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Sample records for human single chain

  1. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    PubMed Central

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  2. Identification of internalizing human single chain antibodies targeting brain tumor sphere cells

    PubMed Central

    Zhu, Xiaodong; Bidlingmaier, Scott; Hashizume, Rintaro; James, C. David; Berger, Mitchel S.; Liu, Bin

    2010-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor and there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive media, and exhibit enhanced tumor initiating ability and resistance to therapy. We report here the identification of internalizing human single chain antibodies (scFvs) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133 positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular non-selective media. Taken together, these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. PMID:20587664

  3. Novel human single chain antibody fragments that are rapidly interalizing effectively target epithelioid and sarcomatoid mesotheliomas

    PubMed Central

    Iyer, Arun K.; Lan, Xiaoli; Zhu, Xiaodong; Su, Yang; Feng, Jinjin; Zhang, Xiaoju; Gao, Dongwei; Seo, Youngho; VanBrocklin, Henry F.; Broaddus, V. Courtney; Liu, Bin; He, Jiang

    2011-01-01

    Human antibodies targeting all subtypes of mesothelioma could be useful to image and treat this deadly disease. Here we report tumor targeting of a novel internalizing human single chain antibody fragment (scFv) labeled with 99mTc (99mTc-M40) in murine models of mesothelioma of both epithelioid (M28) and sarcomatoid (VAMT-1) origins. 99mTc-M40 was taken up rapidly and specifically by both subtype tumor cells in vitro, with 68–92% internalized within 1h. The specificity of binding was evidenced by blocking (up to 95%) with 10-fold excess of unlabeled M40. In animal studies, tumors of both subtypes were clearly visualized by SPECT/CT as early as 1h post-injection of 99mTc-M40. Tumor uptake measured as percent of injected dose per gram tissue (%ID/g) at 3h was 4.38 and 5.84 for M28 and VAMT-1 tumors respectively, significantly greater than all organs or tissues studied (liver, 2.62%ID/g; other organs or tissues <1.7%ID/g), except the kidneys (130.7%ID/g), giving tumor-to-blood ratios of 5:1 and 7:1 and tumor-to-muscle ratios of 45:1 and 60:1, for M28 and VAMT-1 respectively. The target-mediated uptake was confirmed by a nearly 70% reduction in tumor activity following administration of 10-fold excess of unlabeled scFv. Taken together, these results indicate that M40 can rapidly and specifically target epithelioid and sarcomatoid tumor cells, demonstrating the potential of this agent as a versatile targeting ligand for imaging and therapy of all subtypes of mesothelioma. PMID:21447742

  4. Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

    NASA Astrophysics Data System (ADS)

    Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

    1994-05-01

    Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

  5. Anti-hepatoma human single-chain Fv antibody and adriamycin conjugates with potent antitumor activity.

    PubMed

    Chen, Lin; Liu, Yan-Hong; Li, Yue-Hui; Jiang, Yan; Xie, Ping-Li; Zhou, Guo-Hua; Li, Guan-Cheng

    2014-01-01

    To construct an improved biological missile, an immunoconjugate ADM-Dex-ScFv-SA3 was synthesized, which was composed of a hepatocellular carcinoma-specific, single-chain Fv antibody (ScFv-SA3) and a highly potent cytotoxic drug, adriamycin (ADM), as the warhead. Oxidized Dextran T10 (Dex-T10) was used as a linker to connect these two moieties. The 40 KD soluble anti-hepatoma human Trx-ScFv-SA3 protein was expressed in E. coli BL21 (DE3), using a prokaryotic expression vector, pET21a (+)-Trx-ScFv-SA3-His. It was purified using a His-Tag Ni-Agarose column and identified by western blot. The activity of Trx-ScFv-SA3 was verified by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to confirm that it specifically binds to the hepatocellular carcinoma cell line HepG2. To prepare ADM-Dex-ScFv-SA3, ADM was conjugated to the antibody at a molar ratio of 14.21:1. The antitumor effect of the conjugate was tested by MTT assay, plate colony formation assay and xenografts in a nude mice experimental model. In vitro experiments revealed that ADM-Dex-ScFv-SA3 could bind to tumor cells selectively and inhibit the proliferation and the colony formation ability of HepG2 cells. In vivo experiments showed that ADM-Dex-ScFv-SA3 suppressed the tumor growth and prolonged the median survival time in tumor-bearing mice. Tumor histology slides indicated a significantly slower tumor tissue proliferation in the ADM-Dex-ScFv-SA3 group. These data indicate that the targeted drug, ADM-Dex-ScFv-SA3, may be a highly potent and selective therapy for the treatment of hepatoma. PMID:24239629

  6. Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

    PubMed

    Pasello, Michela; Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Picci, Piero; Cianfriglia, Maurizio; Scotlandi, Katia

    2016-04-20

    Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated. PMID:26945728

  7. Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase.

    PubMed

    Cui, Yongliang; Li, Dongyang; Morisseau, Christophe; Dong, Jie-Xian; Yang, Jun; Wan, Debin; Rossotti, Martín A; Gee, Shirley J; González-Sapienza, Gualberto G; Hammock, Bruce D

    2015-09-01

    The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research. PMID:26229025

  8. Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.

    PubMed

    Khantasup, Kannika; Chantima, Warangkana; Sangma, Chak; Poomputsa, Kanokwan; Dharakul, Tararaj

    2015-12-01

    Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma. PMID:26683180

  9. Single Chain Antibodies Against gp55 of Human Cytomegalovirus (HCMV) for Prophylaxis and Treatment of HCMV Infections

    PubMed Central

    Moazen, Bahareh; Ebrahimi, Elahe; Nejatollahi, Foroogh

    2016-01-01

    Background: Immunotherapy is a promising prospective new treatment for cytomegalovirus (CMV) infections. Neutralizing effects have been reported using monoclonal antibodies. Recombinant single chain antibodies (scFvs) due to their advantages over monoclonal antibodies are potential alternatives and provide valuable clinical agents. Objectives: The aim of this study was to select specific single chain antibodies against gp55 of CMV and to evaluate their neutralizing effects. In the present study, we selected specific single chain antibodies against glycoprotein 55 (gp55) of CMV for their use in treatment and diagnosis. Materials and Methods: Single chain antibodies specific against an epitope located in the C-terminal part of gp55 were selected from a phage antibody display library. After four rounds of panning, twenty clones were amplified by the polymerase chain reaction (PCR) and fingerprinted by MvaI restriction enzyme. The reactivities of the specific clones were tested by the enzyme-linked immunosorbent assay (ELISA) and the neutralizing effects were evaluated by the plaque reduction assay. Results: Fingerprinting of selected clones revealed three specific single chain antibodies (scFv1, scFv2 and scFv3) with frequencies 25%, 20 and 20%. The clones produced positive ELISA with the corresponding peptide. The percentages of plaque reduction for scFv1, scFv2 and scFv3 were 23.7, 68.8 and 11.6, respectively. Conclusions: Gp55 of human CMV is considered as an important candidate for immunotherapy. In this study, we selected three specific clones against gp55. The scFvs reacted only with the corresponding peptide in a positive ELISA. The scFv2 with 68.8% neutralizing effect showed the potential to be considered for prophylaxis and treatment of CMV infections, especially in solid organ transplant recipients, for whom treatment of CMV is urgently needed. The scFv2 with neutralizing effect of 68.8%, has the potential to be considered for treatment of these patients

  10. Anti-Human Endoglin (hCD105) Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    PubMed Central

    Barriuso, Begoña; Antolín, Pilar; Arias, F. Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-01-01

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M. PMID:27294959

  11. Design, expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor (EGFR).

    PubMed

    Akbari, Bahman; Farajnia, Safar; Zarghami, Nosratollah; Mahdieh, Nejat; Rahmati, Mohammad; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

    2016-11-01

    Various strategies have been attempted for targeting of epidermal growth factor receptor (EGFR), as an essential biomarker in a variety of cancers. Several anti-EGFR antibodies including cetuximab are used in clinics for treatment of EGFR-overexpressing colorectal and head and neck cancers but the efficiency of these antibodies is threatened by their large size and chimeric nature. Humanized single chains antibodies (huscFv) are smaller generation of antibodies with lower immunogenicity may overcome these limitations. This article reports production and evaluation of a novel humanized anti-EGFR scFv. The CDRs of cetuximab heavy and light chains were grafted onto human antibody frameworks as framework donors. To maintain the antigen binding affinity of murine antibody, the murine vernier zone residues were retained in framework regions of huscFv. Additionally, two point mutations in CDR-L1 and CDR-L3 and three point mutations in CDR-H2 and CDR-H3 loops of the humanized scFv (huscFv) were introduced to increase affinity of the huscFv to EGFR. Analysis of results demonstrated that the humanness degree of resultant huscFv was increased as 19%. HuscFv was expressed in BL21 (DE3) and affinity purified via Ni-NTA column. The reactivity of huscFv with EGFR was evaluated by ELISA and dot blot techniques. Analysis by ELISA and dot blot showed that the huscFv was able to recognize and react with EGFR. Toxicity analysis by MTT assay indicated an inhibitory effect on growth of EGFR-overexpressing A431 cells. In conclusion, the huscFv produced in this study revealed decreased immunogenicity while retained growth inhibitory effect on EGFR-overexpressing tumor cells. PMID:27298212

  12. The effect of internalizing human single chain antibody fragment on liposome targeting to epithelioid and sarcomatoid mesothelioma

    PubMed Central

    Iyer, Arun K.; Su, Yang; Feng, Jinjin; Lan, Xiaoli; Zhu, Xiaodong; Liu, Yue; Gao, Dongwei; Seo, Youngho; VanBrocklin, Henry F.; Broaddus, V. Courtney; Liu, Bin; He, Jiang

    2011-01-01

    Immunoliposomes (ILs) anchored with internalizing human antibodies capable of targeting all subtypes of mesothelioma can be useful for targeted imaging and therapy of this malignant disease. The objectives of this study were to evaluate both the in vitro and in vivo tumor targeted internalization of novel internalizing human single chain antibody (scFv) anchored ILs on both epithelioid (M28) and sarcomatoid (VAMT-1) subtypes of human mesothelioma. ILs were prepared by post-insertion of mesothelioma-targeting human scFv (M1) onto preformed liposomes and radiolabeled with 111In (111In-IL-M1), along with control non-targeted liposomes (111In-CL). Incubation of 111In-IL-M1 with M28, VAMT-1, and a control non-tumorigenic cell-line (BPH-1) at 37°C for 24 h revealed efficient binding and rapid internalization of ILs into both subtypes of tumor cells but not into the BPH-1 cells; internalization accounted for approximately 81-94% of total cell accumulation in mesothelioma cells compared to 37-55% in control cells. In tumor bearing mice intravenous (i.v.) injection of 111In-IL-M1 led to remarkable tumor accumulation: 4 % and 4.7% injected dose per gram (% ID/g) for M28 and VAMT-1 tumors, respectively, 48 h after injection. Furthermore, tumor uptake of 111In-IL-M1 in live xenograft animal models was verified by single photon emission computed tomography (SPECT/CT). In contrast, i.v. injection of 111In-CL in tumor-bearing mice revealed very low uptake in both subtypes of mesothelioma, 48 h after injection. In conclusion, M1 scFv-anchored ILs showed selective tumor targeting and rapid internalization into both epithelioid and sarcomatoid subtypes of human mesothelioma, demonstrating its potential as a promising vector for enhanced tumor drug targeting. PMID:21255833

  13. Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem.

    PubMed Central

    Sugahara, T; Pixley, M R; Minami, S; Perlas, E; Ben-Menahem, D; Hsueh, A J; Boime, I

    1995-01-01

    One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common alpha subunit and a hormone-specific beta subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormone-specific posttranslational modifications, and signal transduction. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide hCG molecule not only was efficiently secreted but also displayed an increased biological activity in vitro and in vivo. These data show that the alpha and hCG beta subunits encoded as a single chain retain a biologically active conformation similar to that seen in the heterodimer. This approach can be used to investigate structure-function relationships of the glycoprotein hormone family that were previously not tractable because of the absolute dependence on assembly for the biological response. Moreover, other bioactive multisubunit ligands can be engineered where the combination efficiency and specificity of heterodimers and homodimers are otherwise difficult to control. Images Fig. 2 Fig. 3 PMID:7892221

  14. Single-Chain Antibody Library

    DOE Data Explorer

    Baird, Cheryl

    Researchers at Pacific Northwest National Laboratory (PNNL) have constructed a nonimmune library consisting of 109 human antibody scFv fragments, which have been cloned and expressed on the surface of yeast. Nanomolar-affinity scFvs are routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010 fold without measurable loss of clonal diversity. This allows for indefinite expansion of the library. All scFv clones can be assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps. The ability to use multiplex library screening demonstrates the utility of this approach for high-throughput antibody isolation for proteomic applications. The yeast library may be used for research projects or teaching performed for U.S. Government purposes only. If you would like to request an aliquot of the single-chain antibody library for your research, please print and fill out the Materials Transfer Agreement (MTA) [PDF, 20K]. The website provides the contact information for mailing the MTA. [copied from http://www.sysbio.org/dataresources/singlechain.stm

  15. Human Single-Chain Fv Immunoconjugates Targeted to a Melanoma-Associated Chondroitin Sulfate Proteoglycan Mediate Specific Lysis of Human Melanoma Cells by Natural Killer Cells and Complement

    NASA Astrophysics Data System (ADS)

    Wang, Baiyang; Chen, Yi-Bin; Ayalon, Oran; Bender, Jeffrey; Garen, Alan

    1999-02-01

    Two antimelanoma immunoconjugates containing a human single-chain Fv (scFv) targeting domain conjugated to the Fc effector domain of human IgG1 were synthesized as secreted two-chain molecules in Chinese hamster ovary and Drosophila S2 cells, and purified by affinity chromatography on protein A. The scFv targeting domains originally were isolated as melanoma-specific clones from a scFv fusion-phage library, derived from the antibody repertoire of a vaccinated melanoma patient. The purified immunoconjugates showed similar binding specificity as did the fusion-phage clones. Binding occurred to human melanoma cells but not to human melanocytes or to several other types of normal cells and tumor cells. A 250-kDa melanoma protein was immunoprecipitated by the immunoconjugates and analyzed by mass spectrometry, using two independent procedures. A screen of protein sequence databases showed an exact match of several peptide masses between the immunoprecipitated protein and the core protein of a chondroitin sulfate proteoglycan, which is expressed on the surface of most human melanoma cells. The Fc effector domain of the immunoconjugates binds natural killer (NK) cells and also the C1q protein that initiates the complement cascade; both NK cells and complement can activate powerful cytolytic responses against the targeted tumor cells. An in vitro cytolysis assay was used to test for an immunoconjugate-dependent specific cytolytic response against cultured human melanoma cells by NK cells and complement. The melanoma cells, but not the human fibroblast cells used as the control, were efficiently lysed by both NK cells and complement in the presence of the immunoconjugates. The in vitro results suggest that the immunoconjugates also could activate a specific cytolytic immune response against melanoma tumors in vivo.

  16. Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.

    PubMed

    Kumano-Kuramochi, Miyuki; Fujimura, Takashi; Komba, Shiro; Maeda-Yamamoto, Mari; Machida, Sachiko

    2016-09-01

    Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (γ+κ and μ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1-2 μg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 μg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations. PMID:27038672

  17. Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody.

    PubMed

    Piazza, Tiziana; Cha, Emanuela; Bongarzone, Italia; Canevari, Silvana; Bolognesi, Andrea; Polito, Letizia; Bargellesi, Antonio; Sassi, Francesca; Ferrini, Silvano; Fabbi, Marina

    2005-04-01

    Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, promotes heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. Here we describe a fully human single-chain antibody fragment (scFv) directed to ALCAM/CD166. We selected the I/F8 scFv from a phage display library of human V-gene segments by cell panning and phage internalization into IGROV-I human ovary carcinoma cells. The I/F8 specificity was identified as ALCAM/CD166 by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) peptide mass fingerprinting of the I/F8-immunoprecipitated protein. The I/F8 scFv reacts with the human, monkey and murine ALCAM/CD166 molecule, indicating that the recognized epitope is highly conserved. The I/F8 scFv completely abolished binding of both ALCAM/Fc and CD6/Fc soluble ligands, whereas it did not compete with the anti-ALCAM/CD166 murine monoclonal antibodies J4-81 and 3A6 and therefore recognizes a different epitope. Engagement through I/F8 scFv, 3A6 monoclonal antibody or CD6/Fc ligand induced ALCAM/CD166 internalization, with a kinetics slower than that of transferrin in the same cells. Newly internalized I/F8-ALCAM complexes colocalized with clathrin but not with caveolin and we demonstrated, using surface biotinylation and recycling assays, that endocytosed ALCAM/CD166 recycles back to the cell surface. Such an endocytic pathway allows the efficient delivery of an I/F8 scFv-saporin immunotoxin into tumor cells, as the conjugates are able to selectively kill cell lines expressing ALCAM/CD166. Altogether these data provide evidence of the suitability of the I/F8 scFv for further functional analysis of ALCAM/CD166 and intracellular delivery of effector moieties. PMID:15769845

  18. Targeting Prostate Cancer Cells In Vivo Using a Rapidly Internalizing Novel Human Single-Chain Antibody Fragment

    PubMed Central

    He, Jiang; Wang, Yong; Feng, Jinjin; Zhu, Xiaodong; Lan, Xiaoli; Iyer, Arun K.; Zhang, Niu; Seo, Youngho; VanBrocklin, Henry F.; Liu, Bin

    2010-01-01

    Human antibodies targeting prostate cancer cell surface epitopes may be useful for imaging and therapy. The objective of this study was to evaluate the tumor targeting of an internalizing human antibody fragment, a small-size platform, to provide high contrast in a mouse model of human prostate carcinoma. Methods A prostate tumor-targeting single-chain antibody fragment (scFv), UA20, along with a nonbinding control scFv, N3M2, were labeled with 99mTc and evaluated for binding and rapid internalization into human prostate tumor cells in vitro and tumor homing in vivo using xenograft models. For the in vitro studies, the labeled UA20 scFv was incubated at 37°C for 1 h with metastatic prostate cancer cells (DU145) to assess the total cellular uptake versus intracellular uptake. For the animal studies, labeled UA20 and N3M2 scFvs were administered to athymic mice implanted subcutaneously with DU145 cells. Mice were imaged with small-animal SPECT/CT with concomitant biodistribution at 1 and 3 h after injection. Results The UA20 scFv was labeled in 55%–65% yield and remained stable in phosphate buffer within 24 h. The labeled UA20 scFv was taken up specifically by prostate tumor cells. Internalization was rapid, because incubation at 37°C for less than 1 h resulted in 93% internalization of total cell-associated scFvs. In animal studies, SPECT/CT showed significant tumor uptake as early as 1 h after injection. At 3 h after injection, tumor uptake was 4.4 percentage injected dose per gram (%ID/g), significantly greater than all organs or tissues studied (liver, 2.7 %ID/g; other organs or tissues, <1 %ID/g), except the kidneys (81.4 %ID/g), giving tumor-to-blood and tumor-to-muscle ratios of 12:1 and 70:1, respectively. In contrast, the control antibody exhibited a tumor uptake of only 0.26 %ID/g, similar to that of muscle and fat. Tumor-specific targeting was evidenced by reduced tumor uptake of nearly 70% on administration of a 10-fold excess of unlabeled UA20 sc

  19. Model protocells from single-chain lipids.

    PubMed

    Mansy, Sheref S

    2009-03-01

    Significant progress has been made in the construction of laboratory models of protocells. Most frequently the developed vesicle systems utilize single-chain lipids rather than the double-chain lipids typically found in biological membranes. Although single-chain lipids yield less robust vesicles, their dynamic characteristics are highly exploitable for protocellular functions. Herein the advantages of using single-chain lipids in the construction of protocells are discussed. PMID:19399223

  20. Model Protocells from Single-Chain Lipids

    PubMed Central

    Mansy, Sheref S.

    2009-01-01

    Significant progress has been made in the construction of laboratory models of protocells. Most frequently the developed vesicle systems utilize single-chain lipids rather than the double-chain lipids typically found in biological membranes. Although single-chain lipids yield less robust vesicles, their dynamic characteristics are highly exploitable for protocellular functions. Herein the advantages of using single-chain lipids in the construction of protocells are discussed. PMID:19399223

  1. Human. cap alpha. /sub 2/-HS-glycoprotein: the A and B chains with a connecting sequence are encoded by a single mRNA transcript

    SciTech Connect

    Lee, C.C.; Bowman, B.H.; Yang, F.

    1987-07-01

    The ..cap alpha../sub 2/-HS-glycoprotein (AHSG) is a plasma protein reported to play roles in bone mineralization and in the immune response. It is composed of two subunits, the A and B chains. Recombinant plasmids containing human cDNA AHSG have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. The cDNA clones containing AHSG inserts span approximately 1.5 kilobase pairs and include the entire AHSG coding sequence, demonstrating that the A and B chains are encoded by a single mRNA transcript. The cDNA sequence predicts an 18-amino-acid signal peptide, followed by the A-chain sequence of AHSG. A heretofore unseen connecting sequence of 40 amino acids was deduced between the A- and B-chain sequences. The connecting sequence demonstrates the unique amino acid doublets and collagen triplets found in the A and B chains; it is not homologous with other reported amino acid sequences. The connecting sequence may be cleaved in a posttranslational step by limited proteolysis before mature AHSG is released into the circulation or may vary in its presence because of alternative processing. The AHSG cDNA was utilized for mapping the AHSG gene to the 3q21..-->..qter region of human chromosome 3. The availability of the AHSG cDNA clone will facilitate the analysis of its genetic control and gene expression during development and bone formation.

  2. Construction and functional analysis of an anti-human cervical carcinoma/anti-human CD3 single-chain bispecific antibody.

    PubMed

    Wu, Hong; Yao, Li; Chou, Lin; Yang, Jin-Hua; Zhang, Yun-Xiu; Li, Xiao-Li; Shan, Bo-Er

    2016-07-01

    The aim of the present study was to construct a single-chain bispecific antibody (scBsAb) against cervical carcinoma and to investigate its biological activities. The scBsAb was constructed using a genetic cloning technique and antigen binding activities were detected by ELISA. The iodogen method was used to analyze the pharmacokinetics. The Rosette formation test was used to detect the binding ability between peripheral blood lymphocytes (PBLs) and Cs1213 cervical cancer cells. In addition, the MTT method was performed to detect the killing effect of PBLs. The molecular weight of the scBsAb was ~60 kDa. The antigen binding activities of scBsAbs were compared with the anti‑human cervical carcinoma antibody single‑chain Fv fragment (CSAs‑1 scFv) and anti‑cluster of differentiation (CD)3 scFv (P>0.05). In addition, a pharmacokinetics assay demonstrated that compared with the two corresponding scFvs, scBsAbs exhibited a significantly prolonged retention time in the body (P<0.01). In addition, the number of rosettes formed by PBLs and Cs1213 cells in the scBsAb group was markedly greater than that in the scFv groups or the RPMI‑1640 group (P<0.05 and P<0.01, respectively). The killing activity of PBLs against scBsAb‑mediated Cs1213 cells was significantly greater than that mediated by the other antibodies (P<0.05). When the concentration of scBsAb was 40 µg/ml, the killing rate was 64.5%. Thus, anti‑human cervical carcinoma/anti‑CD3 scBsAbs may possess two types of antigen binding activity, prolong the duration in vivo and improve the killing activity of PBLs against cancer cells. PMID:27220396

  3. Ingestion of a single serving of saury alters postprandial levels of plasma n-3 polyunsaturated fatty acids and long-chain monounsaturated fatty acids in healthy human adults

    PubMed Central

    2012-01-01

    Background Saury oil contains considerable amounts of n-3 polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA) with long aliphatic tails (>18C atoms). Ingestion of saury oil reduces the risk of developing metabolic syndrome concomitant with increases in n-3 PUFA and long-chain MUFA in plasma and organs of mice. We therefore evaluated changes in postprandial plasma fatty acid levels and plasma parameters in healthy human subjects after ingestion of a single meal of saury. Findings Five healthy human adults ingested 150 g of grilled saury. Blood was collected before the meal and at 2, 6, and 24 hr after the meal, and plasma was prepared. Plasma levels of eicosapentaenoic acid, docosahexaenoic acid, and long-chain MUFA (C20:1 and C22:1 isomers combined) increased significantly throughout the postprandial period compared with the pre-meal baseline. Postprandial plasma insulin concentration increased notably, and plasma levels of glucose and free fatty acids decreased significantly and subsequently returned to the pre-meal levels. Conclusions Our study suggests that a single saury meal may alter the postprandial plasma levels of n-3 PUFA and long-chain MUFA in healthy human subjects. PMID:22846384

  4. Increased Stability and DNA Site Discrimination of Single Chain Variants of the Dimeric beta-Barrel DNA Binding Domain of the Human Papillomavirus E2 Transcriptional Regulator

    SciTech Connect

    Dellarole,M.; Sanchez, I.; Freire, E.; de Prat-Gay, G.

    2007-01-01

    Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric {beta}-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.

  5. Human laminin B2 chain

    SciTech Connect

    Pikkarainen, T.; Kallunki, T.; Tryggvason, K.

    1988-05-15

    The complete amino acid sequence of the human laminin B2 chains has been determined by sequencing of cDNA clones. The six overlapping clones studied cover approximately 7.5 kilobases of which 5312 nucleotides were sequenced from the 5' end. The open reading frame codes for a 33-residue signal peptide and a 1576-residue B2 chain proper, which is 189 residues less than in the highly homologous B1 chain. Computer analysis revealed that the B2 chain consists of distinct domains that contain helical structures, cysteine-rich repeats, and globular regions, as does the B1 chain. However, domain ..cap alpha.. and domain ..beta.. of the B1 chain have no counterpart in B2, and the number of cysteine-rich repeats is 12, or 1 less than in the B1 chain. The degree of homology between the two chains is highest in the cysteine repeat-containing domains III and V where 40% of the residues match. However, in helical domains I/II only 16% of residues match. The results demonstrate that the B1 and B2 chains of laminin are highly homologous proteins that are probably the products of related genes.

  6. Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123.

    PubMed

    Moradi-Kalbolandi, Shima; Habibi-Anbouhi, Mahdi; Golkar, Majid; Behdani, Mahdi; Rezaei, Gashin; Ghazizadeh, Leila; Abolhassani, Mohsen; Shokrgozar, Mohammad Ali

    2016-09-01

    Current therapies for acute myeloid leukemia (AML), are associated with high relapse rates. Hence, development of new therapeutic strategies is crucial to circumvent this problem. Bivalent antibody technology has been used to engineer novel antibody fragments with increased avidity, by assembling two scFv in a single molecule. Here, we present accompanying data from construction and characterization experiments of a biscFv antibody targeting CD123, the most important biomarker of leukemic cancer stem cells which play a key role in relapsed AML after chemotherapy. Data in this article are related to the research paper "Development of a novel engineered antibody targeting human CD123" Moradi-Kalbolandi S. et al. (2016) [1]. PMID:27536714

  7. Detection of single amino acid mutation in human breast cancer by disordered plasmonic self-similar chain

    PubMed Central

    Coluccio, Maria Laura; Gentile, Francesco; Das, Gobind; Nicastri, Annalisa; Perri, Angela Mena; Candeloro, Patrizio; Perozziello, Gerardo; Proietti Zaccaria, Remo; Gongora, Juan Sebastian Totero; Alrasheed, Salma; Fratalocchi, Andrea; Limongi, Tania; Cuda, Giovanni; Di Fabrizio, Enzo

    2015-01-01

    Control of the architecture and electromagnetic behavior of nanostructures offers the possibility of designing and fabricating sensors that, owing to their intrinsic behavior, provide solutions to new problems in various fields. We show detection of peptides in multicomponent mixtures derived from human samples for early diagnosis of breast cancer. The architecture of sensors is based on a matrix array where pixels constitute a plasmonic device showing a strong electric field enhancement localized in an area of a few square nanometers. The method allows detection of single point mutations in peptides composing the BRCA1 protein. The sensitivity demonstrated falls in the picomolar (10−12 M) range. The success of this approach is a result of accurate design and fabrication control. The residual roughness introduced by fabrication was taken into account in optical modeling and was a further contributing factor in plasmon localization, increasing the sensitivity and selectivity of the sensors. This methodology developed for breast cancer detection can be considered a general strategy that is applicable to various pathologies and other chemical analytical cases where complex mixtures have to be resolved in their constitutive components. PMID:26601267

  8. Detection of single amino acid mutation in human breast cancer by disordered plasmonic self-similar chain.

    PubMed

    Coluccio, Maria Laura; Gentile, Francesco; Das, Gobind; Nicastri, Annalisa; Perri, Angela Mena; Candeloro, Patrizio; Perozziello, Gerardo; Proietti Zaccaria, Remo; Gongora, Juan Sebastian Totero; Alrasheed, Salma; Fratalocchi, Andrea; Limongi, Tania; Cuda, Giovanni; Di Fabrizio, Enzo

    2015-09-01

    Control of the architecture and electromagnetic behavior of nanostructures offers the possibility of designing and fabricating sensors that, owing to their intrinsic behavior, provide solutions to new problems in various fields. We show detection of peptides in multicomponent mixtures derived from human samples for early diagnosis of breast cancer. The architecture of sensors is based on a matrix array where pixels constitute a plasmonic device showing a strong electric field enhancement localized in an area of a few square nanometers. The method allows detection of single point mutations in peptides composing the BRCA1 protein. The sensitivity demonstrated falls in the picomolar (10(-12) M) range. The success of this approach is a result of accurate design and fabrication control. The residual roughness introduced by fabrication was taken into account in optical modeling and was a further contributing factor in plasmon localization, increasing the sensitivity and selectivity of the sensors. This methodology developed for breast cancer detection can be considered a general strategy that is applicable to various pathologies and other chemical analytical cases where complex mixtures have to be resolved in their constitutive components. PMID:26601267

  9. A human cytokine/single-chain antibody fusion protein for simultaneous delivery of GM-CSF and IL-2 to Ep-CAM overexpressing tumor cells.

    PubMed

    Schanzer, Juergen M; Baeuerle, Patrick A; Dreier, Torsten; Kufer, Peter

    2006-01-01

    Pro-inflammatory cytokines regulate the growth, differentiation, and activation of immune cells and can play a role in antitumor responses. GM-CSF and IL-2 induce tumor rejection in animal models when expressed by tumor cells, and IL-2 is used for the treatment of melanoma and renal cell cancer. However, high doses of GM-CSF and IL-2 are associated with severe side effects in cancer patients. We generated a dual cytokine fusion protein for simultaneous targeted delivery of human GM-CSF and IL-2 to human tumors. The fusion protein is based on a heterodimeric core structure formed by human CH1 and C kappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused human single-chain Ab fragments (scFv) specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). The dual cytokine heterominibody (DCH) was well expressed and secreted by CHO cells, preserved the specific proliferative activities of the two cytokines, and showed Ep-CAM-specific binding to tumor cells. DCH induced potent tumor cell lysis in vitro by two distinct mechanisms. One was activating PBMCs to lyse tumor cells, which was superior to cytotoxicity induced by equimolar ratios of free recombinant human IL-2 and GM-CSF. The other mechanism was redirected lysis, as seen with isolated human T cells, which was solely dependent on the IL-2 fusion part. The therapeutic principle of dual cytokine targeting may warrant in vivo testing of murine-specific analogues in appropriate mouse models and further preclinical development of the less immunogenic, human cytokine- and human Ep-CAM-specific DCH molecule described here. PMID:16483188

  10. Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans.

    PubMed

    Greineder, Colin F; Hood, Elizabeth D; Yao, Anning; Khoshnejad, Makan; Brenner, Jake S; Johnston, Ian H; Poncz, Mortimer; Gottstein, Claudia; Muzykantov, Vladimir R

    2016-03-28

    Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such "vascular immunotargeting" to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans. PMID:26855052

  11. Investigation of the structure of anti-human seminal plasma protein single-chain antibody and its association with linker peptide length

    PubMed Central

    JIANG, XIN; ZHAI, JUN; SONG, DONGKUI; QU, QINGSHAN; LI, MING; XING, LI; MIAO, SHUZHAI

    2015-01-01

    To enhance the activity of seminoprotein single-chain variable fragment (γ-Sm-ScFv) antibodies, modulation of the length of the linker peptide, which connects the variable region of the heavy chain (VH) and the light chain (VL) of single-chain antibodies, was performed in the present study. Homologous modeling of single VH and VL were performed, respectively. Subsequently, modeling of the whole ScFv sequence, which was previously modified with added linkers of different lengths was also performed, and the (Gly4Ser)n peptide chain structure was used as the linker. The similarities between VH and VL prior to and following the addition of the linker were compared by applying the algorithm of protein similarity, based on spherical coordinates layering. In addition, changes in the fore and aft distance, and diffusion radius were calculated using a MATLAB tool, based on which changes in structural stability were analyzed. Finally, the single-chain antibody was assessed in a nude mouse model. When n=3 or n=6, the similarity between the original distance and VH and VL were the highest, and the fore and aft distance and diffusion radius were relatively close. In addition, the nude mouse model indicated that, when n=3 or n=6, the inhibitory rate of the single-chain antibody against tumor cells was significantly higher, compared with the other linker peptides of different lengths. The effect of structural changes of the linker peptides in the single-chain antibodies on the whole antibody molecule was examined at different levels using a combination of mathematical modeling, bioinformatics methods and biological experiments. The findings of the present study may provide a foundation for further investigation into the preparation of single-chain antibodies. PMID:26099852

  12. Single-Chain Semiconducting Polymer Dots

    PubMed Central

    2015-01-01

    This work describes the preparation and validation of single-chain semiconducting polymer dots (sPdots), which were generated using a method based on surface immobilization, washing, and cleavage. The sPdots have an ultrasmall size of ∼3.0 nm as determined by atomic force microscopy, a size that is consistent with the anticipated diameter calculated from the molecular weight of the single-chain semiconducting polymer. sPdots should find use in biology and medicine as a new class of fluorescent probes. The FRET assay this work presents is a simple and rapid test to ensure methods developed for preparing sPdot indeed produced single-chain Pdots as designed. PMID:25521606

  13. Targeted therapy against human lung cancer in nude mice by high-affinity recombinant antimesothelin single-chain Fv immunotoxin.

    PubMed

    Fan, Dominic; Yano, Seiji; Shinohara, Hisashi; Solorzano, Carmen; Van Arsdall, Melissa; Bucana, Corazon D; Pathak, Sen; Kruzel, Ewa; Herbst, Roy S; Onn, Amir; Roach, Jennifer S; Onda, Masanori; Wang, Qing-cheng; Pastan, Ira; Fidler, Isaiah J

    2002-06-01

    Several tumors, including mesothelioma and ovarian cancer, can overexpress mesothelin, a glycosylphosphatidylinositol-linked differentiation glycoprotein. The membrane-bound type of mesothelin is found in the blood of cancer patients at a very low level, which makes mesothelin a good candidate for targeted therapy of certain cancers. An antimesothelin disulfide-linked Fv (SS1 Fv) was fused to a truncated mutant of Pseudomonas exotoxin A to produce the recombinant immunotoxin SS1(dsFv)-PE38, which has a high binding affinity to mesothelin (Kd = 0.7 nM). Our studies in vitro showed that SS1(dsFv)-PE38 is significantly more cytotoxic to the high-mesothelin-producing NCI-H226 human non-small cell lung cancer cells than to human lung adenocarcinoma PC14PE6 cells, which do not express mesothelin. When administered at a nontoxic dose of 500 microg/kg on days 7, 9, and 11 to nude mice injected i.v. with the two human lung cancer cell lines, SS1(dsFv)-PE38 selectively inhibited experimental lung metastases produced by the mesothelin-producing NCI-H226 cells. Our data indicate that mesothelin-producing squamous cell carcinoma of the lung may be a good target for this immunotoxin. PMID:12479219

  14. Production of a recombinant anti-human CD4 single-chain variable-fragment antibody using phage display technology and its expression in Escherichia coli.

    PubMed

    Babaei, Arash; Zarkesh-Esfahani, Sayyed Hamid; Gharagozloo, Marjan

    2011-05-01

    Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications. PMID:21617352

  15. Human single-chain urokinase is activated by the omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.

    PubMed

    Järvinen, Hanna M; Laakkonen, Liisa; Haiko, Johanna; Johansson, Tiira; Juuti, Katri; Suomalainen, Marjo; Buchrieser, Carmen; Kalkkinen, Nisse; Korhonen, Timo K

    2013-08-01

    Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in omptin proteolysis. PMID:23763588

  16. Targeting human immunodeficiency virus type 1 reverse transcriptase by intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle.

    PubMed Central

    Shaheen, F; Duan, L; Zhu, M; Bagasra, O; Pomerantz, R J

    1996-01-01

    Novel molecular approaches to inhibit human immunodeficiency virus type 1 (HIV-1) infection have received increasing attention because of the lack of effective antiviral drug therapies in vivo. We now demonstrate that cells can be intracellularly immunized by cytoplasmic expression of single-chain variable antibody fragments (SFv) which bind to the HIV-1 reverse transcriptase (RT) enzyme. The expression of anti-RT SFv in T-lymphocytic cells specifically neutralizes the RT activity in the preintegration stage and affects the reverse transcription process, an early event of the HIV-1 life cycle. Blocking the virus at these early stages dramatically decreased HIV-1 propagation, as well as the HIV-1-induced cytopathic effects in susceptible human T lymphocytes, by impeding the formation of the proviral DNA. These data also demonstrate that intracellular, complete SFvs may gain access to viral proteins of the HIV-1 preintegration complex. These SFvs will provide a tool with which to better understand the molecular mechanisms involved in restricting viral replication in HIV-1-infected cells. PMID:8648670

  17. Recruitment of Oligoclonal Viral-Specific T cells to Kill Human Tumor Cells Using Single-Chain Antibody-Peptide-HLA Fusion Molecules.

    PubMed

    Noy, Roy; Haus-Cohen, Maya; Oved, Kfir; Voloshin, Tali; Reiter, Yoram

    2015-06-01

    Tumor progression is often associated with the development of diverse immune escape mechanisms. One of the main tumor escape mechanism is HLA loss, in which human solid tumors exhibit alterations in HLA expression. Moreover, tumors that present immunogenic peptides via class I MHC molecules are not susceptible to CTL-mediated lysis, because of the relatively low potency of the tumor-specific CLTs. Here, we present a novel cancer immunotherapy approach that overcomes these problems by using the high affinity and specificity of antitumor antibodies to recruit potent antiviral memory CTLs to attack tumor cells. We constructed a recombinant molecule by genetic fusion of a cytomegalovirus (CMV)-derived peptide pp65 (NLVPMVATV) to scHLA-A2 molecules that were genetically fused to a single-chain Fv Ab fragment specific for the tumor cell surface antigen mesothelin. This fully covalent fusion molecule was expressed in E. coli as inclusion bodies and refolded in vitro. The fusion molecules could specifically bind mesothelin-expressing cells and mediate their lysis by NLVPMVATV-specific HLA-A2-restricted human CTLs. More importantly, these molecules exhibited very potent antitumor activity in vivo in a nude mouse model bearing preestablished human tumor xenografts that were adoptively transferred along with human memory CTLs. These results represent a novel and powerful approach to immunotherapy for solid tumors, as demonstrated by the ability of the CMV-scHLA-A2-SS1(scFv) fusion molecule to mediate specific and efficient recruitment of CMV-specific CTLs to kill tumor cells. PMID:25852061

  18. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    PubMed Central

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  19. Studies on the synthesis, characterization, human serum albumin binding and biological activity of single chain surfactant-cobalt(III) complexes.

    PubMed

    Vignesh, G; Sugumar, K; Arunachalam, S; Vignesh, S; Arthur James, R; Arun, R; Premkumar, K

    2016-03-01

    The interaction of surfactant-cobalt(III) complexes [Co(bpy)(dien)TA](ClO4)3 · 3H2O (1) and [Co(dien)(phen)TA](ClO4)3 · 4H2O (2), where bpy = 2,2'-bipyridine, dien = diethylenetriamine, phen = 1,10-phenanthroline and TA = tetradecylamine with human serum albumin (HSA) under physiological conditions was analyzed using steady state, synchronous, 3D fluorescence, UV/visabsorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of HSA through a static mechanism. The binding constant (Kb ) and number of binding-sites (n) were obtained at different temperatures. The corresponding thermodynamic parameters (∆G°, ∆H° and ∆S°) and Ea were also obtained. According to Förster's non-radiation energy transfer theory, the binding distance (r) between the complexes and HSA were calculated. The results of synchronous and 3D fluorescence spectroscopy indicate that the binding process has changed considerably the polarity around the fluorophores, along with changes in the conformation of the protein. The antimicrobial and anticancer activities of the complexes were tested and the results show that the complexes have good activities against pathogenic microorganisms and cancer cells. PMID:26250655

  20. High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

    PubMed

    Zarei, Najmeh; Vaziri, Behrouz; Shokrgozar, Mohammad Ali; Mahdian, Reza; Fazel, Ramin; Khalaj, Vahid

    2014-12-01

    Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies. PMID:25239038

  1. Expression and purification of a human anti-cyclin D1 single-chain variable fragment antibody AD5 and its characterization.

    PubMed

    Wu, Yan; Zou, Desheng; Cao, Yuhua; Yao, Nannan; Wang, Junye; Wang, Wenhan; Jiang, Hongyu; Li, Guiying

    2013-12-01

    Cyclin D1 plays an important role in cell cycle progression. Increasing evidence indicates that cyclin D1 is overexpressed in the majority of tumor cells and has become a potential target for tumor therapy. However, little research has been done on the specific inhibition of cyclin D1 for cancer therapy. With the rapid development of the phage display antibody library technique, single-chain variable fragment (scFv) antibodies have emerged, which have tremendous application prospects in cancer therapy and diagonosis. In this study, a human scFv binding specifically to cyclin D1 (AD5) that was derived from a human semi-synthetic scFv phage library was expressed in the soluble form in Escherichia coli (E. coli) HB2151 cells. To characterize AD5, soluble AD5 was purified successfully through ammonium sulfate precipitation and affinity chromatography from the culture supernatant of AD5/HB2151. ELISA assay revealed that purified soluble AD5 could specifically bind to human recombinant cyclin D1 with approximately (1.19±0.056) x 107 M-1 affinity constant and showed approximately 52% competitive inhibition with the anti-cyclin D1 polyclonal antibody for binding to cyclin D1 in vitro. These results suggest that the scFv antibody against cyclin D1 may be a novel potential tool for targeting cyclin D1 in cancer therapy and diagnosis. PMID:24127128

  2. Method for preparation of single chain antibodies

    SciTech Connect

    Cheung, Nai-Kong V.; Guo, Hong-fen

    2012-04-03

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  3. Improved biological activity of a single chain antibody fragment against human epidermal growth factor receptor 2 (HER2) expressed in the periplasm of Escherichia coli.

    PubMed

    Akbari, Vajihe; Sadeghi, Hamid Mir Mohammad; Jafarian-Dehkordi, Abbas; Abedi, Daryoush; Chou, C Perry

    2015-12-01

    A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta®) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFv was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications. PMID:26166178

  4. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    SciTech Connect

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  5. The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

    PubMed

    Yang, Ju-Lun; Pan, Xin-Yan; Zhao, Wen-Xing; Hu, Qi-Chan; Ding, Feng; Feng, Qiang; Li, Gui-Yun; Luo, Ying

    2016-03-01

    Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB‑231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression. PMID:26780944

  6. Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

    PubMed Central

    Patterson, Kelcey G.; Dixon Pittaro, Jennifer L.; Bastedo, Peter S.; Hess, David A.; Haeryfar, S. M. Mansour; McCormick, John K.

    2014-01-01

    Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC-II binding interface was generated (SpeCD203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4+ cancer cells, we engineered a humanized single chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4). Specific targeting of scFv5T4 was verified. SpeCD203A fused to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colorectal cancer cells. Using a xenograft model of established human colon cancer, we demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumors in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as ‘next-generation’ TTSs for cancer immunotherapy. PMID:24736661

  7. Control of established colon cancer xenografts using a novel humanized single chain antibody-streptococcal superantigen fusion protein targeting the 5T4 oncofetal antigen.

    PubMed

    Patterson, Kelcey G; Dixon Pittaro, Jennifer L; Bastedo, Peter S; Hess, David A; Haeryfar, S M Mansour; McCormick, John K

    2014-01-01

    Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC-II binding interface was generated (SpeCD203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4+ cancer cells, we engineered a humanized single chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4). Specific targeting of scFv5T4 was verified. SpeCD203A fused to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colorectal cancer cells. Using a xenograft model of established human colon cancer, we demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumors in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as 'next-generation' TTSs for cancer immunotherapy. PMID:24736661

  8. Chemotactic Signaling by Single-Chain Chemoreceptors

    PubMed Central

    Mowery, Patricia; Ames, Peter; Reiser, Rebecca H.; Parkinson, John S.

    2015-01-01

    Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit

  9. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

    PubMed

    Bublin, Merima; Kostadinova, Maria; Fuchs, Julian E; Ackerbauer, Daniela; Moraes, Adolfo H; Almeida, Fabio C L; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R; Valente, Ana Paula; Breiteneder, Heimo

    2015-01-01

    Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes. PMID:26579717

  10. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope

    PubMed Central

    Fuchs, Julian E.; Ackerbauer, Daniela; Moraes, Adolfo H.; Almeida, Fabio C. L.; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R.; Valente, Ana Paula; Breiteneder, Heimo

    2015-01-01

    Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes. PMID:26579717

  11. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA

    PubMed Central

    Minczuk, Michal; Papworth, Monika A.; Miller, Jeffrey C.; Murphy, Michael P.; Klug, Aaron

    2008-01-01

    The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA molecules and thereby alleviate the defective mitochondrial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases conjugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the selective degradation of particular mtDNA sequences. Typically, pairs of complementary ZFNs are used that heterodimerize on the target DNA sequence; however, conventional ZFNs were ineffective in our system. To overcome this, we created single-chain ZFNs by conjugating two FokI nuclease domains, connected by a flexible linker, to a ZFP with an N-terminal mitochondrial targeting sequence. Here we show that these ZFNs are efficiently transported into mitochondria in cells and bind mtDNA in a sequence-specific manner discriminating between two 12-bp long sequences that differ by a single base pair. Due to their selective binding they cleave dsDNA at predicted sites adjacent to the mutation. When expressed in heteroplasmic cells containing a mixture of mutated and wt mtDNA these ZFNs selectively degrade mutated mtDNA, thereby increasing the proportion of wt mtDNA molecules in the cell. Therefore, mitochondria-targeted single-chain ZFNs are a promising candidate approach for the treatment of mtDNA diseases. PMID:18511461

  12. Mesenchymal Stem Cells Modified with a Single-Chain Antibody against EGFRvIII Successfully Inhibit the Growth of Human Xenograft Malignant Glioma

    PubMed Central

    Balyasnikova, Irina V.; Ferguson, Sherise D.; Sengupta, Sadhak; Han, Yu; Lesniak, Maciej S.

    2010-01-01

    Background Glioblastoma multiforme is the most lethal brain tumor with limited therapeutic options. Antigens expressed on the surface of malignant cells are potential targets for antibody-mediated gene/drug delivery. Principal Findings In this study, we investigated the ability of genetically modified human mesenchymal stem cells (hMSCs) expressing a single-chain antibody (scFv) on their surface against a tumor specific antigen, EGFRvIII, to enhance the therapy of EGFRvIII expressing glioma cells in vivo. The growth of U87-EGFRvIII was specifically delayed in co-culture with hMSC-scFvEGFRvIII. A significant down-regulation was observed in the expression of pAkt in EGFRvIII expressing glioma cells upon culture with hMSC-scFvEGFRvIII vs. controls as well as in EGFRvIII expressing glioma cells from brain tumors co-injected with hMSC-scFvEGFRvIII in vivo. hMSC expressing scFvEGFRvIII also demonstrated several fold enhanced retention in EGFRvIII expressing flank and intracranial glioma xenografts vs. control hMSCs. The growth of U87-EGFRvIII flank xenografts was inhibited by 50% in the presence of hMSC-scFvEGFRvIII (p<0.05). Moreover, animals co-injected with U87-EGFRvIII and hMSC-scFvEGFRvIII intracranially showed significantly improved survival compared to animals injected with U87-EGFRvIII glioma cells alone or with control hMSCs. This survival was further improved when the same animals received an additional dosage of hMSC-scFvEGFRvIII two weeks after initial tumor implantation. Of note, EGFRvIII expressing brain tumors co-injected with hMSCs had a lower density of CD31 expressing blood vessels in comparison with control tumors, suggesting a possible role in tumor angiogenesis. Conclusions/Significance The results presented in this study illustrate that genetically modified MSCs may function as a novel therapeutic vehicle for malignant brain tumors. PMID:20305783

  13. Increase of EPA-derived hydroxy, epoxy and dihydroxy fatty acid levels in human plasma after a single dose of long-chain omega-3 PUFA

    PubMed Central

    Schuchardt, Jan Philipp; Schneider, Inga; Willenberg, Ina; Yang, Jun; Hammock, Bruce D.; Hahn, Andreas; Schebb, Nils Helge

    2014-01-01

    Introduction Several supplementation studies with long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) describe an increase of EPA-derived hydroxy, epoxy and dihydroxy fatty acids in blood, while changes in levels of other LC n-3 and n-6 PUFA-derived oxylipins were minor. In order to investigate the kinetics of changes in oxylipin levels in response to LC n-3 PUFA ingestion, we conducted a single dose treatment study with healthy subjects. Subjects and methods In the present kinetic study, we compared patterns of hydroxy, epoxy and dihydroxy fatty acids in plasma of 6 healthy men before and after 6, 8, 24, and 48 h of fish oil (1008 mg EPA and 672 mg DHA) ingestion. Levels of EPA- as well as other LC PUFA-derived hydroxy, epoxy and dihydroxy fatty acids were analyzed in plasma by LC–MS. Additionally, levels of these oxylipins were compared with their parent PUFA levels in plasma phospholipids. Results All EPA-derived oxylipin levels were significantly increased 6 h after LC n-3 PUFA ingestion and gradually drop thereafter reaching the baseline levels about 48 h after treatment. The relative increase in EPA plasma phospholipid levels highly correlated with the increase of plasma EPA-derived oxylipin levels at different time points. In contrast, plasma levels of arachidonic acid- and DHA-derived oxylipins as well as parent PUFA levels in plasma phospholipids were hardly changed. Discussion and conclusions Our findings demonstrate that a single dose of LC n-3 PUFAs can rapidly induce a shift in the EPA oxylipin profile of healthy subjects within a few hours. Taking the high biological activity of the EPA-derived epoxy fatty acids into account, even short-term treatment with LC n-3 PUFAs may cause systemic effects, which warrant further investigation. PMID:24667634

  14. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.

    PubMed

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis. PMID:24675419

  15. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

    PubMed Central

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis. PMID:24675419

  16. Structure of Human Ferritin L Chain

    SciTech Connect

    Wang,Z.; Li, C.; Ellenburg, M.; Soistman, E.; Ruble, J.; Wright, B.; Ho, J.; Carter, D.

    2006-01-01

    Ferritin is the major iron-storage protein present in all cells. It generally contains 24 subunits, with different ratios of heavy chain (H) to light chain (L), in the shape of a hollow sphere hosting up to 4500 ferric Fe atoms inside. H-rich ferritins catalyze the oxidation of iron(II), while L-rich ferritins promote the nucleation and storage of iron(III). Several X-ray structures have been determined, including those of L-chain ferritins from horse spleen (HoSF), recombinant L-chain ferritins from horse (HoLF), mouse (MoLF) and bullfrog (BfLF) as well as recombinant human H-chain ferritin (HuHF). Here, structures have been determined of two crystal forms of recombinant human L-chain ferritin (HuLF) obtained from native and perdeuterated proteins. The structures show a cluster of acidic residues at the ferrihydrite nucleation site and at the iron channel along the threefold axis. An ordered Cd{sup 2+} structure is observed within the iron channel, offering further insight into the route and mechanism of iron transport into the capsid. The loop between helices D and E, which is disordered in many other L-chain structures, is clearly visible in these two structures. The crystals generated from perdeuterated HuLF will be used for neutron diffraction studies.

  17. Single chain stochastic polymer modeling at high strain rates.

    SciTech Connect

    Harstad, E. N.; Harlow, Francis Harvey,; Schreyer, H. L.

    2001-01-01

    Our goal is to develop constitutive relations for the behavior of a solid polymer during high-strain-rate deformations. In contrast to the classic thermodynamic techniques for deriving stress-strain response in static (equilibrium) circumstances, we employ a statistical-mechanics approach, in which we evolve a probability distribution function (PDF) for the velocity fluctuations of the repeating units of the chain. We use a Langevin description for the dynamics of a single repeating unit and a Lioville equation to describe the variations of the PDF. Moments of the PDF give the conservation equations for a single polymer chain embedded in other similar chains. To extract single-chain analytical constitutive relations these equations have been solved for representative loading paths. By this process we discover that a measure of nonuniform chain link displacement serves this purpose very well. We then derive an evolution equation for the descriptor function, with the result being a history-dependent constitutive relation.

  18. Nanoscience of single polymer chains revealed by nanofishing.

    PubMed

    Nakajima, Ken; Nishi, Toshio

    2006-01-01

    The invention of atomic force microscopy (AFM) enabled us to study the statistical properties of single polymer chains by a method called "nanofishing," which stretches a single polymer chain adsorbed on a substrate with its one end by picking it at the other end. A force-extension curve obtained for a single polystyrene chain in a Theta solvent (cyclohexane) shows good agreement with a worm-like chain model and, therefore, gives microscopic information about entropic elasticity. Furthermore, the nanofishing technique can be used for dynamic viscoelastic measurement of single polymer chains. An AFM cantilever is mechanically oscillated at its resonant frequency during the stretching process. This technique enables the estimation of quantitative and simultaneous elongation-dependent changes of stiffness and viscosity of a single chain with the use of a phenomenological model. In this study, the effect of solvent on viscosity in low extension regions reveals that the viscosity is attributed to monomer-solvent friction. Thus, static and dynamic nanofishing techniques are shown to give powerful experimental proofs for several basic questions in polymer physics. The techniques are expected to reveal hidden properties of polymer chains or polymer solutions by any types of macroscopic measurements in the future. PMID:17099889

  19. Specific dimerization of the light chains of human immunoglobulin.

    PubMed

    Stevenson, G T; Straus, D

    1968-07-01

    1. The light chains of human immunoglobulin were allowed to dimerize in vitro on removal of the dispersing agents acetic acid or urea. 2. On electrophoresis in polyacrylamide gel at pH8.8 the dimers yielded up to nine regularly spaced bands. This approximates to the number of electrophoretic components known to occur among the monomers. 3. Single electrophoretic components of the dimers were isolated from the gel, dissociated into monomers, and subjected as such to electrophoresis in urea-containing gels. Each gave two adjacent bands. 4. Similarly, after all the light chains as monomers had been subjected to electrophoresis in urea-containing gels, single electrophoretic components were isolated and allowed to dimerize. When examined now as dimers in the absence of urea, each component gave two adjacent bands. 5. These findings are explicable on the following basis. (a) The dimerization of the light chains is specific, at least inasmuch as it occurs between monomers of the same electrophoretic mobilities. (b) With the buffer constant, different light chains undergo different changes in net charge on being transferred from urea-containing to urea-free solution; in this way two different chains of the same initial charge can acquire a charge difference of 1. 6. Experiments with Bence-Jones proteins and other homogeneous light chains gave results substantiating the conclusions (a) and (b). PMID:4174431

  20. Phase Transitions of Single Semistiff Polymer Chains

    NASA Astrophysics Data System (ADS)

    Bastolla, Ugo; Grassberger, Peter

    1997-12-01

    We study numerically a lattice model of semiflexible homopolymers with nearest neighbor (nn) attraction and energetic preference for straight joints between bonded monomers. For this we use a new Monte Carlo algorithm, the “prunedenriched Rosenbluth Method” (PERM). It is very efficient both for relatively open configurations at high temperatures and for compact and frozen-in low- T states. This allows us to study in detail the phase diagram as a function of nn attraction ɛ and stiffness x. It shows a θ-collapse line with a transition from open coils (small ɛ) to molten compact globules (large ɛ) and a freezing transition toward a state with orientational global order (large stiffness x). Qualitatively this is similar to a recently studied mean-field theory [S. Doniach, T. Garel, and H. Orland (1996), J. Chem. Phys. 105(4), 1601], but there are important differences in details. In contrast to the mean-field theory and to naive expectations, the θ-temperature increases with stiffness x. The freezing temperature increases even faster, and reaches the θ-line at a finite value of x. For even stiffer chains, the freezing transition takes place directly, without the formation of an intermediate globular state. Although being in conflict with mean-field theory, the latter had been conjectured already by Doniach et al. on the basis of heuristic arguments and of low-statistics Monte Carlo simulations. Finally, we discuss the relevance of the present model as a very crude model for protein folding.

  1. Highly Ordered Single Conjugated Polymer Chain Rod Morphologies

    SciTech Connect

    Adachi, Takuji; Brazard, Johanna; Chokshi, Paresh; Ganesan, Venkat; Bolinger, Joshua; Barbara, Paul F.

    2010-10-15

    We have reexamined the fluorescence polarization anisotropy of single polymer chains of the prototypical conjugated polymer poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) isolated in a poly(methyl methacrylate) (PMMA) matrix employing improved synthetic samples that contain a much smaller number of tetrahedral chemical defects per chain. The new measurements reveal a much larger fraction of highly anisotropic MEH-PPV chains, with >70% of the chains exhibiting polarization anisotropy values falling in the range of 0.6-0.9. High anisotropy is strong evidence for a rod-shaped conformation. A comparison of the experimental results with coarse grain, beads on a chain simulations reveals that simulations with the usual bead-bead pairwise additive potentials cannot reproduce the observed large fraction of high polarization values. Apparently, this type of potential lacks some yet to be identified molecular feature that is necessary to accurately simulate the experimental results.

  2. Single-Chain Probes for Illuminating Androgenicity of Chemicals.

    PubMed

    Kim, Sung-Bae; Tao, Hiroaki

    2016-01-01

    The present protocol introduces a single-chain probe carrying a functional peptide in the N-terminal domain of the androgen receptor (AR NTD) for illuminating androgenicity of ligands. In the single-chain probe, a functional peptide in the AR NTD was genetically fused to the ligand-binding domain of AR (AR LBD) via a flexible linker, and then sandwiched between the N- and C-terminal fragments of split-firefly luciferase (FLuc) dissected at D415. This single-chain probe exerts (1) a high signal-to-background ratio and (2) sensitive discrimination between agonists and antagonists, where the dimerization of AR LBD is not involved. The present protocol guides a fundamental methodology on how to discriminate weak protein-protein (peptide) binding, and provides a new insight into the intramolecular folding inside monomeric AR. PMID:27424901

  3. The novel anti-CD19 chimeric antigen receptors with humanized scFv (single-chain variable fragment) trigger leukemia cell killing.

    PubMed

    Qian, Liren; Li, Dan; Ma, Lie; He, Ting; Qi, Feifei; Shen, Jianliang; Lu, Xin-An

    2016-01-01

    The molecular design of CARs (Chimeric Antigen Receptors), especially the scFv, has been a major part to use of CAR-T cells for targeted adoptive immunotherapy. To address this issue, we chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR. Next, we generated a panel of humanized scFvs and tested in vitro for their ability to direct CAR-T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. Furthermore, in a xenograft model of lymphoma, human T cells expressing humanized scFvs exhibited the same anti-tumor efficacy as those expressing murine scFv and prolonged survival compared with cells expressing control CAR. Therefore, we uncovered CARs expressing humanized scFv domain that contribute the similar enhanced antileukemic efficacy and survival in tumor bearing mice. These results provide the basis for the future clinical studies of CAR-T cells transduced with humanized scFv directed to CD19. PMID:26996927

  4. Entropic forces of single-chain confinement in spherical cavities.

    PubMed

    Jin, Zhehui; Zhao, Shuangliang; Wu, Jianzhong

    2010-10-01

    Thermodynamic properties of a single chain in a confined space have been studied before with the polymer scaling theory and computer simulations. However, a comprehensive understanding of the entropic effects due to the molecular excluded volume and chain connectivity is emerging only recently, especially in the limit of large polymer packing densities as often encountered in biological systems. In this work, we propose a polymer density functional theory (DFT) to study the entropic forces for the confinement of single polymer chains in spherical cavities. At conditions accessible to Monte Carlo simulations, we show that the DFT predictions are in excellent agreement with the simulation results for the distributions of polymer segments as well as the free energy of confinement. The numerical efficiency of the DFT allows us to unify key conclusions from various theoretical analyses and experimental observations. PMID:21230306

  5. Expression, purification and characterization of a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen in Pichia pastoris.

    PubMed

    Wang, Ding-ding; Su, Man-man; Sun, Yan; Huang, Shu-lin; Wang, Ju; Yan, Wei-qun

    2012-11-01

    Because the demand for rabies post exposure prophylaxis (PEP) treatment has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide an adequate amount of the required passive immune component in PEP in countries where canine rabies is endemic. The replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for the treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen to develop a product that can be used as a component of the PEP cocktail. We cloned the ScFv fragment from a human ScFv library that was established previously and inserted this fragment into the expression vector pPICZαC/Fc. An active recombinant ScFv-Fc fusion protein was successfully expressed in Pichia pastoris. The production of ScFv-Fc was optimized and scaled up in an 80L fermentor with yields exceeding 60mg/L. The ScFv-Fc protein was purified to more than 95% purity using a two-step scheme: ammonium sulfate fractionation and Protein A Sepharose CL-4B. The ScFv-Fc fusion protein neutralized rabies virus in a standard in vivo neutralization assay in which the virus was incubated with the ScFv-Fc molecules before intracranial inoculation in mice. Our results suggest that functional antibodies can be produced in P. pastoris and that ScFv-Fc fusion proteins have the potential to serve as therapeutic candidates. PMID:22982755

  6. Single-copy entanglement in a gapped quantum spin chain.

    PubMed

    Hadley, Christopher

    2008-05-01

    The single-copy entanglement of a given many-body quantum system is defined [J. Eisert and M. Cramer, Phys. Rev. A 72, 042112 (2005)10.1103/PhysRevA.72.042112] as the maximal entanglement deterministically distillable from a bipartition of a single specimen of that system. For critical (gapless) spin chains, it was recently shown that this is exactly half the von Neumann entropy [R. Orús, J. I. Latorre, J. Eisert, and M. Cramer, Phys. Rev. A 73, 060303(R) (2006)], itself defined as the entanglement distillable in the asymptotic limit-i.e., given an infinite number of copies of the system. It is an open question as to what the equivalent behavior for gapped systems is. In this Letter, I show that for the paradigmatic spin-S Affleck-Kennedy-Lieb-Tasaki chain (the archetypal gapped chain), the single-copy entanglement is equal to the von Neumann entropy; i.e., all the entanglement present may be distilled from a single specimen. PMID:18518329

  7. Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli.

    PubMed

    Sun, Xue-Wen; Wang, Xiao-Hua; Yao, Yan-Bing

    2014-12-01

    Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb. PMID:25256416

  8. Stretching of Single Polymer Chains Using the Atomic Force Microscope

    NASA Astrophysics Data System (ADS)

    Ortiz, C.; van der Vegte, E. W.; van Swieten, E.; Robillard, G. T.; Hadziioannou, G.

    1998-03-01

    A variety of macroscopic phenomenon involve "nanoscale" polymer deformation including rubber elasticity, shear yielding, strain hardening, stress relaxation, fracture, and flow. With the advent of new and improved experimental techniques, such as the atomic force microscope (AFM), the probing of physical properties of polymers has reached finer and finer scales. The development of mixed self-assembling monolayer techniques and the chemical functionalization of AFM probe tips has allowed for mechanical experiments on single polymer chains of molecular dimensions. In our experiments, mixed monolayers are prepared in which end-functionalized, flexible polymer chains of thiol-terminated poly(methacrylic acid) are covalently bonded, isolated, and randomly distributed on gold substrates. The coils are then imaged, tethered to a gold-coated AFM tip, and stretched between the tip and the substrate in a conventional force / distance experiment. An increase in the attractive force due to entropic, elastic resistance to stretching, as well as fracture of the polymer chain is observed. The effect of chain stiffness, topological constraints, strain rate, mechanical hysteresis, and stress relaxation were investigated. Force modulation techniques were also employed in order to image the viscoelastic character of the polymer chains. Parallel work includes similar studies of biological systems such as wheat gluten proteins and polypeptides.

  9. Single-copy entanglement in critical quantum spin chains

    NASA Astrophysics Data System (ADS)

    Eisert, J.; Cramer, M.

    2005-10-01

    We consider the single-copy entanglement as a quantity to assess quantum correlations in the ground state in quantum many-body systems. We show for a large class of models that already on the level of single specimens of spin chains, criticality is accompanied with the possibility of distilling a maximally entangled state of arbitrary dimension from a sufficiently large block deterministically, with local operations and classical communication. These analytical results—which refine previous results on the divergence of block entropy as the rate at which maximally entangled pairs can be distilled from many identically prepared chains—are made quantitative for general isotropic translationally invariant spin chains that can be mapped onto a quasifree fermionic system, and for the anisotropic XY model. For the XX model, we provide the asymptotic scaling of ˜(1/6)log2(L) , and contrast it with the block entropy.

  10. Dynamics of Single Chains of Suspended Ferrofluid Particles

    NASA Technical Reports Server (NTRS)

    Cutillas, S.; Liu, J.

    1999-01-01

    We present an experimental study of the dynamics of isolated chains made of super-paramagnetic particles under the influence of a magnetic field. The motivation of this work is to understand if the chain fluctuations exist and, if it does, how does the fluctuation affect chain aggregation. We find that single chains strongly fluctuate and that the characteristic frequency of their fluctuations is inversely proportional to the magnetic field strength. The higher the field the lower the characteristic frequency of the chain fluctuations. In the high magnetic field limit, chains behave like rigid rods without any internal motions. In this work, we used ferrofluid particles suspended in water. These particles do not have any intrinsic magnetization. Once a magnetic field is applied, a dipole moment is induced in each particle, proportional to the magnetic field. A dipolar magnetic interaction then occurs between particles. If dipole-dipole magnetic energy is higher than the thermal energy, the result is a structure change inside the dipolar fluid. The ratio of these two energies is expressed by a coupling constant lambda as: lambda = (pi(a(exp 3))(chi(exp 2))(mu(sub 0))(H(sub 0))(exp 2))/18kT Where a is the particle radius, mu(sub 0) is the vacuum magnetic permeability, H(sub 0) the applied magnetic field, k the Boltzmann constant and T the absolute temperature. If lambda > 1, magnetic particles form chains along the field direction. The lateral coalescence of several chains may form bigger aggregates especially if the particle volume fraction is high. While many studies and applications deal with the rheological properties and the structural changes of these dipolar fluids, this work focuses on the understanding of the chain dynamics. In order to probe the chain dynamics, we used dynamic light scattering (DLS) in self-beating mode as our experimental technique. The experimental geometry is such that the scattering plane is perpendicular to the magnetic field

  11. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    SciTech Connect

    Surinder Batra, Ph D

    2006-02-27

    its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  12. Single-chain technology using discrete synthetic macromolecules

    NASA Astrophysics Data System (ADS)

    Ouchi, Makoto; Badi, Nezha; Lutz, Jean-François; Sawamoto, Mitsuo

    2011-12-01

    Fundamental polymer science is undergoing a profound transformation. As a result of recent progress in macromolecular chemistry and physics, synthetic polymer chains are becoming much more than just the modest building blocks of traditional 'plastics'. Promising options for controlling the primary and secondary structures of synthetic polymers have been proposed and, therefore, similarly to biopolymers, synthetic macromolecules may now be exploited as discrete objects with carefully engineered structures and functions. Although it is not possible today to reach the high level of complexity found in biomaterials, these new chemical possibilities open interesting avenues for applications in microelectronics, photovoltaics, catalysis and biotechnology. Here, we describe in detail these recent advances in macromolecular science and emphasize the possible emergence of technologies based on single-chain devices.

  13. Dynamics of Single Chains of Suspended Ferrofluid Particles

    NASA Technical Reports Server (NTRS)

    Cutillas, S.; Liu, J.

    1999-01-01

    We present an experimental study of the dynamics of isolated chains made of super-paramagnetic particles under the influence of a magnetic field. The motivation of this work is to understand if the chain fluctuations exist and, if it does, how does the fluctuation affect chain aggregation. We find that single chains strongly fluctuate and that the characteristic frequency of their fluctuations is inversely proportional to the magnetic field strength. The higher the field the lower the characteristic frequency of the chain fluctuations. In the high magnetic field limit, chains behave like rigid rods without any internal motions. In this work, we used ferrofluid particles suspended in water. These particles do not have any intrinsic magnetization. Once a magnetic field is applied, a dipole moment is induced in each particle, proportional to the magnetic field. A dipolar magnetic interaction then occurs between particles. If dipole-dipole magnetic energy is higher than the thermal energy, the result is a structure change inside the dipolar fluid. The ratio of these two energies is expressed by a coupling constant lambda as: lambda = (pi(a(exp 3))(chi(exp 2))(mu(sub 0))(H(sub 0))(exp 2))/18kT Where a is the particle radius, mu(sub 0) is the vacuum magnetic permeability, H(sub 0) the applied magnetic field, k the Boltzmann constant and T the absolute temperature. If lambda > 1, magnetic particles form chains along the field direction. The lateral coalescence of several chains may form bigger aggregates especially if the particle volume fraction is high. While many studies and applications deal with the rheological properties and the structural changes of these dipolar fluids, this work focuses on the understanding of the chain dynamics. In order to probe the chain dynamics, we used dynamic light scattering (DLS) in self-beating mode as our experimental technique. The experimental geometry is such that the scattering plane is perpendicular to the magnetic field

  14. Improving human skeletal muscle myosin heavy chain fiber typing efficiency.

    PubMed

    Murach, Kevin A; Bagley, James R; McLeland, Kathryn A; Arevalo, Jose A; Ciccone, Anthony B; Malyszek, Kylie K; Wen, Yuan; Galpin, Andrew J

    2016-04-01

    Single muscle fiber sodium dodecyl sulfate polyacrylamide gel-electrophoresis (SDS-PAGE) is a sensitive technique for determining skeletal muscle myosin heavy chain (MHC) composition of human biopsy samples. However, the number of fibers suitable to represent fiber type distribution via this method is undefined. Muscle biopsies were obtained from the vastus lateralis (VL) of nine resistance-trained males (25 ± 1 year, height = 179 ± 5 cm, mass = 82 ± 8 kg). Single fiber MHC composition was determined via SDS-PAGE. VL fiber type distribution [percent MHC I, I/IIa, IIa, IIa/IIx, and total "hybrids" (i.e. I/IIa + IIa/IIx)] was evaluated according to number of fibers analyzed per person (25 vs. 125). VL fiber type distribution did not differ according to number of fibers analyzed (P > 0.05). VL biopsy fiber type distribution of nine subjects is represented by analyzing 25 fibers per person. These data may help minimize cost, personnel-time, and materials associated with this technique, thereby improving fiber typing efficiency in humans. PMID:26842420

  15. Targeting nanodisks via a single chain variable antibody - Apolipoprotein chimera

    SciTech Connect

    Iovannisci, David M.; Beckstead, Jennifer A.; Ryan, Robert O.

    2009-02-06

    Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that {alpha}-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

  16. Design, expression and characterization of single chain Fv, Mms13 and the single chain Fv‑mms13 fusion protein.

    PubMed

    Kong, Deng; Wang, Xiaoke; Wang, Xiaohong; Wang, Xueyun; Chen, Xiaoli; Ji, Guoqiang; Fu, Xinhua; Wang, Shouxun

    2015-07-01

    Single chain Fv (scFv) antibodies are attractive as tumor-targeting vehicles due to their smaller size compared with intact antibody molecules. Mms13 is a putative membrane anchor protein of magnetosome. The present study fused the scFV gene of type Ⅳ collagenase to mms13 using the splicing by overlap extension polymerase chain reaction technique. The genes of scFv, mms13 and the scFv-mms13 fusion gene were cloned into a pET30a(+) vector to construct pET30a(+)-scFv, pET30a(+)-mms13 and pET30a(+)-scFv-mms13 expression vectors. The three protein compositions were confirmed by DNA sequencing and western blot analysis, and their cellular locations were determined using SDS-PAGE. The results of enzyme-linked immunosorbent assays and immunofluorescence demonstrated that the ScFv and ScFv-mms13 fusion proteins bound to the type Ⅳ collagenase and the antigen-associated cancer cells SMMC-7721, MCF-7 and HepG2 cells, in a dose-dependent and saturable manner. Although the immunoreactivities of ScFv-mms13 to the type Ⅳ collagenase and associated tumor cells were marginally lower than the corresponding scFv (3G11), considerable binding ability to the antigen by ScFv-mms13 remained. PMID:25824464

  17. Force extension response of single self-associating polymer chains

    NASA Astrophysics Data System (ADS)

    Sing, Charles; Alexander-Katz, Alfredo

    2012-02-01

    The structure and dynamics of polymeric molecules plays a crucial role in a number of synthetic and biological processes. Great progress has been made in using force spectroscopy methods, such as optical tweezers and atomic force microscopy, to probe these properties of single molecules in novel ways. While there has been significant advances at using analytical theory to model the behavior of, for example, single domain unfolding or multi-domain unfolding of identical domains, we consider for the first time the force-extension behavior of self-associating homopolymers. We use a Brownian dynamics simulation with a Bell-like association model that represents, in a coarse-grained fashion, biological polymers such as von Willebrand Factor that use domain-domain interactions to modulate a larger quaternary structure. We also present a theoretical description of pulling that utilizes a master equation description of the relevant coordinate of the polymer network's ``shortest chain.'' We can accurately reproduce the force-extension features, notably the appearance of a dissipative plateau upon the increase of the association time scale, and provide a clear conceptual description of the appearance of this feature. Qualitative comparison to von Willebrand pulling in experiment is shown.

  18. Solution NMR assignment of the heavy chain complex of the human cardiac myosin regulatory light chain.

    PubMed

    Rostkova, Elena; Gautel, Mathias; Pfuhl, Mark

    2015-04-01

    The regulatory light chain (RLC) of striated and cardiac muscle myosin plays a complex role in muscle function and regulation. Together with the essential light chain it provides stability to the lever arm, which is essential for force generation. Furthermore, phosphorylation and interaction with myosin binding protein C (MyBP-C) suggest an additional role in the regulation of muscle contraction. The former is of particular importance in the heart, where RLC phosphorylation appears to be correlated to the wringing motion of heart contraction. To address these questions and because of a lack of mammalian RLC structures, we initiated an NMR study of the human cardiac regulatory myosin light chain. PMID:24414277

  19. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease

    PubMed Central

    Grizot, Sylvestre; Smith, Julianne; Daboussi, Fayza; Prieto, Jesús; Redondo, Pilar; Merino, Nekane; Villate, Maider; Thomas, Séverine; Lemaire, Laetitia; Montoya, Guillermo; Blanco, Francisco J.; Pâques, Frédéric; Duchateau, Philippe

    2009-01-01

    Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches. PMID:19584299

  20. EARLINET Single Calculus Chain - overview on methodology and strategy

    NASA Astrophysics Data System (ADS)

    D'Amico, G.; Amodeo, A.; Baars, H.; Binietoglou, I.; Freudenthaler, V.; Mattis, I.; Wandinger, U.; Pappalardo, G.

    2015-11-01

    In this paper we describe the EARLINET Single Calculus Chain (SCC), a tool for the automatic analysis of lidar measurements. The development of this tool started in the framework of EARLINET-ASOS (European Aerosol Research Lidar Network - Advanced Sustainable Observation System); it was extended within ACTRIS (Aerosol, Clouds and Trace gases Research InfraStructure Network), and it is continuing within ACTRIS-2. The main idea was to develop a data processing chain that allows all EARLINET stations to retrieve, in a fully automatic way, the aerosol backscatter and extinction profiles starting from the raw lidar data of the lidar systems they operate. The calculus subsystem of the SCC is composed of two modules: a pre-processor module which handles the raw lidar data and corrects them for instrumental effects and an optical processing module for the retrieval of aerosol optical products from the pre-processed data. All input parameters needed to perform the lidar analysis are stored in a database to keep track of all changes which may occur for any EARLINET lidar system over the time. The two calculus modules are coordinated and synchronized by an additional module (daemon) which makes the whole analysis process fully automatic. The end user can interact with the SCC via a user-friendly web interface. All SCC modules are developed using open-source and freely available software packages. The final products retrieved by the SCC fulfill all requirements of the EARLINET quality assurance programs on both instrumental and algorithm levels. Moreover, the manpower needed to provide aerosol optical products is greatly reduced and thus the near-real-time availability of lidar data is improved. The high-quality of the SCC products is proven by the good agreement between the SCC analysis, and the corresponding independent manual retrievals. Finally, the ability of the SCC to provide high-quality aerosol optical products is demonstrated for an EARLINET intense observation

  1. Recombinant human fibrinogen and sulfation of the. gamma. prime chain

    SciTech Connect

    Farrell, D.H.; Huang, S.; Chung, D.W.; Davie, E.W. ); Mulvihill, E.R. )

    1991-10-01

    Human fibrinogen and the homodimeric {gamma}{prime}-chain-containing variant have been expressed in BHK cells using cDNAs coding for the {alpha},{beta}, and {gamma} (or {gamma}{prime}) chains. The fibrinogens were secreted at levels greater than 4 {mu}g (mg of total cell protein){sup {minus}1}day{sup {minus}1} and were biologically active in clotting assays. Recombinant fibrinogen containing the {gamma}' chain incorporated {sup 35}SO{sub 4} into its chains during biosynthesis, while no incorporation occurred in the protein containing the {gamma} chain. The identity of the sulfated {gamma}{prime} chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase {Upsilon} digestion of the recombinant fibrinogen containing the {gamma}{prime} chain released 96% of the {sup 35}S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.

  2. Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

    PubMed Central

    Li, Fangfang; Meng, Fanping; Jin, Quanxin; Sun, Changyuan; Li, Yingxin; Li, Honghua; Jin, Songzhu

    2014-01-01

    Single-chain variable domain fragment (scFv) 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion protein was expressed in Pichia pastoris. The affinity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunofluorescence staining. The ability of the fusion protein to block myasthenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for specific immunosuppressive therapy of myasthenia gravis. PMID:25206900

  3. Single-chain urokinase in empyema induced by Pasturella multocida.

    PubMed

    Idell, Steven; Jun Na, Moon; Liao, Huai; Gazar, A E; Drake, Wonder; Lane, Kirk B; Koenig, Kathy; Komissarov, Andrey; Tucker, Torry; Light, Richard W

    2009-10-01

    Intrapleural fibrin deposition and subsequent fibrosis characterize evolving empyema and contribute to the morbidity associated with this condition. Single-chain urokinase (scuPA) is proenzyme form of the urokinase plasminogen activator, which has recently been shown to effectively clear intrapleural loculation in tetracycline-induced pleurodesis in rabbits. The authors therefore hypothesized that scuPA could likewise improve intrapleural injury associated with empyema. The authors used a rabbit model of empyema induced by intrapleural administration of Pasturella multocida to test this hypothesis and determined the effects of intrapleural scuPA on pleural fluids indices of inflammation and intrapleural fibrosis. The authors found that intrapleural administration of scuPA was well tolerated, generated readily detectable fibrinolytic activity in the empyema fluids and did not induce intrapleural or systemic bleeding. Pleural fluid volume, intrapleural protein, and D-dimer concentrations were increased at 24 and 48 hours (P < .01, respectively) after induction of empyema. Intrapleural loculation did not occur in the scuPA- or vehicle control-treated animals and there was no significant change in the pleural empyema or thickening scores. These findings confirm that intrapleural scuPA generates fibrinolysis in empyema fluids but does not alter fibrotic repair at the pleural surface or the intensity of intrapleural inflammation in this empyema model. PMID:19895321

  4. Purification, characterization, and biotinylation of single-chain antibodies.

    PubMed

    Kipriyanov, S M

    1998-01-01

    The variable region (Fv) portion of an antibody is comprised of the antibody V(H) and V(L) domains and is the smallest antibody fragment containing a complete antigen-binding site. To stabilize the association of the recombinant V(H) and V(L) domains, they have been linked in single-chain Fv constructs with a short peptide that bridges the approx 3.5 nm between the carboxy terminus of one domain and the ammo terminus of the other (1-3). An NMR comparison of the unlinked Fv fragment of the antibody McPC603 with the corresponding scFv containing a V(H)-(Gly(4)Ser)(3)-V(L) linker has shown no perturbation of the folding of the variable domains by the linker (4,5). In comparison to the much larger Fab', F(ab')(2), and IgG forms of monoclonal antibody (MAb) from which they are derived, scFvs have lower retention times in nontarget tissues, more rapid blood clearance, and better tumor penetration (6-8). ScFvs, therefore, represent potentially very useful molecules for the targeted delivery of drugs, toxins, or radionuclides to a tumor site. PMID:21390870

  5. Surface Diffusion of Single Polymer Chain Using Molecular Dynamics SIMULATION*

    NASA Astrophysics Data System (ADS)

    Desai, Tapan; Keblinski, Pawel; Kumar, Sanat; Granick, Steve

    2004-05-01

    Results of recent experiments on polymer chains adsorbed from dilute solution at solid-liquid interface show the power scaling law dependence of the chain diffusivity, D, as a function of the degree of polymerization, N, D ˜ N^3/2. By contrast, DNA molecules bound to fluid cationic lipid bilayers follows Rouse dynamics with D ˜ N^1. We used molecular dynamics simulations to gain an understanding of these dissimilar scaling behaviors. Our model systems contain chains comprised of N monomers connected by anharmonic springs described by the finite extendible nonlinear elastic, FENE potential, embedded into a solvent of N=1 monomers. Two types of simulations we performed: (i) the chain is confined to two dimensions, (ii) the three dimensional chain in the solvent is confined between two solids plates. With randomly placed impenetrable obstacles on the surface, the diffusion of 2D chains exhibits, D ˜ N^3/2 behavior, when the chain radius of gyration, Rg, is larger than half the distance between obstacles, and D ˜ N^1 for shorter chains. In the presence of an athermal solvent, the scaling exponent is 0.75 due to hydrodynamic forces, for the two-dimensional system. We will also discuss the nature of dynamic adsorption transition and effects of hydrodynamics forces on chain diffusion for the three-dimensional simulations.

  6. Linker peptide and affinity tag for detection and purification of single-chain Fv fragments.

    PubMed

    Küttner, Gabriele; Giessmann, Elke; Wessner, Helga; Scholz, Christa; Reinhardt, Dina; Winkler, Karsten; Marx, Uwe; Höhne, Wolfgang

    2004-05-01

    The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs. PMID:15152607

  7. Immobilization strategies for single-chain antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Miller, Keith D.; Pefaur, Noah B.; Gonzalez, Rachel M.; Apiyo, David O.; Engelmann, Heather E.; Srinivastava, Sudhir; Kagan, Jacob; Rodland, Karin D.; Zangar, Richard C.

    2008-06-01

    Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays have great potential for validating biomarkers of disease. ELISA relies on robust affinity reagents that retain activity when immobilized or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional immunoglobin G (IgG). Unfortunately, scFv are typically less stabile than IgG and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv structural modifications to see if we could develop a more robust scFv reagent. Two promising strategies that emerged from these studies: 1) the precapture of epitope-tagged scFv using an anti-epitope antibody and 2) the direct printing of a thioredoxin/scFv fusion protein on glass slides. The use of either strategy improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the anti-epitope precapture method had a risk of reagent transfer. Using the direct printing method, we show that anti-PSA scFv are highly specific when tested against 21 different IgG-based assays. Finally, the scFv microarray PSA assay gave comparable results (R2 = 0.95) to a commercial 96-well ELISA when tested using serum samples. Overall, these results suggest that minor modifications of the scFv protein structure are sufficiently to produce reagents that are suitable for use in multiplex assay systems.

  8. Chain Dynamics in Single Chain Limit by Rheological and Diffusion Measurements

    NASA Astrophysics Data System (ADS)

    Wang, Shi-Qing; Wang, Shanfeng

    2004-03-01

    Our recent trace and self diffusion measurements , indicate (a) (b) that the molecular weight scaling of the self-diffusion coefficient is non-reptative for moderately entangled polymer melts as noted previously (c) but asymptotically approaches the reptative exponent of -2.0 for sufficiently entangled polymers, whereas the trace diffusion coefficient, measured by immersing a dilute amount of probe chains in a matrix of sufficiently entangled polymer of the same species, scales reptatively even for the probe chains of moderate entanglement. To further understand the behavior of probe chain dynamics in a matrix, we have measured the intrinsic viscosity and intrinsic storage and loss moduli of dilute solutions made of long chains (in dilution) and short chains, where both chain lengths can be much longer than the entanglement chain length. A rich variety of chain dynamics is observed including Stokes-Zimm behavior and Rouse like behavior as a function of the long and short chain lengths and concentration. (a) S.Q. Wang, Highlight Article, J. Polym. Sci. Polym. Phys., 41, 1589 (2003). (b) "Diffusion and Rheology of Binary Polymer Mixtures", S. Wang et al, Macromolecules, in press (2003). (c) T.P. Lodge, Phys. Rev. Lett. 83, 3218 (1999).

  9. Promoter region of the human platelet-derived growth factor A-chain gene

    SciTech Connect

    Takimoto, Yasuo; Wang, Zhao Yi; Kobler, K.; Deuel, T.F. )

    1991-03-01

    The platelet-derived growth factor (PDGF) A- and B-chain genes are widely expressed in mammalian tissues and their homodimeric gene products appear to regulate the autocrine growth of both normal and transformed cells. In this study, we analyzed the 5{prime} flanking sequences of the human PDGF A-chain gene to seek elements important to regulating its transcription. The promoter reigon was exceptionally G + C-rich and contained a TATA box but no CAAT box. The transcription start site was identified 845 base pairs 5{prime} to the translation initiation site by S1 nuclease mapping and by primer extension. Both in vitro transcription and transient expression of the chloramphenicol acetyltransferase gene linked to the PDGF A-chain 5{prime} flanking sequences established that the putative promoter region was active, and RNase H mapping established that the three characteristic mRNAs used the same transcription start site, which was used in normal endothelial cells and in two human tumor cell lines that express high levels of A-chain transcripts. The results extablished an exceptionally G + C-rich promoter region and a single transcription start site active for each of the three mRNAs of the PDGF A-chain gene. DNA sites of potential importance in mediating the activation of the PDGF A-chain gene in normal cells and in transformed cell lines expressing high levels of PDGF A-chain were identified.

  10. Markov chain for estimating human mitochondrial DNA mutation pattern

    NASA Astrophysics Data System (ADS)

    Vantika, Sandy; Pasaribu, Udjianna S.

    2015-12-01

    The Markov chain was proposed to estimate the human mitochondrial DNA mutation pattern. One DNA sequence was taken randomly from 100 sequences in Genbank. The nucleotide transition matrix and mutation transition matrix were estimated from this sequence. We determined whether the states (mutation/normal) are recurrent or transient. The results showed that both of them are recurrent.

  11. Glycosylation pattern of human inter-alpha-inhibitor heavy chains.

    PubMed Central

    Flahaut, C; Capon, C; Balduyck, M; Ricart, G; Sautiere, P; Mizon, J

    1998-01-01

    Human inter-alpha-inhibitor (IalphaI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan chain: a light chain named bikunin carrying the anti-proteinase activity and two heavy chains, H1 and H2, which exhibit specific properties, e.g. they interact with hyaluronan thus stabilizing the extracellular matrix. In this study, using matrix-assisted laser desorption ionization-time-of-flight MS and amino acid sequencing of tryptic peptides, we provide a detailed analysis of the glycosylation pattern of both heavy chains. H1 carries two complex-type N-glycans of predominantly biantennary structure linked to asparagine residues at positions 256 and 559 respectively. In contrast, the oligosaccharides attached to H2 are a complex-type N-glycan in the N-terminal region of the protein (Asn64) and three to four type-1 core-structure O-glycans mono- or di-sialylated, clustered in the C-terminal region. We propose that these O-glycans might function as a recognition signal for the H2 heavy chain. The biological implications of this hypothesis, notably for the biosynthetic pathway of IalphaI, are discussed. PMID:9677337

  12. Origin of metastable knots in single flexible chains.

    PubMed

    Dai, Liang; Renner, C Benjamin; Doyle, Patrick S

    2015-01-23

    Recent theoretical progress has explained the physics of knotting of semiflexible polymers, yet knotting of flexible polymers is relatively unexplored. We herein develop a new theory for the size distribution of knots on a flexible polymer and the existence of metastable knots. We show the free energy of a flexible molecule in a tube can be mapped to quantitatively reproduce the free energy distribution of a knot on a flexible chain. The size distribution of knots on flexible chains is expected to be universal and might be observed at a macroscopic scale, such as a string of hard balls. PMID:25659023

  13. Nonlinear Elasticity: From Single Chain to Networks and Gels

    NASA Astrophysics Data System (ADS)

    Dobrynin, Andrey; Carrillo, Jan-Michael; Mackintosh, Fred

    2014-03-01

    Biological and polymeric networks show highly nonlinear stress-strain behavior leading to material hardening with increasing deformation. Using a combination of theoretical analysis and molecular dynamics simulations we develop a model of network deformation that describes nonlinear mechanical properties of a broad variety of biological and polymeric networks and gels by relating their macroscopic strain-hardening behavior with molecular parameters of the network strands. The starting point of our approach is a nonlinear force/elongation relation for discrete chain model with varying bending rigidity. This theory provides a universal relationship between the strain-dependent network modulus and the network deformation as a function of the first invariant and chain elongation ratio that depends on a ratio of the unperturbed chain size to chain dimension in a fully extended conformation. The model predictions for the nonlinear shear modulus and differential shear modulus for uniaxial and shear deformations are in a very good agreement with both the results of molecular dynamics simulations of networks and with experimental data for biopolymer networks of actin, collagen, fibrin, vimentin, neurofilaments, and pectin. NSF-DMR-1004576

  14. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    SciTech Connect

    Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

    2015-01-15

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture.

  15. Crystallization behavior of single isotactic poly(methyl methacrylate) chains visualized by atomic force microscopy.

    PubMed

    Anzai, Takahiro; Kawauchi, Mariko; Kawauchi, Takehiro; Kumaki, Jiro

    2015-01-01

    We have, for the first time, successfully visualized the crystallization behavior of a single isolated polymer chain at the molecular level by atomic force microscopy (AFM). Previously, we found that isotactic poly(methyl methacrylate) (it-PMMA) formed two-dimensional folded chain crystals composed of double-stranded helices upon compression of its Langmuir monolayer on a water surface, and the molecular images of the crystals deposited on mica were clearly visualized by AFM (Kumaki, J.; et al. J. Am. Chem. Soc. 2005, 127, 5788). In the present study, a high-molecular-weight it-PMMA was diluted in a monolayer of an it-PMMA oligomer which cannot crystallize at the experimental temperature due to its low molecular weight. At a low surface pressure, isolated amorphous chains of the high-molecular-weight it-PMMA solubilized in the oligomer monolayer were observed. On compression, the isolated chains converted to crystals composed of a single chain, typically some small crystallites linked by an amorphous chain like a necklace. Detailed AFM observations of the crystals indicated that the crystalline nuclei preferentially formed at the ends of the chains, and the size of the nuclei was almost independent of the molecular weight of it-PMMA over a wide range. At an extremely slow compression, crystallization was promoted, resulting in crystallization of the whole chain. The crystallization behavior of a single isolated chain provides new insights in understanding the polymer crystallization process. PMID:25496047

  16. Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were developed that contain single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or suga...

  17. Serologically defined V region subgroups of human lambda light chains.

    PubMed

    Solomon, A; Weiss, D T

    1987-08-01

    The availability of numerous antisera prepared against lambda-type Bence Jones proteins and lambda chains of known amino acid sequence has led to the differentiation and classification of human lambda light chains into one of five V lambda subgroups. The five serologically defined subgroups, V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI, correspond to the chemical classification that is based on sequence homologies in the first framework region (FR1). Proteins designated by sequence as lambda V react with specific anti-lambda II antisera and are thus included in the V lambda II subgroup classification. The isotypic nature of the five V lambda subgroups was evidenced through analyses of lambda-type light chains that were isolated from the IgG of normal individuals. Based on analyses of 116 Bence Jones proteins, the frequency of distribution of the lambda I, lambda II/V, lambda III, lambda IV, and lambda VI proteins in the normal lambda chain population is estimated to be 27%, 37%, 23%, 3%, and 10%, respectively. This distribution of V lambda subgroups was comparable to that found among 82 monoclonal Ig lambda proteins. Considerable V lambda intragroup antigenic heterogeneity was also apparent. At least two sub-subgroups were identified among each of the five major V lambda subgroups, implying the existence of multiple genes in the human V lambda genome. The V lambda classification of 54 Ig lambda proteins obtained from patients with primary or multiple myeloma-associated amyloidosis substantiated the preferential association of lambda VI light chains with amyloidosis AL and the predominance of the normally rare V lambda VI subgroup in this disease. PMID:3110284

  18. Reversible single-chain selective point folding via cyclodextrin driven host-guest chemistry in water.

    PubMed

    Willenbacher, Johannes; Schmidt, Bernhard V K J; Schulze-Suenninghausen, David; Altintas, Ozcan; Luy, Burkhard; Delaittre, Guillaume; Barner-Kowollik, Christopher

    2014-07-01

    In the present communication we introduce a new platform technology for the reversible folding of single polymer chains in aqueous environment on the basis of cyclodextrin (CD) host-guest chemistry and controlled radical polymerization protocols. The single-chain folding of adamantyl-β-CD α-ω-functionalized poly(N,N-dimethylacrylamide) and its reversion at elevated temperatures were monitored by DLS and nuclear Overhauser enhancement spectroscopy (NOESY). PMID:24850295

  19. Primitive-path statistics of entangled polymers: mapping multi-chain simulations onto single-chain mean-field models

    NASA Astrophysics Data System (ADS)

    Steenbakkers, Rudi J. A.; Tzoumanekas, Christos; Li, Ying; Liu, Wing Kam; Kröger, Martin; Schieber, Jay D.

    2014-01-01

    We present a method to map the full equilibrium distribution of the primitive-path (PP) length, obtained from multi-chain simulations of polymer melts, onto a single-chain mean-field ‘target’ model. Most previous works used the Doi-Edwards tube model as a target. However, the average number of monomers per PP segment, obtained from multi-chain PP networks, has consistently shown a discrepancy of a factor of two with respect to tube-model estimates. Part of the problem is that the tube model neglects fluctuations in the lengths of PP segments, the number of entanglements per chain and the distribution of monomers among PP segments, while all these fluctuations are observed in multi-chain simulations. Here we use a recently proposed slip-link model, which includes fluctuations in all these variables as well as in the spatial positions of the entanglements. This turns out to be essential to obtain qualitative and quantitative agreement with the equilibrium PP-length distribution obtained from multi-chain simulations. By fitting this distribution, we are able to determine two of the three parameters of the model, which govern its equilibrium properties. This mapping is executed for four different linear polymers and for different molecular weights. The two parameters are found to depend on chemistry, but not on molecular weight. The model predicts a constant plateau modulus minus a correction inversely proportional to molecular weight. The value for well-entangled chains, with the parameters determined ab initio, lies in the range of experimental data for the materials investigated.

  20. Chemical Synthesis of Human Syndecan-3 Glycopeptides Bearing Two Heparan Sulfate Glycan Chains

    PubMed Central

    Yoshida, Keisuke; Yang, Bo; Yang, Weizhun; Zhang, Zeren; Zhang, Jicheng

    2014-01-01

    Despite the ubiquitous presence of proteoglycans in mammalian systems, methodologies to synthesize this class of glycopeptides with homogeneous glycans are not well developed. Herein, we report the first synthesis of glycosaminoglycan family glycopeptides containing two different heparan sulfate chains from human syndecan-3. With the large sizes and tremendous structural complexities, multiple unexpected obstacles were encountered in synthesis, which include the high sensitivity to base treatment and the instability of glycopeptides with two glycan chains towards catalytic hydrogenation conditions. To overcome these challenges, after many trials, a successful strategy was established by constructing the partially deprotected single glycan chain containing glycopeptides first followed by union of the glycan bearing fragments and cleavage of ester type protecting groups. This work has laid the foundation to prepare other members of this important class of molecules. PMID:24981920

  1. Optimization modeling of single-chain antibody against hepatoma based on similarity algorithm.

    PubMed

    Zhao, Zhi-Jun; Chen, Jing-Tao; Yuan, Jia-Ying; Yin, Xiao-Xiang; Song, Hua-Yong; Wang, Xin-Chun

    2015-01-01

    The purposes was to establish optimal modeling of single-chain antibody molecules based on similarity algorithm and seek the connecting peptides that had the minimal effect on the structure and bioactivity of the variable region of heavy chain (VH) and that of light chain (VL) in a single-chain antibody against liver cancer. After the Linker with different lengths (n=0~7) had been added into single chain fragment variable (ScFv), modeling of the overall sequences of VH, VL and ScFv were conducted respectively. Meanwhile, the peptide chain structure of (Gly4Ser)n was adopted for the connecting peptide. Then the spatial spherical shell layer alignment algorithm based on spherical polar coordinates was utilized for comparing the structural similarity of VH and VL before and after adding connecting peptide. Equally, in order to determine the stability of VH and VL, MATLAB was applied for analysis of the fore and aft distances and the diffusion radius. Indirect ELISA method was used to detect single-chain antibody immunological activity of Linker with different lengths. The MTT assay was utilized for the examination of the inhibition rate of single-chain antibody with different lengths of Linker to liver cancer cell. When n=4, the structural similarity between VH together with VL and their original ones was the highest. When n=3, the influence of connecting peptide on the stability of VH and VL was minimum. When n>3, the fore and aft distances changed little due to the increase and fold of the length of peptide chain. The results of ELISA detection showed that when n=4, affinity of single chain antibody to liver cancer cells was much higher. The MTT test also indicated that when n=4, the inhibition rate of the connecting peptide on hepatoma carcinoma cell reached the highest, and that came second when n=3. When n=4, the structural stability and biological functions of anti-hepatoma single-chain antibody were both favorable. This study has provided a basis for the design

  2. Single polymer chains in poor solvent: Using the bond fluctuation method with explicit solvent

    NASA Astrophysics Data System (ADS)

    Jentzsch, Christoph; Werner, Marco; Sommer, Jens-Uwe

    2013-03-01

    We use the bond fluctuation model with explicit solvent to study single polymer chains under poor solvent conditions. Static and dynamic properties of the bond fluctuation model with explicit solvent are compared with the implicit solvent model, and the Θ-temperatures are determined for both solvent models. We show that even in the very poor solvent regime, dynamics is not frozen for the explicit solvent model. We investigate some aspects of the structure of a single collapsed globule and show that rather large chain lengths are necessary to reach the scaling regime of a dense sphere. The force-extension curve of a single polymer chain under poor solvent conditions in the fixed end-to-end distance ensemble is analyzed. We find that the transition of the tadpole conformation to the stretched chain conformation is rather smooth because of fluctuation effects, which is in agreement with recent experimental results.

  3. Single polymer chains in poor solvent: using the bond fluctuation method with explicit solvent.

    PubMed

    Jentzsch, Christoph; Werner, Marco; Sommer, Jens-Uwe

    2013-03-01

    We use the bond fluctuation model with explicit solvent to study single polymer chains under poor solvent conditions. Static and dynamic properties of the bond fluctuation model with explicit solvent are compared with the implicit solvent model, and the Θ-temperatures are determined for both solvent models. We show that even in the very poor solvent regime, dynamics is not frozen for the explicit solvent model. We investigate some aspects of the structure of a single collapsed globule and show that rather large chain lengths are necessary to reach the scaling regime of a dense sphere. The force-extension curve of a single polymer chain under poor solvent conditions in the fixed end-to-end distance ensemble is analyzed. We find that the transition of the tadpole conformation to the stretched chain conformation is rather smooth because of fluctuation effects, which is in agreement with recent experimental results. PMID:23485321

  4. Supramolecular Nanoparticles via Single-Chain Folding Driven by Ferrous Ions.

    PubMed

    Wang, Fei; Pu, Hongting; Jin, Ming; Wan, Decheng

    2016-02-01

    Single-chain nanoparticles can be obtained via single-chain folding assisted by intramolecular crosslinking reversibly or irreversibly. Single-chain folding is also an efficient route to simulate biomacromolecules. In present study, poly(N-hydroxyethylacrylamide-co-4'-(propoxy urethane ethyl acrylate)-2,2':6',2''-terpyridine) (P(HEAm-co-EMA-Tpy)) is synthesized via reversible addition fragmentation chain transfer polymerization. Single-chain folding and intramolecular crosslinking of P(HEAm-co-EMA-Tpy) are achieved via metal coordination chemistry. The intramolecular interaction is characterized on ultraviolet/visible spectrophotometer (UV-vis spectroscopy), proton nuclear magnetic resonance ((1)H NMR), and differential scanning calorimetry (DSC). The supramolecular crosslinking mediated by Fe(2+) plays an important role in the intramolecular collapsing of the single-chain and the formation of the nanoparticles. The size and morphology of the nanoparticles can be controlled reversibly via metal coordination chemistry, which can be characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), and atomic force microscope (AFM). PMID:26748641

  5. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    SciTech Connect

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; Foster, Carmen M.; Kennel, Stephen J.; Wall, Jonathan S.

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

  6. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    DOE PAGESBeta

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; et al

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presencemore » of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.« less

  7. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    PubMed Central

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; Foster, Carmen M.; Kennel, Stephen J.; Wall, Jonathan S.

    2015-01-01

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils. PMID:26393799

  8. Tertiary structure of human {Lambda}6 light chains.

    SciTech Connect

    Pokkuluri, P. R.; Solomon, A.; Weiss, D. T.; Stevens, F. J.; Schiffer, M.; Center for Mechanistic Biology and Biotechnology; Univ. of Tennessee Medical Center /Graduate School of Medicine

    1999-01-01

    AL amyloidosis is a disease process characterized by the pathologic deposition of monoclonal light chains in tissue. To date, only limited information has been obtained on the molecular features that render such light chains amyloidogenic. Although protein products of the major human V kappa and V lambda gene families have been identified in AL deposits, one particular subgroup--lambda 6--has been found to be preferentially associated with this disease. Notably, the variable region of lambda 6 proteins (V lambda 6) has distinctive primary structural features including the presence in the third framework region (FR3) of two additional amino acid residues that distinguish members of this subgroup from other types of light chains. However, the structural consequences of these alterations have not been elucidated. To determine if lambda 6 proteins possess unique tertiary structural features, as compared to light chains of other V lambda subgroups, we have obtained x-ray diffraction data on crystals prepared from two recombinant V lambda 6 molecules. These components, isolated from a bacterial expression system, were generated from lambda 6-related cDNAs cloned from bone marrow-derived plasma cells from a patient (Wil) who had documented AL amyloidosis and another (Jto) with multiple myeloma and tubular cast nephropathy, but no evident fibrillar deposits. The x-ray crystallographic analyses revealed that the two-residue insertion located between positions 68 and 69 (not between 66 and 67 as previously surmised) extended an existing loop region that effectively increased the surface area adjacent to the first complementarity determining region (CDR1). Further, an unusual interaction between the Arg 25 and Phe 2 residues commonly found in lambda 6 molecules was noted. However, the structures of V lambda 6 Wil and Jto also differed from each other, as evidenced by the presence in the latter of certain ionic and hydrophobic interactions that we posit increased protein

  9. Light chain editors of anti-DNA receptors in human B cells

    PubMed Central

    Kalinina, Olga; Wang, Yue; Sia, Kevin; Radic, Marko; Cazenave, Pierre-André

    2014-01-01

    Receptor editing is a mechanism of self-tolerance used in newly generated B cells. The expressed heavy (H) or light (L) chain of an autoreactive receptor is replaced by upstream V genes which eliminate or modify autoreactivity. Editing of anti-DNA receptors has been characterized in anti-DNA transgenic mouse models including 3H9, 3H9/56R, and their revertant 3H9GL. Certain L chains, termed editors, rescue anti-DNA B cells by neutralizing or modifying DNA binding of the H chain. This editing mechanism acts on the natural H chain repertoire; endogenous H chains with anti-DNA features are expressed primarily in combination with editor L chains. We ask whether a similar set of L chains exists in the human repertoire, and if so, do they edit H chains with anti-DNA signatures? We compared the protein sequences of mouse editors to all human L chains and found several human L chains similar to mouse editors. These L chains diminish or veto anti-DNA binding when expressed with anti-DNA H chains. The human H chains expressed with these L chains also have relatively high arginine (Arg) content in the H chain complementarity determining region (H3), suggesting that receptor editing plays a role in establishing tolerance to DNA in humans. PMID:24470445

  10. Brownian Dynamics Simulations of Flow Induced Conformation of Single Polymer Chains

    NASA Astrophysics Data System (ADS)

    Oztekin, Alparslan; Webb, Edward; Zhang, Frank; Cheng, Xuanhong

    2012-11-01

    Coarse-grained molecular dynamics simulation of single polymer chains is conducted to examine flow induced conformational changes of single polymer chains in shear and extensional flows. Dynamic properties of polymeric molecules are described using bead-spring models; these models put coarse grain groups of atoms into single particles, connected to adjacent particles via spring like interactions. A common representation of the bonded interaction, or spring, is given by the Finitely Extensible Non-linear Elastic model. The constants used in the model are determined form single-molecule force spectroscopic experiments. Simulations and experiments will help to achieve a clear picture of how von Willebrand Factor, a blood clotting protein, model system for flow-induced activation, achieves flow sensing at the single protein/polymer level. The model includes bead-bead interactions, hydrodynamic interaction, the volume of the beads and spring-spring interaction for finitely extensible dumbbell and other spring models. The effects of walls on the dynamics of polymer chains are also presented. The results are presented for various chain lengths and flow conditions. Hydrodynamic interaction and the presence of wall have strong influences on the dynamics of the polymer chain.

  11. Phase Behavior of a Single Structured Ionomer Chain in Solution

    DOE PAGESBeta

    Aryal, Dipak; Etampawala, Thusitha; Perahia, Dvora; Grest, Gary S.

    2014-08-14

    Structured polymers offer a means to tailor transport pathways within mechanically stable manifolds. Here we examine the building block of such a membrane, namely a single large pentablock co-polymer that consist of a center block of a randomly sulfonated polystyrene, designed for transport, tethered to poly-ethylene-r-propylene and end-capped by poly-t-butyl styrene, for mechanical stability,using molecular dynamics simulations. The polymer structure in a cyclohexane-heptane mixture, a technologically viable solvent, and in water, a poor solvent for all segments and a ubiquitous substance is extracted. In all solvents the pentablock collapsed into nearly spherical aggregates where the ionic block is segregated. Inmore » hydrophobic solvents, the ionic block resides in the center, surrounded by swollen intermix of flexible and end blocks. In water all blocks are collapsed with the sulfonated block residing on the surface. Our results demonstrate that solvents drive different local nano-segregation, providing a gateway to assemble membranes with controlled topology.« less

  12. Phase Behavior of a Single Structured Ionomer Chain in Solution

    SciTech Connect

    Aryal, Dipak; Etampawala, Thusitha; Perahia, Dvora; Grest, Gary S.

    2014-08-14

    Structured polymers offer a means to tailor transport pathways within mechanically stable manifolds. Here we examine the building block of such a membrane, namely a single large pentablock co-polymer that consist of a center block of a randomly sulfonated polystyrene, designed for transport, tethered to poly-ethylene-r-propylene and end-capped by poly-t-butyl styrene, for mechanical stability,using molecular dynamics simulations. The polymer structure in a cyclohexane-heptane mixture, a technologically viable solvent, and in water, a poor solvent for all segments and a ubiquitous substance is extracted. In all solvents the pentablock collapsed into nearly spherical aggregates where the ionic block is segregated. In hydrophobic solvents, the ionic block resides in the center, surrounded by swollen intermix of flexible and end blocks. In water all blocks are collapsed with the sulfonated block residing on the surface. Our results demonstrate that solvents drive different local nano-segregation, providing a gateway to assemble membranes with controlled topology.

  13. Applications of constant denaturant capillary electrophoresis/high-fidelity polymerase chain reaction to human genetic analysis.

    PubMed

    Li-Sucholeiki, X C; Khrapko, K; André, P C; Marcelino, L A; Karger, B L; Thilly, W G

    1999-06-01

    Constant denaturant capillary electrophoresis (CDCE) permits high-resolution separation of single-base variations occurring in an approximately 100 bp isomelting DNA sequence based on their differential melting temperatures. By coupling CDCE for highly efficient enrichment of mutants with high-fidelity polymerase chain reaction (hifi PCR), we have developed an analytical approach to detecting point mutations at frequencies equal to or greater than 10(-6) in human genomic DNA. In this article, we present several applications of this approach in human genetic studies. We have measured the point mutational spectra of a 100 bp mitochondrial DNA sequence in human tissues and cultured cells. The observations have led to the conclusion that the primary causes of mutation in human mitochondrial DNA are spontaneous in origin. In the course of studying the mitochondrial somatic mutations, we have also identified several nuclear pseudogenes homologous to the analyzed mitochondrial DNA fragment. Recently, through developments of the means to isolate the desired target sequences from bulk genomic DNA and to increase the loading capacity of CDCE, we have extended the CDCE/hifi PCR approach to study a chemically induced mutational spectrum in a single-copy nuclear sequence. Future applications of the CDCE/hifi PCR approach to human genetic analysis include studies of somatic mitochondrial mutations with respect to aging, measurement of mutational spectra of nuclear genes in healthy human tissues and population screening for disease-associated single nucleotide polymorphisms (SNPs) in large pooled samples. PMID:10380762

  14. Chain stiffness regulates entropy-templated perfect mixing at single-nanoparticle level

    NASA Astrophysics Data System (ADS)

    Huang, Zihan; Lu, Ce; Dong, Bojun; Xu, Guoxi; Ji, Chengcheng; Zhao, Kongyin; Yan, Li-Tang

    2015-12-01

    The mixing on a single-particle level of chemically incompatible nanoparticles is an outstanding challenge for many applications. Burgeoning research activity suggests that entropic templating is a potential strategy to address this issue. Herein, using systematic computer simulations of model nanoparticle systems, we show that the entropy-templated interfacial organization of nanoparticles significantly depends on the stiffness of tethered chains. Unexpectedly, the optimal chain stiffness can be identified wherein a system exhibits the most perfect mixing for a certain compression ratio. Our simulations demonstrate that entropic templating regulated by chain stiffness precisely reflects various entropic repulsion states that arise from typical conformation regimes of semiflexible chains. The physical mechanism of the chain stiffness effect is revealed by analyzing the entropic repulsion states of tethered chains and quantitatively estimating the resulting entropy penalties, which provides direct evidence that supports the key role of entropic transition in the entropic templating strategy, as suggested in experiments. Moreover, the model nanoparticle systems are found to evolve into binary nanoparticle superlattices by remixing at extremely high stiffness. The findings facilitate the wide application of the entropic templating strategy in creating interfacially reactive nanomaterials with ordered structures on the single-nanoparticle level as well as mechanomutable responses.The mixing on a single-particle level of chemically incompatible nanoparticles is an outstanding challenge for many applications. Burgeoning research activity suggests that entropic templating is a potential strategy to address this issue. Herein, using systematic computer simulations of model nanoparticle systems, we show that the entropy-templated interfacial organization of nanoparticles significantly depends on the stiffness of tethered chains. Unexpectedly, the optimal chain stiffness can

  15. A recombinant, soluble, single-chain class I major histocompatibility complex molecule with biological activity.

    PubMed Central

    Mage, M G; Lee, L; Ribaudo, R K; Corr, M; Kozlowski, S; McHugh, L; Margulies, D H

    1992-01-01

    Heterodimeric class I major histocompatibility complex molecules, which consist of a 45-kDa heavy-chain and a 12-kDa beta 2-microglobulin (beta 2m) light chain, bind endogenously synthesized peptides for presentation to antigen-specific T cells. We have synthesized a gene encoding a single-chain, soluble class I molecule derived from mouse H-2Dd, in which the carboxyl terminus of beta 2m is linked via a peptide spacer to the amino terminus of the heavy chain. The chimeric protein is secreted efficiently from transfected L cells, is thermostable, and when loaded with an appropriate antigenic peptide, stimulates an H-2Dd-restricted antigen-specific T-cell hybridoma. Thus, functional binding of peptide does not require the complete dissociation of beta 2m, implying that a heavy chain/peptide complex is not an obligate intermediate in the assembly of the heavy-chain/beta 2m/peptide heterotrimer. Single-chain major histocompatibility complex molecules uniformly loaded with peptide have potential uses for structural studies, toxin or fluor conjugates, and vaccines. Images PMID:1438262

  16. Isolation of cardiac myosin light-chain isotypes by chromatofocusing. Comparison of human cardiac atrial light-chain 1 and foetal ventricular light-chain 1.

    PubMed

    Vincent, N D; Cummins, P

    1985-04-01

    Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow-range, shallow and stable pH gradients using ion-exchange resins and buffers with even buffering capacity over the required pH range. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of delta pH = 0.004-0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development. Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC-2 and VLC-2* light chains. The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in foetal ventricles and the atrial light chain 1 (ALC-1) in adult atria. Comparative peptide mapping studies and amino acid analyses were carried out on FLC-1 and ALC-1. No differences were detected between FLC-1 and ALC-1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC-1 and ALC-1 are homologous, and possibly identical, light chains. Comparison of human FLC-1/ALC-1 with VLC-1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman-Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207-212]. The latter three residues are known to be rich in the N-termini of cardiac and

  17. Subclustering of human immunoglobulin kappa light chain variable region genes

    SciTech Connect

    Kurth, J.H.; Mountain, J.L.; Cavalli-Sforza, L.L. )

    1993-04-01

    The human immunoglobulin kappa light chain (IgK) locus includes multiple variable region gene segments (V[sub k]) that can be divided into four subgroups. Oligonucleotide primers were designed to amplify specifically gene segments of the V[sub k]I, V[sub k]II, and V[sub k]III subgroups using the polymerase chain reaction (PCR). Product sequences were subcloned, sequenced, and compared. Phylogenetic analyses of sequences within each subgroup indicate that some subgroups can be subdivided further into [open quotes]sub-subgroups.[close quotes] The history of V[sub k] segment duplications apparently includes at least two separate periods, the first giving rise to the subgroups and the second generating further complexity within each subgroup. Duplications of large pieces of DNA (demonstrated by others through pulsed-field gel electrophoresis) also played a role. Rates of synonymous and nonsynonymous base changes between pairs of sequences suggest that natural selection has played a major role in the evolution of the V[sub k] variable gene segments, leading to sequence conservation in some regions and to increased diversity in others. 34 refs., 4 figs., 3 tabs.

  18. A single-chain bispecific Fv2 molecule produced in mammalian cells redirects lysis by activated CTL.

    PubMed

    Jost, C R; Titus, J A; Kurucz, I; Segal, D M

    1996-02-01

    Single-chain Fv (sFv) molecules consist of the two variable domains of an antibody (Ab) connected by a polypeptide spacer and contain the binding activities of their parental antibodies (Abs). In this paper we have attached the C-terminus of 2C11-sFv (anti-mouse CD3 epsilon-chain) to the N-terminus of OKT9-sFv (anti-human transferrin receptor [TfR]) through a 23 amino acid inter-sFv linker consisting primarily of CH1 region residues from 2C11, to form a single-chain bispecific Fv2 [bs(sFv)2] molecule. The bs(sFv)2 was expressed in COS-7 cells, and was secreted at the same rate as the two parental sFvs. The secreted protein had both anti-CD3 and anti-TfR binding activities. Essentially all of the secreted bs(sFv)2 molecules bound TfR and the binding affinity of the bs(sFv)2 was comparable to that of OKT9 sFv and Fab. Thus, the attachment of the inter-sFv linker to the N-terminus of OKT9-sFv did not impair its binding function. The bs(sFv)2 retained both binding specificities after long-term storage at 4 degrees C or overnight incubation at 37 degrees C. It redirected activated mouse CTL to specifically lyse human TfR+ target cells at low (ng/ml) concentrations and was much more active than a chemically cross-linked heteroconjugate prepared from the same parental mAbs. Because bs(sFv)2 molecules secreted by mammalian cells are homogeneous proteins containing two binding sites in a single polypeptide chain, they hold great promise as an easily obtainable, economic source of a bispecific molecule suitable for in vivo use. PMID:8649442

  19. Blood Clotting-Inspired Control of Single-Chain Molecules in Flows

    NASA Astrophysics Data System (ADS)

    Sing, Charles; Alexander-Katz, Alfredo

    2011-03-01

    Recent experimental evidence has demonstrated a clear link between mechanical stimuli and the activation of von Willebrand Factor (vWF), a protein that plays a critical role in the blood clotting cascade. This protein exhibits counter-intuitive conformational and adsorption responses that suggest novel ways of controlling the single-chain dynamics of polymer chains. Specifically, we are using simulation and theoretical approaches to elucidate the fundamental physics that govern globule-stretch transitions in collapsed polymers due to the effect of fluid flows. We begin to extend this general approach to the case of globule adsorption-desorption transitions in the presence of fluid flows, and demonstrate how kinetic considerations must be taken into account to describe the basic features of these transitions. We expect that these results will both allow the development of novel techniques for single-chain targeting and assembly and offer insight into the physiological behavior of vWF.

  20. An Entirely Cell-based System to Generate Single-Chain Antibodies Against Cell Surface Receptors

    PubMed Central

    Lipes, Barbara D.; Chen, Yu-Hsun; Ma, HongZheng; Staats, Herman F.; Kenan, Daniel J.; Gunn, Michael Dee

    2008-01-01

    Summary The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen (Ag). Traditionally, the generation of single chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high throughput screening of arrayed phage clones, and characterization of recombinant single chain variable regions (scFvs). This strategy was used to generate a panel of single chain Abs specific for the innate immunity receptor Toll-like receptor 2 (TLR2). Once generated, individual scFvs were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. PMID:18455737

  1. Structural and electronic properties of a single Si chain doped zigzag AlN nanoribbon

    NASA Astrophysics Data System (ADS)

    Zhang, Jian-Min; Zhang, Jing; Xu, Ke-Wei

    2015-04-01

    The first-principles projector-augmented wave (PAW) potentials within the density function theory (DFT) framework have been used to determine the geometry structures and electronic properties of the zigzag edge AlN nanoribbons (ZAlNNRs) doped with a single Si chain under generalized gradient approximation (GGA). The average Al-Si, Si-Si, Al-N, Si-N, Al-H and N-H bond lengths are 2.39, 2.16, 1.83, 1.74, 1.59 and 1.03 Å, respectively. Pure 7-ZAlNNR is an indirect semiconductor with a large band gap of 2.235 eV, while a semiconductor to metal transformation is taken place after a single Si chain substituting for a single Al-N chain at various positions. In pure 7-ZAlNNR, the HVB and LCB are mainly attributed to the edge N and Al atoms, respectively, while in a single Si chain substituting doped 7-ZAlNNR, the HVB and LCB are mainly attributed to the Si atoms. The Al-N, Al-H and Al-Si bonds are ionic bond, the Si-Si and Si-H bonds are covalent bond, the N-H and N-Si bonds are covalent bond modified ionic bond.

  2. Choice between Single and Multiple Reinforcers in Concurrent-Chains Schedules

    ERIC Educational Resources Information Center

    Mazur, James E.

    2006-01-01

    Pigeons responded on concurrent-chains schedules with equal variable-interval schedules as initial links. One terminal link delivered a single reinforcer after a fixed delay, and the other terminal link delivered either three or five reinforcers, each preceded by a fixed delay. Some conditions included a postreinforcer delay after the single…

  3. Single Multiplex Polymerase Chain Reaction To Detect Diverse Loci Associated with Diarrheagenic Escherichia coli

    PubMed Central

    López-Saucedo, Catalina; Cerna, Jorge F.; Villegas-Sepulveda, Nicolas; Thompson, Rocío; Velazquez, F. Raul; Torres, Javier; Tarr, Phillip I.

    2003-01-01

    We developed and tested a single multiplex polymerase chain reaction (PCR) that detects enterotoxigenic, enteropathogenic, enteroinvasive, and Shiga-toxin–producing Escherichia coli. This PCR is specific, sensitive, and rapid in detecting target isolates in stool and food. Because of its simplicity, economy, and efficiency, this protocol warrants further evaluation in large, prospective studies of polymicrobial substances. PMID:12533296

  4. Production and characterization of a single chain variable fragment (scFv) for the mycotoxin deoxynivalenol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deoxynivalenol (DON)is a mycotoxin produced by certain fungi that infest cereal grains worldwide. A hybridoma cell line producing a monoclonal antibody (Mab) recognizing DON was used as the starting point in the development of a recombinant single chain variable fragment (scFv) antibody. The scFv wa...

  5. Single-photon transport through an atomic chain coupled to a one-dimensional nanophotonic waveguide

    NASA Astrophysics Data System (ADS)

    Liao, Zeyang; Zeng, Xiaodong; Zhu, Shi-Yao; Zubairy, M. Suhail

    2015-08-01

    We study the dynamics of a single-photon pulse traveling through a linear atomic chain coupled to a one-dimensional (1D) single mode photonic waveguide. We derive a time-dependent dynamical theory for this collective many-body system which allows us to study the real time evolution of the photon transport and the atomic excitations. Our analytical result is consistent with previous numerical calculations when there is only one atom. For an atomic chain, the collective interaction between the atoms mediated by the waveguide mode can significantly change the dynamics of the system. The reflectivity of a photon can be tuned by changing the ratio of coupling strength and the photon linewidth or by changing the number of atoms in the chain. The reflectivity of a single-photon pulse with finite bandwidth can even approach 100 % . The spectrum of the reflected and transmitted photon can also be significantly different from the single-atom case. Many interesting physical phenomena can occur in this system such as the photonic band-gap effects, quantum entanglement generation, Fano-like interference, and superradiant effects. For engineering, this system may serve as a single-photon frequency filter, single-photon modulation, and may find important applications in quantum information.

  6. Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology.

    PubMed

    Akbari, V; Sadeghi, H Mir Mohammad; Jafarian-Dehkordi, A; Chou, C Perry; Abedi, D

    2015-01-01

    Human epidermal growth factor receptor (HER) family plays an important role in various types of cancers. As a result, antibodies against HER and the mechanism of antigen-antibody binding action are under active investigation. We previously constructed a single-chain variable fragment (ScFv) against HER2, i.e. anti-Her2 ScFv, for expressing in the Escherichia coli. In the present study, we report the optimization of anti-Her2 ScFv expression in an E. coli host of BL21 (DE3) pLysS using response surface methodology based on tuning of three cultivation variables, including isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration, temperature and post-induction time. A model for protein expression according to the Box-Behnken design predicted a maximal anti-Her2 ScFv expression at 37 °C, a post-induction time of 10.45 h and 0.75 mM IPTG. In addition, strategies based on inclusion body isolation and affinity chromatography were applied to purify anti-Her2 ScFv. The purity of the final product for inclusion bodies isolation and purification by Ni-NTA resin were 70 % and 95 %, respectively. The solubilization of the inclusion bodies was carried out using two denaturant agents, guanidine hydrochloride and urea. The present study showed that guanidine hydrochloride was more effective than urea in solubilizing the inclusion bodies. PMID:26430460

  7. A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors

    PubMed Central

    Kim, Julius W.; Young, Jacob S.; Solomaha, Elena; Kanojia, Deepak; Lesniak, Maciej S.; Balyasnikova, Irina V.

    2015-01-01

    The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10−9 M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies. PMID:26656559

  8. A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors.

    PubMed

    Kim, Julius W; Young, Jacob S; Solomaha, Elena; Kanojia, Deepak; Lesniak, Maciej S; Balyasnikova, Irina V

    2015-01-01

    The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10(-9) M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies. PMID:26656559

  9. Generation and characterization of a single-chain anti-EphA2 antibody

    PubMed Central

    Goldgur, Yehuda; Susi, Petri; Karelehto, Eveliina; Sanmark, Hanna; Lamminmäki, Urpo; Oricchio, Elisa; Wendel, Hans-Guido; Nikolov, Dimitar B; Himanen, Juha P

    2015-01-01

    Recombinant antibody phage library technology provides multiple advantages, including that human antibodies can be generated against proteins that are highly conserved between species. We used this technology to isolate and characterize an anti-EphA2 single-chain antibody. We show that the antibody binds the antigen with 1:1 stoichiometry and has high specificity for EphA2. The crystal structure of the complex reveals that the antibody targets the same receptor surface cavity as the ephrin ligand. Specifically, a lengthy CDR-H3 loop protrudes deep into the ligand-binding cavity, with several hydrophobic residues at its tip forming an anchor-like structure buried within the hydrophobic Eph pocket, in a way similar to the ephrin receptor-binding loop in the Eph/ephrin structures. Consequently, the antibody blocks ephrin binding to EphA2. Furthermore, it induces apoptosis and reduces cell proliferation in lymphoma cells lines. Since Ephs are important mediators of tumorigenesis, such antibodies could have applications both in research and therapy. PMID:25494541

  10. Multi-channeled single chain variable fragment (scFv) based microfluidic device for explosives detection.

    PubMed

    Charles, Paul T; Davis, Jasmine; Adams, André A; Anderson, George P; Liu, Jinny L; Deschamps, Jeffrey R; Kusterbeck, Anne W

    2015-11-01

    The development of explosives detection technologies has increased significantly over the years as environmental and national security agencies implement tighter pollution control measures and methods for improving homeland security. 2, 4, 6-Trinitrotoluene (TNT), known primarily as a component in munitions, has been targeted for both its toxicity and carcinogenic properties that if present at high concentrations can be a detriment to both humans, marine and plant ecosystems. Enabling end users with environmental detection and monitoring systems capable of providing real-time, qualitative and quantitative chemical analysis of these toxic compounds would be extremely beneficial. Reported herein is the development of a multi-channeled microfluidic device immobilized with single chain fragment variable (scFv) recombinant proteins specific for the explosive, TNT. Fluorescence displacement immunoassays performed under constant flow demonstrated trace level sensitivity and specificity for TNT. The utility of three multi-channeled devices immobilized with either (1) scFv recombinant protein, (2) biotinylated-scFv (bt-scFv) and (3) monoclonal anti-TNT (whole IgG molecule) were investigated and compared. Fluorescence dose response curves, crossreactivity measurements and limits of detection (LOD) for TNT were determined. Fluorescence displacement immunoassays for TNT in natural seawater demonstrated detection limits at sub-parts-per-billion levels (0.5 ppb) utilizing the microfluidic device with immobilized bt-scFv. PMID:26452845

  11. Characterization of human cardiac myosin heavy chain genes

    SciTech Connect

    Yamauchi-Takihara, K.; Sole, M.J.; Liew, J.; Ing, D.; Liew, C.C. )

    1989-05-01

    The authors have isolated and analyzed the structure of the genes coding for the {alpha} and {beta} forms of the human cardiac myosin heavy chain (MYHC). Detailed analysis of four overlapping MYHC genomic clones shows that the {alpha}-MYHC and {beta}-MYHC genes constitute a total length of 51 kilobases and are tandemly linked. The {beta}-MYHC-encoding gene, predominantly expressed in the normal human ventricle and also in slow-twitch skeletal muscle, is located 4.5 kilobases upstream of the {alpha}-MYHC-encoding gene, which is predominantly expressed in normal human atrium. The authors have determined the nucleotide sequences of the {beta} form of the MYHC gene, which is 100% homologous to the cardiac MYHC cDNA clone (pHMC3). It is unlikely that the divergence of a few nucleotide sequences from the cardiac {beta}-MYHC cDNA clone (pHMC3) reported in a MYHC cDNA clone (PSMHCZ) from skeletal muscle is due to a splicing mechanism. This finding suggests that the same {beta} form of the cardiac MYHC gene is expressed in both ventricular and slow-twitch skeletal muscle. The promoter regions of both {alpha}- and {beta}-MYHC genes, as well as the first four coding regions in the respective genes, have also been sequenced. The sequences in the 5{prime}-flanking region of the {alpha}- and {beta}-MYHC-encoding genes diverge extensively from one another, suggesting that expression of the {alpha}- and {beta}-MYHC genes is independently regulated.

  12. Complete amino acid sequence of the Mu heavy chain of a human IgM immunoglobulin.

    PubMed

    Putnam, F W; Florent, G; Paul, C; Shinoda, T; Shimizu, A

    1973-10-19

    The amino acid sequence of the micro, chain of a human IgM immunoglobulin, including the location of all disulfide bridges and oligosaccharides, has been determined. The homology of the constant regions of immunoglobulin micro, gamma, alpha, and epsilon heavy chains reveals evolutionary relationships and suggests that two genes code for each heavy chain. PMID:4742735

  13. Stepwise Unfolding of Single-Chain Nanoparticles by Chemically Triggered Gates.

    PubMed

    Fischer, Tobias S; Schulze-Sünninghausen, David; Luy, Burkhard; Altintas, Ozcan; Barner-Kowollik, Christopher

    2016-09-01

    The orthogonal, stepwise, and order-independent unfolding of single-chain nanoparticles (SCNPs) is introduced as a key step towards actively controlling the folding dynamics of SCNPs. The SCNPs are compacted by multiple hydrogen bonds and host-guest interactions. Well-defined diblock (AB) and tetrablock (ABCD) copolymers are equipped with orthogonal recognition motifs via modular ligation along the lateral chain. Initially, single-chain folding of the diblock copolymer was induced by the host-guest complexation of benzo-21-crown-7 (B21C7, host) and a secondary ammonium salt (AS, guest), representing an efficient avenue for single-chain collapse. Next, both orthogonal Hamilton wedge (HW) and cyanuric acid (CA) as well as B21C7-AS motifs were employed to generate SCNPs based on the ABCD polymer system. Subsequently, the stepwise dual-gated and order-independent unfolding of the SCNPs was investigated by the addition of external stimuli. The folding and unfolding were explored by 1D (1) H NMR spectroscopy, dynamic light scattering (DLS), and diffusion-ordered NMR spectroscopy (DOSY). PMID:27357944

  14. A Single-Chain Magnet Tape Based on Hexacyanomanganate(III).

    PubMed

    Zhang, Yuan-Zhu; Zhao, Han-Hua; Funck, Edward; Dunbar, Kim R

    2015-05-01

    The tape-like chain {[(tptz)Mn(II) (H2 O)Mn(III) (CN)6 ]2 Mn(II) (H2 O)2 }n ⋅4n MeOH⋅2n H2 O based on the anisotropic building block hexacyanomanganate(III) exhibits long-range magnetic ordering below 5.1 K as well as single-chain magnetic behavior at lower temperatures with an effective energy barrier of 40.5(7) K. PMID:25784624

  15. Conductance of single microRNAs chains related to the autism spectrum disorder

    NASA Astrophysics Data System (ADS)

    Oliveira, J. I. N.; Albuquerque, E. L.; Fulco, U. L.; Mauriz, P. W.; Sarmento, R. G.; Caetano, E. W. S.; Freire, V. N.

    2014-09-01

    The charge transport properties of single-stranded microRNAs (miRNAs) chains associated to autism disorder were investigated. The computations were performed within a tight-binding model, together with a transfer matrix technique, with ionization energies and hopping parameters obtained by quantum chemistry method. Current-voltage (I× V) curves of twelve miRNA chains related to the autism spectrum disorders were calculated and analysed. We have obtained both semiconductor and insulator behavior, and a relationship between the current intensity and the autism-related miRNA bases sequencies, suggesting that a kind of electronic biosensor can be developed to distinguish different profiles of autism disorders.

  16. Stable double helical iodine chains inside single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Yao, Zhen; Liu, Chun-Jian; Lv, Hang; Liu, Bing-Bing

    2016-08-01

    The helicity of stable double helical iodine chains inside single-walled carbon nanotubes (SWCNTs) is studied by calculating the systematic interaction energy. Our results present clear images of stable double helical structures inside SWCNTs. The optimum helical radius and helical angle increase and decrease with increasing diameter, respectively. The tube's diameter plays a leading role in the helicity of encapsulated structures, while the tube's chirality may induce different metastable structures. This study indicates that the observed double helical iodine chains in experiments are not necessarily the optimum structures, but may also be metastable structures.

  17. Comparison of chain sampling plans with single and double sampling plans

    NASA Technical Reports Server (NTRS)

    Stephens, K. S.; Dodge, H. F.

    1976-01-01

    The efficiency of chain sampling is examined through matching of operating characteristics (OC) curves of chain sampling plans (ChSP) with single and double sampling plans. In particular, the operating characteristics of some ChSP-0, 3 and 1, 3 as well as ChSP-0, 4 and 1, 4 are presented, where the number pairs represent the first and the second cumulative acceptance numbers. The fact that the ChSP procedure uses cumulative results from two or more samples and that the parameters can be varied to produce a wide variety of operating characteristics raises the question whether it may be possible for such plans to provide a given protection with less inspection than with single or double sampling plans. The operating ratio values reported illustrate the possibilities of matching single and double sampling plans with ChSP. It is shown that chain sampling plans provide improved efficiency over single and double sampling plans having substantially the same operating characteristics.

  18. Engineered single-chain variable fragment antibody for immunodiagnosis of groundnut bud necrosis virus infection.

    PubMed

    Maheshwari, Yogita; Vijayanandraj, S; Jain, R K; Mandal, Bikash

    2015-05-01

    Few studies have been done on engineered antibodies for diagnosis of tospovirus infections. The present study was undertaken to develop a single-chain variable fragment (scFv) for specific diagnosis of infection by groundnut bud necrosis virus (GBNV), the most prevalent serogroup IV tospovirus in India. Heavy chain (372 nucleotide [nt]) and light chain (363 nt) variable region clones obtained from a hybridoma were used to make an scFv construct that expressed a ~29-kDa protein in E. coli. The scFv specifically detected GBNV in field samples of cowpea, groundnut, mung bean, and tomato, and it did not recognize watermelon bud necrosis virus, a close relative of GBNV belonging to tospovirus serogroup IV. This study for the first time demonstrated the application of a functional scFv against a serogroup-IV tospovirus. PMID:25698103

  19. Critical single domain grain sizes in chains of interacting greigite particles: Implications for magnetosome crystals

    NASA Astrophysics Data System (ADS)

    Muxworthy, Adrian R.; Williams, Wyn; Roberts, Andrew P.; Winklhofer, Michael; Chang, Liao; Pósfai, Mihály

    2013-12-01

    Magnetotactic bacteria contain chains of magnetically interacting crystals (magnetosomes), which aid navigation (magnetotaxis). To improve the efficiency of magnetotaxis, magnetosome crystals (which can consist of magnetite or greigite) should be magnetically stable single domain (SD) particles. Larger particles subdivide into nonuniform multidomain (MD) magnetic structures that produce weaker magnetic signals, while small SD particles become magnetically unstable due to thermal fluctuations and exhibit superparamagnetic (SP) behavior. In this study, we determined the stable SD range as a function of grain elongation and interparticle separation for chains of identical greigite grains using fundamental parameters recently determined for greigite. Interactions significantly increase the stable SD range. For example, for cube-shaped greigite grains the upper stable SD threshold size is increased from 107 nm for isolated grains to 204 nm for touching grains arranged in chains. The larger critical SD grain size for greigite means that, compared to magnetite magnetosomes, greigite magnetosomes can produce larger magnetic signals without the need for intergrain interactions.

  20. A first-principles study of a single C-chain doped AlN nanoribbons

    NASA Astrophysics Data System (ADS)

    Rao, Qing-lei; Wang, Yong-xin; Chen, Zheng; Du, Xiu-juan; Sun, Ting-ting

    2015-08-01

    Under the generalized gradient approximation (GGA), the structural and electronic properties are studied for both zigzag (ZAlNNRs) and armchair (AAlNNRs) AlN nanoribbons terminated with H atoms at both edges by using the first-principles projector-augmented wave (PAW) potential within the density function theory (DFT) framework. The results show that the Al-N, Al-C and Al-H bonds are ionic bonds while the C-C and C-H bonds are typical covalent bonds, and the N-C and N-H bonds have a degree covalent character. The systems of both perfect 7-ZAlNNR and perfect 7-AAlNNR with a single C-chain doped are still nonmagnetic semiconductors, and the C-chain reduces the band gap. The C-chain can change the band gap of 7-ZAlNNR from direct to indirect independent of the position of the C-chain, which is important in the practical application as light emitting devices. For NZ-ZAlNNR-C(n) with NZ = 3, 5, 6, 10, the band gap decrease successively for C-chain position n from 2 to 3, 5, 6, 7 and 10, respectively. For NA-AAlNNR-C(n) of arbitrary width NA, except NA-AAlNNR-C(1) and NA-AAlNNR-C(n = NA) have a larger band gap, the band gap of the rest of the NA-AAlNNR-C(n) are about 2.0 eV. Furthermore, the maximum band gap gradually decrease with the increase of the width NA. The C-chain substituting Al-N chain process is endothermic for both 7-ZAlNNR and 7-AAlNNR.

  1. Elastic coefficient of a single polymer chain by using Brownian dynamics analysis

    NASA Astrophysics Data System (ADS)

    Horinaka, J.; Maniwa, T.; Oharada, K.; Takigawa, T.

    2007-08-01

    The elastic coefficient of a single polystyrene chain has been experimentally evaluated by using Brownian dynamics analysis. The Brownian motion of the chain is probed using a particle trapped by optical tweezers with a negligibly small spring constant. The displacement of the particle due to Brownian motion is measured by an interferometer assembled using the same laser beam as the optical tweezers. Two methods are employed for Brownian dynamics analysis: (1) the analysis of the time course of the displacement of the particle and (2) the fitting of the power spectrum of Brownian motion with a Lorentzian. The elastic constant of a polystyrene chain in dichloromethane at 21 °C is estimated to be 6.4×10-6 and 1.1×10-5 N/m when methods (1) and (2) are employed, respectively. The elastic constant obtained by approximating the polystyrene chain to a freely jointed chain is in agreement with the experimentally evaluated elastic constant.

  2. Adsorption of a single polymer chain on a surface: effects of the potential range.

    PubMed

    Klushin, Leonid I; Polotsky, Alexey A; Hsu, Hsiao-Ping; Markelov, Denis A; Binder, Kurt; Skvortsov, Alexander M

    2013-02-01

    We investigate the effects of the range of adsorption potential on the equilibrium behavior of a single polymer chain end-attached to a solid surface. The exact analytical theory for ideal lattice chains interacting with a planar surface via a box potential of depth U and width W is presented and compared to continuum model results and to Monte Carlo (MC) simulations using the pruned-enriched Rosenbluth method for self-avoiding chains on a simple cubic lattice. We show that the critical value U(c) corresponding to the adsorption transition scales as W(-1/ν), where the exponent ν=1/2 for ideal chains and ν≈3/5 for self-avoiding walks. Lattice corrections for finite W are incorporated in the analytical prediction of the ideal chain theory U(c)≈(π(2)/24)(W+1/2)(-2) and in the best-fit equation for the MC simulation data U(c)=0.585(W+1/2)(-5/3). Tail, loop, and train distributions at the critical point are evaluated by MC simulations for 1≤W≤10 and compared to analytical results for ideal chains and with scaling theory predictions. The behavior of a self-avoiding chain is remarkably close to that of an ideal chain in several aspects. We demonstrate that the bound fraction θ and the related properties of finite ideal and self-avoiding chains can be presented in a universal reduced form: θ(N,U,W)=θ(NU(c),U/U(c)). By utilizing precise estimations of the critical points we investigate the chain length dependence of the ratio of the normal and lateral components of the gyration radius. Contrary to common expectations this ratio attains a limiting universal value /=0.320±0.003 only at N~5000. Finite-N corrections for this ratio turn out to be of the opposite sign for W=1 and for W≥2. We also study the N dependence of the apparent crossover exponent φ(eff)(N). Strong corrections to scaling of order N(-0.5) are observed, and the extrapolated value φ=0.483±0.003 is found for all values of W. The strong correction

  3. Affinity measurement of single chain antibodies: a mathematical method facilitated by statistical software SigmaPlot.

    PubMed

    Safdari, Yaghoub; Farajnia, Safar; Asgharzadeh, Mohammad; Khalili, Masoumeh; Jaliani, Hossein Zarei

    2014-02-01

    Because they are monovalent for antigen, single chain antibodies display a different antibody-antigen interaction pattern from that of full-length antibodies. Using the law of mass action and considering the antibody-antigen binding pattern at OD-100% and OD-50% points, we introduced a formula for estimating single chain antibody affinity. Sigmoid curves of optical density values versus antibody concentrations were drawn and used to determine antibody concentrations at OD-50% points using statistical software SigmaPlot. The OD-50% points were then used to calculate the affinity via the mathematical formula. A software-adapted format of the equation is also presented for further facilitation of the calculation process. The accuracy of this method for affinity calculation was proved by surface plasma resonance. This method offers a precise evaluation of antibody affinity without requiring special material or apparatus, making it possible to be performed in any biological laboratory with minimum facilities. PMID:24555931

  4. Methods of preparing and using single chain anti-tumor antibodies

    SciTech Connect

    Cheung, Nai-Kong; Guo, Hong-Fen

    2010-02-23

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  5. Injection chaining of diode-pumped single-frequency ring lasers for free-space communication

    NASA Technical Reports Server (NTRS)

    Cheng, E. A. P.; Kane, T. J.; Wallace, R. W.; Cornwell, D. M., Jr.

    1991-01-01

    A high-power three-stage laser suitable for use in a space communication system has been built. This laser uses three diode-pumped Nd:YAG oscillators coherently combined using the technique of injection chaining. All three oscillators are in one compact and permanently aligned package, and are actively frequency locked to provide CW single frequency output. The three stages provide the redundancy desirable for space communications.

  6. Structural organization of the human cardiac [alpha]-myosin heavy chain gene (MYH6)

    SciTech Connect

    Epp, T.A.; Dixon, I.M.C.; Wang, H.Y.; Sole, M.J.; Liew, C.C. )

    1993-12-01

    The human myocardium expresses two cardiac myosin heavy chain (MyHC) isoforms, [alpha] and [beta], that exist in tandem array on chromosome 14q12. The authors have previously sequenced the entire human cardiac [beta]-MyHC gene and now report the complete nucleotide sequence of the human cardiac [alpha]-MyHC, encompassing 26,159 bp as well as the entire 4484-bp 5'-flanking intergenic region. The gene (MYH6) consists of 39 exons, 37 of which contain coding information. The 5'-untranslated region is split into 3 exons, with the third exon containing the AUG translocation initiation codon. With the exception of the 13th intron of the human cardiac [beta]-MyHC, which is not present within the [alpha]-isogene, all exon/intron boundaries are conserved. Conspicuous sequence motifs contained within the [alpha]-MyHC gene include four Alu repeats, a single (GT)[sub n] element, and a homopurine-homopyrimidine tract containing 23 GAA repeating units followed by 10 GAG repeating units. Comparison of the encoded amino acid sequence with a previously reported human [alpha]-MyHC cDNA sequence reveals several potential polymorphisms. 29 refs., 1 fig., 1 tab.

  7. From single Debye-Hückel chains to polyelectrolyte solutions: Simulation results

    NASA Astrophysics Data System (ADS)

    Kremer, Kurt

    1996-03-01

    This lecture will present results from simulations of single weakly charged flexible chains, where the electrostatic part of the interaction is modeled by a Debye-Hückel potential,( with U. Micka, IFF, Forschungszentrum Jülich, 52425 Jülich, Germany) as well as simulations of polyelectrolyte solutions, where the counterions are explicitly taken into account( with M. J. Stevens, Sandia Nat. Lab., Albuquerque, NM 87185-1111) ( M. J. Stevens, K. Kremer, JCP 103), 1669 (1995). The first set of the simulations is meant to clear a recent contoversy on the dependency of the persistence length LP on the screening length Γ. While the analytic theories give Lp ~ Γ^x with either x=1 or x=2, the simulations find for all experimentally accessible chain lengths a varying exponent, which is significantly smaller than 1. This causes serious doubts on the applicability of this model for weakly charged polyelectrolytes in general. The second part deals with strongly charged flexible polyelectrolytes in salt free solution. These simulations are performed for multichain systems. The full Coulomb interactions of the monomers and counterions are treated explicitly. Experimental measurements of the osmotic pressure and the structure factor are reproduced and extended. The simulations reveal a new picture of the chain structure based on calculations of the structure factor, persistence length, end-to-end distance, etc. Even at very low density, the chains show significant bending. Furthermore, the chains contract significantly before they start to overlap. We also show that counterion condensation dramatically alters the chain structure, even for a good solvent backbone.

  8. Choice between Single and Multiple Reinforcers in Concurrent-Chains Schedules

    PubMed Central

    Mazur, James E

    2006-01-01

    Pigeons responded on concurrent-chains schedules with equal variable-interval schedules as initial links. One terminal link delivered a single reinforcer after a fixed delay, and the other terminal link delivered either three or five reinforcers, each preceded by a fixed delay. Some conditions included a postreinforcer delay after the single reinforcer to equate the total durations of the two terminal links, but other conditions did not include such a postreinforcer delay. With short initial links, preference for the single-reinforcer alternative decreased when a postreinforcer delay was present, but with long initial links, the postreinforcer delays had no significant effect on preference. In conditions with a postreinforcer delay, preference for the single-reinforcer alternative frequently switched from above 50% to below 50% as the initial links were lengthened. This pattern of results was consistent with delay-reduction theory (Squires & Fantino, 1971), but not with the contextual-choice model (Grace, 1994) or the hyperbolic value-added model (Mazur, 2001) as they have usually been applied. However, the hyperbolic value-added model could account for the results if its calculations were expanded to include reinforcers delivered in later terminal links. The implications of these findings for models of concurrent-chains performance are discussed. PMID:17002228

  9. A light-induced spin crossover actuated single-chain magnet

    NASA Astrophysics Data System (ADS)

    Liu, Tao; Zheng, Hui; Kang, Soonchul; Shiota, Yoshihito; Hayami, Shinya; Mito, Masaki; Sato, Osamu; Yoshizawa, Kazunari; Kanegawa, Shinji; Duan, Chunying

    2013-11-01

    Both spin-crossover complexes and molecular nanomagnets display bistable magnetic states, potentially behaving as elementary binary units for information storage. It is a challenge to introduce spin-crossover units into molecular nanomagnets to switch the bistable state of the nanomagnets through external stimuli-tuned spin crossover. Here we report an iron(II) spin-crossover unit and paramagnetic iron(III) ions that are incorporated into a well-isolated double-zigzag chain. The chain exhibits thermally induced reversible spin-crossover and light-induced excited spin-state trapping at the iron(II) sites. Single-chain magnet behaviour is actuated accompanying the synergy between light-induced excited spin-state trapping at the iron(II) sites and ferromagnetic interactions between the photoinduced high-spin iron(II) and low-spin iron(III) ions in the chain. The result provides a strategy to switch the bistable state of molecular nanomagnets using external stimuli such as light and heat, with the potential to erase and write information at a molecular level.

  10. Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

    PubMed

    Hojo, Keiko; Hossain, Mohammed Akhter; Tailhades, Julien; Shabanpoor, Fazel; Wong, Lilian L L; Ong-Pålsson, Emma E K; Kastman, Hanna E; Ma, Sherie; Gundlach, Andrew L; Rosengren, K Johan; Wade, John D; Bathgate, Ross A D

    2016-08-25

    Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3. PMID:27464307

  11. Conformation and translational diffusion of a xanthan polyelectrolyte chain: Brownian dynamics simulation and single molecule tracking

    NASA Astrophysics Data System (ADS)

    Chun, Myung-Suk; Kim, Chongyoup; Lee, Duck E.

    2009-05-01

    In our recent Brownian dynamics (BD) simulation study, the structure and dynamics of anionic polyelectrolyte xanthan in bulk solution as well as confined spaces of slitlike channel were examined by applying a coarse-grained model with nonlinear bead-spring discretization of a whole chain [J. Jeon and M.-S. Chun, J. Chem. Phys. 126, 154904 (2007)]. This model goes beyond other simulations as they did not consider both long-range electrostatic and hydrodynamic interactions between pairs of beads. Simulation parameters are obtained from the viscometric method of rheology data on the native and sonicated xanthan polysaccharides, which have a contour length less than 1μm . The size of the semiflexible polyelectrolyte can be well described by the wormlike chain model once the electrostatic effects are taken into account by the persistence length measured at a long length scale. For experimental verifications, single molecule visualization was performed on fluorescein-labeled xanthan using an inverted fluorescence microscope, and the motion of an individual molecule was quantified. Experimental results on the conformational changes in xanthan chain in the electrolyte solution have a reasonable trend to agree with the prediction by BD simulations. In the translational diffusion induced by the Debye screening effect, the simulation prediction reveals slightly higher values compared to those of our measurements, although it agrees with the literature data. Considering the experimental restrictions, our BD simulations are verified to model the single polyelectrolyte well.

  12. A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells.

    PubMed

    Epel, Malka; Ellenhorn, Joshua D; Diamond, Don J; Reiter, Yoram

    2002-11-01

    Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy. PMID:12384808

  13. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    PubMed

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%. PMID:26792375

  14. The establishment of a human myeloma cell line elaborating lambda-light chain protein.

    PubMed

    Niho, Y; Shibuya, T; Yamasaki, K; Kimura, N

    1984-05-01

    A human myeloma cell line (KMM-56) producing lambda-light chain protein was established in vitro by cultivation of the cells in the pleural effusion obtained from a patient with IgD-lambda-myeloma. The cells proliferate in suspension and do not aggregate or attach to the culture dish. Surface marker analysis revealed that the cells were negative for E-rosette, and surface immunoglobulin. Immunoelectrophoresis, immunodiffusion, and immunofluorescence with various antibodies demonstrated no heavy chains, while lambda-light chains were detected in the cytoplasm of the cells. Using the immunodiffusion technique, only lambda-light chains were detected in the frozen and thawed cell extract, the concentrated supernatant of the cell culture, and the urine of the patient. Electron microscopic examination revealed the plasmablastoid appearance of the cells. This cell line may be useful for future studies of human immunoglobulin genes and for the material of human-human hybridoma, which could produce monoclonal human immunoglobulin. PMID:6429256

  15. Coil-globule transition of a single semiflexible chain in slitlike confinement

    PubMed Central

    Dai, Liang; Renner, C. Benjamin; Yan, Jie; Doyle, Patrick S.

    2015-01-01

    Single polymer chains undergo a phase transition from coiled conformations to globular conformations as the effective attraction between monomers becomes strong enough. In this work, we investigated the coil-globule transition of a semiflexible chain confined between two parallel plates, i.e. a slit, using the lattice model and Pruned-enriched Rosenbluth method (PERM) algorithm. We find that as the slit height decreases, the critical attraction for the coil-globule transition changes non-monotonically due to the competition of the confinement free energies of the coiled and globular states. In wide (narrow) slits, the coiled state experiences more (less) confinement free energy, and hence the transition becomes easier (more difficult). In addition, we find that the transition becomes less sharp with the decreasing slit height. Here, the sharpness refers to the sensitivity of thermodynamic quantities when varying the attraction around the critical value. The relevant experiments can be performed for DNA condensation in microfluidic devices. PMID:26679086

  16. Coil-globule transition of a single semiflexible chain in slitlike confinement

    NASA Astrophysics Data System (ADS)

    Dai, Liang; Renner, C. Benjamin; Yan, Jie; Doyle, Patrick S.

    2015-12-01

    Single polymer chains undergo a phase transition from coiled conformations to globular conformations as the effective attraction between monomers becomes strong enough. In this work, we investigated the coil-globule transition of a semiflexible chain confined between two parallel plates, i.e. a slit, using the lattice model and Pruned-enriched Rosenbluth method (PERM) algorithm. We find that as the slit height decreases, the critical attraction for the coil-globule transition changes non-monotonically due to the competition of the confinement free energies of the coiled and globular states. In wide (narrow) slits, the coiled state experiences more (less) confinement free energy, and hence the transition becomes easier (more difficult). In addition, we find that the transition becomes less sharp with the decreasing slit height. Here, the sharpness refers to the sensitivity of thermodynamic quantities when varying the attraction around the critical value. The relevant experiments can be performed for DNA condensation in microfluidic devices.

  17. Light Chain Escape in 3 Cases: Evidence of Intraclonal Heterogeneity in Multiple Myeloma from a Single Institution in Poland.

    PubMed

    Kraj, Maria; Kruk, Barbara; Endean, Kelly; Warzocha, Krzysztof; Budziszewska, Katarzyna; Dąbrowska, Monika

    2015-01-01

    We report three cases of light chain escape (LCE) at a single institution in Poland, including an interesting case of biclonal monoclonal gammopathy of undetermined significance (MGUS) that satisfied the criteria for progression to light chain multiple myeloma (LCMM) with a rapid rise in serum free light chain (FLC) levels, following steroidal treatment for simultaneous temporal artery inflammation and polymyalgia rheumatica (PMR). In the three cases discussed, progression of the disease by light chain escape was associated with rapid and severe renal impairment, highlighting the necessity for prompt detection of such free light chain-only producing clones in order to prevent the possible development of irreversible end-organ damage. Interestingly, monitoring of these three patients by serum free light chain assay (sFLC) and retrospective heavy/light chain analysis (HLC) detected this clonal evolution prior to clinical relapse and suggests that these assays represent important additional tools for more accurate monitoring of multiple myeloma patients. PMID:26881153

  18. Light Chain Escape in 3 Cases: Evidence of Intraclonal Heterogeneity in Multiple Myeloma from a Single Institution in Poland

    PubMed Central

    Kraj, Maria; Kruk, Barbara; Endean, Kelly; Warzocha, Krzysztof; Budziszewska, Katarzyna; Dąbrowska, Monika

    2015-01-01

    We report three cases of light chain escape (LCE) at a single institution in Poland, including an interesting case of biclonal monoclonal gammopathy of undetermined significance (MGUS) that satisfied the criteria for progression to light chain multiple myeloma (LCMM) with a rapid rise in serum free light chain (FLC) levels, following steroidal treatment for simultaneous temporal artery inflammation and polymyalgia rheumatica (PMR). In the three cases discussed, progression of the disease by light chain escape was associated with rapid and severe renal impairment, highlighting the necessity for prompt detection of such free light chain-only producing clones in order to prevent the possible development of irreversible end-organ damage. Interestingly, monitoring of these three patients by serum free light chain assay (sFLC) and retrospective heavy/light chain analysis (HLC) detected this clonal evolution prior to clinical relapse and suggests that these assays represent important additional tools for more accurate monitoring of multiple myeloma patients. PMID:26881153

  19. Using Digital Polymerase Chain Reaction to Detect Single-Nucleotide Substitutions Induced by Genome Editing.

    PubMed

    Miyaoka, Yuichiro; Chan, Amanda H; Conklin, Bruce R

    2016-01-01

    This protocol is designed to detect single-nucleotide substitutions generated by genome editing in a highly sensitive and quantitative manner. It uses a combination of allele-specific hydrolysis probes and a new digital polymerase chain reaction (dPCR) technology called droplet digital PCR (ddPCR). ddPCR partitions a reaction into more than 10,000 nanoliter-scale water-in-oil droplets. As a result, each droplet contains only a few copies of the genome so that ddPCR is able to detect rare genome-editing events without missing them. PMID:27250210

  20. Polyelectrolyte properties of single stranded DNA measured using SAXS and single molecule FRET: beyond the wormlike chain model

    PubMed Central

    Meisburger, Steve P.; Sutton, Julie L.; Chen, Huimin; Pabit, Suzette A.; Kirmizialtin, Serdal; Elber, Ron; Pollack, Lois

    2013-01-01

    Nucleic acids are highly charged polyelectrolytes that interact strongly with salt ions. Rigid, base-paired regions are successfully described with worm like chain models, but non base-paired single stranded regions have fundamentally different polymer properties because of their greater flexibility. Recently, attention has turned to single stranded nucleic acids due to the growing recognition of their biological importance, as well as the availability of sophisticated experimental techniques sensitive to the conformation of individual molecules. We investigate polyelectrolyte properties of poly(dT), an important and widely studied model system for flexible single stranded nucleic acids, in physiologically important mixed mono- and di-valent salt. We report measurements of the form factor and interparticle interactions using SAXS, end to end distances using smFRET, and number of excess ions using ASAXS. We present a coarse-grained model that accounts for flexibility, excluded volume, and electrostatic interactions in these systems. Predictions of the model are validated against experiment. We also discuss the state of all-atom, explicit solvent Molecular Dynamics simulations of poly(dT), the next step in understanding the complexities of ion interactions with these highly charged and flexible polymers. PMID:23606337

  1. New Factorization Techniques and Parallel (log N) Algorithms for Forward Dynamics Solution of Single Closed-Chain Robot Manipulators

    NASA Technical Reports Server (NTRS)

    Fijany, Amir

    1993-01-01

    In this paper parallel 0(log N) algorithms for dynamic simulation of single closed-chain rigid multibody system as specialized to the case of a robot manipulatoar in contact with the environment are developed.

  2. Human genetic mapping studies using single sperm typing

    SciTech Connect

    Hubert, R.S.

    1993-01-01

    Sperm typing is a powerful technique that uses the polymerase chain reaction (PCR) to analyze DNA sequences within single sperm cells in order to construct genetic maps. This methodology was used to estimate the recombination fraction between D3S2 and D3S2 which was found to be 0.28 (95% CI = 0.20-0.36). Pedigree analysis was unable to determine genetic distance between these two markers due to their low informativeness. We also showed that dinucleotide and tetranucleotide repeat polymorphisms can be analyzed in single cells without using radioactivity or denaturing gels. This provides a rich new source of DANA polymorphisms for genetic mapping by sperm typing. In addition, an approach that uses the sperm typing methodology is described that can define the physical boundaries of meiotic recombination hotspots. The hotspot at 4p16.3 near the Huntington disease gene was localized to an interval between D4S10 and D4S126. These studies demonstrated the usefulness of sperm typing as a tool for the study of human genetic.

  3. Preparative isolation by high performance liquid chromatography of human insulin B chain produced in escherichia coli

    SciTech Connect

    Cruz, N.; Antonio, S.; De Anda, R.; Gosset, G.; Bolivar, F. )

    1990-01-01

    This paper reports on a simple method developed for the analytical and preparative purification of human insulin B chain from recombinant origin. Three solvent systems: acetonitrile, isopropanol and methanol, were studied to determine their capacity to resolve the insulin B chain from a mixture of cyanogen bromide generated bacterial peptides. Using a {mu}Bondapak C18 column, it was possible to resolve the insulin B chain in all three systems. On a preparative scale, using a PrePak 500 C18 column with the isopropanol system, it was possible to purify insulin B chain and to obtain a 95% protein recovery.

  4. Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

    NASA Technical Reports Server (NTRS)

    di Maso, N. A.; Caiozzo, V. J.; Baldwin, K. M.

    2000-01-01

    The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

  5. Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

    PubMed Central

    Song, Yunke; Zhang, Yi; Wang, Tza-Huei

    2014-01-01

    Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and tedious assay processes. In this report, we propose an assay technology which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single molecule coincidence detection and superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. PMID:23239594

  6. Effect of coil-globule transition on the single-chain crystallization.

    PubMed

    Wang, Mao-Xiang

    2013-05-30

    The folding process of a single chain including coil-globule transition and crystallization has been investigated through dynamic Monte Carlo simulations. The results based upon ensemble averaging illustrated three distinct states: coil, molten globule, and globule states. Furthermore, the crystallization process from these collapsed states demonstrated various characteristics and it also verified the thermodynamic partitions. The isothermal crystallization in the three states showed the folding rates, and the final crystallite morphologies strongly depended on the collapsed states. Especially, the onset temperature of crystallization in the intermediate molten globule state demonstrated the strongest sensitivity to the solvent qualities in the three different states. Moreover, the crystallization in this intermediate state illustrated a two-step folding mechanism with the prior dense core serving as a precursor to induce the subsequent crystallization. Our observations would help in understanding the thermodynamics and kinetics of phase transition of a single macromolecule. Possible relations to the protein folding were also discussed. PMID:23646890

  7. Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates.

    PubMed

    Neumann-Schaal, Meina; Messerschmidt, Katrin; Grenz, Nicole; Heilmann, Katja

    2013-03-01

    Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps. PMID:23453960

  8. Characterization of the 5'-flanking region of the gene for the alpha chain of human fibrinogen.

    PubMed

    Hu, C H; Harris, J E; Davie, E W; Chung, D W

    1995-11-24

    The 5'-flanking region of the gene coding for the alpha chain of human fibrinogen was isolated, sequenced, and characterized. The principal site of transcription initiation was determined by primer extension analysis and the RNase protection assay and shown to be at an adenine residue located 55 nucleotides upstream from the initiator methionine codon, or 13,399 nucleotides down-stream from the polyadenylation site of the gene coding for the gamma chain. Transient expression of constructs containing sequentially deleted 5'-flanking sequences of the alpha chain gene fused to the chloramphenicol acetyltransferase reporter gene showed that the promoter was liver-specific and inducible by interleukin 6 (IL-6). The shortest DNA fragment with significant promoter activity and full response to IL-6 stimulation encompassed the region from -217 to +1 base pairs (bp). Although six potential IL-6 responsive sequences homologous to the type II IL-6 responsive element were present, a single sequence of CTGGGA localized from -122 to -127 bp was shown to be a functional element in IL-6 induction. A hepatocyte nuclear factor 1 (HNF-1) binding site, present from -47 to -59 bp, in combination with other upstream elements, was essential for liver-specific expression of the gene. A functional CCAAT/enhancer binding protein site (C/EBP, -134 to -142 bp) was also identified within 217 bp from the transcription initiation site. An additional positive element (-1393 to -1133 bp) and a negative element (-1133 to -749 bp) were also found in the upstream region of the alpha-fibrinogen gene. PMID:7499335

  9. Optical Nanofluidic Piston: Assay for Dynamic Force-Compression of Single Confined Polymer Chains

    NASA Astrophysics Data System (ADS)

    Khorshid, Ahmed; Zimny, Philip; Macos, Patrick; Massarelli, Geremia; Tétreault-La Roche, David; Reisner, Walter

    2014-03-01

    While single-molecule approaches now have a long-history in polymer physics, past methodology has a key limitation : it is not currently possible to apply well-defined forces to a precise number of chains in a well-defined volume. To this end,we have developed a nanofluidic assay for the study of DNA compression in vitro, the optical nanofluidic piston. The optical nanofluidic piston is a nanofluidic analog of a macroscopic piston-cylinder apparatus based on a nanosphere (``the piston'') optically trapped inside a 200-400nm nanochannel with embedded barrier (the ``cylinder''). The nanofluidic piston enables quantification of force required to compress single or multiple chains within a defined volume. We present combined fluorescence and force-measurements for the compression of T4 DNA under a variety of compression rates. Surprisingly, we find that compression occurs on a force-scale roughly 100x higher than that predicted by equilibrium theories, suggesting that the DNA is present in highly entangled states during the compression. Moreover, we observe that compression at high rates induces a ``shock-wave'' of high-polymer concentration near the bead, suggesting that our setup can quantitatively access novel non-equilibrium polymer phenomena.

  10. Latex agglutination test based on single-chain Fv recombinant antibody fragment.

    PubMed

    Golchin, M; Khalili-Yazdi, A; Karamouzian, M; Abareghi, A

    2012-01-01

    Recombinant antibodies have been proposed as invaluable tools for various therapeutic and diagnostic purposes. Here, we describe the development of a novel latex agglutination test (LAT) using single-chain Fv recombinant antibody fragment for the detection of K99(+) enterotoxigenic Escherichia coli strains. For the production of a single-chain Fv antibody fragment (scFv) against the major colonization factor (FanC) of K99 antigen, the scFv gene was integrated into a bacterial expression vector under the control of T7 promoter. After high-level expression of soluble scFv (approximately 50 mg/l) in flask cultivation of E. coli DE3 and purification, scFv was immobilized on different latex particles, and then, these sensitized beads were used in LAT. Results obtained with our latex reagents revealed that the recombinant antibody-coated particles were able to give a good agglutination signal with purified antigen, intact cells displaying this protein and clinical specimens. The strength of agglutination of scFv-coated beads for antigen was comparable to that of polyclonal anti-K99-coated particles. However, the assay proved to be simple and rapid, similar to conventional LATs, and owing to more convenient and economical production of recombinant antibodies, they can be considered as a useful reagent for replacing monoclonal antibodies in LATs. PMID:21916915

  11. Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.

    PubMed

    Sato, Yusui; Tanaka, Yusuke; Inaba, Satomi; Sekiguchi, Hiroshi; Maruno, Takahiro; Sasaki, Yuji C; Fukada, Harumi; Kobayashi, Yuji; Azuma, Takachika; Oda, Masayuki

    2016-10-01

    Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events. PMID:27222286

  12. An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody.

    PubMed

    Cogotzi, Laura; Giampetruzzi, Annalisa; Nölke, Greta; Orecchia, Martin; Elicio, Vito; Castellano, Maria Antonietta; Martelli, Giovanni P; Fischer, Rainer; Schillberg, Stefan; Saldarelli, Pasquale

    2009-01-01

    Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevine. A previously described single-chain fragment variable (scFv) antibody (scFvLR3), directed against the coat protein (CP) of GLRaV-3, was expressed in Escherichia coli and used to develop a diagnostic ELISA kit. The antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent C(L)-LR3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. The sensitivity of the reagent against recombinant GLRaV-3 CP was 0.1 ng. The sensitivity, specificity and durability of the reagent was similar to a commercial kit. The C(L)-LR3 showed a weak cross-reaction in immune electron microscopy assays to GLRaV-1 and -7, but not with the phylogenetically more distant GLRaV-2. A fully recombinant kit was developed with the inclusion of a recombinant GLRaV-3 CP expressed in bacteria, thus avoiding problems associated with virus propagation and purification. This system represents a rapid, simple, sensitive and standardized diagnostic protocol for GLRaV-3 detection. PMID:19082687

  13. A conceptual framework for economic optimization of single hazard surveillance in livestock production chains.

    PubMed

    Guo, Xuezhen; Claassen, G D H; Oude Lansink, A G J M; Saatkamp, H W

    2014-06-01

    Economic analysis of hazard surveillance in livestock production chains is essential for surveillance organizations (such as food safety authorities) when making scientifically based decisions on optimization of resource allocation. To enable this, quantitative decision support tools are required at two levels of analysis: (1) single-hazard surveillance system and (2) surveillance portfolio. This paper addresses the first level by presenting a conceptual approach for the economic analysis of single-hazard surveillance systems. The concept includes objective and subjective aspects of single-hazard surveillance system analysis: (1) a simulation part to derive an efficient set of surveillance setups based on the technical surveillance performance parameters (TSPPs) and the corresponding surveillance costs, i.e., objective analysis, and (2) a multi-criteria decision making model to evaluate the impacts of the hazard surveillance, i.e., subjective analysis. The conceptual approach was checked for (1) conceptual validity and (2) data validity. Issues regarding the practical use of the approach, particularly the data requirement, were discussed. We concluded that the conceptual approach is scientifically credible for economic analysis of single-hazard surveillance systems and that the practicability of the approach depends on data availability. PMID:24630402

  14. Mitochondrial DNA mutations in single human blood cells.

    PubMed

    Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

    2015-09-01

    Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. PMID:26149767

  15. Myosin Heavy Chain Composition of the Human Genioglossus Muscle

    ERIC Educational Resources Information Center

    Daugherty, Megan; Luo, Qingwei; Sokoloff, Alan J.

    2012-01-01

    Background: The human tongue muscle genioglossus (GG) is active in speech, swallowing, respiration, and oral transport, behaviors encompassing a wide range of tongue shapes and movement speeds. Studies demonstrate substantial diversity in patterns of human GG motor unit activation, but whether this is accompanied by complex expression of muscle…

  16. A Comparison of In-Context and Traditional Instructional Approaches: Total Task, Single Trial versus Backward Chaining, Multiple Trials.

    ERIC Educational Resources Information Center

    Kayser, Joan E.; And Others

    1986-01-01

    Total task (single trial instruction) and backward chaining (multiple trials instruction) were used to teach eight children with severe handicaps. Total task, single trial instruction resulted in superior acquisition of independent steps in the training setting for three students. In all cases, instructional time was substantially less for total…

  17. Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue.

    PubMed Central

    Jancso, A; Szent-Györgyi, A G

    1994-01-01

    Specific Ca2+ binding and Ca2+ activation of ATPase activity in scallop myosin require a regulatory light chain (RLC) from regulated (molluscan or vertebrate smooth) myosin; hybrids containing vertebrate skeletal RLCs do not bind Ca2+ and their ATPase activity is inhibited. Chimeras between scallop and chicken skeletal RLCs restore Ca2+ sensitivity to RLC-free myosin provided that residues 81-117 are derived from scallop. Six mutants (R90M, A94K, D98P, N105K, M116Q, and G117C) were generated by replacing amino acids of the scallop RLC with the corresponding skeletal RLC residues in positions conserved in either regulated or nonregulated myosins. Ca2+ binding was abolished by a G117C and a G117A mutation; however, these mutants have a decreased affinity for the heavy chain. None of the other mutations affected RLC function. Replacement of the respective cysteine with glycine in the skeletal RLC has markedly changed the regulatory properties of the molecule. The single cysteine to glycine mutation conferred to this light chain the ability to restore Ca2+ binding and regulated ATPase activity, although Ca2+ activation of the actin-activated ATPase was lower than with scallop RLC. The presence of amino acids other than glycine at this position in vertebrate skeletal myosin RLCs may explain why these are not fully functional in the scallop system. The results are in agreement with x-ray crystallography data showing the central role of G117 in stabilizing the Ca(2+)-binding site of scallop myosin. Images PMID:8090720

  18. Engineering tandem single-chain Fv as cell surface reporters with enhanced properties of fluorescence detection.

    PubMed

    Gallo, Eugenio; Snyder, Avin C; Jarvik, Jonathan W

    2015-10-01

    A recently described fluorescence biosensor platform utilizes single-chain Fv (scFvs) that selectively bind and activate fluorogen molecules. In this report we investigated the display of tandem scFv biosensors at the surface of mammalian cells with the aim of advancing current fluorescence detection strategies. We initially screened different peptide linkers to separate each scFv unit, and discovered that tandem proteins joined by either flexible or α-helical linkers properly fold and display at the surface of mammalian cells. Accordingly, we performed a combinatorial scFv-dimer study and identified that fluorescence activation correlated with the cellular location (membrane distal versus proximal) and selections of the different scFvs. Furthermore, in vitro measurements showed that the stability of each scFv monomer unit influenced the folding and cell surface activities of tandem scFvs. Additionally, we investigated the absence or poor signals from some scFv-dimer combinations and discovered that intramolecular and intermolecular scFv chain mispairings led to protein misfolding and/or secretory-pathway-mediated degradation. Furthermore, when tandem scFvs were utilized as fluorescence reporter tags with surface receptors, the biosensor unit and target protein showed independent activities. Thus, the live cell application of tandem scFvs permitted advanced detection of target proteins via fluorescence signal amplification, Förster resonance energy transfer resulting in the increase of Stokes shift and multi-color vesicular traffic of surface receptors. PMID:25843939

  19. Analytical solutions for reactive transport of N-member radionuclide chains in a single fracture.

    PubMed

    Sun, Yunwei; Buscheck, Thomas A

    2003-01-01

    Several numerical codes have been used to simulate radionuclide transport in fractured rock systems. The validation of such numerical codes can be accomplished by comparison of numerical simulations against appropriate analytical solutions. In this paper, we present analytical solutions for the reactive transport of N-member radionuclide chains (i.e., multiple species of radionuclides and their daughter species) through a discrete fracture in a porous rock matrix applying a system decomposition approach. We consider the transport of N-member radionuclide chains in a single-fracture-matrix system as a starting point to simulate more realistic and complex systems. The processes considered are advection along the fracture, lateral diffusion in the matrix, radioactive decay of multiple radionuclides, and adsorption in both the fracture and matrix. Different retardation factors can be specified for the fracture and matrix. However, all species are assumed to share the same retardation factors for the fracture and matrix, respectively. Although a daughter species may penetrate farther along the fracture than its parent species when a constant-concentration boundary condition is applied, our results indicate that all species retain the same transport speed in the fracture if a pulse of the first species is released into the fracture. This solution scheme provides a way to validate numerical computer codes of radionuclide transport in fractured rock, such as those being used to assess the performance of a potential nuclear-waste repository at Yucca Mountain. PMID:12714317

  20. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles.

    PubMed

    Serna, Naroa; Céspedes, María Virtudes; Saccardo, Paolo; Xu, Zhikun; Unzueta, Ugutz; Álamo, Patricia; Pesarrodona, Mireia; Sánchez-Chardi, Alejandro; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio; Ferrer-Miralles, Neus

    2016-07-01

    A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles. PMID:26949165

  1. Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs.

    PubMed

    Wang, Mugui; Wei, Chuchu; Ye, Xiufen; Liu, Jianping; Zhang, Cuicui; Chen, Hao; Zhang, Xiaobo; Tu, Jumin

    2015-04-15

    We developed an effective protocol for generating high-purity heteroduplexes via annealing single-stranded DNAs (ssDNAs) derived from plasmid DNA by asymmetric polymerase chain reaction (A-PCR). With the addition of dimethyl sulfoxide, a one-step A-PCR procedure can generate ssDNAs stably at a range of reaction temperatures. Several annealing buffers can anneal two ssDNAs into heteroduplexes effectively. We further developed a simple strategy to create d(GATC) hemimethylated heteroduplexes by annealing fully methylated homoduplexes in the presence of excessive unmethylated ssDNAs. The constructed heteroduplexes have been well tested as substrates for mismatch repair in Escherichia coli and, thus, can be used in various biotechnology applications. PMID:25575760

  2. A modular approach to introduce function into single-chain polymeric nanoparticles.

    PubMed

    Huerta, Elisa; van Genabeek, Bas; Stals, Patrick J M; Meijer, E W; Palmans, Anja R A

    2014-08-01

    Here, a modular approach is reported to introduce a specific function into single-chain polymeric nanoparticles (SCPNs). Hereto, an amphiphilic polymer with pendant benzene-1,3,5-tricarboxamide (BTA) units is mixed with a "free" BTA that contains a functional group, either a fluorescent naphthalimide or a catalytically active l-proline. Taking advantage of hydrophobic interactions and self-recognition properties of the BTA units, the "free" BTAs are captured into the interior of the SCPN in water as evidenced by fluorescence studies. To illustrate that function can be readily introduced using a modular approach, l-proline-based BTAs are incorporated to procure a catalytically active SCPN in water. The aldol reaction between p-nitrobenzaldehyde and cyclohexanone shows good conversions at low catalyst loadings and substrate concentrations, and high stereoselectivities are obtained (de = 91% and ee = 98%). PMID:24962087

  3. Rotational relaxation time of polyelectrolyte xanthan chain via single molecule tracking method

    NASA Astrophysics Data System (ADS)

    Lee, Jeong Yong; Jung, Hyun Wook; Hyun, Jae Chun

    2012-12-01

    Effect of solvent viscosity on the longest rotational relaxation time of xanthan molecule has been examined using a single molecule tracking method. Incorporating inverted epi-fluorescence microscope and chargedcoupled device (CCD) camera, various features of xanthan ( i.e., radius of gyration, orientation angle, etc.) were interpreted by image processing algorithm from the captured real xanthan images. From the best-fit of the autocorrelation function on the orientation angle, the longest rotational relaxation time was effectively determined. Rotational relaxation time increases with the medium solvent viscosity due to the slow movement of xanthan molecule. It is confirmed that there is a good agreement between experiments and Brownian dynamics simulations on the relaxation patterns of xanthan chain.

  4. The finite scaling for S = 1 XXZ chains with uniaxial single-ion-type anisotropy

    NASA Astrophysics Data System (ADS)

    Wang, Honglei; Xiong, Xingliang

    2014-03-01

    The scaling behavior of criticality for spin-1 XXZ chains with uniaxial single-ion-type anisotropy is investigated by employing the infinite matrix product state representation with the infinite time evolving block decimation method. At criticality, the accuracy of the ground state of a system is limited by the truncation dimension χ of the local Hilbert space. We present four evidences for the scaling of the entanglement entropy, the largest eigenvalue of the Schmidt decomposition, the correlation length, and the connection between the actual correlation length ξ and the energy. The result shows that the finite scalings are governed by the central charge of the critical system. Also, it demonstrates that the infinite time evolving block decimation algorithm by the infinite matrix product state representation can be a quite accurate method to simulate the critical properties at criticality.

  5. Antigen nature and complexity influence human antibody light chain usage and specificity.

    PubMed

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies. PMID:27113164

  6. Fluorescence excitation and emission spectroscopy on single MEH-PPV chains at low temperature.

    PubMed

    Feist, Florian A; Basché, Thomas

    2008-08-14

    Fluorescence emission and excitation spectra of single MEH-PPV polymer molecules dispersed in thin PMMA films have been recorded at 1.2 and 20 K. We observe single as well as multichromophore emission in single chain emission spectra, whereby the relative fractions depend on the two different molecular weights (50 and 350 kDa) studied. The molecular weight also affects the distribution of peak emission maxima, which is monomodal (bimodal) for the low (high) molecular weight. The appearance of an additional "red" subpopulation for the high molecular weight sample is attributed to interactions of multiple chromophores from a sufficiently flexible single chain. The comparison of emission spectra appearing in the "blue" as well as "red" subpopulations suggests that these intrachain interactions rather lead to ground-state aggregates than excimers. Independent of the molecular weight, large variations in spectral shape and apparent line width in the emission spectra have been observed. Occasionally, we find very narrow purely electronic zero-phonon lines both in emission and in excitation spectra, with line widths down to the instrumental resolution. In accordance with earlier literature data it is argued that linear electron-phonon coupling should be quite strong for MEH-PPV in PMMA, leading to only a small fraction of chromophores exhibiting zero-phonon lines. In addition, spectral diffusion, which manifests itself by several time-dependent line shifting and broadening phenomena, contributes to the substantial variations of spectral shapes. Excitation experiments with particularly stable chromophores provide an upper limit for the optical line width (approximately 0.1 cm(-1)), which at 1.2 K can actually approach the lifetime-limited homogeneous width. Raising the temperature to 20 K leads to line broadening and typically, to disappearance of zero-phonon lines. The failure to observe zero-phonon lines of chromophores supposedly serving as donors in intramolecular

  7. Differentiation between Shallow and Deep Charge Trap States on Single Poly(3-hexylthiophene) Chains through Fluorescence Photon Statistics.

    PubMed

    Grußmayer, Kristin S; Steiner, Florian; Lupton, John M; Herten, Dirk-Peter; Vogelsang, Jan

    2015-12-01

    Blinking of the photoluminescence (PL) emitted from individual conjugated polymer chains is one of the central observations made by single-molecule spectroscopy (SMS). Important information, for example regarding excitation energy transfer, can be extracted by evaluating dynamic quenching. However, the nature of trap states, which are responsible for PL quenching, often remains obscured. We present a detailed investigation of the photon statistics of single poly(3-hexylthiophene) (P3HT) chains obtained by SMS. The photon statistics provide a measure of the number and brightness of independently emitting areas on a single chain. These observables can be followed during blinking. A decrease in PL intensity is shown to be correlated with either 1) a decrease in the average brightness of the emitting sites; or 2) a decrease in the number of emitting regions. We attribute these phenomena to the formation of 1) shallow charge traps, which can weakly affect all emitting areas of a single chain at once; and 2) deep traps, which have a strong effect on small regions within the single chains. PMID:26490757

  8. Heterologous Antigen Selection of Camelid Heavy Chain Single Domain Antibodies against Tetrabromobisphenol A

    PubMed Central

    2015-01-01

    Tetrabromobisphenol A (TBBPA) is a ubiquitous flame retardant. A high-throughput immunoassay would allow for monitoring of human and environmental exposures as a part of risk assessment. Naturally occurring antibodies in camelids that are devoid of light chain, show great promise as an efficient tool in monitoring environmental contaminants, but they have been rarely used for small molecules. An alpaca was immunized with a TBBPA hapten coupled to thyroglobulin and a variable domain of heavy chain antibody (VHH) T3–15 highly selective for TBBPA was isolated from a phage displayed VHH library using heterologous coating antigens. Compared to the VHHs isolated using homologous antigens, VHH T3–15 had about a 10-fold improvement in sensitivity in an immunoassay. This assay, under the optimized conditions of 10% methanol in the assay buffer (pH 7.4), had an IC50 for TBBPA of 0.40 ng mL–1 and negligible cross reactivity (<0.1%) with other tested analogues. After heating the VHH at 90 °C for 90 min about 20% of the affinity for coating antigen T3-BSA remained. The recoveries of TBBPA from spiked soil and fetal bovine serum samples ranged from 90.3% to 110.7% by ELISA and agreed well with a liquid chromatography–tandem mass spectrometry method. We conclude the many advantages of VHH make them attractive for the development of immunoassays to small molecules. PMID:25068372

  9. Variable domain structure of {kappa}IV human light chain len : high homology to the murine light chain McPC603.

    SciTech Connect

    Huang, D.-B.; Chang, C.-H.; Ainsworth, C.; Johnson, G.; Solomon, A.; Stevens, F. J.; Schiffer, M.; Center for Mechanistic Biology and Biotechnology; Univ. of Tennessee Medical Center

    1997-12-01

    Antibody light chains of the {kappa} subgroup are the predominant light chain component in human immune responses and are used almost exclusively in the antibody repertoire of mice. Human {kappa} light chains comprise four subgroups. To date, all crystallographic studies of human {kappa} light chains were carried out on proteins of the {kappa}I subgroup. The light chain produced by multiple myeloma patient Len, was of the {kappa}IV subgroup, it differed by only one residue from the germ-line gene encoded protein. The variable domain fragment of the light chain was crystallized from ammonium sulfate in space group C222{sub 1}. The crystal structure was determined by molecular replacement and refined at 1.95 Angstrom resolution to an R-factor of 0.15. Protein Len has six additional residues in its CDR1 segment compared to the {kappa}I proteins previously characterized. The {kappa}IV variable domain. Len, differs in only 23 of 113 residues from murine {kappa} light chain McPC603. The RMS deviation upon superimposing their {alpha}-carbons was 0.69 Angstrom. The CDR1 segment of the human and murine variable domains have the same length and conformation although their amino acid sequences differ in 5 out of 17 residues. Structural features were identified that could account for the significantly higher stability of the human {kappa}IV protein relative to its murine counterpart. This human {kappa}IV light chain structure is the closest human homolog to a murine light chain and can be expected to facilitate detailed structural comparisons necessary for effective humanization of murine antibodies.

  10. Anti-Aβ single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease.

    PubMed

    Fernandez-Funez, Pedro; Zhang, Yan; Sanchez-Garcia, Jonatan; de Mena, Lorena; Khare, Swati; Golde, Todd E; Levites, Yona; Rincon-Limas, Diego E

    2015-11-01

    Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities. PMID:26253732

  11. On the dynamics of chain systems. [applications in manipulator and human body models

    NASA Technical Reports Server (NTRS)

    Huston, R. L.; Passerello, C. E.

    1974-01-01

    A computer-oriented method for obtaining dynamical equations of motion for chain systems is presented. A chain system is defined as an arbitrarily assembled set of rigid bodies such that adjoining bodies have at least one common point and such that closed loops are not formed. The equations of motion are developed through the use of Lagrange's form of d'Alembert's principle. The method and procedure is illustrated with an elementary study of a tripod space manipulator. The method is designed for application with systems such as human body models, chains and cables, and dynamic finite-segment models.

  12. Role of chain entropy in an analytic model of protein binding in single-DNA stretching experiments.

    PubMed

    Lam, Pui-Man; Neumann, Richard M

    2011-09-01

    We show that the simple analytical model proposed by Zhang and Marko [Phys. Rev. E 77, 031916 (2008)] to illustrate Maxwell relations for single-DNA experiments can be improved by including the zero-force entropy of a Gaussian chain. The resulting model is in excellent agreement with the discrete persistent-chain model and is in a form convenient for analyzing experimental data. PMID:22060437

  13. Single Muscle Immobilization Decreases Single-Fibre Myosin Heavy Chain Polymorphism: Possible Involvement of p38 and JNK MAP Kinases

    PubMed Central

    Derbré, Frédéric; Droguet, Mickaël; Léon, Karelle; Troadec, Samuel; Pennec, Jean-Pierre; Giroux-Metges, Marie-Agnès; Rannou, Fabrice

    2016-01-01

    Purpose Muscle contractile phenotype is affected during immobilization. Myosin heavy chain (MHC) isoforms are the major determinant of the muscle contractile phenotype. We therefore sought to evaluate the effects of muscle immobilization on both the MHC composition at single-fibre level and the mitogen-activated protein kinases (MAPK), a family of intracellular signaling pathways involved in the stress-induced muscle plasticity. Methods The distal tendon of female Wistar rat Peroneus Longus (PL) was cut and fixed to the adjacent bone at neutral muscle length. Four weeks after the surgery, immobilized and contralateral PL were dissociated and the isolated fibres were sampled to determine MHC composition. Protein kinase 38 (p38), extracellular signal-regulated kinases (ERK1/2), and c-Jun- NH2-terminal kinase (JNK) phosphorylations were measured in 6- and 15-day immobilized and contralateral PL. Results MHC distribution in immobilized PL was as follows: I = 0%, IIa = 11.8 ± 2.8%, IIx = 53.0 ± 6.1%, IIb = 35.3 ± 7.3% and I = 6.1 ± 3.9%, IIa = 22.1 ± 3.4%, IIx = 46.6 ± 4.5%, IIb = 25.2 ± 6.6% in contralateral muscle. The MHC composition in immobilized muscle is consistent with a faster contractile phenotype according to the Hill’s model of the force-velocity relationship. Immobilized and contralateral muscles displayed a polymorphism index of 31.1% (95% CI 26.1–36.0) and 39.3% (95% CI 37.0–41.5), respectively. Significant increases in p38 and JNK phosphorylation were observed following 6 and 15 days of immobilization. Conclusions Single muscle immobilization at neutral length induces a shift of MHC composition toward a faster contractile phenotype and decreases the polymorphic profile of single fibres. Activation of p38 and JNK could be a potential mechanism involved in these contractile phenotype modifications during muscle immobilization. PMID:27383612

  14. Structural and Functional Characterization of a Single-Chain Form of the Recognition Domain of Complement Protein C1q

    PubMed Central

    Moreau, Christophe; Bally, Isabelle; Chouquet, Anne; Bottazzi, Barbara; Ghebrehiwet, Berhane; Gaboriaud, Christine; Thielens, Nicole

    2016-01-01

    Complement C1q is a soluble pattern recognition molecule comprising six heterotrimeric subunits assembled from three polypeptide chains (A–C). Each heterotrimer forms a collagen-like stem prolonged by a globular recognition domain. These recognition domains sense a wide variety of ligands, including pathogens and altered-self components. Ligand recognition is either direct or mediated by immunoglobulins or pentraxins. Multivalent binding of C1q to its targets triggers immune effector mechanisms mediated via its collagen-like stems. The induced immune response includes activation of the classical complement pathway and enhancement of the phagocytosis of the recognized target. We report here, the first production of a single-chain recombinant form of human C1q globular region (C1q-scGR). The three monomers have been linked in tandem to generate a single continuous polypeptide, based on a strategy previously used for adiponectin, a protein structurally related to C1q. The resulting C1q-scGR protein was produced at high yield in stably transfected 293-F mammalian cells. Recombinant C1q-scGR was correctly folded, as demonstrated by its X-ray crystal structure solved at a resolution of 1.35 Å. Its interaction properties were assessed by surface plasmon resonance analysis using the following physiological C1q ligands: the receptor for C1q globular heads, the long pentraxin PTX3, calreticulin, and heparin. The 3D structure and the binding properties of C1q-scGR were similar to those of the three-chain fragment generated by collagenase digestion of serum-derived C1q. Comparison of the interaction properties of the fragments with those of native C1q provided insights into the avidity component associated with the hexameric assembly of C1q. The interest of this functional recombinant form of the recognition domains of C1q in basic research and its potential biomedical applications are discussed. PMID:26973654

  15. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus.

    PubMed Central

    Gyllensten, U B; Erlich, H A

    1988-01-01

    Single-copy sequences can be enzymatically amplified from genomic DNA by the polymerase chain reaction. By using unequal molar amounts of the two amplification primers, it is possible in a single step to amplify a single-copy gene and produce an excess of single-stranded DNA of a chosen strand for direct sequencing or for use as a hybridization probe. Further, individual alleles in a heterozygote can be sequenced directly by using allele-specific oligonucleotides either in the amplification reaction or as sequencing primers. By using these methods, we have studied the allelic diversity at the HLA-DQA locus and its association with the serologically defined HLA-DR and -DQ types. This analysis has revealed a total of eight alleles and three additional haplotypes. This procedure has wide applications in screening for mutations in human genes and facilitates the linking of enzymatic amplification of genes to automated sequencing. Images PMID:3174659

  16. Single molecule spectroscopy of conjugated polymer chains in an electric field-aligned liquid crystal.

    PubMed

    Chang, Wei-Shun; Link, Stephan; Yethiraj, Arun; Barbara, Paul F

    2008-01-17

    Using single molecule polarization spectroscopy, we investigated the alignment of a polymer solute with respect to the liquid crystal (LC) director in an LC device while applying an external electric field. The polymer solute is poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (or MEH-PPV), and the LC solvent is 5CB. The electric field induces a change in the LC director orientation from a planar alignment (no electric field) to a perpendicular (homeotropic) alignment with an applied field of 5.5 x 103 V/cm. We find that the polymer chains align with the LC director in both planar and homeotropic alignment when measured in the bulk of the LC solution away from the device interface. Single molecule polarization distributions measured as a function of distance from the LC device interface reveal a continuous change of the MEH-PPV alignment from planar to homeotropic. The observed polarization distributions are modeled using a conventional elastic model that predicts the depth profile of the LC director orientation for the applied electric field. The excellent agreement between experiment and simulations shows that the alignment of MEH-PPV follows the LC director throughout the LC sample. Furthermore, our results suggest that conjugated polymers such as MEH-PPV can be used as sensitive local probes to explore complex (and unknown) structures in anisotropic media. PMID:17975912

  17. Pricing and ordering policies for price-dependent demand in a supply chain of a single retailer and a single manufacturer

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Hong, Yushin; Kim, Taebok

    2011-01-01

    This article discusses joint pricing and ordering policies for price-dependent demand in a supply chain consisting of a single retailer and a single manufacturer. The retailer places orders for products according to an EOQ policy and the manufacturer produces them on a lot-for-lot basis. Four mechanisms with differing levels of coordination are presented. Mathematical models are formulated and solution procedures are developed to determine the optimal retail prices and order quantities. Through extensive numerical experiments, we analyse and compare the behaviours and characteristics of the proposed mechanisms, and find that enhancing the level of coordination has important benefits for the supply chain.

  18. A Laboratory Exercise for Genotyping Two Human Single Nucleotide Polymorphisms

    ERIC Educational Resources Information Center

    Fernando, James; Carlson, Bradley; LeBard, Timothy; McCarthy, Michael; Umali, Finianne; Ashton, Bryce; Rose, Ferrill F., Jr.

    2016-01-01

    The dramatic decrease in the cost of sequencing a human genome is leading to an era in which a wide range of students will benefit from having an understanding of human genetic variation. Since over 90% of sequence variation between humans is in the form of single nucleotide polymorphisms (SNPs), a laboratory exercise has been devised in order to…

  19. The complete cDNA sequence of laminin alpha 4 and its relationship to the other human laminin alpha chains.

    PubMed

    Richards, A; Al-Imara, L; Pope, F M

    1996-06-15

    We previously localised the gene (LAMA4) encoding a novel laminin alpha 4 chain to chromosome 6q21. In this study, we describe the complete coding sequence and compare the protein with the other three known human laminin alpha chains. Although closely linked to LAMA2, the LAMA4 product most closely resembles laminin alpha 3, a constituent of laminin 5. Like laminin alpha 3A, the alpha 4 chain is a truncated version of the alpha 1 and alpha 2 chains, with a much reduced short arm. While the alpha 4 molecule is most similar to alpha 3, it shares some features of the C-terminal domains G4 and G5 in common with alpha 2. Unlike the LAMA3 gene, LAMA4 appears to encode only a single transcript, as determined by 5' rapid amplification of cDNA ends. The cDNA sequence encodes 1816 amino acids, which include a 24-residue signal peptide. The gene is expressed in skin, placenta, heart, lung, skeletal muscle, and pancreas. We have also shown that the mRNA can be readily reverse transcribed and amplified from cultured dermal fibroblasts. PMID:8706685

  20. Crystal engineering to control the magnetic interaction between weak ferromagnetic single-chain magnets assembled in a 3D framework.

    PubMed

    Su, Lei; Song, Wei-Chao; Zhao, Jiong-Peng; Liu, Fu-Chen

    2016-07-01

    A new single-chain-magnet (SCM), [Co4(OMe)3(HCO2)2(L)3·DMF]n, (L = 4-(pyridin-4-yl)benzolate) (2), was constructed by changing the spacers of a weak ferromagnetic single-chain magnet [Co8(OMe)6(HCO2)4(isonic)6·H2O]n (1). By contrasting the magnetism of the two complexes, it is found that the longer the linker the stronger the magnetic properties. PMID:27333437

  1. The solution structure of a superpotent B-chain-shortened single-replacement insulin analogue.

    PubMed Central

    Kurapkat, G.; Siedentop, M.; Gattner, H. G.; Hagelstein, M.; Brandenburg, D.; Grötzinger, J.; Wollmer, A.

    1999-01-01

    This paper reports on an insulin analogue with 12.5-fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy. The analogue is [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. C-terminal truncation of the B-chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of D-Ala for the wild-type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild-type hormone when the B-chain is present in full length. The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the "active" conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three-dimensional structure of [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface. PMID:10091652

  2. Comprehensive variation discovery in single human genomes

    PubMed Central

    Weisenfeld, Neil I.; Yin, Shuangye; Sharpe, Ted; Lau, Bayo; Hegarty, Ryan; Holmes, Laurie; Sogoloff, Brian; Tabbaa, Diana; Williams, Louise; Russ, Carsten; Nusbaum, Chad; Lander, Eric S.; MacCallum, Iain; Jaffe, David B.

    2014-01-01

    Complete knowledge of the genetic variation in individual human genomes is a crucial foundation for understanding the etiology of disease. Genetic variation is typically characterized by sequencing individual genomes and comparing reads to a reference. Existing methods do an excellent job of detecting variants in approximately 90% of the human genome, however calling variants in the remaining 10% of the genome (largely low-complexity sequence and segmental duplications) is challenging. To improve variant calling, we developed a new algorithm, DISCOVAR, and examined its performance on improved, low-cost sequence data. Using a newly created reference set of variants from finished sequence of 103 randomly chosen Fosmids, we find that some standard variant call sets miss up to 25% of variants. We show that the combination of new methods and improved data increases sensitivity several-fold, with the greatest impact in challenging regions of the human genome. PMID:25326702

  3. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    NASA Astrophysics Data System (ADS)

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-10-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

  4. The food chain as a source of human exposure from municipal waste combustion: An uncertainty analysis

    SciTech Connect

    Belcher, G.D.; Travis, C.C.; Bruins, R.J.F.; Environmental Protection Agency, Cincinnati, OH . Office of Environmental Criteria and Assessment)

    1989-01-01

    The current installed incineration capacity of municipal waste combustors (MWCs) in the United States has reached approximately 50 thousand tons per day and is projected to triple over the next decade. As the use of MWCs as a waste management alternative has increased, public concern over possible environmental and human health effects has also increased. Of particular concern are health risks associated with potential exposures through the food chain. The food chain is the primary pathway of human exposure for a large class of organics, such as dioxin, PCBs, DDT, and other pesticides. Because many pollutants emitted by MWCs are lipophilic, extremely persistent compounds, they tend to sorb strong to air particles, soil, and sediment and to bioaccumulate in living organisms. As a result, the food chain can be a major pathway of exposure to pollutants emitted by MWCs. It is the purpose of this paper to assess the magnitude of human exposure through the food chain for two pollutants released by MWCs: cadmium and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as dioxin. These pollutants were chosen as representative of two chemical classes: metals and organics. Cadmium is a metal which presumably will enter the food chain primarily through vegetative root uptake, while dioxin is an extremely lipophilic compound which will bioconcentrate in beef and milk. 57 refs., 7 figs., 14 tabs.

  5. [Ontogenic peculiarities of the human tympanic ossicular chain].

    PubMed

    Whyte Orozco, J; Cisneros Gimeno, A I; Urieta Carpi, J J; Yus Gotor, C; Gañet Solé, J; Torres del Puerto, A; Sarrat Torreguitart, R

    2003-01-01

    We have studied the development of the tympanic ossicles in 40 embryo-foetal human series aged between 32 days (6 mm) and newborn. Once performed the measurements to date chronologically embryos and foetuses, we did a meticulous dissection of temporal bones. After fix in 10% formol, decalcified with 2% nitric acid, embedded in Paraplast, sectioned in a sequence of 7 mm, and stained with Martin's trichrome. The tympanic ossicles are developed in the mesenchyme of the two first pharyngeal archs. The head of the malleus, the body and the short limb of the incus arise from the first arch while the handle of the malleus, the long limb of the incus and the mass of the stapes arise from the second arch. The vestibular side of the stapedial footplate develops in the otic capsule. The tympanic ossicles develop from endochondral ossification, while anterior process of the malleus has the membranous ossification. In their ontogenia 6 stages are observed. First stage, the formation of their sketch by mesenchimal condensation, in the second stage, "pre-cartilaginous", the cells of the primordia are differentiated into condroblasts, in the third stage "cartilaginous" the ossicles show a cartilaginous structure, in the forth stage the primary ossification centers are developed, in the fifth stage the ossicles arise in the periostic annulus and inside the endochondral bone, and in the last stage the osseous tissue grows until it acquires a compact osseous structure. PMID:12733315

  6. Accumulation of potentially toxic elements in plants and element transfer to human food chain

    SciTech Connect

    Dudka, S.; Miller, W.P.

    1995-12-31

    This paper summarizes the biological pathways of cadmium, mercury, and lead into the human food chain; major sources of bioaccumulation; and exposure limits. For occupationally non-exposed persons and non-smokers, food is the main source of cadmium. About one-third of the total Cd burden originates from animal products and two-thirds from plant products. Consumption of fish and other aquatic animals is the main source of Hg intake by humans. The Provisional Tolerable Weekly Intake (PTWI) of Hg is achieved through occupational exposure or by consumption of large amounts of contaminated fish. About half of human Pb intake comes from food, of which more than half originates from plants. Drinking water and ingestion of Pb-rich soil and dust make up the other half of the Pb burden in humans. Cases of cadmium and methylmercury poisoning have been reported in Japan. No acute hazard from lead in the food chain has been determined so far.

  7. General model of phospholipid bilayers in fluid phase within the single chain mean field theory

    SciTech Connect

    Guo, Yachong; Baulin, Vladimir A.; Pogodin, Sergey

    2014-05-07

    Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120°, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins.

  8. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    SciTech Connect

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F. )

    1990-11-15

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of {sup 125}I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

  9. Molecular identification of Amazonian stingless bees using polymerase chain reaction single-strand conformation polymorphism.

    PubMed

    Souza, M T; Carvalho-Zilse, G A

    2014-01-01

    In countries containing a mega diversity of wildlife, such as Brazil, identifying and characterizing biological diversity is a continuous process for the scientific community, even in face of technological and scientific advances. This activity demands initiatives for the taxonomic identification of highly diverse groups, such as stingless bees, including molecular analysis strategies. This type of bee is distributed in all of the Brazilian states, with the highest species diversity being found in the State of Amazônia. However, the estimated number of species diverges among taxonomists. These bees are considered the main pollinators in the Amazon rainforest, in which they obtain food and shelter; however, their persistence is constantly threatened by deforestation pressure. Hence, it is important to classify the number and abundance of bee specie, to measure their decline and implement meaningful, priority conservation strategies. This study aims to maximize the implementation of more direct, economic and successful techniques for the taxonomic identification of stingless bees. Specifically, the genes 16S rRNA and COI from mitochondrial DNA were used as molecular markers to differentiate 9 species of Amazonian stingless bees based on DNA polymorphism, using the polymerase chain reaction-single-strand conformation polymorphism technique. We registered different, exclusive SSCP haplotypes for both genes in all species analyzed. These results demonstrate that SSCP is a simple and cost-effective technique that is applicable to the molecular identification of stingless bee species. PMID:25117306

  10. PURE mRNA display for in vitro selection of single-chain antibodies.

    PubMed

    Nagumo, Yu; Fujiwara, Kei; Horisawa, Kenichi; Yanagawa, Hiroshi; Doi, Nobuhide

    2016-05-01

    mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low. To establish an efficient platform for the PURE mRNA display of scFv, we performed affinity selection of a library of scFv antibodies with a C-terminal random sequence and obtained C-terminal sequences that increased the formation of mRNA-protein conjugates. We also identified unexpected common substitution mutations around the start codon of scFv antibodies, which were inferred to destabilize the mRNA secondary structure. This destabilization causes an increase in protein expression and the efficiency of the formation of mRNA-protein conjugates. We believe these improvements should make the PURE mRNA display more efficient for selecting antibodies for diagnostic and therapeutic applications. PMID:26711234

  11. Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans.

    PubMed

    Hagmann, Anna; Hunkeler, Moritz; Stuttfeld, Edward; Maier, Timm

    2016-08-01

    Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes. PMID:27396827

  12. Single-Chain Magnets Based on Octacyanotungstate with the Highest Energy Barriers for Cyanide Compounds

    NASA Astrophysics Data System (ADS)

    Wei, Rong-Min; Cao, Fan; Li, Jing; Yang, Li; Han, Yuan; Zhang, Xiu-Ling; Zhang, Zaichao; Wang, Xin-Yi; Song, You

    2016-04-01

    By introducing large counter cations as the spacer, two isolated 3, 3-ladder compounds, (Ph4P)[CoII(3-Mepy)2.7(H2O)0.3WV(CN)8]·0.6H2O (1) and (Ph4As)[CoII(3-Mepy)3WV(CN)8] (2, 3-Mepy = 3-methylpyridine), were synthesized and characterized. Static and dynamic magnetic characterizations reveal that compounds 1 and 2 both behave as the single-chain magnets (SCMs) with very high energy barriers: 252(9) K for 1 and 224(7) K for 2, respectively. These two compounds display the highest relaxation barriers for cyano-bridged SCMs and are preceded only by two cobalt(II)-radical compounds among all SCMs. Meanwhile, a large coercive field of 26.2 kOe (1) and 22.6 kOe (2) were observed at 1.8 K.

  13. Impact of charge carrier injection on single-chain photophysics of conjugated polymers

    NASA Astrophysics Data System (ADS)

    Hofmann, Felix J.; Vogelsang, Jan; Lupton, John M.

    2016-06-01

    Charges in conjugated polymer materials have a strong impact on the photophysics and their interaction with the primary excited state species has to be taken into account in understanding device properties. Here, we employ single-molecule spectroscopy to unravel the influence of charges on several photoluminescence (PL) observables. The charges are injected either stochastically by a photochemical process or deterministically in a hole-injection sandwich device configuration. We find that upon charge injection, besides a blue-shift of the PL emission and a shortening of the PL lifetime due to quenching and blocking of the lowest-energy chromophores, the non-classical photon arrival time distribution of the multichromophoric chain is modified towards a more classical distribution. Surprisingly, the fidelity of photon antibunching deteriorates upon charging, whereas one would actually expect the opposite: the number of chromophores to be reduced. A qualitative model is presented to explain the observed PL changes. The results are of interest to developing a microscopic understanding of the intrinsic charge-exciton quenching interaction in devices.

  14. MegaTevs: single-chain dual nucleases for efficient gene disruption.

    PubMed

    Wolfs, Jason M; DaSilva, Matthew; Meister, Sarah E; Wang, Xu; Schild-Poulter, Caroline; Edgell, David R

    2014-07-01

    Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications. PMID:25013171

  15. MegaTevs: single-chain dual nucleases for efficient gene disruption

    PubMed Central

    Wolfs, Jason M.; DaSilva, Matthew; Meister, Sarah E.; Wang, Xu; Schild-Poulter, Caroline; Edgell, David R.

    2014-01-01

    Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications. PMID:25013171

  16. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    PubMed

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1. PMID:27173568

  17. An optimized antibody-single-chain TRAIL fusion protein for cancer therapy.

    PubMed

    Siegemund, Martin; Seifert, Oliver; Zarani, Maria; Džinić, Tamara; De Leo, Valentino; Göttsch, Doris; Münkel, Sabine; Hutt, Meike; Pfizenmaier, Klaus; Kontermann, Roland E

    2016-07-01

    Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumor-associated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N- and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the N-terminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics. PMID:27064440

  18. Single-Chain Magnets Based on Octacyanotungstate with the Highest Energy Barriers for Cyanide Compounds

    PubMed Central

    Wei, Rong-Min; Cao, Fan; Li, Jing; Yang, Li; Han, Yuan; Zhang, Xiu-Ling; Zhang, Zaichao; Wang, Xin-Yi; Song, You

    2016-01-01

    By introducing large counter cations as the spacer, two isolated 3, 3-ladder compounds, (Ph4P)[CoII(3-Mepy)2.7(H2O)0.3WV(CN)8]·0.6H2O (1) and (Ph4As)[CoII(3-Mepy)3WV(CN)8] (2, 3-Mepy = 3-methylpyridine), were synthesized and characterized. Static and dynamic magnetic characterizations reveal that compounds 1 and 2 both behave as the single-chain magnets (SCMs) with very high energy barriers: 252(9) K for 1 and 224(7) K for 2, respectively. These two compounds display the highest relaxation barriers for cyano-bridged SCMs and are preceded only by two cobalt(II)-radical compounds among all SCMs. Meanwhile, a large coercive field of 26.2 kOe (1) and 22.6 kOe (2) were observed at 1.8 K. PMID:27071451

  19. Single-Chain Magnets Based on Octacyanotungstate with the Highest Energy Barriers for Cyanide Compounds.

    PubMed

    Wei, Rong-Min; Cao, Fan; Li, Jing; Yang, Li; Han, Yuan; Zhang, Xiu-Ling; Zhang, Zaichao; Wang, Xin-Yi; Song, You

    2016-01-01

    By introducing large counter cations as the spacer, two isolated 3, 3-ladder compounds, (Ph4P)[Co(II)(3-Mepy)2.7(H2O)0.3W(V)(CN)8]·0.6H2O (1) and (Ph4As)[Co(II)(3-Mepy)3W(V)(CN)8] (2, 3-Mepy = 3-methylpyridine), were synthesized and characterized. Static and dynamic magnetic characterizations reveal that compounds 1 and 2 both behave as the single-chain magnets (SCMs) with very high energy barriers: 252(9) K for 1 and 224(7) K for 2, respectively. These two compounds display the highest relaxation barriers for cyano-bridged SCMs and are preceded only by two cobalt(II)-radical compounds among all SCMs. Meanwhile, a large coercive field of 26.2 kOe (1) and 22.6 kOe (2) were observed at 1.8 K. PMID:27071451

  20. Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery*

    PubMed Central

    Ramirez, Arturo B.; Loch, Christian M.; Zhang, Yuzheng; Liu, Yan; Wang, Xiaohong; Wayner, Elizabeth A.; Sargent, Jonathon E.; Sibani, Sahar; Hainsworth, Eugenie; Mendoza, Eliseo A.; Eugene, Ralph; LaBaer, Joshua; Urban, Nicole D.; McIntosh, Martin W.; Lampe, Paul D.

    2010-01-01

    The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis. PMID:20467042

  1. Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To improve expression efficiency of the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse in the methylotrophic yeast Pichia pastoris (P. pastoris) GS115, the DNA sequence encoding for CBL-scFv was designed and synthesized based on the codon bias of P. p...

  2. Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ‘Ca. Liberibacter asiaticus’ is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vec...

  3. Bioaccumulation and Toxicity of Single-Walled Carbon Nanotubes to Benthic Organisms at the Base of the Marine Food Chain

    EPA Science Inventory

    As the use of single-walled carbon nanotubes (SWNTs) increases over time, so does the potential for environmental release. This research aimed to determine the toxicity, bioavailability, and bioaccumulation of SWNTs in marine benthic organisms at the base of the food chain. The t...

  4. Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

    PubMed

    Thatikonda, Naresh; Delfani, Payam; Jansson, Ronnie; Petersson, Linn; Lindberg, Diana; Wingren, Christer; Hedhammar, My

    2016-03-01

    Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes. PMID:26470853

  5. Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue

    NASA Astrophysics Data System (ADS)

    Lee, Hyun Chul; Kim, Su-Jin; Kim, Kyung-Sup; Shin, Hang-Cheol; Yoon, Ji-Won

    2000-11-01

    A cure for diabetes has long been sought using several different approaches, including islet transplantation, regeneration of β cells and insulin gene therapy. However, permanent remission of type 1 diabetes has not yet been satisfactorily achieved. The development of type 1 diabetes results from the almost total destruction of insulin-producing pancreatic β cells by autoimmune responses specific to β cells. Standard insulin therapy may not maintain blood glucose concentrations within the relatively narrow range that occurs in the presence of normal pancreatic β cells. We used a recombinant adeno-associated virus (rAAV) that expresses a single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter, which regulates SIA expression in response to blood glucose levels. Here we show that SIA produced from the gene construct rAAV-LPK-SIA caused remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time without any apparent side effects. This new SIA gene therapy may have potential therapeutic value for the cure of autoimmune diabetes in humans.

  6. Molecular Cytogenetics of Human Single Pronucleated Zygotes

    PubMed Central

    Azevedo, Ana Raquel; Pinho, Maria João; Silva, Joaquina; Sá, Rosália; Thorsteinsdóttir, Sólveig; Barros, Alberto

    2014-01-01

    The aim of the present study was to use fluorescence in situ hybridization to analyze the chromosome status of zygotes with a single pronucleus from in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles. In addition, we performed immunocytochemical detection of nuclear lamins and histone H3 trimethylated at lysine-9, Me(3)H3K9. Zygotes were processed 24 hours after insemination or injection to assure the absence of asynchrony. In opposition to previous results, we observed 2 pronuclei in 16 of 18 IVF zygotes and 40 of 64 ICSI zygotes, suggesting premature pronuclear breakdown. In IVF and ICSI zygotes, the rate of normal diploidy was only 6 of 16 and 27 of 56, respectively, suggesting that monopronucleated zygotes should not be used in assisted reproductive treatments. The possible mechanisms are discussed and compared to previous studies of monopronucleated zygotes. PMID:24717739

  7. Effect of single mismatches at 3′–end of primers on polymerase chain reaction

    PubMed Central

    Simsek, M; Adnan, H

    2000-01-01

    Objective and Method To investigate the effect of three different mismatches (G/T, G/A or G/G) at the 3′–end of a primer to amplify a 268 bp (base pair) region of the human β–globin gene using different annealing temperatures (45 to 65°C). Results The primer with the G/T mismatch was as efficient as the normal primer (G/C match) in the amplification of a 268 bp product at all temperatures tested. However, the primers having G/A or G/G mismatches at the 3′-end did not produce any specific polymerase chain reaction (PCR) fragment at all the annealing temperatures used, except a barely detectable 268 bp product for the G/G mismatch at 45 and 50°C. Conclusion We conclude that our PCR system was refractory to amplification when one of the primers contained a G/A or G/G mismatch at the 3′–end with template DNA. PMID:24019700

  8. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains*

    PubMed Central

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2015-01-01

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL. PMID:25505244

  9. Development and Preclinical Testing of a High Affinity Single Chain Antibody against (+)-Methamphetamine

    PubMed Central

    Peterson, Eric C.; Laurenzana, Elizabeth M.; Atchley, William T.; Hendrickson, Howard; Owens, S. Michael

    2009-01-01

    Chronic or excessive (+)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κ light chain, KD = 11 nM) and found to have similar ligand affinity (KD = 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified and formulated as a naturally occurring mixture of monomer (~75%) and dimer (~25%). To test the in vivo efficacy of the scFv6H4, male Sprague Dawley rats (n=5) were implanted with 3-day sc osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [3H]-scFv6H4 tracer. Serum pharmacokinetic (PCKN) analysis of METH and [3H]-scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0–480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t1/2λz of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t1/2λz (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum. PMID:18192498

  10. In silico experiments of single-chain antibody fragment against drugs of abuse

    PubMed Central

    Hu, Guodong; Chen, L. Y.

    2010-01-01

    SUMMARY Three sets of in silico experiments have been conducted to elucidate the binding mechanics of two drugs, (+)-methamphetamine (METH) and amphetamine (AMP) to the single-chain variable fragment (scFv) recently engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κ light chain, Kd = 11nM). The first set of in silico experiments are long time equilibration runs of scFv:drug complexes and of drug-free scFv both in solution. They demonstrate how the solution structures of scFv deviate from its crystallographic form with or without drug molecules bound to it. And they lead to the prediction that the Arrhenius activation barrier is nearly zero for transitions from the dissociated state to the bound state. The second set of in silico experiments are nonequilibrium dynamics of pulling the drug molecules out of the binding pocket of scFv and the equilibration runs for drugs to fall back into binding pocket. They demonstrate that extra water molecules (in addition to the two crystallographic waters) exist inside the binding pocket, underneath the drug molecules. These extra waters must have been evaporated from the binding pockets during the crystallization process of the in vitro experiments of structural determination. The third set of in silico experiments are nonequilibrium steered molecular dynamics simulations to determine the absolute binding free energies of METH and AMP to scFv. The center-of-mass of a drug molecule (METH or AMP) is steered (pulled) towards (forward) and away from (reverse) the binding site, sampling forward and reverse pulling paths. Mechanic work is measured along the pulling paths. The work measurements are averaged through the Brownian dynamics fluctuation dissipation theorem to produce the free-energy profiles of the scFv:drug complexes as a function of the drug-scFv separation. These experiments lead to the theoretical prediction of absolute binding energies of METH and AMP that are in agreement with the in vitro experimental

  11. Single and multiple objective biomass-to-biofuel supply chain optimization considering environmental impacts

    NASA Astrophysics Data System (ADS)

    Valles Sosa, Claudia Evangelina

    Bioenergy has become an important alternative source of energy to alleviate the reliance on petroleum energy. Bioenergy offers diminishing climate change by reducing Green House Gas Emissions, as well as providing energy security and enhancing rural development. The Energy Independence and Security Act mandate the use of 21 billion gallons of advanced biofuels including 16 billion gallons of cellulosic biofuels by the year 2022. It is clear that Biomass can make a substantial contribution to supply future energy demand in a sustainable way. However, the supply of sustainable energy is one of the main challenges that mankind will face over the coming decades. For instance, many logistical challenges will be faced in order to provide an efficient and reliable supply of quality feedstock to biorefineries. 700 million tons of biomass will be required to be sustainably delivered to biorefineries annually to meet the projected use of biofuels by the year of 2022. Approaching this complex logistic problem as a multi-commodity network flow structure, the present work proposes the use of a genetic algorithm as a single objective optimization problem that considers the maximization of profit and the present work also proposes the use of a Multiple Objective Evolutionary Algorithm to simultaneously maximize profit while minimizing global warming potential. Most transportation optimization problems available in the literature have mostly considered the maximization of profit or the minimization of total travel time as potential objectives to be optimized. However, on this research work, we take a more conscious and sustainable approach for this logistic problem. Planners are increasingly expected to adopt a multi-disciplinary approach, especially due to the rising importance of environmental stewardship. The role of a transportation planner and designer is shifting from simple economic analysis to promoting sustainability through the integration of environmental objectives. To

  12. Allele-specific polymerase chain reaction for the detection of Alzheimer’s disease-related single nucleotide polymorphisms

    PubMed Central

    2013-01-01

    Background The incidence of Alzheimer’s disease, particularly in developing countries, is expected to increase exponentially as the population ages. Continuing research in this area is essential in order to better understand this disease and develop strategies for treatment and prevention. Genome-wide association studies have identified several loci as genetic risk factors of AD aside from apolipoprotein E such as bridging integrator (BIN1), clusterin (CLU), ATP-binding cassette sub-family A member 7 (ABCA7), complement receptor 1 (CR1) and phosphatidylinositol binding clathrin assembly protein (PICALM). However genetic research in developing countries is often limited by lack of funding and expertise. This study therefore developed and validated a simple, cost effective polymerase chain reaction based technique to determine these single nucleotide polymorphisms. Methods An allele-specific PCR method was developed to detect single nucleotide polymorphisms of BIN1 rs744373, CLU rs11136000, ABCA7 rs3764650, CR1 rs3818361 and PICALM rs3851179 in human DNA samples. Allele-specific primers were designed by using appropriate software to permit the PCR amplification only if the nucleotide at the 3’-end of the primer complemented the base at the wild-type or variant-type DNA sample. The primers were then searched for uniqueness using the Basic Local Alignment Search Tool search engine. Results The assay was tested on a hundred samples and accurately detected the homozygous wild-type, homozygous variant-type and heterozygous of each SNP. Validation was by direct DNA sequencing. Conclusion This method will enable researchers to carry out genetic polymorphism studies for genetic risk factors associated with late-onset Alzheimer’s disease (BIN1, CLU, ABCA7, CR1 and PICALM) without the use of expensive instrumentation and reagents. PMID:23419238

  13. High-level secretory expression and purification of unhydroxylated human collagen α1(III) chain in Pichia pastoris GS115.

    PubMed

    Li, Linbo; Fan, Daidi; Ma, Xiaoxuan; Deng, Jianjun; He, Jing

    2015-01-01

    Recombinant collagen and gelatin have been applied in biomedical materials field because of products from genetically engineered microorganisms with improved safety, traceability, reproducibility, and homogeneous quality. To obtain high-level secretory expression of single-chain full-length human collagen α1(III) chain (COL3A1) without the N and C telopeptides, the cDNA coding for the human COL3A1 gene was cloned into the secretory expression vector pPIC9K and integrated into Pichia pastoris GS115. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis of culture supernatant from the recombinant methylotrophic yeast suggested that the unhydroxylated recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium, and exhibited an apparent molecular mass of approximately 130 kDa, which is 1.4 times higher than the theoretical one. Finally, the unhydroxylated rhCOL3A1 was purified to greater than 90% purity using a four-step approach. In addition, methylthiazolydiphenyl-tetrazolium bromide experiments indicated that low concentration of rhCOL3A1 could promote Baby hamster kidney cell (BHK21) proliferation effectively. The production and purification of rhCOL3A1 described in this study offer a new method for obtaining high level of rhCOL3A1 in relatively pure form, which is suitable for biomedical materials application. PMID:25231012

  14. Direct detection of a single photon by humans.

    PubMed

    Tinsley, Jonathan N; Molodtsov, Maxim I; Prevedel, Robert; Wartmann, David; Espigulé-Pons, Jofre; Lauwers, Mattias; Vaziri, Alipasha

    2016-01-01

    Despite investigations for over 70 years, the absolute limits of human vision have remained unclear. Rod cells respond to individual photons, yet whether a single-photon incident on the eye can be perceived by a human subject has remained a fundamental open question. Here we report that humans can detect a single-photon incident on the cornea with a probability significantly above chance. This was achieved by implementing a combination of a psychophysics procedure with a quantum light source that can generate single-photon states of light. We further discover that the probability of reporting a single photon is modulated by the presence of an earlier photon, suggesting a priming process that temporarily enhances the effective gain of the visual system on the timescale of seconds. PMID:27434854

  15. Direct detection of a single photon by humans

    PubMed Central

    Tinsley, Jonathan N.; Molodtsov, Maxim I.; Prevedel, Robert; Wartmann, David; Espigulé-Pons, Jofre; Lauwers, Mattias; Vaziri, Alipasha

    2016-01-01

    Despite investigations for over 70 years, the absolute limits of human vision have remained unclear. Rod cells respond to individual photons, yet whether a single-photon incident on the eye can be perceived by a human subject has remained a fundamental open question. Here we report that humans can detect a single-photon incident on the cornea with a probability significantly above chance. This was achieved by implementing a combination of a psychophysics procedure with a quantum light source that can generate single-photon states of light. We further discover that the probability of reporting a single photon is modulated by the presence of an earlier photon, suggesting a priming process that temporarily enhances the effective gain of the visual system on the timescale of seconds. PMID:27434854

  16. Coexistence of coil and globule domains within a single confined DNA chain

    PubMed Central

    Sung, Baeckkyoung; Leforestier, Amélie; Livolant, Françoise

    2016-01-01

    The highly charged DNA chain may be either in an extended conformation, the coil, or condensed into a highly dense and ordered structure, the toroid. The transition, also called collapse of the chain, can be triggered in different ways, for example by changing the ionic conditions of the solution. We observe individual DNA molecules one by one, kept separated and confined inside a protein shell (the envelope of a bacterial virus, 80 nm in diameter). For subcritical concentrations of spermine (4+), part of the DNA is condensed and organized in a toroid and the other part of the chain remains uncondensed around. Two states coexist along the same DNA chain. These ‘hairy’ globules are imaged by cryo-electron microscopy. We describe the global conformation of the chain and the local ordering of DNA segments inside the toroid. PMID:26704970

  17. EARLINET Single Calculus Chain - technical - Part 1: Pre-processing of raw lidar data

    NASA Astrophysics Data System (ADS)

    D'Amico, G.; Amodeo, A.; Mattis, I.; Freudenthaler, V.; Pappalardo, G.

    2015-10-01

    In this paper we describe an automatic tool for the pre-processing of lidar data called ELPP (EARLINET Lidar Pre-Processor). It is one of two calculus modules of the EARLINET Single Calculus Chain (SCC), the automatic tool for the analysis of EARLINET data. The ELPP is an open source module that executes instrumental corrections and data handling of the raw lidar signals, making the lidar data ready to be processed by the optical retrieval algorithms. According to the specific lidar configuration, the ELPP automatically performs dead-time correction, atmospheric and electronic background subtraction, gluing of lidar signals, and trigger-delay correction. Moreover, the signal-to-noise ratio of the pre-processed signals can be improved by means of configurable time integration of the raw signals and/or spatial smoothing. The ELPP delivers the statistical uncertainties of the final products by means of error propagation or Monte Carlo simulations. During the development of the ELPP module, particular attention has been payed to make the tool flexible enough to handle all lidar configurations currently used within the EARLINET community. Moreover, it has been designed in a modular way to allow an easy extension to lidar configurations not yet implemented. The primary goal of the ELPP module is to enable the application of quality-assured procedures in the lidar data analysis starting from the raw lidar data. This provides the added value of full traceability of each delivered lidar product. Several tests have been performed to check the proper functioning of the ELPP module. The whole SCC has been tested with the same synthetic data sets, which were used for the EARLINET algorithm inter-comparison exercise. The ELPP module has been successfully employed for the automatic near-real-time pre-processing of the raw lidar data measured during several EARLINET inter-comparison campaigns as well as during intense field campaigns.

  18. EARLINET Single Calculus Chain - technical - Part 2: Calculation of optical products

    NASA Astrophysics Data System (ADS)

    Mattis, Ina; D'Amico, Giuseppe; Baars, Holger; Amodeo, Aldo; Madonna, Fabio; Iarlori, Marco

    2016-07-01

    In this paper we present the automated software tool ELDA (EARLINET Lidar Data Analyzer) for the retrieval of profiles of optical particle properties from lidar signals. This tool is one of the calculus modules of the EARLINET Single Calculus Chain (SCC) which allows for the analysis of the data of many different lidar systems of EARLINET in an automated, unsupervised way. ELDA delivers profiles of particle extinction coefficients from Raman signals as well as profiles of particle backscatter coefficients from combinations of Raman and elastic signals or from elastic signals only. Those analyses start from pre-processed signals which have already been corrected for background, range dependency and hardware specific effects. An expert group reviewed all algorithms and solutions for critical calculus subsystems which are used within EARLINET with respect to their applicability for automated retrievals. Those methods have been implemented in ELDA. Since the software was designed in a modular way, it is possible to add new or alternative methods in future. Most of the implemented algorithms are well known and well documented, but some methods have especially been developed for ELDA, e.g., automated vertical smoothing and temporal averaging or the handling of effective vertical resolution in the case of lidar ratio retrievals, or the merging of near-range and far-range products. The accuracy of the retrieved profiles was tested following the procedure of the EARLINET-ASOS algorithm inter-comparison exercise which is based on the analysis of synthetic signals. Mean deviations, mean relative deviations, and normalized root-mean-square deviations were calculated for all possible products and three height layers. In all cases, the deviations were clearly below the maximum allowed values according to the EARLINET quality requirements.

  19. EARLINET Single Calculus Chain - technical - Part 1: Pre-processing of raw lidar data

    NASA Astrophysics Data System (ADS)

    D'Amico, Giuseppe; Amodeo, Aldo; Mattis, Ina; Freudenthaler, Volker; Pappalardo, Gelsomina

    2016-02-01

    In this paper we describe an automatic tool for the pre-processing of aerosol lidar data called ELPP (EARLINET Lidar Pre-Processor). It is one of two calculus modules of the EARLINET Single Calculus Chain (SCC), the automatic tool for the analysis of EARLINET data. ELPP is an open source module that executes instrumental corrections and data handling of the raw lidar signals, making the lidar data ready to be processed by the optical retrieval algorithms. According to the specific lidar configuration, ELPP automatically performs dead-time correction, atmospheric and electronic background subtraction, gluing of lidar signals, and trigger-delay correction. Moreover, the signal-to-noise ratio of the pre-processed signals can be improved by means of configurable time integration of the raw signals and/or spatial smoothing. ELPP delivers the statistical uncertainties of the final products by means of error propagation or Monte Carlo simulations. During the development of ELPP, particular attention has been payed to make the tool flexible enough to handle all lidar configurations currently used within the EARLINET community. Moreover, it has been designed in a modular way to allow an easy extension to lidar configurations not yet implemented. The primary goal of ELPP is to enable the application of quality-assured procedures in the lidar data analysis starting from the raw lidar data. This provides the added value of full traceability of each delivered lidar product. Several tests have been performed to check the proper functioning of ELPP. The whole SCC has been tested with the same synthetic data sets, which were used for the EARLINET algorithm inter-comparison exercise. ELPP has been successfully employed for the automatic near-real-time pre-processing of the raw lidar data measured during several EARLINET inter-comparison campaigns as well as during intense field campaigns.

  20. Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein.

    PubMed

    Yuan, Ruosen; Chen, Xiaoxu; Chen, Yan; Gu, Tiejun; Xi, Hualong; Duan, Ye; Sun, Bo; Yu, Xianghui; Jiang, Chunlai; Liu, Xintao; Wu, Chunlai; Kong, Wei; Wu, Yongge

    2014-02-01

    Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes. PMID:24241896

  1. Localized Single Frequency Lasing States in a Finite Parity-Time Symmetric Resonator Chain

    NASA Astrophysics Data System (ADS)

    Phang, Sendy; Vukovic, Ana; Creagh, Stephen C.; Sewell, Phillip D.; Gradoni, Gabriele; Benson, Trevor M.

    2016-02-01

    In this paper a practical case of a finite periodic Parity Time chain made of resonant dielectric cylinders is considered. The paper analyzes a more general case where PT symmetry is achieved by modulating both the real and imaginary part of the material refractive index along the resonator chain. The band-structure of the finite periodic PT resonator chains is compared to infinite chains in order to understand the complex interdependence of the Bloch phase and the amount of the gain/loss in the system that causes the PT symmetry to break. The results show that the type of the modulation along the unit cell can significantly affect the position of the threshold point of the PT system. In all cases the lowest threshold is achieved near the end of the Brillouin zone. In the case of finite PT-chains, and for a particular type of modulation, early PT symmetry breaking is observed and shown to be caused by the presence of termination states localized at the edges of the finite chain resulting in localized lasing and dissipative modes at each end of the chain.

  2. Mechanism of Polyubiquitin Chain Recognition by the Human Ubiquitin Conjugating Enzyme Ube2g2*

    PubMed Central

    Bocik, William E.; Sircar, Aroop; Gray, Jeffrey J.; Tolman, Joel R.

    2011-01-01

    Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 μm in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ∼3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length. PMID:21098018

  3. Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies*

    PubMed Central

    Rouet, Romain; Dudgeon, Kip; Christie, Mary; Langley, David; Christ, Daniel

    2015-01-01

    Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike “camelized” human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies. PMID:25737448

  4. Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody.

    PubMed

    Du, Xin-Jun; Zhou, Xiao-Nan; Li, Ping; Sheng, Wei; Ducancel, Frédéric; Wang, Shuo

    2016-04-13

    Specific antibodies are essential for the immune detection of small molecule contaminants. In the present study, the heavy and light variable regions (VH and VL) of the immunoglobulin genes from a hybridoma secreting a chloramphenicol (CAP)-specific monoclonal antibody (mAb) were cloned and sequenced. In addition, the light and heavy chains obtained from the monoclonal antibody were separated using SDS-PAGE and analyzed using Orbitrap mass spectrometry. The results of DNA sequencing and mass spectrometry analysis were compared, and the VH and VL chains specific for CAP were determined and used to construct a single-chain variable fragment (scFv). This fragment was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein and used to develop a direct competitive ELISA. Compared with the parent mAb, scFv exhibits lower sensitivity but better food matrix resistance. This work highlights the application of engineered antibodies for CAP detection. PMID:27003441

  5. Detection of gene expression in single neurons by patch-clamp and single-cell reverse transcriptase polymerase chain reaction.

    PubMed

    Chiang, L W

    1998-05-01

    Detection and quantitation of gene expression in single cells is especially important in the central nervous system where, at the cellular level, the synapse can be considered the single functional unit. For example, the consolidation of long-term memories may be mediated by persistent changes in the strength of synaptic transmission at individual synapses. In order to investigate the requirement for de novo RNA synthesis during long-term potentiation in individual neurons, we have combined single-cell electrophysiology with single-cell gene-expression methodology. Described are methods combining whole-cell patch-clamp and single-cell RT-PCR for the detection of a single mRNA species for nitric oxide synthase, or, through a multiplex strategy, for the simultaneous detection of several mRNAs including heme oxygenase 2, protein phosphatase inhibitor 1 protein, and several isoforms of the calcium/calmodulin dependent protein kinase II. PMID:9639890

  6. Brain white matter development is associated with a human-specific haplotype increasing the synthesis of long chain fatty acids.

    PubMed

    Peters, Bart D; Voineskos, Aristotle N; Szeszko, Philip R; Lett, Tristram A; DeRosse, Pamela; Guha, Saurav; Karlsgodt, Katherine H; Ikuta, Toshikazu; Felsky, Daniel; John, Majnu; Rotenberg, David J; Kennedy, James L; Lencz, Todd; Malhotra, Anil K

    2014-04-30

    The genetic and molecular pathways driving human brain white matter (WM) development are only beginning to be discovered. Long chain polyunsaturated fatty acids (LC-PUFAs) have been implicated in myelination in animal models and humans. The biosynthesis of LC-PUFAs is regulated by the fatty acid desaturase (FADS) genes, of which a human-specific haplotype is strongly associated with ω-3 and ω-6 LC-PUFA concentrations in blood. To investigate the relationship between LC-PUFA synthesis and human brain WM development, we examined whether this FADS haplotype is associated with age-related WM differences across the life span in healthy individuals 9-86 years of age (n = 207). Diffusion tensor imaging was performed to measure fractional anisotropy (FA), a putative measure of myelination, of the cerebral WM tracts. FADS haplotype status was determined with a single nucleotide polymorphism (rs174583) that tags this haplotype. Overall, normal age-related WM differences were observed, including higher FA values in early adulthood compared with childhood, followed by lower FA values across older age ranges. However, individuals homozygous for the minor allele (associated with lower LC-PUFA concentrations) did not display these normal age-related WM differences (significant age × genotype interactions, p(corrected) < 0.05). These findings suggest that LC-PUFAs are involved in human brain WM development from childhood into adulthood. This haplotype and LC-PUFAs may play a role in myelin-related disorders of neurodevelopmental origin. PMID:24790207

  7. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein

    PubMed Central

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses. PMID:27119101

  8. The food chain as a source of human exposure to organic chemicals

    SciTech Connect

    Hattemer-Frey, H.A.; Travis, C.C.

    1989-01-01

    Although human exposure to environmentally released pollutants can occur via several pathways including, inhalation, ingestion of contaminated food items, infant consumption of mother's milk, and dermal absorption, of particular concern are potential exposures from ingesting contaminated food items, since the food chain has been shown to be a primary source of human exposure to a large class of organics, including DDT, TCDD, pentachlorophenol, benzo(a)pyrene, and most pesticides. For risk assessment purposes, an important objective in evaluating the environmental behavior and fate of various pollutants is predicting the major pathways and extent of human exposure. Many chemicals cycle in the environment with cross-media transfers occurring between air, water, soil, and biota. As a result of this cycling behavior and a chemical's presence in various environmental media, human exposure often results from multiple sources. We found that 50--80% of all chemicals released into the environment result in human exposure through multiple media. The purpose of this presentation is to provide a perspective on the food chain as a source of human exposure to organic chemicals chronically released into the environment. 121 refs., 3 figs., 9 tabs.

  9. A comparative study on the binding of single and double chain surfactant-cobalt(III) complexes with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Vignesh, G.; Sugumar, K.; Arunachalam, S.; Vignesh, S.; Arthur James, R.

    2013-09-01

    The comparative binding effect of single and double aliphatic chain containing surfactant-cobalt(III) complexes cis-[Co(bpy)2(DA)2](ClO4)3ṡ2H2O (1), cis-[Co(bpy)2(DA)Cl](ClO4)2ṡ2H2O (2), cis-[Co(phen)2(CA)2](ClO4)3ṡ2H2O (3), and cis-[Co(phen)2(CA)Cl](ClO4)2ṡ2H2O (4) with bovine serum albumin (BSA) under physiological condition was analyzed by steady state, time resolved fluorescence, synchronous, three-dimensional fluorescence, UV-Visible absorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of BSA through a static mechanism. The binding constants (Kb) and the number of binding sites were calculated and binding constant values are found in the range of 104-105 M-1. The results indicate that compared to single chain complex, double chain surfactant-cobalt(III) complex interacts strongly with BSA. Also the sign of thermodynamic parameters (ΔG°, ΔH°, and ΔS°) indicate that all the complexes interact with BSA through hydrophobic force. The binding distance (r) between complexes and BSA was calculated using Förster non-radiation energy transfer theory and found to be less than 7 nm. The results of synchronous, three dimensional fluorescence and circular dichroism spectroscopic methods indicate that the double chain surfactant-cobalt(III) complexes changed the conformation of the protein considerably than the respective single chain surfactant-cobalt(III) complexes. Antimicrobial studies of the complexes showed good activities against pathogenic microorganisms.

  10. A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

    PubMed Central

    Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

    2014-01-01

    Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

  11. Conductance and spin-filter effects of oxygen-incorporated Au, Cu, and Fe single-atom chains

    NASA Astrophysics Data System (ADS)

    Zheng, Xiaolong; Xie, Yi-Qun; Ye, Xiang; Ke, San-Huang

    2015-01-01

    We studied the spin-polarized electron transport in oxygen-incorporated Au, Cu, and Fe single-atom chains (SACs) by first-principles calculations. We first investigated the mechanism responsible for the low conductance (<1G0) of the Au and Cu SACs in an oxygen environment reported in recent experiments. We found that for the Au SACs, the low conductance plateau around 0.6G0 can be attributed to a distorted chain doped with a single oxygen atom, while the 0.1G0 conductance comes from a linear chain incorporated with an oxygen molecule and is caused by an antibonding state formed by oxygen's occupied frontier orbital with dz orbitals of adjacent Au atoms. For the Cu SACs, the conductance about 0.3G0 is ascribed to a special configuration that contains Cu and O atoms in an alternating sequence. This exhibits an even-odd conductance oscillation with an amplitude of ˜0.1G0. In contrast, for the alternating Fe-O SACs, conductance overall decreases with an increase in O atoms and it approaches nearly zero for the chain with more than four O atoms. While the Cu-O SACs behave as perfect spin filters for one spin channel due to the half metallic nature, the Fe-O SACs can serve as perfect spin filters for two spin channels depending on the polarity of the applied gate voltage.

  12. A one year experience of an operationnal streamflow ensemble forecasting chain taking into account human expertise

    NASA Astrophysics Data System (ADS)

    Mathevet, T.; Moulin, L.; Gailhard, J.; Garçon, R.; Bernard, P.; Le Lay, M.; Zalachori, I.; Chardon, J.

    2012-04-01

    In the context of a national energy company (EDF : Electricité de France), hydro-meteorological forecasts are necessary to ensure safety of both power plants and dams, they also enables to meet environmental standards and to improve water resources management and decision making. Indeed, hydrometeorological ensemble forecasts allow a better representation of uncertainties from both meteorological and hydrological forecasts, which is essential to synthesize available information, coming from different meteorological and hydrological models and from human experience. An operational hydrological ensemble forecasting chain has been developed at EDF since 2008 and has been in use since 2011: last year more than 1200 ensemble streamflow forecasts have been made on more than 30 watersheds covering different spatial scales. This chain is specific because in one hand it takes into account both meteorological and hydrological uncertainties by pre/post-processing, and on the other hand, the human expertise is encouraged : the forecasters can modify forecasted distributions of mean daily rainfall, mean daily air temperature and streamflow. Firstly, this paper presents the different components of this operational hydrometeorological ensemble forecasting chain. Secondly, performances of this chain are assessed on some particular cases. The main steps of this chain are the following: (1) Pre-processing of meteorological ensembles (temperature and rainfall bias and reliability correction), (2) streamflow forecasts using a rainfall-runoff model and streamflow data assimilation and (3) post-processing of streamflow ensembles. The pre-processing of meteorological input is based on the correction (bias and reliability) of EPS-ECMWF forecasts by statistical analog forecasts based on past observed geopotential fields. The post-processing of streamflow forecasts is based on a simple statistical modelisation of the empirical model error by streamflow class and lead-time. The performance

  13. Alternative types of molecule-decorated atomic chains in Au-CO-Au single-molecule junctions.

    PubMed

    Balogh, Zoltán; Makk, Péter; Halbritter, András

    2015-01-01

    We investigate the formation and evolution of Au-CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference. PMID:26199840

  14. Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

    SciTech Connect

    Meramveliotaki, Chrysi; Kotsifaki, Dina; Androulaki, Maria; Hountas, Athanasios; Eliopoulos, Elias; Kokkinidis, Michael

    2007-10-01

    PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4{sub 2}, with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement.

  15. Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

    PubMed Central

    Balogh, Zoltán; Makk, Péter

    2015-01-01

    Summary We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference. PMID:26199840

  16. Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

    PubMed Central

    Shou, Yulin; Graves, Steven W.; Bradbury, Andrew R. M.

    2011-01-01

    Background Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1). F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection. Methods Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA. Results Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA. Conclusions Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of sc

  17. Single molecule magnet behavior observed in a 1-D dysprosium chain with quasi-D5h symmetry.

    PubMed

    Huang, Xing-Cai; Zhang, Ming; Wu, Dayu; Shao, Dong; Zhao, Xin-Hua; Huang, Wei; Wang, Xin-Yi

    2015-12-28

    Two one-dimensional (1-D) chain complexes with pentagonal bipyramidal Dy(III) centers have been synthesized and magnetically characterized. Field-induced single molecule magnet behavior has been revealed in both compounds, which is still rarely reported in a lanthanide compound with a pentagonal bipyramidal coordination geometry. Their crystal field parameters and orientations of the magnetic easy axes were obtained from the simulation of the magnetic data and the electrostatic model calculation. PMID:26593051

  18. Injectable formulations for an intravitreal sustained-release application of a novel single-chain VEGF antibody fragment.

    PubMed

    Asmus, Lutz R; Grimshaw, John P A; Richle, Philipp; Eicher, Barbara; Urech, David M; Gurny, Robert; Möller, Michael

    2015-09-01

    Sustained-release formulations of a single-chain anti-VEGF-A antibody fragment were investigated in vitro toward their potential use for intravitreal applications. The hydrophobic polyester hexylsubstituted poly(lactic acid) (hexPLA) was selected as the sustained-release excipient for its biodegradability and semi-solid aggregate state, allowing an easy and mild formulation procedure. The lyophilized antibody fragment ESBA903 was micronized and incorporated into the liquid polymer matrix by cryo-milling, forming homogeneous and injectable suspensions. The protein showed excellent compatibility with the hexPLA polymer and storage stability at 4°C for 10 weeks. Additionally, hexPLA shielded the incorporated active substance from the surrounding medium, resulting in a better stability of ESBA903 inside the polymer than after its release in the buffer solution. Formulations of ESBA903 with hexPLA having drug loadings between 1.25% and 5.0% and polymer molecular weights of 1500 g/mol, 2500 g/mol, 3500 g/mol and 5000 g/mol were investigated regarding their in vitro release. All formulations except with the highest molecular weight formed spherical depots in aqueous buffer solutions and released the antibody fragment for at least 6-14 weeks. The polymer viscosity derived from the molecular weight strongly influenced the release rate, while the drug loading had minor influence, allowing customization of the release profile and the daily drug release. Size exclusion chromatography and SDS-PAGE revealed that the antibody fragment structure was kept intact during incorporation and release from the liquid matrix. Furthermore, the released protein monomer maintained its high affinity to human VEGF-A, as measured by surface plasmon resonance analysis. Formulations of ESBA903 in hexPLA meet the basic needs to be used for intravitreal sustained-release applications in age-related macular degeneration treatment. PMID:25779352

  19. Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins.

    PubMed

    Goyal, A; Batra, J K

    2000-01-15

    Chimaeric toxins have considerable therapeutic potential to treat various malignancies. We have previously used the fungal ribonucleolytic toxin restrictocin to make chimaeric toxins in which the ligand was fused at either the N-terminus or the C-terminus of the toxin. Chimaeric toxins containing ligand at the C-terminus of restrictocin were shown to be more active than those having ligand at the N-terminus of the toxin. Here we describe the further engineering of restrictocin-based chimaeric toxins, anti-TFR(scFv)-restrictocin and restrictocin-anti-TFR(scFv), containing restrictocin and a single chain fragment variable (scFv) of a monoclonal antibody directed at the human transferrin receptor (TFR), to enhance their cell-killing activity. To promote the independent folding of the two proteins in the chimaeric toxin, a linear flexible peptide, Gly-Gly-Gly-Gly-Ser, was inserted between the toxin and the ligand to generate restrictocin-linker-anti-TFR(scFv) and anti-TFR(scFv)-linker-restrictocin. A 12-residue spacer, Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu, containing the recognition site for the protease furin, was incorporated between the toxin and the ligand to generate restrictocin-spacer-anti-TFR(scFv) and anti-TFR(scFv)-spacer-restrictocin. The incorporation of the proteolytically cleavable spacer enhanced the cell-killing activity of both constructs by 2-30-fold depending on the target cell line. However, the introduction of linker improved the cytotoxic activity only for anti-TFR(scFv)-linker-restrictocin. The proteolytically cleavable spacer-containing chimaeric toxins had similar cytotoxic activities irrespective of the location of the ligand on the toxin and they were found to release the restrictocin fragment efficiently on proteolysis in vitro. PMID:10620501

  20. Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis

    PubMed Central

    Kazuma, Soraya M; Cavalcante, Marcela F; Telles, Andréia ER; Maranhão, Andrea Queiroz; Abdalla, Dulcineia SP

    2013-01-01

    The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr-/-mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr-/- mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-). PMID:23924793

  1. Peanut consumption increases levels of plasma very long chain fatty acids in humans.

    PubMed

    Lam, Christina; Wong, Derek; Cederbaum, Stephen; Lim, Bennie; Qu, Yong

    2012-11-01

    Peanut consumption has been suspected of raising plasma very long chain fatty acid (VLCFA) levels in humans. The effect of peanut consumption on VLCFAs was studied in six human subjects. After 3 to 4h of peanut butter ingestion, plasma C26:0 and C26:0/C22:0 were found to be significantly elevated to levels seen in patients with peroxisomal disorders. These levels returned to normal within 12h. Peanut consumption needs to be accounted for when interpreting VLCFAs. PMID:22864056

  2. Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance.

    PubMed

    Patel, Rekha; Andrien, Bruce A

    2010-01-01

    Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field. PMID:19720041

  3. Blocking monocyte transmigration in in vitro system by an anti-CD99 human antibody in single chain fragment variable (scFv) format. Efficient large scale purification of biological active scFv from inclusion bodies in E. coli expression system

    PubMed Central

    Moricoli, Diego; Muller, William A.; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Watson, Richard; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

    2015-01-01

    Migration of leukocytes into a site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies under GMP conditions and hence, the absence of toxic reagents utilized for the solubilization and refolding steps of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting we herein describe an efficient and large scale production of the antibody fragments expressed in E.coli as insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signalling. Thanks to the original purification protocol that can be extended to other scFvs that are expressed as inclusion bodies in bacterial systems, the scFv anti-CD99 C7A herein described represents the first step towards the construction of new antibody therapeutic. PMID:24798881

  4. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    PubMed Central

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  5. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain.

    PubMed

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10(-10) M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  6. Carbohydrate chains of human thyrotropin are differentially susceptible to endoglycosidase removal on combined and free polypeptide subunits

    SciTech Connect

    Ronin, C.; Papandreou, M.; Canonne, C.; Weintraub, B.D.

    1987-09-08

    The accessibility of the asparagine-linked carbohydrate chains of human thyrotropin (hTSH) and free ..cap alpha.. and ..beta.. subunits was investigated by their susceptibility to endoglycosidases H and F as well as to peptide:N-glycosidase F. Iodinated hTSH or subunits were incubated with a commercial enzyme preparation containing both endoglycosidase F and N-glycosidase F activities and further analyzed by sodium dodecyl sulfate gel electrophoresis followed by quantitative autoradiography. The authors show that, working at the optimum of the N-glycosidase activity, the relative amount of endoglycosidase required for half-deglycosylation was 20-fold higher for native hTSH than for the reduced and dissociated subunits. Under nondenaturing conditions, the 18K ..beta.. subunit of hTSH could be readily deglycosylated to a 14K species while the 22K ..cap alpha.. subunit was largely resistant. However, both subunits were converted to an apoprotein of similar apparent molecular weight of 14K following reduction of disulfide bonds. In contrast, the free ..cap alpha.. subunit of human choriogonadotropin appeared fully sensitive to carbohydrate removal under nonreducing conditions despite the presence of a partially deglycosylated 18K intermediate at low concentration of endoglycosidase. Similarly, both hTSH-..cap alpha.. and hTSH-..beta.. could be completely deglycosylated after acid dissociation of the native hormone. While all three carbohydrate chains of hTSH are sensitive to pure peptide:N-glycosidase F, only one on ..cap alpha.. and the single oligosaccharide present on ..beta.. in hTSH appeared to be cleaved by pure endoglycosidase F. These findings indicate that while the carbohydrate chain on ..beta.. is not involved in ..cap alpha..BETA association, the oligosaccharides on ..cap alpha.. are hindered when hTSH subunits are combined.

  7. Aberrant immunoglobulin synthesis in light chain amyloidosis. Free light chain and light chain fragment production by human bone marrow cells in short-term tissue culture.

    PubMed Central

    Buxbaum, J

    1986-01-01

    Bone marrow cells obtained from 14 patients with light chain amyloid (AL) deposition were examined by biosynthetic labeling techniques. These analyses identified free monoclonal light chain (L-chain) synthesis even in those patients whose serum or urine contained no M protein or free L-chains or only an intact M protein. The experiments also identified a subset of patients whose plasma cells synthesized polypeptides bearing constant region antigenic determinants that migrated more rapidly than intact L-chains on polyacrylamide gels. Since most AL fibrils contain L-chain fragments rather than intact L-chains, these studies suggested that the genesis of the fibril components may reflect aberrant synthesis, proteolytic processing, or both. We also noted that in some individuals the pattern of Ig synthesis normalized after several courses of cytotoxic therapy. Thus, we could use bone marrow Ig synthesis as a sensitive biochemical parameter for monitoring therapy. Finally, the presence of aberrant synthetic products in these clones raised questions about their origin with respect to the normal processes of transcription, translation, and posttranslational modification in Ig-producing cells. Images PMID:3091637

  8. Effects of resistant starch type III polymorphs on human colon microbiota and short chain fatty acids in human gut models.

    PubMed

    Lesmes, Uri; Beards, Emma J; Gibson, Glenn R; Tuohy, Kieran M; Shimoni, Eyal

    2008-07-01

    This study probed the possible effects of type III resistant starch (RS) crystalline polymorphism on RS fermentability by human gut microbiota and the short chain fatty acids production in vitro. Human fecal pH-controlled batch cultures showed RS induces an ecological shift in the colonic microbiota with polymorph B inducing Bifidobacterium spp. and polymorph A inducing Atopobium spp. Interestingly, polymorph B also induced higher butyrate production to levels of 0.79 mM. In addition, human gut simulation demonstrated that polymorph B promotes the growth of bifidobacteria in the proximal part of the colon and double their relative proportion in the microbiota in the distal colon. These findings suggest that RS polymorph B may promote large bowel health. While the findings are limited by study constraints, they do raise the possibility of using different thermal processing to delineate differences in the prebiotic capabilities of RS, especially its butryrogenicity in the human colon. PMID:18543927

  9. Genetic Modification of Mesenchymal Stem Cells to Express a Single-Chain Antibody Against EGFRvIII on the Cell Surface

    PubMed Central

    Balyasnikova, Irina V.; Franco-Gou, Rosa; Mathis, J. Michael; Lesniak, Maciej S.

    2010-01-01

    Human adult mesenchymal stem cells (hMSC) are under active investigation as cellular carriers for gene therapy. hMSC possess natural tropism toward tumors, however, the targeting of hMSC to specific cell populations within tumors is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumors express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSC to express a single-chain antibody (scFv) to EGFRvIII on their surface. Nucleofection was used to transfect hMSC with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSC was selected based on antibiotic resistance and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSC. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSC expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and at least 7-fold increased retention in human U87-EGFRvIII expressing tumors in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSC with a single-chain antibody against an antigen expressed on the surface of tumor cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. PMID:19937911

  10. Establishing a value chain for human factors in nuclear power plantcontrol room modernization

    SciTech Connect

    Joe, Jeffrey Clark; Thomas, Kenneth David; Boring, Ronald Laurids

    2015-07-01

    Commercial nuclear power plants in the United States (U.S.) have operated reliably and efficiently for decades. With the life extensions of plants now being planned for operation beyond their original operating licenses, there are opportunities to achieve even greater efficiencies, while maintaining high operational reliabilities, with strategic, risk- and economically-informed, upgrades to plant systems and infrastructure. The U.S. Department of Energy’s Light Water Reactor Sustainability (LWRS) program supports the commercial nuclear industry’s modernization efforts through research and development (R&D) activities across many areas to help establish the technical and economic bases for modernization activities. The Advanced Instrumentation, Information, and Control Systems Technologies pathway is one R&D focus area for the LWRS program, and has researchers at Idaho National Laboratory working with select utility partners to use human factors and instrumentation and controls R&D to help modernize the plant’s main control room. However, some in the nuclear industry have not been as enthusiastic about using human factors R&D to inform life extension decision making. Part of the reason for this may stem from uncertainty decision-makers have regarding how human factors fits into the value chain for nuclear power plant control room modernization. This paper reviews past work that has attempted to demonstrate the value of human factors, and then describes the value chain concept, how it applies to control room modernization, and then makes a case for how and why human factors is an essential link in the modernization value chain.

  11. Detection of Merkel cell polyomavirus in the human tissues from 41 Japanese autopsy cases using polymerase chain reaction.

    PubMed

    Matsushita, Michiko; Kuwamoto, Satoshi; Iwasaki, Takeshi; Higaki-Mori, Hiromi; Yashima, Shoji; Kato, Masako; Murakami, Ichiro; Horie, Yasushi; Kitamura, Yukisato; Hayashi, Kazuhiko

    2013-01-01

    It has recently been shown that approximately 80% of Merkel cell carcinomas harbor a novel polyomavirus named Merkel cell polyomavirus (MCPyV). MCPyV has been detected in human tissue samples. However, detailed distribution of MCPyV in non-neoplastic Japanese human tissues remains unclear. To address this, we used single or real-time quantitative polymerase chain reaction (PCR) for 41 autopsy cases. PCR revealed MCPyV-DNA in non-neoplastic samples: total, 29/41 (71%); adult, 29/39 (74%); fetus or infant, 0/2; men, 24/28 (86%); women, 5/13 (38%); total human tissues, 66/572 (12%); skin, 8/15 (53%); adrenal gland, 9/33 (27%), and other 16 organs (4-25%). This study first reported the presence of MCPyV-DNA in non-neoplastic tissues of thyroid gland, adrenal gland, spleen, bone marrow, stomach, gallbladder, pancreas, heart, and aorta. PCR revealed that viral load ranged from 0.00026 to 0.22 in all MCPyV-positive tissues compared with Merkel cell carcinoma samples. These detailed PCR data showed higher prevalence of MCPyV infection in Japanese men than women (p = 0.004) and broad distribution of MCPyV with low viral load in more non-neoplastic human tissues than in the previous reports. These data provide valuable insights for further studies of MCPyV infection and MCPyV-related diseases. PMID:22986833

  12. Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

    PubMed Central

    Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

    2013-01-01

    Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

  13. Anti-metastatic effects of DNA vaccine encoding single-chain trimer composed of MHC I and vascular endothelial growth factor receptor 2 peptide.

    PubMed

    Chen, Ruiling; Wang, Shengchao; Yao, Yunliang; Zhou, Yun; Zhang, Chong; Fang, Jie; Zhang, Dayong; Zhang, Lihuang; Pan, Jianping

    2015-05-01

    Vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling is the key rate-limiting step in angiogenesis. VEGFR2 serves as the most important target of anti-angiogenic therapy for cancers. Single-chain trimer (SCT) comprising antigen peptide, β2-microglobulin (β2m), and major histocompatibility complex (MHC) class I heavy chain was a particularly powerful strategy involved in the increase of the potency of DNA vaccine against tumors and infections. In the present study, we constructed an SCT-encoding VEGFR2 antigen peptide [aa400-408, also known as kinase insert domain-containing receptor (KDR2)], β2m, and mouse MHC class I heavy chain H-2Db [pcDNA3.1(+)-KDR2-β2m-H-2Db, or SCT-KDR2]. The constructed SCT-KDR2 DNA was efficiently expressed in the human A293 embryonic kidney cell line. Intradermal immunization of C57BL/6 mice with SCT-KDR2 DNA was able to successfully break self-immunological tolerance and induce robust cytotoxic T‑lymphocyte (CTL) response to VEGFR2, leading to marked suppression of tumor cell‑induced angiogenesis and metastasis in murine models of B16 melanoma and 3LL Lewis lung carcinoma. Taken together, the results showed that VEGFR2-targeted SCT vaccination is an effective modality that can be utilized in anti-angiogenic active immunotherapy for various types of cancer. PMID:25739076

  14. Single-molecule imaging of hyaluronan in human synovial fluid

    NASA Astrophysics Data System (ADS)

    Kappler, Joachim; Kaminski, Tim P.; Gieselmann, Volkmar; Kubitscheck, Ulrich; Jerosch, Jörg

    2010-11-01

    Human synovial fluid contains a high concentration of hyaluronan, a high molecular weight glycosaminoglycan that provides viscoelasticity and contributes to joint lubrication. In osteoarthritis synovial fluid, the concentration and molecular weight of hyaluronan decrease, thus impairing shock absorption and lubrication. Consistently, substitution of hyaluronan (viscosupplementation) is a widely used treatment for osteoarthritis. So far, the organization and dynamics of hyaluronan in native human synovial fluid and its action mechanism in viscosupplementation are poorly characterized at the molecular level. Here, we introduce highly sensitive single molecule microscopy to analyze the conformation and interactions of fluorescently labeled hyaluronan molecules in native human synovial fluid. Our findings are consistent with a random coil conformation of hyaluronan in human synovial fluid, and point to specific interactions of hyaluronan molecules with the synovial fluid matrix. Furthermore, single molecule microscopy is capable of detecting the breakdown of the synovial fluid matrix in osteoarthritis. Thus, single molecule microscopy is a useful new method to probe the structure of human synovial fluid and its changes in disease states like osteoarthritis.

  15. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  16. Baculovirus display of single chain antibody (scFv) using a novel signal peptide

    PubMed Central

    2010-01-01

    Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV vector resulted in

  17. Peptide docking of HIV-1 p24 with single chain fragment variable (scFv) by CDOCKER algorithm

    NASA Astrophysics Data System (ADS)

    Karim, Hana Atiqah Abdul; Tayapiwatana, Chatchai; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abdul; Lee, Vannajan Sanghiran

    2014-10-01

    In search for the important residues that might have involve in the binding interaction between the p24 caspid protein of HIV-1 fragment (MET68 - PRO90) with the single chain fragment variable (scFv) of FAB23.5, modern computational chemistry approach has been conducted and applied. The p24 fragment was initially taken out from the 1AFV protein molecule consisting of both light (VL) and heavy (VH) chains of FAB23.5 as well as the HIV-1 caspid protein. From there, the p24 (antigen) fragment was made to dock back into the protein pocket receptor (antibody) by using the CDOCKER algorithm to conduct the molecular docking process. The score calculated from the CDOCKER gave 15 possible docked poses with various docked ligand's positions, the interaction energy as well as the binding energy. The best docked pose that imitates the original antigen's position was determined and further processed to the In Situ minimization to obtain the residues interaction energy as well as to observe the hydrogen bonds interaction in the protein-peptide complex. Based on the results demonstrated, the specific residues in the complex that have shown immense lower interaction energies in the 5Å vicinity region from the peptide are from the heavy chain (VH:TYR105) and light chain (VL: ASN31, TYR32, and GLU97). Those residues play vital roles in the binding mechanism of Antibody-Antigen (Ab-Ag) complex of p24 with FAB23.5.

  18. A uranium-based UO2(+)-Mn2+ single-chain magnet assembled trough cation-cation interactions.

    PubMed

    Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Caciuffo, Roberto; Colineau, Eric; Tuna, Floriana; Magnani, Nicola; de Geyer, Arnaud; Pécaut, Jacques; Mazzanti, Marinella

    2014-01-13

    Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO2(salen)(py)][M(py)4](NO3)}]n, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO2(salen)py][Cp*2Co] (Cp*=pentamethylcyclopentadienyl) with Cd(NO3)2 or Mn(NO3)2 in pyridine. The infinite UMn chain displays a high relaxation barrier of 134±0.8 K (93±0.5 cm(-1)), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T<6 K, with an impressive coercive field of 3.4 T at 2 K. PMID:24311434

  19. Role of single-point mutations and deletions on transition temperatures in ideal proteinogenic heteropolymer chains in the gas phase.

    PubMed

    Olivares-Quiroz, L

    2016-07-01

    A coarse-grained statistical mechanics-based model for ideal heteropolymer proteinogenic chains of non-interacting residues is presented in terms of the size K of the chain and the set of helical propensities [Formula: see text] associated with each residue j along the chain. For this model, we provide an algorithm to compute the degeneracy tensor [Formula: see text] associated with energy level [Formula: see text] where [Formula: see text] is the number of residues with a native contact in a given conformation. From these results, we calculate the equilibrium partition function [Formula: see text] and characteristic temperature [Formula: see text] at which a transition from a low to a high entropy states is observed. The formalism is applied to analyze the effect on characteristic temperatures [Formula: see text] of single-point mutations and deletions of specific amino acids [Formula: see text] along the chain. Two probe systems are considered. First, we address the case of a random heteropolymer of size K and given helical propensities [Formula: see text] on a conformational phase space. Second, we focus our attention to a particular set of neuropentapeptides, [Met-5] and [Leu-5] enkephalins whose thermodynamic stability is a key feature on their coupling to [Formula: see text] and [Formula: see text] receptors and the triggering of biochemical responses. PMID:26818963

  20. Conformational changes in single carboxymethylcellulose chains on a highly oriented pyrolytic graphite surface under different salt conditions.

    PubMed

    Ueno, Tomotsugu; Yokota, Shingo; Kitaoka, Takuya; Wariishi, Hiroyuki

    2007-05-21

    Conformational changes in individual carboxymethylcellulose (CMC) chains deposited on a highly oriented pyrolytic graphite (HOPG) surface were investigated by atomic force microscopy (AFM). A small amount of CMC solution with various salt concentrations was deposited onto the HOPG surface. The CMC molecular chains adsorbed onto the HOPG surface were clearly visualized using tapping-mode AFM under ambient conditions, as compared with those on a hydrophilic mica surface. Each CMC chain was distinguishable at the molecular level based on the vertical profiles of the AFM images, and probably aligned along the HOPG crystal lattice. Higher NaCl concentrations brought about dramatic conformational changes from aligned single chains to globular aggregates via the molecular network structure only on the HOPG surface through electrostatic screening of the CM groups. Although CMC is a water-soluble hydrophilic polyelectrolyte, some interaction, possibly due to a CH-pi bonding between the glucopyranosic axial plane of CMC and the aromatic rings of HOPG, is considered to be effective and dominant for the unique molecular attachment. These phenomena would imply the potential use of HOPG as a substrate for not only molecular imaging, but also for nano-scale morphological control of cellulosic polymers and other structural polysaccharides. PMID:17316582

  1. Organization of polymer chains onto long, single-wall carbon nano-tubes: effect of tube diameter and cooling method.

    PubMed

    Kumar, Sunil; Pattanayek, Sudip K; Pereira, Gerald G

    2014-01-14

    We use molecular dynamics simulations to investigate the arrangement of polymer chains when absorbed onto a long, single-wall carbon nano-tube (SWCNT). We study the conformation and organization of the polymer chains on the SWCNT and their dependence on the tube's diameter and the rate of cooling. We use two types of cooling processes: direct quenching and gradual cooling. The radial density distribution function and bond orientational order parameter are used to characterize the polymer chain structure near the surface. In the direct cooling process, the beads of the polymer chain organize in lamella-like patterns on the surface of the SWCNT with the long axis of the lamella parallel to the axis of the SWCNT. In a stepwise, gradual cooling process, the polymer beads form a helical pattern on the surface of a relatively thick SWCNT, but form a lamella-like pattern on the surface of a very thin SWCNT. We develop a theoretical (free energy) model to explain this difference in pattern structures for the gradual cooling process and also provide a qualitative explanation for the pattern that forms from the direct cooling process. PMID:24437908

  2. Novel Monoclonal Antibodies for Studies of Human and Rhesus Macaque Secretory Component and Human J-Chain.

    PubMed

    Zhang, Ruijun; Alam, S Munir; Yu, Jae-Sung; Scearce, Richard; Lockwood, Bradley; Hwang, Kwan-Ki; Parks, Robert; Permar, Sallie; Brandtzaeg, Per; Haynes, Barton F; Liao, Hua-Xin

    2016-08-01

    Immunoglobulin A (IgA) antibodies exist in monomeric, dimeric, and secretory forms. Dimerization of IgA depends on a 15-kD polypeptide termed "joining (J) chain," which is also part of the binding site for an epithelial glycoprotein called "secretory component (SC)," whether this after apical cleavage on secretory epithelia is ligand bound in secretory IgA (SIgA) or in a free form. Uncleaved membrane SC, also called the "polymeric Ig receptor," is thus crucial for transcytotic export of SIgA to mucosal surfaces, where it interacts with and modulates commensal bacteria and mediates protective immune responses against exogenous pathogens. To evaluate different forms of IgA, we have produced mouse monoclonal antibodies (MAbs) against human J-chain and free SC. We found that J-chain MAb 9A8 and SC MAb 9H7 identified human dimeric IgA and SIgA in enzyme-linked immunoassay and western blot analysis, as well as functioning in immunohistochemistry to identify cytoplasmic IgA of intestinal lamina propria plasmablasts/plasma cells and crypt epithelium of distal human intestine. Finally, we demonstrated that SC MAb 9H7 cross-reacted with rhesus macaque SIgA. These novel reagents should be of use in the study of the biology of various forms of IgA in humans and SIgA in macaques, as well as in monitoring the production and/or isolation of these forms of IgA. PMID:27386924

  3. Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

    PubMed Central

    Yin, Wai-Fong; Purmal, Kathiravan; Chin, Shenyang; Chan, Xin-Yue; Chan, Kok-Gan

    2012-01-01

    We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium. PMID:23202161

  4. Targeted inactivation of murine laminin gamma2-chain gene recapitulates human junctional epidermolysis bullosa.

    PubMed

    Meng, Xianmin; Klement, John F; Leperi, Dominic A; Birk, David E; Sasaki, Takako; Timpl, Rupert; Uitto, Jouni; Pulkkinen, Leena

    2003-10-01

    Junctional forms of epidermolysis bullosa (JEB) are associated with mutations in six distinct genes expressed in the cutaneous basement membrane zone; these include LAMA3, LAMB3, and LAMC2, which encode laminin 5 subunit polypeptides, the alpha3-, beta3-, and gamma2-chains, respectively. Here we generated a mouse model for JEB by inactivating the laminin gamma2-chain gene by targeted frameshift deletion of exon 8 in Lamc2. Heterozygous mice were phenotypically normal, whereas the majority of Lamc2-/- mice showed blistering phenotype on days 1 to 2 and died within 5 days of birth. The Lamc2-/- mice demonstrated absent expression of laminin gamma2-chain on the basement membrane zone as well as attenuated expression of alpha3- and beta3-chains of laminin. Transmission electron microscopy revealed rudimentary, poorly developed hemidesmosomes. The epidermis of the Lamc2-/- mice revealed induced apoptosis in the basal cells of the blistered skin, suggesting that cell-matrix adhesion provided by laminin 5 plays a role in cell survival in vivo. Cultured Lamc2-/- keratinocytes demonstrated slightly positive staining with gamma2-chain-specific antibodies, which could be explained by the presence of a transcript with partial restoration of the reading frame owing to alternative splicing in vitro. These cells proliferated in different matrices and attached to type IV collagen and Matrigel as efficiently as the wild-type keratinocytes, whereas their attachment on plastic and laminin was significantly weaker. In summary, Lamc2-/- mouse recapitulates human JEB and provides novel insight into the role of laminin 5 in keratinocyte biology. PMID:14632187

  5. Preparation and identification of a single-chain variable fragment antibody against Newcastle diseases virus F48E9.

    PubMed

    Li, Benqiang; Ye, Jiaxin; Lin, Yuan; Wang, Man; Zhu, Jianguo

    2014-10-15

    This article describes a proposed method for convenient and efficient detection of Newcastle diseases virus (NDV) that uses the fusion of single-chain variable fragment (scFv) and pOPE101 vector. In order to select the single chain variable fragment (scFv) against NDV F48E9, the total RNA was extracted from the spleen of immunized chicken, and then was converted into cDNA via the reverse transcription. The scFv was spliced by using splice-overlap extension polymerase chain reaction (SOE-PCR). The scFv gene was cloned into a pOPE101 vector and expressed in E. coli. Under the optimized conditions, antibody affinity was studied by indirect ELISA. One positive clone was selected by ELISA screening, named ZL.6. Based on the positive clone and the germline sequence, the results of sequence analysis showed that there are more variation in CDR of VH and VL. In addition, BHK21 cell culture was conducted to examine the potential antiviral activity of ZL.6. The experimental result demonstrated that ZL.6 was able to neutralize NDV F48E9 which infected BHK21 cells. So ZL.6 will be proved useful for further characterization of NDV as potential diagnostic tool and therapeutic agent. PMID:25183016

  6. Variability of intracellular lactate dehydrogenase isoenzymes in single human erythrocytes

    SciTech Connect

    Xue, Q.; Yeung, E.S. Iowa State Univ., Ames, IA )

    1994-04-01

    Trace amounts of enzymes within single human erythrocytes can be quantified by a combination of on-column reaction and capillary electrophoresis. A detection limit of 1.3 x 10[sup [minus]21] mol of LDH was achieved with laser-induced fluorescence by monitoring the product of the enzyme-catalyzed reaction between lactate and NAD[sup +]. Single erythrocyte analysis clearly isolates the major forms of LDH. The variation of total LDH activity in a population of cells from a single individual is large, but the relative activities of the isoenzymes LDH-1 and LDH-2 are fairly constant. This can be explained by the distribution of cell age in the population. A lower enzyme activity is indicative of senescence. The efficient separation of different LDH forms and the high detection sensitivity opens up the possibility of multiple-enzyme assays with a single mammalian cell. 41 refs., 5 figs.

  7. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  8. Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism.

    PubMed

    Morrison, Douglas J; Preston, Tom

    2016-05-01

    The formation of SCFA is the result of a complex interplay between diet and the gut microbiota within the gut lumen environment. The discovery of receptors, across a range of cell and tissue types for which short chain fatty acids SCFA appear to be the natural ligands, has led to increased interest in SCFA as signaling molecules between the gut microbiota and the host. SCFA represent the major carbon flux from the diet through the gut microbiota to the host and evidence is emerging for a regulatory role of SCFA in local, intermediary and peripheral metabolism. However, a lack of well-designed and controlled human studies has hampered our understanding of the significance of SCFA in human metabolic health. This review aims to pull together recent findings on the role of SCFA in human metabolism to highlight the multi-faceted role of SCFA on different metabolic systems. PMID:26963409

  9. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells.

    PubMed

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  10. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

    PubMed Central

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  11. Comparing single and cumulative dosing procedures in human triazolam discriminators.

    PubMed Central

    Smith, B J; Bickel, W K

    1999-01-01

    This study evaluated a cumulative dosing procedure for drug discrimination with human participants. Four participants learned to discriminate triazolam (0.35 mg/70 kg) from placebo. A crossover design was used to compare the results under a single dosing procedure with results obtained under a cumulative dosing procedure. Under the single dosing procedure, a dose of triazolam (0, 0.05, 0.15, or 0.35 mg/70 kg) or secobarbital (0, 25, 75, or 175 mg/70 kg) was administered 45 min before assessment. Determining each dose-effect curve thus required four sessions. Under the cumulative dosing procedure, four doses of triazolam (0, 0.05, 0.10, and 0.20 mg/70 kg) or secobarbital (0, 25, 50, and 100 mg/70 kg) were administered approximately 55 min apart, producing a complete dose-effect curve in one four-trial session. Regardless of procedure, triazolam and secobarbital produced discriminative stimulus and self-reported effects similar to previous single dosing studies in humans. Shifts to the right in cumulative dose-effect curves compared to single dose-effect curves occurred on several self-report measures. When qualitative stimulus functions rather than quantitative functions are of interest, application of cumulative dosing may increase efficiency in human drug discrimination. PMID:10344022

  12. A chain modeling approach to estimate the impact of soil cadmium pollution on human dietary exposure.

    PubMed

    Franz, Eelco; Römkens, Paul; van Raamsdonk, Leo; van der Fels-Klerx, Ine

    2008-12-01

    Cadmium in soil poses a risk for human health, due to its accumulation in food and feed crops. The extent of accumulation depends strongly on soil type and the degree of pollution. The objective of the present study was to develop a predictive model to estimate human dietary cadmium exposure from soil characteristics. This chain model consists of three basic steps: (i) calculation of plant cadmium levels from soil contamination levels and soil characteristics, (ii) calculation of animal transfer from consumption and contamination levels, and (iii) human exposure from both plant and animal products. Six soil scenarios were assessed, reflecting a specific contaminated region and ranging from 0.5 mg/kg of Cd (pH 4.5) to 2.5 mg/kg of Cd (pH 5.5). Cadmium levels in feed crops and vegetables were estimated with regression and mathematical models. Animal exposure and transfer to cattle kidneys, livers, and meat were calculated using a consumption database and a parameterized linear simulation model. Human exposure was estimated by Monte Carlo simulation, using a consumption database. The median human exposure for the different scenarios ranged from 0.24 to 0.98 microg/kg of body weight per day, which is comparable to results obtained from exposure levels based on observed field contamination data. The study shows that a chain model approach from soil contamination to human exposure, including animal exposure and transfer to animal products, can successfully be applied. The model can be used for fast evaluation of dietary cadmium exposure and the identification of risk areas based on soil conditions. PMID:19244905

  13. Single-chain folding of polymers for catalytic systems in water.

    PubMed

    Terashima, Takaya; Mes, Tristan; De Greef, Tom F A; Gillissen, Martijn A J; Besenius, Pol; Palmans, Anja R A; Meijer, E W

    2011-04-01

    Enzymes are a source of inspiration for chemists attempting to create versatile synthetic catalysts. In order to arrive at a polymeric chain carrying catalytic units separated spatially, it is a prerequisite to fold these polymers in water into well-defined compartmentalized architectures thus creating a catalytic core. Herein, we report the synthesis, physical properties, and catalytic activity of a water-soluble segmented terpolymer in which a helical structure in the apolar core is created around a ruthenium-based catalyst. The supramolecular chirality of this catalytic system is the result of the self-assembly of benzene-1,3,5-tricarboxamide side chains, while the catalyst arises from the sequential ruthenium-catalyzed living radical polymerization of the different monomers followed by ligand exchange. The polymers exhibit a two-state folding process and show transfer hydrogenation in water. PMID:21405022

  14. INFLATING A CHAIN OF X-RAY-DEFICIENT BUBBLES BY A SINGLE JET ACTIVITY EPISODE

    SciTech Connect

    Refaelovich, Michael; Soker, Noam E-mail: soker@physics.technion.ac.il

    2012-08-10

    We show that a continuous jet with time-independent launching properties can inflate a chain of close and overlapping X-ray deficient bubbles. Using the numerical code PLUTO we run 2.5D (i.e., a spherical coordinate system with cylindrical symmetry) hydrodynamic simulations and study the interaction of the jets with the intracluster medium. A key process is vortex fragmentation due to several mechanisms, including vortex-shedding and Kelvin-Helmholtz instabilities. Our results can account for the structure of two opposite chains of close bubbles as observed in the galaxy cluster Hydra A. Our results imply that the presence of multiple pairs of bubbles does not necessarily imply several jet-launching episodes. This finding might have implications for feedback mechanisms operating by jets.

  15. Dynamic Human Body Modeling Using a Single RGB Camera

    PubMed Central

    Zhu, Haiyu; Yu, Yao; Zhou, Yu; Du, Sidan

    2016-01-01

    In this paper, we present a novel automatic pipeline to build personalized parametric models of dynamic people using a single RGB camera. Compared to previous approaches that use monocular RGB images, our system can model a 3D human body automatically and incrementally, taking advantage of human motion. Based on coarse 2D and 3D poses estimated from image sequences, we first perform a kinematic classification of human body parts to refine the poses and obtain reconstructed body parts. Next, a personalized parametric human model is generated by driving a general template to fit the body parts and calculating the non-rigid deformation. Experimental results show that our shape estimation method achieves comparable accuracy with reconstructed models using depth cameras, yet requires neither user interaction nor any dedicated devices, leading to the feasibility of using this method on widely available smart phones. PMID:26999159

  16. Dynamic Human Body Modeling Using a Single RGB Camera.

    PubMed

    Zhu, Haiyu; Yu, Yao; Zhou, Yu; Du, Sidan

    2016-01-01

    In this paper, we present a novel automatic pipeline to build personalized parametric models of dynamic people using a single RGB camera. Compared to previous approaches that use monocular RGB images, our system can model a 3D human body automatically and incrementally, taking advantage of human motion. Based on coarse 2D and 3D poses estimated from image sequences, we first perform a kinematic classification of human body parts to refine the poses and obtain reconstructed body parts. Next, a personalized parametric human model is generated by driving a general template to fit the body parts and calculating the non-rigid deformation. Experimental results show that our shape estimation method achieves comparable accuracy with reconstructed models using depth cameras, yet requires neither user interaction nor any dedicated devices, leading to the feasibility of using this method on widely available smart phones. PMID:26999159

  17. Conductance and spin-filter effects of oxygen-incorporated Au, Cu, and Fe single-atom chains

    SciTech Connect

    Zheng, Xiaolong; Xie, Yi-Qun Ye, Xiang; Ke, San-Huang

    2015-01-28

    We studied the spin-polarized electron transport in oxygen-incorporated Au, Cu, and Fe single-atom chains (SACs) by first-principles calculations. We first investigated the mechanism responsible for the low conductance (<1G{sub 0}) of the Au and Cu SACs in an oxygen environment reported in recent experiments. We found that for the Au SACs, the low conductance plateau around 0.6G{sub 0} can be attributed to a distorted chain doped with a single oxygen atom, while the 0.1G{sub 0} conductance comes from a linear chain incorporated with an oxygen molecule and is caused by an antibonding state formed by oxygen's occupied frontier orbital with d{sub z} orbitals of adjacent Au atoms. For the Cu SACs, the conductance about 0.3G{sub 0} is ascribed to a special configuration that contains Cu and O atoms in an alternating sequence. This exhibits an even-odd conductance oscillation with an amplitude of ∼0.1G{sub 0}. In contrast, for the alternating Fe-O SACs, conductance overall decreases with an increase in O atoms and it approaches nearly zero for the chain with more than four O atoms. While the Cu-O SACs behave as perfect spin filters for one spin channel due to the half metallic nature, the Fe-O SACs can serve as perfect spin filters for two spin channels depending on the polarity of the applied gate voltage.

  18. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII. PMID:23244324

  19. Divergence of human [alpha]-chain constant region gene sequences: A novel recombinant [alpha]2 gene

    SciTech Connect

    Chintalacharuvu, K. R.; Morrison, S.L. ); Raines, M. )

    1994-06-01

    IgA is the major Ig synthesized in humans and provides the first line of defense at the mucosal surfaces. The constant region of IgA heavy chain is encoded by the [alpha] gene on chromosome 14. Previous studies have indicated the presence of two [alpha] genes, [alpha]1 and [alpha]2 existing in two allotypic forms, [alpha]2 m(1) and [alpha]2 m(2). Here the authors report the cloning and complete nucleotide sequence determination of a novel human [alpha] gene. Nucleotide sequence comparison with the published [alpha] sequences suggests that the gene arose as a consequence of recombination or gene conversion between the two [alpha]2 alleles. The authors have expressed the gene as a chimeric protein in myeloma cells indicating that it encodes a functional protein. The novel IgA resembles IgA2 m(2) in that disulfide bonds link H and L chains. This novel recombinant gene provides insights into the mechanisms of generation of different constant regions and suggests that within human populations, multiple alleles of [alpha] may be present providing IgAs of different structures.

  20. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

    PubMed

    Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan; He, Ting-Fang; Greenbaum, Alon; Sohn, Chang Ho; Lignell, Antti; Choi, Harry M T; Gradinaru, Viviana; Pierce, Niles A; Cai, Long

    2016-08-01

    Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. PMID:27342713

  1. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing

    PubMed Central

    Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan; He, Ting-Fang; Greenbaum, Alon; Sohn, Chang Ho; Lignell, Antti; Choi, Harry M. T.; Gradinaru, Viviana; Pierce, Niles A.

    2016-01-01

    Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas – from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. PMID:27342713

  2. Promoter for the human ferritin heavy chain-encoding gene (FERH): structural and functional characterization.

    PubMed

    Bevilacqua, M A; Giordano, M; D'Agostino, P; Santoro, C; Cimino, F; Costanzo, F

    1992-02-15

    We conducted a functional analysis of the promoter for the human ferritin heavy chain-encoding gene (pFERH) in HepG2 and HeLa cells. The activity of pFERH is equivalent in both cell types, despite their different ferritin (Fer) isotypes. Transfections of a series of 5'-deletion mutants indicate that pFERH activity is essentially dependent on two motifs. One of them, accounting for about 50% of the total transcriptional activity, is recognized by the RNA polymerase II transcription factor, Sp1, and the other by a low-affinity factor present in both the cell types analyzed. PMID:1541403

  3. Interaction of Human Immunodeficiency Virus Type 2 Vpx and Invariant Chain

    PubMed Central

    Pancio, Heather A.; Vander Heyden, Nancy; Kosuri, Kavitha; Cresswell, Peter; Ratner, Lee

    2000-01-01

    Vpx is a virion-associated protein of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency viruses. The yeast two-hybrid system was used to identify invariant chain (Ii) as a cellular protein that interacts with HIV-2 Vpx. Vpx-Ii interaction was confirmed in cell-free reactions using bacterially expressed glutathione S-transferase fusion proteins and by coimmunoprecipitation in transfected and infected cells. In chronically infected cells expressing Vpx, Ii levels were markedly decreased, presumably due to enhanced degradation. These findings suggest that Vpx may disrupt major histocompatibility complex class II antigen presentation. PMID:10846101

  4. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.

    PubMed

    Rut, Wioletta; Drag, Marcin

    2016-09-01

    The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal. PMID:27176742

  5. A single session of open kinetic chain movements emphasizing speed improves speed of movement and modifies postural control in stroke.

    PubMed

    Gray, Vicki L; Ivanova, Tanya D; Garland, S Jayne

    2016-01-01

    Little attention has been given to training speed of movement, even though functional activities require quick submaximal contractions. Closed kinetic chain (CKC) exercises are considered more functional; however, the best method for training speed is not known. A single bout of open kinetic chain (OKC) exercises emphasizing speed was performed to determine whether movement velocity and muscle activation would improve in a single session and whether the improvements transfer to a physiological balance task. Eleven participants <1 year post-stroke performed an arm raise task before and after a single session of fast OKC exercises. Surface electromyography (EMG) from soleus (SOL), tibialis anterior (TA), biceps femoris (BF) and rectus femoris (RF) muscles, peak velocity and average power were recorded during the OKC exercises. EMG from SOL, TA, BF and RF and center of pressure (COP) velocity were measured during arm raise task. At the end of the OKC exercises, velocity, power and TA, BF and RF EMG area increased. The arm acceleration and BF EMG area increased significantly during the arm raise. The improvements observed at the end of the OKC exercises transferred to the arm raise task. The improvements in balance were comparable to those previously seen after CKC exercises. PMID:26863374

  6. Quantitative detection of tumor necrosis factor-α by single molecule counting based on a hybridization chain reaction.

    PubMed

    Dai, Shuang; Feng, Chunjing; Li, Wei; Jiang, Wei; Wang, Lei

    2014-10-15

    This work reports a novel and sensitive quantitative method for detection of tumor necrosis factor-α (TNF-α) based on single molecule counting and hybridization chain reaction (HCR). In the presence of TNF-α, sandwich-type immunocomplex was formed on the surface of glass substrate. The streptavidin acted as a bridge bounded to the biotinylated immunocomplex, which provided three sites to fixate the biotinylated initiator strands. The initiator strands triggered the chain reaction of hybridization to form a long double-helix polymer and SYBR Green I, acted as the fluorescence label, intercalated into the grooves of the long dsDNA polymer. Then, the quantitative detection of TNF-α was realized by single molecule counting. Under the optimal conditions, HCR-based single molecule counting quantitative method could successfully detect TNF-α in the range of 50 fM to 1 pM, and it revealed a reliable result for TNF-α detection in real serum. Moreover, the proposed immunosensor exhibited excellent specificity. These results greatly demonstrated that the proposed method possessed the potentiality in clinical application and it was suitable for quantification of biomarker under low concentration. PMID:24800682

  7. A Single Arabinan Chain Is Attached to the Phosphatidylinositol Mannosyl Core of the Major Immunomodulatory Mycobacterial Cell Envelope Glycoconjugate, Lipoarabinomannan*

    PubMed Central

    Kaur, Devinder; Angala, Shiva K.; Wu, Sz-Wei; Khoo, Kay-Hooi; Chatterjee, Delphi; Brennan, Patrick J.; Jackson, Mary; McNeil, Michael R.

    2014-01-01

    Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5–7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10–12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl “primer.” Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM. PMID:25231986

  8. Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

    PubMed Central

    Moreira, Gustavo M. S. G.; Conceição, Fabricio R.; McBride, Alan J. A.; Pinto, Luciano da S.

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins. PMID:24260572

  9. Mutagenesis of the human IgA1 heavy chain tailpiece that prevents dimer assembly.

    PubMed

    Atkin, J D; Pleass, R J; Owens, R J; Woof, J M

    1996-07-01

    The structural features of the human IgA1 tailpiece required for interaction with J chain in IgA dimer assembly were investigated using a protein engineering approach. Wild-type and mutant forms of IgA1 were expressed in the mouse myeloma cell line, J558L, which endogenously expresses J chain. Wild-type IgA1 was secreted as a mixture of dimers and monomers. Deletion of the entire tailpiece by stop codon introduction completely prevented dimer formation. Similarly, substitution of the penultimate residue of the tailpiece, Cys471, with serine resulted in the secretion of IgA monomers alone. Substitution of Asn459 with alanine to prevent attachment of N-linked carbohydrate to the tailpiece also resulted in markedly reduced dimer assembly. These results indicate the critical role played by the tailpiece, and Cys471 in particular, in IgA dimerization. In addition, we found tailpiece-deleted IgA1 and the Cys to Ser471 mutant IgA1 were secreted as mixtures of covalently associated monomers (alpha 2L2) and alpha L half-molecules. The tailpiece may thus play some role in promoting the association of alpha-chains required for IgA monomer assembly. PMID:8683109

  10. Gene structure for the. alpha. 1 chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and translation termination codon at the 5' end of the last exon

    SciTech Connect

    Tikka, L.; Pihlajaniemi, T.; Henttu, P.; Prockop, D.J.; Tryggvason, K. )

    1988-10-01

    Two overlapping human genomic clones that encode a short-chain collagen, designated {alpha}1(XIII), were isolated by using recently described cDNA clones. Characterization of the cosmid clones that span {approx} 65,000 base pairs (bp) of the 3' end of the gene established several unusual features of this collagen gene. The last exon encodes solely the 3' untranslated region and it begins with a complete stop codon. The 10 adjacent exons vary in size from 27 to 87 bp and two of them are 54 bp. Therefore, the {alpha}1-chain gene of type XIII collagen has some features found in genes for fibrillar collagens but other features that are distinctly different. Previous analysis of overlapping cDNA clones and nuclease S1 mapping of mRNAs indicated one alternative splicing site causing a deletion of 36 bp from the mature mRNA. The present study showed that the 36 bp is contained within the gene as a single exon and also that the gene has a 45-bp -Gly-Xaa-Xaa- repeat coding exon not found in the cDNA clones previously characterized. Nuclease S1 mapping experiments indicated that this 45-bp exon is found in normal human skin fibroblast mRNAs. Accordingly, the data demonstrate that there is alternative splicing of at least two exons of the type {alpha}1(XIII)-chain gene.

  11. Mechanically durable and highly conductive elastomeric composites from long single-walled carbon nanotubes mimicking the chain structure of polymers.

    PubMed

    Ata, Seisuke; Kobashi, Kazufumi; Yumura, Motoo; Hata, Kenji

    2012-06-13

    By using long single-walled carbon nanotubes (SWNTs) as a filler possessing the highest aspect ratio and small diameter, we mimicked the chain structure of polymers in the matrix and realized a highly conductive elastomeric composite (30 S/cm) with an excellent mechanical durability (4500 strain cycles until failure), far superior to any other reported conductive elastomers. This exceptional mechanical durability was explained by the ability of long and traversing SWNTs to deform in concert with the elastomer with minimum stress concentration at their interfaces. The conductivity was sufficient to operate many active electronics components, and thus this material would be useful for practical stretchable electronic devices. PMID:22546049

  12. Sub-diffusive electronic transport in a DNA single-strand chain with electron-phonon coupling.

    PubMed

    Sales, M O; Lyra, M L; de Moura, F A B F; Fulco, U L; Albuquerque, E L

    2015-01-28

    We investigate the electronic wavepacket dynamics in a finite segment of a DNA single-strand chain considering the electron-phonon coupling. Our theoretical approach makes use of an effective tight-binding Hamiltonian to describe the electron dynamics, together with a classical harmonic Hamiltonian to treat the intrinsic DNA vibrations. An effective time-dependent Schrödinger equation is then settled up and solved numerically for an initially localized wave-packet using the standard Dormand-Prince eighth-order Runge-Kutta method. Our numerical results indicate the presence of a sub-diffusive electronic wavepacket spread mediated by the electron-phonon interaction. PMID:25564495

  13. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    PubMed Central

    Galeffi, Patrizia; Lombardi, Alessio; Pietraforte, Immacolata; Novelli, Flavia; Di Donato, Monica; Sperandei, Maria; Tornambé, Andrea; Fraioli, Rocco; Martayan, Aline; Natali, Pier Giorgio; Benevolo, Maria; Mottolese, Marcella; Ylera, Francisco; Cantale, Cristina; Giacomini, Patrizio

    2006-01-01

    Background Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system. Results An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. Conclusion ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for

  14. Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody

    PubMed Central

    Saeed, Mesha; van Brakel, Mandy; Zalba, Sara; Schooten, Erik; Rens, Joost AP; Koning, Gerben A; Debets, Reno; ten Hagen, Timo LM

    2016-01-01

    Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with nonenhanced affinity of M1/A1, but not the one with enhanced affinity, was exclusively bound to and internalized by melanoma tumor cells expressing M1/A1. Taken together, antigen-mediated targeting of tumor cells as well as promoting internalization of nanoparticles by these tumor cells is mediated by TCR-like scFv and can contribute to melanoma-specific targeting. PMID:27022262

  15. Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq.

    PubMed

    Redmond, David; Poran, Asaf; Elemento, Olivier

    2016-01-01

    Accurate characterization of the repertoire of the T-cell receptor (TCR) alpha and beta chains is critical to understanding adaptive immunity. Such characterization has many applications across such fields as vaccine development and response, clone-tracking in cancer, and immunotherapy. Here we present a new methodology called single-cell TCRseq (scTCRseq) for the identification and assembly of full-length rearranged V(D)J T-cell receptor sequences from paired-end single-cell RNA sequencing reads. The method allows accurate identification of the V(D)J rearrangements for each individual T-cell and has the novel ability to recover paired alpha and beta segments. Source code is available at https://github.com/ElementoLab/scTCRseq . PMID:27460926

  16. Adaptive Markov chain Monte Carlo forward projection for statistical analysis in epidemic modelling of human papillomavirus.

    PubMed

    Korostil, Igor A; Peters, Gareth W; Cornebise, Julien; Regan, David G

    2013-05-20

    A Bayesian statistical model and estimation methodology based on forward projection adaptive Markov chain Monte Carlo is developed in order to perform the calibration of a high-dimensional nonlinear system of ordinary differential equations representing an epidemic model for human papillomavirus types 6 and 11 (HPV-6, HPV-11). The model is compartmental and involves stratification by age, gender and sexual-activity group. Developing this model and a means to calibrate it efficiently is relevant because HPV is a very multi-typed and common sexually transmitted infection with more than 100 types currently known. The two types studied in this paper, types 6 and 11, are causing about 90% of anogenital warts. We extend the development of a sexual mixing matrix on the basis of a formulation first suggested by Garnett and Anderson, frequently used to model sexually transmitted infections. In particular, we consider a stochastic mixing matrix framework that allows us to jointly estimate unknown attributes and parameters of the mixing matrix along with the parameters involved in the calibration of the HPV epidemic model. This matrix describes the sexual interactions between members of the population under study and relies on several quantities that are a priori unknown. The Bayesian model developed allows one to estimate jointly the HPV-6 and HPV-11 epidemic model parameters as well as unknown sexual mixing matrix parameters related to assortativity. Finally, we explore the ability of an extension to the class of adaptive Markov chain Monte Carlo algorithms to incorporate a forward projection strategy for the ordinary differential equation state trajectories. Efficient exploration of the Bayesian posterior distribution developed for the ordinary differential equation parameters provides a challenge for any Markov chain sampling methodology, hence the interest in adaptive Markov chain methods. We conclude with simulation studies on synthetic and recent actual data. PMID

  17. Limited Expression of Slow Tonic Myosin Heavy Chain in Human Cranial Muscles

    PubMed Central

    Sokoloff, Alan J.; Li, Haiyan; Burkholder, Thomas J.

    2013-01-01

    Recent reports of slow tonic myosin heavy chain (MHCst) in human masticatory and laryngeal muscles suggest that MHCst may have a wider distribution in humans than previously thought. Because of the novelty of this finding, we sought to confirm the presence of MHCst in human masticatory and laryngeal muscles by reacting tissue from these muscles and controls from extraocular, intrafusal, cardiac, appendicular and developmental muscle with antibodies (Abs) ALD-58 and S46 considered highly specific for MHCst. At Ab dilutions producing minimal reaction to muscle fibers positive for MHCI, only extraocular, intrafusal and fetal tongue tissue reacted with Ab S46 had strong immunoreaction in an appreciable number of muscle fibers. In immunoblots Ab S46, but not Ab ALD-58, labeled adult extraocular muscles; no other muscles were labeled with either Ab. We conclude that, in humans, Ab S46 has greater specificity for MHCst than does Ab ALD-58. We suggest that reports of MHCst in human masticatory and laryngeal muscles reflect false-positive identification of MHCst due to cross-reactivity of Ab ALD-58 with another MHC isoform. PMID:17486578

  18. Polypeptide chain composition diversity of hexagonal-bilayer haemoglobins within a single family of annelids, the alvinellidae.

    PubMed

    Zal, F; Green, B N; Martineu, P; Lallier, F H; Toulmond, A; Vinogradov, S N; Childress, J J

    2000-08-01

    Following previous analysis of the structure of Alvinella pompejana heaxagonal-bilayer haemoglobin (HBL Hb) [1], we report in this paper the structure of three other HBL Hbs belonging to Alvinella caudata, Paralvinella grasslei and Paralvinella palmiformis, members of the Alvinellidae, annelid family strictly endemic to deep-sea hydrothermal vents located on the ridge crests in the Pacific ocean. The multi-angle laser light scattering (MALLS) and fast protein liquid chromatography (FPLC) analysis revealed a broad range of molecular masses for the extracellular Hb molecules, 3517 +/- 14 kDa (A. caudata), 3822 +/- 28 kDa (P. grasslei) and 3750 +/- 150 kDa (P. palmiformis). Native and derivative Hbs (reduced, carbamidomethylated and deglycosylated) were analysed by electrospray ionization mass spectroscopy (ESI-MS) and the data was processed by the maximum entropy deconvolution system (MaxEnt). The most important difference between alvinellid HBL Hbs was the variation in their composition, from two to four monomeric globin chains, and from one to four linker chains. Therefore, despite the fact that all these species belong to a single family, notable differences in the polypeptide chain composition of their HBL Hbs were observed, probably accounting for their different functional properties as previously reported by this group Toulmond, A., El Idrissi Slitine, F., De Frescheville, J. & Jouin, C. (1990) Biol. Bull. 179, 366-373. PMID:10931208

  19. Origin of the red sites and energy transfer rates in single MEH-PPV chains at low temperature.

    PubMed

    Feist, Florian A; Zickler, Martin F; Basché, Thomas

    2011-06-01

    Single poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) molecules dispersed in thin poly(methylmethacrylate) films have been investigated by fluorescence emission, excitation and time-resolved spectroscopy at 1.2 K. For the molecular weight studied (∼200 kDa) a bimodal distribution of emission maxima is observed. Based on a comparison of the spectroscopic properties of blue and red sites and on polarisation-resolved measurements, we argue in agreement with recent quantum-chemical calculations that the red subpopulation most probably does not arise from interchromophoric excitation delocalisation but is to be attributed to longer chromophoric units originating from ordered regions of a polymer chain, where due to constraints on the chain conformation larger conjugation lengths can be realised. In excitation spectra within the red spectral region we can identify multiple chromophoric units, among them chromophores without correspondence in the emission spectrum-donors of the intramolecular energy transfer. Zero-phonon lines of donor chromophores proved to be significantly broadened, indicating fast excited-state population decay due to energy transfer. Thus, a distribution of energy transfer times within MEH-PPV chains could be determined from donor zero-phonon line widths, with an average value of 3.9 ps. Our study represents the first direct measurement of energy transfer times in conjugated polymers, parameters that are crucial for the performance of many technical applications based on this class of material. PMID:21472962

  20. Path Integral Approach to a Single Polymer Chain with Random Media

    NASA Astrophysics Data System (ADS)

    Kunsombat, Ch.; Sa-Yakanit, V.

    In this paper we consider the problem of a polymer chain in random media with finite correlation. We show that the mean square end-to-end distance of a polymer chain can be obtained using the Feynman path integral developed by Feynman for treating the polaron problem and successfuly applied to the theory of heavily doped semiconductor. We show that for short-range correlation or the white Gaussian model we derive the results obtained by Edwards and Muthukumar using the replica method and for long-range correlation we obtain the result of Yohannes Shiferaw and Yadin Y. Goldschimidt. The main idea of this paper is to generalize the model proposed by Edwards and Muthukumar for short-range correlation to finite correlation. Instead of using a replica method, we employ the Feynman path integral by modeling the polymer Hamiltonian as a model of non-local quadratic trial Hamiltonian. This non-local trial Hamiltonian is essential as it will reflect the translation invariant of the original Hamiltonian. The calculation is proceeded by considering the differences between the polymer propagator and the trial propagator as the first cumulant approximation. The variational principle is used to find the optimal values of the variational parameters and the mean square end-to-end distance is obtained. Several asymptotic limits are considered and a comparison between this approaches and replica approach will be discussed.

  1. Quantifying the atomic-level mechanics of single long physisorbed molecular chains

    PubMed Central

    Kawai, Shigeki; Koch, Matthias; Gnecco, Enrico; Sadeghi, Ali; Pawlak, Rémy; Glatzel, Thilo; Schwarz, Jutta; Goedecker, Stefan; Hecht, Stefan; Baratoff, Alexis; Grill, Leonhard; Meyer, Ernst

    2014-01-01

    Individual in situ polymerized fluorene chains 10–100 nm long linked by C–C bonds are pulled vertically from an Au(111) substrate by the tip of a low-temperature atomic force microscope. The conformation of the selected chains is imaged before and after manipulation using scanning tunneling microscopy. The measured force gradient shows strong and periodic variations that correspond to the step-by-step detachment of individual fluorene repeat units. These variations persist at constant intensity until the entire polymer is completely removed from the surface. Calculations based on an extended Frenkel–Kontorova model reproduce the periodicity and magnitude of these features and allow us to relate them to the detachment force and desorption energy of the repeat units. The adsorbed part of the polymer slides easily along the surface during the pulling process, leading to only small oscillations as a result of the high stiffness of the fluorenes and of their length mismatch with respect to the substrate surface structure. A significant lateral force also is caused by the sequential detachment of individual units. The gained insight into the molecule–surface interactions during sliding and pulling should aid the design of mechanoresponsive nanosystems and devices. PMID:24591611

  2. Preparation and Identification of a Single-chain Variable Fragment Antibody Against Canine Distemper Virus.

    PubMed

    Yi, Li; Cheng, Shipeng

    2015-08-01

    The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 1N8, which secretes the monoclonal antibody against CDV N protein (aa 277-471). The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/1N8). After sequence analysis, the scFv/1N8 gene was cloned into the prokaryotic expression vector PET32a with a His-tag. The recombinant scFv/1N8 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the binding activity and specificity of the scFv were determined by indirect ELISA (His-tag) and competitive ELISA. The recombinant scFv/1N8 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule. PMID:26301925

  3. Directed Evolution of Human Heavy Chain Variable Domain (VH) Using In Vivo Protein Fitness Filter

    PubMed Central

    Nam, Hyo Jung; Kim, Sung-Geun; Park, Young-Seoub; Park, Jae-Chan; Woo, Eui-Jeon; Lim, Hyung-Kwon

    2014-01-01

    Human immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one VH protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display. PMID:24892548

  4. SCRL-Model for Human Space Flight Operations Enterprise Supply Chain

    NASA Technical Reports Server (NTRS)

    Tucker, Brian

    2010-01-01

    Standard approach to evaluate and configure adaptable and sustainable program and mission supply chains at an enterprise level. End-to-end view. Total Lifecycle. Evaluate the readiness of the supply chain during the supply chain development phase.

  5. Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction

    SciTech Connect

    Noonan, K.E.; Beck, C.; Holzmayer, T.A.; Chin, J.E.; Roninson, I.B. ); Wunder, J.S.; Andrulis, I.L. ); Gazdar, A.F. ); Willman, C.L.; Griffith, B. ); Von Hoff, D.D. )

    1990-09-01

    The resistance of tumor cells ot chemotheraprutic drugs is a major obstacle to successful cancer chemotherapy. In human cells, expression of the MDR1 gene, encoding a transmembrane efflux pump (P-glycoprotein), leads to decreased intracellular accumulation and resistance to a variety of lipophilic drugs (multidrug resistance; MDR). The levels of MDR in cell lines selected in bitro have been shown to correlate with the steady-state levels of MDR1 mRNA and P-glycoprotein. In cells with a severalfold increase in cellular drug resistance, MDR1 expression levels are close to the limits of detection by conventional assays. MDR1 expression has been frequently observed in human tumors after chemotherapy and in some but not all types of clinically refactory tumors untreated with chemotherapeutic drugs. The authors have devised a highly sensitive, specific, and quantitative protocol for measuring the levels of MDR1 mRNA in clincal samples, based on the polymerase chain reaction. They have used this assay to measure MDR1 gene expression in MDR cell lines and >300 normal tissues, tumor-derived cell lines, and clinical specimens of untreated tumors of the types in which MDR1 expression was rarely observed by standard assays. Low levels of MDR1 expression were found by polymerase chain reaction in most solid tumors and leukemias tested. The frequency of samples without detectable MDR1 expression varied among different types of tumors; MDR1-negative samples were ost common among tumor types known to be relatively responsive to chemotherapy.

  6. Expression, purification, and characterization of recombinant human L-chain ferritin.

    PubMed

    Zou, Wenyan; Liu, Xiaoyu; Zhao, Xi; Wang, Jie; Chen, Dianhua; Li, Jiahuang; Ji, Lina; Hua, Zichun

    2016-03-01

    Ferritins form nanocage architectures and demonstrate their potential to serve as functional nanomaterials with potential applications in medical imaging and therapy. In our study, the cDNA of human L-chain ferritin was cloned into plasmid pET-28a for its overexpression in Escherichia coli. However, the recombinant human L-chain ferritin (rLF) was prone to form inclusion bodies. Molecular chaperones were co-expressed with rLF to facilitate its correct folding. Our results showed that the solubility of rLF was increased about 3-fold in the presence of molecular chaperones, including GroEL, GroES and trigger factor. Taking advantage of its N-terminal His-tag, rLF was then purified with Ni-affinity chromatography. With a yield of 10 mg/L from bacterial culture, the purified rLF was analyzed by circular dichroism spectrometry for its secondary structure. Furthermore, the rLF nanocages were characterized using dynamic light scattering and transmission electron microscopy. PMID:26621552

  7. Dexamethasone modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human T lymphocytes.

    PubMed

    Nambiar, M P; Enyedy, E J; Fisher, C U; Warke, V G; Juang, Y T; Tsokos, G C

    2001-02-25

    Dexamethasone is a potent anti-inflammatory and immunosupressive agent that has complex, yet incompletely defined, effects on the immune response. Here, we explored the effect of dexamethasone on the expression of TCR zeta chain and TCR/CD3-induced early signaling events in human T lymphocytes. Immunoblotting studies using TCR zeta chain specific mAb showed a dose-dependent biphasic effect of dexamethasone on TCR zeta chain expression, that is, it was increased when cells were incubated with 10 nM, whereas the expression was decreased when incubated with 100 nM dexamethasone. The dose-dependent biphasic effect of dexamethsone on the TCR zeta chain expression was also revealed by FACS analysis of permeabilized cells. Time course studies showed that upregulation of the TCR zeta chain at 10 nM dexamethasone reached maximum levels at 24 h and remained elevated up to 48 h. Other subunits of the TCR/CD3 complex were minimally affected under these conditions. The increased expression of the TCR zeta chain following treatment with 10 nM dexamethasone correlated with increased anti-CD3 antibody-induced tyrosine phosphorylation of the TCR zeta chain and downstream signaling intermediate ZAP-70 and PLC gamma with faster kinetics. Similarly, the induction of TCR zeta chain expression at 10 nM dexamethasone correlated with increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response. Reporter gene assays using TCR zeta chain promoter-driven luciferase gene constructs in Jurkat cells showed that treatment with 10 nM dexamethasone increased TCR zeta chain promoter activity and that the region between -160 and +58 was responsible for the observed effect. These results suggest that dexamethasone primarily acts at the transcriptional level and differentially modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human peripheral T lymphocytes. PMID:11277620

  8. Purification of single-chain antibody fragments exploiting pH-gradients in simulated moving bed chromatography.

    PubMed

    Martínez Cristancho, Carlos Andrés; Seidel-Morgenstern, Andreas

    2016-02-19

    This paper deals with the theoretical design and experimental validation of an affinity-based continuous multi-column chromatography process for the purification of single-chain Fragment variable (scFv) antibodies. An open-loop 3-zone pH-gradient simulated moving bed (SMB) process was investigated exploiting the highly specific affinity of metal ions toward histidine-tagged recombinant proteins. The separation problem was simplified by considering the cell culture supernatant as a pseudo-binary mixture. The influence of mobile phase pH on the adsorption isotherm parameters was estimated by the inverse method using recorded pH-gradient batch elution profiles. Suitable operating parameters for the SMB process were identified using an equilibrium stage model and subsequently validated in a lab-scale SMB unit. Finally, the performance of the pH-gradient SMB process was compared against a non-optimized batch process. Biologically active single-chain Fragment variable antibody formats were purified continuously with 9% more recovery, 11 times more productivity (576 mg of purified scFv per day and liter stationary phase in SMB) and enriched by a factor of 2.5 compared to those obtained in the non-optimized batch process. PMID:26810806

  9. New strategy for expression of recombinant hydroxylated human collagen α1(III) chains in Pichia pastoris GS115.

    PubMed

    He, Jing; Ma, Xiaoxuan; Zhang, Fenglong; Li, Linbo; Deng, Jianjun; Xue, Wenjiao; Zhu, Chenhui; Fan, Daidi

    2015-01-01

    Type III collagen is one of the most abundant proteins in the human body, which forms collagen fibrils and provides the stiff, resilient characteristics of many tissues. In this paper, a new method for secretory expression of recombinant hydroxylated human collagen α1(III) chain in Pichia pastoris GS115 was applied. The gene encoding for full-length human collagen α1(III) chain (COL3A1) without N-terminal propeptide and C-terminal propeptide was cloned in the pPIC9K expression vector. The prolyl 4-hydroxylase (P4H, EC 1.14.11.2) α-subunit (P4Hα) and β-subunit (P4Hβ) genes were cloned in the same expression vector, pPICZB. Fluorogenic quantitative PCR indicates that COL3A1 and P4H genes have been expressed in mRNA level. SDS-PAGE shows that secretory expression of recombinant human collagen α1(III) chain was successfully achieved in P. pastoris GS115. In addition, the result of amino acids composition analysis shows that the recombinant human collagen α1(III) chain contains hydroxyproline by coexpression with the P4H. Furthermore, liquid chromatography coupled with tandem mass spectrometry analysis demonstrates that proline residues of the recombinant human collagen α1(III) chain were hydroxylated in the X or Y positions of Gly-X-Y triplets. PMID:24953863

  10. Phase-coexistence simulations of fluid mixtures by the Markov Chain Monte Carlo method using single-particle models

    SciTech Connect

    Li, Jun; Calo, Victor M.

    2013-09-15

    We present a single-particle Lennard–Jones (L-J) model for CO{sub 2} and N{sub 2}. Simplified L-J models for other small polyatomic molecules can be obtained following the methodology described herein. The phase-coexistence diagrams of single-component systems computed using the proposed single-particle models for CO{sub 2} and N{sub 2} agree well with experimental data over a wide range of temperatures. These diagrams are computed using the Markov Chain Monte Carlo method based on the Gibbs-NVT ensemble. This good agreement validates the proposed simplified models. That is, with properly selected parameters, the single-particle models have similar accuracy in predicting gas-phase properties as more complex, state-of-the-art molecular models. To further test these single-particle models, three binary mixtures of CH{sub 4}, CO{sub 2} and N{sub 2} are studied using a Gibbs-NPT ensemble. These results are compared against experimental data over a wide range of pressures. The single-particle model has similar accuracy in the gas phase as traditional models although its deviation in the liquid phase is greater. Since the single-particle model reduces the particle number and avoids the time-consuming Ewald summation used to evaluate Coulomb interactions, the proposed model improves the computational efficiency significantly, particularly in the case of high liquid density where the acceptance rate of the particle-swap trial move increases. We compare, at constant temperature and pressure, the Gibbs-NPT and Gibbs-NVT ensembles to analyze their performance differences and results consistency. As theoretically predicted, the agreement between the simulations implies that Gibbs-NVT can be used to validate Gibbs-NPT predictions when experimental data is not available.

  11. Heisenberg magnetic chain with single-ion easy-plane anisotropy: Hubbard operators approach

    NASA Astrophysics Data System (ADS)

    Spirin, D. V.; Fridman, Yu. A.

    2003-03-01

    We investigate the gap in excitation spectrum of one-dimensional S=1 ferro- and antiferromagnets with easy-plane single-ion anisotropy. The self-consistent modification of Hubbard operators approach which enables to account single-site term exactly is used. For antiferromagnetic model we found Haldane phase that exists up to point D=4 J (where D is anisotropy parameter, J is exchange coupling), while quadrupolar phase realizes at larger values of anisotropy. Our results specify those of Golinelli et al. (Phys. Rev. B. 45 (1992) 9798), where similar model was studied. Besides the method gives gap value closer to numerical estimations than usual spin-wave theories.

  12. Magnetization plateaus in the antiferromagnetic Ising chain with single-ion anisotropy and quenched disorder.

    PubMed

    Neto, Minos A; de Sousa, J Ricardo; Branco, N S

    2015-05-01

    We have studied the presence of plateaus on the low-temperature magnetization of an antiferromagnetic spin-1 chain, as an external uniform magnetic field is varied. A crystal-field interaction is present in the model and the exchange constants follow a random quenched (Bernoulli or Gaussian) distribution. Using a transfer-matrix technique we calculate the largest Lyapunov exponent and, from it, the magnetization at low temperatures as a function of the magnetic field, for different values of the crystal field and the width of the distributions. For the Bernoulli distribution, the number of plateaus increases, with respect to the uniform case [Litaiff et al., Solid State Commun. 147, 494 (2008)] and their presence can be linked to different ground states, when the magnetic field is varied. For the Gaussian distributions, the uniform scenario is maintained, for small widths, but the plateaus structure disappears as the width increases. PMID:26066165

  13. Mapping single macromolecule chains using the colloid deposition method: PDADMAC on mica.

    PubMed

    Michna, Aneta; Adamczyk, Zbigniew; Batys, Piotr

    2015-07-15

    Monolayers of the cationic polyelectrolyte poly(diallyldimethylammonium chloride) (PDADMAC) on mica were thoroughly characterized using the streaming potential and the colloid deposition methods. Initially, the stability of the monolayers was determined by performing desorption experiments carried out under diffusion-controlled regime. It was shown that the desorption of the polyelectrolyte at the ionic strength range 0.01-0.15 M is negligible over the time of 20 h. The structure of PDADMAC monolayers and orientation of molecules were evaluated using the colloid deposition measurements involving negatively charged polystyrene latex microspheres, 820 nm in diameter. The functional relationships between the polyelectrolyte coverage and latex coverage deposited within 20 h were acquired by direct optical microscope. In this way the influence of ionic strength varied in the range 0.15-0.01 M on the molecule orientation in monolayers was determined. It was shown that for ionic strength of 0.15 M nearly one to one mapping of polyelectrolyte chains by colloid particles can be achieved for PDADMAC coverage below 0.1%. In this way, because of a considerable surface area ratio between the macromolecule and the colloid particle, an enhancement factor of 10(3) can be attained. This behavior was quantitatively interpreted in terms of the random site adsorption model whereas the classical mean-field theory proved inadequate. On the other hand, for lower ionic strength, it was confirmed that an irreversible immobilization of latex particles can only occur at a few closely spaced PDADMAC chains. It was shown that these experimental results were consistent with the side-on adsorption mechanisms of PDADMAC at mica for the above ionic strength. PMID:25801136

  14. Harnessing the Risk-Related Data Supply Chain: An Information Architecture Approach to Enriching Human System Research and Operations Knowledge

    NASA Technical Reports Server (NTRS)

    Buquo, Lynn E.; Johnson-Throop, Kathy A.

    2011-01-01

    An Information Architecture facilitates the understanding and, hence, harnessing of the human system risk-related data supply chain which enhances the ability to securely collect, integrate, and share data assets that improve human system research and operations. By mapping the risk-related data flow from raw data to useable information and knowledge (think of it as a data supply chain), the Human Research Program (HRP) and Space Life Science Directorate (SLSD) are building an information architecture plan to leverage their existing, and often shared, IT infrastructure.

  15. High-throughput procedure for single pollen grain collection and polymerase chain reaction in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single pollen grain PCR has succeeded in several species, however only a limited number (up to 60) pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput procedure for dete...

  16. Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage.

    PubMed

    Ronsoni, Marcelo Fernando; Remor, Aline Pertile; Lopes, Mark William; Hohl, Alexandre; Troncoso, Iris H Z; Leal, Rodrigo Bainy; Boos, Gustavo Luchi; Kondageski, Charles; Nunes, Jean Costa; Linhares, Marcelo Neves; Lin, Kátia; Latini, Alexandra Susana; Walz, Roger

    2016-04-01

    Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity to study human brain tissue surgically removed to treat drug resistant epilepsies. Epilepsy surgeries are done with hemodynamic and laboratory parameters to maintain physiology, but there are no studies analyzing the association among these parameters and MRCCE activities in the human brain tissue. We determined the intra-operative parameters independently associated with MRCCE activities in middle temporal neocortex (Cx), amygdala (AMY) and head of hippocampus (HIP) samples of patients (n = 23) who underwent temporal lobectomy using multiple linear regressions. MRCCE activities in Cx, AMY and HIP are differentially associated to trans-operative mean arterial blood pressure, O2 saturation, hemoglobin, and anesthesia duration to time of tissue sampling. The time-course between the last seizure occurrence and tissue sampling as well as the sample storage to biochemical assessments were also associated with enzyme activities. Linear regression models including these variables explain 13-17 % of MRCCE activities and show a moderate to strong effect (r = 0.37-0.82). Intraoperative hemodynamic and laboratory parameters as well as the time from last seizure to tissue sampling and storage time are associated with MRCCE activities in human samples from the Cx, AMYG and HIP. Careful control of these parameters is required to minimize confounding biases in studies using human brain samples collected from elective neurosurgery. PMID:26586405

  17. The effect of short-chain fatty acids on human monocyte-derived dendritic cells.

    PubMed

    Nastasi, Claudia; Candela, Marco; Bonefeld, Charlotte Menné; Geisler, Carsten; Hansen, Morten; Krejsgaard, Thorbjørn; Biagi, Elena; Andersen, Mads Hald; Brigidi, Patrizia; Ødum, Niels; Litman, Thomas; Woetmann, Anders

    2015-01-01

    The gut microbiota is essential for human health and plays an important role in the pathogenesis of several diseases. Short-chain fatty acids (SCFA), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients that distribute systemically via the blood. The aim of this study was to investigate the transcriptional response of immature and LPS-matured human monocyte-derived DC to SCFA. Our data revealed distinct effects exerted by each individual SCFA on gene expression in human monocyte-derived DC, especially in the mature ones. Acetate only exerted negligible effects, while both butyrate and propionate strongly modulated gene expression in both immature and mature human monocyte-derived DC. An Ingenuity pathway analysis based on the differentially expressed genes suggested that propionate and butyrate modulate leukocyte trafficking, as SCFA strongly reduced the release of several pro-inflammatory chemokines including CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11. Additionally, butyrate and propionate inhibited the expression of lipopolysaccharide (LPS)-induced cytokines such as IL-6 and IL-12p40 showing a strong anti-inflammatory effect. This work illustrates that bacterial metabolites far from the site of their production can differentially modulate the inflammatory response and generally provides new insights into host-microbiome interactions. PMID:26541096

  18. Antibody light chain variable domains and their biophysically improved versions for human immunotherapy.

    PubMed

    Kim, Dae Young; To, Rebecca; Kandalaft, Hiba; Ding, Wen; van Faassen, Henk; Luo, Yan; Schrag, Joseph D; St-Amant, Nadereh; Hefford, Mary; Hirama, Tomoko; Kelly, John F; MacKenzie, Roger; Tanha, Jamshid

    2014-01-01

    We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (T(m)), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their T(m)s, by 5.5-17.5 ° C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach's acidic pH and pepsin. PMID:24423624

  19. Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

    PubMed Central

    Kim, Dae Young; To, Rebecca; Kandalaft, Hiba; Ding, Wen; van Faassen, Henk; Luo, Yan; Schrag, Joseph D; St-Amant, Nadereh; Hefford, Mary; Hirama, Tomoko; Kelly, John F; MacKenzie, Roger; Tanha, Jamshid

    2014-01-01

    We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (Tm), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their Tms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin. PMID:24423624

  20. Toward in vivo imaging of heart disease using a radiolabeled single-chain Fv fragment targeting tenascin-C.

    PubMed

    Kobayashi, Norihiro; Odaka, Kenichi; Uehara, Tomoya; Imanaka-Yoshida, Kyoko; Kato, Yoshinori; Oyama, Hiroyuki; Tadokoro, Hiroyuki; Akizawa, Hiromichi; Tanada, Shuji; Hiroe, Michiaki; Fukumura, Toshimitsu; Komuro, Issei; Arano, Yasushi; Yoshida, Toshimichi; Irie, Toshiaki

    2011-12-01

    Antibodies specific to a particular target molecule can be used as analytical reagents, not only for in vitro immunoassays but also for noninvasive in vivo imaging, e.g., immunoscintigraphies. In the latter case, it is important to reduce the size of antibody molecules in order to achieve suitable in vivo "diagnostic kinetics" and generate higher-resolution images. For these purposes, single-chain Fv fragments (scFvs; M(r) < 30 kDa) have greater potential than intact immunoglobulins (~150 kDa) or Fab (or Fab') fragments (~50 kDa). Our recent observation of enhanced tenascin-C (Tnc) expression at sites of cardiac repair after myocardial infarction prompted us to develop a radiolabeled scFv against Tnc for in vivo imaging of heart disease. We cloned the genes encoding the heavy and light chain variable domains of the mouse anti-Tnc monoclonal antibody 4F10, and combined them to create a single gene. The resulting scFv-4F10 gene was expressed in E. coli cells to produce soluble scFv proteins. scFv-4F10 has an affinity for Tnc (K(a) = 3.5 × 10(7) M(-1)), similar to the Fab fragment of antibody 4F10 (K(a) = 1.3 × 10(7) M(-1)) and high enough to be of practical use. A cysteine residue was then added to the C-terminus to achieve site-specific (111)In labeling via a chelating group. The resulting (111)In-labeled scFv was administered to a rat model of acute myocardial infarction. Biodistribution and quantitative autoradiographic studies indicated higher uptake of the radioactivity at the infarcted myocardium than the noninfarcted one. Single photon emission computed tomography (SPECT) provided in vivo cardiac images that coincided with the ex vivo observations. Our results will promote advances in diagnostic strategies for heart disease. PMID:22074352

  1. Single-friction-surface triboelectric generator with human body conduit

    SciTech Connect

    Meng, Bo; Cheng, Xiaoliang; Zhang, Xiaosheng; Han, Mengdi; Liu, Wen; Zhang, Haixia

    2014-03-10

    We present a transparent single-friction-surface triboelectric generator (STEG) employing human body as the conduit, making the applications of STEG in portable electronics much more practical and leading to a significant output improvement. The STEG with micro-patterned polydimethylsiloxane surface achieved an output voltage of over 200 V with a current density of 4.7 μA/cm{sup 2}. With human body conduit, the output current increased by 39% and the amount of charge that transferred increased by 34% compared to the results with grounded electrode. A larger increment of 210% and 81% was obtained in the case of STEG with a large-size flat polyethylene terephthalate surface.

  2. Intestinal Short Chain Fatty Acids and their Link with Diet and Human Health.

    PubMed

    Ríos-Covián, David; Ruas-Madiedo, Patricia; Margolles, Abelardo; Gueimonde, Miguel; de Los Reyes-Gavilán, Clara G; Salazar, Nuria

    2016-01-01

    The colon is inhabited by a dense population of microorganisms, the so-called "gut microbiota," able to ferment carbohydrates and proteins that escape absorption in the small intestine during digestion. This microbiota produces a wide range of metabolites, including short chain fatty acids (SCFA). These compounds are absorbed in the large bowel and are defined as 1-6 carbon volatile fatty acids which can present straight or branched-chain conformation. Their production is influenced by the pattern of food intake and diet-mediated changes in the gut microbiota. SCFA have distinct physiological effects: they contribute to shaping the gut environment, influence the physiology of the colon, they can be used as energy sources by host cells and the intestinal microbiota and they also participate in different host-signaling mechanisms. We summarize the current knowledge about the production of SCFA, including bacterial cross-feedings interactions, and the biological properties of these metabolites with impact on the human health. PMID:26925050

  3. Intestinal Short Chain Fatty Acids and their Link with Diet and Human Health

    PubMed Central

    Ríos-Covián, David; Ruas-Madiedo, Patricia; Margolles, Abelardo; Gueimonde, Miguel; de los Reyes-Gavilán, Clara G.; Salazar, Nuria

    2016-01-01

    The colon is inhabited by a dense population of microorganisms, the so-called “gut microbiota,” able to ferment carbohydrates and proteins that escape absorption in the small intestine during digestion. This microbiota produces a wide range of metabolites, including short chain fatty acids (SCFA). These compounds are absorbed in the large bowel and are defined as 1-6 carbon volatile fatty acids which can present straight or branched-chain conformation. Their production is influenced by the pattern of food intake and diet-mediated changes in the gut microbiota. SCFA have distinct physiological effects: they contribute to shaping the gut environment, influence the physiology of the colon, they can be used as energy sources by host cells and the intestinal microbiota and they also participate in different host-signaling mechanisms. We summarize the current knowledge about the production of SCFA, including bacterial cross-feedings interactions, and the biological properties of these metabolites with impact on the human health. PMID:26925050

  4. Effect of Lysine Modification on the Stability and Cellular Binding of Human Amyloidogenic Light Chains

    SciTech Connect

    O'Neill, Hugh Michael; Davern, Sandra M.; Murphy, Charles L.; Wall, Jonathan; Deborah, Weiss T.; Solomon, Alan

    2011-01-01

    AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescent microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichorism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

  5. Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the beta-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain.

    PubMed

    Yoshioka, N; Atassi, M Z

    1986-03-01

    A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146. PMID:3718478

  6. Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

    PubMed

    Mehalick, Leslie A; Poulsen, Christopher; Fischer, Carol L; Lanzel, Emily A; Bates, Amber M; Walters, Katherine S; Cavanaugh, Joseph E; Guthmiller, Janet M; Johnson, Georgia K; Wertz, Philip W; Brogden, Kim A

    2015-12-01

    Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine) for human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), dendritic cells (DC), and squamous cell carcinoma (SCC) cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2-10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0-80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections. PMID:26550599

  7. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    PubMed

    Toleikis, Lars; Frenzel, André

    2012-01-01

    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli. PMID:22907345

  8. A novel disorder reveals clathrin heavy chain-22 is essential for human pain and touch development

    PubMed Central

    Al-Gazali, Lihadh; Hertecant, Jozef; Owen, David J.; Borner, Georg H. H.; Chen, Ya-Chun; Benn, Caroline L.; Carvalho, Ofélia P.; Shaikh, Samiha S.; Phelan, Anne; Robinson, Margaret S.; Royle, Stephen J.

    2015-01-01

    Congenital inability to feel pain is very rare but the identification of causative genes has yielded significant insights into pain pathways and also novel targets for pain treatment. We report a novel recessive disorder characterized by congenital insensitivity to pain, inability to feel touch, and cognitive delay. Affected individuals harboured a homozygous missense mutation in CLTCL1 encoding the CHC22 clathrin heavy chain, p.E330K, which we demonstrate to have a functional effect on the protein. We found that CLTCL1 is significantly upregulated in the developing human brain, displaying an expression pattern suggestive of an early neurodevelopmental role. Guided by the disease phenotype, we investigated the role of CHC22 in two human neural crest differentiation systems; human induced pluripotent stem cell-derived nociceptors and TRKB-dependant SH-SY5Y cells. In both there was a significant downregulation of CHC22 upon the onset of neural differentiation. Furthermore, knockdown of CHC22 induced neurite outgrowth in neural precursor cells, which was rescued by stable overexpression of small interfering RNA-resistant CHC22, but not by mutant CHC22. Similarly, overexpression of wild-type, but not mutant, CHC22 blocked neurite outgrowth in cells treated with retinoic acid. These results reveal an essential and non-redundant role for CHC22 in neural crest development and in the genesis of pain and touch sensing neurons. PMID:26068709

  9. Human Immune Disorder Arising from Mutation of the α Chain of the Interleukin-2 Receptor

    NASA Astrophysics Data System (ADS)

    Sharfe, Nigel; Dadi, Harjit K.; Shahar, Michal; Roifman, Chaim M.

    1997-04-01

    Profound cellular immunodeficiency occurs as the result of mutations in proteins involved in both the differentiation and function of mature lymphoid cells. We describe here a novel human immune aberration arising from a truncation mutation of the interleukin-2 receptor α chain (CD25), a subunit of the tripartite high-affinity receptor for interleukin 2. This immunodeficiency is characterized by decreased numbers of peripheral T cells displaying abnormal proliferation but normal B cell development. Extensive lymphocytic infiltration of tissues, including lung, liver, gut, and bone, is observed, accompanied by tissue atrophy and inflammation. Although mature T cells are present, the absence of CD25 does affect the differentiation of thymocytes. While displaying normal development of CD2, CD3, CD4, and CD8 expression, CD25-deficient cortical thymocytes do not express CD1, and furthermore they fail to normally down-regulate levels of the anti-apoptotic protein bcl-2.

  10. Procedure optimization for extracting short-chain fatty acids from human faeces.

    PubMed

    Dobrowolska Iwanek, Justyna; Zagrodzki, Paweł; Woźniakiewicz, Michał; Woźniakiewicz, Aneta; Zwolińska Wcisło, Małgorzata; Winnicka, Diana; Paśko, Paweł

    2016-05-30

    Short-chain fatty acids play an important role in the physiology and metabolism of the colon. Disturbed balance of such compounds in human gut can significantly contribute to etiological factors of various gastrointestinal disorders and it may also increase the risk of developing cancer or cardiovascular diseases. The aim of the study was to select the optimal parameters for acetic, propionic and butyric acids extraction from stool samples. The experimental conditions were optimized with respect to the solvent sample shacking time, sample ultrasounds (Ultrasound-Assisted Extraction, UAE) exposure time and the number of extractions from the particulate stool samples. The screening of experimental parameters was conducted with fractional factorial design of experiments, namely 3(3-1). The optimal conditions for UAE were found, namely ultrasound digestion time of 40min (at 35°C), shaking time of 4min, and the three subsequent extractions. PMID:26977586

  11. [Use of the nested polymerase chain reaction in the differential diagnosis of human herpes simplex virus].

    PubMed

    Glukhov, A I; Gordeev, S A; Al'tshuler, M L; Severin, S E

    2003-02-01

    Herpes is one of the most widespread human viral pathologies. The article depicts a special modification of polymerized chain reaction--(PCR)--(referred to as "nested"), which has a higher sensitivity, specificity and reliability as compared to the ordinary PCR, and which is designed to detect the herpes viruses. The method was initially tested at purified preparation of viral DNA, and later--at clinical materials obtained from patients and healthy donors. Secretions from the urogenital tract (smears), scrapes from the urogenital tracts and urinal cellular samples were examined in patients. Herpes simplex was detected in all cases. As for the healthy people, the identical examinations produced in them mainly the negative findings. Therefore, the nested PCR is a simple, sensitive and effective instrument in the diagnostics and prevention of herpes; it can be recommended for a comprehensive usage in medical practice. PMID:12688217

  12. Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

    PubMed

    Bühler, R; Hempel, J; Kaiser, R; de Zalenski, C; von Wartburg, J P; Jörnvall, H

    1984-12-17

    The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. PMID:6391921

  13. Human Lymphoid Development in the Absence of Common Gamma Chain Receptor Signaling

    PubMed Central

    Kohn, Lisa A.; Seet, Christopher S.; Scholes, Jessica; Codrea, Felicia; Chan, Rebecca; Zaidi-Merchant, Sania; Zhu, Yuhua; De Oliveira, Satiro; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Kohn, Donald B.; Crooks, Gay M.

    2014-01-01

    Despite the power of model systems to reveal basic immunologic mechanisms, critical differences exist between species that necessitate the direct study of human cells. Illustrating this point is the difference in phenotype between patients with Severe Combined Immune Deficiency (SCID) caused by mutations affecting the common gamma chain (γc) cytokine signaling pathway and mice with similar mutations. Although in both species, null mutations in either IL2RG (which encodes γc), or its direct downstream signaling partner JAK3, result in T and NK cell deficiency, an associated B cell deficiency is seen in mice but not in humans with these genetic defects. In this study, we applied recent data that have revised our understanding of the earliest stages of lymphoid commitment in human bone marrow (BM), to determine the requirement for signaling through IL2RG and JAK3 in normal development of human lymphoid progenitors. BM samples from SCID patients with IL2RG (n=3) or JAK3 deficiency (n=2), which produce similar “T-NK-B+” clinical phenotypes, were compared to normal BM and umbilical cord blood as well as BM from children on enzyme treatment for adenosine deaminase deficient SCID (n=2). In both IL2RG- and JAK3-SCID patients, the early stages of lymphoid commitment from HSC were present with development of Lymphoid-primed Multipotent Progenitors, common lymphoid progenitors and B cell progenitors, and normal expression patterns of IL7RA and TLSPR, and the DNA recombination genes DNTT and RAG1. Thus, in humans, signaling through the γc pathway is not required for pre-thymic lymphoid commitment or for DNA rearrangement. PMID:24771849

  14. Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

    NASA Astrophysics Data System (ADS)

    Reed, Jason; Hsueh, Carlin; Mishra, Bud; Gimzewski, James K.

    2008-09-01

    We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing.

  15. Differential modulation of human (Caco-2) colon cancer cell line phenotype by short chain fatty acids.

    PubMed

    Basson, M D; Emenaker, N J; Hong, F

    1998-04-01

    Fermentation of dietary fiber within the colonic lumen yields short chain fatty acids (SCFA) such as butyrate, which may modulate colonic mucosal biology and inhibit the development of a malignant phenotype. However, different fibers yield varying proportions of various SCFA. We studied the effects of the three most common SCFA, acetate, butyrate, and propionate, on the proliferation, adhesion, and motility of the human intestinal Caco-2 cell line, as well as the effects of these SCFA on alkaline phosphatase and dipeptidyl dipeptidase specific activity (common laboratory markers of differentiation). In addition, we examined the modulation of c-myc protein and the tyrosine phosphorylation of cellular proteins by these SCFA in order to determine whether the variations in the potency of these three SCFA for phenotypic change extended to variations in effects on intracellular signaling and protooncogene expression. All three SCFA tended to slow proliferation, promote brush border enzyme activity, and inhibit both adhesion to and motility across a type I collagen matrix substrate. However, we observed substantial differences in the potency of these three SCFA with regard to these effects. In particular, butyrate was uniformly more potent than an equimolar concentration of acetate whereas equimolar propionate achieved comparable effects with regard to proliferation and brush border enzyme activity but was intermediate between butyrate and acetate with regard to modulation of cell-matrix interactions. Similarly, the SCFA downregulated c-myc protein levels and modulated the phosphorylation of several intracellular tyrosine phosphoproteins, but the effects of the three SCFA varied substantially for these parameters. These results suggest that the common short chain fatty acids are not equipotent in their effects on human Caco-2 colon cancer cell biology. Such differences in potency could contribute to the observed differences in effects of different dietary fibers in vivo

  16. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    PubMed

    He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

    2016-06-15

    Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection. PMID:26772159

  17. Study of mitochondrial DNA depletion in muscle by single-fiber polymerase chain reaction.

    PubMed

    Sciacco, M; Gasparo-Rippa, P; Vu, T H; Tanji, K; Shanske, S; Mendell, J R; Schon, E A; DiMauro, S; Bonilla, E

    1998-11-01

    We studied muscle biopsies from 3 children with a mitochondrial myopathy characterized histochemically by the presence of ragged-red fibers (RRF) and various numbers of cytochrome c oxidase (COX)-negative fibers. We quantitated the absolute amounts of total mitochondrial DNA (mtDNA) in isolated normal COX-positive muscle fibers and in COX-negative RRF. There was severe mtDNA depletion in all fibers from the two most severe cases. In the third case mtDNA depletion could not be established with conventional diagnostic tools, but it was documented in single COX-negative fibers; COX-positive fibers showed the same amounts of mtDNA as fibers from aged-matched controls. Our observations indicate that mtDNA single-fiber PCR quantitation is a highly sensitive and specific method for diagnosing cases with focal mtDNA depletion. This method also allows one to correlate amounts of mtDNA with histochemical phenotypes in individual fibers from patients and age-matched controls, thereby providing important information about the functional role of residual mtDNA. PMID:9771659

  18. Dimerization of single selenium chains confined in nanochannels of cancrinite: An x-ray absorption study

    NASA Astrophysics Data System (ADS)

    Kolobov, A. V.; Oyanagi, H.; Poborchii, V. V.; Tanaka, K.

    1999-04-01

    Local structure of selenium confined in nanochannels of cancrinite (Can-Se) single crystal and powder samples have been studied by polarized x-ray absorption. The spectra for a single crystal are strongly anisotropic implying linear arrangement of Se species. Polarization dependence of extended x-ray absorption fine structure (EXAFS) data provides direct evidence that dimers with the bond length of 2.40+/-0.01 Å are formed. Polarized x-ray absorption near-edge structure (XANES) spectra demonstrate that they are aligned along the channel of cancrinite. Deconvolution of XANES spectra into Lorentzians (localized states) and the remaining steplike function (continuous states) shows that two localized state peaks are present. While the one polarized parallel to the cancrinite axis is strongly polarized, the other one is essentially isotropic. Comparison of XANES peak positions for Can-Se with that for bulk selenium provides evidence for negative charge on dimers. Despite strong temperature dependence of Raman-scattering spectra found earlier, EXAFS data do not exhibit any noticeable temperature dependence. Possible mechanisms for dimer stabilization are discussed.

  19. The gamma 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers.

    PubMed Central

    Pyke, C.; Rømer, J.; Kallunki, P.; Lund, L. R.; Ralfkiaer, E.; Danø, K.; Tryggvason, K.

    1994-01-01

    All known laminin isoforms are cross-shaped heterotrimeric molecules, consisting of one heavy alpha chain and two light beta and gamma chains. Recently, a cDNA encoding a new gamma chain from laminin 5 (also known as kalinin) was sequenced. This chain, named gamma 2, showed extended homology to the classical gamma 1 chain but differed from this by lacking the terminal globular domain. Recent data, indicating an important role of the gamma 2 chain gene in establishing adhesion contacts between epithelial cells and basement membranes, prompted us to investigate whether the gamma 2 chain gene is aberrantly expressed in cancer tissue, and if so whether its localization could provide clues to its possible role in cancer dissemination. Routinely processed tissue specimens from 36 cases of human cancer were investigated, including 16 cases of colon adenocarcinoma, 7 ductal mammary carcinomas, 4 squamous cell carcinomas, 3 malignant melanomas and 6 sarcomas. In situ hybridization for the detection of mRNAs for the gamma 2 chain and for the classical laminin chains alpha 1, beta 1, and gamma 1 was performed using S-35 labeled antisense RNA probes. As positive control of the specificity of the gamma 2 chain mRNA detection, two different anti-sense probes derived from two nonoverlapping cDNA clones were used. Malignant cells were found to express the gamma 2 chain in 29 of the 30 carcinomas studied and the expression was particularly high in cancer cells located at the invasion front. In contrast, mesenchymally derived cancer cells in three different types of sarcomas did not express the gamma 2 chain. In colon cancer there was a clear histological correlation between the expression of gamma 2 chain by cancer cells and their engagement in tumor budding processes. Laminin chains alpha 1, beta 1, and gamma 1 were weakly expressed throughout cancerous areas with no apparent correlation to sites of invasion. The aberrant expression of the gamma 2 chain gene seen in invasively

  20. Human Temporal Cortical Single Neuron Activity during Language: A Review

    PubMed Central

    Ojemann, George A.

    2013-01-01

    Findings from recordings of human temporal cortical single neuron activity during several measures of language, including object naming and word reading are reviewed and related to changes in activity in the same neurons during recent verbal memory and verbal associative learning measures, in studies conducted during awake neurosurgery for the treatment of epilepsy. The proportion of neurons changing activity with language tasks was similar in either hemisphere. Dominant hemisphere activity was characterized by relative inhibition, some of which occurred during overt speech, possibly to block perception of one’s own voice. However, the majority seems to represent a dynamic network becoming active with verbal memory encoding and especially verbal learning, but inhibited during performance of overlearned language tasks. Individual neurons are involved in different networks for different aspects of language, including naming or reading and naming in different languages. The majority of the changes in activity were tonic sustained shifts in firing. Patterned phasic activity for specific language items was very infrequently recorded. Human single neuron recordings provide a unique perspective on the biologic substrate for language, for these findings are in contrast to many of the findings from other techniques for investigating this. PMID:24961418

  1. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    PubMed

    Lin, Yuan; Li, Benqiang; Ye, Jiaxin; Wang, Man; Wang, Jianhua; Zhang, Ying; Zhu, Jianguo

    2015-09-01

    Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression. PMID:26090700

  2. Development of single-chain Fv against the nucleoprotein of type A influenza virus and its use in ELISA.

    PubMed

    Sengupta, Devyani; Shaikh, Asma; Bhatia, S; Pateriya, A K; Khandia, R; Sood, R; Prakash, A; Pattnaik, B; Pradhan, H K

    2014-11-01

    Single chain fragment variable (ScFv) antibodies specific to the nucleoprotein (NP) of avian influenza virus (AIV) were developed using a phage display system. The variable heavy (VH) and the variable light (VL) chain gene fragments were derived from spleen cells of Balb/c mouse immunized with a recombinant NP (rNP) antigen (∼63 kDa) of H5N1 influenza virus. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in a phagemid to express ScFv protein in Escherichia coli cells. The specific reactivity of the ScFv with the rNP antigen and viral antigen (H5N1) was confirmed by Western blot and ELISA. A competitive inhibition ELISA (CI-ELISA) was developed using the rNP and the anti-NP ScFv for detection of type-specific antibodies to AIV in chicken sera. The ScFv based CI-ELISA was compared with hemagglutination inhibition (HI) test and agar gel immunodiffusion (AGID) test over 850 sera. Sensitivity of the CI-ELISA was 100% with HI and AGID and specificity was 98.7% with HI and 100% with AGID. PMID:25152529

  3. Deciphering the role of individual acyl chains in the interaction network between phosphatidylserines and a single-spanning membrane protein.

    PubMed

    Mousson, Florence; Coïc, Yves-Marie; Baleux, Françoise; Beswick, Veronica; Sanson, Alain; Neumann, Jean-Michel

    2002-11-19

    PMP1 is a small single-spanning membrane protein functioning as a regulatory subunit of the yeast plasma membrane H(+)-ATPase. This protein forms a unique helix and exhibits a positively charged cytoplasmic domain that is able to specifically segregate phosphatidylserines (PSs). A marked groove formed at the helix surface is thought to play a major role in the related lipid-protein interaction network. Mutational analysis and (1)H NMR experiments were therefore performed on a synthetic PMP1 fragment using DPC-d(38) micelles as a membrane-like environment, in the presence of small amounts of POPS. A mutation designed for altering the helix groove was shown to disfavor the POPS binding specificity as much as that affecting the electrostatic interaction network. From POPS titration experiments monitored by a full set of one- and two-dimensional NOESY spectra, the association between the phospholipids and the PMP1 peptide has been followed. Our data reveal that the clustering of POPS molecules is promoted from a stabilized framework obtained by coupling the PMP1 helix groove to a POPS sn-2 chain. To our knowledge, the NOE-based titration plots displayed in this report constitute the first NMR data that directly distinguish the role of the sn-1 and sn-2 acyl chains in a lipid-protein interaction. The results are discussed while taking into account our accurate knowledge of the yeast plasma membrane composition and its ability to form functional lipid rafts. PMID:12427022

  4. Entanglement entropy and fidelity susceptibility in the one-dimensional spin-1 XXZ chains with alternating single-site anisotropy.

    PubMed

    Ren, Jie; Liu, Guang-Hua; You, Wen-Long

    2015-03-18

    We study the fidelity susceptibility in an antiferromagnetic spin-1 XXZ chain numerically. By using the density-matrix renormalization group method, the effects of the alternating single-site anisotropy D on fidelity susceptibility are investigated. Its relation with the quantum phase transition is analyzed. It is found that the quantum phase transition from the Haldane spin liquid to periodic Néel spin solid can be well characterized by the fidelity. Finite size scaling of fidelity susceptibility shows a power-law divergence at criticality, which indicates the quantum phase transition is of second order. The results are confirmed by the second derivative of the ground-state energy. We also study the relationship between the entanglement entropy, the Schmidt gap and quantum phase transitions. Conclusions drawn from these quantum information observables agree well with each other. PMID:25707024

  5. Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology.

    PubMed

    Singh, Pawan Kumar; Agrawal, Ranu; Kamboj, D V; Singh, Lokendra

    2016-01-01

    Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology. PMID:26676049

  6. Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris.

    PubMed

    Chang, Hyun-Joo; Choi, Sung-Wook; Chun, Hyang Sook

    2008-10-01

    A synthetic gene coding for single-chain variable domain fragment antibody against mycotoxin zearalenone (scFv-ZEN) has been designed, constructed and expressed in Pichia pastoris. The native scFv-ZEN sequence was optimized to Pichia preference codon usage. The expression level of codon-optimized scFv-ZEN was slightly higher than that of native scFv-ZEN, and its maximum yield reached 328 mg total protein/l in flask culture. The binding activities of two selected clones to ZEN using surface plasmon resonance analysis were comparable or better than that of monoclonal antibody. Our results demonstrate the potential of soluble scFv-ZEN for developing a rapid and affordable immunoassay for detection of ZEN in food and feedstuff. PMID:18575809

  7. The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant.

    PubMed

    Li, Ting-Feng; Painter, Richard G; Ban, Bhupal; Blake, Robert C

    2015-07-24

    Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm(2) in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s(-1). The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment. PMID:26041781

  8. The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant*

    PubMed Central

    Li, Ting-Feng; Painter, Richard G.; Ban, Bhupal; Blake, Robert C.

    2015-01-01

    Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm2 in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s−1. The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment. PMID:26041781

  9. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity

    PubMed Central

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A.; Motamedi-Shad, Neda; Irving, James A.; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J.; Miranda, Elena; Lomas, David A.

    2015-01-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity. PMID:25757566

  10. Gene expression of single human mesenchymal stem cell in response to fluid shear

    PubMed Central

    Zhang, Hu; Kay, Alasdair; Forsyth, Nicholas R; Liu, Kuo-Kang

    2012-01-01

    Stem cell therapy may rely on delivery and homing through the vascular system to reach the target tissue. An optical tweezer model has been employed to exert different levels of shear stress on a single non-adherent human bone marrow–derived mesenchymal stem cell to simulate physiological flow conditions. A single-cell quantitative polymerase chain reaction analysis showed that collagen type 1, alpha 2 (COL1A2), heat shock 70-kDa protein 1A (HSPA1A) and osteopontin (OPN) are expressed to a detectable level in most of the cells. After exposure to varying levels of shear stress, there were significant variations in gene transcription levels across human mesenchymal stem cells derived from four individual donors. Significant trend towards upregulation of COL1A2 and OPN gene expression following shear was observed in some donors with corresponding variations in HSPA1A gene expression. The results indicate that shear stress associated with vascular flow may have the potential to significantly direct non-adherent stem cell expression towards osteogenic phenotypic expression. However, our results demonstrate that these results are influenced by the selection process and donor variability. PMID:22798982

  11. The possible role of long-chain, omega-3 fatty acids in human brain phylogeny.

    PubMed

    Chamberlain, J G

    1996-01-01

    I propose that one of the key factors in human encephalization was increased HUFA intake, especially long-chain, omega-3 fatty acids from aquatic and terrestial meat source. This provided the needed neural membrane fluidity and transmitter/receptor functions for rapid acquisition of more advanced human traits and allowed the expansion of H. erectus into more northern climates. The human brain initially could build ecophenotypically, or adaptive/directed mutationally upon previously evolved mammalian sensor/motor structures, and could rapidly expand cognitive functions within a few million years; as more niches were invaded, more brain diversity was needed to guarantee reproductive success. The metabolically expensive and expanding brain was nutritionally and biochemically set, as it were, for rapid accommodation to tool making, rock throwing, culture language, electronics, and the eventual endless discussion and writings about the brain itself, the evolution of consciousness, and the mid-bran problem [107]. All of this fits, no matter which theory of human evolution one adheres to--i.e., out of Africa, multiregional, etc.--or even the precis fossil chronology [108]. This proposal, based as it is on known facts and certain assumptions appears logical, simple, and satisfying, but it may be wrong. Yet Charles Darwin himself would have approved, because as he so aptly said: false facts are highly injurious to the progress of science, for they often endure long; but false views, if supported by some evidence do little harm for everyone takes a salutory pleasure in providing their falseness; and when this is done our path toward error is closed and the road to truth is often opened. [109]. PMID:8657555

  12. Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection.

    PubMed

    Venkatesan, Vasuki; Hoti, Sugeerappa Laxmanappa; Kamaraj, Nagalakshmi; Ghosh, Somnath; Rajaram, Kaushik

    2013-09-01

    Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring. PMID:24037206

  13. Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

    PubMed Central

    Venkatesan, Vasuki; Hoti, Sugeerappa Laxmanappa; Kamaraj, Nagalakshmi; Ghosh, Somnath; Rajaram, Kaushik

    2013-01-01

    Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring. PMID:24037206

  14. Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS).

    PubMed

    Mejías, Maria P; Hiriart, Yanina; Lauché, Constanza; Fernández-Brando, Romina J; Pardo, Romina; Bruballa, Andrea; Ramos, María V; Goldbaum, Fernando A; Palermo, Marina S; Zylberman, Vanesa

    2016-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome. PMID:27118524

  15. Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS)

    PubMed Central

    Mejías, Maria P.; Hiriart, Yanina; Lauché, Constanza; Fernández-Brando, Romina J.; Pardo, Romina; Bruballa, Andrea; Ramos, María V.; Goldbaum, Fernando A.; Palermo, Marina S.; Zylberman, Vanesa

    2016-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome. PMID:27118524

  16. On-chip real-time single-copy polymerase chain reaction in picoliter droplets

    SciTech Connect

    Beer, N R; Hindson, B; Wheeler, E; Hall, S B; Rose, K A; Kennedy, I; Colston, B

    2007-04-20

    The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 10{sup 6} smaller than commercial real-time PCR systems. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermal cycled through the PCR protocol without droplet motion. With this system a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of {approx}18, twenty cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.

  17. Biodetection of potential genotoxic pollutants entering the human food chain through ashes used in livestock diets.

    PubMed

    Sanchez-Vicente, Laura; Herraez, Elisa; Briz, Oscar; Nogales, Rogelio; Molina-Alcaide, Eduarda; Marin, Jose J G

    2016-08-15

    Ash derived from energy generation is used as a source of minerals in livestock feeds. The microbial biosensor recApr-Luc2 was built to detect genotoxic hazard in recycled ash. Escherichia coli SOS gene (recA, lexA, dinI and umuC) expression in response to cisplatin-induced DNA damage led to the selection of the recA promoter. The biosensor required functional RecA expression to respond to genotoxic heavy metals (Cr>Cd≈Pb), and polluted ash induced a strong recApr-Luc2 response. In human liver and intestinal cells, heavy metals induced acute toxicity (Cr>Cd>Pb) at concentrations sufficient to activate recApr-Luc2. Cytostatic effects, including genotoxicity, were cell- and metal-dependent, apart from Cr. In agreement with the recApr-Luc2 bioassay, Cr had the strongest effect in all cells. In conclusion, recApr-Luc2 could be useful for evaluating the genotoxic risk of pollutants present in ash that might be concentrated in animal products and, thus, entering the human food chain. PMID:27006217

  18. Polymerase chain reaction–based assays for the diagnosis of human brucellosis

    PubMed Central

    2014-01-01

    Polymerase chain reaction (PCR) is an in vitro technique for the nucleic acid amplification, which is commonly used to diagnose infectious diseases. The use of PCR for pathogens detection, genotyping and quantification has some advantages, such as high sensitivity, high specificity, reproducibility and technical ease. Brucellosis is a common zoonosis caused by Brucella spp., which still remains as a major health problem in many developing countries around the world. The direct culture and immunohistochemistry can be used for detecting infection with Brucella spp. However, PCR has the potential to address limitations of these methods. PCR are now one of the most useful assays for the diagnosis in human brucellosis. The aim of this review was to summarize the main PCR techniques and their applications for diagnosis and follow-up of patients with brucellosis. Moreover, advantages or limitation of the different PCR methods as well as the evaluation of PCR results for treatment and follow-up of human brucellosis were also discussed. PMID:25082566

  19. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis

    PubMed Central

    Guo, Xi-zhi J; Dash, Pradyot; Calverley, Matthew; Tomchuck, Suzanne; Dallas, Mari H; Thomas, Paul G

    2016-01-01

    Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα–β–) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. PMID:26858965

  20. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis.

    PubMed

    Guo, Xi-Zhi J; Dash, Pradyot; Calverley, Matthew; Tomchuck, Suzanne; Dallas, Mari H; Thomas, Paul G

    2016-01-01

    Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα(-)β(-)) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. PMID:26858965

  1. Escherichia coli Surface Display of Single-Chain Antibody VRC01 against HIV-1 infection

    PubMed Central

    Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. PMID:25482819

  2. Transient overexpression of human H- and L-ferritin chains in COS cells.

    PubMed Central

    Corsi, B; Perrone, F; Bourgeois, M; Beaumont, C; Panzeri, M C; Cozzi, A; Sangregorio, R; Santambrogio, P; Albertini, A; Arosio, P; Levi, S

    1998-01-01

    The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism. PMID:9461525

  3. Omega-3 long-chain fatty acids strongly induce angiopoietin-like 4 in humans.

    PubMed

    Brands, Myrte; Sauerwein, Hans P; Ackermans, Mariette T; Kersten, Sander; Serlie, Mireille J

    2013-03-01

    Angiopoietin-like 4 (ANGPTL4) is a regulator of LPL activity. In this study we examined whether different fatty acids have a differential effect on plasma ANGPTL4 levels during hyperinsulinemia in healthy lean males. In 10 healthy lean males, 3 hyperinsulinemic euglycemic clamps were performed during concomitant 6 h intravenous infusion of soybean oil (Intralipid® rich in PUFA), olive oil (Clinoleic® rich in MUFA) and control saline. In 10 other healthy lean males, 2 hyperinsulinemic clamps were performed during infusion of a mixed lipid emulsion containing a mixture of fish oil (FO), medium-chain triglycerides (MCTs), and long-chain triglycerides (LCTs) (FO/MCT/LCT; SMOFlipid®) or saline. FFA levels of approximately 0.5 mmol/l were reached during each lipid infusion. Plasma ANGPTL4 decreased during hyperinsulinemia by 32% (18-52%) from baseline. This insulin-mediated decrease in ANGPTL4 concentrations was partially reduced during concomitant infusion of olive oil and completely blunted during concomitant infusion of soybean oil and FO/MCT/LCT. The reduction in insulin sensitivity was similar between all lipid infusions. In accordance, incubation of rat hepatoma cells with the polyunsaturated fatty acid C22:6 increased ANGPTL4 expression by 70-fold, compared with 27-fold by the polyunsaturated fatty acid C18:2, and 15-fold by the monounsaturated fatty acid C18:1. These results suggest that ANGPTL4 is strongly regulated by fatty acids in humans, and is also dependent on the type of fatty acid. PMID:23319744

  4. Human temporal cortical single neuron activity during working memory maintenance.

    PubMed

    Zamora, Leona; Corina, David; Ojemann, George

    2016-06-01

    The Working Memory model of human memory, first introduced by Baddeley and Hitch (1974), has been one of the most influential psychological constructs in cognitive psychology and human neuroscience. However the neuronal correlates of core components of this model have yet to be fully elucidated. Here we present data from two studies where human temporal cortical single neuron activity was recorded during tasks differentially affecting the maintenance component of verbal working memory. In Study One we vary the presence or absence of distracting items for the entire period of memory storage. In Study Two we vary the duration of storage so that distractors filled all, or only one-third of the time the memory was stored. Extracellular single neuron recordings were obtained from 36 subjects undergoing awake temporal lobe resections for epilepsy, 25 in Study one, 11 in Study two. Recordings were obtained from a total of 166 lateral temporal cortex neurons during performance of one of these two tasks, 86 study one, 80 study two. Significant changes in activity with distractor manipulation were present in 74 of these neurons (45%), 38 Study one, 36 Study two. In 48 (65%) of those there was increased activity during the period when distracting items were absent, 26 Study One, 22 Study Two. The magnitude of this increase was greater for Study One, 47.6%, than Study Two, 8.1%, paralleling the reduction in memory errors in the absence of distracters, for Study One of 70.3%, Study Two 26.3% These findings establish that human lateral temporal cortex is part of the neural system for working memory, with activity during maintenance of that memory that parallels performance, suggesting it represents active rehearsal. In 31 of these neurons (65%) this activity was an extension of that during working memory encoding that differed significantly from the neural processes recorded during overt and silent language tasks without a recent memory component, 17 Study one, 14 Study two

  5. Side-chain control of porosity closure in single- and multiple-peptide-based porous materials by cooperative folding

    NASA Astrophysics Data System (ADS)

    Martí-Gastaldo, C.; Antypov, D.; Warren, J. E.; Briggs, M. E.; Chater, P. A.; Wiper, P. V.; Miller, G. J.; Khimyak, Y. Z.; Darling, G. R.; Berry, N. G.; Rosseinsky, M. J.

    2014-04-01

    Porous materials are attractive for separation and catalysis—these applications rely on selective interactions between host materials and guests. In metal-organic frameworks (MOFs), these interactions can be controlled through a flexible structural response to the presence of guests. Here we report a MOF that consists of glycyl-serine dipeptides coordinated to metal centres, and has a structure that evolves from a solvated porous state to a desolvated non-porous state as a result of ordered cooperative, displacive and conformational changes of the peptide. This behaviour is driven by hydrogen bonding that involves the side-chain hydroxyl groups of the serine. A similar cooperative closure (reminiscent of the folding of proteins) is also displayed with multipeptide solid solutions. For these, the combination of different sequences of amino acids controls the framework's response to the presence of guests in a nonlinear way. This functional control can be compared to the effect of single-point mutations in proteins, in which exchange of single amino acids can radically alter structure and function.

  6. Phase diagram and magnetization structures of spin-3/2 bond-alternating Ising chains with single-ion anisotropy

    NASA Astrophysics Data System (ADS)

    Liu, Guang-Hua; Dou, Jun-Ya; Tian, Guang-Shan

    2016-02-01

    By the infinite time-evolving block decimation (iTEBD) algorithm, the magnetization process of the spin-3/2 bond-alternating Ising chain with single-ion anisotropy (D) is investigated. Magnetization plateaus including detailed magnetization structures of three different cases are uncovered, and three rich ground-state phase diagrams are explicitly determined. Especially, for the uniform antiferromagnetic case, a phase transition line at D=J, which divides the Mz=0 (Mz =1/2) plateau into two phases, are detected by the magnetization structure and the ground-state energy, and a updated phase diagram is proposed. Such a transition line was not recognized by the average magnetization previously. A same transition line (D=J) is also detected in the phase diagram of the antiferromagnetic-ferromagnetic alternating case. Magnetization plateaus are found to be easily induced for the classical Ising systems without quantum fluctuations, and the single-ion anisotropy plays a key role in the formation of Mz = 1/2 and 1 plateaus in the present model.

  7. Reverse transcription-polymerase chain reaction detection of transcribed sequences on human chromosome 21

    SciTech Connect

    Cheng, J.F.; Zhu, Y. )

    1994-03-15

    Seventy-four pairs of oligonucleotides derived from sequence-tagged sites (STSs) on the long arm of human chromosome 21, specifically from bands 21q22.1 to 21q22.3, were used in reverse transcription-polymerase chain reactions (RT-PCR) to detect the presence of expressed sequences in a fetal brain. These STSs included 69 that had not been related to transcribed sequences and 5 that had detected two known genes and three previously isolated cDNA clones. Of the 69 STSs analyzed in RT-PCR, 25 allowed amplification of specific cDNA fragments. The sizes of amplified cDNA fragments match those amplified from either human genomic DNA or somatic hybrid cells containing human chromosome 21. Of the 11 cDNA analyzed in Northern blot hybridizations, 6 hybridized to specific RNA species. The rapid screening for cDNA using previously mapped STSs has provided insight into the distribution of expressed sequences in this region of chromosome 21. Northern blot analysis of the amplified cDNA fragments has revealed interesting candidate genes in two disease loci. The marker D21S267 was previously mapped in the Down syndrome region of chromosome 21, and the marker D21S113 is closely linked to progressive myoclonus epilepsy. The cDNA fragments amplified using the primer sequences derived from D21S267 and D21S113 hybridized to 7- and 6.5-kb transcripts, respectively, which seems to express predominantly in brain. 37 refs., 3 figs., 1 tab.

  8. A novel antiinflammatory role for the short-chain fatty acids in human labor.

    PubMed

    Voltolini, Chiara; Battersby, Sharon; Etherington, Sophie L; Petraglia, Felice; Norman, Jane E; Jabbour, Henry N

    2012-01-01

    Human parturition is an inflammatory process that can be activated prematurely by pathological stimuli. This study investigated the expression of G protein-coupled receptors GPR43 and GPR41 receptors in human uteroplacental tissues and the role of short-chain fatty acids (SCFA) in modulating inflammatory pathways in fetal membranes. Expression of GPR43 and GPR41 was investigated in uteroplacental tissues collected from women delivering at term or preterm after ethical approval and patient informed consent. The effect of SCFA on expression of inflammatory genes was assessed in amnion explants after culture with a mimetic of infection (lipopolysaccharide, LPS). Sodium propionate effect on LPS-induced neutrophil chemotaxis was evaluated by transwell assay. GPR43 and GPR41 mRNA expression was higher in myometrium and fetal membranes collected from women after the onset of labor. GPR43 protein expression localized to immune cells and vascular endothelium in the myometrium and epithelium of fetal membranes. Treatment with LPS significantly increased mRNA expression of GPR43 and inflammatory genes. Cotreatment with LPS and sodium propionate decreased LPS-induced expression of inflammatory genes including IL-6, IL-8, cyclooxygenase-2, IL-1α, intercellular adhesion molecule-1, and platelet endothelial cell adhesion molecule-1 but not IL-1β or lymphocyte function-associated antigen-1. Sodium propionate reduced LPS-induced neutrophil chemotaxis and protein secretion of the neutrophil chemoattractant IL-8. Finally, fetal membrane expression of GPR43 was significantly higher in women delivering preterm with evidence of infection. GPR43-SCFA interactions may represent novel pathways that regulate inflammatory processes involved in human labor. Suppression of inflammatory pathways by SCFA may be therapeutically beneficial for pregnant women at risk of pathogen-induced preterm delivery. PMID:22186417

  9. Short-chain fatty acids stimulate mucosal cell proliferation in the closed human rectum after Hartmann's procedure.

    PubMed

    Mortensen, F V; Langkilde, N C; Joergensen, J C; Hessov, I

    1999-08-01

    Hartmann's procedure in humans results in a closed rectum deprived of its natural short-chain fatty acid source. This induces atrophy of the entire rectal wall. Ki-67 is a monoclonal antibody directed towards proteins in the cell nucleus that are present only during cell proliferation. This study investigated the effects of short-chain fatty acids on mucosal cell proliferation in the human rectum after Hartmann's procedure by means of Ki-67. Eight patients in whom Hartmann's procedure was performed were treated with placebo and then short-chain fatty acids for 14 days. Biopsies specimens were taken from the rectum before and after treatment; these were prepared with Ki-67 and labeling index was determined. The treatment was found significantly to increase proliferative activity in the rectal mucosa (P<0.01); the increase was principally in the middle (P<0.01) and upper crypt (P<0.05) compartments. PMID:10460905

  10. ENGINES: exploring single nucleotide variation in entire human genomes

    PubMed Central

    2011-01-01

    Background Next generation ultra-sequencing technologies are starting to produce extensive quantities of data from entire human genome or exome sequences, and therefore new software is needed to present and analyse this vast amount of information. The 1000 Genomes project has recently released raw data for 629 complete genomes representing several human populations through their Phase I interim analysis and, although there are certain public tools available that allow exploration of these genomes, to date there is no tool that permits comprehensive population analysis of the variation catalogued by such data. Description We have developed a genetic variant site explorer able to retrieve data for Single Nucleotide Variation (SNVs), population by population, from entire genomes without compromising future scalability and agility. ENGINES (ENtire Genome INterface for Exploring SNVs) uses data from the 1000 Genomes Phase I to demonstrate its capacity to handle large amounts of genetic variation (>7.3 billion genotypes and 28 million SNVs), as well as deriving summary statistics of interest for medical and population genetics applications. The whole dataset is pre-processed and summarized into a data mart accessible through a web interface. The query system allows the combination and comparison of each available population sample, while searching by rs-number list, chromosome region, or genes of interest. Frequency and FST filters are available to further refine queries, while results can be visually compared with other large-scale Single Nucleotide Polymorphism (SNP) repositories such as HapMap or Perlegen. Conclusions ENGINES is capable of accessing large-scale variation data repositories in a fast and comprehensive manner. It allows quick browsing of whole genome variation, while providing statistical information for each variant site such as allele frequency, heterozygosity or FST values for genetic differentiation. Access to the data mart generating scripts and to

  11. Diffusion of human Replication Protein A along single stranded DNA

    PubMed Central

    Nguyen, Binh; Sokoloski, Joshua; Galletto, Roberto; Elson, Elliot L.; Wold, Marc S.; Lohman, Timothy M.

    2014-01-01

    Replication Protein A (RPA) is a eukaryotic single stranded (ss) DNA binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA-DNA interactions, we combined ensemble and single molecule fluorescence approaches to examine human RPA diffusion along ssDNA and find that an hRPA hetero-trimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1 ~5000 nucleotide2s−1 at 37°C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA. PMID:25058683

  12. Exploring human epileptic activity at the single-neuron level.

    PubMed

    Tankus, Ariel

    2016-05-01

    Today, localization of the seizure focus heavily relies on EEG monitoring (scalp or intracranial). However, current technology enables much finer resolutions. The activity of hundreds of single neurons in the human brain can now be simultaneously explored before, during, and after a seizure or in association with an interictal discharge. This technology opens up new horizons to understanding epilepsy at a completely new level. This review therefore begins with a brief description of the basis of the technology, the microelectrodes, and the setup for their implantation in patients with epilepsy. Using these electrodes, recent studies provide novel insights into both the time domain and firing patterns of epileptic activity of single neurons. In the time domain, seizure-related activity may occur even minutes before seizure onset (in its current, EEG-based definition). Seizure-related neuronal interactions exhibit complex heterogeneous dynamics. In the seizure-onset zone, changes in firing patterns correlate with cell loss; in the penumbra, neurons maintain their spike stereotypy during a seizure. Hence, investigation of the extracellular electrical activity is expected to provide a better understanding of the mechanisms underlying the disease; it may, in the future, serve for a more accurate localization of the seizure focus; and it may also be employed to predict the occurrence of seizures prior to their behavioral manifestation in order to administer automatic therapeutic interventions. PMID:26994366

  13. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

    PubMed

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An; Chang, Ya-Chun

    2015-10-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  14. Influence of Tacticity on Hydrophobicity of Poly(N-isopropylacrylamide): A Single Chain Molecular Dynamics Simulation Study.

    PubMed

    Chiessi, Ester; Paradossi, Gaio

    2016-04-21

    Several pieces of experimental evidence show that the water affinity of poly(N-isopropylacrylamide), PNIPAM, decreases with the increase of the degree of isotacticity. To give a rationale to such effect we carried out atomistic molecular dynamics simulations of PNIPAM 30-mers with content of meso dyads, m, of 45% and 59%, assuming a Bernoullian dyad distribution. The single chain behavior of these stereoisomers in water was modeled at 283 and 323 K, i.e., below and above the lower critical solution temperature, LCST. Simulation results show that the dyad composition has influence on size and conformation of the oligomer below the LCST, the isotactic-rich stereoisomer preferring conformations with lower radius of gyration. With reference to the cooperative hydration model of PNIPAM, we analyzed the organization and the dynamics of water in the surroundings of the polymer. Below the LCST the number of hydrogen bonds per water molecule in the first hydration shell and the water surface concentration around PNIPAM are affected by the meso/racemo dyad ratio, showing the higher hydrophobicity of the isotactic-rich system. Above the LCST the subtle effects leading to the modulation of conformation and hydration by means of tacticity are overtaken, and the chain collapse is observed for both systems up to a similar globular state. The comparison of simulation findings of the m 45% stereoisomer with available experimental results of atactic PNIPAM highlights additional molecular details of this macromolecule in aqueous environment. The characteristic time for motion of water molecules in the PNIPAM first hydration shell at 283 K is about 34 ps, in agreement with the experimental value. PMID:27031404

  15. Studies on recombinant single chain Jacalin lectin reveal reduced affinity for saccharides despite normal folding like native Jacalin

    PubMed Central

    Sahasrabuddhe, Anagh A.; Gaikwad, Sushama M.; Krishnasastry, M.V.; Khan, M. Islam

    2004-01-01

    Sugar binding studies, inactivation, unfolding, and refolding of native Jacalin (nJacalin) from Artocarpus integrifolia and recombinant single-chain Jacalin (rJacalin) expressed in Escherichia coli were studied by intrinsic fluorescence and thermal and chemical denaturation approaches. Interestingly, rJacalin does not undergo any proteolytic processing in an E. coli environment. It has 100fold less affinity for methyl-α-galactose (Ka: 2.48 × 102) in comparison to nJacalin (Ka: 1.58 × 104), and it also binds Thomsen-Friedenreich (TF) disaccharide (Galβ1–3GalNAc) with less affinity. Overall sugar binding characteristics of rJacalin are qualitatively similar to that of nJacalin (Galchains is probably not a prerequisite for sugar binding but may be required for higher affinity. PMID:15557267

  16. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    PubMed Central

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  17. Human and animal health risk assessments of chemicals in the food chain: Comparative aspects and future perspectives

    SciTech Connect

    Dorne, J.L.C.M.; Fink-Gremmels, J.

    2013-08-01

    Chemicals from anthropogenic and natural origins enter animal feed, human food and water either as undesirable contaminants or as part of the components of a diet. Over the last five decades, considerable efforts and progress to develop methodologies to protect humans and animals against potential risks associated with exposure to such potentially toxic chemicals have been made. This special issue presents relevant methodological developments and examples of risk assessments of undesirable substances in the food chain integrating the animal health and the human health perspective and refers to recent Opinions of the Scientific Panel on Contaminants in the Food Chain (CONTAM) of the European Food Safety Authority (EFSA). This introductory review aims to give a comparative account of the risk assessment steps used in human health and animal health risk assessments for chemicals in the food chain and provides a critical view of the data gaps and future perspectives for this cross-disciplinary field. - Highlights: ► Principles of human and animal health risk assessment. ► Data gaps for each step of animal health risk assessment. ► Implications of animal risk assessment on human risk assessment. ► Future perspectives on chemical risk assessment.

  18. Sequential assignment of proton resonances in the NMR spectrum of Zn-substituted alpha chains from human hemoglobin. Ligand-induced tertiary changes in the heme pocket.

    PubMed

    Martineau, L; Craescu, C T

    1993-06-01

    We constructed an artificial holoprotein as a complex between alpha globin from human adult hemoglobin and the protoporphyrin IX-Zn(II). The prosthetic group is bound in a single conformation to the apoglobin via a coordinative bond between Zn(II) ion and the proximal histidine (His87). The complex is diamagnetic and does not bind either CO nor O2 thus representing a diamagnetic model of deoxygenated alpha chains. In the present paper we report extensive resonance assignment in the proton nuclear magnetic resonance spectrum of the Zn-substituted alpha chains in phosphate buffer pH 5.6. A large number of aromatic and aliphatic side chain spin systems were identified in the two-dimensional homonuclear COSY spectra. Based on the assigned resonances of heme substituent protons and their NOE cross-peaks, we assigned the majority of resonances representing the heme pocket side chains. Using the main-chain-directed assignment strategy, we could establish several continuous patterns of sequential assignment and identify partial or total spin systems for a large number of side chains. The final assignment corresponds to 73% of the amino acids. Analysis of chemical shift of assigned resonances and of nuclear Overhauser enhancement connectivities provides structural information on the global and local tertiary conformation in solution and on the ligand-induced conformational changes. Comparison of observed and calculated ring current shifts enabled us to compare the solution structure with the X-ray crystal structure of alpha subunits in deoxy and carbonmonoxy hemoglobin. The global tertiary structure of unliganded chains is highly similar to both ligand and unliganded counterparts in the crystalline state. On the distal side of the heme pocket. Val62 is significantly closer to the heme center, in agreement with its conformation in the crystallographic structure. In contrast, the position of the proximal histidine (His87) relative to the heme is clearly more closely related

  19. Single Chain Antibodies as Tools to Study transforming growth factor-β-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens.

    PubMed

    Blokzijl, Andries; Zieba, Agata; Hust, Michael; Schirrmann, Thomas; Helmsing, Saskia; Grannas, Karin; Hertz, Ellen; Moren, Anita; Chen, Lei; Söderberg, Ola; Moustakas, Aristidis; Dübel, Stefan; Landegren, Ulf

    2016-06-01

    The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-β signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-β signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-β signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors

  20. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG

    PubMed Central

    Fischer, Nicolas; Elson, Greg; Magistrelli, Giovanni; Dheilly, Elie; Fouque, Nicolas; Laurendon, Amélie; Gueneau, Franck; Ravn, Ulla; Depoisier, Jean-François; Moine, Valery; Raimondi, Sylvain; Malinge, Pauline; Di Grazia, Laura; Rousseau, François; Poitevin, Yves; Calloud, Sébastien; Cayatte, Pierre-Alexis; Alcoz, Mathias; Pontini, Guillemette; Fagète, Séverine; Broyer, Lucile; Corbier, Marie; Schrag, Delphine; Didelot, Gérard; Bosson, Nicolas; Costes, Nessie; Cons, Laura; Buatois, Vanessa; Johnson, Zoe; Ferlin, Walter; Masternak, Krzysztof; Kosco-Vilbois, Marie

    2015-01-01

    Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies. PMID:25672245

  1. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG.

    PubMed

    Fischer, Nicolas; Elson, Greg; Magistrelli, Giovanni; Dheilly, Elie; Fouque, Nicolas; Laurendon, Amélie; Gueneau, Franck; Ravn, Ulla; Depoisier, Jean-François; Moine, Valery; Raimondi, Sylvain; Malinge, Pauline; Di Grazia, Laura; Rousseau, François; Poitevin, Yves; Calloud, Sébastien; Cayatte, Pierre-Alexis; Alcoz, Mathias; Pontini, Guillemette; Fagète, Séverine; Broyer, Lucile; Corbier, Marie; Schrag, Delphine; Didelot, Gérard; Bosson, Nicolas; Costes, Nessie; Cons, Laura; Buatois, Vanessa; Johnson, Zoe; Ferlin, Walter; Masternak, Krzysztof; Kosco-Vilbois, Marie

    2015-01-01

    Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies. PMID:25672245

  2. A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

    SciTech Connect

    Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.; Guetarni, D.; Bachir, D.; Colonna, P.; Beuzard, Y. )

    1989-11-15

    The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with ({sup 3}H)N-ethylmaleimide. This pool of soluble alpha chains was 0.067 {plus minus} 0.017% of hemoglobin in blood of normal adult, 0.11 {plus minus} 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using ({sup 3}H)N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.

  3. Activated Platelets in Carotid Artery Thrombosis in Mice Can Be Selectively Targeted with a Radiolabeled Single-Chain Antibody

    PubMed Central

    Goldschmidt, Jürgen; Pethe, Annette; Hagemeyer, Christoph E.; Neudorfer, Irene; Zirlik, Andreas; Weber, Wolfgang A.; Bode, Christoph; Meyer, Philipp T.

    2011-01-01

    Background Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. Methodology/Principal Findings LIBS as well as an unspecific control single-chain antibody were labeled with 111Indium (111In) via bifunctional DTPA ( = 111In-LIBS/111In-control). Autoradiography after incubation with 111In-LIBS on activated platelets in vitro (mean 3866±28 DLU/mm2, 4010±630 DLU/mm2 and 4520±293 DLU/mm2) produced a significantly higher ligand uptake compared to 111In-control (2101±76 DLU/mm2, 1181±96 DLU/mm2 and 1866±246 DLU/mm2) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of 111In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630±10650 DLU/mm2 vs. 17390±7470 DLU/mm2; P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with 111In-LIBS resulted in a significant increase of the target-to-background ratio compared to 111In-control (1.99±0.36 vs. 1.1±0.24; P<0.01). Conclusions/Significance Nuclear imaging with 111In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis

  4. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A.

    PubMed

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I; Fischer, Kathelijn; Gill, Joan C; P'Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St Ledger, Katie; Pabinger, Ingrid

    2016-08-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain-truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

  5. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

    PubMed Central

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V.; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N.; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I.; Fischer, Kathelijn; Gill, Joan C.; P’Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A.; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St. Ledger, Katie

    2016-01-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

  6. Immunohistochemistry and Polymerase Chain Reaction for Detection Human Papilloma Virus in Warts: A Comparative Study

    PubMed Central

    Lee, Hong Sun; Lee, Ji Hyun; Choo, Ji Yoon; Byun, Hee Jin; Jun, Jin Hyun

    2016-01-01

    Background Immunohistochemistry and polymerase chain reaction (PCR) are the most widely used methods for the detection of viruses. PCR is known to be a more sensitive and specific method than the immunohistochemical method at this time, but PCR has the disadvantages of high cost and skilled work to use widely. With the progress of technology, the immunohistochemical methods used in these days has come to be highly sensitive and actively used in the diagnostic fields. Objective To evaluate and compare the usefulness of immunohistochemistry and PCR for detection human papilloma virus (HPV) in wart lesions. Methods Nine biopsy samples of verruca vulgaris and 10 of condyloma accuminatum were examined. Immunohistochemical staining using monoclonal antibody to HPV L1 capsid protein and PCR were done for the samples. DNA sequencing of the PCR products and HPV genotyping were also done. Results HPV detection rate was 78.9% (88.9% in verruca vulgaris, 70.0% in condyloma accuminatum) on immunohistochemistry and 100.0% for PCR. HPV-6 genotype showed a lower positivity rate on immunohistochemistry (50.0%) as compared to that of the other HPV genotypes. Conclusion Immunohistochemistry for HPV L1 capsid protein showed comparable sensitivity for detection HPV. Considering the high cost and great effort needed for the PCR methods, we can use immunohistochemistry for HPV L1 capsid protein with the advantage of lower cost and simple methods for HPV detection. PMID:27489431

  7. Kinematics of the human knee using an open chain cadaver model.

    PubMed

    Blaha, J David; Mancinelli, Corrie A; Simons, William H; Kish, Vincent L; Thyagarajan, Ganesh

    2003-05-01

    There continues to be controversy about the kinematics of the human knee. This study used seven knees from cadavers moved by pulling on the quadriceps tendon in an open chain fashion using video motion analysis to determine the instantaneous helical axis of movement. Computed tomography scans of the specimens allowed the axes to be related to condyles. The parameter beta was defined by the relationship of the helical axis to the center of the condyle (pure spinning motion) and the contact point of the condyle on the tibia (pure rolling motion). Axes above the center of the condyle represent countertranslation, those between the center and the contact point combined spinning and rolling, and those below represent concordant translation. If the motion of the knee is guided by the crossed four-bar link then this model, that allows the knee to 'seek its own path' throughout the range of motion, should show the rollback that commonly is thought to be an important feature of knee motion. The results of this study show that the medial side of the knee stays stable in spinning kinematics whereas the lateral side has a rolling motion in full flexion progressing to a spinning motion in midflexion and counter-translation near full extension. The kinematics that would be expected from rollback were not observed. PMID:12771814

  8. Human mesenchymal stem cell behavior on segmented polyurethanes prepared with biologically active chain extenders.

    PubMed

    Kavanaugh, Taylor E; Clark, Amy Y; Chan-Chan, Lerma H; Ramírez-Saldaña, Maricela; Vargas-Coronado, Rossana F; Cervantes-Uc, José M; Hernández-Sánchez, Fernando; García, Andrés J; Cauich-Rodríguez, Juan V

    2016-02-01

    The development of elastomeric, bioresorbable and biocompatible segmented polyurethanes (SPUs) for use in tissue-engineering applications has attracted considerable interest because of the existing need of mechanically tunable scaffolds for regeneration of different tissues, but the incorporation of osteoinductive molecules into SPUs has been limited. In this study, SPUs were synthesized from poly (ε-caprolactone)diol, 4,4'-methylene bis(cyclohexyl isocyanate) using biologically active compounds such as ascorbic acid, L-glutamine, β-glycerol phosphate, and dexamethasone as chain extenders. Fourier transform infrared spectroscopy (FTIR) revealed the formation of both urethanes and urea linkages while differential scanning calorimetry, dynamic mechanical analysis, X-ray diffraction and mechanical testing showed that these polyurethanes were semi-crystalline polymers exhibiting high deformations. Cytocompatibility studies showed that only SPUs containing β-glycerol phosphate supported human mesenchymal stem cell adhesion, growth, and osteogenic differentiation, rendering them potentially suitable for bone tissue regeneration, whereas other SPUs failed to support either cell growth or osteogenic differentiation, or both. This study demonstrates that modification of SPUs with osteogenic compounds can lead to new cytocompatible polymers for regenerative medicine applications. PMID:26704555

  9. In Vitro Cytotoxicity of Benzopyranone Derivatives with Basic Side Chain Against Human Lung Cell Lines

    PubMed Central

    Musa, Musiliyu A.; Badisa, Veera L.D.; Latinwo, Lekan M.; Waryoba, Caroline; Ugochukwu, Ngozi

    2013-01-01

    Background Coumarins belong to an important group of useful drugs with diverse pharmacological properties. In the present study, the in vitro cytotoxicity of new coumarin-based benzopyranone derivatives containing diethylaminoethoxy (5), dimethylaminoethoxy (6), morpholinoethoxy (7), piperidinylethoxy (8) and pyrrolidinylethoxyl (9) amino side chain against human carcinoma (A549) and normal (LL47) lung cell lines was evaluated. Materials and Methods The cytotoxicity was evaluated by crystal violet dye binding assay. The effect of compound 9 on different phases of the cell cycle was determined using flow cytometry. Results In A549 cells, the 50% lethal dose (LD50) for compounds 5–9 were found to be 7.08, 5.0, 34.2, 8.33 and 5.83 µM, respectively, while in LL47 cells, the LD50 values were found to be 16.7, 20.4, 34.6, 15.4 and 8.75 µM, respectively after 48 h treatment. Cell cycle data indicated that A549 cells were arrested at different phases depending on the concentration. Conclusion Compounds 5–9 showed anticancer activity against lung cancer cell lines, while compound 6 showed highly selective anticancer activity. PMID:21115914

  10. Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

    PubMed Central

    Adekar, Sharad P.; Takahashi, Tsuyoshi; Jones, R. Mark; Al-Saleem, Fetweh H.; Ancharski, Denise M.; Root, Michael J.; Kapadnis, B. P.; Simpson, Lance L.; Dessain, Scott K.

    2008-01-01

    Background Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure. PMID:18714390

  11. Use of polymerase chain reaction in human African trypanosomiasis stage determination and follow-up.

    PubMed Central

    Truc, P.; Jamonneau, V.; Cuny, G.; Frézil, J. L.

    1999-01-01

    Stage determination of human African trypanosomiasis is based on the detection of parasites and measurements of biological changes in the cerebrospinal fluid (CSF) (concentration of white blood cells > 5 cells per mm3 and increased total protein levels). The patient is treated accordingly. Demonstration of the absence or presence of trypanosomes by the double centrifugation technique is still the only test available to clinicians for assessing treatment success. In this study, however, we evaluate the polymerase chain reaction (PCR) as a tool for assessing the disease stage of trypanosomiasis and for determining whether treatment has been successful. All 15 study patients considered to be in the advanced stage of the disease were PCR positive; however, trypanosomes were demonstrated by double centrifugation in only 11 patients. Of the five remaining patients, who were considered to be in the early stage, PCR and double centrifugation were negative. Following treatment, 13 of the 15 second-stage patients were found to be negative for the disease in at least two samples by PCR and double centrifugation. Two others were still positive by PCR immediately and one month after the treatment. Trypanosome DNA detection using PCR suggested that the two positive patients were not cured but that their possible relapse could not be identified by a search for parasites using the double centrifugation technique. Further evaluation of the PCR method is required, in particular to determine whether PCR assays could be used in studies on patients who fail to respond to melarsoprol, as observed in several foci. PMID:10534898

  12. GPI-anchored single chain Fv - an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike

    PubMed Central

    2010-01-01

    Background Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development. A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes. However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify. Results In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal. We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes. In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency. Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested. Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart. Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4+ T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs. Conclusions Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses. The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy. PMID:20923574

  13. The complete amino acid sequence of the A-chain of human plasma alpha 2HS-glycoprotein.

    PubMed

    Yoshioka, Y; Gejyo, F; Marti, T; Rickli, E E; Bürgi, W; Offner, G D; Troxler, R F; Schmid, K

    1986-02-01

    Normal human plasma alpha 2HS-glycoprotein has earlier been shown to be comprised of two polypeptide chains. Recently, the amino acid and carbohydrate sequences of the short chain were elucidated (Gejyo, F., Chang, J.-L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R.F., van Halbeck, H., Dorland, L., Gerwig, G. J., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 4966-4971). In the present study, the amino acid sequence of the long chain of this protein, designated A-chain, was determined and found to consist of 282 amino acid residues. Twenty-four amino acid doublets were found; the most abundant of these are Pro-Pro and Ala-Ala which each occur five times. Of particular interest is the presence of three Gly-X-Pro and one Gly-Pro-X sequences that are characteristic of the repeating sequences of collagens. Chou-Fasman evaluation of the secondary structure suggested that the A-chain contains 29% alpha-helix, 24% beta-pleated sheet, and 26% reverse turns and, thus, approximately 80% of the polypeptide chain may display ordered structure. Four glycosylation sites were identified. The two N-glycosidic oligosaccharides were found in the center region (residues 138 and 158), whereas the two O-glycosidic heterosaccharides, both linked to threonine (residues 238 and 252), occur within the carboxyl-terminal region. The N-glycans are linked to Asn residues in beta-turns, while the O-glycans are located in short random segments. Comparison of the sequence of the amino- and carboxyl-terminal 30 residues with protein sequences in a data bank demonstrated that the A-chain is not significantly related to any known proteins. However, the proline-rich carboxyl-terminal region of the A-chain displays some sequence similarity to collagens and the collagen-like domains of complement subcomponent C1q. PMID:3944104

  14. Isothermal growth, thickening and melting of poly(ethylene-oxide) single crystals in the bulk. III. Bilayer crystals and the effect of chain ends

    NASA Astrophysics Data System (ADS)

    Kovacs, A. J.; Straupe, C.

    1980-02-01

    Growth, thickening and melting of poly(ethylene-oxide) single crystals, grown from the melt, have been investigated using three fractions of nearly the same chain length. The data are compared to and complement previous work, which is briefly recalled. The new results mainly concern the behavior of siamese twin-lamellae simultaneously grown from the same seed, modelling shish kebabs. On the other hand, the impact of a diphenyl group at one chain end is investigated in a systematic manner. The results show that these small modifications in the constitution of the crystals and in one of the chain ends give rise to rather large and often spectacular effects. The implications of these are discussed in terms of the detailed molecular conformations and mobility in the crystal lattice, in connection with current understanding of chain folding in lamellar polymer crystals.

  15. Adiposity, gut microbiota and faecal short chain fatty acids are linked in adult humans

    PubMed Central

    Fernandes, J; Su, W; Rahat-Rozenbloom, S; Wolever, T M S; Comelli, E M

    2014-01-01

    Background/Objectives: High dietary fibre intakes may protect against obesity by influencing colonic fermentation and the colonic microbiota. Though, recent studies suggest that increased colonic fermentation contributes to adiposity. Diet influences the composition of the gut microbiota. Previous research has not evaluated dietary intakes, body mass index (BMI), faecal microbiota and short chain fatty acid (SCFA) in the same cohort. Our objectives were to compare dietary intakes, faecal SCFA concentrations and gut microbial profiles in healthy lean (LN, BMI⩽25) and overweight or obese (OWOB, BMI>25) participants. Design: We collected demographic information, 3-day diet records, physical activity questionnaires and breath and faecal samples from 94 participants of whom 52 were LN and 42 OWOB. Results: Dietary intakes and physical activity levels did not differ significantly between groups. OWOB participants had higher faecal acetate (P=0.05), propionate (P=0.03), butyrate (P=0.05), valerate (P=0.03) and total short chain fatty acid (SCFA; P=0.02) concentrations than LN. No significant differences in Firmicutes to Bacteroides/Prevotella (F:B) ratio was observed between groups. However, in the entire cohort, Bacteroides/Prevotella counts were negatively correlated with faecal total SCFA (r=−0.32, P=0.002) and F:B ratio was positively correlated with faecal total SCFA (r=0.42, P<0.0001). Principal component analysis identified distinct gut microbiota and SCFA–F:B ratio components, which together accounted for 59% of the variation. F:B ratio loaded with the SCFA and not with the microbiota suggesting that SCFA and F:B ratio vary together and may be interrelated. Conclusions: The results support the hypothesis that colonic fermentation patterns may be altered, leading to different faecal SCFA concentrations in OWOB compared with LN humans. More in-depth studies looking at the metabolic fate of SCFA produced in LN and OWOB participants are needed in order to

  16. SCRL-Model for Human Space Flight Operations Enterprise Supply Chain

    NASA Technical Reports Server (NTRS)

    Tucker, Brian; Paxton, Joseph

    2010-01-01

    This paper will present a Supply Chain Readiness Level (SCRL) model that can be used to evaluate and configure adaptable and sustainable program and mission supply chains at an enterprise level. It will also show that using SCRL in conjunction with Technology Readiness Levels (TRLs), Manufacturing Readiness Levels (MRLs) and National Aeronautics Space Administrations (NASA s) Project Lifecycle Process will provide a more complete means of developing and evaluating a robust sustainable supply chain that encompasses the entire product, system and mission lifecycle. In addition, it will be shown that by implementing the SCRL model, NASA can additionally define supplier requirements to enable effective supply chain management (SCM). Developing and evaluating overall supply chain readiness for any product, system and mission lifecycle is critical for mission success. Readiness levels are presently being used to evaluate the maturity of technology and manufacturing capability during development and deployment phases of products and systems. For example, TRLs are used to support the assessment of the maturity of a particular technology and compare maturity of different types of technologies. MRLs are designed to assess the maturity and risk of a given technology from a manufacturing perspective. In addition, when these measurement systems are used collectively they can offer a more comprehensive view of the maturity of the system. While some aspects of the supply chain and supply chain planning are considered in these familiar metric systems, certain characteristics of an effective supply chain, when evaluated in more detail, will provide an improved insight into the readiness and risk throughout the supply chain. Therefore, a system that concentrates particularly on supply chain attributes is required to better assess enterprise supply chain readiness.

  17. Utility of a single nasal polymerase chain reaction assay in predicting absence of skin and environmental contamination in hospitalized patients with past methicillin-resistant Staphylococcus aureus.

    PubMed

    Guerrero, Dubert M; Wagner, Matthew; Carson, Grace; Hanish, Christine; Thompson, Jody; Orr, Megan; Roth, Felix; Carson, Paul J

    2016-06-01

    We evaluated hospitalized patients with a history of methicillin-resistant Staphylococcus aureus (MRSA) for persistent colonization and need for contact precautions. Up to 3 daily cultures of nares, skin, and any present wounds were compared with a single nasal polymerase chain reaction (PCR) assay. Most patients (76.2%) were no longer colonized with MRSA. A single PCR assay was sufficient to exclude persistent colonization and environmental contamination and remove the contact precautions. PMID:26874408

  18. Scanning probe microscopy characterization of single chains based on a one-dimensional oxalato-bridged manganese(II) complex with 4-aminotriazole.

    PubMed

    García-Couceiro, Urko; Olea, David; Castillo, Oscar; Luque, Antonio; Román, Pascual; de Pablo, Pedro J; Gómez-Herrero, Julio; Zamora, Félix

    2005-11-14

    The compound [Mn(mu-ox)(4atr)2]n (1) (ox = oxalato and 4atr = 4-amine-1,2,4-triazole) has been synthesized and characterized by FT-IR spectroscopy, thermal analysis, variable-temperature magnetic measurements, and X-ray single-crystal diffraction methods. The crystal structure of compound 1 consists of one-dimensional linear chains in which trans-[Mn(4atr)2]2+ units are sequentially bridged by centrosymmetric bis-bidentate oxalato ligands. Cryomagnetic measurements show an overall antiferromagnetic behavior of the compound. Isolated chains of this polymer have been obtained by sonication of 1 in ethanol or treatment of the polymer with NaOH and morphologically characterized on highly oriented pyrolitic graphite and mica surfaces by atomic force microscopy and scanning tunneling microscopy. The procedures employed to obtain single chains of this coordination polymer open a route for future nanotechnological applications of these types of materials. PMID:16270972

  19. Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics.

    PubMed

    Fujiki, Yuya; Kumada, Yoichi; Kishimoto, Michimasa

    2015-08-01

    The proteomics technique, which consists of two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), gel image analysis, and multivariate statistics, was applied to the phase analysis of a fed-batch culture for the production of a single-chain variable fragment (scFv) of an anti-C-reactive protein (CRP) antibody by Pichia pastoris. The time courses of the fed-batch culture were separated into three distinct phases: the growth phase of the batch process, the growth phase of the fed-batch process, and the production phase of the fed-batch process. Multivariate statistical analysis using 2-DE gel image analysis data clearly showed the change in the culture phase and provided information concerning the protein expression, which suggested a metabolic change related to cell growth and production during the fed-batch culture. Furthermore, specific proteins, such as alcohol oxidase, which is strongly related to scFv expression, and proteinase A, which could biodegrade scFv in the latter phases of production, were identified via the PMF method. The proteomics technique provided valuable information about the effect of the methanol concentration on scFv production. PMID:25636980

  20. Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

    PubMed

    Chen, Weifeng; Hu, Li; Liu, Aiping; Li, Jinquan; Chen, Fusheng; Wang, Xiaohong

    2014-11-01

    The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1). PMID:25322256

  1. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay.

    PubMed

    Sakamoto, Seiichi; Tanizaki, Yusuke; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-05-01

    A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv. PMID:21277981

  2. Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

    PubMed

    Sun, Yanan; Ning, Baoan; Liu, Ming; Gao, Xianjun; Fan, Xianjun; Liu, Jianqing; Gao, Zhixian

    2012-01-01

    Single chain variable fragments (scFvs) against diethylstilbestrol (DES) were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM) complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3) for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of one clone (30-1). The measured K(D) was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies. PMID:22427984

  3. Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display.

    PubMed

    Min, Won-Ki; Kim, Sung-Gun; Seo, Jin-Ho

    2015-12-01

    As aflatoxin B1 is one of the most toxic mycotoxins, it is important to detect and to quantify aflatoxin B1 accurately by immunological methods. To enhance aflatoxin B1-binding affinity of the single-chain variable fragment, yeast surface display technique combined with fluorescence-activated cell sorting was applied. A randomly mutated scFv library was subjected to 4 rounds of fluorescence-activated cell sorting, resulting in isolation of 5 scFv variants showing an affinity improvement compared to the parental wild type scFv. The best scFv with a 9-fold improvement in affinity for aflatoxin B1 exhibited similar specificity to the monoclonal antibody. Most of the mutations in scFv-M37 were located outside of the canonical antigen-contact loops, suggesting that its affinity improvement might be driven by an allosteric effect inducing scFv-M37 to form a more favorable binding pocket for aflatoxin B1 than the wild type scFv. PMID:26041237

  4. Single chain antibody fragment with serine protease inhibitory property capable of neutralizing toxicity of Trimeresurus mucrosquamatus venom.

    PubMed

    Lee, Yu-Ching; Chen, Wang-Chuan; Liang, Meng-Huei; Lee, Chi-Hsin; Tsai, Keng-Chang; Chiang, Jen-Ron; Chiang, Liao-Chun; Chen, Chi-Ching; Chang, Chang-Yu; Lee, Ching-Hsiao; Leu, Sy-Jye; Yang, Yi-Yuan

    2015-05-01

    Trimeresurus mucrosquamatus (TM) is one of majorities of snake envenomation with necrotic and hemorrhagic toxin in Taiwan. In this study, chickens were used as an alternative animal model for immunization with TM venom. Using phage display technology to process four rounds of panning, selected single chain variable fragments (scFv) could specifically recognize TM venom proteins, which were later identified as a group of homogeneous venom serine protease. The specific scFv antibodies showed various inhibitory effects on sheep RBC lysis induced by TM venom using an indirect hemolytic assay in vitro. In addition, the survival times of mice were extended to certain degrees when treated with these scFv antibodies individually or in a combination. To elucidate the inhibitory mechanism, we used molecular modeling to build up the serine protease structure to simulate the possible interactions with scFv antibodies. The results suggested that the CDR-loop of the scFv antibodies (3S10 or 4S1) might bind at the 99-loop of venom serine protease so as to affect substrate access due to the partial collapse of the subsite S2 and the partial movement of the subsite S4. It is hoped these chicken-derived antibodies could be applied to develop diagnostic and therapeutic agents against snakebites. PMID:25769957

  5. Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus.

    PubMed

    Pyo, Hyun-Mi; Kim, In-Joong; Kim, Seong-Hee; Kim, Hyun-Soo; Cho, Soo-Dong; Cho, In-Soo; Hyun, Bang-Hun

    2009-03-23

    Porcine epidemic diarrhea virus (PEDV) is a causative agent of severe diarrhea which leads to death in piglets. Because of the high mortality which is up to 100% in suckling piglets, PED is an important porcine disease in Korea. In this study, we developed a prophylactic candidate using single-chain Fvs to prevent the PEDV infection. ScFvs of mouse monoclonal antibody which was verified to neutralize PEDV was expressed in Escherichia coli expression system. After the confirmation of PEDV neutralizing activity of purified recombinant scFvs by VN test, scFvs were expressed on the surface of E. coli cells. The signal sequence and autotransporter beta domain of protease IgA (IgAP) of Neisseria gonorrhoeae were introduced to endow scFvs with the direction to the cell surface and the support as a transmembrane domain. 5x10(6)CFU of E. coli expressing scFvs against PEDV showed promising result of 94% foci reduction compared to wild type E. coli. This result demonstrated that E. coli expressing scFvs on the cell surface retained functional potency of parent antibody and therefore blocked PEDV infection into target cells in vitro. This in vitro assay result proposes the perspective of recombinant E. coli cells expressing scFvs as a novel prophylactic against PEDV infection. PMID:19428826

  6. wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2

    PubMed Central

    Rideout, Marc C.; Naili, Ilham; Boldt, Jeffrey L.; Flores-Fujimoto, America; Patra, Sukanya; Rostron, Jason E.; Segall, Anca M.

    2013-01-01

    DNA repair pathways in bacteria that use homologous recombination involve the formation and subsequent resolution of Holliday junction (HJ) intermediates. We have previously identified several hexameric peptides that bind to HJs and interfere with HJ processing enzymes in vitro. The peptide WRWYCR and its D-amino acid stereoisomer wrwycr, are potent antibacterial agents. These hexapeptides must form homodimers in order to interact stably with HJs, and inhibit bacterial growth, and this represents a potential limitation. Herein we describe a disulfide bond-independent inhibitor, WRWYRGGRYWRW and its D-stereoisomer wrwyrggrywrw. We have characterized these single-chain, linear analogs of the hexapeptides, and show that in addition to effectively binding to HJs, and inhibiting the activity of DNA repair enzymes that process HJs, they have equal or greater potency against Gram-positive and Gram-negative bacterial growth. The analogs were also shown to cause DNA damage in bacteria, and disrupt the integrity of the bacterial cytoplasmic membrane. Finally, we found that they have little toxicity toward several eukaryotic cell types at concentrations needed to inhibit bacterial growth. PMID:23291222

  7. A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

    PubMed Central

    Cox, Dianne; Hodgson, Louis

    2014-01-01

    Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized. PMID:24798463

  8. Isolation of alpaca anti-idiotypic heavy-chain single-domain antibody for the aflatoxin immunoassay.

    PubMed

    Wang, Yanru; Li, Peiwu; Majkova, Zuzana; Bever, Candace R S; Kim, Hee Joo; Zhang, Qi; Dechant, Julie E; Gee, Shirley J; Hammock, Bruce D

    2013-09-01

    Anti-idiotypic antibodies recognize the antigenic determinants of an antibody, thus they can be used as surrogate antigens. Single-domain antibodies from camlid heavy-chain antibodies with the benefit features of small size, thermostability, and ease in expression, are leading candidates to produce anti-idiotypic antibodies. In this work, we constructed an antibody phage library from the mRNA of an alpaca immunized with an antiaflatoxin monoclonal antibody (mAb) 1C11. Three anti-idiotypic VHH antibodies were isolated and applied to immunoassay toward aflatoxin as a coating antigen. The best immunoassay developed with one of these VHH antibodies shows an IC50 of 0.16 ng/mL toward aflatoxin B1 and cross-reactivity toward aflatoxin B2, G1, and G2 of 90.4%, 54.4%, and 37.7%, respectively. The VHH-based immunoassay was successfully applied to the analysis of peanuts, corn, and rice, which are the predominant commodities regularly contaminated by aflatoxins. A good correlation (r(2) = 0.89) was found between the data obtained from the conventional ELISA and the ELISA based on a VHH coating antigen for the analysis of aflatoxins in peanuts and feedstuff. The use of biotechnology in developing the surrogate, the absence of standard aflatoxin and organic solvents in the synthesis procedures, and the reproducibility of the VHH antibody makes it an ideal strategy for replacing conventional synthesized antigens. PMID:23965250

  9. Selection of bisphenol A - single-chain antibodies from a non-immunized mouse library by ribosome display.

    PubMed

    Zhao, Li; Ning, Baoan; Bai, Jialei; Chen, Xiang; Peng, Yuan; Sun, Siming; Li, Guimin; Fan, Xianjun; Liu, Yuanyuan; Liu, Jianqing; Sun, Yanan; Gao, Zhixian; Zhang, Juankun

    2015-11-01

    Developing reagents with high affinity and specificity are critical to detect the environmental hormones or toxicants. Ribosome display technology has been widely used in functional protein or peptide screening and in directed evolution of protein molecules in vitro. In this study, single-chain variable fragments (scFvs) against bisphenol A (BPA) were selected from a library constructed from splenocytes of non-immunized mice. After five rounds of selection, the selected scFvs bound to BPA with high affinity. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was introduced to screen the antibody affinity and specificity to BPA. The equilibrium dissociation constants (KDS) of one clone was 1.76μM as determined by surface plasmon resonance (SPR). This study indicated that ribosome display can isolate binders to small molecules from a non-immunized naive library without any in vivo steps and can generate recombinant antibodies efficiently and rapidly. In addition, this study provides a methodological framework for detection of small molecules using recombinant antibodies. PMID:24269893

  10. CD176 single-chain variable antibody fragment inhibits the adhesion of cancer cells to endothelial cells and hepatocytes.

    PubMed

    Liu, Jiangnan; Yi, Bin; Zhang, Zhe; Cao, Yi

    2016-06-01

    CD176 (Thomsen-Friedenreich antigen) is a tumor-associated carbohydrate epitope (glycotope) functionally involved in blood spread and liver metastasis of cancer cells by mediating the adhesion of cancer cells to endothelial cells and hepatocytes, respectively. CD176 could be a promising target for antitumor immunotherapy. We applied B lymphocytes obtained from mice immunized with CD176 antigen and constructed a phage display library. A positive clone of CD176 single-chain variable antibody fragment (scFv) was successfully screened from this library. The CD176 scFv was expressed in Escherichia coli and purified. The purified scFv can bind to the natural CD176 expressed on the surface of cancer cells. Furthermore, the CD176 scFv inhibits the adhesion of CD176(+) cancer cells to endothelial cells and hepatocytes. This CD176 scFv provides a basis for future development of recombinant CD176-specific antibodies that can be used in therapeutic application. PMID:27090911

  11. A single base mutation in an I-A alpha-chain gene alters T-cell recognition.

    PubMed

    Griffith, I J; Choi, E M; Glimcher, L H

    1987-02-01

    The interaction between the clonally selected T-cell antigen receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered I-Ak alpha-chain (Ak alpha) molecule has been examined in order to provide more information about the relationship between Ia structure and function. The cell line, 3J9, was derived from the TA3 B-cell hybridoma through a series of negative and positive immunoselection steps. The 3J9 mutant lacked the binding site recognized by the Ak alpha-specific monoclonal antibody 39J and failed to present antigen to two T-cell hybridomas out of a large panel of I-Ak-restricted T-cell hybridomas examined. Sequence analysis of the mutant Ak alpha gene showed a single base transition (G----A) that resulted in a glutamic acid to lysine substitution at amino acid 75 of the alpha 1 domain. This mutation confirms the importance of amino acid 75 in the expression of the Ia.19 epitope, demonstrates the involvement of this region in the presentation of antigen to specific T cells, and provides a further example of the multiple functional domains on the Ia molecule that are involved in antigen presentation. PMID:3493486

  12. A single base mutation in an I-A alpha-chain gene alters T-cell recognition.

    PubMed Central

    Griffith, I J; Choi, E M; Glimcher, L H

    1987-01-01

    The interaction between the clonally selected T-cell antigen receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered I-Ak alpha-chain (Ak alpha) molecule has been examined in order to provide more information about the relationship between Ia structure and function. The cell line, 3J9, was derived from the TA3 B-cell hybridoma through a series of negative and positive immunoselection steps. The 3J9 mutant lacked the binding site recognized by the Ak alpha-specific monoclonal antibody 39J and failed to present antigen to two T-cell hybridomas out of a large panel of I-Ak-restricted T-cell hybridomas examined. Sequence analysis of the mutant Ak alpha gene showed a single base transition (G----A) that resulted in a glutamic acid to lysine substitution at amino acid 75 of the alpha 1 domain. This mutation confirms the importance of amino acid 75 in the expression of the Ia.19 epitope, demonstrates the involvement of this region in the presentation of antigen to specific T cells, and provides a further example of the multiple functional domains on the Ia molecule that are involved in antigen presentation. PMID:3493486

  13. Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

    PubMed Central

    Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

    2016-01-01

    A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768

  14. Single-chain Fv fragment antibodies selected from an intrabody library as effective mono- or bivalent reagents for in vitro protein detection.

    PubMed

    Desplancq, Dominique; Rinaldi, Anne-Sophie; Stoessel, Audrey; Sibler, Annie-Paule; Busso, Didier; Oulad-Abdelghani, Mustapha; Van Regenmortel, Marc H; Weiss, Etienne

    2011-06-30

    In spite of their many potential applications, recombinant antibody molecules selected by phage display are rarely available commercially, one reason being the absence of robust bacterial expression systems that yield sufficient quantities of reagents for routine applications. We previously described the construction and validation of an intrabody library that allows the selection of single-chain Fv (scFv) fragments solubly expressed in the cytoplasm. Here, we show that it is possible to obtain monomeric scFvs binding specifically to human papillomavirus type 16 E6 and cellular gankyrin oncoproteins in quantities higher than 0.5 g/L of shake-flask culture in E. coli cytoplasm after auto-induction. In addition, stable bivalent scFvs of increased avidity were produced by tagging the scFvs with the dimeric glutathione-S-transferase enzyme (GST). These minibody-like molecules were further engineered by fusion with green fluorescent protein (GFPuv), leading to high yield of functional bivalent fluorescent antibody fragments. Our results demonstrate that scFvs selected from an intrabody library can be engineered into cost-effective bivalent reagents suitable for many biomedical and industrial applications. PMID:21501618

  15. Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6

    PubMed Central

    Stadler, Christiane R.; Bähr-Mahmud, Hayat; Plum, Laura M.; Schmoldt, Kathrin; Kölsch, Anne C.; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    abstract The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need. We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties. Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cells in vitro with EC50 values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicated by 6PHU3 treatment and survival of mice is significantly prolonged. Tumors of 6PHU3-treated mice are strongly infiltrated with activated CD4+, CD8+ T cells and TEM type cells but not Tregs and display a general activation of a mostly inflammatory phenotype. These effects are only observed upon bispecific but not monospecific engagement of 6PHU3. Together with the exceptionally cancer cell selective expression of the oncofetal tumor marker CLDN6, this provides a safeguard with regard to toxicity. In summary, our data shows that the concept of T-cell redirection combined with that of highly selective targeting of CLDN6-positive solid tumors is effective. Thus, exploring 6PHU3 for clinical therapy is warranted. PMID:27141353

  16. Immunohistochemical Characterization of Slow and Fast Myosin Heavy Chain Composition of Muscle Fibres in the Styloglossus Muscle of the Human and Macaque (M. rhesus)

    PubMed Central

    Sokoloff, Alan J.; Yang, Betty; Li, Haiyan; Burkholder, Thomas J.

    2007-01-01

    Objective Muscle fibre contractile diversity is thought to be increased by the hybridization of multiple myosin heavy chain (MHC) isoforms in single muscle fibres. Reports of hybrid fibres composed of MHCI and MHCII isoforms in human, but not macaque, tongue muscles, suggest a human adaptation for increased tongue muscle contractile diversity. Here we test whether hybrid fibres composed of MHCI and MHCII are unique to human tongue muscles or are present as well in the macaque. Methods MHC composition of the macaque and human styloglossus was characterized with antibodies that allowed identification of three muscle fibre phenotypes, a slow phenotype composed of MHCI, a fast phenotype composed of MHCII and a hybrid phenotype composed of MHCI and MHCII. Results The fast phenotype constitutes 68.5% of fibres in the macaque and 43.4% of fibres in the human (P<0001). The slow phenotype constitutes 20.2% of fibres in the macaque and 39.3% of fibres in the human (P<0001). The hybrid phenotype constitutes 11.2% of fibres in the macaque and 17.3% of fibres in the human (P=0002). Macaques and humans do not differ in fiber size (cross-sectional area, diameter). However, measures of fibre size differ by phenotype such that fast > hybrid > slow (P<0.05). Conclusion These data demonstrate differences in the relative percent of muscle fibre phenotypes in the macaque and human styloglossus but also demonstrate that all three phenotypes are present in both species. These data suggest a similar range of mechanical properties in styloglossus muscle fibres of the macaque and human. PMID:17210117

  17. Efficient human pose estimation from single depth images.

    PubMed

    Shotton, Jamie; Girshick, Ross; Fitzgibbon, Andrew; Sharp, Toby; Cook, Mat; Finocchio, Mark; Moore, Richard; Kohli, Pushmeet; Criminisi, Antonio; Kipman, Alex; Blake, Andrew

    2013-12-01

    We describe two new approaches to human pose estimation. Both can quickly and accurately predict the 3D positions of body joints from a single depth image without using any temporal information. The key to both approaches is the use of a large, realistic, and highly varied synthetic set of training images. This allows us to learn models that are largely invariant to factors such as pose, body shape, field-of-view cropping, and clothing. Our first approach employs an intermediate body parts representation, designed so that an accurate per-pixel classification of the parts will localize the joints of the body. The second approach instead directly regresses the positions of body joints. By using simple depth pixel comparison features and parallelizable decision forests, both approaches can run super-real time on consumer hardware. Our evaluation investigates many aspects of our methods, and compares the approaches to each other and to the state of the art. Results on silhouettes suggest broader applicability to other imaging modalities. PMID:24136424

  18. Efficient Human Pose Estimation from Single Depth Images.

    PubMed

    Shotton, Jamie; Girshick, Ross; Fitzgibbon, Andrew; Sharp, Toby; Cook, Mat; Finocchio, Mark; Moore, Richard; Kohli, Pushmeet; Criminisi, Antonio; Kipman, Alex; Blake, Andrew

    2012-10-26

    We describe two new approaches to human pose estimation. Both can quickly and accurately predict the 3D positions of body joints from a single depth image, without using any temporal information. The key to both approaches is the use of a large, realistic, and highly varied synthetic set of training images. This allows us to learn models that are largely invariant to factors such as pose, body shape, field-of-view cropping, and clothing. Our first approach employs an intermediate body parts representation, designed so that an accurate per-pixel classification of the parts will localize the joints of the body. The second approach instead directly regresses the positions of body joints. By using simple depth pixel comparison features, and parallelizable decision forests, both approaches can run super-realtime on consumer hardware. Our evaluation investigates many aspects of our methods, and compares the approaches to each other and to the state of the art. Results on silhouettes suggest broader applicability to other imaging modalities. PMID:23109523

  19. ATP synthase: from single molecule to human bioenergetics

    PubMed Central

    KAGAWA, Yasuo

    2010-01-01

    ATP synthase (FoF1) consists of an ATP-driven motor (F1) and a H+-driven motor (Fo), which rotate in opposite directions. FoF1 reconstituted into a lipid membrane is capable of ATP synthesis driven by H+ flux. As the basic structures of F1 (α3β3γδε) and Fo (ab2c10) are ubiquitous, stable thermophilic FoF1 (TFoF1) has been used to elucidate molecular mechanisms, while human F1Fo (HF1Fo) has been used to study biomedical significance. Among F1s, only thermophilic F1 (TF1) can be analyzed simultaneously by reconstitution, crystallography, mutagenesis and nanotechnology for torque-driven ATP synthesis using elastic coupling mechanisms. In contrast to the single operon of TFoF1, HFoF1 is encoded by both nuclear DNA with introns and mitochondrial DNA. The regulatory mechanism, tissue specificity and physiopathology of HFoF1 were elucidated by proteomics, RNA interference, cytoplasts and transgenic mice. The ATP synthesized daily by HFoF1 is in the order of tens of kilograms, and is primarily controlled by the brain in response to fluctuations in activity. PMID:20689227

  20. Plural light chains in a single plasma cell of a monoclonal gammopathy undetermined significance case: an ultrastructural study.

    PubMed

    Saito, Nagahito; Konishi, Kohei; Ohta, Shuichi; Kondo, Takeshi; Kato, Mototsugu; Hashino, Satoshi; Takeda, Hiroshi; Asaka, Masahiro; Ooi, Hong-Kean

    2007-02-01

    A 44-year-old man was found to have M-proteins of IgG consisting of kappa- and lambda-chains in serum without lymphadenopathy or splenomegaly. The serum concentrations of IgG, IgA and IgM were within normal limits. Bone marrow examination showed normal cellular marrow containing 6.3% of plasma cells with no abnormal features. No chromosomal abnormality was observed at all. The patient was diagnosed as having monoclonal gammopathy of undetermined significance. The bone marrow plasma cells possessed free kappa- and lambda-chains in Golgi apparatus, rough endoplasmic reticula and cytoplasmic matrices. Plural light chains were simultaneously produced with the same heavy chain in a plasma cell by immunoelectron microscopy. This is the first report in the world of a monoclonal gammopathy of undetermined significance producing plural light chains with the same heavy chain. PMID:17506772

  1. Purification and partial characterization of single-chain ribosome-inactivating proteins from the seeds of Phytolacca dioica L.

    PubMed

    Parente, A; De Luca, P; Bolognesi, A; Barbieri, L; Battelli, M G; Abbondanza, A; Sande, M J; Gigliano, G S; Tazzari, P L; Stirpe, F

    1993-10-19

    Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405-412) were purified from the seeds of Phytolacca dioica. These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M(r) approx. 30,000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana. PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30 + Hodgkin's lymphoma-derived L540 cell line. PMID:8218414

  2. The Rouse-Mooney model for coherent quasielastic neutron scatterings of single chains well entangled in polymer melts

    NASA Astrophysics Data System (ADS)

    Lin, Y.-H.; Huang, C.-F.

    2008-06-01

    The dynamic structure factor (DSF) for single (labeled) chains well entangled in polymer melts has been developed based on the Rouse-Mooney picture; the DSF functions derived from the Langevin equations of the model in both discrete and continuous forms are given. It is shown that for all practical purposes, it is sufficient to use the continuous form to analyze experimental results in the ``safe'' q region (q being the magnitude of the scattering wave vector q) where the Rouse-segment-based theories are applicable. The DSF form reduces to the same limiting form as that of the free Rouse chain as q2a2 or q2R2-->∞ (a and R being the entanglement distance and the root mean square end-to-end distance, respectively), confirming what has been expected physically. The natural reduction to the limiting form allows the full range of DSF curves to be displayed in terms of the reduced Rouse variable q2(Zdt)0.5 in a unified way. The displayed full range represents a framework or ``map,'' with respect to which effects occurring in different regions of the DSF may be located and studied in a consistent manner. One effect is the significant or noticeable deviations of the theoretical DSF curves from the limiting curve in the region ~4>q2(Zdt)0.5>~0.1 (a time region where t<τ1e) to the faster side as qa is in the range 1-5. This is supported by the comparison of the experimental results of an entangled poly(vinylethylene) sample with the theoretical curves. The DSF functional forms predict plateaus with heights depending on the value of q-q-split plateaus-as can be experimentally observed in the time region greater than the relaxation time τ1e of the lowest Rouse-Mooney mode, when qa falls between ~1 and ~7. High sensitivity of the distribution of the q-split plateaus to a enables its value to be extracted from matching the calculated with the experimental results. The thus obtained a value for a well-entangled poly(ethylene-co-butene) polymer is in close agreement with the

  3. TCTEX1D4 Interactome in Human Testis: Unraveling the Function of Dynein Light Chain in Spermatozoa

    PubMed Central

    Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; da Cruz e Silva, Edgar

    2014-01-01

    Abstract Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function. PMID:24606217

  4. Human von Willebrand factor gene and pseudogene: Structural analysis and differentiation by polymerase chain reaction

    SciTech Connect

    Mancuso, D.J.; Tuley, E.A.; Westfield, L.A.; Lester-Mancuso, T.L.; Sorace, J.M.; Sadler, J.E. ); Le Beau, M.M. )

    1991-01-01

    Structural analysis of the von Willebrand factor gene located on chromosome 12 is complicated by the presence of a partial unprocessed pseudogene on chromosome 22q11-13. The structures of the von Willebrand factor pseudogene and corresponding segment of the gene were determined, and methods were developed for the rapid differentiation of von Willebrand factor gene and pseudogene sequences. The pseudogene is 21-29 kilobases in length and corresponds to 12 exons (exons 23-34) of the von Willebrand factor gene. Approximately 21 kilobases of the gene and pseudogene were sequenced, including the 5{prime} boundary of the pseudogene. The 3{prime} boundary of the pseudogene lies within an 8-kb region corresponding to intron 34 of the gene. The presence of splice site and nonsense mutations suggests that the pseudogene cannot yield functional transcripts. The pseudogene has diverged {approximately}3.1{percent} in nucleotide sequence from the gene. This suggests a recent evolutionary origin {approximately}19-29 million years ago, near the time of divergence of humans and apes from monkeys. Several repetitive sequences were identified, including 4 Alu, one Line-1, and several short simple sequence repeats. Several of these simple repeats differ in length between the gene and pseudogene and provide useful markers for distinguishing these loci. Sequence differences between the gene and pseudogene were exploited to design oligonucleotide primers for use in the polymerase chain reaction to selectivity amplify sequences corresponding to exons 23-34 from either the von Willebrand factor gene or the pseudogene. This method is useful for the analysis of gene defects in patients with von Willebrand disease, without interference from homologous sequences in the pseudogene.

  5. Evidence for Greater Production of Colonic Short Chain Fatty Acids in Overweight than Lean Humans

    PubMed Central

    Rahat-Rozenbloom, Sari; Fernandes, Judlyn; Gloor, Gregory B; Wolever, Thomas MS

    2014-01-01

    Background Short-chain fatty acids (SCFA) are produced by colonic microbiota from dietary carbohydrates and proteins that reach the colon. It has been suggested that SCFA may promote obesity via increased colonic energy availability. Recent studies suggest obese humans have higher faecal SCFA than lean, but it is unclear if this difference is due to increased SCFA production or reduced absorption. Objectives To compare rectal SCFA absorption, dietary intake and faecal microbial profile in lean (LN) versus overweight and obese (OWO) individuals. Design Eleven (11) LN and 11 OWO individuals completed a 3-day diet record, provided a fresh faecal sample and had SCFA absorption measured using the rectal dialysis bag method. The procedures were repeated after two weeks. Results Age-adjusted faecal SCFA concentration was significantly higher in OWO than LN (81.3 ± 7.4 vs. 64.1 ± 10.4 mmol/kg, P = 0.023). SCFA absorption (24.4 ± 0.8 vs 24.7 ± 1.2%, respectively, P =0.787) and dietary intakes were similar between the groups, except for a higher fat intake in OWO. However, fat intake did not correlate with SCFAs or bacterial abundance. OWO had higher relative Firmicutes abundance (83.1 ± 4.1 vs 69.5 ± 5.8%, respectively, P = 0.008) and a higher Firmicutes:Bacteriodetes ratio (P = 0.023) than LN. There was a positive correlation between Firmicutes and faecal SCFA within the whole group (r =0.507, P =0.044), with a stronger correlation after adjusting for available carbohydrate (r = 0.615, P =0.005). Conclusions The higher faecal SCFA in OWO subjects is not due to differences in SCFA absorption or diet. Our results are consistent with the hypothesis that OWO subjects produce more colonic SCFA than LN due to differences in colonic microbiota. However, further studies are needed to prove this. PMID:24642959

  6. Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.

    PubMed Central

    Bottinelli, R; Canepari, M; Pellegrino, M A; Reggiani, C

    1996-01-01

    1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force-velocity curves of sixty-seven fibres were obtained by load-clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross-sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack-test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I-IIA and IIA-IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty-one fibres in the range 12-22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol-1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12-17 degrees C range, but less dependent between 17 and 22 degrees C. Images Figure 1 Figure 3 Figure 6 PMID:8887767

  7. Targeted gene walking by low stringency polymerase chain reaction: Assignment of a putative human brain sodium channel gene (SCN3A) to chromosome 2q24-31

    SciTech Connect

    Malo, M.S.; Srivastava, K.; Andresen, J.M.; Ingram, V.M.; Chen, X.N.; Korenberg, J.R.

    1994-04-12

    The authors have developed a low stringency polymerase chain reaction (LSPCR) to isolate the unknown neighboring region around a known DNA sequence, thus allowing efficient targeted gene walking. The method involves the polymerase chain reaction (PCR) with a single primer under conditions of low stringency for primer annealing (40{degrees}C) for the first few cycles followed by more cycles at high stringency (55{degrees}C). Nested PCRs with end-labeled primers are then used to generate a ladder of radioactive bands, which accurately identifies the targeted fragment(s). They performed LSPCR on human placental DNA using a highly conserved sodium channel-specific primer for 5 cycles at 40{degrees}C followed by 27 cycles at 55{degrees}C for primer annealing. Subsequently, using higher stringency (55{degrees}C) PCR with radiolabeled nested primers for 8 cycles, they have isolated a 0.66-kb fragment of a putative human sodium channel gene. Partial sequence (325 bp) of this fragment revealed a 270-bp region (exon) with homology to the rat brain sodium channel III{alpha} (RBIII) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, the authors putatively assign this sequence as a part of a gene coding the {alpha}-subunit of a human brain type III sodium channel (SCN3A). Using PCR on two human/rodent somatic cell hybrid panels with primers specific to this putative SCN3A gene, they have localized this gene to chromosome 2. Fluorescence in situ hybridization to human metaphase chromosomes was used to sublocalize the SCN3A gene to chromosome at 2q24-31. In conclusion, LSPCR is an efficient and sensitive method for targeted gene walking and is also useful for the isolation of homologous genes in related species.

  8. Physical mapping of the human T-cell antigen receptor (TCR) {beta}-chain gene complex

    SciTech Connect

    Yashim, Y.; So, A.K.

    1994-09-01

    The genetic variation of the TCR loci and their contribution to autoimmune diseases is poorly defined, in direct contrast to the clear examples of disease association with the Class I and II alleles of the major histocompatibility complex. We have therefore started to determine the gene organization and polymorphism of the TCR {beta} locus. Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human TCR {beta}-chain gene complex. Variable gene (V{beta}) sequences for the 25 known V{beta} subfamilies were amplified by PCR and were used as probes to screen a YAC library. Five positive YACs were identified. YACs designated B3, E11 and H11 of sizes 820, 400 and 600 kbp, respectively, were analyzed for their V{beta} content by pulse-field gel electrophoresis (PFGE). YAC B3 was found to contain all 25 V{beta} subfamilies, E11 for 14 and H11 for 7. B3 was also positive for the constant region genes. Restriction enzyme mapping of B3 located V{beta} and C{beta} gene regions to four Sfi I fragments of 280, 110, 90 and 125 kbp, and was in accordance with published data. The data thus showed that YAC B3 encoded a complete and unrearranged TCR {beta}-gene locus. The map was further resolved by locating restriction sites for Sal I and Bssll II on B3. Fluorescent in situ hybridization to human metaphase chromosomes localized B3 to chromosome 7q35. However, two additional signals were obtained: one attributable to V{beta} orphon cluster on chromosome 9q21; the second to the long arm of chromosome 2. PCR amplification of a chromosome 2 somatic cell hybrid using primers for all 25 V{beta} gene families revealed the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCR {beta} locus flanked by chromosome 2 sequences. The determination of the TCR genomic organization will help extend studies of the role T-cells play in autoimmune diseases.

  9. Suppression of pancreatic tumor growth by targeted arsenic delivery with anti-CD44v6 single chain antibody conjugated nanoparticles.

    PubMed

    Qian, Chenchen; Wang, Yong; Chen, Yinting; Zeng, Linjuan; Zhang, Qiubo; Shuai, Xintao; Huang, Kaihong

    2013-08-01

    Arsenic trioxide (As2O3) is a promising anticancer agent for solid tumors. However, the high toxicity to normal tissues resulting from the lack of tumor specificity remains a huge challenge in its systemic application. Targeted vectors enabling drug delivery to specific cancer cells bring about great potential for better therapeutic efficacy whereas low side effects in cancer treatments. Our previous work has demonstrated that the anti-CD44v6 single chain variable fragment (scFv(CD44v6)) screened out from the human phage-displayed scFv library possesses high specificity and affinity to membrane antigen CD44v6 over-expressing in a subset of epithelium-derived cancers, such as pancreatic, hepatocellular, colorectal and gastric cancers. Herein, a maleimide-functionalized amphiphilic diblock copolymer of poly (ethylene glycol) and poly (D, L-lactide) (mal-PEG-PDLLA) was synthesized and assembled to vesicles with arsenite ion (As) encapsulated in their cores (As-NPs). Conjugation of scFv(CD44v6) with mal-PEG-PDLLA (scFv-As-NPs) enabled more efficient delivery of As and exhibited higher cytotoxic activity than non-targeted ones (As-NPs) in human pancreatic cancer cells PANC-1. Furthermore, the targeted delivery of As induced more significant gene suppression in terms of the expression of anti-apoptotic Bcl-2 protein. Consequently, the expression level of cleaved caspase-3 which is a molecular indicator of cell apoptosis was remarkably elevated. In animal tests, scFv-As-NPs were found to greatly increase accumulation of drug in tumor site and potentiate the efficacy of As in inhibiting tumor growth owing to the enhanced cell apoptosis. These results imply that our tumor specific nanocarriers provide a highly efficient and safe platform for pancreatic cancer therapy. PMID:23721794

  10. Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency.

    PubMed

    Turki, Imène; Hammami, Akil; Kharmachi, Habib; Mousli, Mohamed

    2014-02-01

    Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10(9)M(-1) and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10(7)M(-1)). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy. PMID:24091293

  11. The human LON protease binds to mitochondrial promoters in a single-stranded, site-specific, strand-specific manner.

    PubMed

    Fu, G K; Markovitz, D M

    1998-02-17

    LON proteases, which are ATP-dependent and exhibit ATPase activity, are found in bacteria, yeast, and humans. In Escherichia coli, LON is known to regulate gene expression by targeting specific regulatory proteins for degradation. The yeast and human LON proteins are encoded in the nucleus but localize to the mitochondrial matrix. In yeast, LON has been shown to be essential for the maintenance of the integrity of the mitochondrial genome. E. coli Lon has long been known to bind DNA, but we have only recently demonstrated that it binds preferentially to a specific TG-rich double-stranded sequence. We now show that human LON recognizes a very similar site in both the light and heavy chain promoters of the mitochondrial genome, in a region which is involved in regulating both DNA replication and transcription. Unlike E. coli Lon, however, human LON specifically binds to the TG-rich element only when it is presented in the context of a single DNA strand. These findings suggest that the human LON protease might regulate mitochondrial DNA replication and/or gene expression using site-specific, single-stranded DNA binding to target the degradation of regulatory proteins binding to adjacent sites in mitochondrial promoters. PMID:9485316

  12. A best on-line algorithm for single machine scheduling the equal length jobs with the special chain precedence and delivery time

    NASA Astrophysics Data System (ADS)

    Gu, Cunchang; Mu, Yundong

    2013-03-01

    In this paper, we consider a single machine on-line scheduling problem with the special chains precedence and delivery time. All jobs arrive over time. The chains chainsi arrive at time ri , it is known that the processing and delivery time of each job on the chain satisfy one special condition CD a forehand: if the job J(i)j is the predecessor of the job J(i)k on the chain chaini, then they satisfy p(i)j = p(i)k = p >= qj >= qk , i = 1,2, ---,n , where pj and qj denote the processing time and the delivery time of the job Jj respectively. Obviously, if the arrival jobs have no chains precedence, it shows that the length of the corresponding chain is 1. The objective is to minimize the time by which all jobs have been delivered. We provide an on-line algorithm with a competitive ratio of √2 , and the result is the best possible.

  13. Characterization and ab initio XRPD structure determination of a novel silicate with Vierer single chains: the crystal structure of NaYSi2O6.

    PubMed

    Többens, Daniel M; Kahlenberg, Volker; Kaindl, Reinhard

    2005-12-12

    The crystal structure of a sodium yttrium silicate with composition NaYSi2O6 has been determined from laboratory X-ray powder diffraction data by simulated annealing, and has been subsequently refined with the Rietveld technique. The compound is monoclinic with space group P2(1)/c and unit cell parameters of a=5.40787(2) A, b=13.69784(5) A, c=7.58431(3) A, and beta=109.9140(3) degrees at 23.5 degrees C (Z=4). The structure was found to be a single-chain silicate with a chain periodicity of four. The two symmetry dependent [Si4O12] chains in the unit cell are parallel to c. A prominent feature is the strong folding of the crankshaft-like chains within the b,c-plane resulting in intrachain Si-Si-Si angles close to 90 degrees. The coordination of the Y3+ ions by O2- is 7-fold in the form of slightly irregular pentagonal bipyramids, with oxygen atoms from four different chains contributing to the coordination polyhedron. Na+ ions are irregularly coordinated by 10 oxygens from two neighboring chains. No disorder of Na+ and Y3+ between the two nontetrahedral cation sites could be observed. Furthermore, micro-Raman spectra have been obtained from the polycrystalline material. PMID:16323944

  14. When Phase Contrast Fails: ChainTracer and NucTracer, Two ImageJ Methods for Semi-Automated Single Cell Analysis Using Membrane or DNA Staining

    PubMed Central

    Syvertsson, Simon; Vischer, Norbert O. E.; Gao, Yongqiang; Hamoen, Leendert W.

    2016-01-01

    Within bacterial populations, genetically identical cells often behave differently. Single-cell measurement methods are required to observe this heterogeneity. Flow cytometry and fluorescence light microscopy are the primary methods to do this. However, flow cytometry requires reasonably strong fluorescence signals and is impractical when bacteria grow in cell chains. Therefore fluorescence light microscopy is often used to measure population heterogeneity in bacteria. Automatic microscopy image analysis programs typically use phase contrast images to identify cells. However, many bacteria divide by forming a cross-wall that is not detectable by phase contrast. We have developed ‘ChainTracer’, a method based on the ImageJ plugin ObjectJ. It can automatically identify individual cells stained by fluorescent membrane dyes, and measure fluorescence intensity, chain length, cell length, and cell diameter. As a complementary analysis method we developed 'NucTracer', which uses DAPI stained nucleoids as a proxy for single cells. The latter method is especially useful when dealing with crowded images. The methods were tested with Bacillus subtilis and Lactococcus lactis cells expressing a GFP-reporter. In conclusion, ChainTracer and NucTracer are useful single cell measurement methods when bacterial cells are difficult to distinguish with phase contrast. PMID:27008090

  15. Single chain morphology and nanofiber-like aggregates of branched β-(1 → 3)-D-glucan in water/dimethylsulfoxide solution.

    PubMed

    Chen, Cong; Meng, Yan; Li, Sheng; Wu, Wenhua; Liu, Chuanjun; Xu, Xiaojuan; Zhang, Lina

    2016-02-10

    A polysaccharide coded as PR-CA was extracted from Polyporus rhinoceros and determined to be a β-(1 → 3)-D-glucan with multiple branches. The weight-average molecular weights (Mw) of PR-CA in dimethylsulfoxide (DMSO) and in water were determined with static light scattering (SLS) to be 3.57 × 10(5) and 1.79 × 10(7), indicating existence of the single chains in DMSO and co-existence of single chains and aggregates in water. Moreover, the stiffness of single chains of PR-CA in water was directly visualized by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The hollow structure of PR-CA nanofibers with width of 30-40 nm and length of ∼ 350 nm formed in the water/DMSO (9:1, v:v) was demonstrated by a fluorescent probe tetraphenylethylene (TPE) via aggregation-induced emission (AIE). The formation of PR-CA nanofibers was ascribed to the parallel aggregation of the extended PR-CA chains due to the hydrogen bonding and hydrophobic interaction. This work offered valuable results for promising applications of natural branched β-glucans in the biological fields of drug inclusion, delivery and disease diagnosis. PMID:26686132

  16. Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli.

    PubMed

    Sonoda, Hiroyuki; Kumada, Yoichi; Katsuda, Tomohisa; Yamaji, Hideki

    2011-04-01

    The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonuclease A single-chain variable fragment (scFv) 3A21 in Escherichia coli were investigated. Co-expression of a cytoplasmic chaperone, GroEL/ES, DnaK/DnaJ/GrpE, trigger factor, or SecB with 3A21 scFv affected the proportions of antigen-binding activity in the cytoplasmic soluble fraction, the periplasmic fraction, and the extracellular medium, but there was no significant difference in the total activity compared to the control without chaperone co-expression. On the other hand, co-expression of a periplasmic chaperone, Skp or FkpA, with the exception of DsbC, greatly increased the binding activity in all the soluble fractions. Co-expression of both Skp and FkpA had no synergistic effect. Combinations of cytoplasmic and periplasmic chaperones decreased the productivity. In shake-flask cultures of cells co-expressing Skp or FkpA, considerable amounts of 3A21 scFv were detected in the extracellular medium by enzyme-linked immunosorbent assay (ELISA) and Western blot, and the extracellular production level of 3A21 scFv was calculated to be around 40mg/l. The binding activity of 3A21 scFv co-expressed with Skp was slightly higher than that with FkpA. These results indicate that the co-expression of periplasmic chaperones Skp and FkpA is extremely useful for the secretory production of scFvs in a culture medium using E. coli, but cytoplasmic chaperones and multiple-chaperone combinations may not be effective. PMID:21324738

  17. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    PubMed

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin. PMID:25757566

  18. Expression, characterization, and evaluation of a RANK-binding single chain fraction variable: an osteoclast targeting drug delivery strategy.

    PubMed

    Newa, Madhuri; Lam, Michael; Bhandari, Krishna Hari; Xu, Biwen; Doschak, Michael R

    2014-01-01

    A single chain Fraction variable (scFv) employs antibody-like target recognition specificity. Osteoclasts, responsible for bone resorption, express Receptor Activator of Nuclear factor Kappa B (RANK) receptors. This study aimed to express, characterize, and evaluate scFv against RANK receptors that may serve as a platform to target osteoclasts. Using phage display technology, scFv against RANK receptor was expressed and characterized by DNA sequencing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption-ionization time-of-flight (MALDI TOF), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunocytochemistry. The potential for cytotoxicity was evaluated using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, and its cross reactivity was evaluated using ELISA. Osteoclast-like cells were generated from RAW 264.7 cells, and the osteoclast targeting ability of scFv was evaluated using immunocytochemistry. ScFv's antiresorptive efficacy was studied using a tartrate-resistant acid phosphatase (TRAP) assay and resorption assay. Anti-RANK scFv was successfully expressed and characterized. No cross reactivity with other tumor necrosis factor receptor (TNFR) members and no cytotoxic effect on a non-RANK bearing cell line were observed. It showed specificity toward a RANK receptor and an inhibitory effect on osteoclast activity. With the increase in development trends for biologics as therapeutics and growing knowledge on the importance of osteoclast targeted therapy, this study may provide a drug delivery strategy to target osteoclasts, thereby leading to a promising therapy for resorptive bone diseases. PMID:24171467

  19. Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis.

    PubMed

    Guo, L; Wang, J; Qian, S; Yan, X; Chen, R; Meng, G

    2000-11-20

    In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme. PMID:11005928

  20. Antiferromagnetic order in single crystals of the S =2 quasi-one-dimensional chain MnCl3(bpy)

    NASA Astrophysics Data System (ADS)

    Shinozaki, Shin-ichi; Okutani, Akira; Yoshizawa, Daichi; Kida, Takanori; Takeuchi, Tetsuya; Yamamoto, Shoji; Risset, Olivia N.; Talham, Daniel R.; Meisel, Mark W.; Hagiwara, Masayuki

    2016-01-01

    A suite of experimental tools, including high-field magnetization and electron spin resonance (ESR) studies in magnetic fields of up to 50 T and heat capacity studies up to 9 T, have revealed antiferromagnetic order in single crystals of the Heisenberg S =2 chain compound MnCl3(bpy), where bpy is 2 ,2'-bipyridine . The Néel temperature, which depends on the strength of the applied magnetic field and its orientation with respect to the crystalline axes that was revealed by heat capacity measurements, is near 11.5 K in zero field. The spin-flop transition is identified in the magnetization curve acquired at 1.7 K and at μoHSFc=24 T along the c axis. The transition field HSF is lower than that expected from the previous antiferromagnetic resonance (AFMR) studies on a powder sample. The identification of the long-range antiferromagnetic order resolves an earlier report by Granroth et al. [Phys. Rev. Lett. 77, 1616 (1996)], 10.1103/PhysRevLett.77.1616 that identified MnCl3(bpy) as an S =2 Haldane system down to 40 mK. The ESR studies identify a wide range of antiferromagnetic resonance modes that provide additional microscopic information about the g values (ga*=2.09 , gb=1.92 , and gc=2.07 ), the zero-field splitting constants, D /kB=-1.5 K and E /kB=-0.17 K when the nearest-neighbor spin interaction J /kB=31.2 K, which is evaluated from fitting the susceptibility, and the anisotropy of this compound (easy axis is the c axis, the second easy-axis is the b axis, and the hard axis is the a* axis), when using a standard (two-sublattice) AFMR analysis that does not quantitatively reproduce the observed HSFc value. The observed resonance mode indicates the frequency minimum at HSFc.

  1. Production and characterization of a biotinylated single-chain variable fragment antibody for detection of parathion-methyl.

    PubMed

    Wang, Huimin; Zhao, Fengchun; Han, Xiao; Yang, Zhengyou

    2016-10-01

    In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM. The selected scFv antibody was fused with a biotin acceptor domain (BAD) and inserted into pET-28a(+) vector for high-level expression in Escherichia coli BL2 (DE3). After optimizing expression conditions, the scFv-BAD antibody was expressed as a soluble protein and biotinylated in vitro by the E. coli biotin ligase (BirA). Subsequently, the biotinylated scFv-BAD antibody was purified with a high yield of 59.2 ± 3.7 mg/L of culture, and was characterized by SDS-PAGE and western blotting. Finally, based on the biotinylated scFv-BAD, a sensitive IC-ELISA for detection of PM was developed, and the 50% inhibition value (IC50) of PM was determined as 14.5 ng/mL, with a limit of detection (LOD, IC10) of 0.9 ng/mL. Cross-reactivity (CR) studies revealed that the scFv antibody showed desirable specificity for PM. PMID:27181246

  2. Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library.

    PubMed

    Xu, Yang; Xiong, Liang; Li, Yanping; Xiong, Yonghua; Tu, Zhui; Fu, Jinheng; Chen, Bo

    2015-07-01

    Compared with peptide-based mimotope, anti-idiotypic antibodies (AIds) are considered as promising biosynthetic surrogate antigen because these antibodies display stable protein conformation. Nevertheless, conventional AIds are generated by immunizing animals with heterologous idiotypic antibody in vivo; isolated AIds commonly exhibit a higher affinity to primary antibodies than target analytes because AIds undergo an affinity-matured process during immune responses, resulting in low sensitivity in competitive immunoassay. In the present study, an anti-citrinin monoclonal antibody (anti-CIT McAb) was designed as primary antibody; one β-type AI alpaca heavy chain single domain antibody (β-AI VHH) was selected as a citrinin (CIT) surrogate from a naive phage-displayed VHH library. The affinity constant (K D) of obtained β-AI VHH to anti-CIT McAb (160 nM) is 2.35 times lower than that of CIT and ovalbumin conjugates (CIT-OVA) to anti-CIT McAb (68 nM). The developed VHH-based enzyme-linked immunosorbent assay (V-ELISA) can be used to perform dynamic linear detection of CIT in 10% (v/v) methanol/PBS from 5.0 to 300.0 ng/mL, with a median inhibitory concentration (IC50) of 44.6 ng/mL (n = 3); this result was twice as good as that of indirect competitive ELISA (ic-ELISA, IC50 = 96.2 ng/mL) with CIT-OVA as a coating antigen. Moreover, the precision of V-ELISA was evaluated by analyzing average recoveries and coefficient of variations of CIT-spiked cereal sample; the reliability of V-ELISA was also validated with a conventional ic-ELISA. In summary, the proposed strategy has a great potential for panning other β-AI VHH toward small organic molecules from a naive VHH library. PMID:25910884

  3. Chemiluminescence resonance energy transfer imaging on magnetic particles for single-nucleotide polymorphism detection based on ligation chain reaction.

    PubMed

    Bi, Sai; Zhang, Zhipeng; Dong, Ying; Wang, Zonghua

    2015-03-15

    A novel ligation chain reaction (LCR) methodology for single-nucleotide polymorphism (SNP) detection was developed based on luminol-H2O2-horseradish peroxidase (HRP)-mimicking DNAzyme-fluorescein chemiluminescence resonance energy transfer (CRET) imaging on magnetic particles. For LCR, four unique target-complement probes (X and X(⁎), YG and Y(⁎)) for the amplification of K-ras (G12C) were designed by modifying G-quadruplex sequence at 3'-end of YG and fluorescein at 5'-end of Y(⁎). After the LCR, the resulting products of XYG/X(⁎)Y(⁎) with biotin-labeled X(⁎) were captured onto streptavidin-coated magnetic particles (SA-MPs) via specific biotin-SA interaction, which stimulated the CRET reaction from hemin/G-quadruplex-catalyzed luminol-H2O2 CL system to fluorescein. By collecting signals by a cooled low-light CCD, a CRET imaging method was proposed for visual detection and quantitative analysis of SNP. As low as 0.86fM mutant DNA was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10,000:1. This high sensitivity and specificity could be attributed to not only the exponential amplification and excellent discrimination of LCR but also the employment of SA-MPs. SA-MPs ensured the feasibility of the proposed strategy, which also simplified the operations through magnetic separation and separated the reaction and detection procedures to improve sensitivity. The proposed LCR-CRET imaging strategy extends the application of signal amplification techniques to SNP detection, providing a promising platform for effective and high-throughput genetic diagnosis. PMID:25461149

  4. Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.

    PubMed

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Minenkova, Olga; Hartung, John

    2016-03-01

    'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications. PMID:26744234

  5. Single Chain Variable Fragment Against Aβ Expressed in Baculovirus Inhibits Abeta Fibril Elongation and Promotes its Disaggregation

    PubMed Central

    Fang, Fang; Song, Lin-Lin; Jiao, Yue-Ying; Wang, He; Peng, Xiang-Lei; Zheng, Yan-Peng; Wang, Jun; He, Jin-Sheng; Hung, Tao

    2015-01-01

    Alzheimer’s disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ “on-pathway” aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of “on-pathway” aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free “on-pathway” Aβ aggregation and will assist in the development of therapeutic strategies for AD. PMID:25919299

  6. Photochemical Modification of Single Crystalline GaN Film Using n-Alkene with Different Carbon Chain Lengths as Biolinker.

    PubMed

    Wang, Chun; Zhuang, Hao; Huang, Nan; Heuser, Steffen; Schlemper, Christoph; Zhai, Zhaofeng; Liu, Baodan; Staedler, Thorsten; Jiang, Xin

    2016-06-14

    As a potential material for biosensing applications, gallium nitride (GaN) films have attracted remarkable attention. In order to construct GaN biosensors, a corresponding immobilization of biolinkers is of great importance in order to render a surface bioactive. In this work, two kinds of n-alkenes with different carbon chain lengths, namely allylamine protected with trifluoroacetamide (TFAAA) and 10-aminodec-1-ene protected with trifluoroacetamide (TFAAD), were used to photochemically functionalize single crystalline GaN films. The successful linkage of both TFAAA and TFAAD to the GaN films is confirmed by time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurement. With increased UV illumination time, the intensity of the secondary ions corresponding to the linker molecules initially increases and subsequently decreases in both cases. Based on the SIMS measurements, the maximum coverage of TFAAA is achieved after 14 h of UV illumination, while only 2 h is required in the case of TFAAD to reach the situation of a fully covered GaN surface. This finding leads to the conclusion that the reaction rate of TFAAD is significantly higher compared to TFAAA. Measurements by atomic force microscopy (AFM) indicate that the coverage of GaN films by a TFAAA layer leads to an increased surface roughness. The atomic terraces, which are clearly observable for the pristine GaN films, disappear once the surface is fully covered by a TFAAA layer. Such TFAAA layers will feature a homogeneous surface topography even for reaction times of 24 h. In contrast to this, TFAAD shows strong cross-polymerization on the surface, this is confirmed by optical microscopy. These results demonstrate that TFAAA is a more suitable candidate as biolinker in context of the GaN surfaces due to its improved controllability. PMID:27217218

  7. EDMA 2000 as a matrix for high-performance liquid chromatography of human haemoglobin chains.

    PubMed

    Suttnar, J; Fortová, H; Brabec, V

    1994-06-01

    An ethylenedimethacrylate polymer-based matrix (EDMA 2000) is described that shortens the time for separation of haemoglobin (Hb) chains to 30 min, compared with the use of C4 large-pore Vydac columns, commonly used for reversed-phase HPLC of Hb variants. EDMA 2000 was successfully used for the isolation of an abnormal beta-chain of the unstable Hb recently characterized as Hb Nottingham. PMID:7952021

  8. Identification of key residues essential for the structural fold and receptor selectivity within the A-chain of human gene-2 (H2) relaxin.

    PubMed

    Chan, Linda J; Rosengren, K Johan; Layfield, Sharon L; Bathgate, Ross A D; Separovic, Frances; Samuel, Chrishan S; Hossain, Mohammed A; Wade, John D

    2012-11-30

    Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin. PMID:23024363

  9. Insight into the structural and biological relevance of the T/R transition of the N-terminus of the B-chain in human insulin.

    PubMed

    Kosinová, Lucie; Veverka, Václav; Novotná, Pavlína; Collinsová, Michaela; Urbanová, Marie; Moody, Nicholas R; Turkenburg, Johan P; Jiráček, Jiří; Brzozowski, Andrzej M; Žáková, Lenka

    2014-06-01

    The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin-insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1-B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the "classical" T-state and that a substantial flexibility of the B1-B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin-IR interaction. PMID:24819248

  10. Insight into the Structural and Biological Relevance of the T/R Transition of the N-Terminus of the B-Chain in Human Insulin

    PubMed Central

    2014-01-01

    The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin–insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1–B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the “classical” T-state and that a substantial flexibility of the B1–B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin–IR interaction. PMID:24819248

  11. Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57.

    PubMed

    Duan, Ye; Gu, Tiejun; Zhang, Xizhen; Jiang, Chunlai; Yuan, Ruosen; Li, Zhuang; Wang, Dandan; Chen, Xiaoxu; Wu, Chunlai; Chen, Yan; Wu, Yongge; Kong, Wei

    2014-06-01

    Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies. PMID:24598312

  12. Medium and Long Chain Fatty Acids Differentially Modulate Apoptosis and Release of Inflammatory Cytokines in Human Liver Cells.

    PubMed

    Li, Lumin; Wang, Baogui; Yu, Ping; Wen, Xuefang; Gong, Deming; Zeng, Zheling

    2016-06-01

    Medium chain fatty acids (MCFA) can be more easily absorbed and supply energy more rapidly than long chain fatty acids (LCFA). However, little is known about the inflammatory response by the treatment of MCFA in human liver cells. Thus this study used human liver cells (LO2) to evaluate the effects of MCFA on apoptosis and inflammatory response. Tetrazolim-based colorimetric assay and lactate dehydrogenase assay were used to measure the viability of LO2 cells, isolated spleens and liver cells from BALB/C mice. Inverted fluorescence microscopy and flow cytometry were used to assess the cell apoptosis. Activity of superoxide dismutase and malondialdehyde level were measured to determine the oxidative damage. mRNA or protein levels of classical pro-inflammatory cytokines were analyzed by quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay and western blotting. The results showed that the liver cells treated with the fatty acids at 200 μM for 24 h exhibited good viability. Fatty acids induced inflammatory cytokines at transcriptional and translational levels to a lesser extent than lipopolysaccharide. LCFA (oleic acid) up-regulated tumor necrosis fator-α, monocyte chemoattractant-1 and interleukin-1β while down-regulated IL-6 and IL-8 secretion to a higher extent than MCFA in mRNA and protein levels. These findings suggested that MCFA may induce apoptosis to a less extent and exert more gentle inflammation than LCFA in human liver cells. PMID:27145239

  13. A novel liquid chromatography tandem mass spectrometry method for simultaneous determination of branched-chain amino acids and branched-chain α-keto acids in human plasma.

    PubMed

    Li, Ruiting; Liu, Peifang; Liu, Peijia; Tian, Yuan; Hua, Yunfei; Gao, Yiqiao; He, Hua; Chen, Jiaqing; Zhang, Zunjian; Huang, Yin

    2016-06-01

    Branched-chain amino acids (BCAAs) and branched-chain α-keto acids (BCKAs) play significant biological roles as they are involved in protein and neurotransmitter synthesis as well as energy metabolism pathways. To routinely and accurately study the dynamics of BCAAs and BCKAs in human diseases, e.g. cerebral infarction, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated. The plasma samples were deproteinized with acetonitrile, and then separated on a reversed phase C18 column with a mobile phase of 0.1 % formic acid (solvent A)-methanol (solvent B) using gradient elution. The detection of BCAAs and BCKAs was conducted in multiple reaction monitoring with positive/negative electrospray ionization switching mode. Biologically relevant isomers such as leucine and isoleucine were individually quantified by combining chromatographic separation and fragmentation. Good linearity (R (2) > 0.99) was obtained for all six analytes with the limits of detection from 0.1 to 0.2 µg/mL. The intra-day and inter-day accuracy ranged from 93.7 to 108.4 % and the relative standard deviation (RSD) did not exceed 15.0 %. The recovery was more than 80 % with RSD less than 14.0 %. The main improvements compared to related, state-of-the-art methods included enhanced sensitivity, enhanced separation of isomers, and reduced complexity of sample processing. Finally, the validated method was applied to analyze the plasma samples of healthy volunteers and patients suffering cerebral infarction, and significant differences in the concentration levels of BCAAs and BCKAs were observed. PMID:26984321

  14. Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation.

    PubMed

    Emelyanova, Larisa; Ashary, Zain; Cosic, Milanka; Negmadjanov, Ulugbek; Ross, Gracious; Rizvi, Farhan; Olet, Susan; Kress, David; Sra, Jasbir; Tajik, A Jamil; Holmuhamedov, Ekhson L; Shi, Yang; Jahangir, Arshad

    2016-07-01

    Mitochondria are critical for maintaining normal cardiac function, and a deficit in mitochondrial energetics can lead to the development of the substrate that promotes atrial fibrillation (AF) and its progression. However, the link between mitochondrial dysfunction and AF in humans is still not fully defined. The aim of this study was to elucidate differences in the functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes and oxidative stress in right atrial tissue from patients without (non-AF) and with AF (AF) who were undergoing open-heart surgery and were not significantly different for age, sex, major comorbidities, and medications. The overall functional activity of the electron transport chain (ETC), NADH:O2 oxidoreductase activity, was reduced by 30% in atrial tissue from AF compared with non-AF patients. This was predominantly due to a selective reduction in complex I (0.06 ± 0.007 vs. 0.09 ± 0.006 nmol·min(-1)·citrate synthase activity(-1), P = 0.02) and II (0.11 ± 0.012 vs. 0.16 ± 0.012 nmol·min(-1)·citrate synthase activity(-1), P = 0.003) functional activity in AF patients. Conversely, complex V activity was significantly increased in AF patients (0.21 ± 0.027 vs. 0.12 ± 0.01 nmol·min(-1)·citrate synthase activity(-1), P = 0.005). In addition, AF patients exhibited a higher oxidative stress with increased production of mitochondrial superoxide (73 ± 17 vs. 11 ± 2 arbitrary units, P = 0.03) and 4-hydroxynonenal level (77.64 ± 30.2 vs. 9.83 ± 2.83 ng·mg(-1) protein, P = 0.048). Our findings suggest that AF is associated with selective downregulation of ETC activity and increased oxidative stress that can contribute to the progression of the substrate for AF. PMID:27199126

  15. Sugar chain of alpha-fetoprotein produced in human yolk sac tumor.

    PubMed

    Yamashita, K; Hitoi, A; Tsuchida, Y; Nishi, S; Kobata, A

    1983-10-01

    The carbohydrate moiety of alpha-fetoprotein purified from human yolk sac tumors grown in nude mice was quantitatively released from the polypeptide chain as oligosaccharides by hydrazinolysis. The oligosaccharides were separated into a neutral and two acidic oligosaccharides by paper electrophoresis. By sequential exoglycosidase digestion in combination with per-O-methylation study and periodate oxidation, their structures were determined to be: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(GlcNAc beta 1 leads to 4) (Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAcOT, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(GlcNAc beta 1 leads to 4) (Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAcOT; and Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(GlcNAc beta 1 leads to 4) (Sia alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAcOT, in which Gal is galactose, GlcNac is N-acetylglucosamine, Man is mannose, Fuc is fucose, Sia is sialic acid, and Subscript OT is the NaB3H4-reduced oligosaccharide. PMID:6192908

  16. Initial stages of chain formation in a single layer of (In,Ga)As quantum dots grown on GaAs (100)

    SciTech Connect

    Schmidbauer, M.; Wang, Zh. M.; Mazur, Yu. I.; Lytvyn, P. M.; Salamo, G. J.; Grigoriev, D.; Schaefer, P.; Koehler, R.; Hanke, M.

    2007-08-27

    The self-organized formation of In{sub 0.40}Ga{sub 0.60}As quantum dot chains was investigated using x-ray scattering. Two samples were compared grown on GaAs(100) by molecular beam epitaxy. The first sample with a single layer of In{sub 0.40}Ga{sub 0.60}As dots shows weak quantum dot alignment and a corresponding elongated shape along [011], while the top layer of a multilayered In{sub 0.40}Ga{sub 0.60}As/GaAs sample exhibits extended and highly regular quantum dot chains oriented along [011]. Numerical calculations of the three-dimensional strain fields are used to explain the initial stages of chain formation by anisotropic strain relaxation induced by the elongated dot shape.

  17. Dynamical universality class of Brownian motion and exact results for a single-impurity s=1/2 XY chain

    NASA Astrophysics Data System (ADS)

    Sen, Surajit

    1996-03-01

    Relaxation phenomena in a class of nondissipative systems with two highly disparate time scales (i.e., Brownian systems) have unique commonalities quantifiable via two scalars. It is shown that the exactly solvable problem of the dynamics of a weakly linked impurity spin in a s=1/2 XY chain belongs to this dynamical universality class and so does that of a heavy mass in an infinite harmonic oscillator chain and a spinless quasi two-dimensional attractive Fermi gas in the long-wavelength limit. The case of the strongly linked impurity in the XY chain is also discussed along with the corresponding limits in the harmonic oscillator chain and the electron-gas problems.

  18. Metabolism of short-chain ceramide by human cancer cells-implications for therapeutic approaches

    PubMed Central

    Chapman, Jacqueline V.; Gouazé-Andersson, Valérie; Messner, Maria C.; Flowers, Margaret; Karimi, Ramin; Kester, Mark; Barth, Brian M.; Liu, Xin; Liu, Yong-Yu; Giuliano, Armando E.; Cabot, Myles C.

    2010-01-01

    Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6 μM) and high-dose (12 μM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [3H]palmitic acid produced a 2-fold increase in 3H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in 3H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer. PMID:20385104

  19. The pharmacokinetic profile of crocetin in healthy adult human volunteers after a single oral administration.

    PubMed

    Umigai, N; Murakami, K; Ulit, M V; Antonio, L S; Shirotori, M; Morikawa, H; Nakano, T

    2011-05-15

    Crocetin, a unique carotenoid with a short carbon chain length, is an active compound of saffron and Gardenia jasminoides Ellis used as traditional herbal medicine. The present study was undertaken to investigate the pharmacokinetic profiles of crocetin in healthy adult subjects. The study was conducted as an open-label, single dose escalation with 10 Filipino volunteers (5 men and 5 women). The subjects received a single dose of crocetin at three doses (7.5, 15 and 22.5 mg) in one week interval. Blood samples were collected from the brachial vein before and at 1, 2, 4, 6, 8, 10 and 24 h after administration. Plasma concentrations of crocetin were determined by high-performance liquid chromatography (HPLC). Crocetin was rapidly absorbed and detected within an hour of administration with a mean time to reach maximum concentration (T(max)) of crocetin ranging from 4.0 to 4.8 h. The mean values of C(max) and AUC(0-24h) ranged from 100.9 to 279.7 ng/ml and 556.5 to 1720.8 ng. h/ml respectively. C(max) and AUC values increased with dose proportional manner. Crocetin was eliminated from human plasma with a mean elimination half life (T(½) of 6.1 to 7.5 h. In summary, there were no serious adverse events up to 22.5 mg dose of crocetin while crocetin was found to be absorbed more quickly than the other carotenoids such as β-carotene, lutein and lycopene. PMID:21112749

  20. Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes.

    PubMed

    Simon, Petra; Omokoko, Tana A; Breitkreuz, Andrea; Hebich, Lisa; Kreiter, Sebastian; Attig, Sebastian; Konur, Abdo; Britten, Cedrik M; Paret, Claudia; Dhaene, Karl; Türeci, Özlem; Sahin, Ugur

    2014-12-01

    The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes. PMID:25245536

  1. Haplotyping the human leukocyte antigen system from single chromosomes

    PubMed Central

    Murphy, Nicholas M.; Burton, Matthew; Powell, David R.; Rossello, Fernando J.; Cooper, Don; Chopra, Abha; Hsieh, Ming Je; Sayer, David C.; Gordon, Lavinia; Pertile, Mark D; Tait, Brian D.; Irving, Helen R.; Pouton, Colin W.

    2016-01-01

    We describe a method for determining the parental HLA haplotypes of a single individual without recourse to conventional segregation genetics. Blood samples were cultured to identify and sort chromosome 6 by bivariate flow cytometry. Single chromosome 6 amplification products were confirmed with a single nucleotide polymorphism (SNP) array and verified by deep sequencing to enable assignment of both alleles at the HLA loci, defining the two haplotypes. This study exemplifies a rapid and efficient method of haplotyping that can be applied to any chromosome pair, or indeed all chromosome pairs, using a single sorting operation. The method represents a cost-effective approach to complete phasing of SNPs, which will facilitate a deeper understanding of the links between SNPs, gene regulation and protein function. PMID:27461731

  2. Haplotyping the human leukocyte antigen system from single chromosomes.