killing activity against multiple serotypes and strains of S. suis, making it a possible tool in the control and prevention of S. suis infections in pigs and humans.

  14. The Phage Lysin PlySs2 Decolonizes Streptococcus suis from Murine Intranasal Mucosa

    PubMed Central

    Gilmer, Daniel B.; Schmitz, Jonathan E.; Thandar, Mya; Euler, Chad W.; Fischetti, Vincent A.

    2017-01-01

    Streptococcus suis infects pigs worldwide and may be zoonotically transmitted to humans with a mortality rate of up to 20%. S. suis has been shown to develop in vitro resistance to the two leading drugs of choice, penicillin and gentamicin. Because of this, we have pursued an alternative therapy to treat these pathogens using bacteriophage lysins. The bacteriophage lysin PlySs2 is derived from an S. suis phage and displays potent lytic activity against most strains of that species including serotypes 2 and 9. At 64 μg/ml, PlySs2 reduced multiple serotypes of S. suis by 5 to 6-logs within 1 hour in vitro and exhibited a minimum inhibitory concentration (MIC) of 32 μg/ml for a S. suis serotype 2 strain and 64 μg/ml for a serotype 9 strain. Using a single 0.1-mg dose, the colonizing S. suis serotype 9 strain was reduced from the murine intranasal mucosa by >4 logs; a 0.1-mg dose of gentamicin reduced S. suis by <3-logs. A combination of 0.05 mg PlySs2 + 0.05 mg gentamicin reduced S. suis by >5-logs. While resistance to gentamicin was induced after systematically increasing levels of gentamicin in an S. suis culture, the same protocol resulted in no observable resistance to PlySs2. Thus, PlySs2 has both broad and high killing activity against multiple serotypes and strains of S. suis, making it a possible tool in the control and prevention of S. suis infections in pigs and humans. PMID:28046082

  15. Differential regulation of cellular functions by the C-termini of transmembrane 4 L six family proteins in 2- or 3-dimensional environment.

    PubMed

    Cheong, Jin-Gyu; Song, Dae-Geun; Song, Haeng Eun; Berditchevski, Fedor; Nam, Seo Hee; Jung, Jae Woo; Kim, Hye-Jin; Kim, Ji Eon; Kim, Somi; Ryu, Jihye; Cho, Chang Yun; Lee, Kyung-Min; Lee, Jung Weon

    2017-02-21

    The transmembrane 4 L six family proteins TM4SF1, TM4SF4, and TM4SF5 share 40-50% overall sequence identity, but their C-terminus identity is limited. It may be likely that the C-termini of the members are important and unique for own regulatory functions. We thus examined how the TM4SF5 C-terminus affected cellular functions differentially from other family members. Using colon cancer cells expressing wildtype (WT), C-terminus-deleted, or chimeric mutants, diverse cellular functions were explored in 2-dimensional (2D) and 3-dimensional (3D) condition. The C-termini of the proteins were relatively comparable with respect to 2D cell proliferation, although each C-terminal-deletion mutant exhibited increased proliferation relative to the WT. Using chimeric constructs, we found that the TM4SF5 C-terminus was critical for regulating the diverse metastatic functions of TM4SF5, and could positively replace the C-termini of other family members. Replacement of the TM4SF1 or TM4SF4 C-terminus with that of TM4SF5 increased spheroids growth, transwell migration, and invasive dissemination from spheroids in 3D collagen gels. TM4SF5-mediated effects required its extracellular loop 2 linked to the C-terminus via the transmembrane domain 4, with causing c-Src activation. Altogether, the C-terminus of TM4SF5 appears to mediate pro-migratory roles, depending on a structural relay from the second extracellular loop to the C-terminus.

  16. Differential regulation of cellular functions by the C-termini of transmembrane 4 L six family proteins in 2- or 3-dimensional environment

    PubMed Central

    Cheong, Jin-Gyu; Song, Dae-Geun; Song, Haeng Eun; Berditchevski, Fedor; Nam, Seo Hee; Jung, Jae Woo; Kim, Hye-Jin; Kim, Ji Eon; Kim, Somi; Ryu, Jihye; Cho, Chang Yun; Lee, Kyung-Min; Lee, Jung Weon

    2017-01-01

    The transmembrane 4 L six family proteins TM4SF1, TM4SF4, and TM4SF5 share 40-50% overall sequence identity, but their C-terminus identity is limited. It may be likely that the C-termini of the members are important and unique for own regulatory functions. We thus examined how the TM4SF5 C-terminus affected cellular functions differentially from other family members. Using colon cancer cells expressing wildtype (WT), C-terminus-deleted, or chimeric mutants, diverse cellular functions were explored in 2-dimensional (2D) and 3-dimensional (3D) condition. The C-termini of the proteins were relatively comparable with respect to 2D cell proliferation, although each C-terminal-deletion mutant exhibited increased proliferation relative to the WT. Using chimeric constructs, we found that the TM4SF5 C-terminus was critical for regulating the diverse metastatic functions of TM4SF5, and could positively replace the C-termini of other family members. Replacement of the TM4SF1 or TM4SF4 C-terminus with that of TM4SF5 increased spheroids growth, transwell migration, and invasive dissemination from spheroids in 3D collagen gels. TM4SF5-mediated effects required its extracellular loop 2 linked to the C-terminus via the transmembrane domain 4, with causing c-Src activation. Altogether, the C-terminus of TM4SF5 appears to mediate pro-migratory roles, depending on a structural relay from the second extracellular loop to the C-terminus. PMID:28129652

  17. Application of Framework for Integrating Safety, Security and Safeguards (3Ss) into the Design Of Used Nuclear Fuel Storage Facility

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

    Badwan, Faris M.; Demuth, Scott F

    Department of Energy’s Office of Nuclear Energy, Fuel Cycle Research and Development develops options to the current commercial fuel cycle management strategy to enable the safe, secure, economic, and sustainable expansion of nuclear energy while minimizing proliferation risks by conducting research and development focused on used nuclear fuel recycling and waste management to meet U.S. needs. Used nuclear fuel is currently stored onsite in either wet pools or in dry storage systems, with disposal envisioned in interim storage facility and, ultimately, in a deep-mined geologic repository. The safe management and disposition of used nuclear fuel and/or nuclear waste is amore » fundamental aspect of any nuclear fuel cycle. Integrating safety, security, and safeguards (3Ss) fully in the early stages of the design process for a new nuclear facility has the potential to effectively minimize safety, proliferation, and security risks. The 3Ss integration framework could become the new national and international norm and the standard process for designing future nuclear facilities. The purpose of this report is to develop a framework for integrating the safety, security and safeguards concept into the design of Used Nuclear Fuel Storage Facility (UNFSF). The primary focus is on integration of safeguards and security into the UNFSF based on the existing Nuclear Regulatory Commission (NRC) approach to addressing the safety/security interface (10 CFR 73.58 and Regulatory Guide 5.73) for nuclear power plants. The methodology used for adaptation of the NRC safety/security interface will be used as the basis for development of the safeguards /security interface and later will be used as the basis for development of safety and safeguards interface. Then this will complete the integration cycle of safety, security, and safeguards. The overall methodology for integration of 3Ss will be proposed, but only the integration of safeguards and security will be applied to the design

  18. Total Skin Electron Beam for Primary Cutaneous T-cell Lymphoma

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

    Elsayad, Khaled; Kriz, Jan; Moustakis, Christos

    Purpose: Recent trials with low-dose total skin electron beam (TSEB) therapy demonstrated encouraging results for treating primary cutaneous T-cell lymphoma (PCTCL). In this study, we assessed the feasibility of different radiation doses and estimated survival rates of different pathologic entities and stages. Methods and Materials: We retrospectively identified 45 patients with PCTCL undergoing TSEB therapy between 2000 and 2015. Clinical characteristics, treatment outcomes, and toxicity were assessed. Results: A total of 49 courses of TSEB therapy were administered to the 45 patients. There were 26 pathologically confirmed cases of mycosis fungoides (MF) lymphoma, 10 cases of Sézary syndrome (SS), andmore » 9 non-MF/SS PCTCL patients. In the MF patients, the overall response rate (ORR) was 92% (50% complete remission [CR]), 70% ORR in SS patients (50% CR), and 89% ORR in non-MF/SS patients (78% CR). The ORR for MF/SS patients treated with conventional dose (30-36 Gy) regimens was 92% (63% CR) and 75% (25% CR) for low-dose (<30-Gy) regimens (P=.09). In MF patients, the overall survival (OS) was 77 months with conventional dose regimens versus 14 months with low-dose regimens (P=.553). In SS patients, the median OS was 48 versus 16 months (P=.219), respectively. Median event-free survival (EFS) for MF in conventional dose patients versus low-dose patients was 15 versus 8 months, respectively (P=.264) and 19 versus 3 months for SS patients (P=.457). Low-dose regimens had shorter treatment time (P=.009) and lower grade 2 adverse events (P=.043). A second TSEB course was administered in 4 MF patients with 100% ORR. There is a possible prognostic impact of supplemental/boost radiation (P<.001); adjuvant treatment (P<.001) and radiation tolerability (P=.021) were detected. Conclusions: TSEB therapy is an efficacious treatment modality in the treatment of several forms of cutaneous T-cell lymphoma. There is a nonsignificant trend to higher and longer clinical

  19. PD-1+Tim-3+ CD8+ T Lymphocytes Display Varied Degrees of Functional Exhaustion in Patients with Regionally Metastatic Differentiated Thyroid Cancer

    PubMed Central

    Severson, Jill J.; Serracino, Hilary S.; Mateescu, Valerica; Raeburn, Christopher D.; C.McIntyre, Robert; Sams, Sharon B.; Haugen, Bryan R.; French, Jena D.

    2015-01-01

    Regional metastatic differentiated thyroid cancer (mDTC) provides a unique model in which to study the tumor-immune interface. These lymph node (LN) metastases persist for years, generally without progression to distant metastases. While the immune system likely impedes disease progression, it is unsuccessful in eliminating disease. Our previous studies revealed that programmed death-1 (PD-1)+ T cells were enriched in tumor-involved lymph nodes (TILN). Tumor-associated leukocytes and tumor cells were collected from grossly involved LNs from 12 patients to further characterize the phenotype and functional potential of mDTC-associated PD-1+ T cells. PD-1+CD4+ and PD-1+CD8+ T cells were enriched in 8/12 TILN samples. PD-1+ T cells co-expressed Tim-3 and CD69 and failed to down-regulate CD27. CD8+ T cells, but not CD4+ T cells, from these samples were variably deficient in their ability to produce effector cytokines when compared to control TILNs that lacked resident PD-1+ T cells. PD-1+CD8+ T cells were capable of exocytosis but lacked intracellular perforin. Surprisingly, T-cell proliferative capacity was largely maintained in all samples. Thus, while PD-1 expression by mDTC-associated CD8+ T cells was associated with dysfunction, exhaustion was not complete. Notably, molecular markers of exhaustion did not translate to dysfunction in all samples or in CD4+ T cells. Regulatory T (Treg) cells, PD-L1, and galectin-9 were commonly found in mDTC and likely contributed to the initiation of T-cell exhaustion and disease progression. Therapies that release the effects of PD-1 and Tim-3 and reduce the suppressive effects of Tregs may encourage tumor elimination in patients with mDTC. PMID:25701326

  20. tPA Prescription and Administration Errors within a Regional Stroke System

    PubMed Central

    Chung, Lee S; Tkach, Aleksander; Lingenfelter, Erin M; Dehoney, Sarah; Rollo, Jeannie; de Havenon, Adam; DeWitt, Lucy Dana; Grantz, Matthew Ryan; Wang, Haimei; Wold, Jana J; Hannon, Peter M; Weathered, Natalie R; Majersik, Jennifer J

    2015-01-01

    Background IV tPA utilization in acute ischemic stroke (AIS) requires weight-based dosing and a standardized infusion rate. In our regional network, we have tried to minimize tPA dosing errors. We describe the frequency and types of tPA administration errors made in our comprehensive stroke center (CSC) and at community hospitals (CHs) prior to transfer. Methods Using our stroke quality database, we extracted clinical and pharmacy information on all patients who received IV tPA from 2010–11 at the CSC or CH prior to transfer. All records were analyzed for the presence of inclusion/exclusion criteria deviations or tPA errors in prescription, reconstitution, dispensing, or administration, and analyzed for association with outcomes. Results We identified 131 AIS cases treated with IV tPA: 51% female; mean age 68; 32% treated at CSC, 68% at CH (including 26% by telestroke) from 22 CHs. tPA prescription and administration errors were present in 64% of all patients (41% CSC, 75% CH, p<0.001), the most common being incorrect dosage for body weight (19% CSC, 55% CH, p<0.001). Of the 27 overdoses, there were 3 deaths due to systemic hemorrhage or ICH. Nonetheless, outcomes (parenchymal hematoma, mortality, mRS) did not differ between CSC and CH patients nor between those with and without errors. Conclusion Despite focus on minimization of tPA administration errors in AIS patients, such errors were very common in our regional stroke system. Although an association between tPA errors and stroke outcomes was not demonstrated, quality assurance mechanisms are still necessary to reduce potentially dangerous, avoidable errors. PMID:26698642

  1. Periodicity Analysis of X-ray Light Curves of SS 433

    NASA Astrophysics Data System (ADS)

    Wang, Jun-yi; Lu, Xiang-long; Zhao, Qiu-wen; Dong, Dian-qiao; Lao, Bao-qiang; Lu, Yang; Wei, Yan-heng; Wu, Xiao-cong; An, Tao

    2017-01-01

    SS 433 is sofar the unique X-ray binary that has the simultaneously detected orbital period, super-orbital period, and nutation period, as well as a bidirectional spiral jet. The study on its X-ray light variability is helpful for understanding the dynamic process of the system, and the correlations between the different wavebands. In this paper, two time-series analysis techniques, i.e., the Lomb-Scargle periodogram and weighted wavelet Z-transform, are employed to search for the periods in the Swift/BAT (Burst Alert Telescope) (15-50 keV) and RXTE/ASM (Rose X-ray Timing Explorer/All Sky Monitor) (1.5-3, 3- 4, and 5-12 keV) light curves of SS 433, and the Monte Carlo simulation is performed for the obtained periodical components. For the 15-50 keV energy band, five significant periodical components are detected, which are P1(∼6.29 d), P2 (∼6.54 d), P3 (∼13.08 d), P4 (∼81.50 d), and P5 (∼162.30 d). For the 3-5 and 5-12 keV energy bands, the periodical components P3 (∼13 d) and P5 (∼162 d) are detected in both energy bands. However, for the 1.5-3 keV energy band, no significant periodic signal is detected. P5 is the strongest periodic signal in the power spectrum for all the energy bands of 3-5, 5-12, and 15-50 keV, and it is consistent with the previous result obtained from the study of optical light curves. Furthermore, in combination with the radio spiral jet of SS 433, it is suggested that the X-ray and optical variability of P5 (∼162 d) is probably related to the precession of its relativistic jet. The high correlation between the X-ray and optical light curves may also imply that the X-ray and optical radiations are of the same physical origin. P3 shows a good agreement with the orbital period (∼13.07 d) obtained by the previous study, and P2 and P4 are respectively the high-frequency harmonics of P3 and P5. P1 is detected only in the power spectrum of the 15-50 keV energy band, and it is consistent with the nutation period of the system. As

  2. KM3NeT

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

    Jong, M. de; Leiden Institute of Physics, Leiden University, Leiden; Collaboration: KM3NeT Collaboration

    2015-07-15

    KM3NeT is a large research infrastructure, that will consist of a network of deep-sea neutrino telescopes in the Mediterranean Sea. The main objective of KM3NeT is the discovery and subsequent observation of high-energy neutrino sources in the Universe. A further physics perspective is the measurement of the mass hierarchy of neutrinos. A corresponding study, ORCA, is ongoing within KM3NeT. A cost effective technology for (very) large water Cherenkov detectors has been developed based on a new generation of low price 3-inch photo-multiplier tubes. Following the successful deployment and operation of two prototypes, the construction of the KM3NeT research infrastructure hasmore » started. The prospects of the different phases of the implementation of KM3NeT are summarised.« less

  3. Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States.

    PubMed

    Tanaka, Koji; Caaveiro, Jose M M; Tsumoto, Kouhei

    2015-11-24

    The bidirectional transformation of a protein between its native water-soluble and integral transmembrane conformations is demonstrated for FraC, a hemolytic protein of the family of pore-forming toxins. In the presence of biological membranes, the water-soluble conformation of FraC undergoes a remarkable structural reorganization generating cytolytic transmembrane nanopores conducive to cell death. So far, the reverse transformation from the native transmembrane conformation to the native water-soluble conformation has not been reported. We describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein.

  4. Restoration of self-awareness of hypoglycemia in adults with long-standing type 1 diabetes: hyperinsulinemic-hypoglycemic clamp substudy results from the HypoCOMPaSS trial.

    PubMed

    Leelarathna, Lalantha; Little, Stuart A; Walkinshaw, Emma; Tan, Horng Kai; Lubina-Solomon, Alexandra; Kumareswaran, Kavita; Lane, Annette P; Chadwick, Thomas; Marshall, Sally M; Speight, Jane; Flanagan, Daniel; Heller, Simon R; Shaw, James A M; Evans, Mark L

    2013-12-01

    Impaired awareness of hypoglycemia (IAH) and defective counterregulation significantly increase severe hypoglycemia risk in type 1 diabetes (T1D). We evaluated restoration of IAH/defective counterregulation by a treatment strategy targeted at hypoglycemia avoidance in adults with T1D with IAH (Gold score ≥4) participating in the U.K.-based multicenter HypoCOMPaSS randomized controlled trial. Eighteen subjects with T1D and IAH (mean ± SD age 50 ± 9 years, T1D duration 35 ± 10 years, HbA1c 8.1 ± 1.0% [65 ± 10.9 mmol/mol]) underwent stepped hyperinsulinemic-hypoglycemic clamp studies before and after a 6-month intervention. The intervention comprised the HypoCOMPaSS education tool in all and randomized allocation, in a 2 × 2 factorial study design, to multiple daily insulin analog injections or continuous subcutaneous insulin infusion therapy and conventional glucose monitoring or real-time continuous glucose monitoring. Symptoms, cognitive function, and counterregulatory hormones were measured at each glucose plateau (5.0, 3.8, 3.4, 2.8, and 2.4 mmol/L), with each step lasting 40 min with subjects kept blinded to their actual glucose value throughout clamp studies. After intervention, glucose concentrations at which subjects first felt hypoglycemic increased (mean ± SE from 2.6 ± 0.1 to 3.1 ± 0.2 mmol/L, P = 0.02), and symptom and plasma metanephrine responses to hypoglycemia were higher (median area under curve for symptoms, 580 [interquartile range {IQR} 420-780] vs. 710 [460-1,260], P = 0.02; metanephrine, 2,412 [-3,026 to 7,279] vs. 5,180 [-771 to 11,513], P = 0.01). Glycemic threshold for deterioration of cognitive function measured by four-choice reaction time was unchanged, while the color-word Stroop test showed a degree of adaptation. Even in long-standing T1D, IAH and defective counterregulation may be improved by a clinical strategy aimed at hypoglycemia avoidance.

  5. SS-mPEG chemical modification of recombinant phospholipase C for enhanced thermal stability and catalytic efficiency.

    PubMed

    Fang, Xian; Wang, Xueting; Li, Guiling; Zeng, Jun; Li, Jian; Liu, Jingwen

    2018-05-01

    PEGylation is one of the most promising and extensively studied strategies for improving the properties of proteins as well as enzymic physical and thermal stability. Phospholipase C, hydrolyzing the phospholipids offers tremendous applications in diverse fields. However, the poor thermal stability and higher cost of production have restricted its industrial application. This study focused on improving the stabilization of recombinant PLC by chemical modification with methoxypolyethylene glycol-Succinimidyl Succinate (SS-mPEG, MW 5000). PLC gene from isolate Bacillus cereus HSL3 was fused with SUMO, a novel small ubiquitin-related modifier expression vector and over expressed in Escherichia coli. The soluble fraction of SUMO-PLC reached 80% of the total recombinant protein. The enzyme exhibited maximum catalytic activity at 80 °C and was relatively thermostable at 40-70 °C. It showed extensive substrate specificity pattern and marked activity toward phosphatidylcholine, which made it a typical non-specific PLC for industrial purpose. SS-mPEG-PLC complex exhibited an enhanced thermal stability at 70-80 °C and the catalytic efficiency (K cat /K m ) had increased by 3.03 folds compared with free PLC. CD spectrum of SS-mPEG-PLC indicated a possible enzyme aggregation after chemical modification, which contributed to the higher thermostability of SS-mPEG-PLC. The increase of antiparallel β sheets in secondary structure also made it more stable than parallel β sheets. The presence of SS-mPEG chains on the enzyme molecule surface somewhat changed the binding rate of the substrates, leading to a significant improvement in catalytic efficiency. This study provided an insight into the addition of SS-mPEG for enhancing the industrial applications of phospholipase C at higher temperature. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Isolation of equine herpesvirus 3 (EHV-3) from equine coital exanthema of two stallions and sero-epidemiology of EHV-3 infection in Japan

    PubMed Central

    KIRISAWA, Rikio; TOISHI, Yuko; AKAMATSU, Ai; SOEJIMA, Kosuke; MIYASHITA, Taisuke; TSUNODA, Nobuo

    2017-01-01

    In the spring of 2015, two stallions reared in Farms A and B in Hokkaido in Japan showed symptoms of equine coital exanthema. Equine herpesvirus 3 (EHV-3) was isolated from penis swab samples of both stallions, and the isolates from each stallion in Farms A and B were designated as SS-1 and YS-1 strains, respectively. BamHI restriction profiles of SS-1 and Japanese reference strain Iwate-1 were indistinguishable, but the BamHI-A fragment of YS-1 was larger than those of SS-1 and Iwate-1 by 1.9 kbp because of the lack of two BamHI sites. Nucleotide sequence analyses of glycoprotein G (gG), gB, gC and VP13/14 coding regions revealed that SS-1 and YS-1 had 99.77% to 100% identities to each other. These results suggested that the origins of SS-1 and YS-1 were different. For a sero-epidemiological survey, serum neutralizing tests using SS-1 against 319 sera of horses from eight farms in Hokkaido were conducted. Six of the eight farms were EHV-3 antibody-positive, and positive rates ranged from 2.6% to 17.6%. To determine the infection time of four EHV-3 antibody-positive horses, a retrospective study was conducted. Infection time of the four horses was in the breeding season, and re-infection or reactivation of latently infected EHV-3 might have occurred in one horse. However, these four horses had never shown any clinical symptoms. The results suggested that several EHV-3 strains are distributed in Japan and that infection is maintained widely in horses without clinical symptoms. PMID:28132964

  7. Computational Identification and Comparative Analysis of Secreted and Transmembrane Proteins in Six Burkholderia Species.

    PubMed

    Nguyen, Thao Thi; Lee, Hyun-Hee; Park, Jungwook; Park, Inmyoung; Seo, Young-Su

    2017-04-01

    As a step towards discovering novel pathogenesis-related proteins, we performed a genome scale computational identification and characterization of secreted and transmembrane (TM) proteins, which are mainly responsible for bacteria-host interactions and interactions with other bacteria, in the genomes of six representative Burkholderia species. The species comprised plant pathogens ( B. glumae BGR1, B. gladioli BSR3), human pathogens ( B. pseudomallei K96243, B. cepacia LO6), and plant-growth promoting endophytes ( Burkholderia sp. KJ006, B. phytofirmans PsJN). The proportions of putative classically secreted proteins (CSPs) and TM proteins among the species were relatively high, up to approximately 20%. Lower proportions of putative type 3 non-classically secreted proteins (T3NCSPs) (~10%) and unclassified non-classically secreted proteins (NCSPs) (~5%) were observed. The numbers of TM proteins among the three clusters (plant pathogens, human pathogens, and endophytes) were different, while the distribution of these proteins according to the number of TM domains was conserved in which TM proteins possessing 1, 2, 4, or 12 TM domains were the dominant groups in all species. In addition, we observed conservation in the protein size distribution of the secreted protein groups among the species. There were species-specific differences in the functional characteristics of these proteins in the various groups of CSPs, T3NCSPs, and unclassified NCSPs. Furthermore, we assigned the complete sets of the conserved and unique NCSP candidates of the collected Burkholderia species using sequence similarity searching. This study could provide new insights into the relationship among plant-pathogenic, human-pathogenic, and endophytic bacteria.

  8. Use of Anti-Idiotypes and Synthetic Peptides for Control of Human T- Lymphotropic Virus Type 3 Infections

    DTIC Science & Technology

    1988-10-28

    peptides, anti-idiotypes, vaccines, 06 03 human immunodeficiency virus, chimpanzees, RAI, Virology, 06 13 HTLV III, Immunology 19. ABSTRACT (Continue on...idiotypes (anti-Id for con- trolling HIV infection. We have identified four regions of the human immunodeficiency virus type I HIV-1 envelope glycoprotein...analogous to amino acid sequences from the feline leukemia virus / transmembrane glycoprotein. Studies have utilized an affinity purified chimpanzee anti

  9. GARP is regulated by miRNAs and controls latent TGF-β1 production by human regulatory T cells.

    PubMed

    Gauthy, Emilie; Cuende, Julia; Stockis, Julie; Huygens, Caroline; Lethé, Bernard; Collet, Jean-François; Bommer, Guido; Coulie, Pierre G; Lucas, Sophie

    2013-01-01

    GARP is a transmembrane protein present on stimulated human regulatory T lymphocytes (Tregs), but not on other T lymphocytes (Th cells). It presents the latent form of TGF-β1 on the Treg surface. We report here that GARP favors the cleavage of the pro-TGF-β1 precursor and increases the amount of secreted latent TGF-β1. Stimulated Tregs, which naturally express GARP, and Th cells transfected with GARP secrete a previously unknown form of latent TGF-β1 that is disulfide-linked to GARP. These GARP/TGF-β1 complexes are possibly shed from the T cell surface. Secretion of GARP/TGF-β1 complexes was not observed with transfected 293 cells and may thus be restricted to the T cell lineage. We conclude that in stimulated human Tregs, GARP not only displays latent TGF-β1 at the cell surface, but also increases its secretion by forming soluble disulfide-linked complexes. Moreover, we identified six microRNAs (miRNAs) that are expressed at lower levels in Treg than in Th clones and that target a short region of the GARP 3' UTR. In transfected Th cells, the presence of this region decreased GARP levels, cleavage of pro-TGF-β1, and secretion of latent TGF-β1.

  10. FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation.

    PubMed

    Kehrmann, Jan; Zeschnigk, Michael; Buer, Jan; Probst-Kepper, Michael

    2011-10-01

    AIM: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25(hi) FOXP3+ T (T(reg)) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (T(h)) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called T(reg)-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. METHODS: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. RESULTS: Although GARP-transduced T(h) cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive T(h) cells. GARP-mediated FOXP3 upregulation in T(h) cells is not associated with T(reg)-specific demethylation of the FOXP3 TSDR. CONCLUSION: Although GARP-engineered T(h) cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards T(reg) cells in vitro, these cells do not completely mimic the epigenotype of natural T(reg) cells. Thus, concepts based on the genetic modification of T(h) cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application.

  11. Is PiSS Alpha-1 Antitrypsin Deficiency Associated with Disease?

    PubMed

    McGee, Dawn; Schwarz, Laura; McClure, Rebecca; Peterka, Lauren; Rouhani, Farshid; Brantly, Mark; Strange, Charlie

    2010-01-01

    Background. Alpha-1 antitrypsin deficiency (AAT) is an inherited condition that predisposes to lung and/or liver disease. Objective. The current study examined the clinical features of the PiSS genotype. Methods. Nineteen study participants (PiSS) and 29 matched control participants (PiMM) were telephone interviewed using a standardized questionnaire. Demographic features, cigarette smoking, vocation, medication history, and clinical diagnoses were compared. Statistical analysis was performed. Finally, a comprehensive literature review was performed by two investigators. Results. 12/19 (63.2%) study participants reported the presence of lung and/or liver disease compared to 12/29 (41.4%) control participants. There trended toward having a higher frequency of medication allergies in the study population (42.11% versus 20.69%). Conclusions. The PiSS genotype was associated with a similar incidence of obstructive lung disease to controls. Selective bias intrinsic in testing for AAT deficiency and the rarity of the PiSS genotype will make future study of this association dependent on population-based tests.

  12. Is PiSS Alpha-1 Antitrypsin Deficiency Associated with Disease?

    PubMed Central

    McGee, Dawn; Schwarz, Laura; McClure, Rebecca; Peterka, Lauren; Rouhani, Farshid; Brantly, Mark; Strange, Charlie

    2010-01-01

    Background. Alpha-1 antitrypsin deficiency (AAT) is an inherited condition that predisposes to lung and/or liver disease. Objective. The current study examined the clinical features of the PiSS genotype. Methods. Nineteen study participants (PiSS) and 29 matched control participants (PiMM) were telephone interviewed using a standardized questionnaire. Demographic features, cigarette smoking, vocation, medication history, and clinical diagnoses were compared. Statistical analysis was performed. Finally, a comprehensive literature review was performed by two investigators. Results. 12/19 (63.2%) study participants reported the presence of lung and/or liver disease compared to 12/29 (41.4%) control participants. There trended toward having a higher frequency of medication allergies in the study population (42.11% versus 20.69%). Conclusions. The PiSS genotype was associated with a similar incidence of obstructive lung disease to controls. Selective bias intrinsic in testing for AAT deficiency and the rarity of the PiSS genotype will make future study of this association dependent on population-based tests. PMID:21687342

  13. Validation of Robotic Surgery Simulator (RoSS).

    PubMed

    Kesavadas, Thenkurussi; Stegemann, Andrew; Sathyaseelan, Gughan; Chowriappa, Ashirwad; Srimathveeravalli, Govindarajan; Seixas-Mikelus, Stéfanie; Chandrasekhar, Rameella; Wilding, Gregory; Guru, Khurshid

    2011-01-01

    Recent growth of daVinci Robotic Surgical System as a minimally invasive surgery tool has led to a call for better training of future surgeons. In this paper, a new virtual reality simulator, called RoSS is presented. Initial results from two studies - face and content validity, are very encouraging. 90% of the cohort of expert robotic surgeons felt that the simulator was excellent or somewhat close to the touch and feel of the daVinci console. Content validity of the simulator received 90% approval in some cases. These studies demonstrate that RoSS has the potential of becoming an important training tool for the daVinci surgical robot.

  14. The Structure of Barmah Forest Virus as Revealed by Cryo-Electron Microscopy at a 6-Angstrom Resolution Has Detailed Transmembrane Protein Architecture and Interactions ▿ †

    PubMed Central

    Kostyuchenko, Victor A.; Jakana, Joanita; Liu, Xiangan; Haddow, Andrew D.; Aung, Myint; Weaver, Scott C.; Chiu, Wah; Lok, Shee-Mei

    2011-01-01

    Barmah Forest virus (BFV) is a mosquito-borne alphavirus that infects humans. A 6-Å-resolution cryo-electron microscopy three-dimensional structure of BFV exhibits a typical alphavirus organization, with RNA-containing nucleocapsid surrounded by a bilipid membrane anchored with the surface proteins E1 and E2. The map allows details of the transmembrane regions of E1 and E2 to be seen. The C-terminal end of the E2 transmembrane helix binds to the capsid protein. Following the E2 transmembrane helix, a short α-helical endodomain lies on the inner surface of the lipid envelope. The E2 endodomain interacts with E1 transmembrane helix from a neighboring E1-E2 trimeric spike, thereby acting as a spacer and a linker between spikes. In agreement with previous mutagenesis studies, the endodomain plays an important role in recruiting other E1-E2 spikes to the budding site during virus assembly. The E2 endodomain may thus serve as a target for antiviral drug design. PMID:21752915

  15. Regional hyperthermia in conjunction with definitive radiotherapy against recurrent or locally advanced prostate cancer T3 pN0 M0.

    PubMed

    Tilly, Wolfgang; Gellermann, Johanna; Graf, Reinhold; Hildebrandt, Bert; Weissbach, Lothar; Budach, Volker; Felix, Roland; Wust, Peter

    2005-01-01

    Since long-term results of the standard treatment of locally advanced or recurrent prostatic carcinoma are unsatisfactory, the role for additional regional hyperthermia was evaluated in a phase I/II study. From 08/1996 to 03/2000, 22 patients were treated by a standard irradiation regimen (68.4 Gy) in combination with regional hyperthermia (weekly, five to six times), and five of 22 patients received short-term (neoadjuvant) hormonal treatment. Of these, 15 patients had primary prostatic carcinoma T3 pN0 M0 and seven a histologically confirmed local recurrence after radical prostatectomy. Feasibility of hyperthermia, and acute/late toxicity as well as long-term follow-up (prostate- specific antigen [PSA] control, overall survival) were analyzed. Clinical endpoints were correlated with thermal parameters. Mean maximum temperatures along the urethra of 41.4 degrees C (41.0 degrees C for the recurrences), and mean T(90) values of 40.7 degrees C could be achieved. Severe acute toxicity of grade 3 occurred at the rectum in three, at the urethra in four, at the intestine in one, and a burn induced by hyperthermia in one of 22 patients. Late toxicity was only observed rectally in one patient (grade 3) and at the urethra in two patients (grade 2). There was no correlation between thermal parameters and any toxicity. The survival curves showed a PSA control for primary prostatic carcinoma > 50% after 6 years, but no long-term PSA control for the recurrences. Overall survival after 6 years was 95% for primary carcinoma, and 60% for the recurrences. There was a clear correlation between higher temperatures or thermal doses with long-term PSA control. Regional hyperthermia might be a low-toxicity approach to increase PSA control of common treatment schedules. Further evaluation, in particular employing improved hyperthermia technology, is worthwhile.

  16. Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix

    PubMed Central

    Öjemalm, Karin; Halling, Katrin K.; Nilsson, IngMarie; von Heijne, Gunnar

    2013-01-01

    Summary α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α-helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α-helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (Ncyt-Clum or Nlum-Ccyt) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the ‘missing hydrophobicity’ in polytopic membrane proteins. PMID:22281052

  17. Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

    PubMed

    Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

    2014-01-01

    The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.

  18. Rapid ammonia gas transport accounts for futile transmembrane cycling under NH3/NH4+ toxicity in plant roots.

    PubMed

    Coskun, Devrim; Britto, Dev T; Li, Mingyuan; Becker, Alexander; Kronzucker, Herbert J

    2013-12-01

    Futile transmembrane NH3/NH4(+) cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4(+) toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4(+)) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope (13)N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4(+) ion. Influx of (13)NH3/(13)NH4(+), which exceeded 200 µmol g(-1) h(-1), was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4(+)), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g(-1) h(-1)). Efflux of (13)NH3/(13)NH4(+) responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.

  19. Genetic diversity of clinical and environmental Vibrio parahaemolyticus strains from the Pacific Northwest.

    PubMed

    Paranjpye, Rohinee; Hamel, Owen S; Stojanovski, Asta; Liermann, Martin

    2012-12-01

    Since 1997, cases of Vibrio parahaemolyticus-related gastroenteritis from the consumption of raw oysters harvested in Washington State have been higher than historical levels. These cases have shown little or no correlation with concentrations of potentially pathogenic V. parahaemolyticus (positive for the thermostable direct hemolysin gene, tdh) in oysters, although significant concentrations of tdh(+) V. parahaemolyticus strains were isolated from shellfish-growing areas in the Pacific Northwest (PNW). We compared clinical and environmental strains isolated from the PNW to those from other geographic regions within the United States and Asia for the presence of virulence-associated genes, including the thermostable direct hemolysin (tdh), the thermostable-related hemolysin (trh), urease (ureR), the pandemic group specific markers orf8 and toxRS, and genes encoding both type 3 secretion systems (T3SS1 and T3SS2). The majority of clinical strains from the PNW were positive for tdh, trh, and ureR genes, while a significant proportion of environmental isolates were tdh(+) but trh negative. Hierarchical clustering grouped the majority of these clinical isolates into a cluster distinct from that including the pandemic strain RIMD2210633, clinical isolates from other geographical regions, and tdh(+), trh-negative environmental isolates from the PNW. We detected T3SS2-related genes (T3SS2β) in environmental strains that were tdh and trh negative. The presence of significant concentrations of tdh(+), trh-negative environmental strains in the PNW that have not been responsible for illness and T3SS2β in tdh- and trh-negative strains emphasizes the diversity in this species and the need to identify additional virulence markers for this bacterium to improve risk assessment tools for the detection of this pathogen.

  20. The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies

    PubMed Central

    Pant, Kiran; Anderson, Brian; Perdana, Hendrik; Malinowski, Matthew A.; Win, Aye T.; Williams, Mark C.

    2018-01-01

    The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes. PMID:29634784

  1. De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies.

    PubMed

    Sundaram, Roshni; Lynch, Marcus P; Rawale, Sharad V; Sun, Yiping; Kazanji, Mirdad; Kaumaya, Pravin T P

    2004-06-04

    Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.

  2. Substitution of lysine-181 to aspartic acid in the third transmembrane region of the endothelin (ET) type B receptor selectively reduces its high-affinity binding with ET-3 peptide.

    PubMed

    Mauzy, C; Wu, L H; Egloff, A M; Mirzadegan, T; Chung, F Z

    1992-01-01

    In the G protein-coupled receptor family, a highly conserved aspartic acid located within the third transmembrane domain has been shown to be involved in ligand binding. Within the endothelin (ET) peptide receptor family, this aspartic acid has been replaced by a lysine. To assess the importance of this residue in ET binding, the lysine (position 181) of rat ET type B receptor was replaced by an aspartic acid. The effects on ligand binding and phosphoinositide turnover of both the wild-type and K181D mutant receptors were examined using transient receptor expression in COS-7 cells. Using [125I]ET-1 as the radioactive peptide ligand in displacement binding studies, the wild-type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three ET peptides (ET-1, ET-2, and ET-3). The mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM). The K181D mutant receptor still elicited full inositol phosphate (IP) accumulation responses in the presence of saturating concentrations of ETs (10 nM of ET-1, 100 nM of ET-2, or 1 microM of ET-3), indicating that the mutation did not affect G protein coupling.

  3. Reconstruction of 7T-Like Images From 3T MRI

    PubMed Central

    Bahrami, Khosro; Shi, Feng; Zong, Xiaopeng; Shin, Hae Won; An, Hongyu

    2016-01-01

    In the recent MRI scanning, ultra-high-field (7T) MR imaging provides higher resolution and better tissue contrast compared to routine 3T MRI, which may help in more accurate and early brain diseases diagnosis. However, currently, 7T MRI scanners are more expensive and less available at clinical and research centers. These motivate us to propose a method for the reconstruction of images close to the quality of 7T MRI, called 7T-like images, from 3T MRI, to improve the quality in terms of resolution and contrast. By doing so, the post-processing tasks, such as tissue segmentation, can be done more accurately and brain tissues details can be seen with higher resolution and contrast. To do this, we have acquired a unique dataset which includes paired 3T and 7T images scanned from same subjects, and then propose a hierarchical reconstruction based on group sparsity in a novel multi-level Canonical Correlation Analysis (CCA) space, to improve the quality of 3T MR image to be 7T-like MRI. First, overlapping patches are extracted from the input 3T MR image. Then, by extracting the most similar patches from all the aligned 3T and 7T images in the training set, the paired 3T and 7T dictionaries are constructed for each patch. It is worth noting that, for the training, we use pairs of 3T and 7T MR images from each training subject. Then, we propose multi-level CCA to map the paired 3T and 7T patch sets to a common space to increase their correlations. In such space, each input 3T MRI patch is sparsely represented by the 3T dictionary and then the obtained sparse coefficients are used together with the corresponding 7T dictionary to reconstruct the 7T-like patch. Also, to have the structural consistency between adjacent patches, the group sparsity is employed. This reconstruction is performed with changing patch sizes in a hierarchical framework. Experiments have been done using 13 subjects with both 3T and 7T MR images. The results show that our method outperforms previous

  4. A Brassica oleracea gene expressed in a variety-specific manner may encode a novel plant transmembrane receptor.

    PubMed

    Palmer, J E; Dikeman, D A; Fujinuma, T; Kim, B; Jones, J I; Denda, M; Martínez-Zapater, J M; Cruz-Alvarez, M

    2001-04-01

    The species Brassica oleracea includes several agricultural varieties characterized by the proliferation of different types of meristems. Using a combination of subtractive hybridization and PCR (polymerase chain reaction) techniques we have identified several genes which are expressed in the reproductive meristems of the cauliflower curd (B. oleracea var. botrytis) but not in the vegetative meristems of Brussels sprouts (B. oleracea var. gemmifera) axillary buds. One of the cloned genes, termed CCE1 (CAULIFLOWER CURD EXPRESSION 1) shows specific expression in the botrytis variety. Preferential expression takes place in this variety in the meristems of the curd and in the stem throughout the vegetative and reproductive stages of plant growth. CCE1 transcripts are not detected in any of the organs of other B. oleracea varieties analyzed. Based on the nucleotide sequence of a cDNA encompassing the complete coding region, we predict that this gene encodes a transmembrane protein, with three transmembrane domains. The deduced amino acid sequence includes motifs conserved in G-protein-coupled receptors (GPCRs) from yeast and animal species. Our results suggest that the cloned gene encodes a protein belonging to a new, so far unidentified, family of transmembrane receptors in plants. The expression pattern of the gene suggests that the receptor may be involved in the control of meristem development/arrest that takes place in cauliflower.

  5. Transmembrane proteins of tight junctions.

    PubMed

    Chiba, Hideki; Osanai, Makoto; Murata, Masaki; Kojima, Takashi; Sawada, Norimasa

    2008-03-01

    Tight junctions contribute to the paracellular barrier, the fence dividing plasma membranes, and signal transduction, acting as a multifunctional complex in vertebrate epithelial and endothelial cells. The identification and characterization of the transmembrane proteins of tight junctions, claudins, junctional adhesion molecules (JAMs), occludin and tricellulin, have led to insights into the molecular nature of tight junctions. We provide an overview of recent progress in studies on these proteins and highlight their roles and regulation, as well as their functional significance in human diseases.

  6. First Optical observation of a microquasar at sub-milliarsec scale: SS 433 resolved by VLTI/GRAVITY

    NASA Astrophysics Data System (ADS)

    Petrucci, P.; Waisberg, I.; Lebouquin, J.; Dexter, J.; Dubus, G.; Perraut, K.; Kervella, P.; Gravity Collaboration

    2017-10-01

    We present the first Optical observation at sub-milliarcsec (mas) scale of the famous microquasar SS 433 obtained with the GRAVITY instrument on the VLTI interferometer. This observation reveals the SS 433 inner regions with unprecedent details: The K-band continuum emitting region is dominated by a marginally resolved point source (< 1 mas) embedded inside a diffuse background accounting for 10% of the total flux. The significant visibility drop across the jet lines present in the K-band spectrum, together with the small and nearly identical phases for all baselines, point toward a jet that is offset by < 0.5 mas from the continuum source and resolved in the direction of propagation, with a size of ˜2 mas. Jet emission so close to the central binary system implies that line locking, if relevant to explain the 0.26c jet velocity, operates on elements heavier than hydrogen. Concerning The Brγ line, it is better resolved than the continuum and the S-shape phase signal present across the line suggests an East-West oriented geometry alike the jet direction and supporting a (polar) disk wind origin. This observation show the potentiality of Optical interferometry to constrain the inner regions of high energy sources like microquasars.

  7. Swine interferon-induced transmembrane protein, sIFITM3, inhibits foot-and-mouth disease virus infection in vitro and in vivo.

    PubMed

    Xu, Jinfang; Qian, Ping; Wu, Qunfeng; Liu, Shasha; Fan, Wenchun; Zhang, Keshan; Wang, Rong; Zhang, Huawei; Chen, Huanchun; Li, Xiangmin

    2014-09-01

    The interferon-induced transmembrane protein 3 (IFITM3) is a widely expressed potent antiviral effector of the host innate immune system. It restricts a diverse group of pathogenic, enveloped viruses, by interfering with endosomal fusion. In this report, the swine IFITM3 (sIFITM3) gene was cloned. It shares the functionally conserved CD225 domain and multiple critical amino acid residues (Y19, F74, F77, R86 and Y98) with its human ortholog, which are essential for antiviral activity. Ectopic expression of sIFITM3 significantly inhibited non-enveloped foot-and-mouth disease virus (FMDV) infection in BHK-21 cells. Furthermore, sIFITM3 blocked FMDV infection at early steps in the virus life cycle by disrupting viral attachment to the host cell surface. Importantly, inoculation of 2-day-old suckling mice with a plasmid expressing sIFITM3 conferred protection against lethal challenge with FMDV. These results suggest that sIFITM3 is a promising antiviral agent and that can safeguard the host from infection with FMDV. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Magnetic field analysis of the bow and terminal shock of the SS 433 jet

    NASA Astrophysics Data System (ADS)

    Sakemi, Haruka; Machida, Mami; Akahori, Takuya; Nakanishi, Hiroyuki; Akamatsu, Hiroki; Kurahara, Kohei; Farnes, Jamie

    2018-03-01

    We report a polarization analysis of the eastern region of W 50, observed with the Australia Telescope Compact Array (ATCA) at 1.4-3.0 GHz. In order to study the physical structures in the region where the SS 433 jet and W 50 interact, we obtain an intrinsic magnetic field vector map of that region. We find that the orientation of the intrinsic magnetic field vectors are aligned along the total intensity structures, and that there are characteristic, separate structures related to the jet, the bow shock, and the terminal shock. The Faraday rotation measures (RMs), and the results of Faraday tomography suggest that a high-intensity, filamentary structure in the north-south direction of the eastern-edge region can be separated into at least two parts to the north and south. The results of Faraday tomography also show that there are multiple components along the line of sight and/or within the beam area. In addition, we analyze the X-ray ring-like structure observed with XMM-Newton. While the possibility still remains that this X-ray ring is "real", it seems that the structure is not ring-like at radio wavelengths. Finally, we suggest that the structure is a part of the helical structure that coils the eastern ear of W 50.

  9. Pancreatic Duct in Autoimmune Pancreatitis: Intraindividual Comparison of Magnetic Resonance Pancreatography at 1.5 T and 3.0 T.

    PubMed

    Kim, Jin Hee; Byun, Jae Ho; Kim, Myung-Hwan; Lee, Sung Koo; Kim, Song Cheol; Kim, Hyoung Jung; Lee, Seung Soo; Kim, So Yeon; Lee, Moon-Gyu

    2017-08-01

    The aim of this study was to intraindividually compare magnetic resonance pancreatography (MRP) image quality at 1.5 T and 3.0 T when demonstrating main pancreatic duct (MPD) abnormalities in patients with autoimmune pancreatitis (AIP). Thirty prospectively enrolled patients with AIP underwent MRP at both 1.5 T and 3.0 T followed by endoscopic retrograde pancreatography before treatment. Two readers independently analyzed the MRP images and graded the visualization of MPD strictures and full-length MPD, using endoscopic retrograde pancreatography as the reference standard, as well as overall image artifacts on a 4-point scale. The contrast between the MPD and periductal area was calculated using a region-of-interest measurement. Visualization scores of MPD strictures and full-length MPD, and summed scores of each qualitative analysis, were significantly greater at 3.0-T MRP than at 1.5-T MRP for both readers (P ≤ 0.02). There were less image artifacts at 3.0 T compared with 1.5 T (P ≤ 0.052). The contrast between the MPD and periductal area was significantly greater at 3.0-T MRP than at 1.5-T MRP (P < 0.001). The MRP at 3.0 T was superior to 1.5-T MRP for demonstrating MPD abnormalities in AIP, with better image contrast and fewer image artifacts. Consequently, 3.0-T MRP may be useful for the diagnosis and management of patients with AIP.

  10. New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome.

    PubMed

    Tsuboi, H; Matsumoto, I; Wakamatsu, E; Nakamura, Y; Iizuka, M; Hayashi, T; Goto, D; Ito, S; Sumida, T

    2010-10-01

    M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca(2+) concentrations [(Ca(2+) )i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca(2+) )i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca(2+) )i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

  11. Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling

    PubMed Central

    North, Justin A.; Javaid, Sarah; Ferdinand, Michelle B.; Chatterjee, Nilanjana; Picking, Jonathan W.; Shoffner, Matthew; Nakkula, Robin J.; Bartholomew, Blaine; Ottesen, Jennifer J.; Fishel, Richard; Poirier, Michael G.

    2011-01-01

    Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA–histone binding by 2 kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2–hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions. PMID:21576235

  12. The APOC3 T-455C and C-482T promoter region polymorphisms are not associated with the severity of liver damage independently of PNPLA3 I148M genotype in patients with nonalcoholic fatty liver.

    PubMed

    Valenti, Luca; Nobili, Valerio; Al-Serri, Ahmad; Rametta, Raffaela; Leathart, Julian B S; Zappa, Marco A; Dongiovanni, Paola; Fracanzani, Anna L; Alterio, Arianna; Roviaro, Giancarlo; Daly, Ann K; Fargion, Silvia; Day, Christopher P

    2011-12-01

    The T-455C and C-482T APOC3 promoter region polymorphisms (SNPs) have recently been reported to predispose to dyslipidemia, insulin resistance, and nonalcoholic fatty liver disease (NAFLD) in Indian subjects, but the association with liver damage has not been evaluated so far. The aim was to assess the association between APOC3 SNPs and liver damage in Caucasian patients. We considered 437 Italian patients with histological diagnosis of NAFLD (including 137 children, 120 morbid obese) and 316 healthy controls, 71 Italian family trios, and 321 patients from the UK. APOC3 SNPs were determined by sequencing, allele-specific oligonucleotide probes and PCR-restriction fragment length polymorphism analysis, hepatic APOC3 mRNA levels by real-time PCR. APOC3 SNPs were not associated with NAFLD in Italian subjects, although a borderline significance for the transmission of the -455T allele was observed in the family study. Homozygosity for the APOC3 wild-type genotype (APOC3 WT) was associated with a more favorable lipid profile in control subjects, and consistently with lower hepatic APOC3 mRNA levels in obese patients without diabetes. However, APOC3 SNPs, alone or in combination, were not associated with insulin resistance, altered lipid levels, liver enzymes, and with liver damage (severity of steatosis, nonalcoholic steatohepatitis, and moderate/severe fibrosis) in Italian as well as in UK patients, and in the whole cohort. Stratification for the I148M PNPLA3 mutation, associated with the susceptibility to NASH, did not alter the results. APOC3 genotype is not associated with progressive liver damage in Caucasian patients with NAFLD. Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  13. Primary and Secondary Dimer Interfaces of the FGFR3 Transmembrane Domain: Characterization via Multiscale Molecular Dynamics Simulations

    PubMed Central

    Reddy, Tyler; Manrique, Santiago; Buyan, Amanda; Hall, Benjamin A.; Chetwynd, Alan; Sansom, Mark S.P.

    2016-01-01

    Receptor tyrosine kinases are single pass membrane proteins which form dimers within the membrane. The interactions of their transmembrane domains (TMDs) play a key role in dimerization and signaling. The fibroblast growth factor receptor 3 (FGFR3) is of interest as a G380R mutation in its TMD is the underlying cause of ~99% of cases of achondroplasia, the most common form of human dwarfism. The structural consequences of this mutation remain uncertain: the mutation shifts the position relative of the TMD relative to the lipid bilayer but does not alter the association free energy. We have combined coarse-grained and all-atom molecular dynamics simulations to study the dimerization of wild-type, heterodimer, and mutant FGFR3 TMDs. The simulations reveal that the helices pack together in the dimer to form a flexible interface. The primary packing mode is mediated by a Gx3G motif. There is also a secondary dimer interface which is more highly populated in heterodimer and mutant configurations which may feature in the molecular mechanism of pathology. Both coarse-grained and atomistic simulations reveal a significant shift of the G380R mutant dimer TMD relative to the bilayer so as to enable interactions of the arginine sidechain with lipid head group phosphates. PMID:24397339

  14. The hyper-diverse ant genus Tetramorium Mayr (Hymenoptera, Formicidae) in the Malagasy region taxonomic revision of the T. naganum, T. plesiarum, T. schaufussii, and T. severini species groups

    PubMed Central

    Hita Garcia, Francisco; Fisher, Brian L.

    2014-01-01

    Abstract The taxonomy of the Tetramorium naganum, T. plesiarum, T. schaufussii, and T. severini species groups are revised for the Malagasy region. A total of 31 species are treated, of which 22 are newly described and nine redescribed. This increases the richness of the hyper-diverse genus Tetramorium in the Malagasy region to 106 species, which makes it the most species-rich genus in the region. Twenty-nine of the treated species are endemic to Madagascar, one is endemic to the Comoros, and one species is found predominantly in Madagascar but also on the island of Reunion. The T. naganum species group contains five species, which are mainly distributed in the rainforests and montane rainforests of eastern and northern Madagascar: T. alperti sp. n., T. dalek sp. n., T. enkidu sp. n., T. gilgamesh sp. n., and T. naganum Bolton, 1979. The T. plesiarum species group holds five species: T. bressleri sp. n., T. hobbit sp. n., T. gollum sp. n., T. mars sp. n., and T. plesiarum Bolton, 1979. All five are arid-adapted species occurring in the southwest and west of Madagascar. The second-most species-rich group in the region is the T. schaufussii species group with 20 species, most of which inhabit rainforests or montane rainforests of eastern and northern Madagascar. This group includes two species complexes each containing ten species: the T. cognatum complex with the species T. aspis sp. n., T. camelliae sp. n., T. cognatum Bolton, 1979, T. freya sp. n., T. gladius sp. n., T. karthala sp. n., T. myrmidon sp. n., T. proximum Bolton, 1979, T. rumo sp. n., and T. tenuinode sp. n.; and the T. schaufussii complex with the species T. merina sp. n., T. monticola sp. n., T. nassonowii Forel, 1892 stat. n., T. obiwan sp. n., T. pseudogladius sp. n., T. rala sp. n., T. schaufussii Forel, 1891, T. sikorae Forel, 1892 (= T. latior (Santschi, 1926)), T. scutum sp. n., T. xanthogaster Santschi, 1911. The last group treated in this study is the T. severini species group, which

  15. Quantitative Mapping of Human Cartilage at 3.0T

    PubMed Central

    Wang, Ligong; Regatte, Ravinder R.

    2014-01-01

    Rationale and Objectives The objectives of this study were to measure the parallel changes of transverse relaxation times (T2), spin-lattice relaxation time in the rotating frame (T1ρ), and the delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC)-T1 mapping of human knee cartilage in detecting cartilage degeneration at 3.0T. Materials and Methods Healthy volunteers (n = 10, mean age 35.6 years) and patients (n = 10, mean age 65 years) with early knee osteoarthritis (OA) were scanned at 3.0T MR using an 8-channel phased array knee coil (transmit–receive). Quantitative assessment of T2, T1ρ, and dGEMRIC-T1 values (global and regional) were correlated between asymptomatic subjects and patients with OA. Results The average T2 (39 ± 2 milliseconds [mean ± standard deviation] vs. 47 ± 6 milliseconds, P < .0007) and T1ρ (48 ± 3 vs. 62 ± 8 milliseconds, P < .0002) values were all markedly increased in all patients with OA when compared to healthy volunteers. The average dGEMRIC-T1 (1244 ± 134 vs. 643 ± 227 milliseconds, P < .000002) value was sharply decreased after intravenous administration of gadolinium contrast agent in all patients with OA. Conclusions The research results showed that all the T2, T1ρ, and dGEMRIC-T1 relaxation times varied with the cartilage degeneration. The dGEMRIC-T1 and T1ρ relaxation times seem to be more sensitive than T2 in detecting early cartilage degeneration. The preliminary study demonstrated that the early biochemical changes in knee osteoarthritic patients could be detected noninvasively in in vivo using T1ρ and dGEMRIC-T1 mapping. PMID:24594416

  16. Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

    PubMed Central

    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

  17. Identification of eight mutations and three sequence variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene

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

    Ghanem, N.; Costes, B.; Girodon, E.

    1994-05-15

    To determine cystic fibrosis (CF) defects in a sample of 224 non-[Delta]F508 CF chromosomes, the authors used denaturing gradient gel multiplex analysis of CF transmembrane conductance regulator gene segments, a strategy based on blind exhaustive analysis rather than a search for known mutations. This process allowed detection of 11 novel variations comprising two nonsense mutations (Q890X and W1204X), a splice defect (405 + 4 A [yields] G), a frameshift (3293delA), four presumed missense mutations (S912L, H949Y, L1065P, Q1071P), and three sequence polymorphisms (R31C or 223 C/T, 3471 T/C, and T1220I or 3791 C/T). The authors describe these variations, together withmore » the associated phenotype when defects on both CF chromosomes were identified. 8 refs., 1 fig., 1 tab.« less

  18. The Role of HuR in the Post-Transcriptional Regulation of Interleukin-3 in T Cells

    PubMed Central

    González-Feliciano, José A.; Hernández-Pérez, Marimar; Estrella, Luis A.; Colón-López, Daisy D.; López, Armando; Martínez, Marina; Maurás-Rivera, Kirla R.; Lasalde, Clarivel; Martínez, Daviana; Araujo-Pérez, Félix; González, Carlos I.

    2014-01-01

    Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells. PMID:24658545

  19. Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

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

    Hajra, A.; Liu, P.; Collins, E.S.

    1994-09-01

    A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicatedmore » by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.« less

  20. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    PubMed Central

    Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

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

    PubMed

    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.

  2. Study of advanced communications satellite systems based on SS-FDMA

    NASA Technical Reports Server (NTRS)

    Kiesling, J.

    1980-01-01

    A satellite communication system based on the use of a multiple, contiguous beam satellite antenna and frequency division multiple access (FDMA) is studied. Emphasis is on the evaluation of the feasibility of SS (satellite switching) FDMA technology, particularly the multiple, contiguous beam antenna, the onboard switch and channelization, and on methods to overcome the effects of severe Ka band fading caused by precipitation. This technology is evaluated and plans for technology development and evaluation are given. The application of SS-FDMA to domestic satellite communications is also evaluated. Due to the potentially low cost Earth stations, SS-FDMA is particularly attractive for thin route applications up to several hundred kilobits per second, and offers the potential for competing with terrestrial facilities at low data rates and over short routes. The onboard switch also provides added route flexibility for heavy route systems. The key beneficial SS-FDMA strategy is to simplify and thus reduce the cost of the direct access Earth station at the expense of increased satellite complexity.

  3. Targeting Fibroblast Activation Protein in Tumor Stroma with Chimeric Antigen Receptor T Cells Can Inhibit Tumor Growth and Augment Host Immunity Without Severe Toxicity

    PubMed Central

    Wang, Liang-Chuan S; Lo, Albert; Scholler, John; Sun, Jing; Majumdar, Rajrupa S; Kapoor, Veena; Antzis, Michael; Cotner, Cody E.; Johnson, Laura A; Durham, Amy C; Solomides, Charalambos C.; June, Carl H; Puré, Ellen; Albelda, Steven M

    2013-01-01

    The majority of chimeric antigen receptor (CAR) T cell research has focused on attacking cancer cells. Here we show that targeting the tumor-promoting, non-transformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single chain Fv FAP (mAb 73.3) with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFNγ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAPhi stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8+ T cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective suggesting that further clinical development of anti-human FAP-CAR is warranted. PMID:24778279

  4. Re-Introduction of Transmembrane Serine Residues Reduce the Minimum Pore Diameter of Channelrhodopsin-2

    PubMed Central

    Richards, Ryan; Dempski, Robert E.

    2012-01-01

    Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2. PMID:23185520

  5. [Anti-M3 muscarinic acetylcholine receptor antibodies and Sjögren's syndrome].

    PubMed

    Tsuboi, Hiroto; Iizuka, Mana; Asashima, Hiromitsu; Sumida, Takayuki

    2013-01-01

    Sjögren's syndrome (SS) is an autoimmune disease that affects exocrine glands including salivary and lacrimal glands. It is characterized by lymphocytic infiltration into exocrine glands, leading to dry mouth and eyes. A number of auto-antibodies are detected in patients with SS. However, no SS-specific pathologic auto-antibodies have yet been found in this condition. M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva. It is reported that some patients with SS carried inhibitory auto-antibodies against M3R. To clarify the epitopes and function of anti-M3R antibodies in SS, we examined antibodies to the extracellular domains (N terminal region, the first, second, and third extracellular loop) of M3R by ELISA using synthesized peptide antigens encoding these domains in 42 SS and 42 healthy controls (HC). Titers and positivity of anti-M3R antibodies to every extracellular domain of M3R were significantly higher in SS than in HC. Our results indicated the presence of several B cell epitopes on M3R in SS. Moreover, we analyzed the functions of anti-M3R antibodies by Ca(2+)-influx assays using a human salivary gland (HSG) cell line. The functional analysis indicated that the influence of such anti-M3R antibodies on Ca(2+)-influx in HSG cells might differ based on the epitopes to which they bind. Interestingly, both IgG from anti-M3R antibodies to the second extracellular loop positive SS and anti-M3R monoclonal antibodies against the second extracellular loop of M3R, which we generated, suppressed Ca(2+)-influx in the HSG cells induced by cevimeline stimulation. These observations suggested that auto-antibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS. These results indicated the presence of several B cell epitopes on M3R in SS and the influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. Thus, anti-M3R antibodies could be not

  6. Seven-pass transmembrane cadherins: roles and emerging mechanisms in axonal and dendritic patterning.

    PubMed

    Berger-Müller, Sandra; Suzuki, Takashi

    2011-12-01

    The Flamingo/Celsr seven-transmembrane cadherins represent a conserved subgroup of the cadherin superfamily involved in multiple aspects of development. In the developing nervous system, Fmi/Celsr control axonal blueprint and dendritic morphogenesis from invertebrates to mammals. As expected from their molecular structure, seven-transmembrane cadherins can induce cell-cell homophilic interactions but also intracellular signaling. Fmi/Celsr is known to regulate planar cell polarity (PCP) through interactions with PCP proteins. In the nervous system, Fmi/Celsr can function in collaboration with or independently of other PCP genes. Here, we focus on recent studies which show that seven-transmembrane cadherins use distinct molecular mechanisms to achieve diverse functions in the development of the nervous system.

  7. Insights into Autoregulation of Notch3 from Structural and Functional Studies of Its Negative Regulatory Region.

    PubMed

    Xu, Xiang; Choi, Sung Hee; Hu, Tiancen; Tiyanont, Kittichoat; Habets, Roger; Groot, Arjan J; Vooijs, Marc; Aster, Jon C; Chopra, Rajiv; Fryer, Christy; Blacklow, Stephen C

    2015-07-07

    Notch receptors are transmembrane proteins that undergo activating proteolysis in response to ligand stimulation. A negative regulatory region (NRR) maintains receptor quiescence by preventing protease cleavage prior to ligand binding. We report here the X-ray structure of the NRR of autoinhibited human Notch3, and compare it with the Notch1 and Notch2 NRRs. The overall architecture of the autoinhibited conformation, in which three LIN12-Notch repeat (LNR) modules wrap around a heterodimerization domain, is preserved in Notch3, but the autoinhibited conformation of the Notch3 NRR is less stable. The Notch3 NRR uses a highly conserved surface on the third LNR module to form a dimer in the crystal. Similar homotypic interfaces exist in Notch1 and Notch2. Together, these studies reveal distinguishing structural features associated with increased basal activity of Notch3, demonstrate increased ligand-independent signaling for disease-associated mutations that map to the Notch3 NRR, and identify a conserved dimerization interface present in multiple Notch receptors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Remote Sensing and Geologic Studies of the Schiller-Schickard Region of the Moon

    NASA Technical Reports Server (NTRS)

    Blewett, David T.; Hawke, B. Ray; Lucey, Paul G.; Taylor, G. Jeffrey; Jaumann, Ralf; Spudis, Paul D.

    1995-01-01

    Near-infrared reflectance spectra, multispectral images, and photogeologic data for the Schiller-Schickard (SS) region were obtained and analyzed in order to determine the composition and origin of a variety of geologic units. These include light plains deposits, Orientale-related deposits, mare units, and dark-haloed impact craters (DHCs). Spectral data indicate that the pre-Orientale highland surface was dominated by noritic anorthosite. Near-IR spectra show that DHCs in the region have excavated ancient (greater than 3.8 Ga) mare basalts from beneath highland-bearing material emplaced by the Orientale impact. Ancient mare basalts were widespread in the SS region prior to the Orientale event, and their distribution appears to have been controlled by the presence of several old impact basins, including the Schiller-Zucchius basin and a basin previously unrecognized. Both near-IR spectra and multispectral images indicate that light plains and other Orientale-related units in the SS region contain major amounts of local, pre-Orientale mare basalt. The amounts of local material in these deposits approach, but seldom exceed, the maximum values predicted by the local mixing hypothesis of Oberbeck and co-workers.

  9. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

    PubMed Central

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-01-01

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

  10. Novel Positive-Sense, Single-Stranded RNA (+ssRNA) Virus with Di-Cistronic Genome from Intestinal Content of Freshwater Carp (Cyprinus carpio)

    PubMed Central

    Pankovics, Péter; Simmonds, Peter

    2011-01-01

    A novel positive-sense, single-stranded RNA (+ssRNA) virus (Halastavi árva RNA virus, HalV; JN000306) with di-cistronic genome organization was serendipitously identified in intestinal contents of freshwater carps (Cyprinus carpio) fished by line-fishing from fishpond “Lőrinte halastó” located in Veszprém County, Hungary. The complete nucleotide (nt) sequence of the genomic RNA is 9565 nt in length and contains two long - non-in-frame - open reading frames (ORFs), which are separated by an intergenic region. The ORF1 (replicase) is preceded by an untranslated sequence of 827 nt, while an untranslated region of 139 nt follows the ORF2 (capsid proteins). The deduced amino acid (aa) sequences of the ORFs showed only low (less than 32%) and partial similarity to the non-structural (2C-like helicase, 3C-like cystein protease and 3D-like RNA dependent RNA polymerase) and structural proteins (VP2/VP4/VP3) of virus families in Picornavirales especially to members of the viruses with dicistronic genome. Halastavi árva RNA virus is present in intestinal contents of omnivorous freshwater carps but the origin and the host species of this virus remains unknown. The unique viral sequence and the actual position indicate that Halastavi árva RNA virus seems to be the first member of a new di-cistronic ssRNA virus. Further studies are required to investigate the specific host species (and spectrum), ecology and role of Halastavi árva RNA virus in the nature. PMID:22195010

  11. Cytosolic T3-binding protein modulates dynamic alteration of T3-mediated gene expression in cells.

    PubMed

    Takeshige, Keiko; Sekido, Takashi; Kitahara, Jun-ichirou; Ohkubo, Yousuke; Hiwatashi, Dai; Ishii, Hiroaki; Nishio, Shin-ichi; Takeda, Teiji; Komatsu, Mitsuhisa; Suzuki, Satoru

    2014-01-01

    μ-Crystallin (CRYM) is also known as NADPH-dependent cytosolic T3-binding protein. A study using CRYM-null mice suggested that CRYM stores triiodothyronine (T3) in tissues. We previously established CRYM-expressing cells derived from parental GH3 cells. To examine the precise regulation of T3-responsive genes in the presence of CRYM, we evaluated serial alterations of T3-responsive gene expression by changing pericellular T3 concentrations in the media. We estimated the constitutive expression of three T3-responsive genes, growth hormone (GH), deiodinase 1 (Dio1), and deiodinase 2 (Dio2), in two cell lines. Subsequently, we measured the responsiveness of these three genes at 4, 8, 16, and 24 h after adding various concentrations of T3. We also estimated the levels of these mRNAs 24 and 48 h after removing T3. The levels of constitutive expression of GH and Dio1 were low and high in C8 cells, respectively, while Dio2 expression was not significantly different between GH3 and C8 cells. When treated with T3, Dio2 expression was significantly enhanced in C8 cells, while there were no differences in GH or Dio1 expression between GH3 and C8 cell lines. In contrast, removal of T3 retained the mRNA expression of GH and Dio2 in C8 cells. These results suggest that CRYM expression increases and sustains the T3 responsiveness of genes in cells, especially with alteration of the pericellular T3 concentration. The heterogeneity of T3-related gene expression is dependent on cellular CRYM expression in cases of dynamic changes in pericellular T3 concentration.

  12. Voxel-based morphometric brain comparison between healthy subjects and major depressive disorder patients in Japanese with the s/s genotype of 5-HTTLPR.

    PubMed

    Igata, Natsuki; Kakeda, Shingo; Watanabe, Keita; Ide, Satoru; Kishi, Taro; Abe, Osamu; Igata, Ryouhei; Katsuki, Asuka; Iwata, Nakao; Yoshimura, Reiji; Korogi, Yukunori

    2017-06-21

    Individuals with s/s genotype of serotonin transporter gene-linked promotor region (5-HTTLPR), which appear with a high frequency in Japanese, exhibit more diagnosable depression in relation to stressful life events than those with the s/l or l/l genotype. We prospectively investigated the brain volume changes in first-episode and medication naïve major depression disorder patients (MDD) with the s/s genotype in Japanese. We assessed the differences between 27 MDD with the s/s genotype and 44 healthy subjects (HS) with the same genotype using a whole-brain voxel-by-voxel statistical analysis of MRI. Gray matter volume in a brain region with significant clusters obtained via voxel-based morphometry analysis were measured and, as an exploratory analysis, evaluated for relationships to the subcategory scores (core, sleep, activity, psychic, somatic anxiety, delusion) of the Hamilton Depression Rating Scale (HAM-D) and the Social Readjustment Rating Scale (SRRS). The brain volume in the left insula lobe was significantly smaller in the MDD than in the HS. The left insula lobe volume correlated negatively with the "psychic" score of HAM-D and the SRRS. In a Japanese population with the s/s genotype, we found an atrophy of the insula in the MDD, which might be associated with "psychic" symptom and stress events.

  13. Structure and mechanism of the phage T4 recombination mediator protein UvsY

    DOE PAGES

    Gajewski, Stefan; Waddell, Michael Brett; Vaithiyalingam, Sivaraja; ...

    2016-03-07

    The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY–ssDNA interaction occurs within the assembly via twomore » distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNA–gp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNA-binding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA–UvsX filament.« less

  14. SS/RCS surface tension propellant acquisition/expulsion tankage technology program

    NASA Technical Reports Server (NTRS)

    1974-01-01

    An evaluation of published propellant physical property data together with bubble point tests of fine-mesh screen in propellants, was conducted. The effort consisted of: (1) the collection and evaluation of pertinent physical property data for hydrazine (N2H4), monomethylhydrazine (MMH), and nitrogen tetroxide (N2O4); (2) testing to determine the effect of dissolved pressurant gas, temperature, purity, and system cleanliness or contamination on system bubble point, and (3) the compilation and publishing of both the literature and test results. The space shuttle reaction control system (SS/RCS) is a bipropellant system using N2O4 and MMH, while the auxiliary power system (SS/APU) employs monopropellant N2H4. Since both the RCS and the APU use a surface tension device for propellant acquisition, the propellant properties of interest are those which impact the design and operation of surface tension systems. Information on propellant density, viscosity, surface tension, and contact angle was collected, compiled, and evaluated.

  15. Molecular characterization of a novel bat-associated circovirus with a poly-T tract in the 3' intergenic region.

    PubMed

    Zhu, Aiwei; Jiang, Tinglei; Hu, Tingsong; Mi, Shijiang; Zhao, Zihan; Zhang, Fuqiang; Feng, Jiang; Fan, Quanshui; He, Biao; Tu, Changchun

    2018-05-02

    The family Circoviridae comprises a large group of small circular single-stranded DNA viruses with several members causing severe pig and poultry diseases. In recent years the number of new viruses within the family has had an explosive increase showing a high level of genetic diversity and a broad host range. In this report we describe two more circoviruses identified from bats in Yunnan and Heilongjiang provinces in China. Full genome sequencing has revealed that these bat associated circoviruses (bat ACV) should be classified as new species within the genus Circovirus based on the demarcation criteria of the International Committee on the Taxonomy of Viruses (ICTV). The most striking result is the novel finding of a 21-28 nt polythymidine (poly-T) tract in the 3' terminal intergenic region of bat ACV isolates from Heilongjiang province. To understand its role in viral replication, a wild type bat ACV and a mutated version with the entire poly-T deleted were rescued through construction of infectious clones. Replication comparison in vitro showed that the poly-T is not essential for viral replication. Identification of additional bat ACV isolates and study of their biological characteristics will be the main task in future to understand the potential roles of bats in transmission of circoviruses to terrestrial mammals and humans. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

    PubMed Central

    Broer, Rene; Boson, Bertrand; Spaan, Willy; Cosset, François-Loïc; Corver, Jeroen

    2006-01-01

    The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity. PMID:16415007

  17. Acanthamoeba belonging to T3, T4, and T11: genotypes isolated from air-conditioning units in Santiago, Chile.

    PubMed

    Astorga, Berbeli; Lorenzo-Morales, Jacob; Martín-Navarro, Carmen M; Alarcón, Verónica; Moreno, Johanna; González, Ana C; Navarrete, Elizabeth; Piñero, José E; Valladares, Basilio

    2011-01-01

    Free-living amoebae (FLA) of the genus Acanthamoeba are widely distributed in the environment, in the air, soil, and water, and have also been isolated from air-conditioning units. The objective of this work was to investigate the presence of this genus of FLA in the air-conditioning equipment at the Institute of Public Health of Chile in Santiago, Chile. Water and air samples were collected from air-conditioning systems and were checked for the presence of Acanthamoeba spp. Positive samples were further classified at the genotype level after sequencing the highly variable diagnostic fragment 3 (DF3) region of the 18S rRNA gene. This is the first report of the T3, T4, and T11 genotypes of Acanthamoeba in air-conditioning units from Chile. Overall, the widespread distribution of potentially pathogenic Acanthamoeba strains in the studied source demands more awareness within the public and health professionals in Chile as this pathogen is emerging as a risk for human health worldwide. © 2011 The Author(s) Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

  18. Transmembrane potential measurements on plant cells using the voltage-sensitive dye ANNINE-6.

    PubMed

    Flickinger, Bianca; Berghöfer, Thomas; Hohenberger, Petra; Eing, Christian; Frey, Wolfgang

    2010-11-01

    The charging of the plasma membrane is a necessary condition for the generation of an electric-field-induced permeability increase of the plasmalemma, which is usually explained by the creation and the growth of aqueous pores. For cells suspended in physiological buffers, the time domain of membrane charging is in the submicrosecond range. Systematic measurements using Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) protoplasts stained with the fast voltage-sensitive fluorescence dye ANNINE-6 have been performed using a pulsed laser fluorescence microscopy setup with a time resolution of 5 ns. A clear saturation of the membrane voltage could be measured, caused by a strong membrane permeability increase, commonly explained by enhanced pore formation, which prevents further membrane charging by external electric field exposure. The field strength dependence of the protoplast's transmembrane potential V (M) shows strong asymmetric saturation characteristics due to the high resting potential of the plants plasmalemma. At the pole of the hyperpolarized hemisphere of the cell, saturation starts at an external field strength of 0.3 kV/cm, resulting in a measured transmembrane voltage shift of ∆V(M) = -150 mV, while on the cathodic (depolarized) cell pole, the threshold for enhanced pore formation is reached at a field strength of approximately 1.0 kV/cm and ∆V(M) = 450 mV, respectively. From this asymmetry of the measured maximum membrane voltage shifts, the resting potential of BY-2 protoplasts at the given experimental conditions can be determined to V(R) = -150 mV. Consequently, a strong membrane permeability increase occurs when the membrane voltage diverges |V(M)| = 300 mV from the resting potential of the protoplast. The largest membrane voltage change at a given external electric field occurs at the cell poles. The azimuthal dependence of the transmembrane potential, measured in angular intervals of 10° along the circumference of the cell, shows a flattening

  19. Damage Analysis of Tensile Deformation of Co-rolled SMATed 304SS

    NASA Astrophysics Data System (ADS)

    Guo, X.; Leung, A. Y. T.; Chen, A.; Ruan, H.; Lu, J.

    2010-05-01

    One of recent experimental progresses in strengthening and toughening metals simultaneously is to adopt techniques of surface mechanical attrition treatment (SMAT) and warm co-rolling to 304 stainless steel (SS). To capture deformation behavior and associated damage initiation/evolution process in the co-rolled SMATed 304SS, cohesive finite element method (CFEM) is employed in this paper and simulation results are in agreement with experimental results. Both strengthening effect due to high yield stress of the nanograin layer and toughening effect due to non-localized damage in the nanograin layer are captured. Effect of energy release rate of nanograin layer on failure strain of layered co-rolled SMATed 304SS is investigated. It is found that the more brittle the nanograin layer is, the more potential necking sites in the nanograin layer are, and the more ductile the layered co-rolled SMATed 304SS is.

  20. A role of carboxy-terminal region of Toxoplasma gondii-heat shock protein 70 in enhancement of T. gondii infection in mice

    PubMed Central

    Mun, Hye-Seong; Norose, Kazumi; Aosai, Fumie; Chen, Mei

    2000-01-01

    We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g.HSP70-NH2-terminal region, or rT.g.HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T. gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region increased the number of T.gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NH2-terminal region did not. These results suggest that T.g.HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya. PMID:10905074

  1. Type II secretion system of Pseudomonas aeruginosa: in vivo evidence of a significant role in death due to lung infection.

    PubMed

    Jyot, Jeevan; Balloy, Viviane; Jouvion, Gregory; Verma, Amrisha; Touqui, Lhousseine; Huerre, Michel; Chignard, Michel; Ramphal, Reuben

    2011-05-15

    The role of toxins secreted by the type II secretion system (T2SS) of Pseudomonas aeruginosa during lung infection has been uncertain despite decades of research. Using a model of pneumonia in Toll-like receptor (TLR) 2,4(-/-) mice, we reexamined the role of the T2SS system. Flagellin-deficient mutants of P. aeruginosa, with mutations in the T2SS and/or T3SS, were used to infect mice. Mice were followed up for survival, with some killed at different intervals to study bacterial clearance, inflammatory responses, and lung pathology. Strains carrying either secretion system were lethal for mice. Double mutants were avirulent. The T3SS(+) strains killed mice within a day, and the T2SS(+) strains killed them later. Mice infected with a strain that had only the T2SS were unable to eradicate the organism from the lungs, whereas those infected with a T2SS-T3SS double deletion were able to clear this mutant. Death caused by the T2SS(+) strain was accompanied by a >50-fold increase in bacterial counts and higher numbers of viable intracellular bacteria. The T2SS of P. aeruginosa may play a role in death from pneumonia, but its action is delayed. These data suggest that antitoxin strategies against this organism will require measures against the toxins secreted by both T2SS and T3SS.

  2. Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ.

    PubMed

    Jeong, Kwang Cheol; Sexton, Jessica A; Vogel, Joseph P

    2015-03-01

    Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.

  3. Structural basis of ligand interaction with atypical chemokine receptor 3

    NASA Astrophysics Data System (ADS)

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

    2017-01-01

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

  4. Evaluation of multimodal segmentation based on 3D T1-, T2- and FLAIR-weighted images - the difficulty of choosing.

    PubMed

    Lindig, Tobias; Kotikalapudi, Raviteja; Schweikardt, Daniel; Martin, Pascal; Bender, Friedemann; Klose, Uwe; Ernemann, Ulrike; Focke, Niels K; Bender, Benjamin

    2018-04-15

    Voxel-based morphometry is still mainly based on T1-weighted MRI scans. Misclassification of vessels and dura mater as gray matter has been previously reported. Goal of the present work was to evaluate the effect of multimodal segmentation methods available in SPM12, and their influence on identification of age related atrophy and lesion detection in epilepsy patients. 3D T1-, T2- and FLAIR-images of 77 healthy adults (mean age 35.8 years, 19-66 years, 45 females), 7 patients with malformation of cortical development (MCD) (mean age 28.1 years,19-40 years, 3 females), and 5 patients with left hippocampal sclerosis (LHS) (mean age 49.0 years, 25-67 years, 3 females) from a 3T scanner were evaluated. Segmentation based on T1-only, T1+T2, T1+FLAIR, T2+FLAIR, and T1+T2+FLAIR were compared in the healthy subjects. Clinical VBM results based on the different segmentation approaches for MCD and for LHS were compared. T1-only segmentation overestimated total intracranial volume by about 80ml compared to the other segmentation methods. This was due to misclassification of dura mater and vessels as GM and CSF. Significant differences were found for several anatomical regions: the occipital lobe, the basal ganglia/thalamus, the pre- and postcentral gyrus, the cerebellum, and the brainstem. None of the segmentation methods yielded completely satisfying results for the basal ganglia/thalamus and the brainstem. The best correlation with age could be found for the multimodal T1+T2+FLAIR segmentation. Highest T-scores for identification of LHS were found for T1+T2 segmentation, while highest T-scores for MCD were dependent on lesion and anatomical location. Multimodal segmentation is superior to T1-only segmentation and reduces the misclassification of dura mater and vessels as GM and CSF. Depending on the anatomical region and the pathology of interest (atrophy, lesion detection, etc.), different combinations of T1, T2 and FLAIR yield optimal results. Copyright © 2017 Elsevier

  5. Influence of magnesium sulfate on HCO3/Cl transmembrane exchange rate in human erythrocytes.

    PubMed

    Chernyshova, Ekaterina S; Zaikina, Yulia S; Tsvetovskaya, Galina A; Strokotov, Dmitry I; Yurkin, Maxim A; Serebrennikova, Elena S; Volkov, Leonid; Maltsev, Valeri P; Chernyshev, Andrei V

    2016-03-21

    Magnesium sulfate (MgSO4) is widely used in medicine but molecular mechanisms of its protection through influence on erythrocytes are not fully understood and are considerably controversial. Using scanning flow cytometry, in this work for the first time we observed experimentally (both in situ and in vitro) a significant increase of HCO3(-)/Cl(-) transmembrane exchange rate of human erythrocytes in the presence of MgSO4 in blood. For a quantitative analysis of the obtained experimental data, we introduced and verified a molecular kinetic model, which describes activation of major anion exchanger Band 3 (or AE1) by its complexation with free intracellular Mg(2+) (taking into account Mg(2+) membrane transport and intracellular buffering). Fitting the model to our in vitro experimental data, we observed a good correspondence between theoretical and experimental kinetic curves that allowed us to evaluate the model parameters and to estimate for the first time the association constant of Mg(2+) with Band 3 as KB~0.07mM, which is in agreement with known values of the apparent Mg(2+) dissociation constant (from 0.01 to 0.1mM) that reflects experiments on enrichment of Mg(2+) at the inner erythrocyte membrane (Gunther, 2007). Results of this work partly clarify the molecular mechanisms of MgSO4 action in human erythrocytes. The method developed allows one to estimate quantitatively a perspective of MgSO4 treatment for a patient. It should be particularly helpful in prenatal medicine for early detection of pathologies associated with the risk of fetal hypoxia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. LoCuSS: comparison of observed X-ray and lensing galaxy cluster scaling relations with simulations

    NASA Astrophysics Data System (ADS)

    Zhang, Y.-Y.; Finoguenov, A.; Böhringer, H.; Kneib, J.-P.; Smith, G. P.; Kneissl, R.; Okabe, N.; Dahle, H.

    2008-05-01

    The Local Cluster Substructure Survey (LoCuSS, Smith et al.) is a systematic multi-wavelength survey of more than 100 X-ray luminous galaxy clusters in the redshift range 0.14-0.3 selected from the ROSAT All Sky Survey. We used data on 37 LoCuSS clusters from the XMM-Newton archive to investigate the global scaling relations of galaxy clusters. The scaling relations based solely on the X-ray data (S-T, S-Y_X, P-Y_X, M-T, M-Y_X, M-M_gas, M_gas-T, L-T, L-Y_X, and L-M) obey empirical self-similarity and reveal no additional evolution beyond the large-scale structure growth. They also reveal up to 17 per cent segregation between all 37 clusters and non-cool core clusters. Weak lensing mass measurements are also available in the literature for 19 of the clusters with XMM-Newton data. The average of the weak lensing mass to X-ray based mass ratio is 1.09± 0.08, setting the limit of the non-thermal pressure support to 9 ± 8 per cent. The mean of the weak lensing mass to X-ray based mass ratio of these clusters is ~1, indicating good agreement between X-ray and weak lensing masses for most clusters, although with 31-51 per cent scatter. The scatter in the mass-observable relations (M-Y_X, M-M_gas, and M-T) is smaller using X-ray based masses than using weak lensing masses by a factor of 2. With the scaled radius defined by the YX profile - r500 Y_X,X, r500YX,wl, and r500Y_X,si, we obtain lower scatter in the weak lensing mass based mass-observable relations, which means the origin of the scatter is M^wl and MX instead of Y_X. The normalization of the M-YX relation using X-ray mass estimates is lower than the one from simulations by up to 18-24 per cent at 3σ significance. This agrees with the M-YX relation based on weak lensing masses, the normalization of the latter being ~20 per cent lower than the one from simulations at ~2σ significance. This difference between observations and simulations is also indicated in the M-M_gas and M-T relations. Despite the large

  7. The global regulator Crc plays a multifaceted role in modulation of type III secretion system in Pseudomonas aeruginosa

    PubMed Central

    Dong, Yi-Hu; Zhang, Xi-Fen; Zhang, Lian-Hui

    2013-01-01

    The opportunistic pathogen Pseudomonas aeruginosa utilizes type III secretion system (T3SS) to translocate effector proteins into eukaryotic host cells that subvert normal host cell functions to the benefit of the pathogen, and results in serious infections. T3SS in P. aeruginosa is controlled by a complex system of regulatory mechanisms and signaling pathways. In this study, we described that Crc, an RNA-binding protein, exerts a positive impact on T3SS in P. aeruginosa, as evidenced by promoter activity assays of several key T3SS genes, transcriptomics, RT-PCR, and immunoblotting in crc mutant. We further demonstrated that the regulatory function of Crc on the T3SS was mediated through the T3SS master regulator ExsA and linked to the Cbr/Crc signaling system. Expression profiling of the crc mutant revealed a downregulation of flagship T3SS genes as well as 16 other genes known to regulate T3SS gene expression in P. aeruginosa. On the basis of these data, we proposed that Crc may exert multifaceted control on the T3SS through various pathways, which may serve to fine-tune this virulence mechanism in response to environmental changes and nutrient sources. PMID:23292701

  8. Search for Doppler-shifted gamma-ray emission from SS 433 using the SMM spectrometer

    NASA Technical Reports Server (NTRS)

    Geldzahler, B. J.; Share, G. H.; Kinzer, R. L.; Magura, J.; Chupp, E. L.

    1989-01-01

    Data accumulated from 1980 to 1983 with the Gamma Ray Spectrometer aboard NASA's Solar Maximum Mission (SMM) satellite were searched for evidence of red and blue Doppler-shifted 1.37 MeV Mg-24 nuclear lines from SS 433. The SMM data base covers 270 days when SS 433 was in the field of view and includes periods of radio flaring and quiescence. No evidence was found for Doppler-shifted line emission in any of the spectra. The range of 3-sigma upper limits for individual 9 day integration periods was 0.0008-0.0023 photons/sq cm per sec for the blue beam, encompassing the reported about 1.5 MeV line, and 0.0008-0.002 photons/sq cm per sec for the red beam, encompassing the reported about 1.2 MeV line; the average 3-sigma upper limit in each beam for shifted about 1.37 MeV lines is 0.0015 photons/sq cm per sec for single 9 day integrations. The 3-sigma upper limit on 1.37 MeV gamma-ray emission over 23 9-day integration intervals for the red beam and 28 intervals for the blue beam is 0.0002 photons/sq cm per sec. These new limits from SMM can be reconciled with the HEAO 3 results only if SS 433 emits gamma radiation at or above the SMM sensitivity limit on rare occasions due to variable physical conditions in the system.

  9. Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis*

    PubMed Central

    Hálová, Ivana; Dráberová, Lubica; Bambousková, Monika; Machyna, Martin; Stegurová, Lucie; Smrž, Daniel; Dráber, Petr

    2013-01-01

    Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca2+ response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)2 or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcϵRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)2 fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcϵRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family. PMID:23443658

  10. Structural and regulatory mutations in Vibrio parahaemolyticus type III secretion systems display variable effects on virulence.

    PubMed

    Calder, Thomas; de Souza Santos, Marcela; Attah, Victoria; Klimko, John; Fernandez, Jessie; Salomon, Dor; Krachler, Anne-Marie; Orth, Kim

    2014-12-01

    The Gram-negative bacterium, Vibrio parahaemolyticus, is a major cause of seafood-derived food poisoning throughout the world. The pathogenicity of V. parahaemolyticus is attributed to several virulence factors, including two type III secretion systems (T3SS), T3SS1 and T3SS2. Herein, we compare the virulence of V. parahaemolyticus POR strains, which harbor a mutation in the T3SS needle apparatus of either system, to V. parahaemolyticus CAB strains, which harbor mutations in positive transcriptional regulators of either system. These strains are derived from the clinical RIMD 2210633 strain. We demonstrate that each mutation affects the virulence of the bacterium in a different manner. POR and CAB strains exhibited similar levels of swarming motility and T3SS effector production and secretion, but the CAB3 and CAB4 strains, which harbor a mutation in the T3SS2 master regulator gene, formed reduced biofilm growth under T3SS2 inducing conditions. Additionally, while the cytotoxicity of the POR and CAB strains was similar, the CAB2 (T3SS1 regulatory mutant) strain was strikingly more invasive than the comparable POR2 (T3SS1 structural mutant) strain. In summary, creating structural or regulatory mutations in either T3SS1 or T3SS2 causes differential downstream effects on other virulence systems. Understanding the biological differences of strains created from a clinical isolate is critical for interpreting and understanding the pathogenic nature of V. parahaemolyticus. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  11. Neutron Diffraction Studies of Fluid Bilayers with Transmembrane Proteins: Structural Consequences of the Achondroplasia Mutation

    PubMed Central

    Han, Xue; Mihailescu, Mihaela; Hristova, Kalina

    2006-01-01

    Achondroplasia, the most common form of human dwarfism, is due to a G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) in >97% of the studied cases. While the molecular mechanism of pathology induction is under debate, the structural consequences of the mutation have not been studied. Here we use neutron diffraction to determine the disposition of FGFR3 transmembrane domain in fluid lipid bilayers, and investigate whether the G380R mutation affects the topology of the protein in the bilayer. Our results demonstrate that, in a model system, the G380R mutation induces a shift in the segment that is embedded in the membrane. The center of the hydrocarbon core-embedded segment in the mutant is close to the midpoint between R380 and R397, supporting previous measurements of arginine insertion energetics into the endoplasmic reticulum. The presented results further our knowledge about basic amino-acid insertion into bilayers, and may lead to new insights into the mechanism of pathogenesis in achondroplasia. PMID:16950849

  12. Modulation of Ca(v)3.1 T-type Ca2+ channels by the ran binding protein RanBPM.

    PubMed

    Kim, Taehyun; Kim, Sunoh; Yun, Hyung-Mun; Chung, Kwang Chul; Han, Ye Sun; Shin, Hee-Sup; Rhim, Hyewhon

    2009-01-02

    In order to study the currently unknown cellular signaling pathways of Ca(v)3.1 T-type Ca(2+) channels (Ca(v)3.1 channels), we performed a yeast two-hybrid screening using intracellular domains of Ca(v)3.1 alpha1 subunit as bait. After screening the human brain cDNA library, several proteins, including RanBPM, were identified as interacting with Ca(v)3.1 channels. RanBPM was found to bind to the cytoplasmic intracellular loop between transmembrane domains I and II of Ca(v)3.1 channels. Using whole-cell patch-clamp techniques, we found that Ca(v)3.1 currents were increased by the expression of RanBPM in HEK293/Ca(v)3.1 cells. We next examined whether RanBPM affected the biophysical properties and plasma membrane expression of Ca(v)3.1 channels. Furthermore, we showed that the PKC activator inhibited Ca(v)3.1 currents, an effect that was abolished by the expression of RanBPM. These results suggest that RanBPM could be a key regulator of Ca(v)3.1 channel-mediated signaling pathways.

  13. RPA homologs and ssDNA processing during meiotic recombination.

    PubMed

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

  14. Chemostat studies of bacteriophage M13 infected Escherichia coli JM109 for continuous ssDNA production.

    PubMed

    Kick, Benjamin; Behler, Karl Lorenz; Severin, Timm Steffen; Weuster-Botz, Dirk

    2017-09-20

    Steady state studies in a chemostat enable the control of microbial growth rate at defined reaction conditions. The effects of bacteriophage M13 infection on maximum growth rate of Escherichia coli JM109 were studied in parallel operated chemostats on a milliliter-scale to analyze the steady state kinetics of phage production. The bacteriophage infection led to a decrease in maximum specific growth rate of 15% from 0.74h -1 to 0.63h -1 . Under steady state conditions, a constant cell specific ssDNA formation rate of 0.15±0.004 mg ssDNA g CDW -1 h -1 was observed, which was independent of the growth rate. Using the estimated kinetic parameters for E. coli infected with bacteriophage M13, the ssDNA concentration in the steady state could be predicted as function of the dilution rate and the glucose concentration in the substrate. Scalability of milliliter-scale data was approved by steady state studies on a liter-scale at a selected dilution rate. An ssDNA space-time yield of 5.7mgL -1 h -1 was achieved with increased glucose concentration in the feed at a dilution rate of 0.3h -1 , which is comparable to established fed-batch fermentation with bacteriophage M13 for ssDNA production. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Very high resolution observations of SS433 at 10.65 GHz

    NASA Technical Reports Server (NTRS)

    Geldzahler, B. J.; Downes, A. J. B.; Shaffer, D. B.

    1981-01-01

    Observations of SS433 made on June 12, 1979, from West Germany, Massachusetts, and West Virginia are discussed. It is noted that SS433 did not show fringes on any baseline although all the calibration sources were seen at their expected strengths. The measured total flux density of SS433 was found to be approximately 0.5 Jy, consistent with previous observations. The source was observed by on-offs at each telescope, which indicates that they were all pointed properly during the observations. The absence of fringes is not attributed to poor observing conditions or instrumental difficulties. It is concluded that if all the 10.65 GHz radiation emanates from a single component, then that component is at least 0.005 arcsec (approximately 10 to the 14th cm) in size. The measurements made on more sensitive intercontinental baselines indicate that there is no component of SS433 smaller than 0.001 arcsec emitting 10.65 GHz radiation above a level of 50 mJy.

  16. Blister Test for Measurements of Adhesion and Adhesion Degradation of Organic Polymers on AA2024-T3

    NASA Astrophysics Data System (ADS)

    Rincon Troconis, Brendy Carolina

    cleaning/desmutting and conversion coating (CC) on the adhesion strength of acetoacetate to AA2024-T3 and the effects of improper water rinse temperature after cleaner were assessed using the BT. The results showed that pretreatments improve the adhesion strength of acetoacetate primer on AA2024-T3, but the comparative behavior depends on the specific treatment. Process control is of paramount importance for the performance of acetoacetate coated systems applied on AA2024-T3. The lack of thermal activity in the water rinse after cleaning step produces deleterious effects on the adhesion and blistering resistance of CC. Finally, a test sample incorporating a coated and scribed Al alloy panel and uncoated through-hole fasteners (Ti, SS316, AA2024-T3) was designed to provide accelerated response during atmospheric corrosion testing in the field (long-term beach exposure) or in laboratory chambers (ASTM B117). The results after only three weeks of exposure to ASTM B117 correlated well with long-term beach exposure, allowing rapid ranking of different coating systems. Of the fastener materials studied, visual observation and volume lost determination indicated that the worst attack occurred near SS316 fasteners its effect was explained by Scanning Kelvin Probe measurements and the available cathodic current measured in chloride solution.

  17. Differential sensitivity of the nicotinic receptor of long (LS) and short (SS) sleep mice to ethanol (E) and forane (F)

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

    McArdle, J.J.; Choi, J.J.

    1989-02-09

    Studies of inbred mice indicate that heredity determines the behavioral response to CNS depressants. For example, LS mice lose their righting reflex at blood levels of E having no effect on this reflex of SS mice. In order to determine if such differential sensitivity extends to the effects of depressants known to alter the mean open time (tau) of the ion channel activated by the nicotinic acetylcholine receptor (AR), we used an extracellular electrode to record miniature end-plate currents (23 C) from the triangularis sterni muscle of adult male LS and SS mice. The average decay time constant (tau) ofmore » 70 currents was calculated before, during and after drug exposure. Tau was the same for LS and SS mice (1.41 {plus minus} 0.03 mS and 1.47 {plus minus} 0.02 mS, respectively) prior to treatment and was reversible prolonged by E and shortened by F as expected. However, tau of SS mice was more responsive. For example, 25 mM of E increased tau by 12.9% and 3.8% in SS and LS mice, respectively. Likewise, the decrease of tau in response to 3 mM F was 18.5% and 9.2%. The net result was that the curve relating tau for LS mice to drug concentration was to the right of the for SS mice. These data suggest that the sensitivity of the peripheral AR to CNS depressants can be genetically controlled.« less

  18. TIMES-SS--recent refinements resulting from an industrial skin sensitisation consortium.

    PubMed

    Patlewicz, G; Kuseva, C; Mehmed, A; Popova, Y; Dimitrova, G; Ellis, G; Hunziker, R; Kern, P; Low, L; Ringeissen, S; Roberts, D W; Mekenyan, O

    2014-01-01

    The TImes MEtabolism Simulator platform for predicting Skin Sensitisation (TIMES-SS) is a hybrid expert system, first developed at Bourgas University using funding and data from a consortium of industry and regulators. TIMES-SS encodes structure-toxicity and structure-skin metabolism relationships through a number of transformations, some of which are underpinned by mechanistic 3D QSARs. The model estimates semi-quantitative skin sensitisation potency classes and has been developed with the aim of minimising animal testing, and also to be scientifically valid in accordance with the OECD principles for (Q)SAR validation. In 2007 an external validation exercise was undertaken to fully address these principles. In 2010, a new industry consortium was established to coordinate research efforts in three specific areas: refinement of abiotic reactions in the skin (namely autoxidation) in the skin, refinement of the manner in which chemical reactivity was captured in terms of structure-toxicity rules (inclusion of alert reliability parameters) and defining the domain based on the underlying experimental data (study of discrepancies between local lymph node assay Local Lymph Node Assay (LLNA) and Guinea Pig Maximisation Test (GPMT)). The present paper summarises the progress of these activities and explains how the insights derived have been translated into refinements, resulting in increased confidence and transparency in the robustness of the TIMES-SS predictions.

  19. T2* Mapping of the Hip in Asymptomatic Volunteers with Normal Cartilage Morphology: An Analysis of Regional and Age-Dependent Distribution.

    PubMed

    Hesper, Tobias; Schleich, Christoph; Buchwald, Alexander; Hosalkar, Harish S; Antoch, Gerald; Krauspe, Rüdiger; Zilkens, Christoph; Bittersohl, Bernd

    2018-01-01

    Objective To assess age-dependent and regional differences in T2* relaxation measurements in hip joint cartilage of asymptomatic volunteers at 3 T. Design Three age cohorts (cohort 1: age 20-30 years, 15 individuals; cohort 2: age 30-40 years, 17 individuals; cohort 3: age 40-50 years, 15 individuals) were enrolled. T2* values were obtained in the central and peripheral cartilage of the acetabulum and the femoral head in 7 regions (anterior to superior and posterior). Results T2* did not differ among age cohorts in acetabular cartilage (cohort 1: 24.65 ± 6.56 ms, cohort 2: 24.70 ± 4.83 ms, cohort 3: 25.81 ± 5.10 ms, P = 0.10) and femoral head cartilage (cohort 1: 27.08 ± 8.24 ms, cohort 2: 25.90 ± 7.82 ms, cohort 3: 26.50 ± 5.61 ms, P = 0.34). Analysis of the regional T2* distribution pattern indicates increased T2* values in the anterior, anterior-superior, superior-anterior, and the posterior-superior aspects of acetabular and femoral head cartilage. For acetabular cartilage, higher values were observed in the central region (25.90 ± 4.80 ms vs. 24.21 ± 4.05 ms, P < 0.0001) whereas femoral head cartilage did not reveal such differences (26.62 ± 5.74 ms vs. 26.37 ± 5.89 ms, P = 0.44). Conclusions The T2* analysis of presumably healthy hip joint cartilage does not seem to be stratified according to age in this population. Regional T2* variation throughout hip joint cartilage is apparent in this modality.

  20. Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

    PubMed

    Wang, Hong; Brautigan, David L

    2006-11-01

    Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

  1. Discrimination against RNA Backbones by a ssDNA Binding Protein.

    PubMed

    Lloyd, Neil R; Wuttke, Deborah S

    2018-05-01

    Pot1 is the shelterin component responsible for the protection of the single-stranded DNA (ssDNA) overhang at telomeres in nearly all eukaryotic organisms. The C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this flexibility, it is unclear how specificity for ssDNA over RNA, an activity required for biological function, is achieved. Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. High-resolution structures of several Pot1pC complexes with RNA-DNA chimeric ligands reveal Pot1pC discriminates against RNA by utilizing non-compensatory binding modes that feature significant rearrangement of the binding interface. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities. These findings suggest that intermolecular interfaces are remarkably sophisticated in their tuning of specificity toward flexible ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Karl Blumenreuter (1881-1969): Himmler's chief pharmacist and medical supply quartermaster of the SS.

    PubMed

    Schmidt, M; Gross, D; Westemeier, J

    2018-04-02

    The present article elucidates the role and function of SS Gruppenführer Karl Blumenreuter (1881-1969), the leading pharmacist of the SS. The aim is to clarify how he participated in the crimes of the Nazi Party and especially the SS during the "Third Reich" and the extent to which he was brought to justice after 1945. Central elements of the study are based on researched archival primary sources. The latter were compared with each other and supplemented with the currently available secondary literature on the subject. It can be established that Blumenreuter was a zealous National Socialist with marked career ambitions who advanced within the SS to the rank of SS Gruppenführer and Lieutenant General of the Waffen SS (Generalleutnant der Waffen SS). He supplied pre-measured phenol ampoules for the murder of prisoners in the concentration camps. Blumenreuter furthermore organised equipment and material supplies for various experiments on humans. After 1945 he succeeded in playing down his participation in the crimes in the time of National Socialism. Blumenreuter died in 1969 without having being brought to justice by the judiciary and without having reflected self-critically on his role in the "Third Reich".

  3. On the Nature of the Compact Object in SS 433: Observational Evidence of X-ray Photon Index Saturation

    NASA Astrophysics Data System (ADS)

    Seifina, Elena; Titarchuk, Lev

    2010-10-01

    We present an analysis of the X-ray spectral properties observed from the black hole candidate (BHC) binary SS 433. We have analyzed Rossi X-ray Timing Explorer data from this source, coordinated with Green Bank Interferometer/RATAN-600. We show that SS 433 undergoes an X-ray spectral transition from the low hard state to the intermediate state (IS). We show that the X-ray broadband energy spectra during all spectral states are well fitted by a sum of the so-called bulk motion Comptonization (BMC) component and by two (broad and narrow) Gaussians for the continuum and line emissions, respectively. In addition to these spectral model components, we also find a strong feature that we identify as a "blackbody-like (BB)" component in which the color temperature is in the range of 4-5 keV in 24 IS spectra during the radio outburst decay in SS 433. Our observational results on the "high-temperature BB" bump lead us to suggest the presence of gravitationally redshifted annihilation line emission in this source. In fact, this spectral feature has been recently reproduced in Monte Carlo simulations by Laurent & Titarchuk. We have also established the photon index saturation at about 2.3 in index versus mass accretion correlation. This index-mass accretion correlation allows us to evaluate the low limit of the black hole (BH) mass of the compact object in SS 433, M_{{bh}}≳ 2 solar masses, using the scaling method using BHC GX 339 - 4 as a reference source. Our estimate of the BH mass in SS 433 is consistent with the recent BH mass measurement using the radial velocity measurements of the binary system by Hillwig & Gies, who find that Mx = (4.3 ± 0.8) solar masses. This is the smallest BH mass found up to now among all BH sources. Moreover, the index saturation effect versus mass accretion rate revealed in SS 433, as in a number of other BH candidates, is strong observational evidence for the presence of a BH in SS 433.

  4. On the Nature of the Compact Object in SS 433. Observational Evidence of X-Ray Photon Index Saturation

    NASA Technical Reports Server (NTRS)

    Seifina, Elena; Titarchuk, Lev

    2010-01-01

    We present an analysis of the X-ray spectral properties observed from black hole , candidate (BHC) binary SS 433. We have analyzed Rossi X-ray Time Explorer (RXTE) data from this source, coordinated with Green Bank Interferometer/RATAN-600. We show that SS 433 undergoes a X-ray spectral transition from the low hard state (LHS) to the intermediate state (IS). We show that the X-ray broad-band energy spectra during all spectral states are well fit by a sum of so called "Bulk Motion Comptonization (BMC) component" and by two (broad and narrow) Gaussians for the continuum and line emissions respectively. In addition to these spectral model components we also find a strong feature that we identify as a" blackbody-like (BB)" component which color temperature is in the range of 4-5 keV in 24 IS spectra during the radio outburst decay in SS 433. Our observational results on the "high temperature BB" bump leads us to suggest the presence of gravitationally redshifted annihilation line emission in this source. In fact this spectral feature has been recently reproduced in Monte Carlo simulations by Laurent and Titarchuk. We have also established the photon index saturation at about 2.3 in index vs mass accretion correlation. This index-mass accretion correlation allows us to evaluate the low limit of black hole (BH) mass of compact object in SS 433, M(sub bh) approximately > 2 solar masses, using the scaling method using BHC GX 339-4 as a reference source. Our estimate of the BH mass in SS 433 is consistent with recent BH mass measurement using the radial-velocity measurements of the binary system by Hillwig & Gies who find that M(sub x)( = (4.3 +/- 0.8) solar masses. This is the smallest BH mass found up to now among all BH sources. Moreover, the index saturation effect versus mass accretion rate revealed in SS 433, like in a number of other BH candidates, is the strong observational evidence for the presence of a BH in SS 433.

  5. Effects of Age, Gender and Hemispheric Location on T2 Hypointensity in the Pulvinar at 3T.

    PubMed

    White, Matthew L; Zhang, Yan; Helvey, Jason T; Yu, Fang; Omojola, Matthew F

    2014-12-01

    Pulvinar signal intensity decrease on T2-weighted images has been reported in some neurological abnormalities. We aimed to define the normal T2 signal hypointensity pattern present in the pulvinar to avoid erroneous radiological interpretation. One hundred and forty-two subjects (54 men and 88 women; age range 9-91 years) with unremarkable brain 3T MR findings were enrolled. MR images were analyzed with regard to signal intensity of the pulvinar relative to the thalamus on fluid attenuated inversion recovery images. Effects of age, gender and hemispheric location on the degree of T2 hypointensity were statistically analyzed. The statistical association was measured between the pattern of signal changes in the pulvinar region and that in the putamen and the globus pallidus. We detected a linear signal decrease in the pulvinar region with age. The male subjects had a more rapid decrease of signal with age than female subjects. The right pulvinar region had a higher chance of hypointensity compared to the left. A positive linear association was found when signal change from the pulvinar region was compared with signal in the putamen and globus pallidus. We detected a linear signal decrease with age in the pulvinar. The physiological signal features of the pulvinar also depend on gender and hemispheric lateralization. The pattern of signal change in the pulvinar is similar to but not the same as that in the putamen and globus pallidus.

  6. The non-conserved region of MRP is involved in the virulence of Streptococcus suis serotype 2

    PubMed Central

    Li, Quan; Fu, Yang; Ma, Caifeng; He, Yanan; Yu, Yanfei; Du, Dechao; Yao, Huochun; Lu, Chengping; Zhang, Wei

    2017-01-01

    ABSTRACT Muramidase-released protein (MRP) of Streptococcus suis serotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Δmrp, Δmrp domain 1 (Δmrp-d1), and Δmrp domain 2 (Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242–596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1* (MRP-D1*, the non-conserved region of MRP from a low virulent strain, a.a. 239–598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848–1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1 mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1 mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection. PMID:28362221

  7. The non-conserved region of MRP is involved in the virulence of Streptococcus suis serotype 2.

    PubMed

    Li, Quan; Fu, Yang; Ma, Caifeng; He, Yanan; Yu, Yanfei; Du, Dechao; Yao, Huochun; Lu, Chengping; Zhang, Wei

    2017-10-03

    Muramidase-released protein (MRP) of Streptococcus suis serotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Δmrp, Δmrp domain 1 (Δmrp-d1), and Δmrp domain 2 (Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242-596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1* (MRP-D1*, the non-conserved region of MRP from a low virulent strain, a.a. 239-598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848-1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1 mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1 mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection.

  8. Studies with pyrethroids (kadethrin and deltamethrin) and lindane in ethanol sensitive (LS) and insensitive (SS) mouse strains

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

    Doherty, J.; Baker, R.C.; Deitrich, R.

    1990-02-26

    Ethanol (E) sensitive (LS) and insensitive (SS) mouse strains are distinguished by their sleeping time to a given dose of E and the locus for this difference is at the level of the neuron. In attempts to understand the neuropharmacological basis of insecticide action and to further define the differences in these mouse lines, LS and SS mice were dosed with type I (kadethrine, K) and II (deltamethrin, D) pyrethroids and lindane (L). These compounds were selected because their proposed modes of action are on the Na+ channel (K and D) and/or the GABA receptor ionophore (D and L). Nomore » consistent differences in the effects of K, D or L in the SS and LS mouse lines were evident. In preliminary studies both SS and LS mice dosed with 50 or 100 {mu}g/brain of L (intracerebroventricularly) but not D slept much longer (2-3X) than when dosed with E alone, an effect opposite of that predicted from L's known excitatory action. These data indicate that as far as can be distinguished by pyrethroids and L, the Na+ channel and GABA receptor/ionophore complex are similar in both the LS and SS mouse lines.« less

  9. The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems

    PubMed Central

    Ma, Jiale; Pan, Zihao; Huang, Jinhu; Sun, Min; Lu, Chengping; Yao, Huochun

    2017-01-01

    ABSTRACT The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3–4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4O1) genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors. PMID:28060574

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

    PubMed Central

    El Hiani, Yassine; Linsdell, Paul

    2015-01-01

    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. PMID:25944907

  11. Detection of the type III secretion system and its phylogenetic and symbiotic characterization in peanut bradyrhizobia isolated from Guangdong Province, China.

    PubMed

    Ruan, Huaqin; Hu, Meijuan; Chen, Jingyu; Li, Xue; Li, Ting; Lai, Yongxiu; Wang, En Tao; Gu, Jun

    2018-04-08

    The distribution of rhcRST and rhcJ-C1 fragments located in different loci of the type III secretion system (T3SS) gene cluster in the peanut-nodulating bradyrhizobia isolated from Guangdong Province, China was investigated by PCR-based sequencing. T3SS was detected in approximately one-third of the peanut bradyrhizobial strains and the T3SS-harboring strains belonging to different Bradyrhizobium genomic species. Diverse T3SS groups corresponding to different symbiotic gene types were defined among the 23 T3SS-harboring strains. The same or similar T3SS genes were detected in different genospecies, indicating that interspecies horizontal transfer of symbiotic genes had occurred in the Bradyrhizobium genus. Copyright © 2018. Published by Elsevier GmbH.

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

    PubMed Central

    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

  13. Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication.

    PubMed

    Steigemann, Birthe; Schulz, Annina; Werten, Sebastiaan

    2013-11-15

    The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors. © 2013.

  14. Comparison of only T3 and T3T4 sympathectomy for axillary hyperhidrosis regarding treatment effect and compensatory sweating

    PubMed Central

    Yuncu, Gökhan; Turk, Figen; Ozturk, Gökhan; Atinkaya, Cansel

    2013-01-01

    OBJECTIVES Patients diagnosed with axillary hyperhidrosis can face psychosocial issues that can ultimately hinder their quality of life both privately and socially. The routine treatment for axillary hyperhidrosis is T3T4 sympathectomy, but compensatory sweating is a serious side effect that is commonly seen with this approach. This study was designed to evaluate whether a T3 sympathectomy was effective for the treatment of axillary hyperhidrosis and whether this treatment led to less compensatory sweating than T3T4 sympathectomies among our 60-patient population. METHODS One hundred and twenty endoscopic thoracic sympathectomies were performed on 60 patients who had axillary hyperhidrosis. The sympathectomies were accomplished by means of a single-lumen endotracheal tube and a single port. The axillary hyperhidrosis patients were randomly divided into two groups with 17 patients in Group 1 undergoing T3T4 sympathectomies and 43 in Group 2 undergoing only T3 sympathectomies. We analysed the data associated with the resolution of axillary hyperhidrosis, the degree of patient satisfaction with the surgical outcome and the quality of life in parallel with compensatory sweating after the procedure as reported by the patient and confirmed by the examiner. Moreover, the results were compared statistically. RESULTS No statistically significant difference was observed between the groups based on age (P = 0.56), gender (P = 0.81), duration of the surgery (P = 0.35) or postoperative satisfaction levels (P = 0.45). However, the incidence and degree of compensatory sweating were lower in the T3 group than the T3T4 group at the 1-year follow-up (P = 0.008). CONCLUSIONS T3 sympathectomy was as effective as T3T4 sympathectomy for the treatment of axillary hyperhidrosis based on the patients’ reported postoperative satisfaction, and the T3 group demonstrated lower compensatory sweating at the 1-year follow-up. PMID:23644731

  15. Middle Level SS&C Energy Series.

    ERIC Educational Resources Information Center

    Crow, Linda W.; Aldridge, Bill G.

    The project on Scope Sequence and Coordination of Secondary School Science (SS&C) was initiated by the National Science Teachers Association (NSTA) and recommends that all students study science every year and advocates carefully sequenced, well-coordinated instruction in biology, chemistry, earth/space science, and physics. This document…

  16. Spectroscopic Observations of the Mass Donor Star in SS 433

    NASA Astrophysics Data System (ADS)

    Hillwig, T. C.; Gies, D. R.

    2008-03-01

    The microquasar SS 433 is an interacting massive binary consisting of an evolved mass donor and a compact companion that ejects relativistic jets. The mass donor was previously identified through spectroscopic observations of absorption lines in the blue part of the spectrum that showed Doppler shifts associated with orbital motion and strength variations related to the orbital modulation of the star-to-disk flux ratio and to disk obscuration. However, subsequent observations revealed other absorption features that lacked these properties and that were probably formed in the disk gas outflow. We present follow-up observations of SS 433 at orbital and precession phases identical to those from several previous studies, with the goals of confirming the detection of the mass donor spectrum and providing more reliable masses for the two system components. We show that the absorption features present as well as those previously observed almost certainly belong to the mass donor star, and find revised masses of 12.3 ± 3.3 and 4.3 ± 0.8 M⊙ for the mass donor and compact object, respectively. Based on observations obtained at the Gemini Observatory, which is operated by the Association of Universities for Research in Astronomy, Inc., under a cooperative agreement with the NSF on behalf of the Gemini partnership: the National Science Foundation (US), the Science and Technology Facilities Council (UK), the National Research Council (Canada), CONICYT (Chile), the Australian Research Council (Australia), CNPq (Brazil), and SECYT (Argentina).

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

    DOE PAGES

    Lv, Chao; Aitchison, Erick W.; Wu, Dongsheng; ...

    2015-06-29

    Hydrogen sulfide (H 2S), 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 H 2S and its structural analogue, water (H 2O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. Here, 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,more » the interface between the polar head-group and nonpolar acyl chain regions. Because the membrane binding affinity of H 2S 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 H2S 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. Lastly, this study not only explains why H 2S 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.« less

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

    PubMed

    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.

  19. The Tropomyosin Binding Region of Cardiac Troponin T Modulates Crossbridge Recruitment Dynamics in Rat Cardiac Muscle Fibers

    PubMed Central

    Gollapudi, Sampath K.; Gallon, Clare E.; Chandra, Murali

    2013-01-01

    The cardiac muscle comprises dynamically interacting components that use allosteric/cooperative mechanisms to yield unique heart-specific properties. An essential protein in this allosteric/cooperative mechanism is cardiac muscle troponin T (cTnT), the central region (CR) and the T2 region of which differ significantly from those of fast skeletal muscle troponin T (fsTnT). To understand the biological significance of such sequence heterogeneity, we replaced the T1 or T2 domain of rat cTnT (RcT1 or RcT2) with its counterpart from rat fsTnT (RfsT1or RfsT2) to generate RfsT1-RcT2 and RcT1-RfsT2 recombinant proteins. In addition to contractile function measurements, dynamic features of RfsT1-RcT2 and RcT1-RfsT2 reconstituted rat cardiac muscle fibers were captured by fitting the recruitment-distortion model to force response of small amplitude (0.5%) muscle length changes. RfsT1-RcT2 fibers showed a ~40% decrease in tension and ~44% decrease in ATPase activity, but RcT1-RfsT2 fibers were unaffected. The magnitude of length-mediated increase in crossbridge recruitment (E0) decreased by ~33% and the speed of crossbridge recruitment (b) increased by ~100% in RfsT1-RcT2 fibers. Our data suggest the following: (1) the CR of cTnT modulates crossbridge recruitment dynamics; (2) the N-terminal end region of cTnT has a synergistic effect on the ability of CR to modulate crossbridge recruitment dynamics; (3) the T2 region is important for tuning the Ca2+ regulation of cardiac thin filaments. The combined effects of CR-Tm interactions and the modulating effect of the N-terminal end of cTnT on CR-Tm interactions may lead to the emergence of a unique property that tunes contractile dynamics to heart rates. PMID:23357173

  20. Maternal predictive factors for fetal congenital heart block in pregnant mothers positive for anti-SS-A antibodies.

    PubMed

    Tsuboi, Hiroto; Sumida, Takayuki; Noma, Hisashi; Yamagishi, Kazumasa; Anami, Ai; Fukushima, Kotaro; Horigome, Hitoshi; Maeno, Yasuki; Kishimoto, Mitsumasa; Takasaki, Yoshinari; Nakayama, Masahiro; Waguri, Masako; Sago, Haruhiko; Murashima, Atsuko

    2016-07-01

    To determine the maternal predictive factors for fetal congenital heart block (CHB) in pregnancy in mothers positive for anti-SS-A antibodies. The Research Team for Surveillance of Autoantibody-Exposed Fetuses and Treatment of Neonatal Lupus Erythematosus, the Research Program of the Japan Ministry of Health, Labor and Welfare, performed a national survey on pregnancy of mothers positive for anti-SS-A antibodies. We analyzed 635 pregnant mothers who tested positive for anti-SS-A antibodies before conception but had no previous history of fetal CHB. We performed univariate and multivariate analysis (models 1, 2, and 3 using different set of independent variables) investigated the relation between risk of fetal CHB and maternal clinical features. Of the 635 pregnant mothers, fetal CHB was detected in 16. Univariate analysis showed that fetal CHB associated with use of corticosteroids before conception (OR 3.72, p = 0.04), and negatively with use of corticosteroids (equivalent doses of prednisolone (PSL), at ≥10 mg/day) after conception before 16-week gestation (OR 0.17, p = 0.03). In multivariate analysis, model 1 identified the use of corticosteroids before conception (OR 4.28, p = 0.04) and high titer of anti-SS-A antibodies (OR 3.58, p = 0.02) as independent and significant risk factors, and model 3 identified use of corticosteroids (equivalent doses of PSL, at ≥10 mg/day) after conception before 16-week gestation as independent protective factor against the development of fetal CHB (OR 0.16, p = 0.03). Other maternal clinical features did not influence the development of fetal CHB. The results identified high titers of anti-SS-A antibodies and use of corticosteroids before conception as independent risk factors, and use of corticosteroids (equivalent doses of PSL, at ≥10 mg/day) after conception before 16-week gestation as an independent protective factor for fetal CHB.

  1. Thermodynamics of membrane insertion and refolding of the diphtheria toxin T-domain

    PubMed Central

    Vargas-Uribe, Mauricio; Rodnin, Mykola V.; Öjemalm, Karin; Holgado, Aurora; Kyrychenko, Alexander; Nilsson, IngMarie; Posokhov, Yevgen O.; Makhatadze, George; von Heijne, Gunnar; Ladokhin, Alexey S.

    2014-01-01

    The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3–5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately −8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between −9.5 to −12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain. PMID:25281329

  2. Previously unknown and highly divergent ssDNA viruses populate the oceans.

    PubMed

    Labonté, Jessica M; Suttle, Curtis A

    2013-11-01

    Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton.

  3. Uneven Distribution of Regional Blood Supply Prompts the Cystic Change of Pituitary Adenoma.

    PubMed

    Zhang, Jianhe; Gu, Jianjun; Ma, Yiming; Huang, Yinxing; Wang, Jiaxing; Wu, Zhifeng; Zhong, Qun; Wang, Shousen

    2017-07-01

    Previous studies have suggested that the cystic change of pituitary adenoma might be related to the blood supply and metabolism of the tumor; however, the exact pathologic mechanism underlying the cystic change remains unknown. We aimed to assess the features of regional blood supply of pituitary adenoma and examine its relationship with the cystic change of pituitary adenoma. Patients (N = 79) with pituitary adenoma admitted to our hospital were divided into the parenchyma group (n = 40) or the cystic change group (n = 39). Dynamic contrast-enhanced magnetic resonance imaging of the pituitary adenoma was conducted for the parenchyma group and the steepest slopes (SS max , reflecting regional blood supply) at different areas were calculated. The location of cystic change of the pituitary adenoma was recorded and analyzed for the cystic change group. The parenchyma group showed an upper SS max of 2.52 ± 1.18, a lower SS max of 2.89 ± 1.46, a left SS max of 2.71 ± 1.31, and a right SS max of 2.66 ± 1.29. The difference between the upper and lower SS max was statistically significant (P < 0.001), with no difference between the left and right regions (P = 0.668). The location of cystic change of the pituitary adenoma was mainly in the upper region, accounting for 48.7% of cases. Regional blood supply is unevenly distributed in the parenchymal pituitary adenoma, with reduced blood supply in the upper than the lower region. Cystic change mainly occurs in the upper region of pituitary adenoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1-specific T-cell effector function in an activation-dependent manner.

    PubMed

    Schmetterer, Klaus G; Haiderer, Daniela; Leb-Reichl, Victoria M; Neunkirchner, Alina; Jahn-Schmid, Beatrice; Küng, Hans J; Schuch, Karina; Steinberger, Peter; Bohle, Barbara; Pickl, Winfried F

    2011-01-01

    Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αβ-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αβ-chains. cDNAs encoding the α and β-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become

  5. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

    PubMed

    Höög, Johanna L; Lacomble, Sylvain; Bouchet-Marquis, Cedric; Briggs, Laura; Park, Kristin; Hoenger, Andreas; Gull, Keith

    2016-01-01

    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.

  6. EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system

    PubMed Central

    Zoltner, Martin; Ng, Wui M.A.V.; Money, Jillian J.; Fyfe, Paul K.; Kneuper, Holger; Palmer, Tracy; Hunter, William N.

    2016-01-01

    The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein. PMID:27130157

  7. Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems*

    PubMed Central

    Durand, Eric; Zoued, Abdelrahim; Spinelli, Silvia; Watson, Paul J. H.; Aschtgen, Marie-Stéphanie; Journet, Laure; Cambillau, Christian; Cascales, Eric

    2012-01-01

    The Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families. PMID:22371492

  8. LOFAR 150-MHz observations of SS 433 and W 50

    NASA Astrophysics Data System (ADS)

    Broderick, J. W.; Fender, R. P.; Miller-Jones, J. C. A.; Trushkin, S. A.; Stewart, A. J.; Anderson, G. E.; Staley, T. D.; Blundell, K. M.; Pietka, M.; Markoff, S.; Rowlinson, A.; Swinbank, J. D.; van der Horst, A. J.; Bell, M. E.; Breton, R. P.; Carbone, D.; Corbel, S.; Eislöffel, J.; Falcke, H.; Grießmeier, J.-M.; Hessels, J. W. T.; Kondratiev, V. I.; Law, C. J.; Molenaar, G. J.; Serylak, M.; Stappers, B. W.; van Leeuwen, J.; Wijers, R. A. M. J.; Wijnands, R.; Wise, M. W.; Zarka, P.

    2018-04-01

    We present Low-Frequency Array (LOFAR) high-band data over the frequency range 115-189 MHz for the X-ray binary SS 433, obtained in an observing campaign from 2013 February to 2014 May. Our results include a deep, wide-field map, allowing a detailed view of the surrounding supernova remnant W 50 at low radio frequencies, as well as a light curve for SS 433 determined from shorter monitoring runs. The complex morphology of W 50 is in excellent agreement with previously published higher frequency maps; we find additional evidence for a spectral turnover in the eastern wing, potentially due to foreground free-free absorption. Furthermore, SS 433 is tentatively variable at 150 MHz, with both a debiased modulation index of 11 per cent and a χ2 probability of a flat light curve of 8.2 × 10-3. By comparing the LOFAR flux densities with contemporaneous observations carried out at 4800 MHz with the RATAN-600 telescope, we suggest that an observed ˜0.5-1 Jy rise in the 150-MHz flux density may correspond to sustained flaring activity over a period of approximately 6 months at 4800 MHz. However, the increase is too large to be explained with a standard synchrotron bubble model. We also detect a wealth of structure along the nearby Galactic plane, including the most complete detection to date of the radio shell of the candidate supernova remnant G 38.7-1.4. This further demonstrates the potential of supernova remnant studies with the current generation of low-frequency radio telescopes.

  9. Redox-sensitive micelles assembled from amphiphilic mPEG-PCL-SS-DTX conjugates for the delivery of docetaxel.

    PubMed

    Zhang, Huiyuan; Wang, Kaiming; Zhang, Pei; He, Wenxiu; Song, Aixin; Luan, Yuxia

    2016-06-01

    Docetaxel (DTX) can produce anti-tumor effects by inhibiting cell growth and inducing apoptosis. However, the poor solubility of DTX restricts its application and its clinical formulation has caused serious adverse reaction due to the use of Tween-80. In the present study, DTX was conjugated to an amphiphilic di-block polymer to solve these problems. Methoxy poly(ethylene glycol)-poly(ε-caprolactone) (mPEG-PCL) was selected as the polymer skeleton and a redox sensitive disulfide bond was used as the linker between DTX and mPEG-PCL. The synthesized mPEG-PCL-SS-DTX conjugates were characterized by (1)H-nuclear magnetic resonance ((1)H NMR) and Fourier transform infrared spectroscopy (FTIR). Interestingly, the mPEG-PCL-SS-DTX conjugates could self-assemble into micelles in aqueous solution. The critical micelle concentration (CMC) of mPEG-PCL-SS-DTX micelles was about 2.3mgL(-1) determined using pyrene molecule fluorescent probe method while the size of mPEG-PCL-SS-DTX micelles was determined to be ca. 17.6nm and 116.0nm with a bimodal distribution by dynamic light scattering (DLS). The in vitro release results indicated that the as-prepared micelles exhibited a sustained release profile with good redox sensitive properties. In particular, the hemolytic toxicity test indicated the as-prepared mPEG-PCL-SS-DTX micelles had negligible hemolytic activity, demonstrating their safety in drug delivery system. Cytotoxicity assay of the mPEG-PCL-SS-DTX micelles verified their highly enhanced cytotoxicity to MCF-7/A and A549 cells. These results thus demonstrated that the present redox-sensitive mPEG-PCL-SS-DTX micelle was an efficient and safe sustained drug delivery system in the biomedical area. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Association analysis of polymorphism in KiSS1 gene with reproductive traits in goats.

    PubMed

    El-Tarabany, Mahmoud S; Zaglool, Asmaa W; El-Tarabany, Akram A; Awad, Ashraf

    2017-05-01

    Understanding the genetic information of related genes is helpful for the selection and breeding course through marker assisted selection. The aim of the current study was to detect polymorphisms of the KiSS1 gene in 137 animals, including Baladi, Zaraibi and Damascus goat breeds by PCR-RFLP, and DNA sequencing and to investigate the association between these variants and reproductive traits. Comparison of the nucleotide sequence indicated the substitution of T with A at position 121 (T121A) in the intron 1 of the KiSS1 gene in all goat breeds. This substitution distorts the restriction site of the XmnI restriction enzyme and consequently two genotypes were detected (TA and TT). The T121A SNP is associated significantly with litter size in Damascus and Zaribi breeds (p=0.025 and 0.001, respectively). The animals with the TT genotype in Damascus and Zaribi breeds had a significantly higher estradiol 17β level than that recorded in TA genotype at estrus phase (p=0.013 and 0.028, respectively) and late-luteal phase (p=0.067 and 0.041, respectively) of the estrus cycle. Furthermore, animals with the TT genotype in Damascus and Zaribi breeds had significant higher progesterone level at mid-luteal (p=0.037 and 0.045, respectively) phase. Meanwhile, there were no significant differences in progesterone level in late-luteal phase between both genotypes in Zaribi breed (p=0.267). The current trial indicated that the prolific TT genotype in both Damascus and Zaribi breeds had superior estradiol 17β level at estrus phase and an eminent progesterone level at both early and mid-luteal phases of the estrous cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Attenuated Salmonella Typhimurium Lacking the Pathogenicity Island-2 Type 3 Secretion System Grow to High Bacterial Numbers inside Phagocytes in Mice

    PubMed Central

    Grant, Andrew J.; Morgan, Fiona J. E.; McKinley, Trevelyan J.; Foster, Gemma L.; Maskell, Duncan J.; Mastroeni, Pietro

    2012-01-01

    Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics. PMID:23236281

  12. A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

    PubMed

    Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

    2018-01-01

    We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. LINKIN, a new transmembrane protein necessary for cell adhesion

    PubMed Central

    Kato, Mihoko; Chou, Tsui-Fen; Yu, Collin Z; DeModena, John; Sternberg, Paul W

    2014-01-01

    In epithelial collective migration, leader and follower cells migrate while maintaining cell–cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG–GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. DOI: http://dx.doi.org/10.7554/eLife.04449.001 PMID:25437307

  14. Steady-State Serum T3 Concentrations for 48 Hours Following the Oral Administration of a Single Dose of 3,5,3'-Triiodothyronine Sulfate (T3S).

    PubMed

    Santini, Ferruccio; Giannetti, Monica; Ricco, Ilaria; Querci, Giorgia; Saponati, Giorgio; Bokor, Daniela; Rivolta, Giovanni; Bussi, Simona; Braverman, Lewis E; Vitti, Paolo; Pinchera, Aldo

    2014-07-01

    Sulfate conjugation of thyroid hormones is an alternate metabolic pathway that facilitates the biliary and urinary excretion of iodothyronines and enhances their deiodination rate, leading to the generation of inactive metabolites. A desulfating pathway reverses this process, and thyromimetic effects have been observed following the parenteral administration of 3,5,3'-triiodothyronine (T3) sulfate (T3S) in rats. The present study investigated whether T3S is absorbed after oral administration in humans and if it represents a source of T3. Twenty-eight hypothyroid patients (7 men and 21 women; mean age, 44 ± 11 years) who had a thyroidectomy for thyroid carcinoma were enrolled. Replacement thyroid hormone therapy was withdrawn (42 days for thyroxine, 14 days for T3) prior to 131I remnant ablation. A single oral dose of 20, 40, 80 (4 patients/group), or 160 μg (16 patients/group) of T3S was administered 3 days before the planned administration of 131I. Blood samples for serum T3S and total T3 (TT3) concentrations were obtained at various times up to 48 hours after T3S administration. At all T3S doses, serum T3S concentrations increased, reaching a peak at 2 to 4 hours and progressively returning to basal levels within 8 to 24 hours. The T3S maximum concentration (Cmax) and area under the 0- to 48-hour concentration-time curve (AUC0-48h) were directly and significantly related to the administered dose. An increase in serum TT3 concentration was observed (significant after 1 hour), and the concentration increased further at 2 and 4 hours and then remained steady up to 48 hours after T3S administration. There was a significant direct correlation between the TT3 AUC0-48h and the administered dose of T3S. No changes in serum free thyroxine (T4) concentrations during the entire study period were observed, whereas serum thyroid-stimulating hormone levels increased slightly at 48 hours, but this was not related to the dose of T3S. No adverse events were reported. (1) T3S is

  15. Characterization of Circular ssDNA Viruses within the Echinoderm Nanobiome

    NASA Astrophysics Data System (ADS)

    Jackson, E.; Bistolas, K. S.; Hewson, I.

    2016-02-01

    Viral metagenomics has revealed a great diversity and presence of circular single-stranded(ss) DNA viruses most similar to the viral family Circoviridae in various environments both ambient and host. These viruses are an emerging paradigm in viral discovery amongst aquatic invertebrates mainly from the sub-phlya Crustacea and to a lesser extent the phylum Echinodermata. This parasite-host relationship is furthered here with the discovery of circo-like viruses extracted from the tissue of members from the family Holothuroidea (sea cucumbers). Verification and presence of these viruses within the tissue of the host was confirmed through rigorous genome architecture screening and PCR amplification of the rep gene from unamplified viral DNA extracts. Phylogenetic analysis of the rep gene reveals high similarity to circular ssDNA viruses from environmental metagenomic surveys of marine habitats. The significance of these findings is changing the perception and understanding of circular ssDNA viruses by broadening the known host range and blurring certain defining characteristics established by their pathogenic counterparts. Aside from discover and characterization, the potential ecological impacts of ssDNA viruses upon their host remains relatively unknown and further investigations should aim to determine the pathology, route of infection, and ecological implications of viral infection.

  16. HIF-1α P582S and A588T polymorphisms and digestive system cancer risk-a meta-analysis.

    PubMed

    Yang, Xi; Zhang, Chi; Zhu, Hong-Cheng; Qin, Qin; Zhao, Lian-Jun; Liu, Jia; Xu, Li-Ping; Zhang, Qu; Cai, Jing; Ma, Jian-Xin; Cheng, Hong-Yan; Sun, Xin-Chen

    2014-03-01

    Hypoxia-inducible factor-1 (HIF-1) influences cancer progression and metastasis through various mechanisms, and HIF-1α polymorphisms are reportedly associated with many cancers; however, the associations of HIF-1α P582S and A588T polymorphisms with the risk of digestive system cancer remain inconclusive. To understand the role of HIF-1α P582S and A588T genotypes in digestive cancer development, we conducted a comprehensive meta-analysis involving 1,517 cases and 3,740 controls. Overall, the P582S polymorphism was not significantly associated with digestive system cancers in all genotypes. By contrast, the A588T polymorphism was significantly associated with digestive system cancers in the dominant model (TT/AT vs. AA: OR = 3.17, 95% CI: 1.21, 8.25; P heterogeneity < 0.001). In subgroup analysis for cancer types, the two polymorphisms were only associated with increased risk of pancreatic cancer (P582S: SS vs. PP: OR = 2.51, 95% CI: 1.31, 4.81; SS vs. OR = 8.73, 95% CI: 1.33, 57.1; A588T: TT vs. AA: OR = 9.30, 95% CI: 1.12, 77.6; P heterogeneity = 0.478; TT vs. OR = 3.14, 95% CI: 1.99, 4.97; P heterogeneity = 0.098; TT/AT vs. AA: OR = 8.65, 95% CI: 1.05, 71.6; P heterogeneity = 0.418). According to the source of ethnicity, the P582S and the A588T polymorphisms are both significantly associated with an increased risk of cancer among Caucasians in the homozygote model (SS vs. PP: OR = 2.41, 95% CI: 1.24, 4.691; P heterogeneity = 0.010; TT vs. AA: OR = 98.6, 95% CI: 4.37, 2,224; P heterogeneity = 0.040) and the recessive model (SS vs. OR = 9.48, 95% CI: 1.12, 80.3; P heterogeneity < 0.001; TT vs. OR = 82.7, 95% CI: 3.79, 1,802; P heterogeneity = 0.041). Our findings suggest that the HIF-1α A588T polymorphism is significantly associated with higher cancer risk and the P582S polymorphism is significantly associated with pancreatic cancer risk. Furthermore, the effect of both polymorphisms on

  17. Predicting Transmembrane Helix Packing Arrangements using Residue Contacts and a Force-Directed Algorithm

    PubMed Central

    Nugent, Timothy; Jones, David T.

    2010-01-01

    Alpha-helical transmembrane proteins constitute roughly 30% of a typical genome and are involved in a wide variety of important biological processes including cell signalling, transport of membrane-impermeable molecules and cell recognition. Despite significant efforts to predict transmembrane protein topology, comparatively little attention has been directed toward developing a method to pack the helices together. Here, we present a novel approach to predict lipid exposure, residue contacts, helix-helix interactions and finally the optimal helical packing arrangement of transmembrane proteins. Using molecular dynamics data, we have trained and cross-validated a support vector machine (SVM) classifier to predict per residue lipid exposure with 69% accuracy. This information is combined with additional features to train a second SVM to predict residue contacts which are then used to determine helix-helix interaction with up to 65% accuracy under stringent cross-validation on a non-redundant test set. Our method is also able to discriminate native from decoy helical packing arrangements with up to 70% accuracy. Finally, we employ a force-directed algorithm to construct the optimal helical packing arrangement which demonstrates success for proteins containing up to 13 transmembrane helices. This software is freely available as source code from http://bioinf.cs.ucl.ac.uk/memsat/mempack/. PMID:20333233

  18. Sequence-structure relationship study in all-α transmembrane proteins using an unsupervised learning approach.

    PubMed

    Esque, Jérémy; Urbain, Aurélie; Etchebest, Catherine; de Brevern, Alexandre G

    2015-11-01

    Transmembrane proteins (TMPs) are major drug targets, but the knowledge of their precise topology structure remains highly limited compared with globular proteins. In spite of the difficulties in obtaining their structures, an important effort has been made these last years to increase their number from an experimental and computational point of view. In view of this emerging challenge, the development of computational methods to extract knowledge from these data is crucial for the better understanding of their functions and in improving the quality of structural models. Here, we revisit an efficient unsupervised learning procedure, called Hybrid Protein Model (HPM), which is applied to the analysis of transmembrane proteins belonging to the all-α structural class. HPM method is an original classification procedure that efficiently combines sequence and structure learning. The procedure was initially applied to the analysis of globular proteins. In the present case, HPM classifies a set of overlapping protein fragments, extracted from a non-redundant databank of TMP 3D structure. After fine-tuning of the learning parameters, the optimal classification results in 65 clusters. They represent at best similar relationships between sequence and local structure properties of TMPs. Interestingly, HPM distinguishes among the resulting clusters two helical regions with distinct hydrophobic patterns. This underlines the complexity of the topology of these proteins. The HPM classification enlightens unusual relationship between amino acids in TMP fragments, which can be useful to elaborate new amino acids substitution matrices. Finally, two challenging applications are described: the first one aims at annotating protein functions (channel or not), the second one intends to assess the quality of the structures (X-ray or models) via a new scoring function deduced from the HPM classification.

  19. Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

    PubMed

    Brent, G A; Williams, G R; Harney, J W; Forman, B M; Samuels, H H; Moore, D D; Larsen, P R

    1992-04-01

    Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common

  20. CMTM3 (CKLF-Like Marvel Transmembrane Domain 3) Mediates Angiogenesis by Regulating Cell Surface Availability of VE-Cadherin in Endothelial Adherens Junctions.

    PubMed

    Chrifi, Ihsan; Louzao-Martinez, Laura; Brandt, Maarten; van Dijk, Christian G M; Burgisser, Petra; Zhu, Changbin; Kros, Johan M; Duncker, Dirk J; Cheng, Caroline

    2017-06-01

    Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1 + endothelial progenitor cells during embryonic development. Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin + vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin + , EEA1 + , Rab11 + , Rab5 + , and Rab7 + vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM

  1. The type III secretion system is necessary for the development of a pathogenic and endophytic interaction between Herbaspirillum rubrisubalbicans and Poaceae

    PubMed Central

    2012-01-01

    Background Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causing the mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with various plants of economic interest in a non pathogenic manner. Results A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization. Conclusions Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice. PMID:22672506

  2. The type III secretion system is necessary for the development of a pathogenic and endophytic interaction between Herbaspirillum rubrisubalbicans and Poaceae.

    PubMed

    Schmidt, Maria Augusta; Balsanelli, Eduardo; Faoro, Hellison; Cruz, Leonardo M; Wassem, Roseli; de Baura, Valter A; Weiss, Vinícius; Yates, Marshall G; Madeira, Humberto M F; Pereira-Ferrari, Lilian; Fungaro, Maria H P; de Paula, Francine M; Pereira, Luiz F P; Vieira, Luiz G E; Olivares, Fábio L; Pedrosa, Fábio O; de Souza, Emanuel M; Monteiro, Rose A

    2012-06-06

    Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causing the mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with various plants of economic interest in a non pathogenic manner. A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization. Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice.

  3. Detailed T1-Weighted Profiles from the Human Cortex Measured in Vivo at 3 Tesla MRI.

    PubMed

    Ferguson, Bart; Petridou, Natalia; Fracasso, Alessio; van den Heuvel, Martijn P; Brouwer, Rachel M; Hulshoff Pol, Hilleke E; Kahn, René S; Mandl, René C W

    2018-04-01

    Studies into cortical thickness in psychiatric diseases based on T1-weighted MRI frequently report on aberrations in the cerebral cortex. Due to limitations in image resolution for studies conducted at conventional MRI field strengths (e.g. 3 Tesla (T)) this information cannot be used to establish which of the cortical layers may be implicated. Here we propose a new analysis method that computes one high-resolution average cortical profile per brain region extracting myeloarchitectural information from T1-weighted MRI scans that are routinely acquired at a conventional field strength. To assess this new method, we acquired standard T1-weighted scans at 3 T and compared them with state-of-the-art ultra-high resolution T1-weighted scans optimised for intracortical myelin contrast acquired at 7 T. Average cortical profiles were computed for seven different brain regions. Besides a qualitative comparison between the 3 T scans, 7 T scans, and results from literature, we tested if the results from dynamic time warping-based clustering are similar for the cortical profiles computed from 7 T and 3 T data. In addition, we quantitatively compared cortical profiles computed for V1, V2 and V7 for both 7 T and 3 T data using a priori information on their relative myelin concentration. Although qualitative comparisons show that at an individual level average profiles computed for 7 T have more pronounced features than 3 T profiles the results from the quantitative analyses suggest that average cortical profiles computed from T1-weighted scans acquired at 3 T indeed contain myeloarchitectural information similar to profiles computed from the scans acquired at 7 T. The proposed method therefore provides a step forward to study cortical myeloarchitecture in vivo at conventional magnetic field strength both in health and disease.

  4. Synergistic Protective Effects of Mitochondrial Division Inhibitor 1 and Mitochondria-Targeted Small Peptide SS31 in Alzheimer's Disease.

    PubMed

    Reddy, P Hemachandra; Manczak, Maria; Yin, XiangLing; Reddy, Arubala P

    2018-01-01

    The purpose of our study was to determine the synergistic protective effects of mitochondria-targeted antioxidant SS31 and mitochondria division inhibitor 1 (Mdivi1) in Alzheimer's disease (AD). Using biochemical methods, we assessed mitochondrial function by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity, mitochondrial ATP, and GTPase Drp1 enzymatic activity in mutant AβPP cells. Using biochemical methods, we also measured cell survival and apoptotic cell death. Amyloid-β (Aβ) levels were measured using sandwich ELISA, and using real-time quantitative RT-PCR, we assessed mtDNA (mtDNA) copy number in relation to nuclear DNA (nDNA) in all groups of cells. We found significantly reduced levels of Aβ40 and Aβ42 in mutant AβPP cells treated with SS31, Mdivi1, and SS31+Mdivi1, and the reduction of Aβ42 levels were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. The levels of mtDNA copy number and cell survival were significantly increased in SS31, Mdivi1, and SS31+Mdivi1 treated mutant AβPP cells; however, the increased levels of mtDNA copy number and cell survival were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. Mitochondrial dysfunction is significantly reduced in SS31, Mdivi1, and SS31+Mdivi1 treated mutant AβPP cells; however, the reduction is much higher in cells treated with both SS31+Mdvi1. Similarly, GTPase Drp1 activity is reduced in all treatments, but reduced much higher in SS31+Mdivi1 treated cells. These observations strongly suggest that combined treatment of SS31+Mdivi1 is effective than individual treatments of SS31 and Mdivi1. Therefore, we propose that combined treatment of SS31+Mdivi1 is a better therapeutic strategy for AD. Ours is the first study to investigate combined treatment of mitochondria-targeted antioxidant SS31 and mitochondrial division inhibitor 1 in AD neurons.

  5. Chemical Synthesis of the Highly Hydrophobic Antiviral Membrane-Associated Protein IFITM3 and Modified Variants.

    PubMed

    Harmand, Thibault J; Pattabiraman, Vijaya R; Bode, Jeffrey W

    2017-10-02

    Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5 kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (>15 mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The global regulator Crc plays a multifaceted role in modulation of type III secretion system in Pseudomonas aeruginosa.

    PubMed

    Dong, Yi-Hu; Zhang, Xi-Fen; Zhang, Lian-Hui

    2013-02-01

    The opportunistic pathogen Pseudomonas aeruginosa utilizes type III secretion system (T3SS) to translocate effector proteins into eukaryotic host cells that subvert normal host cell functions to the benefit of the pathogen, and results in serious infections. T3SS in P. aeruginosa is controlled by a complex system of regulatory mechanisms and signaling pathways. In this study, we described that Crc, an RNA-binding protein, exerts a positive impact on T3SS in P. aeruginosa, as evidenced by promoter activity assays of several key T3SS genes, transcriptomics, RT-PCR, and immunoblotting in crc mutant. We further demonstrated that the regulatory function of Crc on the T3SS was mediated through the T3SS master regulator ExsA and linked to the Cbr/Crc signaling system. Expression profiling of the crc mutant revealed a downregulation of flagship T3SS genes as well as 16 other genes known to regulate T3SS gene expression in P. aeruginosa. On the basis of these data, we proposed that Crc may exert multifaceted control on the T3SS through various pathways, which may serve to fine-tune this virulence mechanism in response to environmental changes and nutrient sources. © 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  7. Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1

    PubMed Central

    Gui, Chunshan; Hagenbuch, Bruno

    2009-01-01

    The liver-specific organic anion transporting polypeptides OATP1B1 and OATP1B3 are highly homologous and share numerous substrates. However, at low concentrations OATP1B1 shows substrate selectivity for estrone-3-sulfate. In this study, we investigated the molecular mechanism for this substrate selectivity of OATP1B1 by constructing OATP1B1/1B3 chimeric transporters and by site-directed mutagenesis. Functional studies of chimeras showed that transmembrane domain 10 is critical for the function of OATP1B1. We further identified four amino acid residues, namely L545, F546, L550, and S554 in TM10, whose simultaneous mutation caused almost complete loss of OATP1B1-mediated estrone-3-sulfate transport. Comparison of the kinetics of estrone-3-sulfate transport confirmed a biphasic pattern for OATP1B1, but showed a monophasic pattern for the quadruple mutant L545S/F546L/L550T/S554T. This mutant also showed reduced transport for other OATP1B1 substrates such as bromosulfophthalein and [d-penicillamine2,5]enkephalin. Helical wheel analysis and molecular modeling suggest that L545 is facing the substrate translocation pathway, whereas F546, L550, and S554 are located inside the protein. These results indicate that L545 might contribute to OATP1B1 function by interacting with substrates, whereas F546, L550, and S554 seem important for protein structure. In conclusion, our results show that TM10 is critical for the function of OATP1B1. PMID:19760661

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

    PubMed

    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.

  9. Surface modification of SS-316L steel using microwave processed Ni/WC based composite clads

    NASA Astrophysics Data System (ADS)

    Kaushal, Sarbjeet; Singh, Dilkaran; Gupta, Dheeraj; Jain, Vivek; Bhowmick, Hiralal

    2018-04-01

    In the present investigation, the claddings of Ni/WC based composite powder were developed on SS-316L steel through microwave hybrid heating method. The experimental trials were carried out inside a domestic microwave oven working at 2.45 GHz and 900 W. The so developed composite clads were characterized using XRD, Vicker's microhardness measurement, and SEM/EDS. The presence of different phases like Co3W3C, NiW, FeNi3, NiSi was confirmed by XRD analysis. Microstructural analysis revealed that the clad of approximately 0.6 mm thickness was developed with no interfacial cracks and negligible porosity. The WC particles were uniformly distributed in the form of cellular structure inside Ni matrix. The average Vicker's microhardness value of the clad section was observed as 925±50 HV, which is three times that of the SS-316L substrate.

  10. Wnt transmembrane signaling and long-term spatial memory

    PubMed Central

    Tabatadze, Nino; Tomas, Caroline; McGonigal, Rhona; Lin, Brian; Schook, Andrew; Routtenberg, Aryeh

    2011-01-01

    Transmembrane signaling mechanisms are critical for regulating the plasticity of neuronal connections underlying the establishment of long-lasting memory (e.g., Linden and Routtenberg, 1989, Brain Res Rev. 14: 279–296; Sossin, 1996, Trends Neurosci 19: 215–218; Mayr and Montminy, 2001, Nat Rev Mol Cell Biol. 2: 599–609; Chen et al., 2011, Nature 469: 491–497). One signaling mechanism that has received surprisingly little attention in this regard is the well-known Wnt transmembrane signaling pathway even though this pathway in the adult plays a significant role, for example, in postsynaptic dendritic spine morphogenesis and presynaptic terminal neurotransmitter release (Inestrosa and Arenas, 2010, Nature Rev Neurosci 11: 77–86). The present report now provides the first evidence of Wnt signaling in spatial information storage processes. Importantly, this Wnt participation is specific and selective. Thus, spatial, but not cued, learning in a water maze selectively elevates the levels in hippocampus of Wnt 7 and Wnt 5a, but not the Wnt 3 isoform, indicating behavioral selectivity and isoform specificity. Wnt 7 elevation is subfield-specific: granule cells show an increase with no detectable change in CA3 neurons. Wnt 7 elevation is temporally specific: increased Wnt signaling is not observed during training, but is seen 7 days and, unexpectedly, 30 days later. If the Wnt elevation after learning is activity-dependent, then it may be possible to model this effect in primary hippocampal neurons in culture. Here we evaluate the consequence of potassium or glutamate depolarization on Wnt signaling. This represents, to our knowledge, the first demonstration of an activation-dependent elevation of Wnt levels. Additionally, the novel finding emerged of an increased number of Wnt-stained puncta in neuritis suggestive of trafficking from the cell body to neuronal processes, probably dendrites. It is proposed that Wnt signaling pathways, both canonical and non

  11. Structural basis of ligand interaction with atypical chemokine receptor 3

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

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje

    2017-01-18

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally drivenmore » models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.« less

  12. Comparison between whole-body and head and neck neurovascular coils for 3-T magnetic resonance proton resonance frequency shift thermography guidance in the head and neck region.

    PubMed

    Ginat, Daniel Thomas; Anthony, Gregory J; Christoforidis, Gregory; Oto, Aytekin; Dalag, Leonard; Sammet, Steffen

    2018-02-01

    The purpose of this study is to compare the image quality of magnetic resonance (MR) treatment planning images and proton resonance frequency (PRF) shift thermography images and inform coil selection for MR-guided laser ablation of tumors in the head and neck region. Laser ablation was performed on an agar phantom and monitored via MR PRF shift thermography on a 3-T scanner, following acquisition of T1-weighted (T1W) planning images. PRF shift thermography images and T2-weighted (T2W) planning images were also performed in the neck region of five normal human volunteers. Signal-to-noise ratios (SNR) and temperature uncertainty were calculated and compared between scans acquired with the quadrature mode body integrated coil and a head and neck neurovascular coil. T1W planning images of the agar phantom produced SNRs of 4.0 and 12.2 for the quadrature mode body integrated coil and head and neck neurovascular coil, respectively. The SNR of the phantom MR thermography magnitude images obtained using the quadrature mode body integrated coil was 14.4 versus 59.6 using the head and neck coil. The average temperature uncertainty for MR thermography performed on the phantom with the quadrature mode body integrated coil was 1.1 versus 0.3 °C with the head and neck coil. T2W planning images of the neck in five human volunteers produced SNRs of 28.3 and 91.0 for the quadrature mode body integrated coil and head and neck coil, respectively. MR thermography magnitude images of the neck in the volunteers obtained using the quadrature mode body integrated coil had a signal-to-noise ratio of 8.3, while the SNR using the head and neck coil was 16.1. The average temperature uncertainty for MR thermography performed on the volunteers with the body coil was 2.5 versus 1.6 °C with the head and neck neurovascular coil. The quadrature mode body integrated coil provides inferior image quality for both basic treatment planning sequences and MR PRF shift thermography compared with a

  13. Functional Effects of Genetic Polymorphisms in the N-acetyltransferase 1 Coding and 3′ Untranslated Regions

    PubMed Central

    Zhu, Yuanqi; States, J. Christopher; Wang, Yang; Hein, David W.

    2011-01-01

    BACKGROUND The functional effects of N-acetyltransferase 1 (NAT1) polymorphisms and haplotypes are poorly understood, compromising the validity of associations reported with diseases including birth defects and numerous cancers. METHODS We investigated the effects of genetic polymorphisms within the NAT1 coding region and the 3′-untranslated region (3′-UTR) and their associated haplotypes on N- and O-acetyltransferase catalytic activities, and NAT1 mRNA and protein levels following recombinant expression in COS-1 cells. RESULTS 1088T>A (rs1057126; 3′-UTR) and 1095C>A (rs15561; 3′-UTR) each slightly reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. A 9-base pair (TAATAATAA) deletion between nucleotides 1065-1090 (3′-UTR) reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. In contrast, a 445G>A (rs4987076; V149I), 459G>A (rs4986990; T153T), 640T>G (rs4986783; S214A) coding region haplotype present in NAT1*11 increased NAT1 catalytic activity and NAT1 protein, but not NAT1 mRNA levels. A combination of the 9-base pair (TAATAATAA) deletion and the 445G>A, 459G>A, 640T>G coding region haplotypes, both present in NAT1*11, appeared to neutralize the opposing effects on NAT1 protein and catalytic activity, resulting in levels of NAT1 protein and catalytic activity that did not differ significantly from the NAT1*4 reference. CONCLUSIONS Since 1095C>A (3′-UTR) is the sole polymorphism present in NAT1*3, our data suggests that NAT1*3 is not functionally equivalent to the NAT1*4 reference. Furthermore, our findings provide biological support for reported associations of 1088T>A and 1095C>A polymorphisms with birth defects. PMID:21290563

  14. Reduction of T cell receptor diversity in NOD mice prevents development of type 1 diabetes but not Sjögren's syndrome.

    PubMed

    Kern, Joanna; Drutel, Robert; Leanhart, Silvia; Bogacz, Marek; Pacholczyk, Rafal

    2014-01-01

    Non-obese diabetic (NOD) mice are well-established models of independently developing spontaneous autoimmune diseases, Sjögren's syndrome (SS) and type 1 diabetes (T1D). The key determining factor for T1D is the strong association with particular MHCII molecule and recognition by diabetogenic T cell receptor (TCR) of an insulin peptide presented in the context of I-Ag7 molecule. For SS the association with MHCII polymorphism is weaker and TCR diversity involved in the onset of the autoimmune phase of SS remains poorly understood. To compare the impact of TCR diversity reduction on the development of both diseases we generated two lines of TCR transgenic NOD mice. One line expresses transgenic TCRβ chain originated from a pathogenically irrelevant TCR, and the second line additionally expresses transgenic TCRαmini locus. Analysis of TCR sequences on NOD background reveals lower TCR diversity on Treg cells not only in the thymus, but also in the periphery. This reduction in diversity does not affect conventional CD4+ T cells, as compared to the TCRmini repertoire on B6 background. Interestingly, neither transgenic TCRβ nor TCRmini mice develop diabetes, which we show is due to lack of insulin B:9-23 specific T cells in the periphery. Conversely SS develops in both lines, with full glandular infiltration, production of autoantibodies and hyposalivation. It shows that SS development is not as sensitive to limited availability of TCR specificities as T1D, which suggests wider range of possible TCR/peptide/MHC interactions driving autoimmunity in SS.

  15. Type three secretion system-mediated escape of Burkholderia pseudomallei into the host cytosol is critical for the activation of NFκB.

    PubMed

    Teh, Boon Eng; French, Christopher Todd; Chen, Yahua; Chen, Isabelle Gek Joo; Wu, Ting-Hsiang; Sagullo, Enrico; Chiou, Pei-Yu; Teitell, Michael A; Miller, Jeff F; Gan, Yunn-Hwen

    2014-05-06

    Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three "injection type" type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFκB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFκB activation is unknown. Known T3SS3 effectors are not responsible for NFκB activation. Furthermore, T3SS3-null mutants are able to activate NFκB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFκB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection. T3SS3 does not directly activate NFκB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFκB activation.

  16. Ecotoxicological and microbiological assessment of sewage sludge associated with sugarcane bagasse.

    PubMed

    Sommaggio, Lais Roberta Deroldo; Mazzeo, Dânia Elisa Christofoletti; Sant' Anna, Débora de Andrade E Silva; Levy, Carlos Emílio; Marin-Morales, Maria Aparecida

    2018-01-01

    Sewage sludge (SS) obtained after sewage treatment process may contain several toxic substances. Bioremediation can decrease the toxicity of the sludge, mainly when it is associated with stimulant agents, such as sugarcane bagasse (B). Samples of pure SS (SSP); SS+B; SS+Soil; and SS+B+Soil were bioremediated for 1, 3, and 6 months (T1, T2, and T3, respectively). After each period, the cytotoxic, genotoxic, and mutagenic potentials of the solid samples and their respective aqueous extracts (aqueous eluate and percolate water) were evaluated by the Allium cepa test. A microbiological analysis of the samples was also performed after each period tested. All solid samples of SS+B (in T1, T2, and T3) and the solid sample of SSP (treatment T3) showed a significant decrease of cell division (cytotoxic effects). The aqueous eluate extracts of SS+B (T1 and T3) and SSP (T2 and T3) induced cytotoxic effect. The solid sample of SS+B (T2 and T3) and aqueous extracts of SSP (T1) were genotoxic, indicating a harmful effect of SS on A. cepa, even after 6 months of bioremediation. There was an alternation in the microbial community both in diversity and in abundance, with the predominance of nonfermenting gram-negative bacilli. The tested bioremediation periods were not sufficient for the complete detoxification of SS, and the use of B did not seem to contribute to the degradation of the pollutants to inert compounds. These data emphasize that a specific relationship should exist between the sludge characteristic and the biostimulating agent used to promote a more efficient bioremediation. These results suggest the necessity to study longer periods of biodegradation and the use of other decomposing agents for greater safety and sustainability for the agricultural use of this residue. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Periodicity Analysis of X-ray Light Curves of SS 433

    NASA Astrophysics Data System (ADS)

    Wang, J. Y.; Lu, X. L.; Zhao, Q. W.; Dong, D. Q.; Lao, B. Q.; Lu, Y.; Wei, Y. H.; Wu, X. C.; An, T.

    2016-03-01

    SS 433 is the only X-ray binary to date that was detected to have a pair of well-collimated jets, and its orbital period, super orbital period, and nutation period were all detected at the same time. The study on the periodic X-ray variabilities is helpful for understanding its dynamic process of the central engine and the correlation with other bands. In the present paper, two time series analysis techniques, Lomb-Scargle periodogram and weighted wavelet Z-transform, are employed to search for the periodicities from the Swift/BAT (Burst Alert Telescope)(15--50 keV) and RXTE/ASM (Rossi X-Ray Timing Explorer/All-Sky Monitor)(1.5--3, 3--5 and 5--12 keV) light curves of SS 433, and the Monte Carlo simulation is performed. For the 15--50 keV energy band, five significant periodic signals are detected, which are P_1(˜6.29 d), P_2 (˜6.54 d), P_3 (˜13.08 d), P_4 (˜81.50 d), and P_5 (˜162.30 d). For the 3--5 and 5--12 keV energy bands, periodic signals P_3 (˜13 d) and P_5 (˜162 d) are detected in both energy bands. However, for the 1.5--3 keV energy band, no significant periodic signal is detected. P_5 has the strongest periodic signal in the power spectrum for all the energy bands of 3--5, 5--12, and 15--50 keV, and it is consistent with that obtained by previous study in optical band. Further, due to the existence of relativistic radio jets, the X-ray and optical band variability of P_5 (˜162 d) is probably related to the precession of the relativistic jets. High coherence between X-ray and optical light curves may also imply that the X-ray and optical emissions are of the same physical origin. P_3 shows a good agreement with the orbital period (˜13.07 d) first obtained by previous study, and P_2 and P_4 are the high frequency harmonic components of P_3 and P_5, respectively. P_1 is detected from the power spectrum of 15--50 keV energy band only, and it is consistent with the systematic nutation period. As the power of energy band decreases (from hard X-ray to

  18. Pharmacology and expression analysis of glycine transporter GlyT1 with [3H]-(N-[3-(4'-fluorophenyl)-3-(4'phenylphenoxy)propyl])sarcosine.

    PubMed

    Mallorga, Pierre J; Williams, Jacinta B; Jacobson, Marlene; Marques, Rosemary; Chaudhary, Ashok; Conn, P Jeffrey; Pettibone, Douglas J; Sur, Cyrille

    2003-10-01

    In the central nervous system, re-uptake of the neurotransmitter glycine is mediated by two different glycine transporters, GlyT1 and GlyT2. GlyT2 is found in brainstem and spinal cord, whereas GlyT1 is expressed in rat forebrain regions where it is responsible for most glycine transport activity. Initially, GlyT1 and GlyT2 were pharmacologically differentiated by sarcosine, a weak selective inhibitor of GlyT1. The recently described selective and potent GlyT1 antagonist, NFPS/ALX-5407 provided an important additional tool to further characterize GlyT1 pharmacology. In the present study, we have radiolabeled the racemic form of NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (also known as ALX-5407) to investigate its interaction with GlyT1, as well as define GlyT1 expression in the rat central nervous system. Kinetic studies indicated that [3H]NFPS binds rapidly to rat forebrain membranes and dissociates with a t(1/2) of 28 +/- 5 min. [3H]NFPS labeled a saturable population of sites in rat forebrain with a Kd of 7.1+/-1.3 nM and a B(max) of 3.14 +/- 0.26 pmol/mg protein. Bound [3H]NFPS was fully and potently displaced by unlabeled NFPS, whereas glycine and sarcosine were weak, Na+-dependent inhibitors with IC50 of 1,008 and 190 microM, respectively. Additional saturation experiments indicated that glycine and sarcosine were non-competitive antagonists of [3H]NFPS binding. Functional studies revealed that NFPS was a non-competitive inhibitor of [3H]glycine uptake and does not interact with Na+ and Cl- binding sites of GlyT1. Overall, this work shows that [3H]NFPS is a valuable tool in studying GlyT1 expression and pharmacology and that NFPS interacts with GlyT1 at a site different from the transporter translocation and ion binding sites.

  19. Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.

    PubMed

    Brennan, Matthew J; Karcz, Jennifer; Vaughn, Nicholas R; Woolwine-Cunningham, Yvonne; DePriest, Adam D; Escalona, Yerko; Perez-Acle, Tomas; Skerrett, I Martha

    2015-07-10

    Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. 3.0 T plaque imaging.

    PubMed

    Hinton-Yates, Denise P; Cury, Ricardo C; Wald, Lawrence L; Wiggins, Graham C; Keil, Boris; Seethmaraju, Ravi; Gangadharamurthy, Dakshinamurthy; Ogilvy, Christopher S; Dai, Guangping; Houser, Stuart L; Stone, James R; Furie, Karen L

    2007-10-01

    The aim of this article is to evaluate 3.0 T magnetic resonance imaging for characterization of vessel morphology and plaque composition. Emphasis is placed on early and moderate stages of carotid atherosclerosis, where increases in signal-to-noise (SNR) and contrast-to-noise (CNR) ratios compared with 1.5 T are sought. Comparison of in vivo 3.0 T imaging to histopathology is performed for validation. Parallel acceleration methods applied with an 8-channel carotid array are investigated as well as higher field ex vivo imaging to explore even further gains. The overall endeavor is to improve prospective assessment of atherosclerosis stage and stability for reduction of atherothrombotic event risk. A total of 10 male and female subjects ranging in age from 22 to 72 years (5 healthy and 5 with cardiovascular disease) participated. Custom-built array coils were used with endogenous and exogenous multicontrast bright and black-blood protocols for 3.0 T carotid imaging. Comparisons were performed to 1.5 T, and ex vivo plaque was stained with hematoxylin and eosin for histology. Imaging (9.4 T) was also performed on intact specimens. The factor of 2 gain in signal-to-noise SNR is realized compared with 1.5 T along with improved wall-lumen and plaque component CNR. Post-contrast black-blood imaging within 5-10 minutes of gadolinium injection is optimal for detection of the necrotic lipid component. In a preliminary 18-month follow-up study, this method provided measurement of a 50% reduction in lipid content with minimal change in plaque size in a subject receiving aggressive statin therapy. Parallel imaging applied with signal averaging further improves 3.0 T black-blood vessel wall imaging. The use of 3.0 T for carotid plaque imaging has demonstrated increases in SNR and CNR compared with 1.5 T. Quantitative prospective studies of moderate and early plaques are feasible at 3.0 T. Continued improvements in coil arrays, 3-dimensional pulse sequences, and the use of novel

  1. Acanthamoeba genotypes T3 and T4 as causative agents of amoebic keratitis in Mexico.

    PubMed

    Omaña-Molina, Maritza; Vanzzini-Zago, Virginia; Hernandez-Martinez, Dolores; Gonzalez-Robles, Arturo; Salazar-Villatoro, Lizbeth; Ramirez-Flores, Elizabeth; Oregon-Miranda, Eric; Lorenzo-Morales, Jacob; Martinez-Palomo, Adolfo

    2016-02-01

    Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection.

  2. ON THE HUBBLE SPACE TELESCOPE TRIGONOMETRIC PARALLAX OF THE DWARF NOVA SS CYGNI

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

    Nelan, Edmund P.; Bond, Howard E., E-mail: nelan@stsci.edu, E-mail: heb11@psu.edu

    SS Cygni is one of the brightest dwarf novae (DNe), and one of the best studied prototypes of the cataclysmic variables. Astrometric observations with the Fine Guidance Sensors (FGSs) on the Hubble Space Telescope (HST), published in 2004, gave an absolute trigonometric parallax of 6.06 {+-} 0.44 mas. However, recent very long baseline interferometry (VLBI), obtained during radio outbursts of SS Cyg, has yielded a significantly larger absolute parallax of 8.80 {+-} 0.12 mas, as well as a large difference in the direction of the proper motion (PM) compared to the HST result. The VLBI distance reduces the implied luminositymore » of SS Cyg by about a factor of two, giving good agreement with predictions based on accretion-disk theory in order to explain the observed DN outburst behavior. This discrepancy raises the possibility of significant systematic errors in FGS parallaxes and PMs. We have reanalyzed the archival HST/FGS data, including (1) a critical redetermination of the parallaxes of the background astrometric reference stars, (2) updated input values of the reference-star PMs, and (3) correction of the position measurements for color-dependent shifts. Our new analysis yields a PM of SS Cyg that agrees well with the VLBI motion, and an absolute parallax of 8.30 {+-} 0.41 mas, also statistically concordant with the VLBI result at the {approx}1.2 {sigma} level. Our results suggest that HST/FGS parallaxes are free of large systematic errors, when the data are reduced using high-quality input values for the astrometry of the reference stars, and when instrumental signatures are properly removed.« less

  3. Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps*

    PubMed Central

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

    2014-01-01

    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. PMID:24876383

  4. Studying Different Binding and Intracellular Delivery Efficiency of ssDNA Single-Walled Carbon Nanotubes and Their Effects on LC3-Related Autophagy in Renal Mesangial Cells via miRNA-382.

    PubMed

    Wang, Guobao; Zhao, Tingting; Wang, Leyu; Hu, Bianxiang; Darabi, Ali; Lin, Jiansheng; Xing, Malcolm M Q; Qiu, Xiaozhong

    2015-11-25

    Single-walled carbon nanotubes (SWCNTs) have been used to deliver single-stranded (ssDNA). ssDNA in oligonucleotide can act as an inhibitor of microRNA to regulate cellular functions. However, these ssDNA are difficult to bind carbon nanotubes with low transferring efficiency to cells. To this end, we designed ssDNA with regulatory and functional units to form ssDNA-SWCNT hybrids to study their binding effects and transferring efficiency. The functional unit on ssDNA mimics the inhibitor (MI) of miRNA-382, which plays a crucial role in the progress of many diseases such as renal interstitial fibrosis. After verification of overexpression of miRNA-382 in a coculture system, we designed oligonucleotide sequences (GCG)5-MI, (TAT)5-MI, and N23-MI as regulatory units added to the 5'-terminal end of the functional DNA fragment, respectively. These regulatory units lead to different secondary structures and thus exhibit different affinity ability to SWCNTs, and finally decide their deliver efficacy to cells. Autophagy, apoptosis and necrosis were observed in renal mesangial cells.

  5. Development in corrosion resistance by microstructural refinement in Zr-16 SS 304 alloy using suction casting technique

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

    Das, N., E-mail: nirupamd@barc.gov.in; Sengupta, P.; Abraham, G.

    Highlights: • Grain refinement was made in Zr–16 wt.% SS alloy while prepared by suction casting process. • Distribution of Laves phase, e.g., Zr{sub 2}(Fe, Cr) was raised in suction cast (SC) Zr–16 wt.% SS. • Corrosion resistance was improved in SC alloy compared to that of arc-melt-cast alloy. • Grain refinement in SC alloy assisted for an increase in its corrosion resistance. - Abstract: Zirconium (Zr)-stainless steel (SS) hybrid alloys are being considered as baseline alloys for developing metallic-waste-form (MWF) with the motivation of disposing of Zr and SS base nuclear metallic wastes. Zr–16 wt.% SS, a MWF alloymore » optimized from previous studies, exhibit significant grain refinement and changes in phase assemblages (soft phase: Zr{sub 2}(Fe, Cr)/α-Zr vs. hard phase: Zr{sub 3}(Fe, Ni)) when prepared by suction casting (SC) technique in comparison to arc-cast-melt (AMC) route. Variation in Cr-distribution among different phases are found to be low in suction cast alloy, which along with grain refinement restricted Cr-depletion at the Zr{sub 2}(Fe, Cr)/Zr interfaces, prone to localized attack. Hence, SC alloy, compared to AMC alloy, showed lower current density, higher potential at the breakdown of passivity and higher corrosion potential during polarization experiments (carried out under possible geological repository environments, viz., pH 8, 5 and 1) indicating its superior corrosion resistance.« less

  6. 32 CFR Appendix E to Part 246 - Stars and Stripes (S&S) Board of Directors

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 2 2013-07-01 2013-07-01 false Stars and Stripes (S&S) Board of Directors E... DEFENSE (CONTINUED) MISCELLANEOUS STARS AND STRIPES (S&S) NEWSPAPER AND BUSINESS OPERATIONS Pt. 246, App. E Appendix E to Part 246—Stars and Stripes (S&S) Board of Directors A. Organization and Management...

  7. 32 CFR Appendix E to Part 246 - Stars and Stripes (S&S) Board of Directors

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 2 2014-07-01 2014-07-01 false Stars and Stripes (S&S) Board of Directors E... DEFENSE (CONTINUED) MISCELLANEOUS STARS AND STRIPES (S&S) NEWSPAPER AND BUSINESS OPERATIONS Pt. 246, App. E Appendix E to Part 246—Stars and Stripes (S&S) Board of Directors A. Organization and Management...

  8. 32 CFR Appendix E to Part 246 - Stars and Stripes (S&S) Board of Directors

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 2 2012-07-01 2012-07-01 false Stars and Stripes (S&S) Board of Directors E... DEFENSE (CONTINUED) MISCELLANEOUS STARS AND STRIPES (S&S) NEWSPAPER AND BUSINESS OPERATIONS Pt. 246, App. E Appendix E to Part 246—Stars and Stripes (S&S) Board of Directors A. Organization and Management...

  9. Effects of potassium iodide in concentrations of TSH, tT3 and tT4 in serum of subjects with sporotrichosis.

    PubMed

    Ramírez Soto, Max Carlos

    2014-08-01

    The saturated potassium iodide solution (SSKI) as treatment for sporotrichosis may cause hypothyroidism by suppressing the synthesis of thyroid hormones (tT3 and tT4 ) and the iodine excess could lead to thyrotoxicosis. Evaluating the changes in serum levels of TSH, tT3 and tT4 in euthyroid patients with sporotrichosis treated with SSKI. For the selection of euthyroid patients, TSH, tT3 and tT4 concentrations were measured for those adults and children diagnosed with sporotrichosis. Each paediatric patient was administered SSKI orally in increasing doses of 2-20 drops/3 times/day and 4-40 drops/3 times/day in adults. Serum concentrations of TSH, tT3 and tT4 were measured 20 days after started the treatment and 15 days posttreatment. Eight euthyroid patients aged between 2 to 65 years old were included. After 20 days of treatment, two suffered subclinical hypothyroidism, one developed subclinical hyperthyroidism, and one hyperthyroxinaemia euthyroid. At 15 days posttreatment only four patients were evaluated and all serum levels of TSH, tT3 and tT4 were normal. Some euthyroid patients with sporotrichosis can develop hyperthyroidism or subclinical iodine-induced hypothyroidism, during the administration of 3 or 6 g SSKI/day. © 2014 Blackwell Verlag GmbH.

  10. Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma

    PubMed Central

    Krenciute, Giedre; Krebs, Simone; Torres, David; Wu, Meng-Fen; Liu, Hao; Dotti, Gianpietro; Li, Xiao-Nan; Lesniak, Maciej S; Balyasnikova, Irina V; Gottschalk, Stephen

    2016-01-01

    Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies. PMID:26514825

  11. Approaches to ab initio molecular replacement of α-helical transmembrane proteins.

    PubMed

    Thomas, Jens M H; Simkovic, Felix; Keegan, Ronan; Mayans, Olga; Zhang, Chengxin; Zhang, Yang; Rigden, Daniel J

    2017-12-01

    α-Helical transmembrane proteins are a ubiquitous and important class of proteins, but present difficulties for crystallographic structure solution. Here, the effectiveness of the AMPLE molecular replacement pipeline in solving α-helical transmembrane-protein structures is assessed using a small library of eight ideal helices, as well as search models derived from ab initio models generated both with and without evolutionary contact information. The ideal helices prove to be surprisingly effective at solving higher resolution structures, but ab initio-derived search models are able to solve structures that could not be solved with the ideal helices. The addition of evolutionary contact information results in a marked improvement in the modelling and makes additional solutions possible.

  12. The role of proline residues in the dynamics of transmembrane helices: the case of bacteriorhodopsin.

    PubMed

    Perálvarez-Marín, Alex; Bourdelande, José-Luis; Querol, Enric; Padrós, Esteve

    2006-01-01

    Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane alpha-helices, as compared to alpha-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F). Compared to wild type, strongly perturbed behaviour has been found for these mutants. In the resting state, increased hydroxylamine accessibility and altered Asp-85 pKa and light-dark adaptation were observed. On light activation, hydroxylamine accessibility was increased and proton transport activity, M formation kinetics and FTIR difference spectra of M and N intermediates showed clear distortions. On the basis of these alterations and the near identity of the crystalline structures of mutants with that of wild type, we conclude that the transmembrane proline residues of bacteriorhodopsin fulfil a dynamic role in both the resting and the light-activated states. Our results are consistent with the notion that mutation of Pro to Ala allows the helix to increase its flexibility towards the direction originally hindered by the steric clash between the ring Cgamma and the carbonyl O of the i-4 residue, at the same time decreasing the mobility towards the opposite direction. Due to their properties, transmembrane Pro residues may serve as transmission elements of conformational changes during the transport process. We propose that these concepts can be extended to other transmembrane proteins.

  13. Simulation of kinematics of SS 433 radio jets that interact with the ambient medium

    NASA Astrophysics Data System (ADS)

    Panferov, A.

    2014-02-01

    Context. The mildly relativistic jets of SS 433 are believed to inflate the surrounding supernova remnant W 50, possibly depositing more than 99% of their kinetic energy in the remnant expansion. Where and how this transformation of the energy occurs is as yet unknown. We can learn from this that the jets decelerate and that this deceleration is non-dissipative. Aims: We uncover the deviation of the arcsecond-scale precessing radio jets of SS 433 from the ballistic locus described by the kinematic model as a signature of the dynamics issuing from the interaction of the jets with the ambient medium. Methods: To do this, we simulated the kinematics of these jets, taking into account the ram pressure on the jets, which we estimated from the profile of brightness of synchrotron radiation along the radio jets, assuming pressure balance in the jets. Results: We found that to fit an observable locus in all scales the radio jets need to be decelerated and twisted in addition to the precession torsion, mostly within the first one-fifth of the precession period, and subsequently they extend in a way that imitates ballistic jets. This jet kinematics implies a smaller distance to SS 433 than the currently accepted 5.5 kpc. The physical parameters of the jet model, which links jets dynamics with radiation, are physically reliable and characteristic for the SS 433 jets. The model proposes that beyond the radio-brightening zone, the jet clouds expand because they are in pressure balance with the intercloud medium, and heat up with distance according to the law T = 2 × 104(r/1015 cm)1.5 K. Conclusions: This model naturally explains and agrees with, the observed properties of the radio jets: a) the shock-pressed morphology; b) the brightness profile; c) the ~10% deflections of the jet kinematics from the standard kinematic model - a magnitude of the jet speed decrement in our simulation; d) the precession-phase deviations from the standard kinematic model predictions; e) the

  14. Multiresolution imaging of mantle reflectivity structure using SS and P'P' precursors

    NASA Astrophysics Data System (ADS)

    Schultz, Ryan; Gu, Yu J.

    2013-10-01

    Knowledge of the mantle reflectivity structure is highly dependent on our ability to efficiently extract, and properly interpret, small seismic arrivals. Among the various data types and techniques, long-period SS/PP precursors and high-frequency receiver functions are routinely utilized to increase the confidence of the recovered mantle stratifications at distinct spatial scales. However, low resolution and a complex Fresnel zone are glaring weaknesses of SS precursors, while over-reliance on receiver distribution is a formidable challenge for the analysis of converted waves from oceanic regions. A promising high frequency alternative to receiver functions is P'P' precursors, which are capable of resolving mantle structures at vertical and lateral resolution of ˜5 and ˜200 km, respectively, owing to their spectral content, shallow angle of incidence and near-symmetric Fresnel zones. This study presents a novel processing method for both SS (or PP) and P'P' precursors based on deconvolution, stacking, Radon transform and depth migration. A suite of synthetic tests is performed to quantify the fidelity and stability of this method under different data conditions. Our multiresolution survey of the mantle at targeted areas near Nazca-South America subduction zone reveal both olivine and garnet related transitions at depths below 400 km. We attribute a depressed 660 to thermal variations, whereas compositional variations atop the upper-mantle transition zone are needed to explain the diminished or highly complex reflected/scattered signals from the 410 km discontinuity. We also observe prominent P'P' reflections within the transition zone, and the anomalous amplitudes near the plate boundary zone indicate a sharp (˜10 km thick) transition that likely resonates with the frequency content of P'P' precursors. The migration of SS precursors in this study shows no evidence of split 660 reflections, but potential majorite-ilmenite (590-640 km) and ilmenite

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

    PubMed

    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.

  16. Immunization with the recombinant antigen Ss-IR induces protective immunity to infection with Strongyloides stercoralis in mice.

    PubMed

    Abraham, David; Hess, Jessica A; Mejia, Rojelio; Nolan, Thomas J; Lok, James B; Lustigman, Sara; Nutman, Thomas B

    2011-10-19

    Human intestinal infections with the nematode Strongyloides stercoralis remain a significant problem worldwide and a vaccine would be a useful addition to the tools available to prevent and control this infection. The goal of this study was to test single antigens for their efficacy in a vaccine against S. stercoralis larvae in mice. Alum was used as the adjuvant in these studies and antigens selected for analysis were either recognized by protective human IgG (Ss-TMY-1, Ss-EAT-6, and Ss-LEC-5) or were known to be highly immunogenic in humans (Ss-NIE-1 and Ss-IR). Only mice immunized with the Ss-IR antigen demonstrated a significant decrease of approximately 80% in the survival of larval parasites in the challenge infection. Antibodies, recovered from mice with protective immunity to S. stercoralis after immunization with Ss-IR, were used to locate the antigen in the larvae. Confocal microscopy revealed that IgG from mice immunized with Ss-IR bound to the surface of the parasites and observations by electron microscopy indicated that IgG bound to granules in the glandular esophagus. Serum collected from mice immunized with Ss-IR passively transferred immunity to naïve mice. These studies demonstrate that Ss-IR, in combination with alum, induces high levels of protective immunity through an antibody dependent mechanism and may therefore be suitable for further development as a vaccine against human strongyloidiasis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Biochemical evaluation of articular cartilage in patients with osteochondrosis dissecans by means of quantitative T2- and T2-mapping at 3T MRI: a feasibility study.

    PubMed

    Marik, W; Apprich, S; Welsch, G H; Mamisch, T C; Trattnig, S

    2012-05-01

    To perform an in vivo evaluation comparing overlying articular cartilage in patients suffering from osteochondrosis dissecans (OCD) in the talocrural joint and healthy volunteers using quantitative T2 mapping at 3.0 T. Ten patients with OCD of Grade II or lower and 9 healthy age matched volunteers were examined at a 3.0 T whole body MR scanner using a flexible multi-element coil. In all investigated persons MRI included proton-density (PD)-FSE and 3D GRE (TrueFisp) sequences for morphological diagnosis and location of anatomical site and quantitative T2 and T2 maps. Region of interest (ROI) analysis was performed for the cartilage layer above the OCD and for a morphologically healthy graded cartilage layer. Mean T2 and T2 values were then statistically analysed. The cartilage layer of healthy volunteers showed mean T2 and T2 values of 29.4 ms (SD 4.9) and 11.8 ms (SD 2.7), respectively. In patients with OCD of grade I and II lesions mean T2 values were 40.9 ms (SD 6.6), 48.7 ms (SD 11.2) and mean T2 values were 16.1 ms (SD 3.2), 16.2 ms (SD 4.8). Therefore statistically significantly higher mean T2 and T2 values were found in patients suffering from OCD compared to healthy volunteers. T2 and T2 mapping can help assess the microstructural composition of cartilage overlying osteochondral lesions. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Integrated Composite Stiffener Structure (ICoSS) Concept for Planetary Entry Vehicles

    NASA Technical Reports Server (NTRS)

    Kellas, Sotiris

    2016-01-01

    Results from the design, manufacturing, and testing of a lightweight Integrated Composite Stiffened Structure (ICoSS) concept, intended for multi-mission planetary entry vehicles are presented. Tests from both component and full-scale tests for a typical Earth Entry Vehicle forward shell manufactured using the ICoSS concept are presented and advantages of the concept for the particular application of passive Earth Entry Vehicles over other structural concepts are discussed.

  19. Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

    PubMed Central

    Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

    2017-01-01

    Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens. PMID:28198457

  20. Partner-facilitating transmembrane penetration of nanoparticles: a biological test in silico.

    PubMed

    Wang, W; Yang, R; Zhang, F; Yuan, B; Yang, K; Ma, Y

    2018-06-21

    Transmembrane penetration of nanoparticles (NPs) promises an effective pathway for cargo delivery into cells, and offers the possibility of organelle-specific targeting for biomedical applications. However, a full understanding of the underlying NP-membrane interaction mechanism is still lacking. In this work, the membrane penetration behavior of NPs is statistically analyzed based on the simulations of over 2.2 ms, which are performed with dissipative particle dynamics (DPD). Influences from multiple factors including the NP concentration, shape and surface chemistry are taken into account. It is interesting to find that, the introduction of a partner NP would greatly facilitate the transmembrane penetration of a host spherical NP. This is probably due to the membrane-mediated cooperation between the NPs. Moreover, the proper selection of a partner NP with specific surface chemistry is of great significance. For example, the best partner for a hydrophilic NP to achieve transmembrane penetration is a Janus-like one, in comparison with the hydrophilic, hydrophobic or randomly surface-decorated NPs. Furthermore, such a partner-facilitating effect in NP translocation also works for a shaped NP although less pronounced. Our results are helpful for a better understanding of the complicated nano-bio interactions, and offer a practical guide to the NP-based drug delivery strategy with high efficiency.