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Sample records for heavy chain antibodies

  1. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen-...... for combating HER2+ breast cancer....

  2. Stability of llama heavy chain antibody fragments under extreme conditions

    NARCIS (Netherlands)

    Dolk, E.

    2004-01-01

    Camelids have next to their normal antibodies, a unique subset of antibodies lacking light chains. The resulting single binding domain, VHH, of these heavy chain antibodies consequently have unique properties. A high stability is one of these properties, which was investigated in this thesis. The a

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

    Science.gov (United States)

    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

  4. Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

    Directory of Open Access Journals (Sweden)

    Christopher Vollmers

    Full Text Available 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.

  5. Generation of heavy-chain-only antibodies in mice.

    NARCIS (Netherlands)

    R. Janssens (Rick); S. Dekker (Sylvia); R.W. Hendriks (Rudi); G. Panayotou (George); A. van Remoortere (Alexandra); J.K. San (John Kong-a); F.G. Grosveld (Frank); D.D. Drabek (Dubravka)

    2006-01-01

    textabstractWe have generated transgenic mice containing hybrid llama/human antibody loci that contain two llama variable regions and the human D, J, and Cmu and/or Cgamma constant regions. Such loci rearrange productively and rescue B cell development efficiently without LC rearrangement. Heavy-cha

  6. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna

    2011-06-28

    The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

  7. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

    NARCIS (Netherlands)

    Joosten, V.; Gouka, R.J.; Hondel, C.A.M.J.J. van den; Verrips, C.T.; Lokman, B.C.

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA level

  8. Improving properties of llama heavy chain antibodies using in silico analysis

    NARCIS (Netherlands)

    Lutje Hulsik, D.

    2009-01-01

    Microbicides offer a promising way to prevent HIV infection in developing countries. Llama heavy chain antibody fragments (VHHs) that neutralize HIV are an ideal candidate for usage as active microbicide component. A VHH is the smallest intact antigen binding domain which allows it to reach for anti

  9. Improved production and function of llama heavy chain antibody fragments by molecular evolution

    NARCIS (Netherlands)

    Linden, van der R.H.; Geus, de B.; Frenken, G.J.; Peters, H.; Verrips, C.T.

    2000-01-01

    The aim of this study was to improve production level of llama heavy chain antibody fragments (V (HH)) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V (HH) fragments specific for the azo-dye reactive red-6. In

  10. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

    NARCIS (Netherlands)

    Joosten, V.; Gouka, R.J.; Hondel, C.A.M.J.J. van den; Verrips, C.T.; Lokman, B.C.

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA

  11. Passive transfer of maternal immunity in the dromedary (Camelus dromedarius), involvement of heavy chain antibodies.

    Science.gov (United States)

    Salhi, Imed; Bessalah, Salma; Mbarek, Sonia Ben; Chniter, Mohamed; Seddik, Mabrouk-Mouldi; Khorchani, Touhami; Hammadi, Mohamed

    2015-03-01

    In many mammalian species, newborns are agammaglobulinemic; thus, colostrum and milk are the main sources of early protective antibodies. These antibodies are produced in the mother's serum and transferred to mammalian glands a few days before parturition. Here, we have studied the transfer of immunity from a she-camel immunized with human serum albumin (HSA) to her calf via colostrum and milk. Our results show that HSA-specific antibodies are produced in the mother's serum and are subsequently transferred to her colostrum. These specific antibodies are then transferred by suckling to the calf. The calf serum did not contain HSA-reactive antibodies at parturition and before the first feed, after suckling, a rise in reactivity was observed peaking at 24 h postpartum. The involvement of heavy chain antibodies (HCAbs) in the process of immunity transfer was also examined, and it was found that they were also transferred from the colostrum to the calf serum like conventional antibodies.

  12. Comparative Analysis of Immune Repertoires between Bactrian Camel's Conventional and Heavy-Chain Antibodies

    Science.gov (United States)

    Yang, Kai; Zhang, Wei; Zhang, Changjiang; Fu, Longfei; Ren, Zhe; Wang, Changxi; Wu, Jinghua; Lu, Ruxue; Ye, Yanrui; He, Mengying; Nie, Chao; Yang, Naibo; Wang, Jian; Yang, Huanming; Liu, Xiao

    2016-01-01

    Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data. PMID:27588755

  13. Porous silicon biosensor for detection of variable domain of heavy-chain of HCAb antibody

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong-yan; Lü Xiao-yi; JIA Zhen-hong; LI Jiang-wei; ZHANG Fu-chun

    2012-01-01

    In this paper,we produce porous silicon (PSi) by electrochemical etching,and it is the first time to evaluate the performance of label-free porous silicon biosensor for detection of variable domain of heavy chain of heavy-chain antibody (VHH).The binding of hen egg white lysozyme (HEWL) and VHH causes a red shift in the reflection spectrum of the biosensor.The red shift is proportional to the VHH concentration in the range from 14 μg · ml-1 to 30μg·ml-1 with a detection limit of 0.648 ng ·ml-1.The research is useful for the development of label-free biosensor applied in the rapid and sensitive determination of small molecules.

  14. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

    Full Text Available Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig heavy chain (HC and kappa light chain (LC was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

  15. Generation and characterization of polyclonal antibody against part of immunoglobulin constant heavy υ chain of goose.

    Science.gov (United States)

    Zhao, Panpan; Guo, Yongli; Ma, Bo; Xing, Mingwei; Wang, Junwei

    2014-08-01

    Immunoglobulin Y (abbreviated as IgY) is a type of immunoglobulin that is the major antibody in bird, reptile, and lungfish blood. IgY consists of two light (λ) and two heavy (υ) chains. In the present study, polyclonal antibody against IgYFc was generated and evaluated. rIgYCυ3/Cυ4 was expressed in Escherichia coli, purified and utilized to raise polyclonal antibody in rabbit. High affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:204,800 for ELISA assay. The antibody can specifically recognize both rIgYCυ3/Cυ4 and native IgY by Western bolt analysis. Furthermore, the serum of Grus japonensis or immunoglobulin of chicken, duck, turkey, and silkie samples and dynamic changes of serum GoIgY after immunogenicity with GPV-VP3-virus-like particles (GPV-VP3-VLPs) can be detected with the anti-GoIgYFc polyclonal antibody. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of GoIgY.

  16. A two-in-one antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions.

    Science.gov (United States)

    Lee, Chingwei V; Koenig, Patrick; Fuh, Germaine

    2014-01-01

    A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.

  17. Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species.

    Science.gov (United States)

    Griffin, Laura M; Snowden, James R; Lawson, Alastair D G; Wernery, Ulrich; Kinne, Jorg; Baker, Terry S

    2014-03-01

    Camel antibodies have been widely investigated, but work has focused upon the unique heavy chain antibodies found across camelid species. These are homodimers, devoid of light chains and the first constant heavy chain domain. Camelid species also display conventional hetero-tetrameric antibodies with identical pairs of heavy and light chains; in Camelus dromedarius these constitute 25% of circulating antibodies. Few investigations have been made on this subset of antibodies and complete conventional camel IgG sequences have not been reported. Here we study the sequence diversity of functional variable and constant regions observed in 57 conventional heavy, 18 kappa and 35 lambda light chains of C. dromedarius and Camelus bactrianus. We detail sequences of the full kappa and lambda light chain, variable and CH1 region for IgG1a and IgG1b and the CH2 and CH3 region for IgG1a. The majority (60%) of IgG1 variable region sequences aligned with the human IgHV3 family (clan III) and had leader sequences beginning with MELG whereas the remaining sequences aligned with the IgHV4 (clan II) and had leader sequences beginning with MRLL. Distinct differences in CDR length were observed between the two; where CDR1 was typically 5 and 7 residues and CDR2 at 17 and 16 residues, respectively. CDR3 length of IgHV4 (range 11 to 20) was closer to that typical of VHH antibodies than that of IgHV3 (range 3 to 18 residues). Designed oligonucleotide primers have enabled identification of paired heavy and light chains of conventional camel antibodies from individual B cell clones.

  18. Cloning,sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

    Institute of Scientific and Technical Information of China (English)

    ZHANGJINSONG; MINGYEH

    1994-01-01

    The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction(PCR) technique.We found that 2 and 3 mAbs utilized genes of the VHIV and VHⅢ families,respectively.The former 2 VH segments were in germline configuration.A common VH segment,with the best similarity of 90.1% to the published VHⅢ germline genes,was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs.This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHⅢ gene.All these polyreactive mAbs displayed a large NDN region(VH-D-JH junction).The entire H chain V regions of these polyreactive mAbs are unusually basic.The analysis of the charge properties of these mAbs as well as those of other poly-and mono-reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites(CDRs),may be an important structural feature involved in antibody polyreactivity.

  19. Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies.

    Science.gov (United States)

    Froning, K J; Leaver-Fay, A; Wu, X; Phan, S; Gao, L; Huang, F; Pustilnik, A; Bacica, M; Houlihan, K; Chai, Q; Fitchett, J R; Hendle, J; Kuhlman, B; Demarest, S J

    2017-07-20

    The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use. © 2017 The Protein Society.

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

    Science.gov (United States)

    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.

  1. N332-Directed broadly neutralizing antibodies use diverse modes of HIV-1 recognition: inferences from heavy-light chain complementation of function.

    Directory of Open Access Journals (Sweden)

    Marie Pancera

    Full Text Available Dozens of broadly neutralizing HIV-1 antibodies have been isolated in the last few years from the sera of HIV-1-infected individuals. Only a limited number of regions on the HIV-1 spike, however, are recognized by these antibodies. One of these regions (N332 is characterized by an N-linked glycan at residue 332 on HIV-1 gp120 and is recognized by antibody 2G12 and by the recently reported antibodies PGT121-137, the latter isolated from three donors. To investigate the diversity in mode of antibody recognition at the N332 site, we used functional complementation between antibody heavy and light chains as a means of assessing similarity in mode of recognition. We examined a matrix of 12 PGT-heavy chains with each of 12 PGT-light chains. Expression in 96-well format for the 144 antibodies (132 chimeric and 12 wild-type was generally consistent (58 ± 10 µg/ml. In contrast, recognition of HIV-1 gp120 was bimodal: when the source of heavy and light chains was from the same donor, recognition was good; when sources of heavy and light chains were from different donors, recognition was poor. Moreover, neutralization of HIV-1 strains SF162.LS and TRO.11 generally followed patterns of gp120 recognition. These results are consistent with published sequence, mutational, and structural findings, all of which indicate that N332-directed neutralizing antibodies from different donors utilize different modes of recognition, and provide support for a correlation between functional complementation of antibody heavy and light chains and similarity in antibody mode of recognition. Overall, our results add to the growing body of evidence that the human immune system is capable of recognizing the N332-region of HIV-1 gp120 in diverse ways.

  2. Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains.

    Science.gov (United States)

    Balabashin, Dmitriy; Kovalenko, Elena; Toporova, Viktoria; Aliev, Teimur; Panina, Anna; Svirshchevskaya, Elena; Dolgikh, Dmitry; Kirpichnikov, Mikhail

    2015-10-01

    Tumor necrosis factor-α (TNF-α) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-α recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

  3. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    Science.gov (United States)

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  4. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

  5. Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

    NARCIS (Netherlands)

    Dolk, E.; Vliet, C. van; Perez, J.M.J.; Vriend, G.; Darbon, H.; Ferrat, G.; Cambillau, C.; Frenken, L.G.J.; Verrips, T.

    2005-01-01

    In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear

  6. Targeting Antibodies to Carbon Nanotube Field Effect Transistors by Pyrene Hydrazide Modification of Heavy Chain Carbohydrates

    Directory of Open Access Journals (Sweden)

    Steingrimur Stefansson

    2012-01-01

    Full Text Available Many carbon nanotube field-effect transistor (CNT-FET studies have used immobilized antibodies as the ligand binding moiety. However, antibodies are not optimal for CNT-FET detection due to their large size and charge. Their size can prevent ligands from reaching within the Debye length of the CNTs and a layer of charged antibodies on the circuits can drown out any ligand signal. In an attempt to minimize the antibody footprint on CNT-FETs, we examined whether pyrene hydrazide modification of antibody carbohydrates could reduce the concentration required to functionalize CNT circuits. The carbohydrates are almost exclusively on the antibody Fc region and this site-specific modification could mediate uniform antibody orientation on the CNTs. We compared the hydrazide modification of anti-E. coli O157:H7 polyclonal antibodies to pyrenebutanoic acid succinimidyl ester-coated CNTs and carbodiimide-mediated antibody CNT attachment. Our results show that the pyrene hydrazide modification was superior to those methods with respect to bacteria detection and less than 1 nM labeled antibody was required to functionalize the circuits.

  7. Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice

    Directory of Open Access Journals (Sweden)

    Min ZHANG,Xueqian CHENG,Dan CHU,Jingwen LIANG,Yi SUN,Li MA,Beilei XU,Min ZHENG,Meili WANG,Liming REN,Xiaoxiang HU,Qingyong MENG,Ran ZHANG,Ying GUO,Yunping DAI,Robert AITKEN,Ning LI,Yaofeng ZHAO

    2014-06-01

    Full Text Available In this study, we introduced the bovine immunoglobulin μ heavy-chain gene (the orphaned gene on BTA11 into mouse germline cells. Bovine IgM was highly expressed in selected transgenic lines, and it largely inhibited rearrangements of the endogenous immunoglobulin heavy chain (IgH genes in these lines. The forced expression of bovine IgM resulted in reduced numbers of pro- and pre-B cells but increased the number of immature B cells in the transgenic mice. Bovine IgM-expressing B cells can migrate from the bone marrow to the spleen, but most of the cells are arrested at the T1 transitional B cell stage, leading to a significantly lower number of T2 transitional and mature B cells in the spleen. Although the serum concentrations of endogenous IgM and IgG in the transgenic mice were significantly decreased, the IgA levels were slightly increased compared to the WT mice. The bovine IgM level in the serum was only one-tenth to one-fifth of that of endogenous mouse IgM, suggesting that most of the serum immunoglobulin were contributed by endogenous IgH gene-expressing B cells. These transgenic mice also exhibited a lower frequency of unique complementarity determining region 3 (CDR3 sequences in their VH repertoire and V&Kgr; repertoire but exhibited an increased frequency of unique CDR3 in their V&Lgr; repertoire. Compared to the WT mice, the transgenic mice had a significantly higher percentage of mouse IgM-expressing B cells that expressed &Lgr; chains. Finally, we showed that the transgenic mice were deficient in a specific antibody response to antigen stimulation.

  8. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variab

  9. Heavy-chain isotype patterns of human antibody-secreting cells induced by Haemophilus influenzae type b conjugate vaccines in relation to age and preimmunity

    DEFF Research Database (Denmark)

    Barington, T; Juul, Lars; Gyhrs, A;

    1994-01-01

    The influence of preexisting immunity on the heavy-chain isotypes of circulating antibody-secreting cells (AbSC) induced by vaccination with Haemophilus influenzae type b (Hib) capsular polysaccharide (HibCP) coupled to tetanus toxoid (TT) or diphtheria toxoid (DT) and by vaccination with TT or DT...

  10. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, van den N.; Goosen, T.; Verrips, C.T.; Hondel, van den C.A.M.J.J.; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V-HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which

  11. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which

  12. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pro

  13. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, van den N.; Goosen, T.; Verrips, C.T.; Hondel, van den C.A.M.J.J.; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V-HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pr

  14. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, van den N.; Goosen, T.; Verrips, C.T.; Hondel, van den C.A.M.J.J.; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V-HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pr

  15. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pro

  16. Improving the accuracy of the structure prediction of the third hypervariable loop of the heavy chains of antibodies.

    KAUST Repository

    Messih, Mario Abdel

    2014-06-13

    MOTIVATION: Antibodies are able to recognize a wide range of antigens through their complementary determining regions formed by six hypervariable loops. Predicting the 3D structure of these loops is essential for the analysis and reengineering of novel antibodies with enhanced affinity and specificity. The canonical structure model allows high accuracy prediction for five of the loops. The third loop of the heavy chain, H3, is the hardest to predict because of its diversity in structure, length and sequence composition. RESULTS: We describe a method, based on the Random Forest automatic learning technique, to select structural templates for H3 loops among a dataset of candidates. These can be used to predict the structure of the loop with a higher accuracy than that achieved by any of the presently available methods. The method also has the advantage of being extremely fast and returning a reliable estimate of the model quality. AVAILABILITY AND IMPLEMENTATION: The source code is freely available at http://www.biocomputing.it/H3Loopred/ .

  17. Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses.

    Science.gov (United States)

    Ferrari, A; Weill, F S; Paz, M L; Cela, E M; González Maglio, D H; Leoni, J

    2012-03-01

    Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a β-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.

  18. High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

    Science.gov (United States)

    Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

    2013-01-01

    Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

  19. The new research and application progress in heavy chain antibody and its genetic engineering single domain antibody%重链抗体及其基因工程单域抗体的研究和应用进展

    Institute of Scientific and Technical Information of China (English)

    蔡家麟; 王颖; 潘欣(通讯作者)

    2013-01-01

      In recent years, researchers found unusual antibodies composed only of heavy chains from camelids and sharks. These peculiar heavy chain antibodies (hcAbs) lack light chains but stil remain dedicated variable domain with the antigen-binding site. Recombinant single-domain antibody is the smalest intact antigen-binding fragment derived from heavy-chain antibodies. The advantageous features of single domain antibody reagents derived from these hcAbs include their smal dimension, high apparent stability, high expression. As expectations, high-affinity, improved solubility without any sign of aggregation single-domain antibodies can be widely used in basic research and improved diagnostic field.%  近年来在骆驼和护士鲨等动物的体内发现了一种重链抗体。重链抗体和普通的抗体相比缺少轻链,仅有可变区,但依然保留了抗原结合能力。通过基因工程改造重链抗体形成的单域抗体不仅保留了分子量小、物理稳定性高、容易表达等特性优点,而且亲和力高、不易聚集,被广泛应用于科研和临床诊断。

  20. Improving the accuracy of the structure prediction of the third hypervariable loop of the heavy chains of antibodies

    DEFF Research Database (Denmark)

    Messih, M. A.; Lepore, R.; Marcatili, Paolo

    2014-01-01

    and specificity. The canonical structure model allows high accuracy prediction for five of the loops. The third loop of the heavy chain, H3, is the hardest to predict because of its diversity in structure, length and sequence composition. Results: We describe a method, based on the Random Forest automatic...

  1. A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG.

    Science.gov (United States)

    Rumfelt, L L; Avila, D; Diaz, M; Bartl, S; McKinney, E C; Flajnik, M F

    2001-02-13

    In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM(1gj), from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells ("germline-joined"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H(1gj) in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H(1gj) chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM(1gj). Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

  2. Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection.

    Science.gov (United States)

    Torán, J L; Sánchez-Pulido, L; Kremer, L; del Real, G; Valencia, A; Martínez-A, C

    2001-01-01

    We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.

  3. A single mutation in framework 2 of the heavy variable domain improves the properties of a diabody and a related single-chain antibody.

    Science.gov (United States)

    Rodríguez-Rodríguez, Everardo Remi; Ledezma-Candanoza, Luis M; Contreras-Ferrat, Luis Gabriel; Olamendi-Portugal, Timoteo; Possani, Lourival D; Becerril, Baltazar; Riaño-Umbarila, Lidia

    2012-10-26

    Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.

  4. Muscle precursor cells in the developing limbs of two isopods (Crustacea, Peracarida): an immunohistochemical study using a novel monoclonal antibody against myosin heavy chain.

    Science.gov (United States)

    Kreissl, S; Uber, A; Harzsch, S

    2008-05-01

    In the hot debate on arthropod relationships, Crustaceans and the morphology of their appendages play a pivotal role. To gain new insights into how arthropod appendages evolved, developmental biologists recently have begun to examine the expression and function of Drosophila appendage genes in Crustaceans. However, cellular aspects of Crustacean limb development such as myogenesis are poorly understood in Crustaceans so that the interpretative context in which to analyse gene functions is still fragmentary. The goal of the present project was to analyse muscle development in Crustacean appendages, and to that end, monoclonal antibodies against arthropod muscle proteins were generated. One of these antibodies recognises certain isoforms of myosin heavy chain and strongly binds to muscle precursor cells in malacostracan Crustacea. We used this antibody to study myogenesis in two isopods, Porcellio scaber and Idotea balthica (Crustacea, Malacostraca, Peracarida), by immunohistochemistry. In these animals, muscles in the limbs originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in other Crustaceans and in insects.

  5. A rice-based soluble form of a murine TNF-specific llama variable domain of heavy-chain antibody suppresses collagen-induced arthritis in mice.

    Science.gov (United States)

    Abe, Michiyo; Yuki, Yoshikazu; Kurokawa, Shiho; Mejima, Mio; Kuroda, Masaharu; Park, Eun Jeong; Scheller, Jürgen; Nakanishi, Ushio; Kiyono, Hiroshi

    2014-04-10

    Tumor necrosis factor alpha (TNF) plays a pivotal role in chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Although anti-TNF antibody therapy is now commonly used to treat patients suffering from these inflammatory conditions, the cost of treatment continues to be a concern. Here, we developed a rice transgenic system for the production of a llama variable domain of a heavy-chain antibody fragment (VHH) specific for mouse TNF in rice seeds (MucoRice-mTNF-VHH). MucoRice-mTNF-VHH was produced at high levels in the rice seeds when we used our most recent transgene-overexpression system with RNA interference technology that suppresses the production of major rice endogenous storage proteins while enhancing the expression of the transgene-derived protein. Production levels of mTNF-VHH in rice seeds reached an average of 1.45% (w/w). Further, approximately 91% of mTNF-VHH was released easily when the powder form of MucoRice-mTNF-VHH was mixed with PBS. mTNF-VHH purified by means of single-step gel filtration from rice PBS extract showed high neutralizing activity in an in vitro mTNF cytotoxicity assay using WEHI164 cells. In addition, purified mTNF-VHH suppressed progression of collagen-induced arthritis in mice. These results show that this rice-expression system is useful for the production of neutralizing VHH antibody specific for mTNF.

  6. Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR.

    Science.gov (United States)

    Okazaki, Fumiyoshi; Aoki, Jun-ichi; Tabuchi, Soichiro; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

    2012-10-01

    We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V(HH)) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V(HH) with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V(HH) reached 73.8 mg/1 in a Sakaguchi flask. The V(HH) fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V(HH) fragment was specifically recognized and could bind to the EGFR with a high affinity.

  7. Molecular characterization of the variable heavy and light chain regions of five HIV-1 specific human monoclonal antibodies.

    NARCIS (Netherlands)

    E.M.M. van der Donk; M. Schutten (Martin); A.D.M.E. Osterhaus (Albert); R.W.J. van der Heijden (Roger)

    1994-01-01

    textabstractWe have reported the generation and characterization of four HIV-1 neutralizing human monoclonal antibodies. Three antibodies recognize a conformational epitope within the CD4-binding site of HIV-1 gp120 and one recognizes a linear epitope located within the hypervariable V3 domain of gp

  8. Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

    Science.gov (United States)

    Jiang, Guoying; Yu, Christopher; Yadav, Daniela B; Hu, Zhilan; Amurao, Annamarie; Duenas, Eileen; Wong, Marc; Iverson, Mark; Zheng, Kai; Lam, Xanthe; Chen, Jia; Vega, Roxanne; Ulufatu, Sheila; Leddy, Cecilia; Davis, Helen; Shen, Amy; Wong, Pin Y; Harris, Reed; Wang, Y John; Li, Dongwei

    2016-07-01

    Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes. Published by Elsevier Inc.

  9. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    Science.gov (United States)

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease.

  10. A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model.

    Science.gov (United States)

    Moayeri, Mahtab; Leysath, Clinton E; Tremblay, Jacqueline M; Vrentas, Catherine; Crown, Devorah; Leppla, Stephen H; Shoemaker, Charles B

    2015-03-06

    Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes.

  11. Differences in modifications of cytoplasmic free Ca2+ concentration and 86Rb+ influx in human neoplastic B cells by antibodies to mu- relative to delta-Ig heavy chains.

    Science.gov (United States)

    Heikkilä, R; Ruud, E; Funderud, S; Godal, T

    1985-01-01

    Cytoplasmic free Ca2+ concentration and influx of 86Rb+ (K+ analogue) were determined during the first minutes after stimulation of neoplastic human B cells and B cell lines by antibodies to surface Ig. The Ca2+ concentration increased in the great majority of samples (41 of 48). All of four B cell lines also responded, providing formal evidence that accessory cells are not required for this early, surface Ig-mediated event. Antibodies to delta as well as mu, heavy chains (anti-delta and anti-mu) could induce both Ca2+ and 86Rb+ responses. 86Rb+ responders were found within the group of Ca2+ responders, but no quantitative relation was observed between the two responses. In cells expressing both sIgM and sIgD, antibodies to delta heavy chains were more potent than those to mu heavy chains in inducing Ca2+ responses, whereas the opposite pattern was seen with regard to 86Rb+ responses. These results demonstrate that sIgM and sIgD can deliver different biochemical signals to the cell. PMID:3921300

  12. Construction and characterization of a non-immune Llama variable heavy chain phage display antibody library%羊驼非免疫重链单域抗体库的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    吴标; 王树军; 夏立亮; 季萍; 葛海良; 赵国屏; 王颖

    2011-01-01

    本研究旨在通过构建羊驼非免疫重链单域抗体库,完成抗体库多样性的鉴定,为进一步筛选抗原特异性重链抗体奠定基础.我们从未经免疫的羊驼外周血中分离外周血单个核细胞(PBMC),抽提RNA后,用RT-PCR方法特异性扩增羊驼重链抗体可变区(VHH)片段;并采用两步连接方法将重链抗体可变区片段与噬菌粒载体pCANTAB5E连接获得重组子,多次电转感受态大肠杆菌TG1后获得VHH抗体基因库;并采用稀释计数法测定抗体库库容量,随机挑取克隆测序验证抗体库多样性.结果显示,我们所构建的羊驼非免疫重链单域抗体库的库容量为1.5×109,随机克隆测序验证多样性良好,独立克隆所占比例为80%,并显示出和人源抗体较高的同源性.上述结果表明,我们已经成功构建获得大容量的羊驼非免疫重链单域抗体库,为进一步筛选抗原特异性重链抗体奠定基础.%To construct a non-immune Llama variable domain of heavy chain antibody(VHH) phage display antibody library (VHH antibody library). Llama peripheral blood mononuclear lymphocytes were isolated from whole blood by Ficoll-hypaque density gradient centrifugation. Total RNA was extracted from PBMCs and reverse-transcripted into cDNA by using specific primers. VHH were amplified by nested PCR. PCR products of VHH fragments were then purified and ligated with phagemid vector pCANTAB5E by a modified two-step ligation method. Recombinant pCANTAB5E-VHH vectors were electroporated into competent TGI E.coli cells to obtain the primary VHH antibody library. The library capacity was titrated through limited dilution. Recombination efficacy and diversity of VHH antibody library was determined by sequencing analysis. Alignment a-nalysis was performed to compare the homology between Llama VHH domain and human/mouse variable regions of heavy chains. By using a modified strategy, we have constructed a non-immune Llama VHH antibody library with 1. 5

  13. Engineered single chain antibody fragments for radioimmunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Huhalov, A.; Chester, K. A. [Cancer Research UK Imaging and Targeting Group Royal Free, London (United Kingdom). Department of Oncology; University College Medical School Royal Free Campus, London (United Kingdom)

    2004-12-01

    An ideal molecule to deliver radioimmunotherapy (RIT) would be target specific and have prolonged residence time at high concentrations in the tumour with rapid clearance from normal tissues. It would also be non-immunogenic. These features can be rationally introduced into recombinant antibody-based proteins using antibody engineering techniques. This reviews focuses on the use of antibody engineering in the design and development of RIT molecules which have single chain Fv (scFv) antibody fragments as building blocks.

  14. Heavy chain-only IgG2b llama antibody effects near-pan HIV-1 neutralization by recognizing a CD4-induced epitope that includes elements of coreceptor- and CD4-binding sites.

    Science.gov (United States)

    Acharya, Priyamvada; Luongo, Timothy S; Georgiev, Ivelin S; Matz, Julie; Schmidt, Stephen D; Louder, Mark K; Kessler, Pascal; Yang, Yongping; McKee, Krisha; O'Dell, Sijy; Chen, Lei; Baty, Daniel; Chames, Patrick; Martin, Loïc; Mascola, John R; Kwong, Peter D

    2013-09-01

    The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

  15. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.

    Science.gov (United States)

    Sun, Shuang; Yang, Xiao; Wang, Haifeng; Zhao, Yun; Lin, Yan; Ye, Chen; Fang, Xiangdong; Hang, Haiying

    2016-07-01

    Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.

  16. Design of Chemical Conjugate for Targeted Therapy of Multiple Sclerosis Based of Constant Fragment of Human Antibody Heavy Chain and Peptoid Analog of Autoantigen MOG35-55.

    Science.gov (United States)

    Lomakin, Y A; Stepanov, A V; Balabashin, D S; Ponomarenko, N A; Smirnov, I V; Belogurov, A A

    2017-04-01

    Elimination of B cells producing autoantibodies to neuroantigens is considered as beneficial in the treatment of multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG) is a significant autoantigen in multiple sclerosis. It was shown that MOG-like peptoid AMogP3 can bind autoantibodies produced by pathological lymphocytes. We propose a structure of an innovative drug for targeted elimination of the pool of autoreactive B cells responsible for multiple sclerosis pathogenesis; this compound is a complex of peptoid AMogP3 with Fc fragment of human immunoglobulin. The obtained Fc-PEG-AMogP3 conjugate effectively interact with autoreactive antibodies, which attests to their high therapeutic potential.

  17. ISOLATION OF ENDOTOXIN-SPECIFIC ANTIBODIES BY SELECTION OF AN SINGLE CHAIN PHAGE ANTIBODY LIBRARY

    Institute of Scientific and Technical Information of China (English)

    陈鸣; 俞丽丽; 张雪; 府伟灵

    2002-01-01

    Objective: To isolate murine anti endotoxin single chain phage antibody from a constructed library. Methods: Total RNA was firstly extracted from murine splenic cells and mRNA was reverse-transcribed into cDNA. Then the designed primers were used to amplify the variable region genes of the heavy and light chain (VH, VL) with polymerase chain reaction. The linker was used to assemble the VH and VL into ScFv, and the NotI and SfiI restriction enzymes were used to digest the ScFv in order to ligate into the pCANTAB5E phagemid vector that was already digested with the same restriction enzymes. The ligated vector was then introduced into competent E.coli TG1 cells to construct a single-chain phage antibody library. After rescued with M13KO7 helper phage, recombinant phages displaying ScFv fragments were harvested from the supernatant and selected with endotoxin. The enriched positive clones were reinfected into TG1 cells. Finally, 190 clones were randomly selected to detect the anti endotoxin antibody with indirect ELISA. Results: The titer of anti endotoxin in murine sera was 1:12,800. The concentration of total RNA was 12.38 μg/ml. 1.9×107 clones were obtained after transformed into TG1. 3×104 colonies were gotten after one round panning. Two positive colonies were confirmed with indirect ELISA among 190 randomly selected colonies. Conclusion: A 1.9×107 murine anti endotoxin single chain phage antibody library was successfully constructed. Two anti endotoxin antibodies were obtained from the library.

  18. Translocations affecting human immunoglobulin heavy chain locus

    Directory of Open Access Journals (Sweden)

    Sklyar I. V.

    2014-03-01

    Full Text Available Translocations involving human immunoglobulin heavy chain (IGH locus are implicated in different leukaemias and lymphomas, including multiple myeloma, mantle cell lymphoma, Burkitt’s lymphoma and diffuse large B cell lymphoma. We have analysed published data and identified eleven breakpoint cluster regions (bcr related to these cancers within the IgH locus. These ~1 kbp bcrs are specific for one or several types of blood cancer. Our findings could help devise PCR-based assays to detect cancer-related translocations, to identify the mechanisms of translocations and to help in the research of potential translocation partners of the immunoglobulin locus at different stages of B-cell differentiation.

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

    Science.gov (United States)

    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.

  20. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  1. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  2. Diversity Analysis of the Immunoglobulin M Heavy Chain Gene in ...

    African Journals Online (AJOL)

    A full-length cDNA encoding the immunoglobulin (IgM) heavy chain gene of Nile tilapia was ... The deduced amino acid sequence of the Nile tilapia IgM heavy chain ... followed by the spleen, intestine and peripheral blood leukocytes (PBLs).

  3. Microinjection of antibodies to the calpactin I light chain in MDBK cells causes precipition of the cytoskeletal calpactin I complex without affecting the distribution of related proteins.

    Science.gov (United States)

    Glenney, J R

    1990-01-01

    The calpactin I complex is composed of two heavy chain (39K) and two light chain (11K) subunits. The heavy chain is a member of a protein family that includes lipocortins, endonexin and chromobindins while the light chain is a member of the S100 family (7 distinct members are known). We have found that the kidney epithelial cell line MDBK expresses four members of the heavy chain family and two members of the light chain protein family. Antibodies to the light chain of calpactin I were found to cause the precipitation of injected antibody together with the associated heavy chain without apparent effect on the distribution of related proteins. This suggests a differential targeting of various members of the calpactin heavy and light chain families even within the same cell.

  4. Immunoglobulin heavy chain exclusion in the shark.

    Directory of Open Access Journals (Sweden)

    Karolina Malecek

    2008-06-01

    Full Text Available The adaptive immune system depends on specific antigen receptors, immunoglobulins (Ig in B lymphocytes and T cell receptors (TCR in T lymphocytes. Adaptive responses to immune challenge are based on the expression of a single species of antigen receptor per cell; and in B cells, this is mediated in part by allelic exclusion at the Ig heavy (H chain locus. How allelic exclusion is regulated is unclear; we considered that sharks, the oldest vertebrates possessing the Ig/TCR-based immune system, would yield insights not previously approachable and reveal the primordial basis of the regulation of allelic exclusion. Sharks have an IgH locus organization consisting of 15-200 independently rearranging miniloci (VH-D1-D2-JH-Cmu, a gene organization that is considered ancestral to the tetrapod and bony fish IgH locus. We found that rearrangement takes place only within a minilocus, and the recombining gene segments are assembled simultaneously and randomly. Only one or few H chain genes were fully rearranged in each shark B cell, whereas the other loci retained their germline configuration. In contrast, most IgH were partially rearranged in every thymocyte (developing T cell examined, but no IgH transcripts were detected. The distinction between B and T cells in their IgH configurations and transcription reveals a heretofore unsuspected chromatin state permissive for rearrangement in precursor lymphocytes, and suggests that controlled limitation of B cell lineage-specific factors mediate regulated rearrangement and allelic exclusion. This regulation may be shared by higher vertebrates in which additional mechanistic and regulatory elements have evolved with their structurally complex IgH locus.

  5. [Human single chain antibodies directed to tumor necrosis factor].

    Science.gov (United States)

    Vikhrova, M A; Batanova, T A; Lebedev, L R; Shingarova, L N; Frank, L A; Kirpichnikov, M P; Tikunova, N V

    2011-01-01

    Six unique phage antibodies to human TNF have been selected from a combinatorial library of human single chain fragment variable. ELISA and Western-blotting was used to study selected phage antibodies binding with TNF. The specificity of selected antibodies was determined by binding with interferon alpha and gamma, bovine serum albumin, ovalbumin and ubiquitin. Two antibodies, sA1 and sB3, were converted into a soluble single-chain antibody form and their affinity was 2.5 and 13.7 nM respectively.

  6. Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells.

    Science.gov (United States)

    Zuhaida, A A; Ali, A M; Tamilselvan, S; Alitheen, N B; Hamid, M; Noor, A M; Yeap, S K

    2013-11-18

    A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications.

  7. λ Light Chain Bias Associated With Enhanced Binding and Function of Anti-HIV Env Glycoprotein Antibodies.

    Science.gov (United States)

    Sajadi, Mohammad M; Farshidpour, Maham; Brown, Eric P; Ouyang, Xin; Seaman, Michael S; Pazgier, Marzena; Ackerman, Margaret E; Robinson, Harriet; Tomaras, Georgia; Parsons, Matthew S; Charurat, Manhattan; DeVico, Anthony L; Redfield, Robert R; Lewis, George K

    2016-01-01

    The humoral response to human immunodeficiency virus (HIV) remains incompletely understood. In this report, we describe biased λ light chain use during the HIV Env glycoprotein (Env) response in HIV infection and vaccination. We examined HIV Env binding (and neutralization) in the context of light chain use in subjects with acute HIV infection, chronic HIV infection, and among HIV vaccinees. In all populations tested, there was a λ chain bias for HIV Env binding antibodies, compared with other HIV antigens (such as p24) or tetanus toxoid. In subjects with chronic HIV infection, a λ bias was noted for neutralization, with λ antibodies accounting for up to 90% of all neutralization activity observed. This is the first report of antibody function in a human infection being tied to light chain use. In HIV infection, antibodies expressing λ light chains tended to have longer CDRL3s, increased light chain contact with HIV Env, and less hypermutation in the heavy chain, compared with antibodies using the κ light chain. These data also support an evolutionary model for the understanding the various κ to λ light chain ratios observed across species and suggest that the λ light chain bias against HIV provides the host an advantage in developing a more efficient humoral response. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  8. A comprehensive analysis of immunoglobulin heavy chain genes in the Bactrian camel (Camelus bactrianus

    Directory of Open Access Journals (Sweden)

    Zuoxiang LIANG,Tao WANG,Yi SUN,Wenlong YANG,Zhihong LIU,Jing FEI,Ying GUO,Qingwei MA,Qingjie PAN,Liming REN

    2015-09-01

    Full Text Available Heavy chain only antibodies (HCAbs represent a rare type of antibody that is devoid of light chains and the CH1 domain that have been reported in cartilaginous fish and camelids. By analyzing transcript data and genome sequences, we conducted a comprehensive analysis of Bactrian camel immunoglobulin heavy chain genes. Based on the transcript data, one μ gene, five γ genes, one α gene and one ε gene were found. Additionally, the variable region of HCAbs (VHH and the conventional antibodies (VH sequences associated with the γ3, γ1a/b and μ genes were amplified. Based on these genome sequences, seven DH, six JH, μ, γ2a, γ2c, α, and ε genes and a portion of a γ3 gene were observed. Different Kozak sequences within different VH families were found in our analysis, and the variability index differed between the VHH3 and VH3 families. Phylogenetic analysis of the constant regions of the camelid immunoglobulin genes indicates that these genes appeared before the evolutionary divergence of Bactrian camels and dromedaries.

  9. Ferritin heavy chain in triple negative breast cancer

    DEFF Research Database (Denmark)

    Liu, Ning Qing; De Marchi, Tommaso; Timmermans, Annemieke M

    2014-01-01

    Ferritin heavy chain (FTH1) is a 21-kDa subunit of the ferritin complex, known for its role in iron metabolism, and which has recently been identified as a favorable prognostic protein for triple negative breast cancer (TNBC) patients. Currently, it is not well understood how FTH1 contributes...

  10. [Cloning and expression of a single human immunoglobulin heavy-chain variable domain with vascular endothelial growth factor binding activity].

    Science.gov (United States)

    Liu, Heng; Liu, Siguo; Wu, Yi; Zili, M; Liu, Yu; Zhang, Aimin; Chen, Jianquan; Cheng, Guoxiang

    2010-11-01

    In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.

  11. Dynein modifiers in C. elegans: light chains suppress conditional heavy chain mutants.

    Directory of Open Access Journals (Sweden)

    Sean M O'Rourke

    2007-08-01

    Full Text Available Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis.

  12. Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant.

    Science.gov (United States)

    Mitsuda, Yukie; Hifumi, Emi; Tsuruhata, Kumi; Fujinami, Hiroko; Yamamoto, Naoki; Uda, Taizo

    2004-04-20

    A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.

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

    Science.gov (United States)

    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.

  14. Characterization of single chain antibody targets through yeast two hybrid

    Directory of Open Access Journals (Sweden)

    Vielemeyer Ole

    2010-08-01

    Full Text Available Abstract Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv, are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID, efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise

  15. Specificity of human anti-variable heavy (VH ) chain autoantibodies and impact on the design and clinical testing of a VH domain antibody antagonist of tumour necrosis factor-α receptor 1.

    Science.gov (United States)

    Cordy, J C; Morley, P J; Wright, T J; Birchler, M A; Lewis, A P; Emmins, R; Chen, Y Z; Powley, W M; Bareille, P J; Wilson, R; Tonkyn, J; Bayliffe, A I; Lazaar, A L

    2015-11-01

    During clinical trials of a tumour necrosis factor (TNF)-R1 domain antibody (dAb™) antagonist (GSK1995057), infusion reactions consistent with cytokine release were observed in healthy subjects with high levels of a novel, pre-existing human anti-VH (HAVH) autoantibody. In the presence of HAVH autoantibodies, GSK1995057 induced cytokine release in vitro due to binding of HAVH autoantibodies to a framework region of the dAb. The epitope on GSK1995057 was characterized and dAbs with reduced binding to HAVH autoantibodies were generated; pharmacological comparability was determined in human in-vitro systems and in-vivo animal experiments. A Phase I clinical trial was conducted to investigate the safety and tolerability of the modified dAb (GSK2862277). A significant reduction in HAVH binding was achieved by adding a single alanine residue at the C-terminus to create GSK2862277. Screening a pool of healthy donors demonstrated a reduced frequency of pre-existing autoantibodies from 51% to 7%; in all other respects, GSK2862277 and the parent dAb were comparable. In the Phase I trial, GSK2862277 was well tolerated by both the inhaled and intravenous routes. One subject experienced a mild infusion reaction with cytokine release following intravenous dosing. Subsequently, this subject was found to have high levels of a novel pre-existing antibody specific to the extended C-terminus of GSK2862277. Despite the reduced binding of GSK2862277 to pre-existing HAVH autoantibodies, adverse effects associated with the presence of a novel pre-existing antibody response specific to the modified dAb framework were identified and highlight the challenge of developing biological antagonists to this class of receptor.

  16. Amino terminal sequence of heavy and light chains from ratfish immunoglobulin.

    Science.gov (United States)

    De Ioannes, A E; Aguila, H L

    1989-01-01

    The ratfish, Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (Mr 960,000), composed of heavy (Mr 71,000), light (Mr 22,500), and J (Mr 16,000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark, Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.

  17. 甲型H1N1流感病毒血凝素单克隆抗体轻链和重链可变区基因的巢式PCR扩增及序列分析%Nested PCR Amplifying and DNA Sequence Analysis of Light and Heavy Chain Variable Region Gene of Monoclonal Antibody Against H1N1 Influenza Virus Hemagglutinin

    Institute of Scientific and Technical Information of China (English)

    李慧瑾; 胡军; 李研; 孙晶莹; 高锦伟; 赵向绒; 封青; 谭天天; 胡巧侠; 李元

    2013-01-01

    Objective: To clone and analysis light and heavy chain genes of monoclonal antibody against H1N1 influenza virus hemagglutinin by Nested PCR, then construct a common method for cloning light and heavy chain variable region genes of mouse anti-human H1N1 influenza virus hemagglutinin monoclonal antibody. Methods:We designed 22 pairs of primers for amplifying Igκ light and heavy chain variable region gene, and cloned light and heavy chain variable region genes of 6 mouse anti-human H1N1 influenza virus hemagglutinin monoclonal anti⁃bodies. Cloning and subsequent sequences analysis of immunoglobulin gene were performed, sequence alignment with mouse immunoglobulin in NCBI. Results: The nucleotide and corresponding amino acid sequences were in line with characteristics of mouse immunoglobulin variable region. Conclusion: In this study, we cloned 6 mouse antibody variable region genes, and found a common method for cloning immunoglobulin light and heavy chain variable region genes. It provides a basis for late amplifying monoclonal antibody variable region, and experimental data for analysis of H1N1 influenza virus hemagglutinin and antibody binding sites.%  目的:采用巢式PCR对甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链基因进行扩增,对获得的基因进行序列分析,并找出克隆鼠Igκ轻链和重链可变区基因的通用方法。方法:设计22对扩增鼠Igκ轻链可变区和重链可变区基因的引物,对6株鼠抗人甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链可变区基因进行克隆并测序,与NCBI公布的鼠免疫球蛋白序列比对分析。结果:巢式PCR方法可以有效避免单克隆抗体克隆过程的假基因,并且得到的单克隆抗体的氨基酸序列均符合鼠免疫球蛋白可变区特征。结论:建立了克隆鼠免疫球蛋白轻链和重链可变区基因的通用方法,为后期克隆鼠源性单克隆抗体的可变区基因提供了

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

    Science.gov (United States)

    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.

  19. Immunoglobulin heavy chain/light chain pairs (HLC, Hevylite™) assays for diagnosing and monitoring monoclonal gammopathies.

    Science.gov (United States)

    Kraj, Maria

    2014-01-01

    Immunofixation (IFE) is a standard method for detecting monoclonal immunoglobulins and characterizing its isotype. Recently clonality can also be determined by using immunoglobulin (Ig) heavy chain/light chain immunoassays - HLC, HevyliteTM. HLC separately measures in pairs light chain types of each intact Ig class generating ratios of monoclonal Ig/uninvolved polyclonal Ig concentrations. Studies have shown that HLC and IFE are complementary methods. HLC assays quantify monoclonal proteins and identify monoclonality. It is possible to predict prognosis in multiple myeloma and to monitor response to treatment using HLC ratio. HLC ratio may serve as a parameter for myeloma induced immunoparesis and serve as a new marker for validating remission depth and relapse probabilities.

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

    Science.gov (United States)

    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 (V(H )and V(L)) 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 V(H) and V(L) 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.

  1. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

    Science.gov (United States)

    Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

    2015-12-23

    Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

  2. Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

    Directory of Open Access Journals (Sweden)

    Anabelle S. Cornachione

    2014-07-01

    Full Text Available Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

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

    Science.gov (United States)

    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.

  4. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    Science.gov (United States)

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property.

  5. Evolutionary conservation of heavy chain protein transfer between glycosaminoglycans.

    Science.gov (United States)

    Sanggaard, Kristian W; Hansen, Lone; Scavenius, Carsten; Wisniewski, Hans-Georg; Kristensen, Torsten; Thøgersen, Ida B; Enghild, Jan J

    2010-04-01

    The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HC*TSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2*bikunin. In addition, Gg pre-alpha-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein-glycosaminoglycan-protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2*bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-alpha-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of

  6. Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Imanaka Tadayuki

    2002-09-01

    Full Text Available Abstract Background Hep27 monoclonal (Hep27 Mab is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102. We attempted to produce a single-chain fragment (scFv, a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques. Results The sequences encoding the variable regions of heavy (VH and light (VL chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser3 and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa. Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab. Conclusion This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.

  7. Ubiquitin Chain Editing Revealed By Polyubiquitin Linkage-Specific Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Newton, K.; Matsumoto, M.L.; Wertz, I.E.; Kirkpatrick, D.S.; Lill, J.R.; Tan, J.; Dugger, D.; Gordon, N.; Sidhu, S.S.; Fellouse, F.A.; Komuves, L.; French, D.M.; Ferrando, R.E.; Lam, C.; Compaan, D.; Yu, C.; Bosanac, I.; Hymowitz, S.G.; Kelley, R.F.; Dixit, V.M.

    2009-05-22

    Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-{kappa}B activation, and IRAK1, which participates in signaling by interleukin-1{beta} and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  9. Expression of muscle-specific myosin heavy chain and myosin light chain 1 in the electric tissue of Electrophorus electricus (L.) in comparison with other vertebrate species.

    Science.gov (United States)

    Ayres Sá, L; Menezes, M A; dos Santos Mermelstein, C

    2001-08-01

    Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus. Copyright 2001 Wiley-Liss, Inc.

  10. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.

    Science.gov (United States)

    Dobhal, S; Chaudhary, V K; Singh, A; Pandey, D; Kumar, A; Agrawal, S

    2013-12-01

    Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

  11. Neurofilament heavy chain expression and neuroplasticity in rat auditory cortex after unilateral and bilateral deafness.

    Science.gov (United States)

    Park, Min-Hyun; Jang, Jeong Hun; Song, Jae-Jin; Lee, Ho Sun; Oh, Seung Ha

    2016-09-01

    Deafness induces many plastic changes in the auditory neural system. For instance, dendritic changes cause synaptic changes in neural cells. SMI-32, a monoclonal antibody reveals auditory areas and recognizes non-phosphorylated epitopes on medium- and high-molecular-weight subunits of neurofilament proteins in cortical pyramidal neuron dendrites. We investigated SMI-32-immunoreactive (-ir) protein levels in the auditory cortices of rats with induced unilateral and bilateral deafness. Adult male Sprague-Dawley rats were divided into unilateral deafness (UD), bilateral deafness (BD), and control groups. Deafness was induced by cochlear ablation. All rats were sacrificed, and the auditory cortices were harvested for real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analyses at 2, 4, 6, and 12 weeks after deafness was induced. Immunohistochemical staining was performed to evaluate the location of SMI-32-ir neurons. Neurofilament heavy chain (NEFH) mRNA expression and SMI-32-ir protein levels were increased in the BD group. In particular, SMI-32-ir protein levels increased significantly 6 and 12 weeks after deafness was induced. In contrast, no significant changes in protein level were detected in the right or left auditory cortices at any time point in the UD group. NEFH mRNA level decreased at 4 weeks after deafness was induced in the UD group, but recovered thereafter. Taken together, BD induced plastic changes in the auditory cortex, whereas UD did not affect the auditory neural system sufficiently to show plastic changes, as measured by neurofilament protein level.

  12. [Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.].

    Science.gov (United States)

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2009-12-25

    Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, Pintein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved

  13. [Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli].

    Science.gov (United States)

    Zhu, Fuxiang; Liu, Zelong; Qu, Huige; Xin, Xiaolin; Dong, Hongxin; Liu, Xiangqin

    2009-07-01

    We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.

  14. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  15. Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

    Science.gov (United States)

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

    2016-10-01

    IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

  16. Construction and Analysis of Three-dimensional Graphic Model of Single-chain Fv Derived from an Anti-human Placental Acidic Isoferritin Monoclonal Antibody by Computer

    Institute of Scientific and Technical Information of China (English)

    ZHOU Chun; SHEN Guanxin; ZHU Huifen; YANG Jing; ZHANG Yue; FENG Jiannan; SHEN Beifen

    2000-01-01

    A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

  17. Production and characterization of a single-chain antibody of anti-CD3%抗CD3单链抗体基因克隆表达及生物活性鉴定

    Institute of Scientific and Technical Information of China (English)

    陈秀芹; 阎锡蕴

    2003-01-01

    The genes encoding antibody heavy and light chain variable regions(VH and VL)were cloned by RT-PCR from OKT3 hybridoma cells,which produced anti-CD3 moleclonal antibody.The VH and VL genes were fused and become a single chain Fv(scFv).The scFv gene was cloned into pCANTAB5E vector and expressed on bacterial phage surface.By three panning rounds,we have obtained two single-chain antibodys that specific for CD3.The antiCD3 scFv wil be a reagent fox diagnosis and therapy of immuno-disorder.

  18. Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching.

    Science.gov (United States)

    Matheson, Louise S; Osborn, Michael J; Smith, Jennifer A; Corcos, Daniel; Hamon, Maureen; Chaouaf, Rima; Coadwell, John; Morgan, Geoff; Oxley, David; Brüggemann, Marianne

    2009-08-01

    Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L(-/-) with silenced kappa and lambda loci) despite a block in B cell development. In murine H-chain IgG, the first Cgamma exon, C(H)1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L(-/-) mice generate a novel class of H-chain Ig with covalently linked alpha chains, not identified in any other healthy mammal. Surprisingly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum and sometimes in milk and saliva. Surface IgA without L-chain is expressed in B220(+) spleen cells, which exhibited a novel B cell receptor, suggesting that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP association seems to be prevented as only alpha chains lacking C(H)1 are released from the cell. This appears to be accomplished by imprecise class-switch recombination (CSR) from Smu into the alpha constant region, which removes all or part of the Calpha1 exon at the genomic level.

  19. The role of CTCF binding sites in the 3' immunoglobulin heavy chain regulatory region.

    Science.gov (United States)

    Birshtein, Barbara K

    2012-01-01

    The immunoglobulin heavy chain locus undergoes a series of DNA rearrangements and modifications to achieve the construction and expression of individual antibody heavy chain genes in B cells. These events affect variable regions, through VDJ joining and subsequent somatic hypermutation, and constant regions through class switch recombination (CSR). Levels of IgH expression are also regulated during B cell development, resulting in high levels of secreted antibodies from fully differentiated plasma cells. Regulation of these events has been attributed primarily to two cis-elements that work from long distances on their target sequences, i.e., an ∼1 kb intronic enhancer, Eμ, located between the V region segments and the most 5' constant region gene, Cμ; and an ∼40 kb 3' regulatory region (3' RR) that is located downstream of the most 3' C(H) gene, Cα. The 3' RR is a candidate for an "end" of B cell-specific regulation of the Igh locus. The 3' RR contains several B cell-specific enhancers associated with DNase I hypersensitive sites (hs1-4), which are essential for CSR and for high levels of IgH expression in plasma cells. Downstream of this enhancer-containing region is a region of high-density CTCF binding sites, which extends through hs5, 6, and 7 and further downstream. CTCF, with its enhancer-blocking activities, has been associated with all mammalian insulators and implicated in multiple chromosomal interactions. Here we address the 3' RR CTCF-binding region as a potential insulator of the Igh locus, an independent regulatory element and a predicted modulator of the activity of 3' RR enhancers. Using chromosome conformation capture technology, chromatin immunoprecipitation, and genetic approaches, we have found that the 3' RR with its CTCF-binding region interacts with target sequences in the V(H), Eμ, and C(H) regions through DNA looping as regulated by protein binding. This region impacts on B cell-specific Igh processes at different stages of B cell

  20. The role of CTCF binding sites in the 3’ immunoglobulin heavy chain regulatory region

    Directory of Open Access Journals (Sweden)

    Barbara K Birshtein

    2012-11-01

    Full Text Available The immunoglobulin heavy chain locus undergoes a series of DNA rearrangements and modifications to achieve the construction and expression of individual antibody heavy chain genes in B cells. These events affect variable regions, through VDJ joining and subsequent somatic hypermutation, and constant regions through class switch recombination. Levels of IgH expression are also regulated during B cell development, resulting in high levels of secreted antibodies from fully-differentiated plasma cells. Regulation of these events has been attributed primarily to two cis-elements that work from long distances on their target sequences, i.e., an ~1 kb intronic enhancer, Eμ, located between the V region segments and the most 5′ constant region gene, Cμ; and an ~40 kb 3′ regulatory region (3′ RR that is located downstream of the most 3′ CH gene, Cα. The 3′ RR is a candidate for an end of B cell-specific regulation of the Igh locus. The 3′ RR contains several B cell-specific enhancers associated with DNase I hypersensitive sites (hs1-4, which are essential for class switch recombination and for high levels of IgH expression in plasma cells. Downstream of this enhancer-containing region is a region of high-density CTCF binding sites, which extends through hs5, 6, and 7 and further downstream. CTCF, with its enhancer-blocking activities, has been associated with all mammalian insulators and implicated in multiple chromosomal interactions. Here we address the 3′ RR CTCF-binding region as a potential insulator of the Igh locus, an independent regulatory element and a predicted modulator of the activity of 3’ RR enhancers. Using chromosome conformation capture technology, chromatin immunoprecipitation and genetic approaches, we have found that the 3’ RR with its CTCF binding region interacts with target sequences in the VH, Eμ and CH regions through DNA looping as regulated by protein binding. This region impacts on B cell-specific Igh

  1. Uses of monoclonial antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2015-06-23

    This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

  2. Antibody elbow angles are influenced by their light chain class

    Energy Technology Data Exchange (ETDEWEB)

    Stanfield, R; Zemla, A; Wilson, I; Rupp, B

    2006-01-12

    We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa-chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195{sup o}) elbow angles. This apparent hyperflexibility of lambda-chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is also described.

  3. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    Full Text Available 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.

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

    Science.gov (United States)

    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...

  5. Myosin heavy chain composition of tiger (Panthera tigris) and cheetah (Acinonyx jubatus) hindlimb muscles.

    Science.gov (United States)

    Hyatt, Jon-Philippe K; Roy, Roland R; Rugg, Stuart; Talmadge, Robert J

    2010-01-01

    Felids have a wide range of locomotor activity patterns and maximal running speeds, including the very fast cheetah (Acinonyx jubatas), the roaming tiger (Panthera tigris), and the relatively sedentary domestic cat (Felis catus). As previous studies have suggested a relationship between the amount and type of activity and the myosin heavy chain (MHC) isoform composition of a muscle, we assessed the MHC isoform composition of selected hindlimb muscles from these three felid species with differing activity regimens. Using gel electrophoresis, western blotting, histochemistry, and immunohistochemistry with MHC isoform-specific antibodies, we compared the MHC composition in the tibialis anterior, medial gastrocnemius (MG), plantaris (Plt), and soleus muscles of the tiger, cheetah, and domestic cat. The soleus muscle was absent in the cheetah. At least one slow (type I) and three fast (types IIa, IIx, and IIb) MHC isoforms were present in the muscles of each felid. The tiger had a high combined percentage of the characteristically slower isoforms (MHCs I and IIa) in the MG (62%) and the Plt (86%), whereas these percentages were relatively low in the MG (44%) and Plt (55%) of the cheetah. In general, the MHC isoform characteristics of the hindlimb muscles matched the daily activity patterns of these felids: the tiger has daily demands for covering long distances, whereas the cheetah has requirements for speed and power. (c) 2009 Wiley-Liss, Inc.

  6. PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    何凤田; 聂勇战; 陈宝军; 乔太东; 韩者艺; 樊代明

    2002-01-01

    Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti -Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL)genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13KO7 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. Thetypesoftheanti-IdScFvdisplayedontheselectedphagecloneswerepreliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γtype anti-Id ScFv.Conclsion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

  7. Production of a human single-chain variable fragment antibody against esophageal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ming-Yan Xu; Xiao-Hu Xu; Geng-Zhen Chen; Xiao-Ling Deng; Jonathan Li; Xiao-Jun Yu; Mei-Zhen Chen

    2004-01-01

    AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFv antibody against esophageal cancer.METHODS: Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed. esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coli HB2151 which was transfected with the positive phage clone, then purified by affinity chromatography.Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.RESULTS: The size of scFv gene library was approximately 9×106 clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells, 25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Eca109 cells, but not to HeLa and NHEEC cells. Variable heavy (VH) gene from one of the positive clones was shown to be derived from the γ chain subgroup Ⅳ of immunoglobulin, and variable light (VL) gene from the κchain subgroup I of immunoglobulin.CONCLUSION: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.

  8. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

  9. Random Mutagenesis by Error-Prone Polymerase Chain Reaction Using a Heavy Water Solvent.

    Science.gov (United States)

    Minamoto, Toshifumi

    2017-01-01

    Heavy water is a form of water that contains a heavier isotope of hydrogen ((2)H, also known as deuterium, D) or oxygen ((17)O or (18)O). When using heavy water as a solvent, error-prone polymerase chain reaction (epPCR) can induce random mutations independent of the polymerase used or the composition of the PCR reaction mixture. This relatively new method can easily be combined with the existing epPCR methods to increase the rate of mutations.

  10. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    OpenAIRE

    Oliinyk O. S.; Kaberniuk A. A.; Kolibo D. V.; Komisarenko S. V.

    2014-01-01

    Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv) antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized) human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, an...

  11. [Advances in the study of natural small molecular antibody].

    Science.gov (United States)

    Zhu, Lei; Zhang, Da-peng

    2012-10-01

    Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called Vnar fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC.

  12. Heavy hadron spectra from spin chains and strings

    CERN Document Server

    Cotrone, A L; Pons, J M; Talavera, P

    2007-01-01

    We study the spectrum of hadronic states made up of very massive complex scalar fields in a confining gauge theory admitting a supergravity dual background. We show that for a sub-sector of operators dual to certain spinning strings, the mass spectrum exhibits an integrable structure equal to the Heisenberg spin chain, up to an overall factor. This result is compared with the corresponding string prediction.

  13. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine.

  14. Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in Sf9 insect cell.

    Science.gov (United States)

    Sakamoto, Seiichi; Taura, Futoshi; Tsuchihashi, Ryota; Putalun, Waraporn; Kinjo, Junei; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-12-01

    Plumbagin (PL; 5-hydroxy-2-methyl-1, 4-naphthoquinone) is an important secondary metabolite, mainly produced in the Plumbago zeylanica L. (Plumbaginaceae). A single-chain variable fragment (scFv) antibody, fusion of the variable regions of the heavy chain and light chain of immunoglobulin against PL (PL-scFv) was expressed by Bac-to-Bac Baculovirus Expression System using Spodoptera frugiperda (Sf9) insect cells and characterized to investigate potential use of PL-scFv as a tool for plant immunomodulation. Functional PL-scFv expressed in the Sf9 insect cells were purified using cation exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). The yields of the purified PL-scFv in the culture supernatant and Sf9 insect cells were 2.0 mg and 5.2 mg per 1 liter of Sf9 culture medium, respectively. Recombinant purified PL-scFv was then characterized by the indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivity and sensitivity of PL-scFv expressed in Sf9 insect cells were compared with PL-scFv expressed in Escherichia coli and its parental anti-plumbagin monoclonal antibody (MAb 3A3) secreted from hybridoma cells. Intriguingly, the specificity of the PL-scFv expressed in Sf9 insect cells was found to be different from that expressed in E. coli and parental MAb 3A3, although the detectable level (0.2-25 μg/mL) was the same in ELISA using each antibody. Even more interestingly, the characteristics of PL-scFv, which have wide cross-reactivity against 1,4-napththoquinone, suggest its potential use as a tool for plant immunomodulation not only for breeding Plumbaginacea family containing PL but also for breeding other medicinal plants containing bioactive naphthoquinones.

  15. Construction of a human functional single-chain variable fragment (scFv) antibody recognizing the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Wajanarogana, Sumet; Prasomrothanakul, Teerawat; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2006-04-01

    Falciparum malaria is one of the most deadly and profound human health problems around the tropical world. Antimalarial drugs are now considered to be a powerful treatment; however, there are drugs currently being used that are resistant to Plasmodium falciparum parasites spreading in different parts of the world. Although the protective immune response against intraerythrocytic stages of the falciparum malaria parasite is still not fully understood, immune antibodies have been shown to be associated with reduced parasite prevalence. Therefore antibodies of the right specificity present in adequate concentrations and affinity are reasonably effective in providing protection. In the present study, VH (variable domain of heavy chain) and VL (variable domain of light chain) were isolated from human blood lymphocytes of P. falciparum in one person who had high serum titre to RESA (ring-infected erythrocyte surface antigen). Equal amounts of VH and VL were assembled together with universal linker (G4S)3 to generate scFvs (single-chain variable fragments). The scFv antibodies were expressed with a phage system for the selection process. Exclusively, an expressed scFv against asynchronous culture of P. falciparum-infected erythrocytes was selected and characterized. Sequence analysis of selected scFv revealed that this clone could be classified into a VH family-derived germline gene (VH1) and Vkappa family segment (Vkappa1). Using an indirect immunofluorescence assay, we could show that soluble expressed scFv reacted with falciparum-infected erythrocytes. The results encourage the further study of scFvs for development as a potential immunotherapeutic agent.

  16. Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57.

    Science.gov (United States)

    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.

  17. The 4C5 cell-impermeable anti-HSP90 antibody with anti-cancer activity, is composed of a single light chain dimer.

    Science.gov (United States)

    Sidera, Katerina; El Hamidieh, Avraam; Mamalaki, Avgi; Patsavoudi, Evangelia

    2011-01-01

    MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics.

  18. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    Science.gov (United States)

    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.

  19. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment.

    Science.gov (United States)

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-12-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies.

  20. The motion planning problem and exponential stabilization of a heavy chain. Part II

    Directory of Open Access Journals (Sweden)

    Piotr Grabowski

    2008-01-01

    Full Text Available This is the second part of paper [P. Grabowski, The motion planning problem and exponential stabilization of a heavy chain. Part I, to appear in International Journal of Control], where a model of a heavy chain system with a punctual load (tip mass in the form of a system of partial differential equations was interpreted as an abstract semigroup system and then analysed on a Hilbert state space. In particular, in [P. Grabowski, The motion planning problem and exponential stabilization of a heavy chain. Part I, to appear in International Journal of Control] we have formulated the problem of exponential stabilizability of a heavy chain in a given position. It was also shown that the exponential stability can be achieved by applying a stabilizer of the colocated-type. The proof used the method of Lyapunov functionals. In the present paper, we give other two proofs of the exponential stability, which provides an additional intrinsic insight into the exponential stabilizability mechanism. The first proof makes use of some spectral properties of the system. In the second proof, we employ some relationships between exponential stability and exact observability.

  1. Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene

    DEFF Research Database (Denmark)

    Amiquel, P; Kristensen, Torsten; D'Eustachio, P;

    1990-01-01

    p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been deter...

  2. Myosin heavy chain expression can vary over the length of jaw and leg muscles

    NARCIS (Netherlands)

    Korfage, J.A.M.; Kwee, K.E.; Everts, V.; Langenbach, G.E.J.

    2016-01-01

    Muscle fiber type classification can be determined by its myosin heavy chain (MyHC) composition based on a few consecutive sections. It is generally assumed that the MyHC expression of a muscle fiber is the same over its length since neural stimulation and systemic influences are supposed to be the

  3. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lin-Xu [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Mellon, Michael [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Bowder, Dane [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Quinn, Meghan [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Shea, Danielle; Wood, Charles [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Xiang, Shi-Hua, E-mail: sxiang2@unl.edu [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States)

    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.

  5. Cynomolgus macaque (Macaca fascicularis) immunoglobulin heavy chain locus description.

    Science.gov (United States)

    Yu, Guo-Yun; Mate, Suzanne; Garcia, Karla; Ward, Michael D; Brueggemann, Ernst; Hall, Matthew; Kenny, Tara; Sanchez-Lockhart, Mariano; Lefranc, Marie-Paule; Palacios, Gustavo

    2016-07-01

    Cynomolgus macaques (Macaca fascicularis) have become an important animal model for biomedical research. In particular, it is the animal model of choice for the development of vaccine candidates associated with emerging dangerous pathogens. Despite their increasing importance as animal models, the cynomolgus macaque genome is not fully characterized, hindering molecular studies for this model. More importantly, the lack of knowledge about the immunoglobulin (IG) locus organization directly impacts the analysis of the humoral response in cynomolgus macaques. Recent advances in next generation sequencing (NGS) technologies to analyze IG repertoires open the opportunity to deeply characterize the humoral immune response. However, the IG locus organization for the animal is required to completely dissect IG repertoires. Here, we describe the localization and organization of the rearranging IG heavy (IGH) genes on chromosome 7 of the cynomolgus macaque draft genome. Our annotation comprises 108 functional genes which include 63 variable (IGHV), 38 diversity (IGHD), and 7 joining (IGHJ) genes. For validation, we provide RNA transcript data for most of the IGHV genes and all of the annotated IGHJ genes, as well as proteomic data to validate IGH constant genes. The description and annotation of the rearranging IGH genes for the cynomolgus macaques will significantly facilitate scientific research. This is particularly relevant to dissect the immune response during vaccination or infection with dangerous pathogens such as Ebola, Marburg and other emerging pathogens where non-human primate models play a significant role for countermeasure development.

  6. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    Science.gov (United States)

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  7. Heavy chain transfer by tumor necrosis factor-stimulated gene 6 to the bikunin proteoglycan.

    Science.gov (United States)

    Lamkin, Elliott; Cheng, Georgiana; Calabro, Anthony; Hascall, Vincent C; Joo, Eun Ji; Li, Lingyun; Linhardt, Robert J; Lauer, Mark E

    2015-02-20

    We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells.

    Science.gov (United States)

    Ren, Weicheng; Grimsholm, Ola; Bernardi, Angelina I; Höök, Nina; Stern, Anna; Cavallini, Nicola; Mårtensson, Inga-Lill

    2015-04-01

    Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

  9. Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish, Raja erinacea.

    Science.gov (United States)

    Harding, F A; Amemiya, C T; Litman, R T; Cohen, N; Litman, G W

    1990-11-11

    Immunoglobulin heavy chain genes in Raja erinacea (little skate) are organized in clusters consisting of VH, DH, JH segments and CH exons (1). An immunoglobulin heavy chain mu-like isotype that exhibits 61-91% nucleotide sequence identity in coding segments to the Heterodontus francisci (horned shark) mu-type immunoglobulin is described. The overall length of the mu-type clusters is approximately 16 kb; transmembrane exons (TM1 and TM2) are located 3 to CH exon 4 (CH4). In three of four TM-containing genomic clones, a significant deletion is present in TM1. A second isotype of Raja immunoglobulin heavy chain genes has been detected by screening a spleen cDNA library with homologous Raja VH- and CH1-specific probes complementing the respective regions of the mu-like isotype. Weak hybridization with VH-specific probes and no discernable hybridization with C mu-specific probes were considered presumptive evidence for a second immunoglobulin isotype that nominally is designated as X-type. The Vx region of the X-type cDNA is approximately 60% identical at the nucleotide (nt) level to other Raja VH segments and thus represents a second VH family. Putative Dx and Jx sequences also have been identified. The constant region of the X-type immunoglobulin heavy chain gene consists of two characteristic immunoglobulin domains and a cysteine-rich carboxy terminal segment that are only partially homologous with the mu-like isotype. Genomic Southern blotting indicates that the V and C segments of both immunoglobulin heavy chain isotypes are encoded by complex multigene families. Vx- and different Cx-specific probes hybridize to different length transcripts in northern blot analyses of Raja spleen RNA suggesting that the regulation of expression of the X-type genes may involve differential RNA processing.

  10. Improved expression of single-chain antibodies in Ustilago maydis.

    Science.gov (United States)

    Sarkari, Parveen; Reindl, Michèle; Stock, Janpeter; Müller, Olaf; Kahmann, Regine; Feldbrügge, Michael; Schipper, Kerstin

    2014-12-10

    To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.

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

    Science.gov (United States)

    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.

  12. Guinea pig line 10 hepatocarcinoma model: characterization of monoclonal antibody and in vivo effect of unconjugated antibody and antibody conjugated to diphtheria toxin A chain.

    Science.gov (United States)

    Bernhard, M I; Foon, K A; Oeltmann, T N; Key, M E; Hwang, K M; Clarke, G C; Christensen, W L; Hoyer, L C; Hanna, M G; Oldham, R K

    1983-09-01

    Monoclonal antibodies were raised against the guinea pig line 10 (L10) hepatocarcinoma, and an IgG1-producing hybridoma (D3) was selected for further study. D3 is a true monoclonal antibody as demonstrated by two-dimensional gel electrophoresis. Radioimmunoassays on live cells revealed no cross-reactivity with normal tissues or with the line 1 hepatocarcinoma which was used as a control. Membrane immunofluorescence assays demonstrated similar specificity. Immunoperoxidase staining of cryostat sections of tumor and normal tissues of both adult animals and fetuses showed that the D3 monoclonal antibody reacted primarily with the L10 tumor, but some cross-reactivity with smooth muscle, placenta, fetal skeletal muscle, and fetal liver was also demonstrated. Radioimmunoprecipitation of detergent extracts of iodinated L10 cells showed that the antigen is present on the cell surface as a dimer of Mr 290,000 (unit size, Mr 148,000). Therapy studies with unconjugated D3 antibody demonstrated a minor dose-dependent effect on tumor growth. D3 antibody conjugated to the A chain of diphtheria toxin (10(-7) M) was cytotoxic to 100% of L10 cells in vitro. Animals treated with a single 1-mg i.v. injection of this immunoconjugate on Day 7 following the intradermal injection of 10(5) tumor cells demonstrated a highly significant inhibition of tumor growth compared to control animals and those treated with unconjugated antibody.

  13. Antibodies against major histocompatibility complex class I-related chain A in transplant recipients

    Institute of Scientific and Technical Information of China (English)

    Yizhou Zou; Peter Stastny

    2011-01-01

    Objective To review the role of polymorphism of major histocompatibility complex class I-related chain A (MICA) gene and antibodies against MICA antigens in transplant immunology.Data sources The data used in this review were mainly from our own results and from the relevant English language literatures published from 1999 to 2010. Some data presented in this review are in press.Study selection Articles regarding MICA gene discovery and pioneering finding of antibodies against MICA antigen and allograft rejection were selected. This review chronicles the development of our understanding of the role that MICA antigens and antibodies may play in organ transplantation.Results Polymorphic glycoprotein MICA antigens were detected on freshly isolated human umbilical cord endothelial cells, but not on peripheral lymphocytes. Antibodies were found and typing of recipients and donors by sequencing the MICA alleles has established that de novo antibodies produced in kidney transplant recipients are directed at mismatched MICA epitopes and are associated with acute rejection and chronic transplant failure. The specificity of antibodies against the epitopes of MICA antigens were well characterized by donor MICA typing, single antigen array testing with antibody absorption and elution. Acute graft-versus-host disease was observed in stem-cell recipients who were mismatched for MICA.Conclusions Immunization against mismatched MICA epitopes encountered in donor organs after transplantation may result in antibodies against MICA alleles. Testing for MICA donor-specific antibodies (DSA) which are associated with early failure of kidney transplants may be helpful for identifying some of the targets of antibodies against antigens other than the human leukocyte antigen (HLA) and for improving transplantation outcome.

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

    Science.gov (United States)

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

    2014-12-01

    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.

  15. Effects of interlinker sequences on the biological properties of bispecific single-chain antibodies

    Institute of Scientific and Technical Information of China (English)

    FANG Min; JIANG Xin; YANG Zhi; YIN Changcheng; LI Hua; ZHAO Rui; ZHANG Zhong; LIN Qing; HUANG Hualiang

    2003-01-01

    Single-chain bispecific antibody (scBsAb) is one of the promising genetic engineering antibody formats for clinical application. But the effects of interlinker sequences on the biological properties of bispecific single-chain antibodies have not been studied in detail. Three interlinker sequences were designed and synthesized, and denominated as Fc, HSA, 205C′, respectively. Universal vectors with these different interlinker sequences for scBsAb expression in E. coli were constructed. A model scBsAb based on a reshaped single-chain antibody (scFv) against human CD3 and a scFv directed against human ovarian carcinoma were generated and expressed in E. coli. The results of SDS-PAGE and Western blot showed that the different interlinker sequences did not affect the expression levelof scBsAb. However, as demonstrated by ELISA and pharmacokinetics studies performed in mice, scBsAbs with different interlinker sequences had difference in the antigen-binding activities and terminal half-life time (T1/2β) in vivo, the interlinker HSA could remarkably prolong the retention time of scBsAb in blood. These results indicated that the peptide sequence of interlinker could affect important biological properties of scBsAb, such as antigen-binding properties and stability in vivo. So, selection of an appropriate interlinker sequence is very important for scBsAb construction. Optimal interlinker can bring scBsAb biologicalproperties more suitable for clinical application.

  16. Mouse Cmu heavy chain immunoglobulin gene segment contains three intervening sequences separating domains.

    Science.gov (United States)

    Calame, K; Rogers, J; Early, P; Davis, M; Livant, D; Wall, R; Hood, L

    1980-04-03

    The IgM molecule is composed of subunits made up of two light chain and two heavy chain (mu) polypeptides. The mu chain is encoded by several gene segments--variable (V), joining (J) and constant (Cmu). The Cmu gene segment is of particular interest for several reasons. First, the mu chain must exist in two very different environments--as an integral membrane protein in receptor IgM molecules (micrometer) and as soluble serum protein in IgM molecules into the blood (mus). Second, the Cmu region in mus is composed of four homology units or domains (Cmu1, Cmu2, Cmu3 and Cmu4) of approximately 110 amino acid residues plus a C-terminal tail of 19 residues. We asked two questions concerning the organisation of the Cmu gene segment. (1) Are the homology units separated by intervening DNA sequences as has been reported for alpha (ref. 5), gamma 1 (ref. 6) and gamma 2b (ref. 7) heavy chain genes? (2) Is the C-terminal tail separated from the Cmu4 domain by an intervening DNA sequence? If so, DNA rearrangements or RNA splicing could generate hydrophilic and hydrophobic C-terminal tails for the mus and micrometer polypeptides, respectively. We demonstrate here that intervening DNA sequences separate each of the four coding regions for Cmu domains, and that the coding regions for the Cmu4 domains and the C-terminal tail are directly contiguous.

  17. Co-deposition of amyloidogenic immunoglobulin light and heavy chains in localized pulmonary amyloidosis.

    Science.gov (United States)

    Kaplan, Batia; Martin, Brian M; Boykov, Olga; Gal, Rivka; Pras, Mordechai; Shechtman, Itzhak; Saute, Milton; Kramer, Mordechai R

    2005-10-01

    Localized pulmonary amyloidosis is a rare condition whose pathogenesis is insufficiently understood. In the present study, we report a case of localized pulmonary amyloidosis associated with lung-restricted lymphoplasmacytoid lymphoma, monoclonal for immunoglobulin (Ig) G lambda (lambda). Biochemical microtechniques have been applied for extraction, purification, and characterization of amyloid proteins. Surprisingly, chemical analysis of these proteins revealed a not-previously-described case of combined deposits containing Ig fragments of gamma heavy chain (variable domain) and lambda light chain (constant domain). In view of the absence of circulating monoclonal Ig, this case supports the hypothesis that localized amyloid is formed by local plasmacytoid cells.

  18. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    Science.gov (United States)

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.

  19. Biotransfer of heavy metals along a soil-plant-insect-chicken food chain:Field study

    Institute of Scientific and Technical Information of China (English)

    ZHUANG Ping; ZOU Huiling; SHU Wensheng

    2009-01-01

    The study investigated the accumulation and transfer of Pb,Zn,Cu,and Cd along a soil-plant-insect-chicken food chain at contaminated sites.The study site nearing the Pb/Zn mine had been contaminated by heavy metals severely.Cadmium and Pb concentrations steadily declined with increasing trophic level (p0.05).The concentrations of heavy metals were the highest in chicken muscle,with lower values in liver and blood.Bioaccumulation of Pb was observed in the chicken livers.The eliminations of Pb,Zn,Cu,and Cd via insect and chicken feces avoid metal bioaccumulation in insect and chicken body.These results suggest that the accumulation of heavy metals in specific animal organ of tissues could not be neglected,although transfer of metals to chicken from plant and insect is limited.

  20. Complete structure and organization of immunoglobulin heavy chain constant region genes in a phylogenetically primitive vertebrate.

    OpenAIRE

    Kokubu, F; Hinds, K.; Litman, R.; Shamblott, M J; Litman, G. W.

    1988-01-01

    Immunoglobulin (Ig) gene organization in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, is unique. Homologous Ig heavy chain variable (VH) and constant region (CH) specific probes were used to screen a spleen cDNA library constructed in lambda gt11. Both secretory (SEC) and transmembrane (TM) cDNA clones were recovered; the latter were identified by a negative selection strategy. The complete sequence of the CH portion of a Heterodontus genomic DNA-lambda clon...

  1. Novel Mutation in the α-Myosin Heavy Chain Gene Is Associated With Sick Sinus Syndrome

    OpenAIRE

    Ishikawa, Taisuke; Jou, Chuanchau J.; Nogami, Akihiko; Kowase, Shinya; Arrington, Cammon B.; Barnett, Spencer M.; Harrell, Daniel T.; Arimura, Takuro; Tsuji, Yukiomi; Kimura, Akinori; Makita, Naomasa

    2015-01-01

    Recent genome-wide association studies have demonstrated an association between MYH6, the gene encoding α-myosin heavy chain (α-MHC), and sinus node function in the general population. Moreover, a rare MYH6 variant, R721W, predisposing susceptibility to sick sinus syndrome has been identified. However, the existence of disease-causing MYH6 mutations for familial sick sinus syndrome and their underlying mechanisms remain unknown. Methods and Results-We screened 9 genotype-negative probands wit...

  2. Complete structure and organization of immunoglobulin heavy chain constant region genes in a phylogenetically primitive vertebrate.

    Science.gov (United States)

    Kokubu, F; Hinds, K; Litman, R; Shamblott, M J; Litman, G W

    1988-07-01

    Immunoglobulin (Ig) gene organization in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, is unique. Homologous Ig heavy chain variable (VH) and constant region (CH) specific probes were used to screen a spleen cDNA library constructed in lambda gt11. Both secretory (SEC) and transmembrane (TM) cDNA clones were recovered; the latter were identified by a negative selection strategy. The complete sequence of the CH portion of a Heterodontus genomic DNA-lambda clone also was determined. The sequences of the individual CH genes differ from each other in all exons. When compared to mammalian prototypes, similarities in exon and intron organization as well as conservation of sequences involved with differential splicing of SEC and TM mRNA indicate that Heterodontus heavy chain genes are of the mu type, although intron lengths are uniformly longer in Heterodontus. Heterodontus genes are not associated, however, with the family of DNA sequences that have been implicated in heavy chain class switching in mammals. Spleen cDNA library screening and RNA blot analyses indicate that mRNAs encoding TM Ig are exceedingly rare. The relationship between this quantitative difference and the distribution of polyadenylation signal sequences suggests that regulation of Ig gene expression in Heterodontus may be highly dependent on position effects.

  3. Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea.

    Science.gov (United States)

    Harding, F A; Cohen, N; Litman, G W

    1990-02-25

    Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.

  4. Anti Zn antibodies: cross reactivity and competitive binding with heavy metals.

    Science.gov (United States)

    Sarada, N C; Thamaraiselvi, K; Vijayalakshmi, M A

    2008-01-15

    Monoclonal antibodies of IgM class, specific to IDA-Zn were used for evaluating their Zn(2+) binding efficiency in the presence of trace metal ions such as Cr(3+) Cr(6+), Cu(2+) and Cd(2+). In the present work, antibody raised against the hapten IDA-Zn(II) was pre-incubated with different metal ions and the binding capacity to the specific hapten was tested using ELISA and immobilized metal ion affinity chromatography (IMAC) techniques. IMAC was carried out with the free antibody and antibody pre-incubated with selected heavy metal ions using Sepharose IDA-Zn(2+) column and the same samples were tested using a hapten specific ELISA with non-protein hapten carrier. Different effects were observed after pre-incubation with metal ions. Cr(3+) exhibited synergistic binding where as antagonism was detected with Cd(2+). The synergistic effect observed with Cr(3+) suggests involvement of binding sites other than that of zinc and conformational changes that result from Cr(3+) binding. It is probable that, this binding event also increases the accessibility of the zinc binding sites on IgM. On the same lines, the antagonism observed with Cd(2+) could be attributed to structural changes resulting in reduced accessibility to zinc binding sites. In case of Cr(6+), no appreciable change in binding to IDA-Zn was observed while Cu(2+) showed competitive binding.

  5. Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia

    NARCIS (Netherlands)

    Derksen, P W; Langerak, A W; Kerkhof, E; Wolvers-Tettero, I L; Boor, P P; Mulder, A H; Vrints, L W; Coebergh, J W; van Krieken, J H; Schuuring, E; Kluin, P M; van Dongen, J J

    1999-01-01

    Several frequently applied polymerase chain reaction strategies for analysis of immunoglobulin heavy-chain gene rearrangements were compared by analyzing 70 B-cell lymphoproliferative disorders and 24 reactive lymphoid lesions. Southern blot analysis was used as the "gold standard" for clonality ass

  6. Construction of Large Human Single-chain Antibody Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A large human naive single chain antibody (scFv) library is constructed from 60 healthy donors via phage display technique. During the period, some methods are employed to optimize the diversity, such as multi donors, different annealing temperature, half-nest PCR, and assembly by two-way fusion PCR. In this stud y, 78 electroporations resulted in 1010 library, diversity of which is assayed by enzyme fingerprint. The efficiency and diversity are all better than other rese arches.

  7. Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

    Science.gov (United States)

    Wang, Yujun; Nakagaki, Masao

    2014-07-18

    The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

  8. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

    Science.gov (United States)

    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.

  9. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    Science.gov (United States)

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun

    2012-10-02

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  10. Construction and Expression of a Single Chain Antibody Mimicing Human Ovarian Cancer Antigen CA125

    Institute of Scientific and Technical Information of China (English)

    Aidong Li; Zheng Li; Yinghong Wang; Yongming Zhang; Jie Ma

    2006-01-01

    One concept for immune therapy of cancer involves induction of antigen mimic antibodies to trigger the immune response against tumor cells. Anti-idiotypic antibodies directed against the antigen-binding site of antibodies specific for tumor antigen may functionally and even structurally mimic antigen and induce anti-anti-idiotypic immune response. Monoclonal antibody WJ02 is one of such anti-idiotypic antibodies, which contains internal image of CA125. In order to improve the immunospecificity of mAb WJ02, we constructed a single chain of mAb WJ02 in Vl-linker-Vh orientation. The scFv-WJ02 could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein with a molecular weight of 30 kD retained the biological activity of mAb WJ02, which was proved by a direct binding assay and inhibition experiment. Our results indicated that the scFv-WJ02 could be used as a possible tool for idiotypic therapy against ovarian cancer, which might enhance the possibility of eliminating nonspecific responses induced by mAb WJ02.

  11. Somatic diversification in the heavy chain variable region genes expressed by human autoantibodies bearing a lupus-associated nephritogenic anti-DNA idiotype

    Energy Technology Data Exchange (ETDEWEB)

    Demaison, C.; Chastagner, P.; Theze, J.; Zouali, M. (Institut Pasteur, Paris (France))

    1994-01-18

    Monoclonal anti-DNA antibodies bearing a lupus nephritis-associated idiotype were derived from five patients with systemic lupus erythematosus (SLE). Genes encoding their heavy (H)-chain variable (V[sub H]) regions were cloned and sequenced. When compared with their closest V[sub h] germ-line gene relatives, these sequences exhibit a number of silent (S) and replacement (R) substitutions. The ratios of R/S mutations were much higher in the complementarity-determining regions (CDRs) of the antibodies than in the framework regions. Molecular amplification of genomic V[sub H] genes and Southern hybridization with somatic CDR2-specific oligonucleotide probes showed that the configuration of the V[sub H] genes corresponding to V[sub H] sequences in the nephritogenic antibodies is not present in the patient's own germ-line DNA, implying that the B-cell clones underwent somatic mutation in vivo. These findings, together with the characteristics of the diversity and junctional gene elements utilized to form the antibody, indicate that these autoantibodies have been driven through somatic selection processes reminiscent of those that govern antibody responses triggered by exogenous stimuli.

  12. Myosin Heavy Chain 2B isoform is expressed in specialized eye muscles but not in trunk and limb muscles of cattle

    Directory of Open Access Journals (Sweden)

    L Maccatrozzo

    2009-06-01

    Full Text Available Myosin heavy chain isoforms (MHC of adult skeletal muscles are codified by four genes named: slow, or type 1, and fast types 2A, 2X and 2B. The slow, 2A and 2X isoforms have been found expressed in all mammalian species studied so far whereas there is a large inter-species variability in the expression of MHC-2B. In this study histochemistry (m- ATPase, immunohistochemistry with the use of specific monoclonal antibodies and RT-PCR were combined together to assess whether the MHC-2B gene is expressed in bovine muscles. ATPase staining and RT-PCR experiments showed that three MHC isoforms (1, 2A, 2X were expressed in trunk and limb muscles. Slow or type 1 expression was confirmed using a specific antibody (BA-F8 whereas the detection of fast MHC isoforms were validate by means of BF-35 antibody although not by the SC-71 antibody. MHC-2B was absent in limb and trunk muscles, but was present in specialized eye muscles (rectus lateralis and retractor bulbi as consistently showed by RT-PCR and reactivity with a specific antibody (BF-F3. Interestingly, a cardiac isoform, MHC-a- cardiac was found to be expressed not only in extraocular muscles but also in masticatory muscles as masseter.

  13. Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Frank; Martiniuk; Seth; Pincus; Sybille; Müller; Heinz; Kohler; Kam-Meng; Tchou-Wong

    2010-01-01

    AIM:To evaluate the ability of anti-ricin A-chain antibodies,delivered intracellularly,to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide.RAW264.7 cells were incubatedwith these antibodies either before or after ricin exposure.The changes in cytotoxicity were estimated by MTT assay.Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS:Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies.Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. CONCLUSION:Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for postexposure treatment of ricin intoxication.

  14. Cloning,expression and characterization of a single-chain antibody PS-9 targeted to pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴; 田波; 李力; 袁玫

    1995-01-01

    Genes encoding single-chain antibodies have been first constructed,which consist of the heavyand light chain variable domains of antibody PS-9 joined together by a flexible peptide linker.The geneswere cloned into coat protein g3p genes of pCANTAB5 phagemids,and expressed as fusion proteins on thephage tips.Immunological assay demonstrated that the engineered antibodies specifically bound to cancer cellsLS-174-T as well as to pure bovine submaxillary gland mucin.Their specificity and affinity appeared the sameas their parent antibodies.Our results supposed that the single-chain antibodies will be a target for thediagnosis and treatment of cancer.

  15. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    Science.gov (United States)

    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.

  16. Immunoglobulin heavy chain/light chain pair measurement is associated with survival in diffuse large B-cell lymphoma.

    Science.gov (United States)

    Jardin, Fabrice; Delfau-Larue, Marie Hélène; Molina, Thierry Jo; Copie-Bergman, Christiane; Brière, Josette; Petrella, Tony; Canioni, Danielle; Fabiani, Bettina; Jais, Jean-Philippe; Figeac, Martin; Leroy, Karen; Mareschal, Sylvain; Salles, Gilles André; Coiffier, Bertrand; Delarue, Richard; Peyrade, Frédéric; Bosly, André; André, Marc; Ketterer, Nicolas; Haioun, Corinne; Tilly, Hervé

    2013-09-01

    Elevated serum free light chains (FLCs) have been associated with an unfavorable prognosis in diffuse large B-cell lymphoma (DLBCL). The aim of this study was to determine the clinical relevance of a quantitative assessment of intact circulating immunoglobulin (Ig), using serum Ig heavy chain/light chain pair (HLC) measurements in patients with DLBCL. FLC and HLC were measured in 409 serum samples of patients with DLBCL included in the LNH03-B clinical trial program of the Groupe d'Etudes des Lymphomes de l'Adulte (GELA). Patients with an abnormal IgMκ/IgMλ ratio or an abnormal FLC ratio more frequently displayed adverse clinical characteristics. Patients with abnormal IgMκ/IgMλ ratios had inferior progression-free survival (PFS) and overall survival (OS) as compared to patients with a normal ratio in the overall cohort (5-year PFS 44.9% vs. 69.3%, p = 0.0003 and 5-year OS 50.8% vs. 78.1%, p = 0.0003) and in the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) cohort (5-year OS 43.5% vs. 70.3%, p = 0.003). In multivariate analysis, including elevated FLC/HLC and International Prognostic Index (IPI), an abnormal IgMκ/IgMλ ratio (hazard ratio [HR] = 1.54, 95% confidence interval [CI] 1.03-2.3, p = 0.03) remained predictive of shorter progression-free survival. Gene expression profile experiments and immunohistochemistry indicate that this measurement is at least partially related to tumor cell secretion. Both elevated serum FLCs and an abnormal IgMκ/IgMλ ratio are associated with unfavorable outcomes in patients with DLBCL treated by R-CHOP.

  17. Creation and Evaluation of a Single-chain Antibody Tetramer that Targets Brain Endothelial Cells.

    Science.gov (United States)

    Zhang, Xiaobin; Wang, Xin Xiang; Shusta, Eric V

    2014-04-01

    Antibodies that target and internalize into blood-brain barrier (BBB) endothelial cells offer promise as drug delivery agents. Previously, we identified a single-chain antibody (scFvA) capable of binding to the BBB. In an attempt to improve the binding and internalization properties of the single chain antibody (scFvA), a biotinylation tag (Avitag) was fused to scFvA and the protein secreted by yeast. The scFvA-Avitag could be biotinylated by yeast-displayed BirA enzyme and biotinylated scFvA-Avitag could be used to create scFv tetramers. Tetramerization of scFvA improved the internalization of scFvA into BBB endothelial cells, and biotinylated scFvA-Avitag could also be used to target streptavidin-coated quantum dots for BBB endothelial cell internalization. Perfusing the rat brain with scFvA-tetramer confirmed that the antigen targeted by scFvA is distributed on blood side of the BBB, suggesting the potential for downstream application of scFvA in brain-targeted drug delivery.

  18. Y-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

    Institute of Scientific and Technical Information of China (English)

    王字玲; 荣康泰; 杨日芳; 恽榴红

    2000-01-01

    A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitro-phenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P6 to the carrier proteins BSA (bovine serum albumin) and LPH (Limulus polyphenus hemocyanin). Mice were immunized with P6-LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P6-BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP6) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number kcatt= 198 min-1 and the rate enhancement kcatkuncat = 122 419. When 0.16 mg · mL-1

  19. Effect of aerobic exercise on the contractile function of gastrocnemius myosin heavy chain

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To study the effect of 4-6 weeks' treadmill training of male SD rats on the contractile function of their gastrocnemius myosin heavy chain (MHC). Methods Forty male SD rats were randomly divided into control group and training group. The treadmill training of the training group rats was incessantly performed for 4-6 weeks at an intensity of about 75% VO2max (18.5-24 m/min,gradient of 0°,each training session lasting 50 minutes,twice a day). The content of gastrocnemius MHC mRNA was tested by rever...

  20. Variable fragment of heavy chain antibody against the terminal protein region of hepatitis B virus polymerase introduced by Tat protein transduction domain inhibits the replication of hepatitis B virus in vitro%蛋白转导结构域介导乙型肝炎病毒聚合酶末端蛋白重链可变区抗体体外抑制病毒的复制

    Institute of Scientific and Technical Information of China (English)

    于俊岩; 兰林; 李俊刚; 张长江; 王宇明

    2009-01-01

    目的 蛋白转导结构域(TAT)介导HBV聚合酶末端蛋白(TP)重链可变区(VH)抗体,研究特异性TAT-VH抗体体外对HBV复制的影响.方法 将TAT-VH基因克隆入原核表达载体pET28a(+),在大肠埃希菌BL21(DE3)LysS内诱导融合蛋白表达并进行纯化.纯化的TAT-VH加入培养的HepG2.2.15细胞,间接免疫荧光法检测其导入HepG2.2.15细胞的效率,四甲基偶氮唑盐(MTT)法检测其对细胞生长代谢的影响,将TAT-VH加入培养的HepG2.2.15细胞,定量PCR法检测HBV DNA水平.数据行单因素方差分析和t检验.结果 成功制备了TAT-VH融合蛋白,间接免疫荧光及MTT证实TAT-VH可以跨膜导入HepG2.2.15细胞,且对细胞生长无影响;加入5 000 nmol/L TAT-VH的HepG2.2.15细胞培养上清液内HBV DNA为(1.211±0.132)lg拷贝/mL,对照组为(5.325±0.041)lg拷贝/mL(t=72.91,P<0.05);细胞内分别为(3.521±0.411)和(8.532±0.132)Ig拷贝/mL(t=28.41,P<0.05).结论 HBV聚合酶TP区特异性TAT-VH抗体在体外可抑制HBV复制,为应用细胞内抗体治疗HBV感染提供了良好的实验基础.%Objective To study a functional variable fragment of heavy chain(VH)antibody against the terminal protein(TP)region of hepatitis B virus(HBV)polymerase introduced by human immunodeficiency virus Tat protein transduction domain(TAT)and the inhibitive activity of TAT-VH on the replication of HBV in vitro.Methods The gene encoding TAT-VH was cloned into prokaryotic expression vector pET28a(+).Recombinant plasmid was transduced into E coli BL21(DE3)LysS,then the protein was expressed and purified.The purified TAT-VH fusion protein was added into HepG2.2.15 cell culture.The transduction efficiency was evaluated by indirect fluorescence assay(IFA).The cytotoxicity of TAT-VH was detected by Methabenzthiazuron(MTT)assay.HBV DNA level in HepG2.2.15 cell culture was measured using quantitative polymerase chain reaction(PCR).The data were analyzed by one-factor analysis of variance and t test.Results TAT

  1. Assay interference caused by antibodies reacting with rat kappa light-chain in human sera.

    Science.gov (United States)

    Degn, Søren E; Andersen, Stig Henrik; Jensen, Lisbeth; Thiel, Steffen; Jensenius, Jens C

    2011-09-30

    The enzyme-linked immunosorbent assay (ELISA) and its derivatives are powerful tools used in research, in the clinic, and in many other analytical and quality control settings. In general, ELISAs are robust, reproducible and reliable. However, a number of pitfalls of ELISAs have been described over the years. The issue of rheumatoid factor (RF), autoantibodies against the Fc portion of IgG, is well recognized (yet often forgotten), as are problems arising from heterophilic antibodies induced by external antigens that cross-react with self-antigens. A few years ago focus was on human anti-mouse antibodies (HAMA) concomitant with the increased use of mouse monoclonal antibody therapy, a problem that is now diminishing due to development of humanized antibodies. Issues pertaining to food antigens or environmentally encountered antigens are less recognized. We report a recently encountered example of the latter resulting in interference in a solid-phase sandwich assay. Due to the set-up employing a monoclonal rat IgG for capture and a monoclonal rat IgM for development the interference had to be human antibodies reacting with rat light-chain. Out of 102 Danish Caucasian blood donors we found a prevalence of anti-rat kappa light chain antibodies of close to 40% (39/102, defined as at least 2-fold elevated measurements), with around 6% (6/102) having very high levels (defined as at least 4-fold elevated measurements), yielding significantly higher measurements in the assay designed to measure the complement component MAp19 in serum samples. The interference could be blocked by the addition of rat immunoglobulin to the sample buffer. An individual, who had been followed over time, demonstrated a periodic increase of interfering antibodies, highlighting that it is an independently varying parameter and thereby a variable interference in assays. Our results highlight a major pitfall of potential relevance to many sandwich-type assays, as well as an approach to rectify such

  2. Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening

    Directory of Open Access Journals (Sweden)

    Hiller Gösta

    2009-06-01

    Full Text Available Abstract Background Deregulated activation of cyclin-dependent kinase-5 (Cdk5 is implicated in neurodegenerative disorders such as Alzheimer's disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems. Methods HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates. Results Western blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B. Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line. Conclusion A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.

  3. Characterization of a complete immunoglobulin heavy-chain variable region germ-line gene of rainbow trout.

    Science.gov (United States)

    Matsunaga, T; Chen, T; Törmänen, V

    1990-10-01

    A germ-line heavy-chain variable region (VH) gene (RTVH431) has been isolated from a rainbow trout (Salmo gairdneri) and characterized by complete nucleotide sequencing. It is characteristic of VH, as shown by the conserved octamer and TATA motif in the 5' region, the heptamernonamer recombination signal sequence in the 3' region, and the 18-amino-acid-long hydrophobic leader interrupted by an intron. The 98-amino-acid-long VH coding region has 50-70% nucleotide sequence homology and 40-60% amino acid sequence homology with VHS of various vertebrate species. We have also found unique or species-specific amino acid residues in the VHS of rainbow trout, amphibia (Xenopus), reptile (Caiman), and shark (Heterodontus) in our sequence analyses. The RTVH431 has an unusual amino acid in the conserved 34th position in complementarity-determining region 1 of VH. Southern hybridization results suggest the presence of a large gene family related to RTVH431 in the trout genome. The complex evolution of antibody V genes is discussed.

  4. Intermediate filament-co-localized molecules with myosin heavy chain epitopes define distinct cellular domains in hair follicles and epidermis

    Directory of Open Access Journals (Sweden)

    Hughes Simon M

    2003-08-01

    Full Text Available Abstract Background Proteins linking intermediate filaments to other cytoskeletal components have important functions in maintaining tissue integrity and cell shape. Results We found a set of monoclonal antibodies raised against specific human sarcomeric myosin heavy chain (MyHC isoforms labels cells in distinct regions of the mammalian epidermis. The antigens co-localize with intermediate filament-containing structures. A slow MyHC-related antigen is punctate on the cell surface and co-localizes with desmoplakin at desmosomal junctions of all suprabasal epidermal layers from rat fœtal day 16 onwards, in the root sheath of the hair follicle and in intercalated disks of cardiomyocytes. A fast MyHC-related antigen occurs in cytoplasmic filaments in a subset of basal cells of skin epidermis and bulb, but not neck, of hair follicles. A fast IIA MyHC-related antigen labels filaments of a single layer of cells in hair bulb. This 230 000 Mr antigen co-purifies with keratin. No obvious candidate for any of the antigens appears in the literature. Conclusions We describe a set of molecules that co-localize with intermediate filament in specific cell subsets in epithelial tissues. These antigens presumably influence intermediate filament structure or function.

  5. Differential sensitivity of myosin-heavy-chain-typed fibers to distinct aggregates of nerve-mediated activation.

    Science.gov (United States)

    Dunn, S E; Michel, R N

    1999-02-01

    We studied the regulatory effects of nerve-mediated activity on the early expression of embryonic and adult myosin heavy chains (MHC) within inactive though still innervated rat plantaris and soleus muscle fibers. To this end, we stimulated motor nerves that were quiescent following treatment with tetrodotoxin (TTX) with paradigms designed to partition the influence of neural activation frequency and assessed the selective expression and accumulation of MHCs within muscle fibers using an array of specific antibodies. We show rapid de novo expression of IIx MHC within select soleus fibers in response to high-frequency activation for more than 0.01% of daily time. High-frequency aggregates were also the most effective in preventing the TTX-induced reexpression of embryonic MHCs within specific fibers. Only configurations that included high-frequency trains for more than 0.01% of daily time or combined with 10 Hz stimulation preserved the size of select fibers, used as a measure of the net cellular content of MHC. The effectiveness of this preservation varied according to the muscle type and MHC expressed, and, in a subset of fibers, was influenced by contractile loading status. Our results demonstrate that distinct subsets of MHC-typed fibers are differentially sensitive to the neural activation cues mediating the cellular expression of these proteins.

  6. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    OpenAIRE

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2010-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Bece...

  7. Molecular characterization of a single-chain antibody variable fragment (scFv) specific for PspA from Streptococcus pneumoniae.

    Science.gov (United States)

    Jang, ShinA; Kim, Gyuhee; Oh, Jihye; Lee, Seungyeop; Kim, Dongho; Kim, Kook-Han; Kim, Yong Ho; Rhee, Dong-Kwon; Lee, Sangho

    2017-01-01

    Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.

  8. The milk delivery chain and presence of Brucella spp. antibodies in bulk milk in Uganda.

    Science.gov (United States)

    Rock, Kim Toeroek; Mugizi, Denis Rwabiita; Ståhl, Karl; Magnusson, Ulf; Boqvist, Sofia

    2016-06-01

    This study examined the influence of informal milk delivery chains on the risk of human exposure to Brucella spp. through milk consumption in two regions of Uganda (Gulu and Soroti Districts). The work involved describing milk delivery chains, investigating brucellosis awareness amongst milk deliverers and determining the presence of Brucella spp. antibodies in cattle milk on delivery to primary collection points (boiling points and dairies). Milk samples (n = 331) were collected from deliverers at primary collection points and from street vendors at point of sale and analysed using indirect enzyme-linked immunosorbent assay (I-ELISA). A written questionnaire was used to collect data from deliverers (n = 279) on their milk delivery chains and their brucellosis awareness. The most common delivery points in Gulu District were small dairies and in Soroti District boiling points. The presence of Brucella spp. antibodies in milk samples was higher in Soroti (40 %) than in Gulu (11 %) (P < 0.0001). There are possible public health risk consequences of this finding as 42 % of deliverers in Soroti District reported drinking raw milk, compared with 15 % in Gulu District (P < 0.0001). Awareness of brucellosis was low, with 70 % of all milk deliverers reporting not having heard of the disease or the bacterium. Application of quality controls for milk (colour and odour) along the delivery chain varied depending upon supply and demand. This study provides evidence of the diversity of informal milk markets in low-income countries and of the potential public health risks of consuming unpasteurised milk. These results can be useful to those planning interventions to reduce brucellosis.

  9. Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2.

    Directory of Open Access Journals (Sweden)

    Xiangjing Fu

    Full Text Available Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×10(6 cfu/3 µg and 2×10(9 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2 Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

  10. Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.

    Science.gov (United States)

    Hosseini, Hossien; Rajabibazl, Masoumeh; Ebrahimizadeh, Walead; Dehbidi, Gholamreza Rafiei

    2015-01-01

    Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10μg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.

  11. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    Energy Technology Data Exchange (ETDEWEB)

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  12. Directed evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter.

    Science.gov (United States)

    Kim, Dong-Sik; Song, Hyung-Nam; 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.

  13. Preparative crystallization of a single chain antibody using an aqueous two-phase system.

    Science.gov (United States)

    Huettmann, Hauke; Berkemeyer, Matthias; Buchinger, Wolfgang; Jungbauer, Alois

    2014-11-01

    A simultaneous crystallization and aqueous two-phase extraction of a single chain antibody was developed, demonstrating process integration. The process conditions were designed to form an aqueous two-phase system, and to favor crystallization, using sodium sulfate and PEG-2000. At sufficiently high concentrations of PEG, a second phase was generated in which the protein crystallization occurred simultaneously. The single chain antibody crystals were partitioned to the top, polyethylene glycol-rich phase. The crystal nucleation took place in the sodium sulfate-rich phase and at the phase boundary, whereas crystal growth was progressing mainly in the polyethylene glycol-rich phase. The crystals in the polyethylene glycol-rich phase grew to a size of >50 µm. Additionally, polyethylene glycol acted as an anti-solvent, thus, it influenced the crystallization yield. A phase diagram with an undersaturation zone, crystallization area, and amorphous precipitation zone was established. Only small differences in polyethylene glycol concentration caused significant shifts of the crystallization yield. An increase of the polyethylene glycol content from 2% (w/v) to 4% (w/v) increased the yield from approximately 63-87%, respectively. Our results show that crystallization in aqueous two-phase systems is an opportunity to foster process integration.

  14. Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3

    Institute of Scientific and Technical Information of China (English)

    刘喜富; 萧飒; 顾征; 王勇; 张卫国; 陈艾; 林晴; 黄华梁; 孙健; 陈润生; 沈倍奋; 陈兴

    1997-01-01

    Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.

  15. Selection and characterization of single-chain recombinant antibodies against phosphoprotein of Newcastle disease virus.

    Science.gov (United States)

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

    2014-09-01

    Phosphoprotein (P), involved in virus RNA replication and transcription, had become a new target for the research on treating Newcastle disease virus (NDV). Here we described the cloning and expression of phosphoprotein from NDV, and then screened the anti-P antibodies from the chicken single chain fragment variable (scFv) library, which were generated from chickens immunized with the ND vaccines. As a first step, the recombinant expression vector pET28a-P was successfully constructed. In a following step, two anti-P positive scFv clones from the scFv library were selected by indirect enzyme-linked immunosorbent assay (ELISA) method. The sequence analysis of two positive clones showed that there were more variation in complementary determine region (CDR) of VH and VL, and the CDR3 in VH exhibited a significant change in amino acid number and type. In another experiment, the purified scFv antibodies used in the assay was shown to be specific for NDV-P by western blot. The results indicated that the strategy we used in this experiment proved to be convenient way for screening scFv antibody, which paved a new way for the immunization diagnosis and the exploration of integrated control of NDV.

  16. γ-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitrophenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P6 to the carrier proteins BSA (bovine serum albumin) and LPH (Limulus polyphenus hemocyanin). Mice were immunized with P6-LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P6-BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP6) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number kcat = 198 minγ1 and the rate enhancement kcat/kuncat = 122 419. When 0.16 mg·mLγ1 EP6 was preincubated in vitro with 0.132 mmol·Lγ1 (220 g·kgγ1 = 1.1×LD95) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP6 could prolong the latent time of spasm and death when mice were passively immunized with EP6 intravenously 15 min before 1×LD95 of soman challenge. These results demonstrate that EP6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.

  17. Bioaccumulative and conchological assessment of heavy metal transfer in a soil-plant-snail food chain

    Directory of Open Access Journals (Sweden)

    Nica Dragos V

    2012-06-01

    Full Text Available Abstract Background Copper (Cu, zinc (Zn, cadmium (Cd, and lead (Pb can pose serious threats to environmental health because they tend to bioaccumulate in terrestrial ecosystems. We investigated under field conditions the transfer of these heavy metals in a soil-plant-snail food chain in Banat area, Romania. The main goal of this paper was to assess the Roman snail (Helix pomatia usefulness in environmental monitoring as bioindicator of heavy metal accumulation. Eight sampling sites, selected by different history of heavy metal (HM exposure, were chosen to be sampled for soil, nettle leaves, and newly matured snails. This study also aimed to identify the putative effects of HM accumulation in the environment on phenotypic variability in selected shell features, which included shell height (SH, relative shell height (RSH, and whorl number (WN. Results Significantly higher amounts of HMs were accumulated in snail hepatopancreas and not in foot. Cu, Zn, and Cd have biomagnified in the snail body, particularly in the hepatopancreas. In contrast, Pb decreased when going up into the food chain. Zn, Cd, and Pb correlated highly with each other at all levels of the investigated food chain. Zn and Pb exhibited an effective soil–plant transfer, whereas in the snail body only foot Cu concentration was correlated with that in soil. There were significant differences among sampling sites for WN, SH, and RSH when compared with reference snails. WN was strongly correlated with Cd and Pb concentrations in nettle leaves but not with Cu and Zn. SH was independent of HM concentrations in soil, snail hepatopancreas, and foot. However, SH correlated negatively with nettle leaves concentrations for each HM except Cu. In contrast, RSH correlated significantly only with Pb concentration in hepatopancreas. Conclusions The snail hepatopancreas accumulates high amounts of HMs, and therefore, this organ can function as a reliable biomarker for tracking HM bioavailability

  18. Dilated cardiomyopathy mutation (R134W in mouse cardiac troponin T induces greater contractile deficits against α-myosin heavy chain than against β-myosin heavy chain

    Directory of Open Access Journals (Sweden)

    Sampath K Gollapudi

    2016-10-01

    Full Text Available Many studies have demonstrated that depressed myofilament Ca2+ sensitivity is common to dilated cardiomyopathy (DCM in humans. However, it remains unclear whether a single determinant — such as myofilament Ca2+ sensitivity — is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT, which is known to modulate crossbridge (XB recruitment dynamics in a myosin heavy chain (MHC-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W of the human DCM mutation (R131W, we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC and transgenic mice (β-MHC. TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca2+ sensitivity to a greater extent in α-MHC (0.14 pCa units than in β-MHC fiber bundles (0.08 pCa units. Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background.

  19. On generic chaining and the smallest singular value of random matrices with heavy tails

    CERN Document Server

    Mendelson, Shahar

    2011-01-01

    We present a very general chaining method which allows one to control the supremum of the empirical process $\\sup_{h \\in H} |N^{-1}\\sum_{i=1}^N h^2(X_i)-\\E h^2|$ in rather general situations. We use this method to establish two main results. First, a quantitative (non asymptotic) version of the classical Bai-Yin Theorem on the singular values of a random matrix with i.i.d entries that have heavy tails, and second, a sharp estimate on the quadratic empirical process when $H=\\{\\inr{t,\\cdot} : t \\in T\\}$, $T \\subset \\R^n$ and $\\mu$ is an isotropic, unconditional, log-concave measure.

  20. Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin

    Indian Academy of Sciences (India)

    Qizhuang Lv; Kangkang Guo; Tao Wang; Chengcheng Zhang; Yanming Zhang

    2015-09-01

    Porcine circovirus type 2 (PCV2) is the primary infectious agent of PCV-associated disease (PCVAD) in swine. ORF4 protein is a newly identified viral protein of PCV2 and is involved in virus-induced apoptosis. However, the molecular mechanisms of ORF4 protein regulation of apoptosis remain unclear, especially given there is no information regarding any cellular partners of the ORF4 protein. Here, we have utilized the yeast two-hybrid assay and identified four host proteins (FHC, SNRPN, COX8A and Lamin C) interacting with the ORF4 protein. Specially, FHC was chosen for further characterization due to its important role in apoptosis. GST pull-down, subcellular co-location and co-immunoprecipitation assays confirmed that the PCV2 ORF4 protein indeed interacted with the heavy-chain ferritin, which is an interesting clue that will allow us to determine the role of the ORF4 protein in apoptosis.

  1. Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Jiong Cai; Fang Li; Shizhen Wang

    2008-01-01

    BACKGROUND: Studies have shown that monoclonal or polyclonal antibody injections ofamyloid β peptide arc effective in removing amyloid β peptide overload in the brain.OBJECTIVE: Based on successful screening of a human single-chain fragment variable antibody specific to amyloid β peptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide.DESIGN, TIME AND SETTING: A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital (Beijing, China) from January to July 2006.MATERIALS: Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library.METHODS: Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a Teasy plasmid for pT-seFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvA β was cut by EcoRl and Notl endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvA β expression vector, which was confirmed by gene sequencing. Linearized pPICgK-scFvA β was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5 % methanol to express human single-chain fragment variable antibody specific to amyloid β peptide.MAIN OUTCOME MEASURES: Protein electrophoresis was used to identify PCR products, gene sequencing was uscd to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris.RESULTS: Gene sequencing confirmed pPICgK-scFvA β orientation. Rccomhinants were obtained by lineadzed pPIC9K-scFvA β transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size

  2. cDNA cloning of the immunoglobulin heavy chain genes in banded houndshark Triakis scyllium.

    Science.gov (United States)

    Honda, Yuka; Kondo, Hidehiro; Caipang, Christopher Marlowe A; Hirono, Ikuo; Aoki, Takashi

    2010-11-01

    In this study, cDNAs encoding the secreted forms of the immunoglobulin (Ig) heavy chains of IgM, IgNAR, and IgW were cloned from the banded houndshark Triakis scyllium. Two clones for the IgM heavy chains encoded 569 and 570 amino acids, whose conserved (C) region showed 47-70% amino acid identities to those reported in other cartilaginous fish. Four clones for the IgNAR encoded 673-670 amino acids with conserved Ig-superfamily domains. The IgNAR C region showed 56-69% amino acid identities to those so far reported. High-throughput sequencing revealed that in most of the IgNAR sequences, the two variable regions (CDR1 and CDR3) each possess a cysteine residue. Three types of IgW were identified; one contained Ig-superfamily domains that are in other cartilaginous fish, one lacks the 3rd domain in the constant region, and one lacks the 3rd to 5th domains. Despite these differences, the IgW isoforms clustered with IgWs of other cartilaginous fishes and the C regions showed 47-89% amino acid identities. mRNAs for IgM and IgNAR were detected in various tissues, while IgW mRNA was mainly detected in pancreas. The banded hounded shark also has IgM, IgW and IgNAR as well as the other cartilaginous fish with unique IgW isoform.

  3. Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition.

    Science.gov (United States)

    Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Suita, Kenji; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

    2015-01-01

    Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition. We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB. Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.

  4. Chronic sleep deprivation alters the myosin heavy chain isoforms in the masseter muscle in rats.

    Science.gov (United States)

    Cao, Ruihua; Huang, Fei; Wang, Peihuan; Chen, Chen; Zhu, Guoxiong; Chen, Lei; Wu, Gaoyi

    2015-05-01

    To investigate the changes in myosin heavy chain (MyHC) isoforms of rat masseter muscle fibres caused by chronic sleep deprivation and a possible link with the pathogenesis of disorders of the temporomandibular joint (TMJ). A total of 180 male rats were randomly divided into three groups (n=60 in each): cage controls, large platform controls, and chronic sleep deprivation group. Each group was further divided into three subgroups with different observation periods (7, 14, and 21 days). We investigated he expression of MyHC isoforms in masseter muscle fibres by real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunohistochemical staining. In rats with chronic sleep deprivation there was increased MyHC-I expression in layers of both shallow and deep muscles at 7 and 21 days compared with the control groups, whereas sleep deprivation was associated with significantly decreased MyHC-II expression. At 21 days, there were no differences in MyHC-I or MyHC-II expression between the groups and there were no differences between the two control groups at any time point. These findings suggest that chronic sleep deprivation alters the expression of MyHC isoforms, which may contribute to the pathogenesis of disorders of the TMJ.

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

    Science.gov (United States)

    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.

  6. Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

    Science.gov (United States)

    SERRANO, A. L.; PÉREZ, MARGARITA; LUCÍA, A.; CHICHARRO, J. L.; QUIROZ-ROTHE, E.; RIVERO, J. L. L.

    2001-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC β-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies. PMID:11554510

  7. Biosynthesis and characterization of a non-repetitive polypeptide derived from silk fibroin heavy chain

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Gaoqiang; Wu, Mingyang; Yi, Honggen; Wang, Jiannan, E-mail: wangjn@suda.edu.cn

    2016-02-01

    Silk fibroin heavy chain is the major protein component of Bombyx mori silk fibroin and is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. In order to determine the biomedical function of the non-repetitive domain or potentially use it to modify hydrophobic biomaterials, high-purity isolation is necessary. Previously, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain. Here, this motif and its multimers are inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif f(1) and multimers f(4) and f(8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography, and single bands of purified fusion proteins GST-F(1), GST-F(4), and GST-F(8), were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Target polypeptides F(1), F(4), and F(8), were cleaved clearly from the GST-fusion tag following thrombin digestion. Mass spectrometry results indicate that the molecular weights associated with fusion proteins GST-F(1), GST-F(4), and GST-F(8) are 31.5, 43.8, and 59.0 kDa, respectively, and with the cleaved polypeptides F(1), F(4), and F(8) are 4.8, 16.8, and 32.8 kDa, respectively. The F(1), F(4), and F(8) polypeptide chains are negatively charged with isoelectric points (pI) of 3.3, 3.2, and 3.0, respectively. The molecular weight and pI values of the polypeptide chains are consistent with the predicted values and the amino acid compositions similar to predicted sequences. FTIR and CD results show the molecular conformation of F(1) was mainly random coil, and more stable α-helix structure formed in longer molecular chain. - Highlights: • A non-repetitive domain and its multimers of silk fibroin were expressed by E. coli. • The corresponding target polypeptides F(1), F(4) and F(8) were cleaved clearly. • Their

  8. ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) is a new acute-phase protein isolated from cattle during experimental infection

    DEFF Research Database (Denmark)

    Pineiro, M.; Andres, M.; Iturralde, M.;

    2004-01-01

    We have isolated from calf serum a protein with an apparent M, of 120,000. The protein was detected by using antibodies against major acute-phase protein in pigs with acute inflammation. The amino acid sequence of an internal fragment revealed that this protein is the bovine counterpart of ITIH4......, the heavy chain 4 of the inter-alpha-trypsin inhibitor family. The response of this protein in the sera was determined for animals during experimental bacterial and viral infections. In the bacterial model, animals were inoculated with a mixture of Actinomyces pyogenes, Fusobacterium necrophorum....... Because of the significant induction of the protein in the animals in the mastitis and BRSV infection models, we can conclude that ITIH4 is a new positive acute-phase protein in cattle....

  9. Enhanced factor VIII heavy chain for gene therapy of hemophilia A.

    Science.gov (United States)

    Chen, Lingxia; Lu, Hui; Wang, Jinhui; Sarkar, Rita; Yang, Xiao; Wang, Hongli; High, Katherine A; Xiao, Weidong

    2009-03-01

    Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a "chain imbalance" problem in which LC secretion is approximately 1-2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC(745)). The optimized HC mutein, HC(HL), including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HC(HL)+AAV8-LC or AAV8-HC(745)+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HC(HL)+AAV8-LC achieved three- to fourfold higher HC expression than AAV8-HC(745)+AAV8-LC, consistent with the FVIII functional assays. HC(HL) should be substituted for HC(745) in a dual AAV vector strategy due to its enhanced expression.

  10. Improved Binding Activity of Antibodies against Major Histocompatibility Complex Class I Chain-Related Gene A by Phage Display Technology for Cancer-Targeted Therapy

    Directory of Open Access Journals (Sweden)

    Achara Phumyen

    2012-01-01

    Full Text Available Major histocompatibility complex class I chain-related gene A (MICA is an NKG2D ligand that is over-expressed under cellular stress including cancer transformation and viral infection. High expression of MICA in cancer tissues or patients' sera is useful for prognostic or follow-up markers in cancer patients. In this study, phage display technology was employed to improve antigen-binding activities of anti-MICA monoclonal antibodies (WW2G8, WW6B7, and WW9B8. The 12 amino acid residues in the complementarity determining regions (CDRs on the V domain of the heavy chain CDR3 (HCDR3 of these anti-MICA antibodies were modified by PCR-random mutagenesis, and phages displaying mutated anti-MICA Fab were constructed. After seven rounds of panning, five clones of phages displaying mutant anti-MICA Fab which exhibited 3–7-folds higher antigen-binding activities were isolated. Two clones of the mutants (phage-displayed mutant Fab WW9B8.1 and phage-displayed mutant Fab WW9B8.21 were confirmed to have antigen-binding specificity for cell surface MICA proteins by flow cytometry. These phage clones are able to recognize MICA in a native form according to positive results obtained by indirect ELISA and flow cytometry. Thus, these phage particles could be potentially used for further development of nanomedicine specifically targeting cancer cells expressing MICA proteins.

  11. Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development.

    Science.gov (United States)

    Isaacs, Alison T; Jasinskiene, Nijole; Tretiakov, Mikhail; Thiery, Isabelle; Zettor, Agnès; Bourgouin, Catherine; James, Anthony A

    2012-07-10

    Anopheles stephensi mosquitoes expressing m1C3, m4B7, or m2A10 single-chain antibodies (scFvs) have significantly lower levels of infection compared to controls when challenged with Plasmodium falciparum, a human malaria pathogen. These scFvs are derived from antibodies specific to a parasite chitinase, the 25 kDa protein and the circumsporozoite protein, respectively. Transgenes comprising m2A10 in combination with either m1C3 or m4B7 were inserted into previously-characterized mosquito chromosomal "docking" sites using site-specific recombination. Transgene expression was evaluated at four different genomic locations and a docking site that permitted tissue- and sex-specific expression was researched further. Fitness studies of docking site and dual scFv transgene strains detected only one significant fitness cost: adult docking-site males displayed a late-onset reduction in survival. The m4B7/m2A10 mosquitoes challenged with P. falciparum had few or no sporozoites, the parasite stage infective to humans, in three of four experiments. No sporozoites were detected in m1C3/m2A10 mosquitoes in challenge experiments when both genes were induced at developmentally relevant times. These studies support the conclusion that expression of a single copy of a dual scFv transgene can completely inhibit parasite development without imposing a fitness cost on the mosquito.

  12. Isolation of BNYVV coat protein-specific single chain Fv from a mouse phage library antibody.

    Science.gov (United States)

    Jahromi, Zahra Moghaddassi; Salmanian, Ali Hatef; Rastgoo, Nasrin; Arbabi, Mehdi

    2009-10-01

    Beet necrotic yellow vein virus (BNYVV) infects sugar beet plants worldwide and is responsible for the rhizomania disease and severe economic losses. Disease severity and lack of naturally occurring resistant plants make it very difficult to control the virus, both from epidemiological and economic standpoints. Therefore, early detection is vital to impose hygiene restrictions and prevent further spread of the virus in the field. Immunoassays are one of the most popular methodologies for the primary identification of plant pathogens including BNYVV since they are robust, sensitive, fast, and inexpensive. In this study, the major coat protein (CP21) of BNYVV was cloned and expressed in Escherichia coli. Thereafter, mice were immunized with purified CP21 and a phage antibody library was constructed from their PCR-amplified immunoglobulin repertoire. Following filamentous phage rescue of the library and four rounds of panning against recombinant CP21 antigen, several specific single chain Fv fragments were isolated and characterized. This approach may pave the way to develop novel immunoassays for a rapid detection of viral infection. Moreover, it will likely provide essential tools to establish antibody-mediated resistant transgenic technology in sugar beet plants.

  13. Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y.

    Science.gov (United States)

    Rouis, Souad; Lafaye, Pierre; Jaoua-Aydi, Leila; Sghaier, Zidani; Ayadi, Hammadi; Gargouri-Bouzid, Radhia

    2006-10-01

    Three single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies.

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

    Science.gov (United States)

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

    2010-07-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which 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 medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) 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 nonselective medium. 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. (c)2010 AACR.

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

    Science.gov (United States)

    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

  16. Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

    Science.gov (United States)

    Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

    2011-11-15

    Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that

  17. ImmunoGlobulin galaxy (IGGalaxy) for simple determination and quantitation of immunoglobulin heavy chain rearrangements from NGS

    NARCIS (Netherlands)

    M.J. Moorhouse (Michael); D. van Zessen (David); H. IJspeert (Hanna); S. Hiltemann (Saskia); S. Horsman (Sebastiaan); P.J. van der Spek (Peter); M. van der Burg (Mirjam); A. Stubbs (Andrew)

    2014-01-01

    textabstractBackground: Sequence analysis of immunoglobulin heavy chain (IGH) gene rearrangements and frequency analysis is a powerful tool for studying the immune repertoire, immune responses and immune dysregulation in health and disease. The challenge is to provide user friendly, secure and repro

  18. Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s

    DEFF Research Database (Denmark)

    Eriksson, H; Nissen, Mogens Holst

    1990-01-01

    into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular...

  19. The role of the ubiquitin system in human cytomegalovirus-mediated degradation of MHC class I heavy chains

    NARCIS (Netherlands)

    Hassink, Gerrit Cornelis

    2006-01-01

    One of the mechanisms used by HCMV to downregulate cell surface expression of the MHC class I complex involves the dislocation of newly synthesized class I heavy chains into the cytosol, where they are degraded by the proteasome. Misfolded ER proteins have been found to be degraded via the same rout

  20. Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease

    DEFF Research Database (Denmark)

    Pecci, A.; Panza, E.; Pujol-Moix, N.

    2008-01-01

    MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness...

  1. Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: neutralization capacity versus thermodynamic stability.

    Science.gov (United States)

    Quintero-Hernández, Veronica; Del Pozo-Yauner, Luis; Pedraza-Escalona, Martha; Juárez-González, Victor R; Alcántara-Recillas, Israel; Possani, Lourival D; Becerril, Baltazar

    2012-04-30

    The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab≫scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.

  2. Single chain Fab (scFab fragment

    Directory of Open Access Journals (Sweden)

    Brenneis Mariam

    2007-03-01

    Full Text Available Abstract Background The connection of the variable part of the heavy chain (VH and and the variable part of the light chain (VL by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd and the light chain (LC, resulting in the formation of a single chain Fab fragment (scFab, can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC connecting the constant part 1 of the heavy chain (CH1 and the constant part of the light chain (CL were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of

  3. Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity.

    Science.gov (United States)

    Eddinger, T J; Meer, D P

    2001-02-01

    Isolated single smooth muscle cells (SMCs) from different regions of the rabbit stomach were used to determine a possible correlation between unloaded shortening velocity and smooth muscle (SM) myosin heavy chain (MHC) S1 head isoform composition (SMA, no head insert; SMB, with head insert). alpha-Toxin-permeabilized isolated single cells were maximally activated to measure unloaded shortening velocity and subsequently used in an RT-PCR reaction to determine the SMA/SMB content of the same cell. SM MHC SMA and SMB isoforms are uniquely distributed in the stomach with cells from the fundic region expressing little SMB (38.1 +/- 7.3% SMB; n = 16); cells from the antrum express primarily SMB (94.9 +/- 1.0% SMB; n = 16). Mean fundic cell unloaded shortening velocity was 0.014 +/- 0.002 cell lengths/s compared with 0.036 +/- 0.002 for the antrum cells. Unloaded shortening velocity in these cells was significantly correlated with their percent SMB expression (r2 = 0.58). Resting cell length does not correlate with the percent SMB expression (n = 32 cells). Previously published assays of purified or expressed SMA and SMB heavy meromyosin show a twofold difference in actin filament sliding speed in in vitro motility assays. Extrapolation of our data to 0-100% SMB would give a 10-fold range of shortening velocity, which is closer to the approximately 20-fold range reported from various SM tissues. This suggests that mechanisms in addition to the MHC S1 head isoforms regulate shortening velocity.

  4. Mitochondrial Electron Transport Chain in Heavy Metal-Induced Neurotoxicity: Effects of Cadmium, Mercury, and Copper

    Directory of Open Access Journals (Sweden)

    Elena A. Belyaeva

    2012-01-01

    Full Text Available To clarify the role of mitochondrial electron transport chain (mtETC in heavy-metal-induced neurotoxicity, we studied action of Cd2+, Hg2+, and Cu2+ on cell viability, intracellular reactive oxygen species formation, respiratory function, and mitochondrial membrane potential of rat cell line PC12. As found, the metals produced, although in a different way, dose- and time-dependent changes of all these parameters. Importantly, Cd2+ beginning from 10 [mu]M and already at short incubation time (3 h significantly inhibited the FCCP-uncoupled cell respiration; besides, practically the complete inhibition of the respiration was reached after 3 h incubation with 50 [mu]M Hg2+ or 500 [mu]M Cd2+, whereas even after 48 h exposure with 500 [mu]M Cu2+, only a 50% inhibition of the respiration occurred. Against the Cd2+-induced cell injury, not only different antioxidants and mitochondrial permeability transition pore inhibitors were protective but also such mtETC effectors as FCCP and stigmatellin (complex III inhibitor. However, all mtETC effectors used did not protect against the Hg2+- or Cu2+-induced cell damage. Notably, stigmatellin was shown to be one of the strongest protectors against the Cd2+-induced cell damage, producing a 15–20% increase in the cell viability. The mechanisms of the mtETC involvement in the heavy-metal-induced mitochondrial membrane permeabilization and cell death are discussed.

  5. Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

    NARCIS (Netherlands)

    Arafat, WO; Gomez-Navarro, J; Buchsbaum, DJ; Xiang, J; Casado, E; Barker, SD; Mahasreshti, PJ; Haisma, HJ; Barnes, MN; Siegal, GP; Alvarez, RD; Hemminki, A; Nettelbeck, DM; Curiel, DT

    2002-01-01

    Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of s

  6. A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo.

    Science.gov (United States)

    Cheng, Yue; Li, Zhuang; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-01-01

    Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

  7. Effect of aerobic exercise on the contractile function of gastrocnemius myosin heavy chain

    Institute of Scientific and Technical Information of China (English)

    Wen-jun Ren

    2009-01-01

    Objective To study the effect of 4- 6 weeks' treadmill training of male SD rats on the contractile function of their gnstroenemius myosin heavy chain (MHC). Methods Forty male SD rats were randomly divided into control group and training group. The treadmill training of the training group rats was incessantly performed for 4- 6 weeks at an intensity of about 75% VO2max (18. 5- 24 m/min, gradient of 0°, each training session lasting 50 minutes, twice a day). The content of gastrocnemlas MHC mRNA was tested by reverse transcription polymernse chain reaction (RT-PCR), and the changes of muscle fibre and its cross-section area (CSA) were measured using immunohistochemistry. Electric stimulation tests were used to determine the maximal tension of isometric contraction of the post-training gastrocnemius. Results ① After continuous treadmill training for 4 - 6 weeks, we found that the content of the total MHC, MHC Ⅰ , MHC Ⅱ x, MHC Ⅱ a mRNAs was 105%, 105%, 109% and 108% of that in the resting control group, respectively, and the MHC Ⅱ b mRNA content did not change significantly. The percentage of MHC Ⅰ mRNA in the total MHC mRNA increased while that of MHC Ⅱ mRNA decreased after aerobic training. ② The slow type of fibre type Ⅰ was the main part of the MHC after training and the CSA of the muscle fibres increased simultaneously. ③ The maximal tension of isometric contraction by pulse stimulation of square wave in the training group increased significantly compared with that in the control group (P<0. 01). Conclusion The findings indicate that aerobic exercise may promote an increase in the contractile function of MHC.

  8. Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

    Science.gov (United States)

    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Morgenstern, Sven; Pavlovic, Dragan; Kunert-Keil, Christiane

    2011-12-01

    The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P muscle (65.7 versus 73.8 per cent; P muscle was lower than in the controls (25.9 versus 30.8 per cent; P muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

  9. Neuronal ferritin heavy chain and drug abuse affect HIV-associated cognitive dysfunction.

    Science.gov (United States)

    Pitcher, Jonathan; Abt, Anna; Myers, Jaclyn; Han, Rachel; Snyder, Melissa; Graziano, Alessandro; Festa, Lindsay; Kutzler, Michele; Garcia, Fernando; Gao, Wen-Jun; Fischer-Smith, Tracy; Rappaport, Jay; Meucci, Olimpia

    2014-02-01

    Interaction of the chemokine CXCL12 with its receptor CXCR4 promotes neuronal function and survival during embryonic development and throughout adulthood. Previous studies indicated that μ-opioid agonists specifically elevate neuronal levels of the protein ferritin heavy chain (FHC), which negatively regulates CXCR4 signaling and affects the neuroprotective function of the CXCL12/CXCR4 axis. Here, we determined that CXCL12/CXCR4 activity increased dendritic spine density, and also examined FHC expression and CXCR4 status in opiate abusers and patients with HIV-associated neurocognitive disorders (HAND), which is typically exacerbated by illicit drug use. Drug abusers and HIV patients with HAND had increased levels of FHC, which correlated with reduced CXCR4 activation, within cortical neurons. We confirmed these findings in a nonhuman primate model of SIV infection with morphine administration. Transfection of a CXCR4-expressing human cell line with an iron-deficient FHC mutant confirmed that increased FHC expression deregulated CXCR4 signaling and that this function of FHC was independent of iron binding. Furthermore, examination of morphine-treated rodents and isolated neurons expressing FHC shRNA revealed that FHC contributed to morphine-induced dendritic spine loss. Together, these data implicate FHC-dependent deregulation of CXCL12/CXCR4 as a contributing factor to cognitive dysfunction in neuroAIDS.

  10. A Mouse Neurodegenerative Dynein Heavy Chain Mutation Alters Dynein Motility and Localization in Neurospora crassa

    Science.gov (United States)

    Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

    2013-01-01

    Cytoplasmic dynein is responsible for the transport and delivery of cargoes in organisms ranging from humans to fungi. Dysfunction of dynein motor machinery due to mutations in dynein or its activating complex dynactin can result in one of several neurological diseases in mammals. The mouse Legs at odd angles (Loa) mutation in the tail domain of the dynein heavy chain has been shown to lead to progressive neurodegeneration in mice. The mechanism by which the Loa mutation affects dynein function is just beginning to be understood. In this work, we generated the dynein tail mutation observed in Loa mice into the Neurospora crassa genome and utilized cell biological and complementing biochemical approaches to characterize how that tail mutation affected dynein function. We determined that the Loa mutation exhibits several subtle defects upon dynein function in N. crassa that were not seen in mice, including alterations in dynein localization, impaired velocity of vesicle transport, and in the biochemical properties of purified motors. Our work provides new information on the role of the tail domain on dynein function and points out areas of future research that will be of interest to pursue in mammalian systems. PMID:22991199

  11. Segmental distribution of myosin heavy chain isoforms within single muscle fibers.

    Science.gov (United States)

    Zhang, Ming; Gould, Maree

    2017-02-18

    Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the 3 EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I&cenveo_unknown_entity_wingdings_F0F3;IIA&cenveo_unknown_entity_wingdings_F0F3;IIX&cenveo_unknown_entity_wingdings_F0F3;IIB, however in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be due to the myonuclear domain. This article is protected by copyright. All rights reserved.

  12. Iron Deposition and Ferritin Heavy Chain (Fth Localization in Rodent Teeth

    Directory of Open Access Journals (Sweden)

    Wen Xin

    2013-01-01

    Full Text Available Abstract Background An iron rich layer on the labial surface is characteristic of the enamel of rodent incisors. In order to address a role for iron content in continuously growing incisors during odontogenesis, we studied iron deposition patterns in enamel and dentine using Perls’ blue staining and ferritin heavy chain (Fth immunolocalization. Fth expression is regulated by iron level; therefore its localization can be used as a sensitive indicator for iron deposition. Results Sagittal sections of 4-week old rat incisors showed a gradual increase in iron level in the enamel organ from secretory to maturation stages. In addition, iron was detected in ameloblasts of erupting third molars of 4-week old rats, suggesting iron plays a role in both incisor and molar development. In odontoblasts, the presence of iron was demonstrated, and this is consistent with iron’s role in collagen synthesis. Using postnatal 3-, 6-, 9-day old mice, the spatial and temporal expression of Fth in tooth development again indicated the presence of iron in mature ameloblasts and odontoblasts. Conclusions While these data do not explain what functional role iron has in tooth formation, it does highlight a significant molecular activity associated with the formation of the rodent dentition.

  13. Plasma neurofilament heavy chain is not a useful biomarker in Charcot-Marie-Tooth disease.

    Science.gov (United States)

    Rossor, Alexander M; Liu, Ching-Hua; Petzold, Axel; Malaspina, Andreas; Laura, Matilde; Greensmith, Linda; Reilly, Mary M

    2016-06-01

    The negative results in trials of vitamin C in Charcot-Marie-Tooth disease (CMT) type 1A have highlighted the lack of sensitive outcome measures. Neurofilaments are abundant neuronal cytoskeletal proteins, and their concentration in blood is likely to reflect axonal breakdown. We therefore examined plasma neurofilament heavy-chain (NfH) concentration as a potential biomarker in CMT. Blood samples were collected from healthy controls and patients with CMT over a 2-year period. Disease severity was measured using the CMT Examination Score. An in-house enzyme-linked immunoabsorbent assay was used to measure plasma NfH levels. There was no significant difference in plasma NfH concentrations between CMT patients and controls (P = 0.449). There was also no significant difference in plasma NfH levels in the CMT group over 1 year (mean difference = -0.02, SEM = 4.44, P = 0.98). Plasma NfH levels are not altered in patients with CMT and are not a suitable biomarker of disease activity. Muscle Nerve 53: 972-975, 2016. © 2016 Wiley Periodicals, Inc.

  14. Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.

    Science.gov (United States)

    Morin, Nicole A; Oakes, Patrick W; Hyun, Young-Min; Lee, Dooyoung; Chin, Y Eugene; Chin, Eugene Y; King, Michael R; Springer, Timothy A; Shimaoka, Motomu; Tang, Jay X; Reichner, Jonathan S; Kim, Minsoo

    2008-01-21

    Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

  15. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    Science.gov (United States)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  16. Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state

    Science.gov (United States)

    Adams, G. R.; McCue, S. A.; Zeng, M.; Baldwin, K. M.

    1999-01-01

    During the postnatal period, rat limb muscles adapt to weight bearing via the replacement of embryonic (Emb) and neonatal (Neo) myosin heavy chains (MHCs) by the adult isoforms. Our aim was to characterize this transition in terms of the six MHC isoforms expressed in skeletal muscle and to determine the importance of innervation and thyroid hormone status on the attainment of the adult MHC phenotype. Neonatal rats were made hypothyroid via propylthiouracil (PTU) injection. In normal and PTU subgroups, leg muscles were unilaterally denervated at 15 days of age. The MHC profiles of plantaris (PLN) and soleus (Sol) muscles were determined at 7, 14, 23, and 30 days postpartum. At day 7, the Sol MHC profile was 55% type I, 30% Emb, and 10% Neo; in the PLN, the pattern was 60% Neo and 25% Emb. By day 30 the Sol and PLN had essentially attained an adult MHC profile in the controls. PTU augmented slow MHC expression in the Sol, whereas in the PLN it markedly repressed IIb MHC by retaining neonatal MHC expression. Denervation blunted the upregulation of IIb in the PLN and of Type I in the Sol and shifted the pattern to greater expression of IIa and IIx MHCs in both muscles. In contrast to previous observations, these findings collectively suggest that both an intact thyroid and innervation state are obligatory for the attainment of the adult MHC phenotype, particularly in fast-twitch muscles.

  17. Effect of myosin heavy chain composition of muscles on meat quality in Laiwu pigs and Duroc

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to explain the mechanism of high meat quality in Laiwu pigs and investigate the relation between myosin heavy chains (MyHC) composition and meat quality, meat quality analysis was conducted and mRNA expression of MyHC I, IIa, IIx, IIb was quantified by real-time fluorescence PCR in longissimus muscle (LM) and semimembranous muscle of Laiwu pigs and Duroc. The result indicated that, compared with Duroc, mRNA expression of MyHC IIa, IIx in LM and semimembranous muscle of Laiwu pigs was significantly increased, mRNA expression of MyHC IIb was dramatically decreased. However, the expression of MyHC I was not significantly affected by breeds. The correlation between mRNA expression of MyHC I, IIa, IIx in LM and meat color, pH value, marbling, intramuscular fat content was positive, but shear value of LM was negative. The relation between MyHC IIb mRNA expression and marbling, intramuscular fat content was dramatically negative, whereas shear value was strikingly positive, as well as fiber diameter, but without reaching statistical significance. Therefore, the composition of MyHC I, IIa, IIx, IIb affected meat quality, furthermore, expression of MyHC I, IIa, IIx, IIb mRNA prominently influenced meat characteristics, especially edible quality of muscle, suggesting that mRNA expression level of MyHC I, IIa, IIx, IIb can exactly and impersonally estimate meat quality.

  18. Effect of myosin heavy chain composition of muscles on meat quality in Laiwu pigs and Duroc

    Institute of Scientific and Technical Information of China (English)

    HU HongMei; WANG JiYing; ZHU RongSheng; GUO JianFeng; WU Ying

    2008-01-01

    In order to explain the mechanism of high meat quality in Laiwu pigs and investigate the relation between myosin heavy chains (MyHC) composition and meat quality, meat quality analysis was conducted and mRNA expression of MyHC I, IIa, IIx, IIb was quantified by real-time fluorescence PCR in Iongissimus muscle (LM) and semimembranous muscle of Laiwu pigs and Duroc. The result indicated that, compared with Duroc, mRNA expression of MyHC IIa, IIx in LM and semimembranous muscle of Laiwu pigs was significantly increased, mRNA expression of MyHC Ilia'was dramatically decreased.However, the expression of MyHC I was not significantly affected by breeds. The correlation between mRNA expression of MyHC I, IIa, IIx in LM and meat color, pH value, marbling, intramuscular fat content was positive, but shear value of LM was negative. The relation between MyHC lib mRNA expression and marbling, intramuscular fat content was dramatically negative, whereas shear value was strikingly positive, as well as fiber diameter, but without reaching statistical significance. Therefore, the composition of MyHC I, IIa, IIx, IIb affected meat quality, furthermore, expression of MyHC I,IIa, IIx, IIb mRNA prominently influenced meat characteristics, especially edible quality of muscle, suggesting that mRNA expression level of MyHC I, IIa, IIx, IIb can exactly and impersonally estimate meat quality.

  19. Analysis and comparison of the mouse and human immunoglobulin heavy chain JH-Cmu-Cdelta locus.

    Science.gov (United States)

    Koop, B F; Richards, J E; Durfee, T D; Bansberg, J; Wells, J; Gilliam, A C; Chen, H L; Clausell, A; Tucker, P W; Blattner, F R

    1996-02-01

    We report here 23,686 bases of contiguous DNA sequences from the mouse germline immunoglobulin heavy chain (H) constant (C) mu delta region. The sequence spans the joining (JH) regions, the mu constant region (C mu), the delta constant region (C delta) coding regions, a domain relic, the mu switch region (S mu), seven blocks of simple sequence repeats, a large unique sequence inverted repeat, a large unique sequence forward repeat, and all of the intervening material. A comparison of this 23.7-kb region with the corresponding human C mu/C delta region reveals clear homology in the coding and introns of C mu but not in the 5' flanking J gene segments nor in the intergenic and C delta regions. This mixed pattern of similarity between the human and the mouse sequences contrasts with high levels of similarity found in the T-cell receptor C alpha/C delta region and alpha and beta myosin genes and the very low levels found in the gamma-crystallin, XRCC1, and beta-globin gene clusters. The human and mouse comparison further suggests the incorporation of novel sequences into expressed genes of IgD.

  20. The diagnostic and prognostic value of neurofilament heavy chain levels in immune-mediated optic neuropathies.

    Science.gov (United States)

    Petzold, Axel; Plant, Gordon T

    2012-01-01

    Background. Loss of visual function differs between immune-mediated optic neuropathies and is related to axonal loss in the optic nerve. This study investigated the diagnostic and prognostic value of a biomarker for neurodegeneration, the neurofilament heavy chain (NfH) in three immune-mediated optic neuropathies. Methods. A prospective, longitudinal study including patients with optic neuritis due to multiple sclerosis (MSON, n = 20), chronic relapsing inflammatory optic neuritis (CRION, n = 19), neuromyelitis optica (NMO, n = 9), and healthy controls (n = 28). Serum NfH-SMI35 levels were quantified by ELISA. Findings. Serum NfH-SMI35 levels were highest in patients with NMO (mean 0.79 ± 1.51 ng/mL) compared to patients with CRION (0.13 ± 0.16 ng/mL, P = 0.007), MSON (0.09 ± 0.09, P = 0.008), and healthy controls (0.01 ± 0.02 ng/mL, P = 0.001). High serum NfH-SMI35 levels were related to poor visual outcome. Conclusions. Blood NfH-SMI35 levels are of moderate diagnostic and more important prognostic value in immune-mediated optic neuropathies. We speculate that longitudinal blood NfH levels may help to identify particular disabling events in relapsing conditions.

  1. An electrophoretic study of myosin heavy chain expression in skeletal muscles of the toad Bufo marinus.

    Science.gov (United States)

    Nguyen, L T; Stephenson, G M

    1999-10-01

    In this study we developed an SDS-PAGE protocol which for the first time separates effectively all myosin heavy chain (MHC) isoforms expected to be expressed in iliofibularis (IF), pyriformis (PYR), cruralis (CRU) and sartorius (SAR) muscles of the toad Bufo marinus on the basis of previously reported fibre type composition. The main feature of the method is the use of alanine instead of glycine both in the separating gel and in the running buffer. The correlation between the MHC isoform composition of IF, SAR and PYR muscles determined in this study and the previously reported fibre type composition of IF and SAR muscles in the toad and of PYR muscle in the frog was used to tentatively identify the MHC isoforms expressed by twitch fibre types 1, 2 and 3 and by tonic fibres. The alanine-SDS electrophoretic method was employed to examine changes in the MHC composition of IF, PYR, CRU and SAR muscles with the ontogenetic growth of the toad from post-natal life (body weight muscle observed in this study are in very good agreement with those in the fibre type composition of the developing IF muscle reported in the literature.

  2. The Diagnostic and Prognostic Value of Neurofilament Heavy Chain Levels in Immune-Mediated Optic Neuropathies

    Directory of Open Access Journals (Sweden)

    Axel Petzold

    2012-01-01

    Full Text Available Background. Loss of visual function differs between immune-mediated optic neuropathies and is related to axonal loss in the optic nerve. This study investigated the diagnostic and prognostic value of a biomarker for neurodegeneration, the neurofilament heavy chain (NfH in three immune-mediated optic neuropathies. Methods. A prospective, longitudinal study including patients with optic neuritis due to multiple sclerosis (MSON, n=20, chronic relapsing inflammatory optic neuritis (CRION, n=19, neuromyelitis optica (NMO, n=9, and healthy controls (n=28. Serum NfH-SMI35 levels were quantified by ELISA. Findings. Serum NfH-SMI35 levels were highest in patients with NMO (mean 0.79±1.51 ng/mL compared to patients with CRION (0.13±0.16 ng/mL, P=0.007, MSON (0.09±0.09, P=0.008, and healthy controls (0.01±0.02 ng/mL, P=0.001. High serum NfH-SMI35 levels were related to poor visual outcome. Conclusions. Blood NfH-SMI35 levels are of moderate diagnostic and more important prognostic value in immune-mediated optic neuropathies. We speculate that longitudinal blood NfH levels may help to identify particular disabling events in relapsing conditions.

  3. Prognostic value of serum heavy/light chain ratios in patients with POEMS syndrome.

    Science.gov (United States)

    Wang, Chen; Su, Wei; Cai, Qian-Qian; Cai, Hao; Ji, Wei; Di, Qian; Duan, Ming-Hui; Cao, Xin-Xin; Zhou, Dao-Bin; Li, Jian

    2016-07-01

    POEMS syndrome is a rare plasma cell dyscrasia. Serum concentrations of the monoclonal protein in this disorder are typically low, and inapplicable to monitor disease activity in most cases, resulting in limited practical and prognostic values. Novel immunoassays measuring isotype-specific heavy/light chain (HLC) pairs showed its utility in disease monitoring and outcome prediction in several plasma cell dyscrasias. We report results of HLC measurements in 90 patients with POEMS syndrome. Sixty-six patients (73%; 95% confidence interval, 63-82%) had an abnormal HLC ratio at baseline. It could stratify the risk of disease relapse and was strongly associated with worse progression-free survival in a multivariate analysis (P = 0.021; hazard ratio [HR] 6.89, 95% CI 1.34-35.43). After therapy, HLC ratios improved, with 43 patients (48%) remaining abnormal. The post-therapeutic HLC ratio, if abnormal, also remained as an independent prognostic factor associated with worse progression-free survival (P = 0.019; HR 4.30, 95% CI 1.27-14.56). These results suggest the prognostic utility of HLC ratios in clinical management of POEMS patients. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Possible deletion of a developmentally regulated heavy-chain variable region gene in autoimmune diseases

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Pei-Ming; Olee, Tsaiwei; Kozin, F.; Carson, D.A.; Chen, P.P. (Research Institute of Scripps Clinic, La Jolla, CA (USA)); Olsen, N.J. (Vanderbilt Univ., Nashville, TN (USA)); Siminovitch, K.A. (Univ. of Toronto (Canada))

    1990-10-01

    Several autoantibody-associated variable region (V) genes are preferentially expressed during early ontogenic development, suggesting strongly that they are of developmental and physiological importance. As such, it is possible that polymorphisms in one or more of these genes may alter susceptibility to autoimmune disease. The authors have searched extensively for a probe related to a developmentally regulated V gene that has the power to differentiate among highly homologous V genes in human populations. Using such a probe (i.e., Humhv3005/P1) related to both anti-DNA and anti-IgG autoantibodies, they studied restriction fragment length polymorphisms in patients with rheumatoid arthritis and systemic lupus erythematosus and found an apparent heavy-chain V (V{sub H}) gene deletion that was nearly restricted to the autoimmune patients. These data suggest that deletions of physiologically important V{sub H} genes may increase the risk of autoimmunity through indirect effects on the development and homeostasis of the B-cell repertoire.

  5. [The role of the assessment of heavy/light chain pairs of immunoglobulin in monoclonal gammopathies].

    Science.gov (United States)

    Ščudla, Vlastimil; Pika, Tomáš; Minařík, Jiří

    2015-01-01

    The aim of the paper is to inform about the contribution of novel, highly sensitive analytic technique for the assessment of serum immunoglobulins (Hevylite), enabling separate quantitative assessment of heavy/light chain pairs of immunoglobulin (HLC), i. e. the monoclonal ("involved") and polyclonal ("noninvolved") isotype including their ratio (HLC-r) in monoclonal gammopathies. We particularly target the characteristics of this technique, the compari-son of its clinical contribution with standard methods used in the diagnostics, course and the detection of relapse and progression of the disease, as well as the stratification, assessment of therapeutic outcome and prognosis in monoclonal gammopathy of undetermined significance, multiple myeloma, Waldenström´s macroglobulinemia, systemic AL-amyloidosis and some non-Hodgkin lymphomas. Present results show that in comparison with existing routinely used techniques the Hevylite method enriches clinical practice with the assessment of serum levels of "uninvolved" Ig. It enables the evaluation of the depth of "immunoparesis", and the determination of HLC-r index that is needful for the stratification of MM into "risk cohorts". It also contributes to prognostic assessment and improvement of the evaluation of the depth of therapeutic response. In MGUS individuals the HLC-r index provides information about the risk of malignant transformation. We await the results of ongoing validation studies that are expected to provide specific indications for Hevylite technique for MG in routine practice.

  6. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    Science.gov (United States)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  7. Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D; Becerril, Baltazar

    2011-02-25

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

  8. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2011-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity. PMID:21156801

  9. Antibody

    Science.gov (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  10. Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

    Institute of Scientific and Technical Information of China (English)

    Dao-Feng Yang; Hui-Fen Zhu; Zhi-Hua Wang; Guan-Xin Shen; De-Ying Tian

    2005-01-01

    AIM: To construct fusion protein of a single-chain antibody(scFv) against transferrin receptor (TfR) with alkalinephosphatase (AP).METHODS: The VH-linker-VL, namely scFv gene, wasprepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by SfiⅠ and NotⅠ, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E. coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and NotⅠ restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E. coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity.CONCLUSION: We have successfully prepared the antihuman TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

  11. Estimating Copy Number and Allelic Variation at the Immunoglobulin Heavy Chain Locus Using Short Reads.

    Directory of Open Access Journals (Sweden)

    Shishi Luo

    2016-09-01

    Full Text Available The study of genomic regions that contain gene copies and structural variation is a major challenge in modern genomics. Unlike variation involving single nucleotide changes, data on the variation of copy number is difficult to collect and few tools exist for analyzing the variation between individuals. The immunoglobulin heavy variable (IGHV locus, which plays an integral role in the adaptive immune response, is an example of a complex genomic region that varies in gene copy number. Lack of standard methods to genotype this region prevents it from being included in association studies and is holding back the growing field of antibody repertoire analysis. Here we develop a method that takes short reads from high-throughput sequencing and outputs a genetic profile of the IGHV locus with the read coverage depth and a putative nucleotide sequence for each operationally defined gene cluster. Our operationally defined gene clusters aim to address a major challenge in studying the IGHV locus: the high sequence similarity between gene segments in different genomic locations. Tests on simulated data demonstrate that our approach can accurately determine the presence or absence of a gene cluster from reads as short as 70 bp. More detailed resolution on the copy number of gene clusters can be obtained from read coverage depth using longer reads (e.g., ≥ 100 bp. Detail at the nucleotide resolution of single copy genes (genes present in one copy per haplotype can be determined with 250 bp reads. For IGHV genes with more than one copy, accurate nucleotide-resolution reconstruction is currently beyond the means of our approach. When applied to a family of European ancestry, our pipeline outputs genotypes that are consistent with the family pedigree, confirms existing multigene variants and suggests new copy number variants. This study paves the way for analyzing population-level patterns of variation in IGHV gene clusters in larger diverse datasets and for

  12. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

    Directory of Open Access Journals (Sweden)

    Kazuhisa Sugimura

    2013-02-01

    Full Text Available An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe.

  13. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments.

    Science.gov (United States)

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C; Johnson, Jennifer L; Entzminger, Kevin; Jain, Avni; Heaner, David P; Morales, Ivan A; Truskett, Thomas M; Maynard, Jennifer A; Lieberman, Raquel L

    2014-09-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three-dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts.

  14. Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies.

    Science.gov (United States)

    Prechl, J; Tchorbanov, A; Horváth, A; Baiu, D C; Hazenbos, W; Rajnavölgyi, E; Kurucz, I; Capel, P J; Erdei, A

    1999-05-01

    Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

  15. A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Juárez-González, Victor Rivelino; Olamendi-Portugal, Timoteo; Ortíz-León, Mauricio; Possani, Lourival Domingos; Becerril, Baltazar

    2005-05-01

    This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

  16. The repertoire of heavy chain immunoglobulin genes in B‑cell chronic lymphocytic leukemia in Russia and Belarus

    Directory of Open Access Journals (Sweden)

    B. V. Biderman

    2012-01-01

    Full Text Available Mutation status of the heavy chain variable region genes has long been known as an important factor in long‑term prognosis in B‑cell chronic lymphocytic leukemia (B‑CLL. A more detailed study of the gene sequences of immunoglobulin heavy chain (IgVH led to the discovery of stereotyped antigen receptors (SAR — receptors that have the same set of VH‑, D‑ and JH‑genes used. Cells with SARs have been found almost in a quarter of all B‑CLL cases. This phenomenon is not observed in other lymphatic tumors. In our study, we confirmed and extended the basic observations concerning the repertoire of IgVH in B‑CLL. Differences in the B‑CLL IgVH gene repertoirs between Russia, Вelarus and other countries are also analysed and discussed.

  17. The repertoire of heavy chain immunoglobulin genes in B‑cell chronic lymphocytic leukemia in Russia and Belarus

    Directory of Open Access Journals (Sweden)

    B. V. Biderman

    2014-07-01

    Full Text Available Mutation status of the heavy chain variable region genes has long been known as an important factor in long‑term prognosis in B‑cell chronic lymphocytic leukemia (B‑CLL. A more detailed study of the gene sequences of immunoglobulin heavy chain (IgVH led to the discovery of stereotyped antigen receptors (SAR — receptors that have the same set of VH‑, D‑ and JH‑genes used. Cells with SARs have been found almost in a quarter of all B‑CLL cases. This phenomenon is not observed in other lymphatic tumors. In our study, we confirmed and extended the basic observations concerning the repertoire of IgVH in B‑CLL. Differences in the B‑CLL IgVH gene repertoirs between Russia, Вelarus and other countries are also analysed and discussed.

  18. Effects of Opiates and HIV Proteins on Neurons: The Role of Ferritin Heavy Chain and a Potential for Synergism

    OpenAIRE

    Festa, Lindsay; Meucci, Olimpia

    2012-01-01

    Human immunodeficiency virus 1 (HIV-1) and its associated proteins can have a profound impact on the central nervous system. Co-morbid abuse of opiates, such as morphine and heroin, is often associated with rapid disease progression and greater neurological dysfunction. The mechanisms by which HIV proteins and opiates cause neuronal damage on their own and together are unclear. The emergence of ferritin heavy chain (FHC) as a negative regulator of the chemokine receptor CXCR4, a co-receptor f...

  19. Large-scale chromatin remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene regulation.

    Science.gov (United States)

    Bolland, Daniel J; Wood, Andrew L; Corcoran, Anne E

    2009-01-01

    V(D)J recombination in lymphocytes is the cutting and pasting together of antigen receptor genes in cis to generate the enormous variety of coding sequences required to produce diverse antigen receptor proteins. It is the key role of the adaptive immune response, which must potentially combat millions of different foreign antigens. Most antigen receptor loci have evolved to be extremely large and contain multiple individual V, D and J genes. The immunoglobulin heavy chain (Igh) and immunoglobulin kappa light chain (Igk) loci are the largest multigene loci in the mammalian genome and V(D)J recombination is one of the most complicated genetic processes in the nucleus. The challenge for the appropriate lymphocyte is one of macro-management-to make all of the antigen receptor genes in a particular locus available for recombination at the appropriate developmental time-point. Conversely, these large loci must be kept closed in lymphocytes in which they do not normally recombine, to guard against genomic instability generated by the DNA double strand breaks inherent to the V(D)J recombination process. To manage all of these demanding criteria, V(D)J recombination is regulated at numerous levels. It is restricted to lymphocytes since the Rag genes which control the DNA double-strand break step of recombination are only expressed in these cells. Within the lymphocyte lineage, immunoglobulin recombination is restricted to B-lymphocytes and TCR recombination to T-lymphocytes by regulation of locus accessibility, which occurs at multiple levels. Accessibility of recombination signal sequences (RSSs) flanking individual V, D and J genes at the nucleosomal level is the key micro-management mechanism, which is discussed in greater detail in other chapters. This chapter will explore how the antigen receptor loci are regulated as a whole, focussing on the Igh locus as a paradigm for the mechanisms involved. Numerous recent studies have begun to unravel the complex and

  20. Interferon-γ causes cardiac myocyte atrophy via selective degradation of myosin heavy chain in a model of chronic myocarditis.

    Science.gov (United States)

    Cosper, Pippa F; Harvey, Pamela A; Leinwand, Leslie A

    2012-12-01

    Interferon-γ (IFN-γ), a proinflammatory cytokine, has been implicated in the pathogenesis of a number of forms of heart disease including myocarditis and congestive heart failure. In fact, overexpression of IFN-γ in mice causes dilated cardiomyopathy. However, the direct effects of IFN-γ on cardiac myocytes and the mechanism by which it causes cardiac dysfunction have not been described. Here, we present the molecular pathology of IFN-γ exposure and its effect on myofibrillar proteins in isolated neonatal rat ventricular myocytes. Treatment with IFN-γ caused cardiac myocyte atrophy attributable to a specific decrease in myosin heavy chain protein. This selective degradation of myosin heavy chain was not accompanied by a decrease in total protein synthesis or by an increase in total protein degradation. IFN-γ increased both proteasome and immunoproteasome activity in cardiac myocytes and their inhibition blocked myosin heavy chain loss and myocyte atrophy, whereas inhibition of the lysosome or autophagosome did not. Collectively, these results provide a mechanism by which IFN-γ causes cardiac pathology in the setting of chronic inflammatory diseases. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. Effect of Fetal Hypothyroidism on Cardiac Myosin Heavy Chain Expression in Male Rats

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    Nasibeh Yousefzadeh

    2016-01-01

    Full Text Available Abstract Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05 and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05 were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, β-MHC expression was higher (201% and α- MHC expression was lower (47% than control. Conclusion: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of β-MHC to α- MHC ratio in the heart.

  2. Myosin heavy chain 15 is associated with bovine pulmonary arterial pressure.

    Science.gov (United States)

    Neary, Marianne T; Neary, Joseph M; Lund, Gretchen K; Holt, Timothy N; Garry, Franklyn B; Mohun, Timothy J; Breckenridge, Ross A

    2014-09-01

    Bovine pulmonary hypertension, brisket disease, causes significant morbidity and mortality at elevations above 2,000 m. Mean pulmonary arterial pressure (mPAP) is moderately heritable, with inheritance estimated to lie within a few major genes. Invasive mPAP measurement is currently the only tool available to identify cattle at risk of hypoxia-induced pulmonary hypertension. A genetic test could allow selection of cattle suitable for high altitude without the need for invasive testing. In this study we evaluated three candidate genes (myosin heavy chain 15 [MYH15], NADH dehydrogenase flavoprotein 2, and FK binding protein 1A) for association with mPAP in 166 yearling Angus bulls grazing at 2,182 m. The T allele (rs29016420) of MYH15 was linked to lower mPAP in a dominant manner (CC 47.2 ± 1.6 mmHg [mean ± standard error of the mean]; CT/TT 42.8 ± 0.7 mmHg; P = 0.02). The proportions of cattle with MYH15 CC, CT, and TT genotypes were 55%, 41%, and 4%, respectively. Given the high frequency of the deleterious allele, it is likely that the relative contribution of MYH15 polymorphisms to pulmonary hypertension is small, supporting previous predictions that the disease is polygenic. We evaluated allelic frequency of MYH15 in the Himalayan yak (Bos grunniens), a closely related species adapted to high altitude, and found 100% prevalence of T allele homozygosity. In summary, we identified a polymorphism in MYH15 significantly associated with mPAP. This finding may aid selection of cattle suitable for high altitude and contribute to understanding human hypoxia-induced pulmonary hypertension.

  3. Role of kinesin heavy chain in Crumbs localization along the rhabdomere elongation in Drosophila photoreceptor.

    Directory of Open Access Journals (Sweden)

    Garrett P League

    Full Text Available BACKGROUND: Crumbs (Crb, a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ, and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc, a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors. METHODOLOGY/PRINCIPAL FINDINGS: Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc's gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors. CONCLUSIONS/SIGNIFICANCE: In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in

  4. Effect of Fetal Hypothyroidism on Cardiac Myosin Heavy Chain Expression in Male Rats

    Science.gov (United States)

    Yousefzadeh, Nasibeh; Jeddi, Sajad; Alipour, Mohammad Reza

    2016-01-01

    Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05) were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, β-MHC expression was higher (201%) and α- MHC expression was lower (47%) than control. Conclusion: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of β-MHC to α- MHC ratio in the heart. PMID:27411095

  5. Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Crew, Jennifer R.; Falzari, Kanakeshwari [Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (United States); DiMario, Joseph X., E-mail: joseph.dimario@rosalindfranklin.edu [Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (United States)

    2010-04-01

    Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-{gamma} co-activator-1 (PGC-1{alpha}) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.

  6. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

    Science.gov (United States)

    Velten, Brandy P; Welch, Kenneth C

    2014-06-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. Copyright © 2014 the American Physiological Society.

  7. Myosin heavy chain 15 is associated with bovine pulmonary arterial pressure

    Science.gov (United States)

    Neary, Joseph M.; Lund, Gretchen K.; Holt, Timothy N.; Garry, Franklyn B.; Mohun, Timothy J.; Breckenridge, Ross A.

    2014-01-01

    Abstract Bovine pulmonary hypertension, brisket disease, causes significant morbidity and mortality at elevations above 2,000 m. Mean pulmonary arterial pressure (mPAP) is moderately heritable, with inheritance estimated to lie within a few major genes. Invasive mPAP measurement is currently the only tool available to identify cattle at risk of hypoxia-induced pulmonary hypertension. A genetic test could allow selection of cattle suitable for high altitude without the need for invasive testing. In this study we evaluated three candidate genes (myosin heavy chain 15 [MYH15], NADH dehydrogenase flavoprotein 2, and FK binding protein 1A) for association with mPAP in 166 yearling Angus bulls grazing at 2,182 m. The T allele (rs29016420) of MYH15 was linked to lower mPAP in a dominant manner (CC 47.2 ± 1.6 mmHg [mean ± standard error of the mean]; CT/TT 42.8 ± 0.7 mmHg; P = 0.02). The proportions of cattle with MYH15 CC, CT, and TT genotypes were 55%, 41%, and 4%, respectively. Given the high frequency of the deleterious allele, it is likely that the relative contribution of MYH15 polymorphisms to pulmonary hypertension is small, supporting previous predictions that the disease is polygenic. We evaluated allelic frequency of MYH15 in the Himalayan yak (Bos grunniens), a closely related species adapted to high altitude, and found 100% prevalence of T allele homozygosity. In summary, we identified a polymorphism in MYH15 significantly associated with mPAP. This finding may aid selection of cattle suitable for high altitude and contribute to understanding human hypoxia-induced pulmonary hypertension. PMID:25621163

  8. Expression of myosin heavy-chain mRNA in cultured myoblasts induced by centrifugal force.

    Science.gov (United States)

    Kurokawa, Katsuhide; Sakiyama, Koji; Abe, Shinichi; Hiroki, Emi; Naito, Kaoru; Nakajima, Kazunori; Takeda, Tomotaka; Inoue, Takashi; Ide, Yoshinobu; Ishigami, Keiichi

    2008-11-01

    Ballistic muscle training leads to hypertrophy of fast type fibers and training for endurance induces that of slow type fibers. Numerous studies have been conducted on electrical, extending and magnetic stimulation of cells, but the effect of centrifugal force on cells remains to be investigated. In this study, we investigated the effect of stimulating cultured myoblasts with centrifugal force at different speeds on cell proliferation and myosin heavy-chain (MyHC) mRNA expression in muscle fiber. Stimulation of myoblasts was carried out at 2 different speeds for 20 min using the Himac CT6D, a desk centrifuge, and cells were observed at 1, 3 and 5 days later. Number of cells 1 and 5 days after centrifugal stimulation was significantly larger in the 62.5 x g and 4,170 x g stimulation groups than in the control group. Expression of MyHC-2b mRNA 1 day after centrifugal stimulation was significantly higher in the 2 stimulation groups than in the control group. Almost no expression of MyHC-2a was observed in any group at 1 and 3 days after centrifugal stimulation. However, 5 days after stimulation, MyHC-2a was strongly expressed in the 2 stimulation groups in comparison to the control group. Three days after centrifugal stimulation, expression of MyHC-1 was significantly higher in the 2 stimulation groups than in the control group. The results of this study clarified the effect of different centrifugal stimulation speeds on muscle fiber characteristics, and suggest that centrifugal stimulation of myoblasts enhances cell proliferation.

  9. Signal peptide-dependent inhibition of MHC class I heavy chain translation by rhesus cytomegalovirus.

    Science.gov (United States)

    Powers, Colin J; Früh, Klaus

    2008-10-03

    The US2-11 region of human and rhesus cytomegalovirus encodes a conserved family of glycoproteins that inhibit MHC-I assembly with viral peptides, thus preventing cytotoxic T cell recognition. Since HCMV lacking US2-11 is no longer able to block assembly and transport of MHC-I, we examined whether this is also observed for RhCMV lacking the corresponding region. Unexpectedly, recombinant RhCMV lacking US2-11 was still able to inhibit MHC-I expression in infected fibroblasts, suggesting the presence of an additional MHC-I evasion mechanism. Progressive deletion analysis of RhCMV-specific genomic regions revealed that MHC-I expression is fully restored upon additional deletion of rh178. The protein encoded by this RhCMV-specific open reading frame is anchored in the endoplasmic reticulum membrane. In the presence of rh178, RhCMV prevented MHC-I heavy chain (HC) expression, but did not inhibit mRNA transcription or association of HC mRNA with translating ribosomes. Proteasome inhibitors stabilized a HC degradation intermediate in the absence of rh178, but not in its presence, suggesting that rh178 prevents completion of HC translation. This interference was signal sequence-dependent since replacing the signal peptide with that of CD4 or murine HC rendered human HCs resistant to rh178. We have identified an inhibitor of antigen presentation encoded by rhesus cytomegalovirus unique in both its lack of homology to any other known protein and in its mechanism of action. By preventing signal sequence-dependent HC translocation, rh178 acts prior to US2, US3 and US11 which attack MHC-I proteins after protein synthesis is completed. Rh178 is the first viral protein known to interfere at this step of the MHC-I pathway, thus taking advantage of the conserved nature of HC leader peptides, and represents a new mechanism of translational interference.

  10. Shifts in the myosin heavy chain isozymes in the mouse heart result in increased energy efficiency

    Science.gov (United States)

    Hoyer, Kirsten; Krenz, Maike; Robbins, Jeffrey; Ingwall, Joanne S.

    2007-01-01

    Cardiac-specific transgenesis in the mouse is widely used to study the basic biology and chemistry of the heart and to model human cardiovascular disease. A fundamental difference between mouse and human hearts is the background motor protein: mouse hearts contain predominantly the αα-myosin heavy chain (MyHC) isozyme while human hearts contain predominantly the ββ-MyHC isozyme. Although the intrinsic differences in mechanical and enzymatic properties of the αα- and ββ-MyHC molecules are well known, the consequences of isozyme shifts on energetic of the intact beating heart remain unknown. Therefore, we compared the free energy of ATP hydrolysis (|ΔG~ATP|) determined by 31P NMR spectroscopy in isolated perfused littermate mouse hearts containing the same amount of myosin comprised of either >95% αα-MyHC or ~83% ββ-MyHC. |ΔG~ATP| was ~2 kJ mol−1 higher in the ββ-MyHC hearts at all workloads. Furthermore, upon inotropic challenge, hearts containing predominantly ββ-MyHC hearts increased developed pressure more than αα-MyHC hearts whereas heart rate increased more in αα-MyHC hearts. Thus, hearts containing predominantly the ββ-MyHC isozyme are more energy efficient than αα-MyHC hearts. We suggest that these fundamental differences in the motor protein energy efficiency at the whole heart level should be considered when interpreting results using mouse-based cardiovascular modeling of normal and diseased human heart. PMID:17054980

  11. Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial

    Science.gov (United States)

    Ackerman, Margaret; Saunders, Kevin O.; Pollara, Justin; Vandergrift, Nathan; Parks, Rob; Michael, Nelson L.; O’Connell, Robert J.; Vasan, Sandhya; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Sinangil, Faruk; Phogat, Sanjay; Alam, S. Munir; Liao, Hua-Xin; Ferrari, Guido; Seaman, Michael S.; Montefiori, David C.; Harrison, Stephen C.; Haynes, Barton F.

    2017-01-01

    The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6–8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains. Trial Registration: ClinicalTrials.gov NCT01435135 PMID:28235027

  12. Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial.

    Science.gov (United States)

    Easterhoff, David; Moody, M Anthony; Fera, Daniela; Cheng, Hao; Ackerman, Margaret; Wiehe, Kevin; Saunders, Kevin O; Pollara, Justin; Vandergrift, Nathan; Parks, Rob; Kim, Jerome; Michael, Nelson L; O'Connell, Robert J; Excler, Jean-Louis; Robb, Merlin L; Vasan, Sandhya; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Sinangil, Faruk; Tartaglia, James; Phogat, Sanjay; Kepler, Thomas B; Alam, S Munir; Liao, Hua-Xin; Ferrari, Guido; Seaman, Michael S; Montefiori, David C; Tomaras, Georgia D; Harrison, Stephen C; Haynes, Barton F

    2017-02-01

    The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6-8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains. ClinicalTrials.gov NCT01435135.

  13. Antiproliferative and Apoptotic Effects of a Specific Antiprostate Stem Cell Single Chain Antibody on Human Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Foroogh Nejatollahi

    2013-01-01

    Full Text Available Prostate stem cell antigen (PSCA is a highly glycosylated cell surface protein which is overexpressed in several malignancies including prostate, pancreas, and urinary bladder cancers. Tumor suppression has been reported by anti-PSCA antibody. Small and high affinity single chain antibodies (scFv have been introduced as effective agents for cancer immunotargeting approaches. In the present study, we used a phage antibody display library of scFv and selected two antibodies against two immunodominant epitopes of PSCA by panning process. The reactivity of the scFvs for the corresponding epitopes was determined by phage ELISA. The binding specificity of antibodies to PSCA-expressing prostate cancer cell line, DU-145, was analyzed by flow cytometry. The antiproliferative and apoptotic induction effects were evaluated by MTT and Annexin-V assays, respectively. Results represented functional scFv C5-II which could bind specifically to DU-145 cells and significantly inhibited the proliferation of these cells (61% with no effect on PSCA-negative cells. The antibody also induced apoptosis in the PSCA expressing cells. The percentage of the apoptotic cells after 24 hrs of exposure to 500 scFv/cell was 33.80%. These results demonstrate that the functional anti-PSCA scFv C5-II has the potential to be considered as a new agent for targeted therapy of prostate cancer.

  14. Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

    Science.gov (United States)

    Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

    2000-03-01

    MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

  15. Evaluation of canine and feline leishmaniasis by the association of blood culture, immunofluorescent antibody test and polymerase chain reaction

    OpenAIRE

    Campos Braga, Audrey Renno; Langoni, Hélio; Lucheis, Simone Baldini [UNESP

    2014-01-01

    Background: This study aimed to evaluate the occurrence of Leishmania spp. in dogs and cats from Botucatu, Sao Paulo state, and Campo Grande, Mato Grosso do Sul state, Brazil, by the association of three diagnostic tests: blood culture in liver infusion tryptose medium, immunofluorescent antibody test and polymerase chain reaction. Fifty blood samples of dogs and cats from the Center for Zoonosis Control in Campo Grande, an area endemic for canine visceral leishmaniasis, were collected random...

  16. Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses.

    Science.gov (United States)

    Hurwitz, Amy M; Huang, Wanzhi; Kou, Baijun; Estes, Mary K; Atmar, Robert L; Palzkill, Timothy

    2017-01-01

    Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)-named NJT-R3-A2 and NJT-R3-A3-that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.

  17. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    Science.gov (United States)

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  18. Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry

    Institute of Scientific and Technical Information of China (English)

    Yan-Wei Zhong; Jun Cheng; Gang Wang; Shuang-Shuang Shi; Li Li; Ling-Xia Zhang; Ju-Mei Chen

    2002-01-01

    AIM: To screen human single chain Fv antibody (scFv)against hepatitis C virus E2 antigen and identify its applicationin immunohistochemistry.METHODS: The phage antibody library was panned by HCVE2 antigen, which was coated in microtiter plate. After fiverounds of biopanning,56 phage clones were identified specificto HCV E2 antigen. The selected scFv clones were digestedby SfiI/NotI and DNA was sequenced. Then it was subclonedinto the vector pCANTABSE for expression as E-taggedsoluble scFv. The liver tissue sections from normal personand patients with chronic hepatitis B and chronic hepatitis Cwere immunostained with HCV E2 scFv antibody.RESULTS: The data of scFv-E2 DNA digestion and DNAsequencing showed that the scFv gene is composed of 750bp. ELISA and immunohistochemistry demonstrated that thehuman single chain Fy antibody against hepatitis C E2 antigenhas a specific binding character with hepatitis virus E2 antigenand paraffin-embedded tissue, but did not react with liver tissuesfrom healthy persons or patients with chronic hepatitis B.CONCLUSION: We have successfully screened andidentified HCV E2 scFv and the scFv could be used in theimmunostaining of liver tissue sections from patients withchronic hepatitis C.

  19. Distinct functions for the glycans of tapasin and heavy chains in the assembly of MHC class I molecules.

    Science.gov (United States)

    Rizvi, Syed Monem; Del Cid, Natasha; Lybarger, Lonnie; Raghavan, Malini

    2011-02-15

    Complexes of specific assembly factors and generic endoplasmic reticulum (ER) chaperones, collectively called the MHC class I peptide-loading complex (PLC), function in the folding and assembly of MHC class I molecules. The glycan-binding chaperone calreticulin (CRT) and partner oxidoreductase ERp57 are important in MHC class I assembly, but the sequence of assembly events and specific interactions involved remain incompletely understood. We show that the recruitments of CRT and ERp57 to the PLC are codependent and also dependent upon the ERp57 binding site and the glycan of the assembly factor tapasin. Furthermore, the ERp57 binding site and the glycan of tapasin enhance β(2)m and MHC class I heavy (H) chain recruitment to the PLC, with the ERp57 binding site having the dominant effect. In contrast, the conserved MHC class I H chain glycan played a minor role in CRT recruitment into the PLC, but impacted the recruitment of H chains into the PLC, and glycan-deficient H chains were impaired for tapasin-independent and tapasin-assisted assembly. The conserved MHC class I glycan and tapasin facilitated an early step in the assembly of H chain-β(2)m heterodimers, for which tapasin-ERp57 or tapasin-CRT complexes were not required. Together, these studies provide insights into how PLCs are constructed, demonstrate two distinct mechanisms by which PLCs can be stabilized, and suggest the presence of intermediate H chain-deficient PLCs.

  20. Frequency patterns of T-cell exposed motifs in immunoglobulin heavy chain peptides presented by MHCs

    Directory of Open Access Journals (Sweden)

    Robert D. Bremel

    2014-10-01

    Full Text Available Immunoglobulins are highly diverse protein sequences that are processed and presented to T-cells by B-cells and other antigen presenting cells. We examined a large dataset of immunoglobulin heavy chain variable regions (IGHV to assess the diversity of T-cell exposed motifs (TCEM. TCEM comprise those amino acids in a MHC-bound peptide which face outwards, surrounded by the MHC histotope, and which engage the T-cell receptor. Within IGHV there is a distinct pattern of predicted MHC class II binding and a very high frequency of re-use of the TCEMs. The re-use frequency indicates that only a limited number of different cognate T-cells are required to engage many different clonal B-cells. The amino acids in each outward-facing TCEM are intercalated with the amino acids of inward-facing MHC groove-exposed motifs (GEM. Different GEM may have differing, allele-specific, MHC binding affinities. The intercalation of TCEM and GEM in a peptide allows for a vast combinatorial repertoire of epitopes, each eliciting a different response. Outcome of T-cell receptor binding is determined by overall signal strength, which is a function of the number of responding T-cells and the duration of engagement. Hence, the frequency of T-cell exposed motif re-use appears to be an important determinant of whether a T-cell response is stimulatory or suppressive. The frequency distribution of TCEMs implies that somatic hypermutation is followed by clonal expansion that develop along repeated pathways. The observations of TCEM and GEM derived from immunoglobulins suggest a relatively simple, yet powerful, mechanism to correlate T-cell polyspecificity, through re-use of TCEMs, with a very high degree of specificity achieved by combination with a diversity of GEMs. The frequency profile of TCEMs also points to an economical mechanism for maintaining T-cell memory, recall, and self-discrimination based on an endogenously generated profile of motifs.

  1. Signal peptide-dependent inhibition of MHC class I heavy chain translation by rhesus cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Colin J Powers

    Full Text Available The US2-11 region of human and rhesus cytomegalovirus encodes a conserved family of glycoproteins that inhibit MHC-I assembly with viral peptides, thus preventing cytotoxic T cell recognition. Since HCMV lacking US2-11 is no longer able to block assembly and transport of MHC-I, we examined whether this is also observed for RhCMV lacking the corresponding region. Unexpectedly, recombinant RhCMV lacking US2-11 was still able to inhibit MHC-I expression in infected fibroblasts, suggesting the presence of an additional MHC-I evasion mechanism. Progressive deletion analysis of RhCMV-specific genomic regions revealed that MHC-I expression is fully restored upon additional deletion of rh178. The protein encoded by this RhCMV-specific open reading frame is anchored in the endoplasmic reticulum membrane. In the presence of rh178, RhCMV prevented MHC-I heavy chain (HC expression, but did not inhibit mRNA transcription or association of HC mRNA with translating ribosomes. Proteasome inhibitors stabilized a HC degradation intermediate in the absence of rh178, but not in its presence, suggesting that rh178 prevents completion of HC translation. This interference was signal sequence-dependent since replacing the signal peptide with that of CD4 or murine HC rendered human HCs resistant to rh178. We have identified an inhibitor of antigen presentation encoded by rhesus cytomegalovirus unique in both its lack of homology to any other known protein and in its mechanism of action. By preventing signal sequence-dependent HC translocation, rh178 acts prior to US2, US3 and US11 which attack MHC-I proteins after protein synthesis is completed. Rh178 is the first viral protein known to interfere at this step of the MHC-I pathway, thus taking advantage of the conserved nature of HC leader peptides, and represents a new mechanism of translational interference.

  2. Characterisation of the sarcomeric myosin heavy chain multigene family in the laboratory guinea pig

    Directory of Open Access Journals (Sweden)

    Bardsley Ronald G

    2010-06-01

    Full Text Available Abstract Background Several chronic conditions leading to skeletal muscle dysfunction are known to be associated with changes in the expression of myosin heavy chain (MHC isoforms at both the mRNA and protein level. Many of these conditions are modelled, pre-clinically, in the guinea pig due to similar disease onset and progression to the human condition, and their generally well-characterised anatomy. MHC composition is amenable to determination by protein and mRNA based methodologies, the latter quantifying the expression of MHC isoform-specific gene transcripts allowing the detection of earlier, and more subtle changes. As such, the MHC mRNAs, and specific oligonucleotide primers of all common laboratory species have been available for some time. However, due to incomplete genomic annotation, assessment of guinea pig MHC mRNA expression has not been previously possible, precluding the full characterisation of early changes in skeletal muscle in response to disease and disease modulation. The purpose of this study was to characterise the multigenic structure of the sarcomeric MHC family in the guinea pig, and to design and validate specific oligonucleotide primers to enable the assessment of the predominant adult-muscle associated MHC mRNAs in relevant disease models. Results Using a combination of ligase-mediated rapid amplification of 5' and 3' cDNA ends (RACE and bioinformatics, mRNAs to the four main skeletal-muscle isoforms of MHC were determined. Specific oligonucleotide primers were designed, and following verification of their specificity, found to successfully determine the expression of each MHC mRNA independently. Conclusions Because of their utilisation in the in vivo modelling of disease, there is a requirement to develop molecular methods that accurately differentiate the different MHC mRNAs in the guinea pig to enable rapid profiling of muscle composition in appropriate disease models. The methods developed here are suitable for

  3. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    Science.gov (United States)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  4. A novel heavy domain antibody library with functionally optimized complementarity determining regions.

    Directory of Open Access Journals (Sweden)

    Ole Aalund Mandrup

    Full Text Available Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.

  5. Distinct functions for the glycans of tapasin and heavy chains in the assembly of major histocompatibility complex (MHC) class I molecules

    Science.gov (United States)

    Rizvi, Syed Monem; Cid, Natasha Del; Lybarger, Lonnie; Raghavan, Malini

    2011-01-01

    Complexes of specific assembly factors and generic endoplasmic reticulum (ER) chaperones collectively called the major histocompatibility complex (MHC) class I peptide loading complex (PLC) function in the folding and assembly of MHC class I molecules. The glycan binding chaperone calreticulin and partner oxidoreductase ERp57 are important in MHC class I assembly, but the sequence of assembly events and specific interactions involved remain incompletely understood. We show that the recruitments of calreticulin and ERp57 to the PLC are co-dependent and also dependent upon the ERp57 binding site and the glycan of the assembly factor tapasin. Furthermore, the ERp57 binding site and the glycan of tapasin enhance β2m and MHC class I heavy chain recruitment to the PLC, with the ERp57 binding site having the dominant effect. On the other hand, the conserved MHC class I heavy chain glycan played a minor role in calreticulin recruitment into the PLC, but impacted the recruitment of heavy chains into the PLC, and glycan-deficient heavy chains were impaired for tapasin-independent and tapasin-assisted assembly. The conserved MHC class I glycan and tapasin facilitated an early step in the assembly of heavy chain-β2m heterodimers, for which tapasin-ERp57 or tapasin-calreticulin complexes were not required. Together, these studies provide insights into how PLCs are constructed, demonstrate two distinct mechanisms by which PLCs can be stabilized, and suggest the presence of intermediate heavy chain-deficient PLCs. PMID:21263072

  6. Generation and characterization of a human single-chain fragment variable (scFv antibody against cytosine deaminase from Yeast

    Directory of Open Access Journals (Sweden)

    Tombesi Marina

    2008-09-01

    Full Text Available Abstract Background The ability of cytosine deaminase (CD to convert the antifungal agent 5-fluorocytosine (5-FC into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT aiming to improve the therapeutic ratio (benefit versus toxic side-effects of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

  7. Human single chain antibodies against heparin: selection, characterization, and effect on coagulation.

    NARCIS (Netherlands)

    Westerlo, E.M.A. van de; Smetsers, T.F.; Dennissen, M.A.B.A.; Linhardt, R.J.; Veerkamp, J.H.; Muijen, G.N.P. van; Kuppevelt, A.H.M.S.M. van

    2002-01-01

    Heparin, located in mast cells and basophilic granulocytes, is widely used as an anticoagulant. It belongs to a class of linear polysaccharides called glycosaminoglycans (GAGs). Using phage display technology, we have selected 19 unique human antiheparin antibodies. Some antibodies react almost excl

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

    Directory of Open Access Journals (Sweden)

    Gonzalez Gaëlle

    2010-11-01

    Full Text Available Abstract 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

  9. Hotspots for Vitamin-Steroid-Thyroid Hormone Response Elements Within Switch Regions of Immunoglobulin Heavy Chain Loci Predict a Direct Influence of Vitamins and Hormones on B Cell Class Switch Recombination.

    Science.gov (United States)

    Hurwitz, Julia L; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Partridge, Janet F; Maul, Robert W; Gearhart, Patricia J

    2016-03-01

    Vitamin A deficiencies are common throughout the world and have a significant negative influence on immune protection against viral infections. Mouse models demonstrate that the production of IgA, a first line of defense against viruses at mucosal sites, is inhibited in the context of vitamin A deficiency. In vitro, the addition of vitamin A to activated B cells can enhance IgA expression, but downregulate IgE. Previous reports have demonstrated that vitamin A modifies cytokine patterns, and in so doing may influence antibody isotype expression by an indirect mechanism. However, we have now discovered hundreds of potential response elements among Sμ, Sɛ, and Sα switch sites within immunoglobulin heavy chain loci. These hotspots appear in both mouse and human loci and include targets for vitamin receptors and related proteins (e.g., estrogen receptors) in the nuclear receptor superfamily. Full response elements with direct repeats are relatively infrequent or absent in Sγ regions although half-sites are present. Based on these results, we pose a hypothesis that nuclear receptors have a direct effect on the immunoglobulin heavy chain class switch recombination event. We propose that vitamin A may alter S site accessibility to activation-induced deaminase and nonhomologous end-joining machinery, thereby influencing the isotype switch, antibody production, and protection against viral infections at mucosal sites.

  10. Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses

    Science.gov (United States)

    Hurwitz, Amy M.; Huang, Wanzhi; Kou, Baijun; Estes, Mary K.; Atmar, Robert L.; Palzkill, Timothy

    2017-01-01

    Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)—named NJT-R3-A2 and NJT-R3-A3—that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics. PMID:28095447

  11. Tuning of shortening speed in coleoid cephalopod muscle: no evidence for tissue-specific muscle myosin heavy chain isoforms.

    Science.gov (United States)

    Shaffer, Justin F; Kier, William M

    2016-03-01

    The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities.

  12. The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats

    DEFF Research Database (Denmark)

    Sarin, C T; Tack, B F; Kristensen, Torsten;

    1986-01-01

    We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and...

  13. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  14. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  15. Sequence Tolerance of a Highly Stable Single Domain Antibody: Comparison of Computational and Experimental Profiles

    Science.gov (United States)

    2016-09-09

    popular construct of the heavy chain is single-domain antibody (sdAb) chains derived from camelids. The interest in sdAbs lies in their biotechnological ...thermal stable lama single domain antibody specific for Staphylococcus aureus enterotoxin B. BMC Biotechnology 11. 3. Zabetakis D, Anderson GP, Bayya N...neighbors from a Dali search of the single-chain conformers of the assembly. (a) Conformers of A3 where the color green denotes the A chain and blue

  16. Species Differences in the Distribution of the Nonmuscle Myosin Heavy Chain IIB Inserted Isoform in the Brain(Biochemistry)

    OpenAIRE

    Shin-ya, Hagiwara; MASAYUKI, TAKAHASHI; Akihiko, Yamagishi; Division of Biological Sciences, Graduate School of Science, Hokkaido University

    2001-01-01

    The alternatively spliced isoform of the nonmuscle myosin heavy chain IIB (MHC-IIB) with an insert of 21 amino acids near the actin-binding region, MHC-IIB (B2), is expressed specifically in the brain and spinal cord in Mammalia and Aves. We performed immunoblot analyses to elucidate the distribution of MHC-IIB (B2) in the brains of various animals. Nearly half of MHC-IIB existed as the B2 inserted isoform (MHC-IIB (B2)) in the cerebrum of the guinea-pig, rabbit and pig, while the non-B2 inse...

  17. Species Differences in the Distribution of the Nonmuscle Myosin Heavy Chain IIB Inserted Isoform in the Brain

    OpenAIRE

    Hagiwara, Shin-ya; Takahashi, Masayuki; Yamagishi, Akihiko

    2001-01-01

    The alternatively spliced isoform of the nonmuscle myosin heavy chain IIB (MHC-IIB) with an insert of 21 amino acids near the actin-binding region, MHC-IIB(B2), is expressed specifically in the brain and spinal cord in Mammalia and Aves. We performed immunoblot analyses to elucidate the distribution of MHC-IIB(B2) in the brains of various animals. Nearly half of MHC-IIB existed as the B2 inserted isoform (MHC-IIB(B2)) in the cerebrum of the guinea-pig, rabbit and pig, while the non-B2 inserte...

  18. Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.

    Science.gov (United States)

    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.

  19. Multiplex genotype determination at a DNA sequence polymorphism cluster in the human immunoglobulin heavy-chain region

    Energy Technology Data Exchange (ETDEWEB)

    Li, H.; Hood, L. [California Institute of Technology, Pasadena, CA (United States)

    1995-03-20

    We have developed a method for multilocus genotype determination. The method involves using restriction fragment length polymorphisms (RFLPs) for allele discrimination. If a polymorphism is not an RFLP, it is converted into an RFLP during the polymerase chain reaction (PCR). After amplification and restriction enzyme digestion, samples are analyzed by sequential gel loading during electrophoresis. The efficiency of this method was demonstrated by determining the genotypes of 108 semen samples at seven DNA sequence polymorphic sites identified in the human immunoglobulin heavy-chain variable region. It was shown that more than 1000 PCR products could be easily analyzed per day per investigator. To show the reliability of this method, some of the typing results were confirmed by DNA sequence analysis. By computer simulation, most (98%) polymorphisms were shown to be natural or convertible (by changing 1 bp close to or next to each polymorphic site) RFLPs for the commercially available 4-base cutters. 47 refs., 4 figs., 3 tabs.

  20. Efficient expression of single chain variable fragment antibody against paclitaxel using the Bombyx mori nucleopolyhedrovirus bacmid DNA system and its characterizations.

    Science.gov (United States)

    Yusakul, Gorawit; Sakamoto, Seiichi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-07-01

    A single chain variable fragment (scFv), the smallest unit of functional recombinant antibody, is an attractive format of recombinant antibodies for various applications due to its small fragment and possibility of genetic engineering. Hybridoma clone 3A3 secreting anti-paclitaxel monoclonal antibody was used to construct genes encoding its variable domains of heavy (VH) and light (VL) chains. The VH and VL domains were linked to be the PT-scFv3A3 using flexible peptide linker in a format of VH-(GGGGS)5-VL. The PT-scFv3A3 was primarily expressed using the pET28a(+) vector in the Escherichia coli system, and was then further expressed by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Interestingly, the reactivity of PT-scFv3A3 expressed in the hemolymph of B. mori using the BmNPV bacmid DNA system was much higher than that expressed in the E. coli system. Using indirect competitive enzyme-linked immunosorbent assay (icELISA), the PT-scFv3A3 (B. mori) reacted not only with immobilized paclitaxel, but also with free paclitaxel in a concentration-dependent manner, with the linear range of free paclitaxel between 0.156 and 5.00 µg/ml. The PT-scFv3A3 (B. mori) exhibited less cross-reactivity (%) than its parental MAb clone 3A3 against paclitaxel-related compounds, including docetaxel (31.1 %), 7-xylosyltaxol (22.1 %), baccatin III (<0.68 %), 10-deacetylbaccatin III (<0.68 %), 1-hydroxybaccatin I (<0.68 %), and 1-acetoxy-5-deacetylbaccatin I (<0.68 %). With the exception of cephalomannine, the cross-reactivity was slightly increased to 8.50 %. The BmNPV bacmid DNA system was a highly efficient expression system of active PT-scFv3A3, which is applicable for PT-scFv3A3-based immunoassay of paclitaxel. In addition, the PT-scFv3A3 can be applied to evaluate its neutralizing property of paclitaxel or docetaxel toxicity.

  1. Arg9 facilitates the translocation and downstream signal inhibition of an anti-HER2 single chain antibody

    Directory of Open Access Journals (Sweden)

    Hu Yi

    2012-07-01

    Full Text Available Abstract Background HER2 plays a critical role in the pathogenesis of many cancers and is linked to poor prognosis or cancer metastases. Monoclonal antibodies, such as Herceptin against HER2-overexpressing cancers, have showed satisfactory clinical therapeutic effect. However, they have difficulty to surmount obstacles to enter cells or blood–brain barrier. Results In this study, a cell-penetrating peptide Arg9 was linked to the C-terminus of anti-HER2 single chain antibody (MIL5scFv. Flow cytometry, confocal microscopy and electron microscopy analysis all revealed that Arg9 peptide facilitated the penetration of MIL5scFv into HER2-negative cell line NIH3T3 and orientate in mitochondria. More interestingly, Western blot assay showed the potential enhanced bioactivity of MIL5scFv-Arg9 in HER2+ cell line SKOV3, indicating that Arg9 could help large molecules (e.g. antibody to penetrate into cells and therefore enhance its anti-neoplastic function. Conclusions Our work represented an attractive by preliminary strategy to enhance the therapeutic effect of existing antibodies by entering cells easier, or more desirable, surmounting the physical barriers, especially in hard-to-reach cancers such as brain metastases cases.

  2. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    Science.gov (United States)

    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.

  3. Functions of chondroitin sulfate/dermatan sulfate chains in brain development. Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7.

    NARCIS (Netherlands)

    Purushothaman, A.; Fukuda, J.; Mizumoto, S.; Dam, G.B. ten; Kuppevelt, A.H.M.S.M. van; Kitagawa, H.; Mikami, T.; Sugahara, K.

    2007-01-01

    Chondroitin sulfate (CS) and dermatan sulfate (DS) have been implicated in the processes of neural development in the brain. In this study, we characterized developmentally regulated brain CS/DS chains using a single chain antibody, GD3G7, produced by the phage display technique. Evaluation of the s

  4. VH Replacement Footprint Analyzer-I, a Java-Based Computer Program for Analyses of Immunoglobulin Heavy Chain Genes and Potential VH Replacement Products in Human and Mouse.

    Science.gov (United States)

    Huang, Lin; Lange, Miles D; Zhang, Zhixin

    2014-01-01

    VH replacement occurs through RAG-mediated secondary recombination between a rearranged VH gene and an upstream unrearranged VH gene. Due to the location of the cryptic recombination signal sequence (cRSS, TACTGTG) at the 3' end of VH gene coding region, a short stretch of nucleotides from the previous rearranged VH gene can be retained in the newly formed VH-DH junction as a "footprint" of VH replacement. Such footprints can be used as markers to identify Ig heavy chain (IgH) genes potentially generated through VH replacement. To explore the contribution of VH replacement products to the antibody repertoire, we developed a Java-based computer program, VH replacement footprint analyzer-I (VHRFA-I), to analyze published or newly obtained IgH genes from human or mouse. The VHRFA-1 program has multiple functional modules: it first uses service provided by the IMGT/V-QUEST program to assign potential VH, DH, and JH germline genes; then, it searches for VH replacement footprint motifs within the VH-DH junction (N1) regions of IgH gene sequences to identify potential VH replacement products; it can also analyze the frequencies of VH replacement products in correlation with publications, keywords, or VH, DH, and JH gene usages, and mutation status; it can further analyze the amino acid usages encoded by the identified VH replacement footprints. In summary, this program provides a useful computation tool for exploring the biological significance of VH replacement products in human and mouse.

  5. Genetic control of the immune response to staphylococcal nuclease. IX. Recombination between genes determining BALB/c antinuclease idiotypes and the heavy chain allotype locus.

    Science.gov (United States)

    Pisetsky, D S; Riordan, S E; Sachs, D H

    1979-03-01

    The genetic linkage relationship of two antinuclease idiotypes produced by the BALB/c strain was investigated in the backcross (BALB/c x CB.20) X CB.20. These two idiotypes were detected by Lewis rat anti-idiotypic antisera prepared against affinity-purified A/J and SJL antinuclease antibodies, termed the A/J and SJL idiotypes, respectively. Both idiotypes were found to be linked to the IgCHa immunoglobulin heavy chain allotype locus. There was, however, a high frequency of recombination observed between both markers and the IgCHa locus, with eight of 83 backcross animals recombinant for the A/J idiotype and five of 83 recombinant for the SJL idiotype. All such recombinant animals were IgCHb/b homozygotes that had gained one or both idiotypes. These results are consistent with a genetic map of VHr region genes in the BALB/c strain in which genes determining the SJL idiotype are closer to the IgCHa allotype locus than are genes determining the A/J idiotype. This high frequency of recombination may indicate that the chromosome segment containing VH region genes is very large or that it has structural features that promote recombination.

  6. Diagnostic value of clonality of surface immunoglobulin light and heavy chains in malignant lymphoproliferative disorders.

    Science.gov (United States)

    Batata, A; Shen, B

    1993-08-01

    Cell suspensions from the peripheral blood of B-chronic lymphoid leukemias (B-CLL, n = 274) and reactive lymphocytosis (RLC, n = 132) and from solid tissue samples of B-non-Hodgkin's lymphoma (B-NHL, n = 466) and reactive lymphadenopathy (RLA, n = 324) were analyzed to evaluate the diagnostic value of clonality of L- and H- chains in B-CLL and B-NHL. Cutoff levels for monoclonal L-chain (mono-L) and monoclonal H-chain (mono-H) were defined. In B-CLL, the association patterns of L- and H- chains were as follows: mono-L/mono-H, 245 cases (89.42%); mono-L/polyclonal H chain (poly-H), 4 (1.46%); polyclonal L chain (poly-L)/mono-H, 2 (0.73%); poly-L/poly-H, 2 (0.73%); undetected (und)-L/mono-H, 6 (2.19%); and und-L/und-H, 15 (5.47%). In B-NHL, the association patterns were mono-L/mono-H, 433 cases (92.92%); mono-L/poly-H, 4 (0.86%); poly-L/mono-H, 8 (1.72%); poly-L/poly-H, 2 (0.43%); und-L/mono-H, 4 (0.86%); and und-L/und-H, 15 (3.22%). Monoclonality of H chains are complementary to L-chain restriction, especially in the cases with poly-L or und-L, and should be considered as a positive criterion in determining surface immunoglobulin (SIg) clonality. Monoclonality of SIg assessed by both L and H chains is both sensitive and specific for the diagnosis of B-CLL and B-NHL, and their differentiation from RLC and RLA, since none of the cases of RLC and RLA showed monoclonal SIg.

  7. Segmental duplication as one of the driving forces underlying the diversity of the human immunoglobulin heavy chain variable gene region

    Directory of Open Access Journals (Sweden)

    Gao Richeng

    2011-01-01

    Full Text Available Abstract Background Segmental duplication and deletion were implicated for a region containing the human immunoglobulin heavy chain variable (IGHV gene segments, 1.9III/hv3005 (possible allelic variants of IGHV3-30 and hv3019b9 (a possible allelic variant of IGHV3-33. However, very little is known about the ranges of the duplication and the polymorphic region. This is mainly because of the difficulty associated with distinguishing between allelic and paralogous sequences in the IGHV region containing extensive repetitive sequences. Inability to separate the two parental haploid genomes in the subjects is another serious barrier. To address these issues, unique DNA sequence tags evenly distributed within and flanking the duplicated region implicated by the previous studies were selected. The selected tags in single sperm from six unrelated healthy donors were amplified by multiplex PCR followed by microarray detection. In this way, individual haplotypes of different parental origins in the sperm donors could be analyzed separately and precisely. The identified polymorphic region was further analyzed at the nucleotide sequence level using sequences from the three human genomic sequence assemblies in the database. Results A large polymorphic region was identified using the selected sequence tags. Four of the 12 haplotypes were shown to contain consecutively undetectable tags spanning in a variable range. Detailed analysis of sequences from the genomic sequence assemblies revealed two large duplicate sequence blocks of 24,696 bp and 24,387 bp, respectively, and an incomplete copy of 961 bp in this region. It contains up to 13 IGHV gene segments depending on haplotypes. A polymorphic region was found to be located within the duplicated blocks. The variants of this polymorphism unusually diverged at the nucleotide sequence level and in IGHV gene segment number, composition and organization, indicating a limited selection pressure in general. However

  8. Anti-DNA antibodies: Sequencing, cloning, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Barry, M.M.

    1992-01-01

    To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

  9. Bacterial extracellular polymeric substances (EPS): A carrier of heavy metals in the marine food-chain

    Digital Repository Service at National Institute of Oceanography (India)

    Bhaskar, P.V.; Bhosle, N.B.

    The ecological implications of metal binding properties of bacterial EPS and its possible role in the bioaccumulation of pollutants in the marine food-chain was investigated using a partially purified and chemically characterized microbial EPS...

  10. Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

    Science.gov (United States)

    Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

    2012-01-01

    Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

  11. Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune.

    Science.gov (United States)

    Brunsch, Melanie; Schubert, Daniela; Gube, Matthias; Ring, Christiane; Hanisch, Lisa; Linde, Jörg; Krause, Katrin; Kothe, Erika

    2015-01-01

    The white-rot fungus Schizophyllum commune (Agaricomycetes) was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.

  12. Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune.

    Directory of Open Access Journals (Sweden)

    Melanie Brunsch

    Full Text Available The white-rot fungus Schizophyllum commune (Agaricomycetes was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.

  13. Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis

    Directory of Open Access Journals (Sweden)

    Pleckaityte Milda

    2011-11-01

    Full Text Available Abstract Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. Results Single-chain variable fragments of immunoglobulins (scFvs were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. Conclusions Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in

  14. SNPs within the beta myosin heavy chain (MYH7 and the pyruvate kinase muscle (PKM2 genes in horse

    Directory of Open Access Journals (Sweden)

    Vincenzo Russo

    2010-01-01

    Full Text Available Two highly expressed skeletal muscle genes (the MYH7 gene encoding the myosin heavy chain slow/β-cardiac isoform and the PKM2 gene encoding the pyruvate kinase muscle isoforms were investigated with the objective to identify DNA markers in horses. A panel of DNA samples from different horse breeds was analysed using a PCR-single strand conformation polymorphism (SSCP approach. Four and two alleles were identified for the MYH7 and PKM2 loci, respectively. Mendelian inheritance of alleles of the two investigated genes was confirmed analysing horse families. Sequencing of PCR products obtained from the MYH7 and PKM2 genes made it possible to characterise two SSCP alleles for each gene. The polymorphisms found in the MYH7 and PKM2 genes were further studied in 61 and 68 horses of three (Italian Heavy Draught Horse, Italian Saddler and Murgese and five (Franches-Montagnes, Haflinger, Italian Heavy Draught Horse, Murgese and Standardbred breeds, respectively. Allele frequencies of the two loci varied among the considered breeds. The SNPs discovery in MYH7 and PKM2 genes makes it possible to locate new molecular markers to ECA1. The identified markers could be used in association analysis with performance traits in horses.

  15. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  16. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...... surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...

  17. A 10-RESIDUE FRAGMENT OF AN ANTIBODY (MINI-ANTIBODY) DIRECTED AGAINST LYSOZYME AS LIGAND IN IMMUNOAFFINITY CHROMATOGRAPHY

    NARCIS (Netherlands)

    WELLING, GW; VANGORKUM, J; DAMHOF, RA; DRIJFHOUT, JW; BLOEMHOFF, W; WELLINGWESTER, S

    1991-01-01

    The interaction between an antibody molecule and a protein antigen is an example of "natural" protein modelling. Amino acids of the antigen-binding site consisting of three hypervariable segments (L1, L2, L3) of the light (L) and three (H1, H2, H3) of the heavy (H) chain of an antibody molecule inte

  18. cDNA sequence and Fab crystal structure of HL4E10, a hamster IgG lambda light chain antibody stimulatory for γδ T cells.

    Directory of Open Access Journals (Sweden)

    Petra Verdino

    Full Text Available Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αβ T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer.

  19. cDNA sequence and Fab crystal structure of HL4E10, a hamster IgG lambda light chain antibody stimulatory for γδ T cells.

    Science.gov (United States)

    Verdino, Petra; Witherden, Deborah A; Podshivalova, Katie; Rieder, Stephanie E; Havran, Wendy L; Wilson, Ian A

    2011-01-01

    Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αβ T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML) protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer.

  20. SMRT sequencing provides insight into the diversity of the bovine immunoglobulin heavy chain repertoire

    Science.gov (United States)

    The vertebrate immune system produces a diverse antibody repertoire capable of responding to a vast array of antigens. This diversity is generated through a multifaceted process of gene segment recombination and somatic hypermutation or gene conversion. Recent advances in high-throughput sequencin...

  1. Design Method Amelioration of Human Immunoglobulin Gene Primer in Heavy Chain Variable Region%人免疫球蛋白重链可变区基因引物设计方法的改良

    Institute of Scientific and Technical Information of China (English)

    姜晶; 孙颖; 刘红艳; 牟玲; 罗恩杰

    2012-01-01

    针对抗体胚系基因数据库的数据不断更新和完善,为获得人全部免疫球蛋白(Ig)重链可变区基因,改进引物设计方法,自主设计针对可变区基因高度保守的框架区1(FR1)和框架区4(FR4)的引物,提取未经免疫的健康人外周血单个核细胞,通过RT-PCR扩增重链可变区基因.其DNA序列与GenBank数据库和IMGT/V-QUEST软件比对,序列分析符合人免疫球蛋白重链基本框架结构,为胚系基因重排产生的序列.多个克隆的测序结果对比分析显示了良好的多样性.获得的重链序列为研制基因工程抗体及构建噬菌体抗体库奠定了物质基础,也为扩增其他物种Ig可变区基因的引物提供新的设计思路.%Aiming at the continuous renovation and consummation of dala of databank on antibody germ line gene, in order to obtain full gene of heavy chain variable region of human immunoglobin (Ig) , to ameliorate primer design method, a primer was independently designed aiming at variable gene highly conservative region framework 1 ( FR1) and framework 4 ( FR4) , and extracted peripheral blood mononuclear cells (PBMCs) from healthy volunteer, and amplified its heavy chain variable region gene through RT-PCR. After comparison with databank in CenBank and IMGT/V-QUEST software and sequence analysis, the DNA sequence accorded with basic framework structure of human Ig heavy chain, and was the sequence produced by the rearrangement of germ line gene. Comparative analysis of multiple clones sequencing showed a good diversity. The obtained data of heavy chain sequence had provided the material foundation for research and development of gene engineering antibodies and phage libraries, and new design ideas for amplifying Ig variable region gene primers of other species.

  2. Simultaneous expression of displayed and secreted antibodies for antibody screen.

    Directory of Open Access Journals (Sweden)

    Yuanping Zhou

    Full Text Available The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS analysis, as well as in conditioned medium in a secreted version for function analysis.

  3. Chemical Chaperones Increasing Expression Level of Soluble Single-chain Fv Antibody(scFv2F3)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Glutathione peroxidase (GPX) is a selenoenzyme that protects the biomembrane and other cellular components against oxidative damage. The selenium-containing single chain Fv fragment of monoclonal antibody 2F3 (SescFv2F3 ) is a kind of GPX mimic and it has a wide clinical applications because of its high activity and low antigenicity. Se-scFv2F3 is generated by the chemical modification of the single chain Fy fragment of monoclonal antibody 2F3 ( scFv2F3 ), which can be expressed in E. coli. In this article, the effect of chemical chaperones, such as glycerol, glucose, and β-cyclodextrin added to the culture medium, on the expression of soluble scFv2F3 was investigated. The expression level was evaluated by the determination of soluble scFv2F3 contents in the whole cell lysates.The results suggest that both glycerol and β-cyclodextrin greatly increase the expression level of soluble scFv2F3, and β-cyclodextrin is found to be more effective compared with glycerol. Glucose has a slight effect on the expression level of soluble scFv2F3. This is the first example, wherein β-cyclodextrin has been used as a chemical chaperone during the cell culture to improve the expression level of recombinant proteins. In addition, chemical chaperones are found to decrease the toxic effect of IPTG on cells.

  4. Evaluation of canine and feline leishmaniasis by the association of blood culture, immunofluorescent antibody test and polymerase chain reaction.

    Science.gov (United States)

    Braga, Audrey Rennó Campos; Langoni, Hélio; Lucheis, Simone Baldini

    2014-02-24

    This study aimed to evaluate the occurrence of Leishmania spp. in dogs and cats from Botucatu, São Paulo state, and Campo Grande, Mato Grosso do Sul state, Brazil, by the association of three diagnostic tests: blood culture in liver infusion tryptose medium, immunofluorescent antibody test and polymerase chain reaction. Fifty blood samples of dogs and cats from the Center for Zoonosis Control in Campo Grande, an area endemic for canine visceral leishmaniasis, were collected randomly, as well as canine and feline blood samples from the Municipal Kennel and Animal Protection Association in Botucatu, currently considered a transmission-free, non-endemic area. Of the 50 dog blood cultures from Botucatu, three (6%) were positive and of the 50 cats, two (4%) were positive. In Campo Grande, 29 dog blood cultures (58%) were positive and all (100%) cats negative by this test. Polymerase chain reaction detected Leishmania spp. in 100% of dog and cat samples from Botucatu but found all the cats from Campo Grande to be negative. On the other hand, 36 dogs from Campo Grande were positive (72%) by the same technique. Immunofluorescent antibody test in Botucatu found 100% of dogs and cats non-reactive, while in Campo Grande, it detected positivity in 32 dogs (64%) and 15 cats (30%). The results show the importance of not only continuous epidemiological surveillance in areas not endemic for leishmaniasis, but also research for accurate diagnosis of this zoonosis.

  5. Serrumab: a novel human single chain-fragment antibody with multiple scorpion toxin-neutralizing capacities.

    Science.gov (United States)

    Pucca, Manuela Berto; Cerni, Felipe Augusto; Peigneur, Steve; Arantes, Eliane Candiani; Tytgat, Jan; Barbosa, José Elpidio

    2014-01-01

    In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds' maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera.

  6. Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin

    Directory of Open Access Journals (Sweden)

    Zilvinas Dapkunas

    2017-07-01

    Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.

  7. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model.

    Directory of Open Access Journals (Sweden)

    Jyoti Gupta

    Full Text Available We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo of Brugia malayi (B. malayi in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+ and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32 against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23 and pcD-Myo (41.6%±2.45. In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC to B. malayi infective larvae (L3. pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ and anti-inflammatory (IL-4, IL-10 cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of

  8. Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Xueling; Zhou, Tongqing; Zhu, Jiang; Zhang, Baoshan; Georgiev, Ivelin; Wang, Charlene; Chen, Xuejun; Longo, Nancy S.; Louder, Mark; McKee, Krisha; O’Dell, Sijy; Perfetto, Stephen; Schmidt, Stephen D.; Shi, Wei; Wu, Lan; Yang, Yongping; Yang, Zhi-Yong; Yang, Zhongjia; Zhang, Zhenhai; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Doria-Rose, Nicole A.; Connors, Mark; Mullikin, James C.; Nabel, Gary J.; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D.; Mascola, John R. (Tumaini); (NIH); (Duke); (Kilimanjaro Repro.); (IAVI)

    2013-03-04

    Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

  9. The protective effects and underlying mechanism of an anti-oligomeric Aβ42 single-chain variable fragment antibody.

    Science.gov (United States)

    Zhang, Yuan; Chen, Xu; Liu, Jinyu; Zhang, Yingjiu

    2015-12-01

    Oligomeric Aβ42 aggregates have been identified as one of the major neurotoxic components of Alzheimer's disease (AD). Immunotherapy targeted against these Aβ42 aggregates has been proposed as an appropriate therapeutic approach for the treatment of AD. Here, we report an anti-oligomeric Aβ42 single-chain variable fragment (scFv) antibody, named MO6, obtained from the human antibody library of a healthy donor. ScFv MO6 specifically recognized and bound to the oligomeric Aβ42 (Aβ42 oligomers and immature protofibrils; 18-37 kDa), and reduced their levels mainly by blocking their formation, although scFv MO6 also induced disaggregation of Aβ42 aggregates. More importantly, scFv MO6 ameliorated or attenuated Aβ42-induced cytotoxicity and increased cell viability by up to 33%. Furthermore, scFv MO6 efficiently passed through an in vitro blood-brain barrier (BBB) model with a delivery efficiency of 66% after 60 min post-administration. ScFv MO6 is a monovalent antibody with an affinity constant (KD) of 5.2×10(-6) M for Aβ42 oligomers. Molecular docking simulations of Aβ42 to scFv MO6 revealed that the approach and specific binding of scFv MO6 to oligomeric Aβ42 aggregates was achieved by conformational recognition and directed induction, which resulted in a more dynamic adaptation of Aβ42 to scFv MO6, occurring mainly in the N-terminal (3-4), middle (12-19) and C-terminal (34-42) regions of Aβ42. This binding mode of scFv MO6 to Aβ42 explains its protective effects against oligomeric Aβ42. Our findings may be applied for the design of a smaller antibody specific for Aβ42 oligermers.

  10. Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures.

    Science.gov (United States)

    Gao, Feng-shan; Bai, Jing; Zhang, Qiang; Xu, Chong-bo; Li, Yanmin

    2012-07-10

    Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and β(2)m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-β(2)m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MBP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-β(2)m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1 kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins, SLA-I monomeric proteins of 41.6 kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an α-helical structure, and the average α-helix, β-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas

    2003-01-01

    conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules...... with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta...

  12. Evidence for gene conversion among immunoglobulin heavy chain variable region genes.

    Science.gov (United States)

    Clarke, S H; Rudikoff, S

    1984-03-01

    We have previously reported that the VH region amino acid sequence of a phosphocholine (PC)-binding hybridoma antibody of CBA/J origin, HP101 6G6 (6G6), differs extensively from the VH regions of other PC-binding antibodies. The sequence of 6G6 VH appears to be derived from a gene homologous to the BALB/c V11 gene, a member of the PC VH (T15 VH) gene family not normally used to encode PC-binding antibodies. The 6G6 VH sequence differs from the translated sequence of V11 by six amino acids, four of which occur at the same position in other members of this gene family. This coincidence led to the proposal that the 6G6 VH gene was derived by gene conversion involving three genes of the PC VH gene family. We report here the nucleic acid sequence of the rearranged VH gene of hybridoma 6G6. This sequence supports our previous suggestion of gene conversion by confirming those differences, relative to the BALB/c V11 gene sequence, that are encoded by other members of this gene family, and extends this correlation to include three silent base pair substitutions as well. In addition, 5' noncoding region sequence and Southern blot analysis using probes derived from the coding and 5' noncoding regions confirm that the 6G6 VH gene is likely to be derived from the V11 homologue in CBA/J mice, and suggest that all three genes believed to be involved in the generation of the 6G6 VH gene are present in the CBA/J genome, a prerequisite for their involvement in gene conversion.

  13. Study on the Prediction Method of the First Heavy Rain in the Spring of Xi an City Based on Markov Chain

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    [Objective] The aim was to study the Markov chain prediction method of first heavy rain in spring in Xi'an City.[Method] According to the dependent random variables of the occurrence of heavy rain,precipitation in spring in seven meteorological stations in Xi'an City from 1959 to 2010 was selected.Its occurrence date was determined by the standard of first heavy rain in spring in meteorology.According to the length of the sequence of the problem and actual situation,six states were divided.And by dint of Ma...

  14. Myosin heavy-chain isoform distribution, fibre-type composition and fibre size in skeletal muscle of patients on haemodialysis

    DEFF Research Database (Denmark)

    Molsted, Stig; Eidemak, Inge; Sorensen, Helle Tauby;

    2007-01-01

    Objective. Chronic uraemia is associated with abnormalities in skeletal muscles, which can affect their working capacity. It is also well known that the fibre-type composition of skeletal muscles influences endurance, muscle strength and power. In this study we therefore determined the size...... and distribution of muscle fibres and the myosin heavy-chain (MHC) isoform composition in patiens on haemodialysis (HD) in order to establish any differences with values for untrained control subjects. Material and methods. Muscle biopsies were obtained from the vastus lateralis muscle of 14 non-diabetic patients...... determined fibre-type composition of the vastus lateralis muscle. The mean fibre area of type 1 and 2 fibres was 3283±873 and 3594±1483 µm2, respectively. The MHC composition and the size of the type 1 fibres of the patients on HD were significantly different from those of the control subjects. Conclusions...

  15. Effects of opiates and HIV proteins on neurons: the role of ferritin heavy chain and a potential for synergism.

    Science.gov (United States)

    Festa, Lindsay; Meucci, Olimpia

    2012-07-01

    Human immunodeficiency virus 1 (HIV-1) and its associated proteins can have a profound impact on the central nervous system. Co-morbid abuse of opiates, such as morphine and heroin, is often associated with rapid disease progression and greater neurological dysfunction. The mechanisms by which HIV proteins and opiates cause neuronal damage on their own and together are unclear. The emergence of ferritin heavy chain (FHC) as a negative regulator of the chemokine receptor CXCR4, a co-receptor for HIV, may prove to be important in elucidating the interaction between HIV proteins and opiates. This review summarizes our current knowledge of central nervous system damage inflicted by HIV and opiates, as well as the regulation of CXCR4 by opiate-induced changes in FHC protein levels. We propose that HIV proteins and opiates exhibit an additive or synergistic effect on FHC/CXCR4, thereby decreasing neuronal signaling and function.

  16. Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

    Science.gov (United States)

    Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

    1999-01-01

    The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

  17. Myosin heavy chain isoform expression in human extraocular muscles: longitudinal variation and patterns of expression in global and orbital layers.

    Science.gov (United States)

    Park, Kyung-Ah; Lim, Jeonghee; Sohn, Seongsoo; Oh, Sei Yeul

    2012-05-01

    We investigated the distribution of myosin heavy chain (MyHC) isoforms along the length of the global and orbital layers of human extraocular muscles (EOMs). Whole muscle tissue extracts of human EOMs were cross-sectioned consecutively and separated into orbital and global layers. The extracts from these layers were subjected to electrophoretic analysis, followed by quantification with scanning densitometry. MyHC isoforms displayed different distributions along the lengths of EOMs. In the orbital and global layers of all EOMs except for the superior oblique muscle, MyHCeom was enriched in the central regions. MyHCIIa and MyHCI were most abundant in the proximal and distal ends. A variation in MyHC isoform expression was apparent along the lengths of human EOMs. These results provide a basis for understanding the molecular mechanisms underlying the functional diversity of EOMs. Copyright © 2012 Wiley Periodicals, Inc.

  18. Repeat organization and epigenetic regulation of the DH-Cmu domain of the immunoglobulin heavy-chain gene locus.

    Science.gov (United States)

    Chakraborty, Tirtha; Chowdhury, Dipanjan; Keyes, Amanda; Jani, Anant; Subrahmanyam, Ramesh; Ivanova, Irina; Sen, Ranjan

    2007-09-07

    The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.

  19. Characterization of the fine specificity of peptide antibodies to HLA-DQ beta-chain molecules

    DEFF Research Database (Denmark)

    Petersen, J S; Atar, D; Karlsen, Alan E

    1990-01-01

    specifically recognized DQw7 beta peptides and two antisera bound only to DQw8 beta peptides from the region containing the amino acid in position 57. To analyze whether the antisera bound to native HLA-DQ beta-chain molecules, FACS analysis was carried out. Seven of the 20 antisera bound to intact EBV...

  20. Caffeine Positively Modulates Ferritin Heavy Chain Expression in H460 Cells: Effects on Cell Proliferation

    Science.gov (United States)

    Battaglia, Anna Martina; Faniello, Maria Concetta; Cuda, Giovanni; Costanzo, Francesco

    2016-01-01

    Both the methylxanthine caffeine and the heavy subunit of ferritin molecule (FHC) are able to control the proliferation rate of several cancer cell lines. While caffeine acts exclusively as a negative modulator of cell proliferation, FHC might reduce or enhance cell viability depending upon the different cell type. In this work we have demonstrated that physiological concentrations of caffeine reduce the proliferation rate of H460 cells: along with the modulation of p53, pAKT and Cyclin D1, caffeine also determines a significant FHC up-regulation through the activation of its transcriptional efficiency. FHC plays a central role in the molecular pathways modulated by caffeine, ending in a reduced cell growth, since its specific silencing by siRNA almost completely abolishes caffeine effects on H460 cell proliferation. These results allow the inclusion of ferritin heavy subunits among the multiple molecular targets of caffeine and open the way for studying the relationship between caffeine and intracellular iron metabolism. PMID:27657916

  1. Serum major-histocompatibility-complex class Ⅰ-related chain A antibody detection for the evaluation of graft dysfunction in renal allograft recipients

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ming; LU Fu-ming; QU Lian-xi; HE Jun; YUAN Xiao-niao; GU Yong

    2011-01-01

    Background In addition to the well-known antibodies against human leukocyte antigens (HLA)-induced kidney-graft rejection, polymorphic major-histocompatibility-complex (MHC) class Ⅰ-related chain A (MICA) antigens can elicit antibodies and have been suggested to play a role in the antibody-mediated allograft rejection (AMR). We carded out a prospective study of MICA antibodies in post-renal transplant patients to determine the association between MICA antibodies, C4d staining, histological features, and graft outcome.Methods We tested 52 patients who had biopsy results due to graft dysfunction. The MICA antibodies in concurrent sera were determined by Luminex. All patients were followed up for one year after renal biopsy. The influence of antibody production on the function of graft was analyzed.Results Antibodies against MICA were positive in 15 out of the 52 patients (28.9%). The presence of MICA antibodies was associated with renal-allograft deterioration. During one-year follow-up, the estimated glomerular filtration rate (eGFR) decreased (24.0±3.4)% among recipients with anti-MICA antibodies. However, among recipients without anti-MICA antibodies, the eGFR has declined only (8.4+3.0)% (P=0.017). The association between C4d staining,histological features and MICA antibody production was found no significant difference.Conclusion Besides anti-HLA antibodies, the presence of post-transplant MICA antibody is associated with poor graft outcome and increases the risk of graft failure.

  2. Heavy chain of Acanthamoeba myosine IB is a fusion of myosin-like and non-myosin-like sequences

    Energy Technology Data Exchange (ETDEWEB)

    Jung, G.; Korn, E.D.; Hammer, J.A. III

    1987-10-01

    Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. The authors present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which they deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approx. 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approx. 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an ..cap alpha..-helical, coiled-coil structure. They conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, they find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. They discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure.

  3. Single-domain antibodies for biomedical applications.

    Science.gov (United States)

    Krah, Simon; Schröter, Christian; Zielonka, Stefan; Empting, Martin; Valldorf, Bernhard; Kolmar, Harald

    2016-01-01

    Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.

  4. Analysis of the 3' Cmu region of the rabbit Ig heavy chain locus.

    Science.gov (United States)

    Lanning, Dennis K; Zhai, Shi-Kang; Knight, Katherine L

    2003-05-08

    The immunoglobulin D (IgD) antibody class was, for many years, identified only in primates, rodents and teleost fish. The limited distribution of IgD among vertebrates suggested that IgD is a functionally redundant antibody class that has been lost by many vertebrate species during evolution. The recent identification of IgD in artiodactyls, however, suggests that IgD might be more widely expressed among vertebrates than previously thought, possibly serving a unique role in immunity. IgD expression has been searched for but not detected in rabbits. In order to search directly for a rabbit Cdelta locus encoding the constant region of IgD, we determined the nucleotide sequence of 13.5 kb of genomic DNA downstream of the rabbit Cmu locus. We did not find a rabbit Cdelta locus in this region, but found instead that this region is densely populated by repetitive elements, including a long interspersed DNA element repeat, six C repeats, and two processed pseudogenes. We conclude that the rabbit probably does not express IgD because there is no Cdelta locus immediately downstream of the rabbit Cmu locus.

  5. Multiple myeloma and multiple plasmacytomas associated with free gamma heavy chain, free kappa light chain and IgGk paraproteins: an unusual triple gammopathy.

    Science.gov (United States)

    Deighan, William I; O'Kane, Maurice J; McNicholl, Feargal P; Keren, David F

    2016-11-01

    Multiple myeloma is a malignant plasma cell dyscrasia that is becoming more prevalent in an increasingly ageing population. It is a complex disease with clinical phases ranging from the premalignant monoclonal gammopathy of undetermined significance to asymptomatic (smouldering) myeloma and then symptomatic myeloma; the latter occasionally terminating in the clonal proliferation of plasma cells outside the bone marrow. We present a patient whose clonally evolved disease from monoclonal gammopathy of undetermined significance to multiple myeloma demonstrated the presence of an unusual combination of monoclonal immunoproteins. Capillary electrophoresis demonstrated the presence of three paraproteins in the gamma region (γ-region), two of which were additional to the IgGk paraprotein which migrated in the slow γ-region at initial diagnosis. Subsequent isotypic identification of the new paraproteins was not possible by immunotyping and initial immunofixation studies failed to definitively characterize the monoclonal proteins. After reduction with beta-mercaptoethanol, two paraproteins were detected by both capillary and gel electrophoresis. However, only immunofixation was able to resolve three distinct monoclonal bands, confirming the presence of free monoclonal kappa light chains in the mid-gamma region and free monoclonal heavy chains in the fast gamma region. Triple gammopathies in themselves are uncommon; this case presents a very unusual combination of paraproteins which required various electrophoretical and immunochemical techniques to identify and characterize them. The change of electrophoretic signature from the monoclonal gammopathy of undetermined significance phase to the diagnosis of multiple myeloma suggested that a number of genetically distinct subclones were present in the pretreatment clonal evolution of the disease.

  6. LUZ-Y, a Novel Platform for the Mammalian Cell Production of Full-length IgG-bispecific Antibodies*

    Science.gov (United States)

    Wranik, Bernd J.; Christensen, Erin L.; Schaefer, Gabriele; Jackman, Janet K.; Vendel, Andrew C.; Eaton, Dan

    2012-01-01

    The ability of bispecific antibodies to simultaneously bind two unique antigens has great clinical potential. However, most approaches utilized to generate bispecific antibodies yield antibody-like structures that diverge significantly from the structure of archetype human IgG, and those that do approach structural similarity to native antibodies are often challenging to engineer and manufacture. Here, we present a novel platform for the mammalian cell production of bispecific antibodies that differ from their parental mAbs by only a single point mutation per heavy chain. Central to this platform is the addition of a leucine zipper to the C terminus of the CH3 domain of the antibody that is sufficient to drive the heterodimeric assembly of antibody heavy chains and can be readily removed post-purification. Using this approach, we developed various antibody constructs including one-armed Abs, bispecific antibodies that utilize a common light chain, and bispecific antibodies that pair light chains to their cognate heavy chains via peptide tethers. We have applied this technology to various antibody pairings and will demonstrate the engineering, purification, and biological activity of these antibodies herein. PMID:23118228

  7. Binding of human beta 2-microglobulin to murine EL4 thymoma cells upregulates MHC class I heavy-chain epitopes, inhibits IL-2 secretion and induces resistance to killing by natural killer cells

    DEFF Research Database (Denmark)

    Claësson, M H; Nissen, Mogens Holst

    1994-01-01

    . EL4 cells which had bound h beta 2m decreased their rate of constitutive IL-2 secretion and became resistant to activated natural killer (NK) cell killing. The present data suggest the binding of h beta 2m to mouse T cells leads to conformational changes of MHC-I heavy chains which influence both......A variety of murine tumor cell lines was studied for its binding of exogeneously added human beta 2-microglobulin (h beta 2m). Three T lymphomas and one IL-2-dependent T-cell line (HT-1) bound substantial amounts of h beta 2m, whereas P815 mastocytoma cells, an Abelson virus-infected pre-B cell...... line (ABLS-8), X63 B-lymphoma cells and YAC cells did not bind h beta 2m. In two of the T lymphomas, EL4 and BW5147, binding of h beta 2m led to an increase in major histocompatibility complex class I (MHC-I) heavy-chain epitope expression as measured by anti-H-2K/D antibody binding and FACS analysis...

  8. Study of the linked dipole chain model in heavy quark production at the Tevatron

    Energy Technology Data Exchange (ETDEWEB)

    Lipatov, Artem V. [Physical Department, M.V. Lomonosov Moscow State University, Moscow (Russian Federation)]. E-mail: lipatov@theory.sinp.msu.ru; Leif Loennblad [Dept. of Theoretical Physics, Lund (Sweden)]. E-mail: Leif.Lonnblad@thep.lu.se; Zotov, Nikolai P. [D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow (Russian Federation)]. E-mail: zotov@theory.sinp.msu.ru

    2004-01-01

    We present calculations of charm and beauty production at Tevatron within the framework of k{sub T} -factorization, using the unintegrated gluon distributions as obtained from the Linked Dipole Chain model. The analysis covers transverse momentum and rapidity distributions and the azimuthal correlations between b and b-bar quarks (or rather muons from their decay) which are powerful tests for the different unintegrated gluon distributions. We compare the theoretical results with recent experimental data taken by D{phi} and CDF collaborations at the Tevatron Run I and II. (author)

  9. Quantification of β region IgA paraproteins - should we include immunochemical "heavy/light chain" measurements? Counterpoint.

    Science.gov (United States)

    Paolini, Lucia

    2016-06-01

    Serum protein electrophoresis (SPE), serum immunofixation (s-IFE), free light chain measurement (FLC) and nephelometric measurements of total immunoglobulin in serum (IgTot) are some of the laboratory tests required for the management of plasma cell proliferative disorders. The monoclonal protein is usually visible on SPE as a spike (M-spike) in the γ region and the derived densitogram is used to quantify it relative to serum total protein concentration. IgA M-protein, however, often migrates in the β region on SPE and its quantification can be masked by other serum proteins that migrate in this region. The immunoassay Hevylite™ (heavy/light chain, HLC) seems to solve this problem: it quantifies the involved/uninvolved isotype, calculating the ratio IgAκ/IgAλ, considered indicative of clonal proliferation. However, this test seems redundant in the case of artifacts on SPE such as obvious hemolysis or lipemia, or if the IgA M-spike is clearly visible in the β region. In conclusion whereas the IgA HLC assay does not represent an alternative to SPE and s-IFE in the diagnostic patient workup, it may prove to be an alternative to SPE, s-IFE and total IgA quantification in risk stratification and evaluation of response to therapy in patients affected by MM and other monoclonal plasma proliferative disorders.

  10. Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus)

    DEFF Research Database (Denmark)

    Kim, Young Kyu; Lee, Jung Seok; Jung, Jae Wook

    2017-01-01

    abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder.......Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used...... successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11...

  11. Human germline antibody gene segments encode polyspecific antibodies.

    Science.gov (United States)

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  12. C terminal half fragment (50 kDa) of heavy chain components of Clostridium botulinum type C and D neurotoxins can be used as an effective vaccine.

    Science.gov (United States)

    Lee, Jae-Chul; Hwang, Hyun-Jung; Sakaguchi, Yoshihiko; Yamamoto, Yumiko; Arimitsu, Hideyuki; Tsuji, Takao; Watanabe, Toshihiro; Ohyama, Tohru; Tsuchiya, Tomofusa; Oguma, Keiji

    2007-01-01

    Recombinant whole heavy chains (H, 100 kDa) and their N-terminal (Hn, 50 kDa) and C-terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST eliminated-preparations of H (10 microg), Hn (5 microg), Hc (5 microg), or a mixture of Hn (5 microg) and Hc (5 microg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 10(6) the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 10(5) to 10(6) MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C-Hc and D-Hc, each 5 microg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused-or GST eliminatedpreparation were quite similar. These results indicate that recombinant GST-fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C-Hc or D-Hc, 100 microg, is useful for vaccine trials in mice.

  13. Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv against human ICAM-1

    Directory of Open Access Journals (Sweden)

    H. Sun

    2014-07-01

    Full Text Available Intercellular adhesion molecule-1 (ICAM-1 is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

  14. Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle.

    Science.gov (United States)

    Zhang, Kathy; Geddie, Melissa L; Kohli, Neeraj; Kornaga, Tad; Kirpotin, Dmitri B; Jiao, Yang; Rennard, Rachel; Drummond, Daryl C; Nielsen, Ulrik B; Xu, Lihui; Lugovskoy, Alexey A

    2015-01-01

    Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

  15. Exchanging murine and human immunoglobulin constant chains affects the kinetics and thermodynamics of antigen binding and chimeric antibody autoreactivity.

    Directory of Open Access Journals (Sweden)

    Marcela Torres

    Full Text Available Mouse-human chimeric antibodies composed of murine variable (V and human (C chains are useful therapeutic reagents. Consequently, we investigated whether heterologous C-regions from mice and humans affected specificity and affinity, and determined the contribution of C(H glycosylation to antigen binding. The interaction of a 12-mer peptide mimetic with monoclonal antibody (mAb 18B7 to Cryptococcus neoformans glucuronoxylomannan, and its chimeric (ch and deglycosylated forms were studied by surface plasmon resonance. The equilibrium and rate association constants for the chAb were higher than for mAb 18B7. V region affinity was not affected by C(H region glycosylation whereas heterologous C region of the same isotype altered the Ab binding affinity and the specificity for self-antigens. Structural models displayed local differences that implied changes on the connectivity of residues. These findings suggest that V region conformational changes can be dictated by the C(H domains through an allosteric effect involving networks of highly connected amino acids.

  16. Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor.

    Science.gov (United States)

    Sotiriadis, A; Keshavarz, T; Keshavarz-Moore, E

    2001-01-01

    A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1). A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1). A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1). Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment.

  17. Modified human beta 2-microglobulin (desLys(58)) displays decreased affinity for the heavy chain of MHC class I and induces nitric oxide production and apoptosis

    DEFF Research Database (Denmark)

    Wang, M; Harhaji, L; Lamberth, K;

    2009-01-01

    -I associated beta2m from the cell surface. In addition, only beta2m is able to restore MHC-I/peptide complex formation on acid-treated cells whereas dbeta2m appears to bind preferentially to denatured MHC-I heavy chains. In cell cultures, exogenously added dbeta2m, but not beta2m, induces apoptotic cell death...

  18. [Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian gerbil (Meriones unguiculatus) after 12-day spaceflight].

    Science.gov (United States)

    Okuneva, A D; Vikhliantsev, I M; Shpagina, M D; Rogachevskiĭ, V V; Khutsian, S S; Poddubnaia, Z A; Grigor'ev, A I

    2012-01-01

    Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus ) were investigated after 12-day spaceflight on board of Russian space vehicle "Foton-M3". In m. psoas and m. soleus in the gerbils from "Flight" group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in "Flight" group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from "Flight" group were observed. In skeletal muscles of the gerbils from "Flight" group the relative content of titin N2A-isoform was reduced (by 1,2-1,7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from "Flight" group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.

  19. High fat/low carbohydrate diet attenuates left ventricular hypertrophy and prevents myosin heavy chain isoform switching induced by chronic hypertenstion

    Science.gov (United States)

    A switch in the expression of myosin heavy chain isoform (MHC) alpha to beta is observed with left ventricular hypertrophy (LVH) and heart failure. This switch is associated with a defect in myocardial energy production and contractile dysfunction. Similar MHC isoform profile is observed in the fe...

  20. Expansion of B-1a cells with germline heavy chain sequence in lupus mice

    Directory of Open Access Journals (Sweden)

    Nichol E Holodick

    2016-03-01

    Full Text Available B6.Sle1.Sle2.Sle3 (B6.TC lupus-prone mice carrying the NZB allele of Cdkn2c, encoding for the cyclin-dependent kinase inhibitor P18INK4, accumulate B-1a cells due to a higher rate of proliferative self-renewal. However, it is unclear whether this affects primarily early appearing B-1a cells of fetal origin or later appearing B-1a cells that emerge from bone marrow. B-1a cells are the major source of natural autoantibodies, and it has been shown that their protective nature is associated with a germline-like sequence, which is characterized by few N-nucleotide insertions and a repertoire skewed towards rearrangements predominated during fetal life, VH11 and VH12. To determine the nature of B-1a cells expanded in B6.TC mice, we amplified immunoglobulin genes by PCR from single cells in mice. Sequencing showed a significantly higher proportion of B-1a cell antibodies display fewer N-additions in B6.TC mice than in B6 control mice. Following this lower number of N-insertions within the CDR-H3 region, the B6.TC B-1a cells display shorter CDR-H3 length than B6 B-1a cells. The absence of N-additions is a surrogate for fetal origin, as TdT expression starts after birth in mice. Therefore, our results suggest that the B-1a cell population is not only expanded in autoimmune B6.TC mice but also qualitatively different with the majority of cells from fetal origin. Accordingly, our sequencing results also demonstrated overuse of VH11 and VH12 in autoimmune B6.TC mice as compared to B6 controls. These results suggest that the development of lupus autoantibodies in these mice is coupled with skewing of the B-1a cell repertoire and possible retention of protective natural antibodies.

  1. Expansion of B-1a Cells with Germline Heavy Chain Sequence in Lupus Mice

    Science.gov (United States)

    Holodick, Nichol E.; Zeumer, Leilani; Rothstein, Thomas L.; Morel, Laurence

    2016-01-01

    B6.Sle1.Sle2.Sle3 (B6.TC) lupus-prone mice carrying the NZB allele of Cdkn2c, encoding for the cyclin-dependent kinase inhibitor P18INK4, accumulate B-1a cells due to a higher rate of proliferative self-renewal. However, it is unclear whether this affects primarily early-appearing B-1a cells of fetal origin or later-appearing B-1a cells that emerge from bone marrow. B-1a cells are the major source of natural autoantibodies, and it has been shown that their protective nature is associated with a germline-like sequence, which is characterized by few N-nucleotide insertions and a repertoire skewed toward rearrangements predominated during fetal life, VH11 and VH12. To determine the nature of B-1a cells expanded in B6.TC mice, we amplified immunoglobulin genes by PCR from single cells in mice. Sequencing showed a significantly higher proportion of B-1a cell antibodies that display fewer N-additions in B6.TC mice than in B6 control mice. Following this lower number of N-insertions within the CDR-H3 region, the B6.TC B-1a cells display shorter CDR-H3 length than B6 B-1a cells. The absence of N-additions is a surrogate for fetal origin, as TdT expression starts after birth in mice. Therefore, our results suggest that the B-1a cell population is not only expanded in autoimmune B6.TC mice but also qualitatively different with the majority of cells from fetal origin. Accordingly, our sequencing results also demonstrated the overuse of VH11 and VH12 in autoimmune B6.TC mice as compared to B6 controls. These results suggest that the development of lupus autoantibodies in these mice is coupled with skewing of the B-1a cell repertoire and possible retention of protective natural antibodies. PMID:27047495

  2. Measurements on Pb27+ Sources for the CERN Heavy Ion Injection Chain

    CERN Document Server

    Chamings, J A

    2004-01-01

    CERN, the world's largest particle physics laboratory near Geneva, is currently in the process of building the Large Hadron Collider (LHC). Lead-208 will be used in this accelerator and to meet the injection requirements much work is required to find a suitable and reliable heavy ion source. The work in this report covers two ion sources, the Laser Ion Source (LIS), and the Electron Cyclotron Resonance Ion Source (ECRIS). An emittance measurement, using a pepper pot and CCD camera, was completed on the recently re-installed LIS, measured and analysed to be 140mm.mrad un-normalised and 0.8mm.mrad normalised 4rms for the maximum intensity charge state, Pb27+. A Visual Basic Program was modified to allow Charge State Distribution (CSD) and other scans of the ECRIS at CERN to be taken. Chapter 5 presents the results of the CSD scans taken. This program provided a new method to take 1 dimensional transverse beam profiles. From this a direct emittance measurement was formed for the first time since the source was o...

  3. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    Science.gov (United States)

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  4. Comparative genomic analysis of the arthropod muscle myosin heavy chain genes allows ancestral gene reconstruction and reveals a new type of 'partially' processed pseudogene

    Directory of Open Access Journals (Sweden)

    Kollmar Martin

    2008-02-01

    Full Text Available Abstract Background Alternative splicing of mutually exclusive exons is an important mechanism for increasing protein diversity in eukaryotes. The insect Mhc (myosin heavy chain gene produces all different muscle myosins as a result of alternative splicing in contrast to most other organisms of the Metazoa lineage, that have a family of muscle genes with each gene coding for a protein specialized for a functional niche. Results The muscle myosin heavy chain genes of 22 species of the Arthropoda ranging from the waterflea to wasp and Drosophila have been annotated. The analysis of the gene structures allowed the reconstruction of an ancient muscle myosin heavy chain gene and showed that during evolution of the arthropods introns have mainly been lost in these genes although intron gain might have happened in a few cases. Surprisingly, the genome of Aedes aegypti contains another and that of Culex pipiens quinquefasciatus two further muscle myosin heavy chain genes, called Mhc3 and Mhc4, that contain only one variant of the corresponding alternative exons of the Mhc1 gene. Mhc3 transcription in Aedes aegypti is documented by EST data. Mhc3 and Mhc4 inserted in the Aedes and Culex genomes either by gene duplication followed by the loss of all but one variant of the alternative exons, or by incorporation of a transcript of which all other variants have been spliced out retaining the exon-intron structure. The second and more likely possibility represents a new type of a 'partially' processed pseudogene. Conclusion Based on the comparative genomic analysis of the alternatively spliced arthropod muscle myosin heavy chain genes we propose that the splicing process operates sequentially on the transcript. The process consists of the splicing of the mutually exclusive exons until one exon out of the cluster remains while retaining surrounding intronic sequence. In a second step splicing of introns takes place. A related mechanism could be responsible for

  5. Epigenetic Regulation of Individual Modules of the immunoglobulin heavy chain locus 3' Regulatory Region.

    Science.gov (United States)

    Birshtein, Barbara K

    2014-01-01

    The Igh locus undergoes an amazing array of DNA rearrangements and modifications during B cell development. During early stages, the variable region gene is constructed from constituent variable (V), diversity (D), and joining (J) segments (VDJ joining). B cells that successfully express an antibody can be activated, leading to somatic hypermutation (SHM) focused on the variable region, and class switch recombination (CSR), which substitutes downstream constant region genes for the originally used Cμ constant region gene. Many investigators, ourselves included, have sought to understand how these processes specifically target the Igh locus and avoid other loci and potential deleterious consequences of malignant transformation. Our laboratory has concentrated on a complex regulatory region (RR) that is located downstream of Cα, the most 3' of the Igh constant region genes. The ~40 kb 3' RR, which is predicted to serve as a downstream major regulator of the Igh locus, contains two distinct segments: an ~28 kb region comprising four enhancers, and an adjacent ~12 kb region containing multiple CTCF and Pax5 binding sites. Analysis of targeted mutations in mice by a number of investigators has concluded that the entire 3' RR enhancer region is essential for SHM and CSR (but not for VDJ joining) and for high levels of expression of multiple isotypes. The CTCF/Pax5 binding region is a candidate for influencing VDJ joining early in B cell development and serving as a potential insulator of the Igh locus. Components of the 3' RR are subject to a variety of epigenetic changes during B cell development, i.e., DNAse I hypersensitivity, histone modifications, and DNA methylation, in association with transcription factor binding. I propose that these changes provide a foundation by which regulatory elements in modules of the 3' RR function by interacting with each other and with target sequences of the Igh locus.

  6. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    Directory of Open Access Journals (Sweden)

    Benevolo Maria

    2006-09-01

    Full Text Available Abstract 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

  7. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    Science.gov (United States)

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.

  8. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

    Science.gov (United States)

    Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

    2017-01-01

    Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

  9. Circulating antibody free light chains and risk of posttransplant lymphoproliferative disorder.

    Science.gov (United States)

    Engels, E A; Preiksaitis, J; Zingone, A; Landgren, O

    2012-05-01

    Posttransplant lymphoproliferative disorder (PTLD) is a major complication of solid-organ transplantation. With human immunodeficiency virus infection (an analogous immunosuppressive state), elevated kappa and lambda immunoglobulin free light chains (FLCs) in peripheral blood are associated with increased risk of lymphoma. To assess the role of B-cell dysfunction in PTLD, we measured circulating FLCs among Canadian transplant recipients, including 29 individuals with PTLD and 57 matched transplant recipients who were PTLD-free. Compared with controls, PTLD cases had higher kappa FLCs (median 1.53 vs. 1.07 times upper limit of normal) and lambda FLCs (1.03 vs. 0.68). Using samples obtained on average 3.5 months before PTLD diagnosis, cases were more likely to have polyclonal FLC elevations (i.e. elevated kappa and/or lambda with normal kappa/lambda ratio: odds ratio [OR] 4.2, 95%CI 1.1-15) or monoclonal elevations (elevated kappa and/or lambda with abnormal ratio: OR 3.0, 95%CI 0.5-18). Strong FLC-PTLD associations were also observed at diagnosis/selection. Among recipients with Epstein-Barr virus (EBV) DNA measured in blood, EBV DNAemia was associated with FLC abnormalities (ORs 6.2 and 3.2 for monoclonal and polyclonal elevations). FLC elevations are common in transplant recipients and associated with heightened PTLD risk. FLCs likely reflect B-cell dysfunction, perhaps related to EBV-driven lymphoproliferation.

  10. CXCR4 and CXCL12 (SDF-1) in prostate cancer: inhibitory effects of human single chain Fv antibodies.

    Science.gov (United States)

    Vaday, Gayle G; Hua, Shao-Bing; Peehl, Donna M; Pauling, Michelle H; Lin, Yu-Huei; Zhu, Li; Lawrence, Diana M; Foda, Hussein D; Zucker, Stanley

    2004-08-15

    Metastasis is a major cause of morbidity in prostate cancer (PCa). Several studies have shown that the chemokine receptor CXCR4 and its ligand, CXCL12 (stromal cell-derived factor-1), regulate tumor cell metastasis to specific organs. Recently, it was demonstrated that CXCL12 enhances PCa cell adhesion, migration, and invasion, implicating CXCR4 in PCa metastasis. In this study, we examined the inhibitory effects of anti-CXCR4 antibodies on CXCL12-mediated PCa cell activities. We developed fully human single chain Fv antibodies (scFv), Ab124 and Ab125, against CXCR4 using the yeast two-hybrid system. We performed immunofluorescent staining, flow cytometry, and ELISA-binding assays to measure scFv binding to PCa cells. We also examined the effects of scFv on CXCL12-mediated calcium mobilization, cell migration, and invasion. Our results confirmed that PCa cell lines express cell-surface CXCR4. Real-time quantitative reverse transcription-PCR and immunohistochemical staining also verified that CXCR4 is expressed in primary cultures of prostate epithelial cells from adenocarcinomas and in human prostate tissues. Ab124 and Ab125 demonstrated specific binding to PCa cell lines by flow cytometry and in binding assays. Preincubation with scFv resulted in significant reduction of CXCL12-induced calcium mobilization in PC-3 and LNCaP cells. Ab124 and Ab125 also inhibited PCa cell migration toward CXCL12, as well as invasion through extracellular matrix gels. Ab124 and Ab125 inhibit CXCL12-mediated cellular activities by binding the receptor CXCR4. Recombinant scFv are an efficient mode of targeting tumor antigens. Considering the high incidence of PCa, the development of fully human scFv may be a useful therapeutic approach in the prevention and treatment of PCa metastasis.

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

    Science.gov (United States)

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

    2011-04-01

    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 (111)In ((111)In-IL-M1), along with control non-targeted liposomes ((111)In-CL). Incubation of (111)In-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 (111)In-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 (111)In-IL-M1 in live xenograft animal models was verified by single photon emission computed tomography (SPECT/CT). In contrast, i.v. injection of (111)In-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.

  12. Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

    Science.gov (United States)

    Shigemori, Suguru; Ihara, Masaki; Sato, Takashi; Yamamoto, Yoshinari; Nigar, Shireen; Ogita, Tasuku; Shimosato, Takeshi

    2017-01-01

    Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

  13. MR molecular imaging of tumours using ferritin heavy chain reporter gene expression mediated by the hTERT promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yan [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China); The First Affiliated Hospital of ChengDu Medical College, Department of Radiology, ChengDu (China); Gong, Ming-fu; Yang, Hua; Zhang, Song; Wang, Guang-xian; Su, Tong-sheng; Wen, Li; Zhang, Dong [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China)

    2016-11-15

    Using the human telomerase reverse transcriptase (hTERT) promoter and the modified ferritin heavy chain (Fth) reporter gene, reporter gene expression for MRI was examined in telomerase positive and negative tumour cells and xenografts. Activity of the reporter gene expression vector Lenti-hTERT-Fth1-3FLAG-Puro was compared to constitutive CMV-driven expression and to the untransfected parental control in five tumour cell lines: A549, SKOV3, 293T, U2OS and HPDLF. In vitro, transfected cells were evaluated for FLAG-tagged protein expression, iron accumulation and transverse relaxation. In vivo, tumours transduced by lentiviral vector injection were imaged using T2*WI. Changes in tumour signal intensity were validated by histology. Only telomerase positive tumour cells expressed FLAG-tagged Fth and displayed an increase in R2* above the parental control, with a corresponding change in T2*WI. In addition, only telomerase positive tumours, transduced by injection of the reporter gene expression construct, exhibited a change in signal intensity on T2*WI. Tumour histology verified the expression of FLAG-tagged Fth and iron accumulation in telomerase positive tissue. Reporter gene expression for MRI, using the Fth reporter and the hTERT promoter, may be a useful strategy for the non-invasive diagnosis of many types of cancer. (orig.)

  14. Molecular cloning and comparative analysis of immunoglobulin heavy chain genes from Phasianus colchicus, Meleagris gallopavo, and Coturnix japonica.

    Science.gov (United States)

    Choi, Jin Won; Kim, Jin-Kyoo; Seo, Hee Won; Cho, Byung Wook; Song, Gwonhwa; Han, Jae Yong

    2010-08-15

    To date, immunoglobulin (Ig) genes have only been fully characterized in a small number of aves, which pose a major obstacle to understanding Ig evolution. Thus, we cloned the cDNAs of three immunoglobulin classes, IgA, IgM, and IgY, from Phasianus colchicus, Coturnix japonica, and Meleagris gallopavo. Multiple sequence alignments revealed that the highest degree of sequence homology in all Ig classes was observed between pheasant and turkey whereas the degree of homology between the galliforms and non-galliforms was relatively low compared to that among the galliforms. When the constant region domains of the four human Ig classes were compared with the corresponding regions in aves, the average percent homology between human CH2 and avian CH3, and between human CH3 and avian CH4, was greater than between identical domains in IgA and IgY, which are in partial agreement with the hypothesis that the avian CH2 domain evolved to form the mammalian hinge via domain condensation. Phylogenetic analysis showed that the galliform Ig heavy chain constant regions were divided into quail and the common ancestor of chicken, turkey, and pheasant, and that chicken was separated from turkey and pheasant, which were grouped together. These results add to our knowledge of galliform Igs and the diversification of these genes.

  15. Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9 and Rac1 activation

    Directory of Open Access Journals (Sweden)

    Mohammad G. Sabbir

    2016-04-01

    Full Text Available The Deleted in liver cancer 1 (Dlc1 gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9, plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype.

  16. Germ line transcription in mice bearing neor gene downstream of Igamma3 exon in the Ig heavy chain locus.

    Science.gov (United States)

    Samara, Maha; Oruc, Zeliha; Dougier, Hei-Lanne; Essawi, Tamer; Cogné, Michel; Khamlichi, Ahmed Amine

    2006-04-01

    Class switch recombination (CSR) is preceded by germ line transcription that initiates from promoters upstream of switch (S) sequences and terminates downstream of associated constant genes. Previous work showed that germ line transcripts and their processing are required for CSR and that germ line transcription is regulated in a major part by a regulatory region located downstream of the Ig heavy chain locus. This long-range, polarized effect can be disturbed by inserting an expressed neomycine resistance (neo(r)) gene. To contribute to a better understanding of the mechanism of such a long-distance regulation, we generated knock-in mice in which a neo(r) gene was inserted downstream of Igamma3 exon leaving intact all the necessary elements for germ line transcription and splicing. We show that the expressed neo(r) gene interferes with transcription initiation from Igamma3, and that it impairs but does not block S recombination to Cgamma3. Moreover, we show for the first time that the neo(r) gene provides through chimeric neo(r)-Cgamma3 transcripts the necessary elements for splicing of germ line transcripts by activating two novel cryptic splice sites, one in the coding region of the intronless neo(r) gene and the other in the Igamma3-Cgamma3 intron.

  17. Sulfasalazine Treatment Suppresses the Formation of HLA-B27 Heavy Chain Homodimer in Patients with Ankylosing Spondylitis.

    Science.gov (United States)

    Yu, Hui-Chun; Lu, Ming-Chi; Huang, Kuang-Yung; Huang, Hsien-Lu; Liu, Su-Qin; Huang, Hsien-Bin; Lai, Ning-Sheng

    2015-12-29

    Human leukocytic antigen-B27 heavy chain (HLA-B27 HC) has the tendency to fold slowly, in turn gradually forming a homodimer, (B27-HC)₂ via a disulfide linkage to activate killer cells and T-helper 17 cells and inducing endoplasmic reticulum (ER) stress to trigger the IL-23/IL-17 axis for pro-inflammatory reactions. All these consequences lead to the pathogenesis of ankylosing spondylitis (AS). Sulfasalazine (SSA) is a common medication used for treatment of patients with AS. However, the effects of SSA treatment on (B27-HC)₂ formation and on suppression of IL-23/IL-17 axis of AS patients remain to be determined. In the current study, we examine the (B27-HC)₂ of peripheral blood mononuclear cells (PBMC), the mean grade of sarcoiliitis and lumbar spine Bath Ankylosing Spondylitis Radiology Index (BASRI) scores of 23 AS patients. The results indicated that AS patients without (B27-HC)₂ on PBMC showed the lower levels of mean grade of sarcoiliitis and the lumbar spine BASRI scores. In addition, after treatment with SSA for four months, the levels of (B27-HC)₂ on PBMCs were significantly reduced. Cytokines mRNA levels, including TNFα, IL-17A, IL-17F and IFNγ, were also significantly down-regulated in PBMCs. However, SSA treatment did not affect the levels of IL-23 and IL-23R mRNAs.

  18. The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain.

    Science.gov (United States)

    Ducret, Axel; Nguyen, Mai; Breckenridge, David G; Shore, Gordon C

    2003-01-01

    BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase-8. Recently, we reported that the caspase-resistant BAP31 inhibited Fas-mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731-6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle gamma-actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis.

  19. SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum.

    Science.gov (United States)

    Huang, Qian; Babu, Gopal J; Periasamy, Muthu; Eddinger, Thomas J

    2013-01-15

    The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

  20. Myosin heavy chain expression and atrophy in rat skeletal muscle during transition from cardiac hypertrophy to heart failure.

    Science.gov (United States)

    Carvalho, Robson Francisco; Cicogna, Antonio Carlos; Campos, Gerson Eduardo Rocha; De Assis, Jeane Marlene Fogaça; Padovani, Carlos Roberto; Okoshi, Marina Politi; Pai-Silva, Maeli Dal

    2003-08-01

    The purpose of this investigation was to determine whether changes in myosin heavy chain (MHC) expression and atrophy in rat skeletal muscle are observed during transition from cardiac hypertrophy to chronic heart failure (CHF) induced by aortic stenosis (AS). AS and control animals were studied 12 and 18 weeks after surgery and when overt CHF had developed in AS animals, 28 weeks after the surgery. The following parameters were studied in the soleus muscle: muscle atrophy index (soleus weight/body weight), muscle fibre diameter and frequency and MHC expression. AS animals presented decreases in both MHC1 and type I fibres and increases in both MHC2a and type IIa fibres during late cardiac hypertrophy and CHF. Type IIa fibre atrophy occurred during CHF. In conclusion, our data demonstrate that skeletal muscle phenotype changes occur in both late cardiac hypertrophy and heart failure; this suggests that attention should be given to the fact that skeletal muscle phenotype changes occur prior to overt heart failure symptoms.

  1. Somatic variation precedes extensive diversification of germline sequences and combinatorial joining in the evolution of immunoglobulin heavy chain diversity.

    Science.gov (United States)

    Hinds-Frey, K R; Nishikata, H; Litman, R T; Litman, G W

    1993-09-01

    In Heterodontus, a phylogenetically primitive shark species, the variable (VH), diversity (DH), joining (JH) segments, and constant (CH) exons are organized in individual approximately 18-20-kb "clusters." A single large VH family with > 90% nucleic acid homology and a monotypic second gene family are identified by extensive screening of a genomic DNA library. Little variation in the nucleotide sequences of DH segments from different germline gene clusters is evident, suggesting that the early role for DH was in promoting junctional diversity rather than contributing unique coding specificities. A gene-specific oligodeoxynucleotide screening method was used to relate specific transcription products (cDNAs) to individual gene clusters and showed that gene rearrangements are intra- rather than intercluster. This provides further evidence for restricted diversity in the immunoglobulin heavy chain of Heterodontus, from which it is inferred that combinatorial diversity is a more recently acquired means for generating diversity. The observed differences between cDNA sequences selected and the sequences of segmental elements derived from conventional genomic libraries as well as from VH segment-specific libraries generated by direct PCR amplification of genomic DNA indicate that the VH repertoire is diversified by both junctional diversity and somatic mutation. Taken together, these findings suggest a heretofore unrecognized contribution of somatic variation that preceded both extensive diversification of the germline repertoire and the combinatorial joining process in the evolution of humoral immunity.

  2. The Florida manatee (Trichechus manatus latirostris) immunoglobulin heavy chain suggests the importance of clan III variable segments in repertoire diversity.

    Science.gov (United States)

    Breaux, Breanna; Deiss, Thaddeus C; Chen, Patricia L; Cruz-Schneider, Maria Paula; Sena, Leonardo; Hunter, Margaret E; Bonde, Robert K; Criscitiello, Michael F

    2017-07-01

    Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent Afrotheria. Manatee health and resistance to infectious disease is of great concern to conservation groups, but little is known about their immune system. To develop manatee-specific tools for monitoring health, we first must have a general knowledge of how the immunoglobulin heavy (IgH) chain locus is organized and transcriptionally expressed. Using the genomic scaffolds of the Florida manatee (Trichechus manatus latirostris), we characterized the potential IgH segmental diversity and constant region isotypic diversity and performed the first Afrotherian repertoire analysis. The Florida manatee has low V(D)J combinatorial diversity (3744 potential combinations) and few constant region isotypes. They also lack clan III V segments, which may have caused reduced VH segment numbers. However, we found productive somatic hypermutation concentrated in the complementarity determining regions. In conclusion, manatees have limited IGHV clan and combinatorial diversity. This suggests that clan III V segments are essential for maintaining IgH locus diversity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Ubiquitin Ligase, MuRF-1 regulates myosin heavy chain type IIa transcripts during muscle atrophy under microgravity conditions

    Science.gov (United States)

    Kagawa, Sachiko

    Skeletal muscles are vulnerable to marked atrophy under microgravity conditions. We previously reported that gastrocnemius muscle atrophy by spaceflight was specifically sensitive to the ubiquitin-proteasome proteolytic pathway. We also screened more over 26,000 skeletal muscle genes in rats exposed to real weightlessness and found that the expression of Ubiquitin Ligase, Muscle specific Ring Finger-1 (MuRF-1) upregulated under microgravity. In the present study, we examined the role of MuRF-1 in microgravity-induced muscle atrophy. The amounts of MuRF-1 transcripts significantly increased in skeletal muscle after denervation, an in vivo model of microgravity-induced unloading. MuRF-1 deficient (MuRF-1-/-) mice significantly inhibited reduction of muscle weight for muscle atrophy, compared with wild type mice. Interestingly, MuRF-1-/- mice significantly inhibited upregulation of myosin heavy chain (MyHC) type IIa transcrips, while wild type mice significantly increased expression of MyHC type IIa transcripts in denervated skeletal muscle. Our present results suggest that MuRF-1 may play an important role in regulation of MyHC type IIa during muscle atrophy under microgravity conditions.

  4. Evolution of the recombination signal sequences in the Ig heavy-chain variable region locus of mammals

    Science.gov (United States)

    Hassanin, Alexandre; Golub, Rachel; Lewis, Susanna M.; Wu, Gillian E.

    2000-01-01

    The Ig and T cell receptor (TCR) loci have an exceptionally dynamic evolutionary history, but the mechanisms responsible remain a subject of speculation. Ig and TCR genes are unique in vertebrates in that they are assembled from V, D, and J segments by site-specific recombination in developing lymphocytes. Here we examine the extent to which the V(D)J recombination in germline cells may have been responsible for remodeling Ig and TCR loci in mammals by asking whether gene segments have evolved as a unit, or whether, instead, recombination signal sequences (RSSs) and coding sequences have different phylogenies. Four distinct types of RSS have been defined in the human Ig heavy-chain variable region (Vh) locus, namely H1, H2, H3, and H5, and no other RSS type has been detected in other mammalian species. There is a well-supported discrepancy between the evolutionary history of the RSSs as compared with the Vh coding sequences: the RSS type H2 of one Vh gene segment has clearly become replaced by a RSS type H3 during mammalian evolution, between 115 and 65 million years ago. Two general models might explain the RSS swap: the first involves an unequal crossing over, and the second implicates germline activation of V(D)J recombination. The Vh-H2/RSS-H3 recombination product has likely been selected during the evolution of mammals because it provides better V(D)J recombination efficiency. PMID:11027341

  5. Activated platelets in carotid artery thrombosis in mice can be selectively targeted with a radiolabeled single-chain antibody.

    Directory of Open Access Journals (Sweden)

    Timo Heidt

    Full Text Available 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/mm(2, 4010 ± 630 DLU/mm(2 and 4520 ± 293 DLU/mm(2 produced a significantly higher ligand uptake compared to (111In-control (2101 ± 76 DLU/mm(2, 1181 ± 96 DLU/mm(2 and 1866 ± 246 DLU/mm(2 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/mm(2 vs. 17390 ± 7470 DLU/mm(2; 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

  6. Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

    Directory of Open Access Journals (Sweden)

    Yang J

    2014-05-01

    Full Text Available Jingjing Yang,1,3,* Xiaoping Huang,1,3,* Fanghong Luo,1 Xiaofeng Cheng,3 Lianna Cheng,3 Bin Liu,4 Lihong Chen,2 Ruyi Hu,1,3 Chunyan Shi,1,3 Guohong Zhuang,1,3 Ping Yin2 1Anti-Cancer Research Center, Medical College, Xiamen University, Fujian, People's Republic of China, 2The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, People's Republic of China, 3Organ transplantation institution, Xiamen University, Xiamen, People's Republic of China, 4Jilin Vocational College of Industry and Technology, Jilin, People's Republic of China  *These authors contributed equally to this work Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action. Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors. Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected

  7. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  8. Effect of linker length between variable domains of single chain variable fragment antibody against daidzin on its reactivity.

    Science.gov (United States)

    Yusakul, Gorawit; Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-07-01

    The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs.

  9. Single-chain antibody-based gene therapy: Inhibition of tumor growth by in situ production of phage-derived antibodies blocking functionally active sites of cell-associated matrices

    DEFF Research Database (Denmark)

    Sanz, Laura; Kristensen, Peter; Blanco, Belén

    2002-01-01

    . This scFv inhibits angiogenesis in vivo in the chick embryo chorioallantoic membrane assay and prevents the establishment and growth of subcutaneous tumors in mice, either when administered as bolus protein therapy or when produced locally by gene-modified tumor cells. Our work represents the first...... demonstration of a direct in vivo therapeutic effect of a single-chain antibody secreted by gene-modified mammalian cells. These results open the way for a new antibody-based gene therapy strategy of cancer....

  10. A Comparative Analysis of Polymerase Chain Reaction and Direct Fluorescent Antibody Test for Diagnosis of Genital Herpes.

    Science.gov (United States)

    Patwardhan, Vrushali; Bhalla, Preena; Rawat, Deepti; Garg, Vijay Kumar; Sardana, Kabir; Sethi, Sumit

    2017-01-01

    To compare laboratory tests that can simultaneously detect and type herpes simplex virus (HSV) directly from the genital ulcer specimens in clinically suspected cases of genital herpes. A study was conducted over 10 months and 44 adult male and female patients clinically suspected with genital herpes were recruited. Genital ulcer swab specimens were subjected to glycoprotein-G gene-based conventional polymerase chain reaction (PCR) and commercially available direct fluorescent antibody (DFA) test and the results were compared. PCR for HSV was positive in 82% (36/44) cases. DFA was positive in 68.2% (30/44) cases. There was 100% agreement between HSV types detected by DFA and PCR. The strength of agreement between the results was better in primary genital herpes than recurrent cases. PCR was found to be better in the detection of HSV in recurrent genital herpes patients. It is a better modality, especially when genital herpes clinically presents with ulcerative or crusted lesions, and is also a cheaper alternative as compared to DFA.

  11. Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

    Science.gov (United States)

    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).

  12. Disulfide-stabilized single-chain antibody-targeted superantigen: Construction of a prokaryotic expression system and its functional analysis

    Institute of Scientific and Technical Information of China (English)

    Jian-Li Wang; Yu-Ling Zheng; Ru Ma; Bao-Li Wang; Ai-Guang Guo; Yong-Qiang Jiang

    2005-01-01

    AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

  13. Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement

    DEFF Research Database (Denmark)

    Zhou, X.G.; Sandvej, K.; Gregersen, Niels

    1999-01-01

    Aims-To evaluate the specificity of standard and fluorescence based (GENESCAN) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. Methods-PCR IgH gene rearrangement...... showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with GENESCAN analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal. Conclusions These results confirm that PCR based IgH gene...

  14. Characterization of productive and sterile transcripts from the immunoglobulin heavy-chain locus: processing of micron and muS mRNA.

    OpenAIRE

    Nelson, K J; Haimovich, J; Perry, R P

    1983-01-01

    An analysis of the sizes and sequence content of nuclear RNA transcripts of the heavy-chain locus in two B-cell lymphomas, 70Z/3 and 38C-13, and in selected hybridoma derivatives of 38C has led to the identification of two distinct precursors of the mRNAs encoding the membrane and secretory forms of mu chain. These precursors, termed Pm1 and Ps1, extend from a common 5' terminus (presumably the cap site) to alternative polyadenylation sites located 3' of the membrane and secretory tailpieces,...

  15. Homologous elements hs3a and hs3b in the 3' regulatory region of the murine immunoglobulin heavy chain (Igh) locus are both dispensable for class-switch recombination.

    Science.gov (United States)

    Yan, Yi; Pieretti, Joyce; Ju, Zhongliang; Wei, Shiniu; Christin, John R; Bah, Fatmata; Birshtein, Barbara K; Eckhardt, Laurel A

    2011-08-01

    Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

  16. A novel monoclonal antibody to human laminin α5 chain strongly inhibits integrin-mediated cell adhesion and migration on laminins 511 and 521.

    Science.gov (United States)

    Wondimu, Zenebech; Omrani, Shahin; Ishikawa, Taichi; Javed, Fawad; Oikawa, Yuko; Virtanen, Ismo; Juronen, Erkki; Ingerpuu, Sulev; Patarroyo, Manuel

    2013-01-01

    Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1) and 521 (α5β2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.

  17. A novel monoclonal antibody to human laminin α5 chain strongly inhibits integrin-mediated cell adhesion and migration on laminins 511 and 521.

    Directory of Open Access Journals (Sweden)

    Zenebech Wondimu

    Full Text Available Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1 and 521 (α5β2γ1. As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.

  18. Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3 in Thai malignant lymphoma by High Resolution Melting curve analysis

    Directory of Open Access Journals (Sweden)

    Pongpruttipan Tawatchai

    2010-05-01

    Full Text Available Abstract Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR and immunoglobulin heavy chain (IgH by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM. The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma. Introduction Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population 1. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year 2. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR and immunoglobulin heavy chain (IgH by polymerase chain reaction (PCR assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal

  19. Recombinant shark natural antibodies to thyroglobulin.

    Science.gov (United States)

    Schluter, Samuel F; Jensen, Ingvill; Ramsland, Paul A; Marchalonis, John J

    2005-01-01

    As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.

  20. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange.

    Science.gov (United States)

    van der Neut Kolfschoten, Marijn; Schuurman, Janine; Losen, Mario; Bleeker, Wim K; Martínez-Martínez, Pilar; Vermeulen, Ellen; den Bleker, Tamara H; Wiegman, Luus; Vink, Tom; Aarden, Lucien A; De Baets, Marc H; van de Winkel, Jan G J; Aalberse, Rob C; Parren, Paul W H I

    2007-09-14

    Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

  1. Transgenes of the mouse immunoglobulin heavy chain locus, lacking distal elements in the 3' regulatory region, are impaired for class switch recombination.

    Directory of Open Access Journals (Sweden)

    Wesley A Dunnick

    Full Text Available The immunoglobulin heavy (H chain class switch is mediated by a deletional recombination event between µ and γ, α, or ε constant region genes. This recombination event is upregulated during immune responses by a regulatory region that lies 3' of the constant region genes. We study switch recombination using a transgene of the entire murine H chain constant region locus. We isolated two lines of mice in which the H chain transgenes were truncated at their 3' ends. The truncation in both transgenic lines results in deletion of the 3'-most enhancer (HS4 and a region with insulator-like structure and activities. Even though both truncated transgenes express the µ H chain gene well, they undergo very low or undetectable switch recombination to transgenic γ and α constant region genes. For both transgenic lines, germline transcription of some H chain constant regions genes is severely impaired. However, the germline transcription of the γ1 and γ2a genes is at wild type levels for the transgenic line with the larger truncation, but at reduced levels for the transgenic line with the smaller truncation. The dramatic reduction in class switch recombination for all H chain genes and the varied reduction in germline transcription for some H chain genes could be caused by (i insertion site effects or (ii deletion of enhancer elements for class switch recombination and transcription, or (iii a combination of both effects.

  2. Shared gene structures and clusters of mutually exclusive spliced exons within the metazoan muscle myosin heavy chain genes.

    Directory of Open Access Journals (Sweden)

    Martin Kollmar

    Full Text Available Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs. The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis. Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both have independently been developed

  3. Myosin heavy chain isoform expression regulates shortening velocity in smooth muscle: studies using an SMB KO mouse line.

    Science.gov (United States)

    Karagiannis, Peter; Babu, G J; Periasamy, M; Brozovich, Frank V

    2004-01-01

    The kinetics of smooth muscle are thought to be partially determined by the level of the expression of the 7 amino acid insert, SMB, in the myosin heavy chain, as SMB is generally expressed at higher levels in faster smooth muscle. In this study, we determined the role of this insert on shortening velocity and force regeneration following rapid reduction in muscle length (k(step)) in bladder tissue from a transgenic mouse line expressing the insert at three different levels: wild type (WT, +/+, SMB/SMB), an SMA homozygous type (SMB KO, -/-), and a heterozygous type (+/-, SMB/SMA). Smooth muscle from +/+ bladder shorten faster than both the +/- and -/- bladder smooth muscle when activated with Ca2+, consistent with SMB determining the shortening velocity of smooth muscle. The addition of Pi to the fully activated skinned bladder strips did not affect the rate of shortening for either the +/+ or -/- bladder types but did significantly decrease the rate of shortening for the +/- type. In contrast, the addition of ADP to fully Ca2+ activated bladder strips increased the rate of shortening for all three bladder types. However after thiophosphorylation, ADP slowed the shortening velocity. These data are consistent with shortening velocity being determined by the level of activation (or crossbridge attachment) in smooth muscle. The rates of force regeneration according to the k(step) protocol showed no differences between bladder types and also proved insensitive to either Pi or ADP. These data suggest that the rates of force regeneration were determined not only by the kinetics of the crossbridge cycle, but also by factors outside the contractile apparatus.

  4. Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix.

    Science.gov (United States)

    Briggs, David C; Birchenough, Holly L; Ali, Tariq; Rugg, Marilyn S; Waltho, Jon P; Ievoli, Elena; Jowitt, Thomas A; Enghild, Jan J; Richter, Ralf P; Salustri, Antonietta; Milner, Caroline M; Day, Anthony J

    2015-11-27

    The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

  5. Correlation between histochemically assessed fiber type distribution and isomyosin and myosin heavy chain content in porcine skeletal muscles.

    Science.gov (United States)

    Bee, G; Solomon, M B; Czerwinski, S M; Long, C; Pursel, V G

    1999-08-01

    Highly sensitive enzyme assays developed to differentiate skeletal muscle fibers allow the recognition of three main fiber types: slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG). Myosin, the predominant contractile protein in mammalian skeletal muscle, can be separated based on the electrophoretic mobility under nondissociating conditions into SM2, SM1, IM, FM3, and FM2 isoforms, or under dissociating conditions into myosin heavy chain (MHC) I, IIb, IIx/d, and IIa. The purpose of the present study was to determine whether the histochemical method of differentiation of fiber types is consistent with the electrophoretically identified isomyosin and MHC isoforms. These comparisons were made using serratus ventralis (SV), gluteus medius (GM), and longissimus muscles (LM) from 13 pigs. Two calculation methods for the histochemical assessed fiber type distribution were adopted. The first method incorporated the number of fibers counted for each fiber type and calculated a percentage of the total fiber number (fiber number percentage: FNP). The second method expressed the cross-sectional area of each fiber type as a percentage of the total fiber area measured per muscle (fiber area percentage: FAP). Independent of the calculation methods, correlation analyses revealed in all muscles a strong relation between SO fibers, the slow isomyosin (SM1 and SM2), and MHCI, as well as between the FG fibers, the fast isomyosin (FM3 and FM2), and MHCIIx/b content (PFOG fiber population assessed by histochemical analysis and intermediate isoform (IM) or MHCIIa content. The present results did not provide conclusive evidence as to which of the calculation methods (FNP or FAP) was more closely related to myosin composition of skeletal muscles. Despite some incompatibility between the methods, the present study shows that histochemical as well as electrophoretic analyses yielded important information about the composition of porcine

  6. A continuum of myofibers in adult rabbit extraocular muscle: force, shortening velocity, and patterns of myosin heavy chain colocalization.

    Science.gov (United States)

    McLoon, Linda K; Park, Han Na; Kim, Jong-Hee; Pedrosa-Domellöf, Fatima; Thompson, Ladora V

    2011-10-01

    Extraocular muscle (EOM) myofibers do not fit the traditional fiber typing classifications normally used in noncranial skeletal muscle, in part, due to the complexity of their individual myofibers. With single skinned myofibers isolated from rectus muscles of normal adult rabbits, force and shortening velocity were determined for 220 fibers. Each fiber was examined for myosin heavy chain (MyHC) isoform composition by densitometric analysis of electrophoresis gels. Rectus muscle serial sections were examined for coexpression of eight MyHC isoforms. A continuum was seen in single myofiber shortening velocities as well as force generation, both in absolute force (g) and specific tension (kN/m(2)). Shortening velocity correlated with MyHCIIB, IIA, and I content, the more abundant MyHC isoforms expressed within individual myofibers. Importantly, single fibers with similar or identical shortening velocities expressed significantly different ratios of MyHC isoforms. The vast majority of myofibers in both the orbital and global layers expressed more than one MyHC isoform, with up to six isoforms in single fiber segments. MyHC expression varied significantly and unpredictably along the length of single myofibers. Thus EOM myofibers represent a continuum in their histological and physiological characteristics. This continuum would facilitate fine motor control of eye position, speed, and direction of movement in all positions of gaze and with all types of eye movements-from slow vergence movements to fast saccades. To fully understand how the brain controls eye position and movements, it is critical that this significant EOM myofiber heterogeneity be integrated into hypotheses of oculomotor control.

  7. Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle.

    Science.gov (United States)

    Andruchova, Olena; Stephenson, Gabriela M M; Andruchov, Oleg; Stephenson, D George; Galler, Stefan

    2006-07-01

    Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis, a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity.

  8. Clonorchis sinensis ferritin heavy chain triggers free radicals and mediates inflammation signaling in human hepatic stellate cells.

    Science.gov (United States)

    Mao, Qiang; Xie, Zhizhi; Wang, Xiaoyun; Chen, Wenjun; Ren, Mengyu; Shang, Mei; Lei, Huali; Tian, Yanli; Li, Shan; Liang, Pei; Chen, Tingjin; Liang, Chi; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing

    2015-02-01

    Clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis, is associated with hepatobiliary damage, inflammation, periductal fibrosis, and the development of cholangiocarcinoma. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators which drive fibrogenesis; however, their endogenous sources and pathophysiological roles in host cells were not determined. C. sinensis ferritin heavy chain (CsFHC) was previously confirmed as a component of excretory/secretory products and exhibited a number of extrahepatic immunomodulatory properties in various diseases. In this study, we investigated the expression pattern and biological role of CsFHC in C. sinensis. CsFHC was expressed throughout life stages of C. sinensis. More importantly, we found that treatment of human hepatic stellate cell line LX-2 with CsFHC triggered the production of free radicals via time-dependent activation of NADPH oxidase, xanthine oxidase, and inducible nitric oxide synthase. The increase in free radicals substantially promoted the degradation of cytosolic IκBα and nuclear translocation of NF-κB subunits (p65 and p50). CsFHC-induced NF-κB activation was markedly attenuated by preincubation with specific inhibitors of corresponding free radical-producing enzyme or the antioxidant. In addition, CsFHC induced an increased expression level of proinflammatory cytokines, IL-1β and IL-6, in NF-κB-dependent manner. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

  9. Cold exposure increases slow-type myosin heavy chain 1 (MyHC1) composition of soleus muscle in rats.

    Science.gov (United States)

    Mizunoya, Wataru; Iwamoto, Yohei; Sato, Yusuke; Tatsumi, Ryuichi; Ikeuchi, Yoshihide

    2014-03-01

    The aim of this study was to examine the effects of cold exposure on rat skeletal muscle fiber type, according to myosin heavy chain (MyHC) isoform and metabolism-related factors. Male Wistar rats (7 weeks old) were housed individually at 4 ± 2°C as a cold-exposed group or at room temperature (22 ± 2°C) as a control group for 4 weeks. We found that cold exposure significantly increased the slow-type MyHC1 content in the soleus muscle (a typical slow-type fiber), while the intermediate-type MyHC2A content was significantly decreased. In contrast to soleus, MyHC composition of extensor digitorum longus (EDL, a typical fast-type fiber) and gastrocnemius (a mix of slow-type and fast-type fibers) muscle did not change from cold exposure. Cold exposure increased mRNA expression of mitochondrial uncoupling protein 3 (UCP3) in both the soleus and EDL. Cold exposure also increased mRNA expression of myoglobin, peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and forkhead box O1 (FOXO1) in the soleus. Upregulation of UCP3 and PGC1α proteins were observed with Western blotting in the gastrocnemius. Thus, cold exposure increased metabolism-related factors in all muscle types that were tested, but MyHC isoforms changed only in the soleus. © 2013 Japanese Society of Animal Science.

  10. Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression.

    Directory of Open Access Journals (Sweden)

    Kelly C Clause

    Full Text Available Skeletal muscle derived stem cells (MDSCs transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC and cardiac specific troponin-I (cTn-I positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13 and 20 (ED20, neonatal day 0 (ND0 and 4 (ND4, postnatal day 10 (PND10, and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle.

  11. Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex.

    Science.gov (United States)

    Gomez Toledo, Alejandro; Nilsson, Jonas; Noborn, Fredrik; Sihlbom, Carina; Larson, Göran

    2015-12-01

    The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)(n), to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally

  12. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

    Science.gov (United States)

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-04-22

    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies.

  13. Targeted therapy with bispecific single chain antibodies for acute leukemia%双特异单链抗体靶向治疗急性白血病

    Institute of Scientific and Technical Information of China (English)

    崔鹤; 张王刚

    2015-01-01

    目前除急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)以外的急性白血病( acute leukemia,AL)疗效堪忧,急需研发新的治疗药物。采用基因工程抗体技术制备的双特异性单链抗体能同时结合两个特异性抗原,与其他抗体药物相比有明显的优越性,是一种有很好临床应用前景的免疫治疗药物,本文对双特异性单链抗体靶向治疗急性白血病的最新进展做一综述。%At present,there is a urgent need to develop new drugs for acute leukemia patients because of their poor effect except for acute promyelocytic leukemia. The bispecific single chain antibodies can combine with two specific antigens using gene engineering antibody technology,show more obvious advantage than other antibody drugs,and po-tential prospect in clinical application. We described the current new targeted therapy with bispecific single chain anti-bodies for acute leukemia in this article.

  14. Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-Methamphetamine and 3,4-methylenedioxymethamphetamine.

    Science.gov (United States)

    Celikel, Reha; Peterson, Eric C; Owens, S Michael; Varughese, Kottayil I

    2009-11-01

    Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(+)-METH therapeutic single chain antibody fragment (scFv6H4, K(D)= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name "ecstasy." The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a C--H...O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 A, and 2.4 A, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.

  15. Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening.

    Science.gov (United States)

    Sydor, Jens R; Mariano, Maria; Sideris, Steve; Nock, Steffen

    2002-01-01

    Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

  16. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

    DEFF Research Database (Denmark)

    Álvarez-Cienfuegos, Ana; Alanes, Natalia Nuñez del Prado; Compte, Marta

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we p...

  17. Stable recombinant alpaca antibodies for detection of Tulip virus X

    NARCIS (Netherlands)

    Beekwilder, M.J.; Houwelingen, van A.M.M.L.; Beckhoven, van J.R.C.M.; Speksnijder, A.G.C.L.

    2008-01-01

    For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage

  18. Single chain FV constructs of anti-ganglioside GD2 antibodies for radioimaging and radioimmumotheraphy. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, N.K.V.; Larson, S.M.

    1993-11-01

    For the past several years, we have studied the anti-G{sub D2} murine monoclonal antibody, 3F8, in radiolabeled form, for diagnosis and therapy of neuroblastoma. The targeting properties of this antibody/antigen system are exceptional, with uptakes consistently in the highest range of reported results for in vivo human studies. The radioiodinated antibody 3F8 is now used by us as our criteria for diagnosis and staging of advanced neuroblastoma. This antibody is showing considerable promise also in our Phase I trials in Stage 4 neuroblastoma, and major responses are being seen at current dose level, with manageable marrow toxicity, but no limiting organ toxicity.

  19. Responses of Myosin Heavy Chain Phenotypes and Gene Expressions in Neck Muscle to Micro- an Hyper-Gravity in Mice

    Science.gov (United States)

    Ohira, Tomotaka; Ohira, Takashi; Kawano, F.; Shibaguchi, T.; Okabe, H.; Ohno, Y.; Nakai, N.; Ochiai, T.; Goto, K.; Ohira, Y.

    2013-02-01

    Neck muscles are known to play important roles in the maintenance of head posture against gravity. However, it is not known how the properties of neck muscle are influenced by gravity. Therefore, the current study was performed to investigate the responses of neck muscle (rhomboideus capitis) in mice to inhibition of gravity and/or increase to 2-G for 3 months to test the hypothesis that the properties of neck muscles are regulated in response to the level of mechanical load applied by the gravitational load. Three male wild type C57BL/10J mice (8 weeks old) were launched by space shuttle Discovery (STS-128) and housed in Japanese Experimental Module “KIBO” on the International Space Station in mouse drawer system (MDS) project, which was organized by Italian Space Agency. Only 1 mouse returned to the Earth alive after 3 months by space shuttle Atlantis (STS-129). Neck muscles were sampled from both sides within 3 hours after landing. Cage and laboratory control experiments were also performed on the ground. Further, 3-month ground-based control experiments were performed with 6 groups, i.e. pre-experiment, 3-month hindlimb suspension, 2-G exposure by using animal centrifuge, and vivarium control (n=5 each group). Five mice were allowed to recover from hindlimb suspension (including 5 cage control) for 3 months in the cage. Neck muscles were sampled bilaterally before and after 3-month suspension and 2-G exposure, and at the end of 3-month ambulation recovery. Spaceflight-associated shift of myosin heavy chain phenotype from type I to II and atrophy of type I fibers were observed. In response to spaceflight, 17 genes were up-regulated and 13 genes were down-regulated vs. those in the laboratory control. Expression of 6 genes were up-regulated and that of 88 genes were down-regulated by 3-month exposure to 2-G vs. the age-matched cage control. In response to chronic hindlimb suspension, 4 and 20 genes were up- or down-regulated. Further, 98 genes responded

  20. Expression profiles of myostatin, myogenin, and Myosin heavy chain in skeletal muscles of two rabbit breeds differing in growth rate.

    Science.gov (United States)

    Kuang, Liangde; Xie, Xiaohong; Zhang, Xiangyu; Lei, Min; Li, Congyan; Ren, Yongjun; Zheng, Jie; Guo, Zhiqiang; Zhang, Cuixia; Yang, Chao; Zheng, Yucai

    2014-01-01

    The purpose of the present study was to compare mRNA levels of myostatin (MSTN), myogenin (MyoG), and fiber type compositions in terms of myosin heavy chain (MyHC) in skeletal muscles of two rabbit breeds with different body sizes and growth rates. Longissimus dorsi and biceps femoris muscles of 16 Californian rabbits (CW) and 16 Germany great line of ZIKA rabbits (GZ) were collected at the ages of 35d and 84d (slaughter age). The results showed that the live weights of GZ rabbits of 35d and 84d old were approximately 36% and 26% greater than those of CW rabbits, respectively. Quantitative real-time PCR analysis revealed that at the age of 84d GZ rabbits contained significantly lower MSTN mRNA level and higher MyoG mRNA level in both longissimus dorsi and biceps femoris muscles than CW rabbits, and mRNA levels of MSTN and MyoG exhibited opposite changes from the age of 35d to 84d, suggesting that GZ rabbits were subjected to less growth inhibition from MSTN at slaughter age, which occurred most possibly in skeletal muscles. Four types of fiber were identified by real-time PCR in rabbit muscles, with MyHC-1 and MyHC-2D, MyHC-2B were the major types in biceps femoris and longissimus dorsi muscles, respectively. At the age of 84d, GZ rabbits contained greater proportion of MyHC-1 and decreased proportion of MyHC-2D and decreased lactate dehydrogenase activity in biceps femoris than CW rabbits, and the results were exactly opposite in longissimus dorsi, suggesting that GZ rabbits show higher oxidative capacity in biceps femoris muscle than CW rabbits. In conclusion, the trends of mRNA levels of MSTN and fiber types in GZ rabbits' skeletal muscles might be consistent with the putative fast growth characteristic of GZ rabbits compared to CW rabbits.

  1. Somatic populations of PGT135-137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics

    Directory of Open Access Journals (Sweden)

    Jiang eZhu

    2012-09-01

    Full Text Available Select HIV-1-infected individuals develop sera capable of neutralizing diverse viral strains. The molecular basis of this neutralization is currently being deciphered by the isolation of HIV-1-neutralizing antibodies. In one infected donor, three neutralizing antibodies, PGT135-137, were identified by assessment of neutralization from individually sorted B cells and found to recognize an epitope containing an N-linked glycan at residue 332 on HIV-1 gp120. Here we use deep sequencing and bioinformatics methods to interrogate the B cell record of this donor to gain a more complete understanding of the humoral immune response. PGT135-137-gene family-specific primers were used to amplify heavy and light chain-variable domain sequences. 454 pyrosequencing produced 141,298 heavy-chain sequences of IGHV4-39 origin and 87,229 light-chain sequences of IGKV3-15 origin. A number of heavy and light chain sequences of ~90% identity to PGT137, several to PGT136, and none of high identity to PGT135 were identified. After expansion of these sequences to include close phylogenetic relatives, a total of 202 heavy-chain sequences and 72 light-chain sequences were identified. These sequences were clustered into populations of 95% identity comprising 15 for heavy chain and 10 for light chain, and a select sequence from each population was synthesized and reconstituted with a PGT137-partner chain. Reconstituted antibodies showed varied neutralization phenotypes for HIV-1 clade A and D isolates. Sequence diversity of the antibody population represented by these tested sequences was notably higher than observed with a 454 pyrosequencing-control analysis on 10 antibodies of defined sequence, suggesting that this diversity results primarily from somatic maturation. Our results thus provide an example of how pathogens like HIV-1 are opposed by a varied humoral immune response, derived from intrinsic mechanisms of antibody development, and embodied by somatic populations

  2. Application of principal component analysis in the pollution assessment with heavy metals of vegetable food chain in the old mining areas

    Directory of Open Access Journals (Sweden)

    Gergen Iosif

    2012-12-01

    Full Text Available Abstract Background The aim of the paper is to assess by the principal components analysis (PCA the heavy metal contamination of soil and vegetables widely used as food for people who live in areas contaminated by heavy metals (HMs due to long-lasting mining activities. This chemometric technique allowed us to select the best model for determining the risk of HMs on the food chain as well as on people's health. Results Many PCA models were computed with different variables: heavy metals contents and some agro-chemical parameters which characterize the soil samples from contaminated and uncontaminated areas, HMs contents of different types of vegetables grown and consumed in these areas, and the complex parameter target hazard quotients (THQ. Results were discussed in terms of principal component analysis. Conclusion There were two major benefits in processing the data PCA: firstly, it helped in optimizing the number and type of data that are best in rendering the HMs contamination of the soil and vegetables. Secondly, it was valuable for selecting the vegetable species which present the highest/minimum risk of a negative impact on the food chain and human health.

  3. Defining the recognition elements of Lewis Y-reactive antibodies.

    Directory of Open Access Journals (Sweden)

    Somdutta Saha

    Full Text Available Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2 response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1-4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens.

  4. Defining the Recognition Elements of Lewis Y-Reactive Antibodies

    Science.gov (United States)

    Saha, Somdutta; Pashov, Anastas; Siegel, Eric R.; Murali, Ramachandran; Kieber-Emmons, Thomas

    2014-01-01

    Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1–4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens. PMID:25117628

  5. Defining the recognition elements of Lewis Y-reactive antibodies.

    Science.gov (United States)

    Saha, Somdutta; Pashov, Anastas; Siegel, Eric R; Murali, Ramachandran; Kieber-Emmons, Thomas

    2014-01-01

    Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1-4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens.

  6. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Zu-Quan [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Li, He-Ping [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zhang, Jing-Bo [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Huang, Tao [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Liu, Jin-Long; Xue, Sheng [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Wu, Ai-Bo [Institute for Agri-food Standards and Testing Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products, Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403 (China); Liao, Yu-Cai, E-mail: ycliao06@yahoo.com.cn [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); National Center of Plant Gene Research, Wuhan 430070 (China)

    2013-02-18

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding

  7. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    Energy Technology Data Exchange (ETDEWEB)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  8. Bone marrow transplantation from unrelated donors: the impact of mismatches with substitutions at position 116 of the human leukocyte antigen class I heavy chain.

    Science.gov (United States)

    Ferrara, G B; Bacigalupo, A; Lamparelli, T; Lanino, E; Delfino, L; Morabito, A; Parodi, A M; Pera, C; Pozzi, S; Sormani, M P; Bruzzi, P; Bordo, D; Bolognesi, M; Bandini, G; Bontadini, A; Barbanti, M; Frumento, G

    2001-11-15

    The hypothesis was tested that amino acid substitutions in specific positions within human leukocyte antigen class I heavy chain would have different impacts on transplant-related mortality (TRM) in patients receiving transplanted bone marrow from unrelated donors. One hundred patients and their unrelated donors were typed by sequence-based typing for the human leukocyte antigen (HLA)-A, -B, and -C loci. All pairs were matched for DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 loci. Forty pairs were also matched at class I, and 60 pairs had one or more mismatches at class I loci. It was found that substitutions at positions 116 and 114 of class I heavy chain significantly increased the risk for TRM in univariate and bivariate Cox analyses. Conversely, no association between number of multiple mismatches or number of amino acid substitutions and TRM was seen when positions 116 and 114 were adjusted for. Variables predictive of TRM in multivariate Cox analysis were number of cells infused, diagnosis (chronic myeloid leukemia [CML] or non-CML), and amino acid substitution at position 116 or 152. The only variable predictive of severe acute graft-versus-host disease (GVHD) in multivariate Cox analysis was substitution at position 116. Actuarial risk for acute GVHD grade III-IV, TRM, and relapse in pairs with substitutions at position 116 (n = 37) compared to other pairs (n = 63) was, respectively, 36% versus 14% (P =.01), 59% versus 28% (P =.001), and 25% versus 31% (P =.4). In conclusion these data suggest that substitutions at position 116 of class I heavy chain increase the risk for acute GVHD and TRM in patients who receive transplanted bone marrow from unrelated donors.

  9. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface.

    Science.gov (United States)

    Lewis, Steven M; Wu, Xiufeng; Pustilnik, Anna; Sereno, Arlene; Huang, Flora; Rick, Heather L; Guntas, Gurkan; Leaver-Fay, Andrew; Smith, Eric M; Ho, Carolyn; Hansen-Estruch, Christophe; Chamberlain, Aaron K; Truhlar, Stephanie M; Conner, Elaine M; Atwell, Shane; Kuhlman, Brian; Demarest, Stephen J

    2014-02-01

    Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

  10. Designing Optimal LNG Station Network for U.S. Heavy-Duty Freight Trucks using Temporally and Spatially Explicit Supply Chain Optimization

    Science.gov (United States)

    Lee, Allen

    The recent natural gas boom has opened much discussion about the potential of natural gas and specifically Liquefied Natural Gas (LNG) in the United States transportation sector. The switch from diesel to natural gas vehicles would reduce foreign dependence on oil, spur domestic economic growth, and potentially reduce greenhouse gas emissions. LNG provides the most potential for the medium to heavy-duty vehicle market partially due to unstable oil prices and stagnant natural gas prices. As long as the abundance of unconventional gas in the United States remains cheap, fuel switching to natural gas could provide significant cost savings for long haul freight industry. Amid a growing LNG station network and ever increasing demand for freight movement, LNG heavy-duty truck sales are less than anticipated and the industry as a whole is less economic than expected. In spite of much existing and mature natural gas infrastructure, the supply chain for LNG is different and requires explicit and careful planning. This thesis proposes research to explore the claim that the largest obstacle to widespread LNG market penetration is sub-optimal infrastructure planning. No other study we are aware of has explicitly explored the LNG transportation fuel supply chain for heavy-duty freight trucks. This thesis presents a novel methodology that links a network infrastructure optimization model (represents supply side) with a vehicle stock and economic payback model (represents demand side). The model characterizes both a temporal and spatial optimization model of future LNG transportation fuel supply chains in the United States. The principal research goal is to assess the economic feasibility of the current LNG transportation fuel industry and to determine an optimal pathway to achieve ubiquitous commercialization of LNG vehicles in the heavy-duty transport sector. The results indicate that LNG is not economic as a heavy-duty truck fuel until 2030 under current market conditions

  11. Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain

    Science.gov (United States)

    Berg, Jorg; Lotscher, Erika; Steimer, Kathelyn S.; Capon, Daniel J.; Baenziger, Jurg; Jack, Hans-Martin; Wabl, Matthias

    1991-06-01

    Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

  12. Molecular cloning and mRNA expression analysis of myosin heavy chain(MyHC)from fast skeletal muscle of grass carp,Ctenopharyngodon idella

    Institute of Scientific and Technical Information of China (English)

    褚武英; 符贵红; 宾石玉; 蒙涛; 周瑞雪; 成嘉; 赵发兰; 张红芳; 张建社

    2010-01-01

    The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequenc...

  13. Distance-Guided Forward and Backward Chain-Growth Monte Carlo Method for Conformational Sampling and Structural Prediction of Antibody CDR-H3 Loops.

    Science.gov (United States)

    Tang, Ke; Zhang, Jinfeng; Liang, Jie

    2017-01-10

    Antibodies recognize antigens through the complementary determining regions (CDR) formed by six-loop hypervariable regions crucial for the diversity of antigen specificities. Among the six CDR loops, the H3 loop is the most challenging to predict because of its much higher variation in sequence length and identity, resulting in much larger and complex structural space, compared to the other five loops. We developed a novel method based on a chain-growth sequential Monte Carlo method, called distance-guided sequential chain-growth Monte Carlo for H3 loops (DiSGro-H3). The new method samples protein chains in both forward and backward directions. It can efficiently generate low energy, near-native H3 loop structures using the conformation types predicted from the sequences of H3 loops. DiSGro-H3 performs significantly better than another ab initio method, RosettaAntibody, in both sampling and prediction, while taking less computational time. It performs comparably to template-based methods. As an ab initio method, DiSGro-H3 offers satisfactory accuracy while being able to predict any H3 loops without templates.

  14. Diagnosis of Avian bornavirus infection in psittaciformes by serum antibody detection and reverse transcription polymerase chain reaction assay using feather calami.

    Science.gov (United States)

    de Kloet, Arne H; Kerski, Anelle; de Kloet, Siwo R

    2011-05-01

    Avian bornavirus (ABV) is the causative agent of proventricular dilatation disease (PDD), a highly devastating and contagious disease of psittacines (parrots and parakeets), which has resulted in the death of many captive birds. Accurate diagnosis of bornavirus infection is therefore important for the identification and isolation of infected birds. The current study showed that nonvascular contour (chest) feather calami provide a ready and minimally invasive source of RNA for the detection of ABV by reverse transcription polymerase chain reaction (RT-PCR). Storage of the feathers at room temperature for at least a month did not affect the results. Serological analysis by enzyme-linked immunosorbent assay (ELISA) showed that identification of anti-bornaviral nucleoprotein P40 antibodies can identify many birds with a past or present infection. The presence of anti-avian bornaviral P24 phosphoprotein and P16 matrix protein antibodies was quite variable, rendering these antibodies less useful for diagnosis of ABV infection. The significance of the present findings is that the use of nonvascular feathers as a source of RNA allows sample collection under conditions where storage of other samples would be difficult. Serum detection by ELISA of anti-P40 antibodies allows the identification of infected birds when RT-PCR fails. © 2011 The Author(s)

  15. 人源抗恶性疟原虫单链抗体库的制备及鉴定%Preparation and identification of a human single chain Fv antibody against Plasmodium falciparum

    Institute of Scientific and Technical Information of China (English)

    何光志; 俞琦; 王乾宇; 曹峰; 黄高; 韩洁; 蔡琨; 安传伟; 田维毅; 邓树轩; 高英; 李世军; 吴玛莉; 王平; 王文佳; 奚锦

    2012-01-01

    OBJECTIVE To construct humanize phage antibody library against Plasmodium falciparum and to screen specificity scFv to Plasmodium falciparum. METHODS Total RN A was extracted from fresh blood of patients with Plasmodium falciparum followed RT-PCR. The heavy-chain and light-chain variable region genes (VH and VL) repertoire of immunoglobulin were amplified respectively by PCR. Then they were linked as VH-linker-VL sequence forming scFv by SOE (splicing by overlap extension) PCR. These fragments were cloned into the phagemid pCANTAB5E and the recombinant phagemids were transformed to susceptible Escherichi a coli TGI by roporation. After rescuing with helper phage M13K07, A natural single-chain antibody library for Plasmodium falciparum was successfully constructed. RESULTS 15 antigen binding clones were identified from 20 individual phagemid clones. CONCLUSION This study shows the strategy of phage display technique and of RT-PCR from peripheral blood lymphocytes mRNA may provide an alternative approach.%目的 通过噬菌体展示技术构建人源抗恶性疟原虫单链抗体库(ScFv),从中筛选出高亲和力的抗体基因片段,为进一步单链抗体表达打下基础.方法 收集患者新鲜外周血,进行淋巴细胞分离和总RNA的提取,提取总RNA经RT-PCR扩增出入源抗体重链和轻链抗体库,采用用重叠延伸拼接法将VH与VL以Linker相连组装成单链抗体基因(ScFv),将ScFv基因片段连接至载体pCANTAB5E,转化大肠杆菌TG1感受态细胞,构建的鼠源性抗恶性疟原虫天然噬菌体抗体库.结果 从20个噬菌体克隆中筛选到15个具有弓形虫可溶性抗原结合活性的阳性克隆.结论 从外周血淋巴细胞中获取可变区基因,利用噬菌体抗体库技术制备人源抗恶性疟原虫单链抗体的策略是可行的.

  16. Screening and identification of neutralizated single-chain antibody of anti-glomerular basement membrane antibody%中和抗肾小球基底膜抗体的单链抗体的筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    肖静; 刘章锁; 王沛; 黄留玉; 宋宏彬; 赵明辉

    2010-01-01

    Objective To screen a human single-chain variable fragments(scFv)against antiGBM antibody.Methods Using phage display technique,the phage antibody library was panned by antiglomerular basement membrane(GBM)antibody which was coated in a micro-titer plate,one clone was found to have high affinity to anti-GBM antibody.The DNA sequence of the positive clone was determined.Results Along with the increase of rounds anti-GBM antibody specific phage antibody was highly enriched and screening efficiency was increased 137 folds than the firest round.ELISA and competition inhibition assay showed that the scFv had a specific combination character with anti-GBM antibody.DNA sequencing confirmed that the whole gene of scFv was 750 bp,and in accordance with humanized single-chain variable region antibody sequence structure.Conclusion The results suggested that the scFv fragment to anti-GBM antibody could be successfully selected by recombinant phage antibody technique,which will laid an experimental foundation for further research of the therapy of Goodpasture syndrome.%目的 制备人抗肾小球基底膜(GBM)抗体的特异性人源化单链可变区抗体.方法 采用噬菌体表面展示技术,获得一个与人抗GBM抗体结合活性较强的单链可变区抗体片段的阳性克隆,并对该克隆进行DNA序列测定分析.结果 对噬菌体单链可变区抗体库经过3轮筛选后,与第1轮相比富集了137倍.噬菌体抗体与人抗GBM抗体的结合活性其中有35株克隆ELISA的吸光度较高.对这些噬菌体抗体进行交叉反应后,确定其中有10株交叉反应较弱.确定1株(C31)阳性克隆提取质粒,进行DNA序列测定,大小为750 bp,并符合人源化单链可变区抗体的序列结构.结论 应用噬菌体展示技术成功获得人-抗GBM抗体的单链可变区抗体基因,为临床上治疗Goodpasture综合征奠定实验基础.

  17. Antithyroglobulin antibody

    Science.gov (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  18. Mouse x pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies.

    Science.gov (United States)

    Jar, Ana M; Osorio, Fernando A; López, Osvaldo J

    2009-01-01

    The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse x pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.

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

    OpenAIRE

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

    2011-01-01

    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 Å resolution. A 15-residue span of the toxin is recognized by th...

  20. Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide. gamma. -chain-specific monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ioannides, C.G.; Itoh, K.; Fox, F.E.; Pahwa, R.; Good, R.A.; Platsoucas, C.D.

    1987-06-01

    The authors developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the lambda chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, they identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from /sup 125/I-labeled denatured lysates of T3/sup +/ WT31/sup -/ lymphocytes expanded in culture from a SCID patient. Chemical crosslinking of /sup 125/I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These experiments were done with polyclonal cell populations. Cloned T3/sup +/ WT31/sup -/ cell populations are required to determine whether the TCR contains two lambda polypeptide chains. Using the same 9D7 anti-P18K mAb and immunoblotting analysis, they identified a 35 kDa ..gamma..-chain polypeptide under reducing conditions expressed on purified L3T4/sup -/ Lyt2/sup -/ BALB/c mouse thymocytes. This ..gamma..-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.

  1. Dietary cholecalciferol regulates the recruitment and growth of skeletal muscle fibers and the expressions of myogenic regulatory factors and the myosin heavy chain in European sea bass larvae.

    Science.gov (United States)

    Alami-Durante, Hélène; Cluzeaud, Marianne; Bazin, Didier; Mazurais, David; Zambonino-Infante, José L

    2011-12-01

    The aim of this study was to determine whether dietary cholecalciferol affects the recruitment and growth of axial skeletal muscle fibers in first-feeding European sea bass. Larvae were fed diets containing 0.28 (VD-L, low dose), 0.69 (VD-C, control dose), or 3.00 (VD-H, high dose) mg cholecalciferol/kg from 9 to 44 d posthatching (dph). Larvae were sampled at 44 dph for quantification of somatic growth, muscle growth, and muscle growth dynamics and at 22 and 44 dph for the relative quantification of transcripts encoded by genes involved in myogenesis, cell proliferation, and muscle structure. The weight increase of the VD-L-fed larvae was less than that of the VD-H-fed group, whereas that of VD-C-fed larvae was intermediate. The level of expression of genes involved in cell proliferation (PCNA) and early myogenesis (Myf5) decreased between 22 and 44 dph, whereas that of the myogenic determination factor MyoD1 and that of genes involved in muscle structure and function (myosin heavy chain, myosin light chains 2 and 3) increased. Dietary cholecalciferol regulated Myf5, MyoD1, myogenin, and myosin heavy chain gene expression, with a gene-specific shape of response. The maximum hypertrophy of white muscle fibers was higher in larvae fed the VD-C and VD-H diets than in larvae fed the VD-L diet. White muscle hyperplasia was highly stimulated in VD-H-fed larvae compared to VD-L- and VD-C-fed ones. These findings demonstrate a dietary cholecalciferol effect on skeletal muscle growth mechanisms of a Teleost species.

  2. Polymorphisms of the T cell receptor CD3delta and CD3varepsilon chains affect anti-CD3 antibody binding and T cell activation

    DEFF Research Database (Denmark)

    Boding, Lasse; Nielsen, Martin Weiss; Bonefeld, Charlotte Menné

    2010-01-01

    T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability...... result in amino acid substitutions in the ectodomains of both the CD3delta and CD3varepsilon chains in 129 mice compared to C57BL/6 mice. The amino acid substitutions do not change the stoichiometry or surface expression level of the TCR complex in 129 T cells but cause reduced anti-CD3 antibody binding...... to 129 T cells. Further, when stimulated with mitogenic anti-CD3 antibodies, T cells from the 129 strains show reduced expression of the activation marker CD69, Ca(2+) flux, IL-2 production and proliferative responses compared to C57BL/6 T cells. These findings demonstrate that polymorphisms of the CD3...

  3. Rational Design of CXCR4 Specific Antibodies with Elongated CDRs

    Science.gov (United States)

    2015-01-01

    The bovine antibody (BLV1H12) which has an ultralong heavy chain complementarity determining region 3 (CDRH3) provides a novel scaffold for antibody engineering. By substituting the extended CDRH3 of BLV1H12 with modified CXCR4 binding peptides that adopt a β-hairpin conformation, we generated antibodies specifically targeting the ligand binding pocket of CXCR4 receptor. These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range. In addition, they inhibit SDF-1-dependent signal transduction and cell migration in a transwell assay. Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a Kd of 0.9 nM. This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future. PMID:25041362

  4. Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

    OpenAIRE

    Hiroaki Matsushita; Akiko Sano; Hua Wu; Jin-An Jiao; Poothappillai Kasinathan; Eddie J. Sullivan; Zhongde Wang; Yoshimi Kuroiwa

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM−/− , bIGHM...

  5. Triple Immunoglobulin Gene Knockout Transchromosomic (Tc) Cattle: Bovine Lambda Cluster Deletion and its Effect on Fully Human Polyclonal Antibody Production

    OpenAIRE

    Matsushita, H.; Sano, A.; Wu, H.; J. Jiao; Kasinathan, P.; Sullivan, E. J.; Wang, Zhongde; Kuroiwa, K

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML...

  6. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  7. Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes.

    Directory of Open Access Journals (Sweden)

    Man Cheng

    Full Text Available Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3. The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx. Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N protein of SARS-associated coronavirus (SCoV were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.

  8. Generation of intracellular single-chain antibodies directed against polypeptide GalNAc-transferase using a yeast two-hybrid system.

    Science.gov (United States)

    Ma, Li; Koyota, Souichi; Myoen, Yu; Yamashita, Tetsuro; Yatabe, Naoki; Koizumi, Yukio; Aosasa, Masayoshi; Nishimichi, Norihisa; Matsuda, Haruo; Sugiyama, Toshihiro

    2012-02-24

    Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.

  9. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

    Science.gov (United States)

    Wesolowski, Janusz; Alzogaray, Vanina; Reyelt, Jan; Unger, Mandy; Juarez, Karla; Urrutia, Mariela; Cauerhff, Ana; Danquah, Welbeck; Rissiek, Björn; Scheuplein, Felix; Schwarz, Nicole; Adriouch, Sahil; Boyer, Olivier; Seman, Michel; Licea, Alexei; Serreze, David V; Goldbaum, Fernando A; Haag, Friedrich; Koch-Nolte, Friedrich

    2009-08-01

    Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

  10. Solid phase synthesis of bifunctional antibodies.

    Science.gov (United States)

    DeSilva, B S; Wilson, G S

    1995-12-15

    Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab')2 fragments thus produced did not contain any IgG impurities. The Fab' fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.

  11. Generation of cytotoxic T lymphocytes specific for B-cell acute lymphoblastic leukemia family-shared peptides derived from immunoglobulin heavy chain framework region

    Institute of Scientific and Technical Information of China (English)

    LIU Ying; ZHU Ping; HU Ya-mei

    2007-01-01

    Background Immunoglobulin heavy chain variable region (IgHV) is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte (CTL) responses. However,complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions (FR) shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia (B-ALL). Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.Methods Seven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A*0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay.Results Complete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed ≥98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature.Twelve nonapeptides of high HLA-A*0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten (83%) of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 (3-11) shared among the IgHV1

  12. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    Science.gov (United States)

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars.

  13. Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae.

    NARCIS (Netherlands)

    Gorlani, A.; Hulsik, D.L.; Adams, H.; Vriend, G.; Hermans, P.; Verrips, T.

    2012-01-01

    Variable domains of llama heavy-chain antibodies (VHH) are becoming a potent tool for a wide range of biotechnological and medical applications. Because of structural features typical of their single-domain nature, they are relatively easy to produce in lower eukaryotes, but it is not uncommon that

  14. Generation of human single-chain variable fragment antibodies specific to dengue virus non-structural protein 1 that interfere with the virus infectious cycle.

    Science.gov (United States)

    Poungpair, Ornnuthchar; Bangphoomi, Kunan; Chaowalit, Prapaipit; Sawasdee, Nunghathai; Saokaew, Nichapatr; Choowongkomon, Kiattawee; Chaicumpa, Wanpen; Yenchitsomanus, Pa-thai

    2014-01-01

    Severe forms of dengue virus (DENV) infection frequently cause high case fatality rate. Currently, there is no effective vaccine against the infection. Clinical cases are given only palliative treatment as specific anti-DENV immunotherapy is not available and it is urgently required. In this study, human single-chain variable fragment (HuScFv) antibodies that bound specifically to the conserved non-structural protein-1 (NS1) of DENV and interfered with the virus replication cycle were produced by using phage display technology. Recombinant NS1 (rNS1) of DENV serotype 2 (DENV2) was used as antigen in phage bio-panning to select phage clones that displayed HuScFv from antibody phage display library. HuScFv from two phagemid transformed E. coli clones, i.e., clones 11 and 13, bound to the rNS1 as well as native NS1 in both secreted and intracellular forms. Culture fluids of the HuScFv11/HuScFv13 exposed DENV2 infected cells had significant reduction of the infectious viral particles, implying that the antibody fragments affected the virus morphogenesis or release. HuScFv epitope mapping by phage mimotope searching revealed that HuScFv11 bound to amino acids 1-14 of NS1, while the HuScFv13 bound to conformational epitope at the C-terminal portion of the NS1. Although the functions of the epitopes and the molecular mechanism of the HuScFv11 and HuScFv13 require further investigations, these small antibodies have high potential for development as anti-DENV biomolecules.

  15. Identification of the specificity of isolated phage display single-chain antibodies using yeast two-hybrid screens

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik

    2009-01-01

    A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a yeast ...... two-hybrid system that additionally allows for reorganization of post-translational modifications to the bait and target proteins. This technique is therefore especially useful for identifying surface-expressed antigens....

  16. Serum immunoglobulin free light chain measurements and heavy chain isotype usage provide insight into disease biology in patients with POEMS syndrome.

    Science.gov (United States)

    Stankowski-Drengler, Trista; Gertz, Morie A; Katzmann, Jerry A; Lacy, Martha Q; Kumar, Shaji; Leung, Nelson; Hayman, Suzanne R; Buadi, Francis; Kyle, Robert A; Rajkumar, S Vincent; Dispenzieri, Angela

    2010-06-01

    POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) syndrome is a rare paraneoplastic syndrome in which nearly all patients have a monoclonal lambda restricted plasma cell disorder. We investigated whether patients with POEMS have abnormal serum immunoglobulin free light chain (FLC) ratios. Fifty patients with newly diagnosed POEMS syndrome were assessed. Cystatin C levels were measured to discern whether subclinical renal insufficiency could account for FLC elevations in the presence of a normal FLC ratio. Forty-five patients (90%) had elevated lambda FLC; however, only nine (18%) had abnormal FLC ratios. The rise in serum FLC of POEMS patients appeared to be multifactorial-both a function of subclinical renal insufficiency and polyclonal activation of medullary and extramedullary plasma cells. Those patients expressing a clonal IgA were more likely to have clonal plasmacytosis observed on iliac crest biopsy than those with IgG. In summary, serum immunoglobulin profiles are unique in POEMS syndrome as compared with other plasma cell disorders.

  17. A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

    DEFF Research Database (Denmark)

    Sylvester-Hvid, C; Buus, S

    1999-01-01

    recombinant MHC-I molecules. A convenient source of MHC-I molecules would be a valuable tool in structural and biochemical analysis of MHC-I, and in experiments using MHC-I molecules to enable specific manipulations of experimental and physiological CTL responses. Here we describe the generation...... of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule...... was denatured, extracted, purified and folded using a recently developed in vitro reiterative refolding strategy. This led to the formation of soluble, recombinant MHC-I molecules, which migrated as monomers of the expected size when submitted to non-reducing sodium dodecyl sulphate-polyacrylamide gel...

  18. Prognostic significance of highly sulfated chondroitin sulfates in ovarian cancer defined by the single chain antibody GD3A11

    NARCIS (Netherlands)

    Steen, S.C.H.A. van der; Tilborg, A.G. van; Vallen, M.J.E.; Bulten, J.; Kuppevelt, T.H. van; Massuger, L.F.

    2016-01-01

    OBJECTIVE: The extracellular matrix (ECM) of ovarian cancer may provide a number of potential biomarkers. Chondroitin sulfate (CS), a class of sulfated polysaccharides, is abundantly present in the ECM of ovarian cancer. Structural alterations of CS chains (i.e. sulfation pattern) have been demonstr

  19. Comparisons of different myosin heavy chain types, AMPK, and PGC-1α gene expression in the longissimus dorsi muscles in Bama Xiang and Landrace pigs.

    Science.gov (United States)

    Huang, Y N; Ao, Q W; Jiang, Q Y; Guo, Y F; Lan, G Q; Jiang, H S

    2016-07-14

    Bama Xiang and Landrace pigs are the local fatty and lean breeds, respectively, in China. We compared differences in carcass traits, meat quality traits, and myosin heavy chain (MyHC) types in the longissimus dorsi muscles between Bama Xiang and Landrace pigs. This was done in pigs of the same age, using real-time PCR, to investigate the relationship between MyHC fiber types and carcass characteristics, meat quality traits, and the key factors regulating muscle fiber type. Bama Xiang pigs exhibited smaller size and slower growth than Landrace pigs (P Landrace pig muscle had a higher glycolytic type IIb muscle fiber content (P Landrace pigs. These results may provide a theoretical basis for further studies of the molecular mechanism underlying the excellent meat quality of the Bama Xiang pig.

  20. Molecular characterization and immunological response analysis of a novel transferrin-like, pacifastin heavy chain protein in giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879).

    Science.gov (United States)

    Toe, Aung; Areechon, Nontawith; Srisapoome, Prapansak

    2012-10-01

    The full-length cDNA of the pacifastin heavy chain gene from giant freshwater prawn (Macrobrachium rosenbergii, Mr-PHC) was cloned and characterized. The full sequence of the Mr-PHC cDNA was 4331 bp and contained a 119-bp 5'-untranslated region (UTR), a 3990-bp open reading frame (ORF) encoding 1329 amino acid residues and a 222-bp 3' UTR. The Mr-PHC protein predicted by its full ORF, exhibited a unique transferrin-like protein structure containing 4 different lobes that have not been previously identified. Three of the four lobes contained highly conserved of iron/anion binding residues. Expression analyses by conventional RT-PCR demonstrated that Mr-PHC was expressed predominantly during postlarval stage 45 and also in the foregut and gills of the adult prawn. Interestingly, dose response analyses that were quantified using quantitative real-time PCR indicated a significant upregulation of Mr-PHC during postlarval stage 45 in prawn grown at hour 24 after challenging with 10(9) cfu/ml of Aeromonas hydrophila, which is a pathogenic bacterium. Mr-HPC in the adult prawn was significantly upregulated at both hour 12 and day 7 after stimulation with A. hydrophila (P < 0.05 and P < 0.01, respectively). Additionally, a delayed induction response of the Mr-PHC gene was observed at 14 days when the experimental adult prawns were fed with β-glucan-supplemented feed. Based on results of this study, the transferrin-like protein encoded by the pacifastin heavy chain gene may exist in all decapod crustaceans. Even though the function as an iron transporter is not proven, immune response studies are clearly indicated that PHC is critically involved in the immune system in these animals.

  1. Recombination events near the immunoglobulin Cmu gene join variable and constant region genes, switch heavy-chain expression, or inactivate the locus.

    Science.gov (United States)

    Cory, S; Webb, E; Gough, J; Adams, J M

    1981-04-28

    Immunoglobulin heavy-chain expression is initiated by recombination between a variable region (VH) gene and one of several joining region (JH) genes located near the mu constant region (Cmu) gene, and the active VH gene can subsequently switch to another CH gene. That the general mechanism for CH switching involves recombination between sites within the JH-Cmu intervening sequence and the 5' flanking region of another CH gene is supported here by Southern blot hybridization analysis of eight IgG- and IgA-secreting plasmacytomas. An alternative model requiring successive VH linkage to similar JH clusters near each CH gene is shown to be very unlikely since the mouse genome appears to contain only one complement of the JH locus and no JH gene was detectable within large cloned sequences flanking germline C gamma 3 and C gamma 1 genes. Thus, VH-JH joining and CH switching are mediated by separate regions of "the joining-switch" or J-S element. In each plasmacytoma examined, the J-S element had undergone recombination within both the JH locus and the switch region and was shown to be linked to the functional CH gene in an IgG3, and IgG1, and three IgA secretors. Both JH joining and CH switching occurred by deletion of DNA. Switch recombination occurred at more than one site within the J-S element in different lines, even for recombination with the same CH gene. Significantly, although heavy-chain expression is restricted to one allele ("allelic exclusion"), all rearranged in each plasmacytoma. Some rearrangements were aberrant, involving, for example, deletion of all JH genes from the allele. Hence, an error-prone recombination machinery may account for allelic exclusion in many plasmacytomas.

  2. Differential effects of docoosahexaenoic and arachidonic acid on fatty acid composition and myosin heavy chain-related genes of slow- and fast-twitch skeletal muscle tissues.

    Science.gov (United States)

    Hashimoto, Michio; Inoue, Takayuki; Katakura, Masanori; Hossain, Shahdat; Mamun, Abdullah Al; Matsuzaki, Kentaro; Arai, Hiroyuki; Shido, Osamu

    2016-04-01

    Myosin heavy chain (MHC) mediates the metabolic and contractile responses of skeletal muscles. MHC displays different isoforms, each of which has different characteristics. To better understand the effect of polyunsaturated fatty acids in skeletal muscles, rats were fed with control-, docosahexaenoic acid (DHA)-, and arachidonic acid (ARA)-oil, and the effects on plasma and muscular fatty acid profile, oxidative stress, mRNA levels of myosin heavy chain isoforms MHC1 of slow-twitch muscle (SO) and MHC2A, MHC2X, and MHCB isoforms of extensor digitorum longus (EDL) of fast-twitch muscle were evaluated. Concomitantly, mRNA levels of anti-oxidative enzymes, such as, catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD were determined. The expressions of MHC1, MHC2A, MHC2X, and MHC2B were lower in the SO of the DHA-fed rats. In the EDL muscles of DHA-fed rats, the expressions of MHC1 and MHC2A increased; however, the expressions of MHC2X increased and that of the MHC2 were not altered. Oxidative stress, as indicated by the levels of LPO, was significantly higher in the plasma of the ARA-fed rats, when compared with that of the DHA-fed rats. The LPO levels were higher both in the SO and EDL muscles of ARA-fed rats. Compared with ARA oil intake, DHA oil showed higher mRNA levels of GPx and SOD. Catalase expression was higher only in the EDL but not in the SO-type muscles. Our studies finally indicate that DHA and ARA differentially affect the regulation of contractile and metabolic properties of slow- and fast-twitch skeletal muscles.

  3. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact......, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides....

  4. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E. (Vanderbilt); (Scripps); (CDC)

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  5. High-Throughput Kinetic Characterization of Ricin Toxin B Chain and Ovalbumin Antibodies Using Surface Plasmon Resonance

    Science.gov (United States)

    2013-10-01

    developing these antibodies.                                                               Calm, A. Microbiological and Molecular Operations; SOP RNB...Bean Proteins in a Ricin Preparation, Castor Bean Extract, and Surface Swabs from a Public Health Investigation. Forensic Sci. Int. 2011, 209(1‒3

  6. Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter-alpha-trypsin inhibitor and saposin B but increased expression of an inter-alpha-trypsin inhibitor heavy chain 4 fragment.

    Science.gov (United States)

    Jayapalan, Jaime J; Ng, Keng L; Shuib, Adawiyah S; Razack, Azad H A; Hashim, Onn H

    2013-06-01

    The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Purification and on-column refolding of a single-chain antibody fragment against rabies virus glycoprotein expressed in Escherichia coli.

    Science.gov (United States)

    Xi, Hualong; Yuan, Ruosen; Chen, Xiaoxu; Gu, Tiejun; Cheng, Yue; Li, Zhuang; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-10-01

    An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.

  8. Utility of recombinant Fragment C for assessment of anti-tetanus antibodies in plasma

    Science.gov (United States)

    Ramakrishnan, Girija; Pedersen, Karl; Guenette, Denis; Sink, Joyce; Haque, Rashidul; Petri, William A.; Herbein, Joel; Gilchrist, Carol A.

    2016-01-01

    Anti-tetanus antibodies in biological samples are typically detected using an ELISA based on toxoided tetanus neurotoxin as antigen. We demonstrate that recombinantly produced Fragment C of the toxin heavy chain (rFragC) is an effective alternative antigen for assessment of tetanus- immune status in plasma samples. PMID:25749462

  9. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

    Science.gov (United States)

    Pecora, Andrea; Malacari, Darío A; Pérez Aguirreburualde, María S; Bellido, Demian; Escribano, José M; Dus Santos, María J; Wigdorovitz, Andrés

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 μg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  10. Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment.

    Science.gov (United States)

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

    2011-06-10

    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 Å 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 β-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.

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

    Science.gov (United States)

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

    2011-01-01

    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 Å 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 β-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. PMID:21489992

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  13. Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer showed that scFv M97 transfection significantly prolonged the survival time of nude mice. The results indicate that intracellular antibody technology represents a novel and efficient way to abrogate selectively the activity of type IV collagenase.

  14. Generation and expression in plants of a single-chain variable fragment antibody against the immunodominant membrane protein of Candidatus phytoplasma aurantifolia.

    Science.gov (United States)

    Shahryari, F; Safarnejad, M R; Shams-Bakhsh, M; Schillberg, S; Nölke, G

    2013-08-01

    Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

  15. Varicella Zoster Virus Myelitis in Two Elderly Patients: Diagnostic Value of Nested Polymerase Chain Reaction Assay and Antibody Index for Cerebrospinal Fluid Specimens

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    Teruyuki Takahashi

    2013-04-01

    Full Text Available Background: Myelitis is one of the rarest neurological complications of the varicella zoster virus (VZV infection. Focal muscle weakness with or without sensory disturbance occurs in approximately 5% of the cases after acute VZV infection, with complete recovery in 50-70%. Case Presentation: This report describes two rare cases of elderly patients with VZV myelitis secondary to dermatomal zoster rash. Patient 1 was a 79-year-old woman who developed paraplegia, numbness and decreased sensation in the left arm and below thoracic (Th-10 after sacral zoster. Spinal cord MRI showed a high-signal-intensity lesion at the cervical spinal nerve 2 on a T2-weighted image. Patient 2 was a 73-year-old man who developed right flaccid leg weakness and urinary retention after right dorsal Th 5-8 zoster. Spinal cord MRI showed a high-signal-intensity lesion at Th 3-4 on a T2-weighted image. In both cases, although the conventional single polymerase chain reaction (PCR assays all showed negative results, the original nested PCR assay detected VZV DNA in the cerebrospinal fluid (CSF specimen collected on admission. In addition, the anti-VZV IgG antibody by enzyme immunoassay and antibody index were elevated in the CSF specimens during the clinical courses of both patients. On the basis of these findings, both patients were diagnosed with VZV myelitis and were treated with high-dose acyclovir and corticosteroid. This combined treatment was appropriate and effective for the improvement of their functional outcomes. Conclusion: The detection of VZV DNA in CSF by nested PCR assay and the evaluation of the antibody index to VZV had significant diagnostic value.

  16. Varicella zoster virus myelitis in two elderly patients: diagnostic value of nested polymerase chain reaction assay and antibody index for cerebrospinal fluid specimens.

    Science.gov (United States)

    Takahashi, Teruyuki; Tamura, Masato; Miki, Kenji; Yamaguchi, Mai; Kanno, Akira; Nunomura, Satoshi; Ra, Chisei; Tamiya, Takashi; Kamei, Satoshi; Takasu, Toshiaki

    2013-01-01

    Myelitis is one of the rarest neurological complications of the varicella zoster virus (VZV) infection. Focal muscle weakness with or without sensory disturbance occurs in approximately 5% of the cases after acute VZV infection, with complete recovery in 50-70%. This report describes two rare cases of elderly patients with VZV myelitis secondary to dermatomal zoster rash. Patient 1 was a 79-year-old woman who developed paraplegia, numbness and decreased sensation in the left arm and below thoracic (Th)-10 after sacral zoster. Spinal cord MRI showed a high-signal-intensity lesion at the cervical spinal nerve 2 on a T2-weighted image. Patient 2 was a 73-year-old man who developed right flaccid leg weakness and urinary retention after right dorsal Th 5-8 zoster. Spinal cord MRI showed a high-signal-intensity lesion at Th 3-4 on a T2-weighted image. In both cases, although the conventional single polymerase chain reaction (PCR) assays all showed negative results, the original nested PCR assay detected VZV DNA in the cerebrospinal fluid (CSF) specimen collected on admission. In addition, the anti-VZV IgG antibody by enzyme immunoassay and antibody index were elevated in the CSF specimens during the clinical courses of both patients. On the basis of these findings, both patients were diagnosed with VZV myelitis and were treated with high-dose acyclovir and corticosteroid. This combined treatment was appropriate and effective for the improvement of their functional outcomes. The detection of VZV DNA in CSF by nested PCR assay and the evaluation of the antibody index to VZV had significant diagnostic value.

  17. Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies.

    Science.gov (United States)

    Su, Yu-Cheng; Al-Qaisi, Talal S; Tung, Hsin-Yi; Cheng, Tian-Lu; Chuang, Kuo-Hsiang; Chen, Bing-Mae; Roffler, Steve R

    2014-01-01

    Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative "cloning-free" approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.

  18. Primary structure of bovine calpactin I heavy chain (p36), a major cellular substrate for retroviral protein-tyrosine kinases: homology with the human phospholipase A2 inhibitor lipocortin

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Sarin, C J; Hunter, T;

    1986-01-01

    An amplified Okayama-Berg plasmid cDNA library was constructed from total poly(A)+ RNA isolated from the Madin-Darby bovine kidney cell line MDBK. This library was screened with a partial murine calpactin I heavy chain (p36) cDNA clone, the identification of which was based on bovine p36 tryptic ...

  19. Heavy metals in the habitat and throughout the food chain of the Neotropical otter, Lontra longicaudis, in protected Mexican wetlands.

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    Ramos-Rosas, Nadia N; Valdespino, Carolina; García-Hernández, Jaqueline; Gallo-Reynoso, Juan P; Olguín, Eugenia J

    2013-02-01

    Top predators like the Neotropical otter, Lontra longicaudis annectens, are usually considered good bioindicators of habitat quality. In this study, we evaluated heavy metal contamination (Hg(tot), Pb, Cd) in the riverine habitat, prey (crustaceans and fish), and otter feces in two Ramsar wetlands with contrasting upstream contamination discharges: Río Blanco and Río Caño Grande in Veracruz, Mexico, during the dry, the wet, and the nortes seasons. Most comparisons revealed no differences between sites while seasonal differences were repeatedly detected for all of the compartments. Higher concentrations of Pb during the dry season and of Cd during the wet season in otter feces mirrored differences detected in the most seasonally consumed prey. Compared with fecal methylmercury values reported for the European otter (0.25-0.75 mg kg(-1)) in unprotected areas, the Hg(tot) levels that we measured were lower (0.02-0.17 mg kg(-1)). However, Pb (117.87 mg kg(-1)) and Cd (9.14 mg kg(-1)) concentrations were higher (Pb, 38.15 mg kg(-1) and Cd, 4.72 mg kg(-1)) in the two Ramsar wetlands. Protected areas may shelter species, but those with water-linked diets may suffer the effect of chemicals used upstream.

  20. PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies.

    Science.gov (United States)

    Tabibian-Keissar, Hilla; Schibby, Ginette; Michaeli, Miri; Rakovsky-Shapira, Aviya; Azogui-Rosenthal, Noemie; Dunn-Walters, Deborah K; Rosenblatt, Kinneret; Mehr, Ramit; Barshack, Iris

    2013-02-01

    The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives.

  1. Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Choudhary, Nandlal; Roy, Avijit; Govindarajulu, A; Nakhla, M K; Levy, L; Brlansky, R H

    2014-09-01

    Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing. Published by Elsevier B.V.

  2. Epigenetic regulation of individual modules of the immunoglobulin heavy chain locus 3’ regulatory region (3’ RR

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    Barbara K Birshtein

    2014-04-01

    Full Text Available The Igh locus undergoes an amazing array of DNA rearrangements and modifications during B cell development. During early stages, the variable region gene is constructed from constituent variable (V, diversity (D and joining (J segments (VDJ joining. B cells that successfully express an antibody can be activated, leading to somatic hypermutation (SHM focused on the variable region, and class switch recombination (CSR, which substitutes downstream constant region genes for the originally used Cμ constant region gene. Many investigators, ourselves included, have sought to understand how these processes specifically target the Igh locus and avoid other loci and potential deleterious consequences of malignant transformation. Our laboratory has concentrated on a complex regulatory region (RR that is located downstream of Cα, the most 3’ of the Igh constant region genes. The ~40 kb 3’ RR, which is predicted to serve as a downstream major regulator of the Igh locus, contains two distinct segments: an ~28 kb region of four enhancers and an adjacent ~12 kb region containing multiple CTCF and Pax5 binding sites. Analysis of targeted mutations in mice by a number of investigators has concluded that the entire 3’ RR enhancer region is essential for SHM and CSR (but not for VDJ joining and for high levels of expression of multiple isotypes. The CTCF/Pax5 binding region is a candidate for influencing VDJ joining early in B cell development and serving as a potential insulator of the Igh locus. Components of the 3’ RR are subject to a variety of epigenetic changes during B cell development, i.e., DNAse I hypersensitivity, histone modifications and DNA methylation, in association with transcription factor binding. We propose that these changes provide a foundation by which regulatory elements in modules of the 3’ RR function by interacting with each other and with target sequences of the Igh locus.

  3. Cloning of a hamster anti-mouse CD79B antibody sequences and identification of a new hamster immunoglobulin lambda constant IGLC gene region.

    Science.gov (United States)

    Haggart, Ryan; Perera, Jason; Huang, Haochu

    2013-06-01

    Anti-CD79 antibodies have been effective at targeting B cell lymphoma cells and depleting B cells in animal models. In order to engineer recombinant antibodies with additional effector functions in mice, we cloned and sequenced the full-length cDNAs of the heavy and light chain of a hamster anti-mouse CD79B antibody. Although hamster antibodies represent a unique source of monoclonal antibodies against mouse, rat, and human antigens, sequence information of hamster immunoglobulins (IG) is sparse. Here, we report a new hamster (Cricetulus migratorius) IG lambda constant (IGLC) gene region that is most homologous to mouse IGLC2 and IGLC3.

  4. Calcineurin-NFAT Signaling and Neurotrophins Control Transformation of Myosin Heavy Chain Isoforms in Rat Soleus Muscle in Response to Aerobic Treadmill Training

    Directory of Open Access Journals (Sweden)

    Wenfeng Liu, Gan Chen, Fanling Li, Changfa Tang

    2014-12-01

    Full Text Available This study elucidated the role of CaN-NFAT signaling and neurotrophins on the transformation of myosin heavy chain isoforms in the rat soleus muscle fiber following aerobic exercise training. To do so, we examined the content and distribution of myosin heavy chain (MyHC isoforms in the rat soleus muscle fiber, the activity of CaN and expression of NFATc1 in these fibers, and changes in the expression of nerve growth factor (NGF, brain-derived neurotrophic factor (BDNF and neutrophin-3 (NT-3 in the soleus and striatum following high-and medium-intensity aerobic treadmill training. Specific pathogen-free 2 month old male Sprague-Dawley (SD rats were randomly divided into three groups: Control group (Con, n = 8, moderate-intensity aerobic exercise group (M-Ex, n = 8 and high-intensity aerobic exercise group (H-Ex, n = 8. We used ATPase staining to identify the muscle fiber type I and II, SDS-PAGE to separate and analyze the isoforms MyHCI, MyHCIIA, MyHCIIB and MyHCIIx, and performed western blots to determine the expression of NFATc1, NGF, BDNF and NT-3. CaN activity was measured using a colorimetric assay. In the soleus muscle, 8 weeks of moderate-intensity exercise can induce transformation of MyHC IIA and MyHC IIB to MyHC IIX and MyHC I (p < 0.01, while high-intensity treadmill exercise can induce transform MyHC IIx to MyHC IIB, MyHC IIA and MyHC I (p < 0.01. In comparison to the control group, CaN activity and NFATcl protein level were significantly increased in both the M-Ex and H-Ex groups (p < 0.05, p < 0.01, with a more pronounced upregulation in the M-Ex group (p < 0.05. Eight weeks of moderate- and high-intensity aerobic exercise induced the expression of NGF, BDNF and NT-3 in the soleus muscle and the striatum (p < 0.01, with the most significant increase in the H-Ex group (p < 0.01. In the rat soleus muscle, (1 CaN–NFATcl signaling contributes to the conversion of MyHC I isoform in response to moderate-intensity exercise; (2

  5. The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

    Science.gov (United States)

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew R M; Kiss, Csaba

    2014-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1-2 million reads can be accomplished in 10-15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.

  6. Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH genes in multiple human solid tumors: A systematic expression analysis

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    Werbowetski-Ogilvie Tamra

    2008-01-01

    Full Text Available Abstract Background The inter-alpha-trypsin inhibitors (ITI are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5, contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. Methods We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas using cDNA dot blot analysis (Cancer Profiling Array, CPA, semiquantitative RT-PCR and immunohistochemistry. Results We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA could be confirmed by real-time PCR in an additional set of breast cancers (n = 36. Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p Conclusion Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies.

  7. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Muscle in Growing Pigs

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    X. M. Men

    2016-04-01

    Full Text Available The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA, to quantity myosin heavy-chains (MyHCs mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (% of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01, and MyHC I mRNA proportion was positively correlated with creatine kinase (CK, succinate dehydrogenase (SDH, malate dehydrogenase (MDH activities (p<0.05. These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics.

  8. Prognostic Impact of Serum Heavy/Light Chain Pairs in Patients With Monoclonal Gammopathy of Undetermined Significance and Smoldering Myeloma: Long-Term Results From a Single Institution.

    Science.gov (United States)

    Magnano, Laura; Fernández de Larrea, Carlos; Elena, Montserrat; Cibeira, María Teresa; Tovar, Natalia; Aróstegui, Juan I; Pedrosa, Fabiola; Rosiñol, Laura; Filella, Xavier; Yagüe, Jordi; Bladé, Joan

    2016-06-01

    Asymptomatic monoclonal gammopathies, such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), are clinical conditions that usually precede symptomatic multiple myeloma. Therefore, risk stratification is crucial owing to the heterogeneous progression rate among these patients. In previous years, suppression of the uninvolved chain of specific heavy/light chain (HLC) pairs in serum has been identified as a new risk factor in MGUS. The aim of the present study was to investigate the prognostic effect of involved and uninvolved HLC pairs and HLC