The Florida manatee (Trichechus manatus latirostris) immunoglobulin heavy chain suggests the importance of clan III variable segments in repertoire diversity

USGS Publications Warehouse

Breaux, Breanna; Deiss, Thaddeus C.; Chen, Patricia L.; Cruz-Schneider, Maria Paula; Sena, Leonardo; Hunter, Margaret E.; Bonde, Robert K.; Criscitiello, Michael F.

2017-01-01

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.

Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

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

Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender

Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocketmore » on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.« less

The Florida manatee (Trichechus manatus latirostris) immunoglobulin heavy chain suggests the importance of clan III variable segments in repertoire diversity.

PubMed

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.

Crystal structure of an antibody bound to an immunodominant peptide epitope: novel features in peptide-antibody recognition.

PubMed

Nair, D T; Singh, K; Sahu, N; Rao, K V; Salunke, D M

2000-12-15

The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 A(2) for the peptide and 625 A(2) for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity.

Multiple productive immunoglobulin heavy chain gene rearrangements in chronic lymphocytic leukemia are mostly derived from independent clones

PubMed Central

Plevova, Karla; Francova, Hana Skuhrova; Burckova, Katerina; Brychtova, Yvona; Doubek, Michael; Pavlova, Sarka; Malcikova, Jitka; Mayer, Jiri; Tichy, Boris; Pospisilova, Sarka

2014-01-01

In chronic lymphocytic leukemia, usually a monoclonal disease, multiple productive immunoglobulin heavy chain gene rearrangements are identified sporadically. Prognostication of such cases based on immunoglobulin heavy variable gene mutational status can be problematic, especially if the different rearrangements have discordant mutational status. To gain insight into the possible biological mechanisms underlying the origin of the multiple rearrangements, we performed a comprehensive immunogenetic and immunophenotypic characterization of 31 cases with the multiple rearrangements identified in a cohort of 1147 patients with chronic lymphocytic leukemia. For the majority of cases (25/31), we provide evidence of the co-existence of at least two B lymphocyte clones with a chronic lymphocytic leukemia phenotype. We also identified clonal drifts in serial samples, likely driven by selection forces. More specifically, higher immunoglobulin variable gene identity to germline and longer complementarity determining region 3 were preferred in persistent or newly appearing clones, a phenomenon more pronounced in patients with stereotyped B-cell receptors. Finally, we report that other factors, such as TP53 gene defects and therapy administration, influence clonal selection. Our findings are relevant to clonal evolution in the context of antigen stimulation and transition of monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia. PMID:24038023

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

PubMed

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.

The Role of Shape Complementarity in the Protein-Protein Interactions

PubMed Central

Li, Ye; Zhang, Xianren; Cao, Dapeng

2013-01-01

We use a dissipative particle dynamic simulation to investigate the effects of shape complementarity on the protein-protein interactions. By monitoring different kinds of protein shape-complementarity modes, we gave a clear mechanism to reveal the role of the shape complementarity in the protein-protein interactions, i.e., when the two proteins with shape complementarity approach each other, the conformation of lipid chains between two proteins would be restricted significantly. The lipid molecules tend to leave the gap formed by two proteins to maximize the configuration entropy, and therefore yield an effective entropy-induced protein-protein attraction, which enhances the protein aggregation. In short, this work provides an insight into understanding the importance of the shape complementarity in the protein-protein interactions especially for protein aggregation and antibody–antigen complexes. Definitely, the shape complementarity is the third key factor affecting protein aggregation and complex, besides the electrostatic-complementarity and hydrophobic complementarity. PMID:24253561

The Role of Shape Complementarity in the Protein-Protein Interactions

NASA Astrophysics Data System (ADS)

Li, Ye; Zhang, Xianren; Cao, Dapeng

2013-11-01

We use a dissipative particle dynamic simulation to investigate the effects of shape complementarity on the protein-protein interactions. By monitoring different kinds of protein shape-complementarity modes, we gave a clear mechanism to reveal the role of the shape complementarity in the protein-protein interactions, i.e., when the two proteins with shape complementarity approach each other, the conformation of lipid chains between two proteins would be restricted significantly. The lipid molecules tend to leave the gap formed by two proteins to maximize the configuration entropy, and therefore yield an effective entropy-induced protein-protein attraction, which enhances the protein aggregation. In short, this work provides an insight into understanding the importance of the shape complementarity in the protein-protein interactions especially for protein aggregation and antibody-antigen complexes. Definitely, the shape complementarity is the third key factor affecting protein aggregation and complex, besides the electrostatic-complementarity and hydrophobic complementarity.

Self-Complementarity within Proteins: Bridging the Gap between Binding and Folding

PubMed Central

Basu, Sankar; Bhattacharyya, Dhananjay; Banerjee, Rahul

2012-01-01

Complementarity, in terms of both shape and electrostatic potential, has been quantitatively estimated at protein-protein interfaces and used extensively to predict the specific geometry of association between interacting proteins. In this work, we attempted to place both binding and folding on a common conceptual platform based on complementarity. To that end, we estimated (for the first time to our knowledge) electrostatic complementarity (Em) for residues buried within proteins. Em measures the correlation of surface electrostatic potential at protein interiors. The results show fairly uniform and significant values for all amino acids. Interestingly, hydrophobic side chains also attain appreciable complementarity primarily due to the trajectory of the main chain. Previous work from our laboratory characterized the surface (or shape) complementarity (Sm) of interior residues, and both of these measures have now been combined to derive two scoring functions to identify the native fold amid a set of decoys. These scoring functions are somewhat similar to functions that discriminate among multiple solutions in a protein-protein docking exercise. The performances of both of these functions on state-of-the-art databases were comparable if not better than most currently available scoring functions. Thus, analogously to interfacial residues of protein chains associated (docked) with specific geometry, amino acids found in the native interior have to satisfy fairly stringent constraints in terms of both Sm and Em. The functions were also found to be useful for correctly identifying the same fold for two sequences with low sequence identity. Finally, inspired by the Ramachandran plot, we developed a plot of Sm versus Em (referred to as the complementarity plot) that identifies residues with suboptimal packing and electrostatics which appear to be correlated to coordinate errors. PMID:22713576

Self-complementarity within proteins: bridging the gap between binding and folding.

PubMed

Basu, Sankar; Bhattacharyya, Dhananjay; Banerjee, Rahul

2012-06-06

Complementarity, in terms of both shape and electrostatic potential, has been quantitatively estimated at protein-protein interfaces and used extensively to predict the specific geometry of association between interacting proteins. In this work, we attempted to place both binding and folding on a common conceptual platform based on complementarity. To that end, we estimated (for the first time to our knowledge) electrostatic complementarity (Em) for residues buried within proteins. Em measures the correlation of surface electrostatic potential at protein interiors. The results show fairly uniform and significant values for all amino acids. Interestingly, hydrophobic side chains also attain appreciable complementarity primarily due to the trajectory of the main chain. Previous work from our laboratory characterized the surface (or shape) complementarity (Sm) of interior residues, and both of these measures have now been combined to derive two scoring functions to identify the native fold amid a set of decoys. These scoring functions are somewhat similar to functions that discriminate among multiple solutions in a protein-protein docking exercise. The performances of both of these functions on state-of-the-art databases were comparable if not better than most currently available scoring functions. Thus, analogously to interfacial residues of protein chains associated (docked) with specific geometry, amino acids found in the native interior have to satisfy fairly stringent constraints in terms of both Sm and Em. The functions were also found to be useful for correctly identifying the same fold for two sequences with low sequence identity. Finally, inspired by the Ramachandran plot, we developed a plot of Sm versus Em (referred to as the complementarity plot) that identifies residues with suboptimal packing and electrostatics which appear to be correlated to coordinate errors. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

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

Demaison, C.; Chastagner, P.; Theze, J.

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 tomore » 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.« less

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

PubMed Central

Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

2013-01-01

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

The I binding specificity of human VH 4-34 (VH 4-21) encoded antibodies is determined by both VH framework region 1 and complementarity determining region 3.

PubMed

Li, Y; Spellerberg, M B; Stevenson, F K; Capra, J D; Potter, K N

1996-03-01

Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.

Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment.

PubMed

Stewart, Alex; Harrison, Joseph S; Regula, Lauren K; Lai, Jonathan R

2013-04-08

Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464-5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1-3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high-affinity interaction involving an antibody from the VH1-69 germline segment.

The three-dimensional structure of a complex of a murine Fab (NC10. 14) with a potent sweetener (NC174): an illustration of structural diversity in antigen recognition by immunoglobulins.

PubMed

Guddat, L W; Shan, L; Broomell, C; Ramsland, P A; Fan, Z; Anchin, J M; Linthicum, D S; Edmundson, A B

2000-09-29

The three-dimensional structure of a complex of an Fab from a murine IgG2b(lambda) antibody (NC10.14) with a high potency sweet tasting hap- ten, N-(p-cyanophenyl)-N'-(diphenylmethyl)-N"-(carboxymethyl)guan idine (NC174), has been determined to 2.6 A resolution by X-ray crystallography. This complex crystallized in the triclinic space group P1, with two molecules in the asymmetric unit. In contrast to a companion monoclonal antibody (NC6.8) with a kappa-type light chain and similar high affinity for the NC174 ligand, the NC10.14 antibody possessed a large and deep antigen combining site bounded primarily by the third complementarity-determining regions (CDR3s) of the light and heavy chains. CDR3 of the heavy chain dominated the site and its crown protruded into the external solvent as a type 1' beta-turn. NC174 was nested against HCDR3 and was held in place by two tryptophan side-chains (L91 and L96) from LCDR3. The diphenyl rings were accommodated on an upper tier of the binding pocket that is largely hydrophobic. At the floor of the site, a positively charged arginine side-chain (H95) stabilized the orientation of the electronegative cyano group of the hapten. The negative charge on the acetate group was partially neutralized by a hydrogen bond with the phenolic hydroxyl group of tyrosine H58. Comparisons of the modes of binding of NC174 to the NC6.8 and NC10.14 antibodies illustrate the enormous structural and mechanistic diversity manifest by immune responses. Copyright 2000 Academic Press.

Enhancement and Analysis of Human Antiaflatoxin B1 (AFB1) scFv Antibody-Ligand Interaction Using Chain Shuffling.

PubMed

Rangnoi, Kuntalee; Choowongkomon, Kiattawee; O'Kennedy, Richard; Rüker, Florian; Yamabhai, Montarop

2018-06-06

A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.

Rational Design of Dual Agonist-Antibody Fusions as Long-acting Therapeutic Hormones.

PubMed

Liu, Yan; Wang, Ying; Zhang, Yong; Liu, Tao; Jia, Haiqun; Zou, Huafei; Fu, Qiangwei; Zhang, Yuhan; Lu, Lucy; Chao, Elizabeth; Parker, Holly; Nguyen-Tran, Van; Shen, Weijun; Wang, Danling; Schultz, Peter G; Wang, Feng

2016-11-18

Recent studies have suggested that modulation of two or more signaling pathways can achieve substantial weight loss and glycemic stability. We have developed an approach to the generation of bifunctional antibody agonists that activate leptin receptor and GLP-1 receptor. Leptin was fused into the complementarity determining region 3 loop of the light chain alone, or in combination with exendin-4 (EX4) fused at the N-terminus of the heavy chain of Herceptin. The antibody fusions exhibit similar or increased in vitro activities on their cognate receptors, but 50-100-fold longer circulating half-lives in rodents compared to the corresponding native peptides/proteins. The efficacy of the leptin/EX4 dual antibody fusion on weight loss, especially fat mass loss, was enhanced in ob/ob mice and DIO mice compared to the antibody fusion of either EX4 or leptin alone. This work demonstrates the versatility of this combinatorial fusion strategy for generating dual antibody agonists with long half-lives.

Experimental investigation of halogen-bond hard-soft acid-base complementarity.

PubMed

Riel, Asia Marie S; Jessop, Morly J; Decato, Daniel A; Massena, Casey J; Nascimento, Vinicius R; Berryman, Orion B

2017-04-01

The halogen bond (XB) is a topical noncovalent interaction of rapidly increasing importance. The XB employs a `soft' donor atom in comparison to the `hard' proton of the hydrogen bond (HB). This difference has led to the hypothesis that XBs can form more favorable interactions with `soft' bases than HBs. While computational studies have supported this suggestion, solution and solid-state data are lacking. Here, XB soft-soft complementarity is investigated with a bidentate receptor that shows similar associations with neutral carbonyls and heavy chalcogen analogs. The solution speciation and XB soft-soft complementarity is supported by four crystal structures containing neutral and anionic soft Lewis bases.

Attempts to locate residues in complementarity-determining regions of antibody combining sites that make contact with antigen.

PubMed

Kabat, E A; Wu, T T; Bilofsky, H

1976-02-01

From collected data on variable region sequences of heavy chains of immunoglobulins, the probability of random associations of any two amino-acid residues in the complementarity-determining segments was computed, and pairs of residues occurring significantly more frequently than expected were selected by computer. Significant associations between Phe 32 and Tyr 33, Phe 32 and Glu 35, and Tyr 33 and Glu 35 were found in six proteins, all of which were mouse myeloma proteins which bound phosphorylcholine (= phosphocholine). From the x-ray structure of McPC603, Tyr 33 and Glu 35 are contacting residues; a seventh phosphorylcholine-binding mouse myeloma protein also contained Phe 32 and Tyr 33 but position 35 had only been determined as Glx and thus this position had not been selected. Met 34 occurred in all seven phosphorylcholine-binding myeoma proteins but was also present at this position in 29 other proteins and thus was not selected; it is seen in the x-ray structure not to be a contacting residue. The role of Phe 32 is not obvious but it could have some conformational influence. A human phosphorylcholine-binding myeloma protien also had Phe, Tyr, and Met at positions 32, 33, and 34, but had Asp instead of Glu at position 35 and showed a lower binding constant. The ability to use sequence data to locate residues in complementarity-determing segments making contact with antigenic determinants and those playing essentially a structural role would contribute substantially to the understanding of antibody specificity.

An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

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

Fanning, Sean W.; Horn, James R.

2014-03-05

Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while themore » crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.« less

Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

PubMed Central

Ghiotto, Fabio; Marcatili, Paolo; Tenca, Claudya; Calevo, Maria Grazia; Yan, Xiao-Jie; Albesiano, Emilia; Bagnara, Davide; Colombo, Monica; Cutrona, Giovanna; Chu, Charles C; Morabito, Fortunato; Bruno, Silvia; Ferrarini, Manlio; Tramontano, Anna; Fais, Franco; Chiorazzi, Nicholas

2011-01-01

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV–diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation. PMID:21785810

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library

PubMed Central

Kim, Sangkyu; Park, Insoo; Park, Seung Gu; Cho, Seulki; Kim, Jin Hong; Ipper, Nagesh S.; Choi, Sun Shim; Lee, Eung Suk; Hong, Hyo Jeong

2017-01-01

We constructed a large naïve human Fab library (3 × 1010 colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and κ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity. PMID:28927259

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology.

PubMed

Desmyter, Aline; Spinelli, Silvia; Payan, Francoise; Lauwereys, Marc; Wyns, Lode; Muyldermans, Serge; Cambillau, Christian

2002-06-28

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library.

PubMed

Kim, Sangkyu; Park, Insoo; Park, Seung Gu; Cho, Seulki; Kim, Jin Hong; Ipper, Nagesh S; Choi, Sun Shim; Lee, Eung Suk; Hong, Hyo Jeong

2017-09-30

We constructed a large naïve human Fab library (3 × 10 10 colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and κ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity.

Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

PubMed

Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

2012-09-01

Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer.

PubMed

He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L; Mann, Colin J; Augst, Ryan; Morris, Charles D; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B; Burton, Dennis R; Zhu, Jiang

2017-01-01

Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.

Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer

PubMed Central

He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L.; Mann, Colin J.; Augst, Ryan; Morris, Charles D.; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B.; Burton, Dennis R.; Zhu, Jiang

2017-01-01

Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development. PMID:28883821

Structural classification of CDR-H3 revisited: a lesson in antibody modeling.

PubMed

Kuroda, Daisuke; Shirai, Hiroki; Kobori, Masato; Nakamura, Haruki

2008-11-15

Among the six complementarity-determining regions (CDRs) in the variable domains of an antibody, the third CDR of the heavy chain (CDR-H3), which lies in the center of the antigen-binding site, plays a particularly important role in antigen recognition. CDR-H3 shows significant variability in its length, sequence, and structure. Although difficult, model building of this segment is the most critical step in antibody modeling. Since our first proposal of the "H3-rules," which classify CDR-H3 structure based on amino acid sequence, the number of experimentally determined antibody structures has increased. Here, we revise these H3-rules and propose an improved classification scheme for CDR-H3 structure modeling. In addition, we determine the common features of CDR-H3 in antibody drugs as well as discuss the concept of "antibody druggability," which can be applied as an indicator of antibody evaluation during drug discovery.

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

PubMed Central

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

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.

PubMed

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.

Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

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

Stanfield, R.L.; Dooley, H.; Verdino, P.

2007-07-13

Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts withmore » antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.« less

Viral receptor-binding site antibodies with diverse germline origins.

PubMed

Schmidt, Aaron G; Therkelsen, Matthew D; Stewart, Shaun; Kepler, Thomas B; Liao, Hua-Xin; Moody, M Anthony; Haynes, Barton F; Harrison, Stephen C

2015-05-21

Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by 11 different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B cell targets. Copyright © 2015 Elsevier Inc. All rights reserved.

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

NASA Technical Reports Server (NTRS)

He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

1992-01-01

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

NASA Technical Reports Server (NTRS)

He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

1992-01-01

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

A binary plasmid system for shuffling combinatorial antibody libraries.

PubMed

Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

1992-11-01

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.

A binary plasmid system for shuffling combinatorial antibody libraries.

PubMed Central

Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

1992-01-01

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis. Images PMID:1438192

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

PubMed

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.

Three-dimensional structure of an antibody-antigen complex.

PubMed

Sheriff, S; Silverton, E W; Padlan, E A; Cohen, G H; Smith-Gill, S J; Finzel, B C; Davies, D R

1987-11-01

We have determined the three-dimensional structure of two crystal forms of an antilysozyme Fab-lysozyme complex by x-ray crystallography. The epitope on lysozyme consists of three sequentially separated subsites, including one long, nearly continuous, site from Gln-41 through Tyr-53 and one from Gly-67 through Pro-70. Antibody residues interacting with lysozyme occur in each of the six complementarity-determining regions and also include one framework residue. Arg-45 and Arg-68 form a ridge on the surface of lysozyme, which binds in a groove on the antibody surface. Otherwise the surface of interaction between the two proteins is relatively flat, although it curls at the edges. The surface of interaction is approximately 26 X 19 A. No water molecules are found in the interface. The positive charge on the two arginines is complemented by the negative charge of Glu-35 and Glu-50 from the heavy chain of the antibody. The backbone structure of the antigen, lysozyme, is mostly unperturbed, although there are some changes in the epitope region, most notably Pro-70. One side chain not in the epitope, Trp-63, undergoes a rotation of approximately 180 degrees about the C beta--C gamma bond. The Fab elbow bends in the two crystal forms differ by 7 degrees.

Single cardiac ventricular myosins are autonomous motors

PubMed Central

Wang, Yihua; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta

2018-01-01

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule. PMID:29669825

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning

PubMed Central

Klimka, A; Matthey, B; Roovers, R C; Barth, S; Arends, J-W; Engert, A; Hoogenboom, H R

2000-01-01

In various clinical studies, Hodgkin’s patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the non-human therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics. © 2000 Cancer Research Campaign PMID:10901379

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen

PubMed Central

1995-01-01

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p- azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment. PMID:7650481

IgM anti-myeloperoxidase antibody-secreting lymphocytes are present in the peripheral repertoire of lupus mice but rarely differentiate into IgG-producing cells

PubMed Central

Vittecoq, O; Brard, F; Jovelin, F; Le Loet, X; Tron, F; Gilbert, D

1999-01-01

Two IgM, κ anti-myeloperoxidase (MPO) monoclonal antibodies, 6D6 and 9B5, bound to MPO in a solid-phase enzyme-linked immunosorbent assay were derived from the splenocytes of (NZB × NZW) F1 and MRL/lpr-lpr mice, respectively. 6D6 gave a characteristic perinuclear immunofluorescence staining pattern on ethanol-fixed human neutrophils, bound to the native form of MPO by immunoblotting and had a high constant affinity for MPO as demonstrated by real-time specific interaction. 9B5 produced a cytoplasmic immunofluorescence staining pattern, reacted with the heavy chain of MPO and had a low constant affinity for MPO. The heavy-and light-chain variable region genes of monoclonal antibodies (mAb) 6D6 and 9B5 were sequenced and found to be highly homologous to germline genes and to contain negatively charged amino acids in the complementarity determining regions. IgM MPO-binding activity was observed in most BW and MRL/lpr-lpr mouse sera, which may correspond to polyclonal activation of B cells, whereas IgG anti-MPO antibodies could be rarely detected. Thus, this study indicates that (i) BW and MRL/lpr-lpr mice do not delete IgM anti-MPO secreting B cells, do not maintain these B cells in a state of anergy, but most individuals are not able to spontaneously induce the class-switching of this autoantibody population; (ii) IgM anti-MPO antibodies can recognize different epitopes on MPO and produce different immunofluorescence staining pattern on ethanol-fixed human neutrophils, as demonstrated by the immunochemical properties of the two lupus-mouse derived mAb. PMID:10540169

The Unusual Genetics and Biochemistry of Bovine Immunoglobulins.

PubMed

Stanfield, Robyn L; Haakenson, Jeremy; Deiss, Thaddeus C; Criscitiello, Michael F; Wilson, Ian A; Smider, Vaughn V

2018-01-01

Antibodies are the key circulating molecules that have evolved to fight infection by the adaptive immune system of vertebrates. Typical antibodies of most species contain six complementarity-determining regions (CDRs), where the third CDR of the heavy chain (CDR H3) has the greatest diversity and often makes the most significant contact with antigen. Generally, the process of V(D)J recombination produces a vast repertoire of antibodies; multiple V, D, and J gene segments recombine with additional junctional diversity at the V-D and D-J joints, and additional combinatorial possibilities occur through heavy- and light-chain pairing. Despite these processes, the overall structure of the resulting antibody is largely conserved, and binding to antigen occurs predominantly through the CDR loops of the immunoglobulin V domains. Bovines have deviated from this general paradigm by having few VH regions and thus little germline combinatorial diversity, but their antibodies contain long CDR H3 regions, with substantial diversity generated through somatic hypermutation. A subset of the repertoire comprises antibodies with ultralong CDR H3s, which can reach over 70 amino acids in length. Structurally, these unusual antibodies form a β-ribbon "stalk" and disulfide-bonded "knob" that protrude far from the antibody surface. These long CDR H3s allow cows to mount a particularly robust immune response when immunized with viral antigens, particularly to broadly neutralizing epitopes on a stabilized HIV gp140 trimer, which has been a challenge for other species. The unusual genetics and structural biology of cows provide for a unique paradigm for creation of immune diversity and could enable generation of antibodies against especially challenging targets and epitopes. © 2018 Elsevier Inc. All rights reserved.

Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

DOE PAGES

D'Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie; ...

2018-03-08

Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with themore » same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding.« less

Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

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

D'Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie

Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with themore » same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding.« less

Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

PubMed Central

Kiehart, D P; Lutz, M S; Chan, D; Ketchum, A S; Laymon, R A; Nguyen, B; Goldstein, L S

1989-01-01

In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis. Images PMID:2498088

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

PubMed

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%.

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

NASA Astrophysics Data System (ADS)

Liu, Hongcheng; Gaza-Bulseco, Georgeen; Chumsae, Chris

2009-12-01

Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly.

Use of Trypanosoma equiperdum infected rabbits as a source of splenic mRNA; construction of cDNA clones and identification of a rabbit mu heavy chain clone.

PubMed

Bernstein, K E; Pavirani, A; Alexander, C; Jacobsen, F; Fitzmaurice, L; Mage, R

1983-01-01

Rabbits were infected by Trypanosoma equiperdum and the splenic mRNA was isolated. In vitro translation of this RNA and immunoprecipitation with anti-light chain, anti-heavy chain, anti-mu and anti-VH antibodies demonstrated that T. equiperdum infection elicits large quantities of splenic mRNA encoding mu and kappa chains. The mu and gamma heavy chains and the kappa light chains synthesized in the cell-free translation system were specifically immunoprecipitated by antisera to heavy chain VHa and light chain kappa b allotypes. In vitro labeling of spleen cells from trypanosome-infected animals demonstrated that the biosynthetically labeled IgM has a mu chain of higher molecular weight than the mu chain synthesized by in vitro translation, a difference that is largely abolished when cellular glycosylation is blocked with the antibiotic tunicamycin. Enrichment for heavy chain or light chain mRNA was achieved by fractionating mRNA from trypanosome-infected animals on a sucrose gradient. cDNA clones carrying mu heavy chain sequences were produced using a 'one tube' protocol and identified by cross species hybridization and hybridization selection. Infection of rabbits with T. equiperdum followed by sucrose gradient enrichment of splenic mRNA has provided sufficient quantities of mRNA encoding mu heavy chain suitable for cDNA cloning.

¹⁵N, ¹³C and ¹H resonance assignments and secondary structure determination of a variable heavy domain of a heavy chain antibody.

PubMed

Prosser, Christine E; Waters, Lorna C; Muskett, Frederick W; Veverka, Vaclav; Addis, Philip W; Griffin, Laura M; Baker, Terry S; Lawson, Alastair D G; Wernery, Ulrich; Kinne, Jorg; Henry, Alistair J; Taylor, Richard J; Carr, Mark D

2014-04-01

Heavy chain antibodies differ in structure to conventional antibodies lacking both the light chain and the first heavy chain constant domain (CH1). Characteristics of the antigen-binding variable heavy domain of the heavy chain antibody (VHH) including the smaller size, high solubility and stability make them an attractive alternative to more traditional antibody fragments for detailed NMR-based structural analysis. Here we report essentially complete backbone and side chain (15)N, (13)C and (1)H assignments for a free VHH. Analysis of the backbone chemical shift data obtained indicates that the VHH is comprised predominantly of β-sheets corresponding to nearly 60% of the protein backbone.

Myosin light chains: Teaching old dogs new tricks

PubMed Central

Heissler, Sarah M; Sellers, James R

2014-01-01

The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin. PMID:26155737

Activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer's patch and spleen and is associated with expansion of the primary antibody repertoire in the absence of exogenous antigens.

PubMed

Liljavirta, J; Ekman, A; Knight, J S; Pernthaner, A; Iivanainen, A; Niku, M

2013-09-01

Due to a limited range of immunoglobulin (Ig) genes, cattle and several other domestic animals rely on postrecombinatorial amplification of the primary repertoire. We report that activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer's patch and spleen but not in fetal bone marrow. The numbers of IGHV (immunoglobulin heavy chain variable) mutations correlate with AID expression. The mutational profile in the fetuses is similar to postnatal and immunized calves, with targeting of complementarity-determining region (CDR) over framework region (FR), preference of replacement over silent mutations in CDRs but not in FRs, and targeting of the AID hotspot motif RGYW/WRCY. Statistical analysis indicates negative selection on FRs and positive selection on CDRs. Our results suggest that AID-mediated somatic hypermutation and selection take place in bovine fetuses, implying a role for AID in the diversification of the primary antibody repertoire in the absence of exogenous antigens.

HIV Envelope Glycoform Heterogeneity and Localized Diversity Govern the Initiation and Maturation of a V2 Apex Broadly Neutralizing Antibody Lineage.

PubMed

Landais, Elise; Murrell, Ben; Briney, Bryan; Murrell, Sasha; Rantalainen, Kimmo; Berndsen, Zachary T; Ramos, Alejandra; Wickramasinghe, Lalinda; Smith, Melissa Laird; Eren, Kemal; de Val, Natalia; Wu, Mengyu; Cappelletti, Audrey; Umotoy, Jeffrey; Lie, Yolanda; Wrin, Terri; Algate, Paul; Chan-Hui, Po-Ying; Karita, Etienne; Ward, Andrew B; Wilson, Ian A; Burton, Dennis R; Smith, Davey; Pond, Sergei L Kosakovsky; Poignard, Pascal

2017-11-21

Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Cloning and molecular characterization of the cDNAs encoding the variable regions of an anti-CD20 monoclonal antibody.

PubMed

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili

2017-01-01

CD20-based targeting of B-cells in hematologic malignancies and autoimmune disorders is associated with outstanding clinical outcomes. Isolation and characterization of VH and VL cDNAs encoding the variable regions of the heavy and light chains of monoclonal antibodies (MAb) is necessary to produce next generation MAbs and their derivatives such as bispecific antibodies (bsAb) and single-chain variable fragments (scFv). This study was aimed at cloning and characterization of the VH and VL cDNAs from a hybridoma cell line producing an anti-CD20 MAb. VH and VL fragments were amplified, cloned and characterized. Furthermore, amino acid sequences of VH, VL and corresponding complementarity-determining regions (CDR) were determined and compared with those of four approved MAbs including Rituximab (RTX), Ibritumomab tiuxetan, Ofatumumab and GA101. The cloned VH and VL cDNAs were found to be functional and follow a consensus pattern. Amino acid sequences corresponding to the VH and VL fragments also indicated noticeable homologies to those of RTX and Ibritumomab. Furthermore, amino acid sequences of the relating CDRs had remarkable similarities to their counterparts in RTX and Ibritumomab. Successful recovery of VH and VL fragments encourages the development of novel CD20 targeting bsAbs, scFvs, antibody conjugates and T-cells armed with chimeric antigen receptors.

Eradication of Borrelia burgdorferi infection in primary marginal zone B-cell lymphoma of the skin.

PubMed

Roggero, E; Zucca, E; Mainetti, C; Bertoni, F; Valsangiacomo, C; Pedrinis, E; Borisch, B; Piffaretti, J C; Cavalli, F; Isaacson, P G

2000-02-01

Primary cutaneous B-cell lymphomas have been associated with Borrelia burgdorferi, the spirochete responsible for Lyme disease. Recently, cutaneous marginal zone B-cell lymphoma has been proposed as a distinct clinical-pathological entity. We report a case of primary cutaneous marginal zone lymphoma, associated with B burgdorferi infection. Polymerase chain reaction (PCR) amplification of the third complementarity determining region (CDR3) of the immunoglobulin heavy chain gene showed the presence of a monoclonal lymphoproliferation, therefore strengthening the histological diagnosis of a malignant process. B burgdorfer-specific hbb gene sequences were detected by PCR in the lymphoma tissue at diagnosis but not after antibiotic treatment. A nearly complete clinical and histological regression was observed after B burgdorferi eradication, with immunohistochemistry studies showing disappearance of plasma cell differentiation and a marked decline in the number of CD3+ T cells and Ki-67+ cells. Our case confirms the link between B burgdorferi and some cutaneous lymphomas. The disappearance of the microorganism accompanied by the unequivocal decrease of most indicators of active T- and B-cell immune response strongly supported a pathogenetic role for B burgdorferi in sustaining an antigen-driven development and growth of this cutaneous marginal zone lymphoma. Antibiotic therapy (analogous to Helicobacter pylori infection in gastric MALT lymphoma) might be helpful with the aim of averting or at least deferring the indication for more aggressive treatment.

Characterization of the interaction between the heavy and light chains of bovine factor Va.

PubMed

Walker, F J

1992-10-05

Bovine factor Va has been previously been shown to consist of heavy (M(r) = 94,000) and light chains (M(r) = 81,000), that interact in a manner dependent upon the presence of either calcium or manganese ions. In an attempt to understand the mechanism of subunit interaction we have studied the effects of temperature and ions on factor Va stability. The rates of formation of factor Va from isolated chains and dissociation were temperature-dependent with an energy of activation of 6.2 and 1.3 kcal mol-1, respectively. The yield of factor Va from isolated chains was inversely related to the amount of time the chains were incubated at 4 degrees C. Incubation of individual chains revealed that the heavy chain is cold-labile, an effect that is reversible. Manganese ion was observed to prevent the conversion to the inactive form. High salt tends to stabilize the two-chain structure of factor Va, but is inhibitory to its formation from isolated chains. High concentrations of either manganese or calcium ions also inhibited reconstitution of activity. The light chain, in particular, was sensitive to the presence of manganese or calcium ion. Heavy chain that had been cleaved by activated protein C had a weakened interaction with the light chain, and the resulting complex had no procoagulant activity. Cooling of the heavy chain to 4 degrees C enhanced its intrinsic fluorescence. Manganese ion prevented some of this enhancement. The heavy chain fluorescence returned to the room temperature value with a half-life of approximately 10 min. In the presence of manganese ion relaxation was accelerated. The intrinsic fluorescence of activated protein C-cleaved heavy chain was not increased when the temperature was decreased. These data suggest that the heavy chain can exist in two forms. Elevated temperature converts it to a form that can bind ions and have a productive interaction with the light chain. However, conditions that prevent the heavy chain from combining with the light chain also stabilize the two subunit structure, suggesting that the high affinity of the complex is due to conformational changes that occur after chain interaction.

Clathrin Heavy Chain Is Important for Viability, Oviposition, Embryogenesis and, Possibly, Systemic RNAi Response in the Predatory Mite Metaseiulus occidentalis

PubMed Central

Wu, Ke; Hoy, Marjorie A.

2014-01-01

Clathrin heavy chain has been shown to be important for viability, embryogenesis, and RNA interference (RNAi) in arthropods such as Drosophila melanogaster. However, the functional roles of clathrin heavy chain in chelicerate arthropods, such as the predatory mite Metaseiulus occidentalis, remain unknown. We previously showed that dsRNA ingestion, followed by feeding on spider mites, induced systemic and robust RNAi in M. occidentalis females. In the current study, we performed a loss-of-function analysis of the clathrin heavy chain gene in M. occidentalis using RNAi. We showed that ingestion of clathrin heavy chain dsRNA by M. occidentalis females resulted in gene knockdown and reduced longevity. In addition, clathrin heavy chain dsRNA treatment almost completely abolished oviposition by M. occidentalis females and the few eggs produced did not hatch. Finally, we demonstrated that clathrin heavy chain gene knockdown in M. occidentalis females significantly reduced a subsequent RNAi response induced by ingestion of cathepsin L dsRNA. The last finding suggests that clathrin heavy chain may be involved in systemic RNAi responses mediated by orally delivered dsRNAs in M. occidentalis. PMID:25329675

Heavy and Light chain amyloidosois presenting as complete heart block: A rare presentation of a rare disease.

PubMed

Priyamvada, P S; Morkhandikar, S; Srinivas, B H; Parameswaran, S

2015-01-01

Amyloidosis is an uncommon disease characterized by deposition of proteinaceous material in the extracellular matrix, which results from abnormal protein folding. Even though more than 25 precursor proteins are identified, majority of systemic amyloidosis results from deposition of abnormal immunoglobulin (Ig) light chains. In heavy chain amyloidosis (AH), deposits are derived from both heavy chain alone, whereas in heavy and light chain amyloidosis (AHL), the deposits are derived from Ig heavy chains and light chains. Both AH and AHL are extremely rare diseases. Here, we report an unusual presentation of IgG (lambda) AHL amyloidosis in the background of multiple myeloma, where the initial clinical presentation was complete heart block, which preceded the definitive diagnosis by 18 months.

Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.

PubMed

Roux, K H; Greenberg, A S; Greene, L; Strelets, L; Avila, D; McKinney, E C; Flajnik, M F

1998-09-29

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (VH) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.

BCR CDR3 length distributions differ between blood and spleen and between old and young patients, and TCR distributions can be used to detect myelodysplastic syndrome

NASA Astrophysics Data System (ADS)

Pickman, Yishai; Dunn-Walters, Deborah; Mehr, Ramit

2013-10-01

Complementarity-determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and responding lymphocytes. There are millions of different TCR Vβ chain or BCR heavy chain CDR3 sequences in human blood. Even now, when high-throughput sequencing becomes widely used, CDR3 length distributions (also called spectratypes) are still a much quicker and cheaper method of assessing repertoire diversity. However, distribution complexity and the large amount of information per sample (e.g. 32 distributions of the TCRα chain, and 24 of TCRβ) calls for the use of machine learning tools for full exploration. We have examined the ability of supervised machine learning, which uses computational models to find hidden patterns in predefined biological groups, to analyze CDR3 length distributions from various sources, and distinguish between experimental groups. We found that (a) splenic BCR CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions; (b) healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young; and (c) a machine learning model based on TCR CDR3 distribution features can detect myelodysplastic syndrome with approximately 93% accuracy. Overall, we demonstrate that using supervised machine learning methods can contribute to our understanding of lymphocyte repertoire diversity.

Shape Complementarity of Protein-Protein Complexes at Multiple Resolutions

PubMed Central

Zhang, Qing; Sanner, Michel; Olson, Arthur J.

2010-01-01

Biological complexes typically exhibit intermolecular interfaces of high shape complementarity. Many computational docking approaches use this surface complementarity as a guide in the search for predicting the structures of protein-protein complexes. Proteins often undergo conformational changes in order to create a highly complementary interface when associating. These conformational changes are a major cause of failure for automated docking procedures when predicting binding modes between proteins using their unbound conformations. Low resolution surfaces in which high frequency geometric details are omitted have been used to address this problem. These smoothed, or blurred, surfaces are expected to minimize the differences between free and bound structures, especially those that are due to side chain conformations or small backbone deviations. In spite of the fact that this approach has been used in many docking protocols, there has yet to be a systematic study of the effects of such surface smoothing on the shape complementarity of the resulting interfaces. Here we investigate this question by computing shape complementarity of a set of 66 protein-protein complexes represented by multi-resolution blurred surfaces. Complexed and unbound structures are available for these protein-protein complexes. They are a subset of complexes from a non-redundant docking benchmark selected for rigidity (i.e. the proteins undergo limited conformational changes between their bound and unbound states). In this work we construct the surfaces by isocontouring a density map obtained by accumulating the densities of Gaussian functions placed at all atom centers of the molecule. The smoothness or resolution is specified by a Gaussian fall-off coefficient, termed “blobbyness”. Shape complementarity is quantified using a histogram of the shortest distances between two proteins' surface mesh vertices for both the crystallographic complexes and the complexes built using the protein structures in their unbound conformation. The histograms calculated for the bound complex structures demonstrate that medium resolution smoothing (blobbyness=−0.9) can reproduce about 88% of the shape complementarity of atomic resolution surfaces. Complexes formed from the free component structures show a partial loss of shape complementarity (more overlaps and gaps) with the atomic resolution surfaces. For surfaces smoothed to low resolution (blobbyness=−0.3), we find more consistency of shape complementarity between the complexed and free cases. To further reduce bad contacts without significantly impacting the good contacts we introduce another blurred surface, in which the Gaussian densities of flexible atoms are reduced. From these results we discuss the use of shape complementarity in protein-protein docking. PMID:18837463

Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

PubMed

Gadermaier, Elisabeth; Flicker, Sabine; Lupinek, Christian; Steinberger, Peter; Valenta, Rudolf

2013-04-01

Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Integrating PCR Theory and Bioinformatics into a Research-oriented Primer Design Exercise

ERIC Educational Resources Information Center

Robertson, Amber L.; Phillips, Allison R.

2008-01-01

Polymerase chain reaction (PCR) is a conceptually difficult technique that embodies many fundamental biological processes. Traditionally, students have struggled to analyze PCR results due to an incomplete understanding of the biological concepts (theory) of DNA replication and strand complementarity. Here we describe the design of a novel…

Excessive amounts of mu heavy chain block B-cell development.

PubMed

Zhu, Lingqiao; Chang, Cheong-Hee; Dunnick, Wesley

2011-09-01

Antigen-independent B-cell development occurs in several stages that depend on the expression of Ig heavy and light chain. We identified a line of mice that lacked mature B cells in the spleen. This mouse line carried approximately 11 copies of a transgene of the murine heavy chain constant region locus, and B-lineage cells expressed excessive amounts of the intracellular μ heavy chain. B-cell development failed in the bone marrow at the pro/pre B-cell transition, and examination of other lines with various copy numbers of the same transgene suggested that deficiencies in B-cell development increased with increased transgene copy number. Expression of a transgenic (Tg) light chain along with the Tg μ heavy chain led to minimal rescue of B-cell development in the bone marrow and B cells in the spleen. There are several potential mechanisms for the death of pro/pre B cells as a consequence of excess heavy chain expression.

Human Anti-V3 HIV-1 Monoclonal Antibodies Encoded by the VH5-51/VL Lambda Genes Define a Conserved Antigenic Structure

PubMed Central

Gorny, Miroslaw K.; Sampson, Jared; Li, Huiguang; Jiang, Xunqing; Totrov, Maxim; Wang, Xiao-Hong; Williams, Constance; O'Neal, Timothy; Volsky, Barbara; Li, Liuzhe; Cardozo, Timothy; Nyambi, Phillipe; Zolla-Pazner, Susan; Kong, Xiang-Peng

2011-01-01

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs. PMID:22164215

Structural and affinity studies of IgM polyreactive natural autoantibodies.

PubMed

Diaw, L; Magnac, C; Pritsch, O; Buckle, M; Alzari, P M; Dighiero, G

1997-01-15

Natural polyreactive autoantibodies (NAA) are an important component of the normal B cell repertoire. One intriguing characteristic of these Abs is their binding to various dissimilar Ags. It has been generally assumed that these Abs bind the Ags with low affinity, and are encoded by germline genes. We have used surface plasmon resonance to determine binding of avidities, and conducted a structural analysis of five murine monoclonal natural autoantibodies displaying a typical polyreactive binding pattern against cytoskeleton Ags and DNA. We show that 1) all the five Abs bind the different Ags with kinetic constants similar to those observed for immune Abs; 2) they express a restricted set of V(H) and V(L) genes, since the same V(H) gene is expressed by three out of the five, and one particular Vkappa gene was expressed twice. In addition, a single D gene segment was used by three of the five Abs; and 3) they express, in most cases, genes in a close germline configuration. Our amino acid sequence and modeling studies show that the distribution of exposed side chains in the NAA paratopes is close to the general pattern observed in the complementarity-determining regions (CDRs) of variable domains from immune Abs. Although CDR3 regions of the heavy chain have been postulated to play a major role in determining polyreactivity on the basis of recombinatorial experiments, our results failed to show any distinctive particularity of this region in terms of length or charge when compared with classical immune Abs.

Function of fusion regulatory proteins (FRPs) in immune cells and virus-infected cells.

PubMed

Tsurudome, M; Ito, Y

2000-01-01

Two molecules that regulate cell fusion have been identified and designated fusion regulatory protein-1 (FRP-1) and FRP-2. FRP-1 is a complex composed of a glycosylated heavy chain and a nonglycosylated light chain that are disulfide linked. FRP-1 heavy chain is identical to 4F2/CD98 heavy chain, whereas FRP-2 is identical to integrin alpha3 subunit. The FRP-1 heavy chain is a multifunctional molecule: that is, fusion regulator, amino acid transporter, integrin regulator, comitogenic factor, Na+-Ca2+ exchanger, oncogenic protein, and so on. Several aspects of the structure and function of the FRP-1 system are reviewed: fusion regulatory molecular mechanisms, cross-talk between the FRP-1 and integrin, the FRP-1 system as amino acid transporter, and FRP-1-mediated T-cell activation. The FRP-1 system is involved in virus-mediated cell fusion and multinucleated giant cell formation of blood monocytes. Monoclonal antibodies against human FRP-1 heavy chain induce polykaryocytes that have properties as osteoclasts. Multiple steps participate in molecular mechanisms regulating cell fusion. The FRP-1 heavy chain supports amino acid transport activity and the FRP-1 light chains have recently been cloned as amino acid transporters that require association with the heavy chain to exhibit their activity. Novel pathways for monocyte-dependent regulation of T-cell activation have recently been found that are mediated by the FRP-1 system. In conclusion, the FRP-1 molecules are essential factors for basic cellular functions.

Uses of monoclonial antibody 8H9

DOEpatents

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.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

PubMed

Harris, Katherine E; Aldred, Shelley Force; Davison, Laura M; Ogana, Heather Anne N; Boudreau, Andrew; Brüggemann, Marianne; Osborn, Michael; Ma, Biao; Buelow, Benjamin; Clarke, Starlynn C; Dang, Kevin H; Iyer, Suhasini; Jorgensen, Brett; Pham, Duy T; Pratap, Payal P; Rangaswamy, Udaya S; Schellenberger, Ute; van Schooten, Wim C; Ugamraj, Harshad S; Vafa, Omid; Buelow, Roland; Trinklein, Nathan D

2018-01-01

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive.

PubMed

Wang, Jin; Muto, Masaki; Yatabe, Rui; Onodera, Takeshi; Tanaka, Masayoshi; Okochi, Mina; Toko, Kiyoshi

2017-09-30

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive

PubMed Central

Wang, Jin; Muto, Masaki; Yatabe, Rui; Onodera, Takeshi; Okochi, Mina; Toko, Kiyoshi

2017-01-01

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT. PMID:28973962

Interpretation Difficulties of Serum Immunofixation Test in Immunoglobulin D Multiple Myeloma with Hidden Lambda Light Chains.

PubMed

Biaz, A; Uwingabiye, J; Rachid, A; Dami, A; Bouhsain, S; Ouzzif, Z; Idrissi, S El Machtani

2018-06-01

We report a case of immunoglobulin (Ig) D myeloma with hidden lambda light chains in a patient whose immunofixation test was very difficult to interpret: the IgD reacts with the anti-δ heavy chain antiserum but does not react with anti-lambda antiserum. The band in the D heavy chain lane is unmatched in light chain lanes and the band in lambda light chain lane migrates higher. To distinguish between heavy chain disease and immunoglobulin with "hidden" light chains, the sample was exposed to a very high concentration of anti-lambda and anti-kappa antisera for 48 hours. The serum immunofixation test of the sample treated with anti-lambda showed a decrease in the intensity of the band corresponding to D heavy chain lane as well as the modification of its mobility confirming the presence of IgD with the hidden lambda light chains. The IgD myeloma with hidden light chains remains a rare entity, hence the interest of sensitizing health professionals to be vigilant and ensure a good diagnosis. The proposed technique is useful, simple, reliable, and less laborious than those previous reported in the literature. Medical laboratories using Sebia-Hydrasys® system should be aware of the described phenomenon in order to avoid identifying an IgD myeloma as a delta heavy chain disease.

Major immunoglobulin classes of the echidna (Tachyglossus aculeatus)

PubMed Central

Atwell, J. L.; Marchalonis, J. J.; Ealey, E. H. M.

1973-01-01

The Australian echidna responds to the antigen Salmonella adelaide flagella by producing antibodies characterized by mol. wt of 900,000 and 150,000. After cleavage of interchain disulphide bonds, both the high and low mol. wt immunoglobulins can be resolved into light and heavy polypeptide chains. In both cases, the light chains resemble those of other vertebrate immunoglobulins in size (22,500 Daltons) and electrophoretic mobility. The 900,000 Dalton immunoglobulin contains heavy chains similar to human μ chains in size (70,000 Daltons) and electrophoretic mobility. The 150,000 Dalton immunoglobulin contains a different class of heavy chain, similar in size (50,000 Daltons) and electrophoretic mobility to human γ chains. Proportional mass contributions of the light and heavy chains to the intact molecule suggest the structure of the intact molecules could be represented by (L2, μ2)5 and (L2, γ2) for the high and low mol. wt immunoglobulins respectively. These configurations are similar to those described for human γM and γG immunoglobulins. The results are relevant to theories of the evolution of the different classes of immunoglobulins. While the echidna is distinctly more primitive than eutherian mammals and still retains structural features characteristic of reptiles, its major immunoglobulin classes are very similar to human IgM and IgG. The striking similarities between the γ-like heavy chain of the echnidna and human IgG heavy chains suggest that the echidna may be the first species in which a γ chain gene directly homologous to mammalian γ chain genes is expressed. ImagesFIG. 4 PMID:4761634

Proximal tubulopathies associated with monoclonal light chains: the spectrum of clinicopathologic manifestations and molecular pathogenesis.

PubMed

Herrera, Guillermo A

2014-10-01

Lesions associated with monoclonal light and heavy chains display a variety of glomerular, tubular interstitial, and vascular manifestations. While some of the entities are well recognized, including light and heavy chain deposition diseases, AL (light chain) and AH (heavy chain) amyloidosis, and light chain ("myeloma") cast nephropathy, other lesions centered on proximal tubules are much less accurately identified, properly diagnosed, and adequately understood in terms of pathogenesis and molecular mechanisms involved. These proximal tubule-centered lesions are typically associated with monoclonal light chains and have not been reported in patients with circulating monoclonal heavy chains. To determine the incidence of proximal tubulopathies in a series of patients with monoclonal light chain-related renal lesions and characterize them with an emphasis on clinical correlations and elucidation of molecular mechanisms involved in their pathogenesis. A study of 5410 renal biopsies with careful evaluation of light microscopic, immunofluorescence, and electron microscopic findings was conducted to identify these monoclonal light/heavy chain-related lesions. In selected cases, ultrastructural immunolabeling was performed to better illustrate and understand molecular mechanisms involved or to resolve specific diagnostic difficulties. In all, 2.5% of the biopsies were diagnosed as demonstrating renal pathology associated with monoclonal light or heavy chains. Of these, approximately 46% were classified as proximal tubule-centered lesions, also referred to as monoclonal light chain-associated proximal tubulopathies. These proximal tubulopathies were divided into 4 groups defined by characteristic immunomorphologic manifestations associated with specific clinical settings. These are important lesions whose recognition in the different clinical settings is extremely important for patients' clinical management, therapeutic purposes, and prognosis. These entities have been segregated into 4 distinct variants, conceptualized morphologically and clinically. Specific mechanisms involved in their pathogenesis are proposed.

Myosin content of individual human muscle fibers isolated by laser capture microdissection.

PubMed

Stuart, Charles A; Stone, William L; Howell, Mary E A; Brannon, Marianne F; Hall, H Kenton; Gibson, Andrew L; Stone, Michael H

2016-03-01

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. Copyright © 2016 the American Physiological Society.

Myosin content of individual human muscle fibers isolated by laser capture microdissection

PubMed Central

Stone, William L.; Howell, Mary E. A.; Brannon, Marianne F.; Hall, H. Kenton; Gibson, Andrew L.; Stone, Michael H.

2015-01-01

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. PMID:26676053

Identification of globular mechanochemical heads of kinesin.

PubMed

Scholey, J M; Heuser, J; Yang, J T; Goldstein, L S

1989-03-23

Kinesin is a mechanoenzyme which uses energy liberated from ATP hydrolysis to transport particles towards the 'plus ends' of microtubules. The enzyme consists of two polypeptide heavy chains of relative molecular mass (Mr) approximately 110,000-140,000 (110K-140K) plus copurifying light chains; these polypeptides are arranged in a structure consisting of two globular heads attached to a fibrous stalk which terminates in a 'feathered' tail. Here we report that a function-disrupting monoclonal antikinesin, which binds to the 45K fragment of the kinesin heavy chain, recognizes an epitope located towards the N-terminal end of the heavy chain, and decorates the two globular heads lying at one end of the intact molecules (one antibody per head). The results show that the two heavy chains of native kinesin are arranged in parallel, and that the 45K fragments, which display nucleotide-sensitive interactions with microtubules, represent mechanochemical 'heads' located at the N-terminal regions of the heavy chains. Thus, it is likely that the kinesin heads are analogous to the subfragment-1 domains of myosin.

The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations.

PubMed

Scavenius, Carsten; Nikolajsen, Camilla Lund; Stenvang, Marcel; Thøgersen, Ida B; Wyrożemski, Łukasz; Wisniewski, Hans-Georg; Otzen, Daniel E; Sanggaard, Kristian W; Enghild, Jan J

2016-02-26

Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg(2+) or Mn(2+), but not Ca(2+), induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg(2+) found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains.

PubMed

Fera, Daniela; Schmidt, Aaron G; Haynes, Barton F; Gao, Feng; Liao, Hua-Xin; Kepler, Thomas B; Harrison, Stephen C

2014-07-15

Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus-antibody "arms race." Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus-antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule.

Force decay evaluation of thermoplastic and thermoset elastomeric chains: A mechanical design comparison.

PubMed

Masoud, Ahmed I; Tsay, T Peter; BeGole, Ellen; Bedran-Russo, Ana K

2014-11-01

To compare the following over a period of 8 weeks: (1) force decay between thermoplastic (TP) and thermoset (TS) elastomeric chains; (2) force decay between light (200-g) and heavy (350-g) initial forces; and (3) force decay between direct chains and chain loops (stretched from one pin around the second pin and back to the first pin). TP and TS chains were obtained from American Orthodontics™ (AOTP, AOTS) and ORMCO™ (OrTP, OrTS). Each of the four chain groups was subdivided into four subgroups with 10 specimens per subgroup: (1) direct chains light force, (2) direct chains heavy force, (3) chain loops light force, and (4) chain loops heavy force. The experiment was performed in artificial saliva (pH of 6.75) at 37°C. A significant difference was found between TP and TS chains, with an average mean difference of around 20% more force decay found in the TP chains (P < .001, α  =  .05). There was no significant difference between direct chains and chain loops except in OrTP, in which direct chains showed more force decay. There was also no significant difference in force decay identified when using light vs heavy forces. TS chains decayed less than TP chains, and chain loop retraction was beneficial only when using OrTP chains. Contrary to the interchangeable use of TP and TS chains in the published literature and in clinical practice, this study demonstrates that they perform differently under stress and that a clear distinction should be made between the two.

CPdock: the complementarity plot for docking of proteins: implementing multi-dielectric continuum electrostatics.

PubMed

Basu, Sankar

2017-12-07

The complementarity plot (CP) is an established validation tool for protein structures, applicable to both globular proteins (folding) as well as protein-protein complexes (binding). It computes the shape and electrostatic complementarities (S m , E m ) for amino acid side-chains buried within the protein interior or interface and plots them in a two-dimensional plot having knowledge-based probabilistic quality estimates for the residues as well as for the whole structure. The current report essentially presents an upgraded version of the plot with the implementation of the advanced multi-dielectric functionality (as in Delphi version 6.2 or higher) in the computation of electrostatic complementarity to make the validation tool physico-chemically more realistic. The two methods (single- and multi-dielectric) agree decently in their resultant E m values, and hence, provisions for both methods have been kept in the software suite. So to speak, the global electrostatic balance within a well-folded protein and/or a well-packed interface seems only marginally perturbed by the choice of different internal dielectric values. However, both from theoretical as well as practical grounds, the more advanced multi-dielectric version of the plot is certainly recommended for potentially producing more reliable results. The report also presents a new methodology and a variant plot, namely CP dock , based on the same principles of complementarity specifically designed to be used in the docking of proteins. The efficacy of the method to discriminate between good and bad docked protein complexes has been tested on a recent state-of-the-art docking benchmark. The results unambiguously indicate that CP dock can indeed be effective in the initial screening phase of a docking scoring pipeline before going into more sophisticated and computationally expensive scoring functions. CP dock has been made available at https://github.com/nemo8130/CPdock . Graphical Abstract An example showing the efficacy of CP dock to be used in the initial screening phase of a protein-protein docking scoring pipeline.

SDU: A Semidefinite Programming-Based Underestimation Method for Stochastic Global Optimization in Protein Docking

DTIC Science & Technology

2007-04-01

optimization methodology we introduce. State-of-the-art protein - protein docking approaches start by identifying conformations with good surface /chemical com...side-chains on the interface ). The protein - protein docking literature (e.g., [8] and the references therein) is predominantly treating the docking...mations by various measures of surface complementarity which can be efficiently computed using fast Fourier correlation tech- niques (FFTs). However, when

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

PubMed Central

1988-01-01

We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459

Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

PubMed Central

Bui, Khanh Huy; Sakakibara, Hitoshi; Movassagh, Tandis; Oiwa, Kazuhiro; Ishikawa, Takashi

2008-01-01

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins. PMID:19029338

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody.

PubMed

Apgar, James R; Mader, Michelle; Agostinelli, Rita; Benard, Susan; Bialek, Peter; Johnson, Mark; Gao, Yijie; Krebs, Mark; Owens, Jane; Parris, Kevin; St Andre, Michael; Svenson, Kris; Morris, Carl; Tchistiakova, Lioudmila

2016-10-01

Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

PubMed Central

Apgar, James R.; Mader, Michelle; Agostinelli, Rita; Benard, Susan; Bialek, Peter; Johnson, Mark; Gao, Yijie; Krebs, Mark; Owens, Jane; Parris, Kevin; St. Andre, Michael; Svenson, Kris; Morris, Carl; Tchistiakova, Lioudmila

2016-01-01

ABSTRACT Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall “humanness” was increased for both the light and heavy chain variable regions. PMID:27625211

A chemical and computational approach to comprehensive glycation characterization on antibodies

PubMed Central

Saleem, Ramsey A; Affholter, Brittany R; Deng, Sihong; Campbell, Phil C; Matthies, Kelli; Eakin, Catherine M; Wallace, Alison

2015-01-01

Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS2 mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS1 based approach, which allowed the identification of a glycation hot spot in the heavy chain complementarity-determining region 3 of the mAb. This targeted approach provided a path forward to developing a structural understanding of the propensity of sites to become glycated on mAbs. Through structural analysis we propose a model in which the number and 3-dimensional distances of carboxylic acid amino acyl residues create a favorable environment for glycation to occur. PMID:26030340

Productive Recognition of Factor IX by Factor XIa Exosites Requires Disulfide Linkage between Heavy and Light Chains of Factor XIa*

PubMed Central

Marcinkiewicz, Mariola M.; Sinha, Dipali; Walsh, Peter N.

2012-01-01

In the intrinsic pathway of blood coagulation factor XIa (FXIa) activates factor IX (FIX) by cleaving the zymogen at Arg145-Ala146 and Arg180-Val181 bonds releasing an 11-kDa activation peptide. FXIa and its isolated light chain (FXIa-LC) cleave S-2366 at comparable rates, but FXIa-LC is a very poor activator of FIX, possibly because FIX undergoes allosteric modification on binding to an exosite on the heavy chain of FXIa (FXIa-HC) required for optimal cleavage rates of the two scissile bonds of FIX. However preincubation of FIX with a saturating concentration of isolated FXIa-HC did not result in any potentiation in the rate of FIX cleavage by FXIa-LC. Furthermore, if FIX binding via the heavy chain exosite of FXIa determines the affinity of the enzyme-substrate interaction, then the isolated FXIa-HC should inhibit the rate of FIX activation by depleting the substrate. However, whereas FXIa/S557A inhibited FIX activation of by FXIa, FXIa-HC did not. Therefore, we examined FIX binding to FXIa/S557A, FXIa-HC, FXIa-LC, FXIa/C362S/C482S, and FXIa/S557A/C362S/C482S. The heavy and light chains are disulfide-linked in FXIa/S557A but not in FXIa/C362S/C482S and FXIa/S557A/C362S/C482S. In an ELISA assay only FXI/S557A ligated FIX with high affinity. Partial reduction of FXIa/S557A to produce heavy and light chains resulted in decreased FIX binding, and this function was regained upon reformation of the disulfide linkage between the heavy and the light chains. We therefore conclude that substrate recognition by the FXIa exosite(s) requires disulfide-linked heavy and light chains. PMID:22207756

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments.

PubMed

Solforosi, Laura; Mancini, Nicasio; Canducci, Filippo; Clementi, Nicola; Sautto, Giuseppe Andrea; Diotti, Roberta Antonia; Clementi, Massimo; Burioni, Roberto

2012-07-01

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.

Lineage-restricted retention of a primitive immunoglobulin heavy chain isotype within the Dipnoi reveals an evolutionary paradox

PubMed Central

Ota, Tatsuya; Rast, Jonathan P.; Litman, Gary W.; Amemiya, Chris T.

2003-01-01

The lineage leading to lungfishes is one of the few major jawed vertebrate groups in which Ig heavy chain isotype structure has not been investigated at the genetic level. In this study, we have characterized three different Ig heavy chain isotypes of the African lungfish, Protopterus aethiopicus, including an IgM-type heavy chain and short and long forms of non-IgM heavy chains. Northern blot analysis as well as patterns of VH utilization suggest that the IgM and non-IgM isotypes are likely encoded in separate loci. The two non-IgM isotypes identified in Protopterus share structural features with the short and long forms of IgX/W/NARC (referred to hereafter as IgW), which were previously considered to be restricted to the cartilaginous fish. It seems that the IgW isotype has a far broader phylogenetic distribution than considered originally and raises questions with regard to the origin and evolutionary divergence of IgM and IgW. Moreover, its absence in other gnathostome lineages implies paradoxically that the IgW-type genes were lost from teleost and tetrapod lineages. PMID:12606718

Gravity changes during animal development affect IgM heavy-chain transcription and probably lymphopoiesis.

PubMed

Huin-Schohn, Cécile; Guéguinou, Nathan; Schenten, Véronique; Bascove, Matthieu; Koch, Guillemette Gauquelin; Baatout, Sarah; Tschirhart, Eric; Frippiat, Jean-Pol

2013-01-01

Our previous research demonstrated that spaceflight conditions affect antibody production in response to an antigenic stimulation in adult amphibians. Here, we investigated whether antibody synthesis is affected when animal development occurs onboard a space station. To answer this question, embryos of the Iberian ribbed newt, Pleurodeles waltl, were sent to the International Space Station (ISS) before the initiation of immunoglobulin heavy-chain expression. Thus, antibody synthesis began in space. On landing, we determined the effects of spaceflight on P. waltl development and IgM heavy-chain transcription. Results were compared with those obtained using embryos that developed on Earth. We find that IgM heavy-chain transcription is doubled at landing and that spaceflight does not affect P. waltl development and does not induce inflammation. We also recreated the environmental modifications encountered by the embryos during their development onboard the ISS. This strategy allowed us to demonstrate that gravity change is the factor responsible for antibody heavy-chain transcription modifications that are associated with NF-κB mRNA level variations. Taken together, and given that the larvae were not immunized, these data suggest a modification of lymphopoiesis when gravity changes occur during ontogeny.

Anatomy of exotic Higgs decays in 2HDM

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

Kling, Felix; No, Jose Miguel; Su, Shufang

Large mass splittings between new scalars in two-Higgs-doublet models (2HDM) open a key avenue to search for these new states via exotic heavy Higgs decays. We discuss in detail the different search channels for these new scalars at the LHC in the presence of a sizable mass splitting, i.e. a hierarchical 2HDM scenario, taking into account the theoretical and experimental constraints. Here, we provide benchmark planes to exploit the complementarity among these searches, analyzing their potential to probe the hierarchical 2HDM parameter space during LHC Run 2.

Anatomy of exotic Higgs decays in 2HDM

NASA Astrophysics Data System (ADS)

Kling, Felix; No, Jose Miguel; Su, Shufang

2016-09-01

Large mass splittings between new scalars in two-Higgs-doublet models (2HDM) open a key avenue to search for these new states via exotic heavy Higgs decays. We discuss in detail the different search channels for these new scalars at the LHC in the presence of a sizable mass splitting, i.e. a hierarchical 2HDM scenario, taking into account the theoretical and experimental constraints. We provide benchmark planes to exploit the complementarity among these searches, analyzing their potential to probe the hierarchical 2HDM parameter space during LHC Run 2.

Anatomy of exotic Higgs decays in 2HDM

DOE PAGES

Kling, Felix; No, Jose Miguel; Su, Shufang

2016-09-16

Large mass splittings between new scalars in two-Higgs-doublet models (2HDM) open a key avenue to search for these new states via exotic heavy Higgs decays. We discuss in detail the different search channels for these new scalars at the LHC in the presence of a sizable mass splitting, i.e. a hierarchical 2HDM scenario, taking into account the theoretical and experimental constraints. Here, we provide benchmark planes to exploit the complementarity among these searches, analyzing their potential to probe the hierarchical 2HDM parameter space during LHC Run 2.

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

PubMed

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.

A potent human anti-eotaxin1 antibody, CAT-213: isolation by phage display and in vitro and in vivo efficacy.

PubMed

Main, Sarah; Handy, Rachel; Wilton, Jane; Smith, Stephen; Williams, Liz; Fou, Leila Du; Andrews, John; Conroy, Louise A; May, Richard; Anderson, Ian; Vaughan, Tristan J

2006-12-01

The CC chemokine, eotaxin1 (CCL11) is an important regulator of eosinophil function. A marked accumulation of eosinophils in tissues has been correlated with the up-regulation of eotaxin1 expression in several diseases. The potential therapeutic value of neutralizing the effects of eotaxin1 in inflammatory conditions (including asthma) is under investigation. A human single-chain fragment variable antibody that neutralizes human eotaxin1 (CAT-212) was produced using antibody phage display and converted to whole antibody IgG4 format (CAT-213). A novel approach to lead optimization in which the length of the variable heavy chain complementarity-determining region 3 was reduced by one amino acid resulted in an increase in potency of >1000-fold compared with the parent anti-eotaxin1 antibody. The optimized antibody binds eotaxin1 with high affinity (80.4 pM) and specificity. CAT-213 and CAT-212 do not bind or neutralize a range of other human proteins including human monocyte chemoattractant protein-1, a structurally similar chemokine. CAT-213 neutralizes the ability of eotaxin1 to cause an increase in intracellular calcium signaling (with an IC(50) value of 2.86 nM), migration of CCR3-expressing L1.2 cells (with an IC(50) value of 0.48 nM), and inhibition of the eotaxin1-evoked shape change of human eosinophils in vitro (with an IC(50) of 0.71 nM). Local administration of CAT-213 to mice (1-100 microg kg(-1)) attenuates dermal eosinophilia induced by human eotaxin1, achieving >90% inhibition of eosinophil influx. CAT-213 may therefore be of therapeutic value in inhibiting diseases in which eotaxin1 and eosinophils play a major role, for example, severe asthma.

Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies

PubMed Central

Hwang, Joyce K.; Wang, Chong; Du, Zhou; Meyers, Robin M.; Kepler, Thomas B.; Neuberg, Donna; Kwong, Peter D.; Mascola, John R.; Joyce, M. Gordon; Bonsignori, Mattia; Haynes, Barton F.; Yeap, Leng-Siew; Alt, Frederick W.

2017-01-01

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1–infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms. PMID:28747530

Quantum delayed-choice experiment with a single neutral atom.

PubMed

Li, Gang; Zhang, Pengfei; Zhang, Tiancai

2017-10-01

We present a proposal to implement a quantum delayed-choice (QDC) experiment with a single neutral atom, such as a rubidium or cesium atom. In our proposal, a Ramsey interferometer is adopted to observe the wave-like or particle-like behaviors of a single atom depending on the existence or absence of the second π/2-rotation. A quantum-controlled π/2-rotation on target atom is realized through a Rydberg-Rydberg interaction by another ancilla atom. It shows that a heavy neutral atom can also have a morphing behavior between the particle and the wave. The realization of the QDC experiment with such heavy neutral atoms not only is significant to understand the Bohr's complementarity principle in matter-wave and matter-particle domains but also has great potential on the quantum information process with neutral atoms.

Continued Expression of Neonatal Myosin Heavy Chain in Adult Dystrophic Skeletal Muscle

NASA Astrophysics Data System (ADS)

Bandman, Everett

1985-02-01

The expression of myosin heavy chain isoforms was examined in normal and dystrophic chicken muscle with a monoclonal antibody specific for neonatal myosin. Adult dystrophic muscle continued to contain neonatal myosin long after it disappeared from adult normal muscle. A new technique involving western blotting and peptide mapping demonstrated that the immunoreactive myosin in adult dystrophic muscle was identical to that found in neonatal normal muscle. Immunocytochemistry revealed that all fibers in the dystrophic muscle failed to repress neonatal myosin heavy chain. These studies suggest that muscular dystrophy inhibits the myosin gene switching that normally occurs during muscle maturation.

J Genes for Heavy Chain Immunoglobulins of Mouse

NASA Astrophysics Data System (ADS)

Newell, Nanette; Richards, Julia E.; Tucker, Philip W.; Blattner, Frederick R.

1980-09-01

A 15.8-kilobase pair fragment of BALB/c mouse liver DNA, cloned in the Charon 4Aλ phage vector system, was shown to contain the μ heavy chain constant region (CHμ ) gene for the mouse immunoglobulin M. In addition, this fragment of DNA contains at least two J genes, used to code for the carboxyl terminal portion of heavy chain variable regions. These genes are located in genomic DNA about eight kilobase pairs to the 5' side of the CHμ gene. The complete nucleotide sequence of a 1120-base pair stretch of DNA that includes the two J genes has been determined.

[Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].

PubMed

Wen, Yangming; Lan, Kaijian; Wang, Junjie; Yu, Jingyi; Qu, Yarong; Zhao, Wei; Zhang, Fuchun; Tan, Wanlong; Cao, Hong; Zhou, Chen

2013-06-01

To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)]. Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

Induction of IgA B cell differentiation of bone marrow-derived B cells by Peyer's patch autoreactive helper T cells.

PubMed

Kihira, T; Kawanishi, H

1995-08-01

The objective of this study was to demonstrate in vitro that bone marrow-derived pro/pre-B cells bearing mu mRNA can switch their Ig heavy-chain isotype to that of alpha mRNA-expressing B cells after contact with Peyer's patches-derived activated autoreactive CD4+ T cells. Bone marrow-derived pro/pre-B cells and activated autoreactive Peyer's patch, mesenteric lymph node, or spleen CD4+ T cells were co-cultured in the presence of recombinant (r) IL-2, rIL-7, and Con A for 3 days. The mixed cultured cells were isolated for preparation of total RNA. Dot/slot hybridization, using murine C mu (pu3741) and C alpha (P alpha J558) Ig heavy-chain cDNA probes, detected C mu and C alpha Ig heavy-chain mRNA transcripts. The magnitude of each mRNA expression was measured demsitometrically. In addition, the secreted class-specific Ig contents from the co-cultured supernatants were measured. The results indicate that activated autoreactive Peyer's patch and mesenteric lymph node CD4+ T cells provide a specific Ig heavy-chain switch from mu to alpha (Peyer's patch CD4+ T cells > mesenteric lymph node CD4+ T cells) in bone marrow-derived pro/pre-B cells and also assist to develop IgA-secreting plasma cells. The alpha heavy-chain switch and IgA production do not occur in the presence of activated autoreactive spleen CD4+ T cells. These results support the view that autoreactive gut Peyer's patch CD4+ T cells, at least, regulate IgA B cell heavy-chain switching and terminal differentiation during gut mucosal B cell development.

Molecular Characterization and Functional Analysis of a Ferritin Heavy Chain Subunit from the Eri-Silkworm, Samia cynthia ricini

PubMed Central

Yu, Hai-Zhong; Zhang, Shang-Zhi; Ma, Yan; Fei, Dong-Qiong; Li, Bing; Yang, Li-Ang; Wang, Jie; Li, Zhen; Muhammad, Azharuddin; Xu, Jia-Ping

2017-01-01

Ferritins are conserved iron-binding proteins that are primarily involved in iron storage, detoxification and the immune response. Despite the importance of ferritin in organisms, little is known about their roles in the eri-silkworm (Samia cynthia ricini). We previously identified a ferritin heavy chain subunit named ScFerHCH in the S. c. ricini transcriptome database. The full-length S. c. ricini ferritin heavy chain subunit (ScFerHCH) was 1863 bp and encoded a protein of 231 amino acids with a deduced molecular weight of 25.89 kDa. Phylogenetic analysis revealed that ScFerHCH shared a high amino acid identity with the Bombyx mori and Danaus plexippus heavy chain subunits. Higher ScFerHCH expression levels were found in the silk gland, fat body and midgut of S. c. ricini by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Injection of Staphylococcus aureus and Pseudomonas aeruginosa was associated with an upregulation of ScFerHCH in the midgut, fat body and hemolymph, indicating that ScFerHCH may contribute to the host’s defense against invading pathogens. In addition, the anti-oxidation activity and iron-binding capacity of recombinant ScFerHCH protein were examined. Taken together, our results suggest that the ferritin heavy chain subunit from eri-silkworm may play critical roles not only in innate immune defense, but also in organismic iron homeostasis. PMID:29036914

Antibody repertoire development in camelids.

PubMed

De Genst, Erwin; Saerens, Dirk; Muyldermans, Serge; Conrath, Katja

2006-01-01

The humoral immune response of the Camelidae is unique as these animals are the only known mammals that seem to possess functional homodimeric heavy-chain antibodies besides the classical heteromeric antibodies composed of heavy (H) and light (L) chains. By definition, the heavy-chain antibodies lack the L-chain, and it was noticed that their H-chain is devoid of the typical first constant domain (CH1) and contains a dedicated variable domain, referred to as VHH. The VHH exon is assembled from separate V-D-J gene segments. The recombined VHH region is subjected to somatic hypermutations; however, the timing and actual mechanism of the class switch from mu to the dedicated gamma-isotype remains elusive. Interestingly, antigen-specific VHHs are easily retrieved after panning of a phage-displayed rearranged V-gene pool cloned from an immunised camelid. These single-domain antigen binding entities possess a number of biophysical properties that offer particular advantages in various medical and biotechnological applications.

Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

PubMed

Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

2018-01-01

Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

Change in IgHV Mutational Status of CLL Suggests Origin From Multiple Clones.

PubMed

Osman, Afaf; Gocke, Christopher D; Gladstone, Douglas E

2017-02-01

Fluorescence in situ hybridization and immunoglobulin (Ig) heavy-chain variable-region (IgHV) mutational status are used to predict outcome in chronic lymphocytic leukemia (CLL). Although DNA aberrations change over time, IgHV sequences and mutational status are considered stable. In a retrospective review, 409 CLL patients, between 2008 and 2015, had IgHV analysis: 56 patients had multiple analyses performed. Seven patients' IgHV results changed: 2 from unmutated to mutated and 5 from mutated to unmutated IgHV sequence. Three concurrently changed their variable heavy-chain sequence. Secondary to allelic exclusion, 2 of the new variable heavy chains produced were biologically nonplausible. The existence of these new nonplausible heavy-chain variable regions suggests either the CLL cancer stem-cell maintains the ability to rearrange a previously silenced IgH allele or more likely that the cancer stem-cell produced at least 2 subclones, suggesting that the CLL cancer stem cell exists before the process of allelic exclusion occurs. Copyright © 2016 Elsevier Inc. All rights reserved.

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library.

PubMed

Liu, Jinny L; Anderson, George P; Goldman, Ellen R

2007-11-19

Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

Clonal expansion and somatic hypermutation of V(H) genes of B cells from cerebrospinal fluid in multiple sclerosis.

PubMed Central

Qin, Y; Duquette, P; Zhang, Y; Talbot, P; Poole, R; Antel, J

1998-01-01

The cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients is characterized by increased concentrations of immunoglobulin (Ig), which on electrophoretic analysis shows restricted heterogeneity (oligoclonal bands). CSF Ig is composed of both serum and intrathecally produced components. To examine the properties of intrathecal antibody-producing B cells, we analyzed Ig heavy-chain variable (V(H)) region genes of B cells recovered from the CSF of 12 MS patients and 15 patients with other neurological diseases (OND). Using a PCR technique, we could detect rearrangements of Ig V(H) genes in all samples. Sequence analysis of complementarity-determining region 3 (CDR3) of rearranged VDJ genes revealed expansion of a dominant clone or clones in 10 of the 12 MS patients. B cell clonal expansion was identified in 3 of 15 OND. The nucleotide sequences of V(H) genes from clonally expanded CSF B cells in MS patients demonstrated the preferential usage of the V(H) IV family. There were numerous somatic mutations, mainly in the CDRs, with a high replacement-to-silent ratio; the mutations were distributed in a way suggesting that these B cells had been positively selected through their antigen receptor. Our results demonstrate that in MS CSF, there is a high frequency of clonally expanded B cells that have properties of postgerminal center memory or antibody-forming lymphocytes. PMID:9727074

Human Peripheral Blood Antibodies with Long HCDR3s Are Established Primarily at Original Recombination Using a Limited Subset of Germline Genes

PubMed Central

Briney, Bryan S.; Willis, Jordan R.; Crowe, James E.

2012-01-01

A number of antibodies that efficiently neutralize microbial targets contain long heavy chain complementarity determining region 3 (HCDR3) loops. For HIV, several of the most broad and potently neutralizing antibodies have exceptionally long HCDR3s. Two broad potently neutralizing HIV-specific antibodies, PG9 and PG16, exhibit secondary structure. Two other long HCDR3 antibodies, 2F5 and 4E10, protect against mucosal challenge with SHIV. Induction of such long HCDR3 antibodies may be critical to the design of an effective vaccine strategy for HIV and other pathogens, however it is unclear at present how to induce such antibodies. Here, we present genetic evidence that human peripheral blood antibodies containing long HCDR3s are not primarily generated by insertions introduced during the somatic hypermutation process. Instead, they are typically formed by processes occurring as part of the original recombination event. Thus, the response of B cells encoding antibodies with long HCDR3s results from selection of unusual clones from the naïve repertoire rather than through accumulation of insertions. These antibodies typically use a small subset of D and J gene segments that are particularly suited to encoding long HCDR3s, resulting in the incorporation of highly conserved genetic elements in the majority of antibody sequences encoding long HCDR3s. PMID:22590602

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

PubMed Central

Liu, Jinny L; Anderson, George P; Goldman, Ellen R

2007-01-01

Background Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (VH), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins. PMID:18021450

cDNA sequences and organization of IgM heavy chain genes in two holostean fish.

PubMed

Wilson, M R; van Ravenstein, E; Miller, N W; Clem, L W; Middleton, D L; Warr, G W

1995-01-01

Immunoglobulin M heavy chain (mu) sequences of two holostean fish, the bowfin, Amia calva, and the longnose gar, Lepisosteus osseus, were amplified from spleen mRNA by RACE-PCR, cloned, and sequenced. Each mu chain showed the conserved four constant domain structure typical of a secreted mu chain. Southern blot analyses with specific heavy chain variable (VH) and constant (CH) region probes suggest that both fish possess an IgH locus that resembles that of the teleosts, amphibians, and mammals in its organization. The overall sequence similarity of gar and bowfin mu chains was 60% and 48% at the nucleotide and amino acid levels, respectively, while similarity to the mu chains of teleosts and elasmobranchs was lower. The bowfin mu chain possesses a distinctive proline-rich sequence at the C mu 1/C mu 2 boundary; a shorter proline-rich sequence is present at this position in the gar mu chain. Both gar and bowfin show, in their C mu 4 sequences, motifs that could serve as cryptic splice donor sites for the production of mRNA encoding the membrane-bound form of the mu chains, and the bowfin also shows a potential cryptic splice donor site in the C mu 3 exon.

Expansion of the Preimmune Antibody Repertoire by Junctional Diversity in Bos taurus

PubMed Central

Liljavirta, Jenni; Niku, Mikael; Pessa-Morikawa, Tiina; Ekman, Anna; Iivanainen, Antti

2014-01-01

Cattle have a limited range of immunoglobulin genes which are further diversified by antigen independent somatic hypermutation in fetuses. Junctional diversity generated during somatic recombination contributes to antibody diversity but its relative significance has not been comprehensively studied. We have investigated the importance of terminal deoxynucleotidyl transferase (TdT) -mediated junctional diversity to the bovine immunoglobulin repertoire. We also searched for new bovine heavy chain diversity (IGHD) genes as the information of the germline sequences is essential to define the junctional boundaries between gene segments. New heavy chain variable genes (IGHV) were explored to address the gene usage in the fetal recombinations. Our bioinformatics search revealed five new IGHD genes, which included the longest IGHD reported so far, 154 bp. By genomic sequencing we found 26 new IGHV sequences that represent potentially new IGHV genes or allelic variants. Sequence analysis of immunoglobulin heavy chain cDNA libraries of fetal bone marrow, ileum and spleen showed 0 to 36 nontemplated N-nucleotide additions between variable, diversity and joining genes. A maximum of 8 N nucleotides were also identified in the light chains. The junctional base profile was biased towards A and T nucleotide additions (64% in heavy chain VD, 52% in heavy chain DJ and 61% in light chain VJ junctions) in contrast to the high G/C content which is usually observed in mice. Sequence analysis also revealed extensive exonuclease activity, providing additional diversity. B-lymphocyte specific TdT expression was detected in bovine fetal bone marrow by reverse transcription-qPCR and immunofluorescence. These results suggest that TdT-mediated junctional diversity and exonuclease activity contribute significantly to the size of the cattle preimmune antibody repertoire already in the fetal period. PMID:24926997

The formation of pyrrolid-2-one-5-carboxylic acid at the N-terminus of immunoglobulin G heavy chain

PubMed Central

Stott, D. I.; Munro, A. J.

1972-01-01

We propose that pyrrolid-2-one-5-carboxyl-tRNA is not involved in the initiation of protein synthesis in eukaryotic cells and that the N-terminal pyrrolid-2-one-5-carboxylic acid group of an IgG (immunoglobulin G) (that secreted by the mouse plasmacytoma Adj PC5) is formed by the enzymic cyclization of the N-terminal glutamine of the heavy chain of the completed IgG molecule and that the cyclization takes place inside the cell. We base these conclusions on the following evidence. (1) Pyrrolidonecarboxyl-tRNA was not found in incorporation experiments with rat liver preparations and [U-14C]-pyrrolidonecarboxylic acid, glutamic acid and glutamine, even though an incorporation extent of less than 2% of the total products could have been detected. (2) Double-labelling experiments showed that less than 8% of the nascent peptides of heavy chains (those obtained by precipitation by the antibody to Fc fragment) began with pyrrolidonecarboxylic acid. (3) Further double-labelling experiments showed that 60–66% of the heavy chains of the completed intracellular IgG molecule began with pyrrolidonecarboxylic acid after both 1 and 5h of labelling. (4) The IgG, after secretion by plasmacytoma Adj PC5, was found to have the sequence [unk]Glu- Val-Gln-Leu- at the N-termini of the heavy chains. PMID:4674626

New Existence Conditions for Order Complementarity Problems

NASA Astrophysics Data System (ADS)

Németh, S. Z.

2009-09-01

Complementarity problems are mathematical models of problems in economics, engineering and physics. A special class of complementarity problems are the order complementarity problems [2]. Order complementarity problems can be applied in lubrication theory [6] and economics [1]. The notion of exceptional family of elements for general order complementarity problems in Banach spaces will be introduced. It will be shown that for general order complementarity problems defined by completely continuous fields the problem has either a solution or an exceptional family of elements (for other notions of exceptional family of elements see [1, 2, 3, 4] and the related references therein). This solves a conjecture of [2] about the existence of exceptional family of elements for order complementarity problems. The proof can be done by using the Leray-Schauder alternative [5]. An application to integral operators will be given.

Functional Consequences of Complementarity-determining Region Deactivation in a Multifunctional Anti-nucleic Acid Antibody*

PubMed Central

Lee, Jiyeon; Kim, Hye-Jin; Roh, Jooho; Seo, Youngsil; Kim, Minjae; Jun, Hye-Ryeong; Pham, Chuong D.; Kwon, Myung-Hee

2013-01-01

Many murine monoclonal anti-DNA antibodies (Abs) derived from mice models for systemic lupus erythematosus have additional cell-penetration and/or nucleic acid-hydrolysis properties. Here, we examined the influence of deactivating each complementarity-determining region (CDR) within a multifunctional anti-nucleic acid antibody (Ab) that possesses these activities, the catalytic 3D8 single chain variable fragment (scFv). CDR-deactivated 3D8 scFv variants were generated by replacing all of the amino acids within each CDR with Gly/Ser residues. The structure of 3D8 scFv accommodated single complete CDR deactivations. Different functional activities of 3D8 scFv were affected differently depending on which CDR was deactivated. The only exception was CDR1, located within the light chain (LCDR1); deactivation of LCDR1 abolished all of the functional activities of 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv. These results suggest that the activity of a multifunctional 3D8 scFv Ab can be modulated by single complete CDR deactivation and that the LCDR1 plays a crucial role in maintaining Ab properties. This study presents a new approach for determining the role of individual CDRs in multifunctional Abs with important implications for the future of Ab engineering. PMID:24155236

Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

PubMed Central

Matsushita, Hiroaki; Sano, Akiko; Wu, Hua; Jiao, Jin-an; Kasinathan, Poothappillai; Sullivan, Eddie J.; Wang, Zhongde; Kuroiwa, Yoshimi

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−/−, bIGHML1−/−; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM−/−, bIGHML1−/− and bIGL−/−; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ). PMID:24603704

Electrostatic complementarity between proteins and ligands. 1. Charge disposition, dielectric and interface effects

NASA Astrophysics Data System (ADS)

Chau, P.-L.; Dean, P. M.

1994-10-01

Electrostatic interactions have always been considered an important factor governing ligand-receptor interactions. Previous work in this field has established the existence of electrostatic complementarity between the ligand and its receptor site. However, this property has not been treated rigorously, and the description remains largely qualitative. In this work, 34 data sets of high quality were chosen from the Brookhaven Protein Databank. The electrostatic complementarity has been calculated between the surface potentials; complementarity is absent between adjacent or neighbouring atoms of the ligand and the receptor. There is little difference between complementarities on the total ligand surface and the interfacial region. Altering the homogeneous dielectric to distance-dependent dielectrics reduces the complementarity slightly, but does not affect the pattern of complementarity.

Assessing potential dietary toxicity of heavy metals in selected vegetables and food crops*

PubMed Central

Islam, Ejaz ul; Yang, Xiao-e; He, Zhen-li; Mahmood, Qaisar

2007-01-01

Heavy metals, such as cadmium, copper, lead, chromium and mercury, are important environmental pollutants, particularly in areas with high anthropogenic pressure. Their presence in the atmosphere, soil and water, even in traces can cause serious problems to all organisms, and heavy metal bioaccumulation in the food chain especially can be highly dangerous to human health. Heavy metals enter the human body mainly through two routes namely: inhalation and ingestion, ingestion being the main route of exposure to these elements in human population. Heavy metals intake by human populations through food chain has been reported in many countries. Soil threshold for heavy metal toxicity is an important factor affecting soil environmental capacity of heavy metal and determines heavy metal cumulative loading limits. For soil-plant system, heavy metal toxicity threshold is the highest permissible content in the soil (total or bioavailable concentration) that does not pose any phytotoxic effects or heavy metals in the edible parts of the crops does not exceed food hygiene standards. Factors affecting the thresholds of dietary toxicity of heavy metal in soil-crop system include: soil type which includes soil pH, organic matter content, clay mineral and other soil chemical and biochemical properties; and crop species or cultivars regulated by genetic basis for heavy metal transport and accumulation in plants. In addition, the interactions of soil-plant root-microbes play important roles in regulating heavy metal movement from soil to the edible parts of crops. Agronomic practices such as fertilizer and water managements as well as crop rotation system can affect bioavailability and crop accumulation of heavy metals, thus influencing the thresholds for assessing dietary toxicity of heavy metals in the food chain. This paper reviews the phytotoxic effects and bioaccumulation of heavy metals in vegetables and food crops and assesses soil heavy metal thresholds for potential dietary toxicity. PMID:17173356

Assessing potential dietary toxicity of heavy metals in selected vegetables and food crops.

PubMed

Islam, Ejaz ul; Yang, Xiao-e; He, Zhen-li; Mahmood, Qaisar

2007-01-01

Heavy metals, such as cadmium, copper, lead, chromium and mercury, are important environmental pollutants, particularly in areas with high anthropogenic pressure. Their presence in the atmosphere, soil and water, even in traces can cause serious problems to all organisms, and heavy metal bioaccumulation in the food chain especially can be highly dangerous to human health. Heavy metals enter the human body mainly through two routes namely: inhalation and ingestion, ingestion being the main route of exposure to these elements in human population. Heavy metals intake by human populations through food chain has been reported in many countries. Soil threshold for heavy metal toxicity is an important factor affecting soil environmental capacity of heavy metal and determines heavy metal cumulative loading limits. For soil-plant system, heavy metal toxicity threshold is the highest permissible content in the soil (total or bioavailable concentration) that does not pose any phytotoxic effects or heavy metals in the edible parts of the crops does not exceed food hygiene standards. Factors affecting the thresholds of dietary toxicity of heavy metal in soil-crop system include: soil type which includes soil pH, organic matter content, clay mineral and other soil chemical and biochemical properties; and crop species or cultivars regulated by genetic basis for heavy metal transport and accumulation in plants. In addition, the interactions of soil-plant root-microbes play important roles in regulating heavy metal movement from soil to the edible parts of crops. Agronomic practices such as fertilizer and water managements as well as crop rotation system can affect bioavailability and crop accumulation of heavy metals, thus influencing the thresholds for assessing dietary toxicity of heavy metals in the food chain. This paper reviews the phytotoxic effects and bioaccumulation of heavy metals in vegetables and food crops and assesses soil heavy metal thresholds for potential dietary toxicity.

Processing of fish lg heavy chain transcripts diverse splicing patterns and unusual nonsense mediated decay

USDA-ARS?s Scientific Manuscript database

Alternate pathways of RNA processing play an important role in the expression of the secreted (S) and membrane (Mb) forms of immunoglobulin (Ig) heavy (H) chain isotypes in all vertebrates. Interestingly, while the differential splicing mechanism and the splice sites that generate the two forms of I...

A single amino acid substitution in the variable region of the light chain specifically blocks immunoglobulin secretion.

PubMed Central

Dul, J L; Argon, Y

1990-01-01

Although immunoglobulin light chains are usually secreted in association with heavy chains, free light chains can be secreted by lymphocytes. To identify the structural features of light chains that are essential for their secretion, we mutated a conserved sequence in the variable domain of a lambda I light chain. The effects of the mutations on secretion were assayed by transient expression in COS-1 cells. One mutant (AV60), which replaced Ala-60 with Val, was secreted as efficiently as wild-type lambda I by transfected COS-1 cells. This result was not surprising because secreted lambda II chains contain valine in this position. However, a second lambda I mutant (AV60FS62), which replaced Phe-62 with Ser as well as Ala-60 with Val, was not secreted. This mutant was arrested in the endoplasmic reticulum, as judged by immunofluorescence and by its association with a lumenal endoplasmic reticulum protein, immunoglobulin heavy chain binding protein (BiP). The defect in secretion was not due to gross misfolding of the lambda I chain, since cells cotransfected with AV60FS62 and an immunoglobulin heavy chain gene produced functional antigen-binding antibodies. These assembled IgM molecules were still not secreted. Hence, the replacement of Phe-62 with Ser specifically affects a determinant on the lambda I light chain that is necessary for the intracellular transport of this molecule. Images PMID:2122454

Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo.

PubMed

Chen, Chih-Ying; Brodsky, Frances M

2005-02-18

Clathrin heavy and light chains form triskelia, which assemble into polyhedral coats of membrane vesicles that mediate transport for endocytosis and organelle biogenesis. Light chain subunits regulate clathrin assembly in vitro by suppressing spontaneous self-assembly of the heavy chains. The residues that play this regulatory role are at the N terminus of a conserved 22-amino acid sequence that is shared by all vertebrate light chains. Here we show that these regulatory residues and others in the conserved sequence mediate light chain interaction with Hip1 and Hip1R. These related proteins were previously found to be enriched in clathrin-coated vesicles and to promote clathrin assembly in vitro. We demonstrate Hip1R binding preference for light chains associated with clathrin heavy chain and show that Hip1R stimulation of clathrin assembly in vitro is blocked by mutations in the conserved sequence of light chains that abolish interaction with Hip1 and Hip1R. In vivo overexpression of a fragment of clathrin light chain comprising the Hip1R-binding region affected cellular actin distribution. Together these results suggest that the roles of Hip1 and Hip1R in affecting clathrin assembly and actin distribution are mediated by their interaction with the conserved sequence of clathrin light chains.

The production and characterization of novel heavy-chain antibodies against the tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdolamir; Sarrami, Ramin; Nasiry, Habib; Sadeghizadeh, Majid

2005-01-01

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH1). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. Ka values of specific polyclonal antipeptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 x 10(10) M(-1) and 1.4 x 10(10) M(-1) respectively.

Studies of thermostability in Camelus bactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia.

PubMed

Omidfar, Kobra; Rasaee, Mohhamad Javad; Kashanian, Soheila; Paknejad, Malieheh; Bathaie, Zahra

2007-01-01

Camelids have a unique immune system capable of producing heavy-chain antibodies lacking the light chains and CH1 (constant heavy-chain domain 1). It has been shown that, in contrast with conventional antibody fragments, the variable domains of these heavy-chain antibodies are functional at or after exposure to high temperatures. In the present study, the VHH (variable domain of heavy-chain antibody) camel antibody was subcloned into vector Ppiczc and expressed in Pichia pastoris. ORB1-83 VHH antibody recognizes the external domain of the mutant EGFR [EGF (epidermal growth factor) receptor], EGFR VIII. This tumour-specific antigen is ligand-independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. We report here that, although expression from P. pastoris resulted in a significantly increased level of expression of the anti-EGFR VIII VHH antibodies compared with Escherichia coli [Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani, Bakhtiari, Paknejad and Kashanian (2004) Tumor Biol. 25, 179-187; Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani and Golmakany (2004) Tumor Biol. 25, 296-305], this antibody selectively bound to the EGFR VIII peptide and reacted specifically with the immunoaffinity-purified antigen from non-small-cell lung cancer. Furthermore, thermal denaturation stability and CD spectra analysis of the Camelus bactrianus (Bactrian camel) VHH and heavy-chain antibodies at different temperature proved reversibility and binding activity after heat denaturation. Our results indicate that the P. pastoris expression system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allowing it to regain binding activity after heat denaturation.

Sequences of heavy and light chain variable regions from four bovine immunoglobulins.

PubMed

Armour, K L; Tempest, P R; Fawcett, P H; Fernie, M L; King, S I; White, P; Taylor, G; Harris, W J

1994-12-01

Oligodeoxyribonucleotide primers based on the 5' ends of bovine IgG1/2 and lambda constant (C) region genes, together with primers encoding conserved amino acids at the N-terminus of mature variable (V) regions from other species, have been used in cDNA and polymerase chain reactions (PCRs) to amplify heavy and light chain V region cDNA from bovine heterohybridomas. The amino acid sequences of VH and V lambda from four bovine immunoglobulins of different specificities are presented.

Future flavour physics experiments

PubMed Central

2015-01-01

The current status of flavour physics and the prospects for present and future experiments will be reviewed. Measurements in B‐physics, in which sensitive probes of new physics are the CKM angle γ, the Bs mixing phase ϕs, and the branching ratios of the rare decays B(s)0→μ+μ− , will be highlighted. Topics in charm and kaon physics, in which the measurements of ACP and the branching ratios of the rare decays K→πνν¯ are key measurements, will be discussed. Finally the complementarity of the future heavy flavour experiments, the LHCb upgrade and Belle‐II, will be summarised. PMID:26877543

Bioethical pluralism and complementarity.

PubMed

Grinnell, Frederick; Bishop, Jeffrey P; McCullough, Laurence B

2002-01-01

This essay presents complementarity as a novel feature of bioethical pluralism. First introduced by Neils Bohr in conjunction with quantum physics, complementarity in bioethics occurs when different perspectives account for equally important features of a situation but are mutually exclusive. Unlike conventional approaches to bioethical pluralism, which attempt in one fashion or another to isolate and choose between different perspectives, complementarity accepts all perspectives. As a result, complementarity results in a state of holistic, dynamic tension, rather than one that yields singular or final moral judgments.

The C-Terminal Amino Acid of the MHC-I Heavy Chain Is Critical for Binding to Derlin-1 in Human Cytomegalovirus US11-Induced MHC-I Degradation

PubMed Central

Cho, Sunglim; Kim, Bo Young; Ahn, Kwangseog; Jun, Youngsoo

2013-01-01

Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation. PMID:23951315

The C-terminal amino acid of the MHC-I heavy chain is critical for binding to Derlin-1 in human cytomegalovirus US11-induced MHC-I degradation.

PubMed

Cho, Sunglim; Kim, Bo Young; Ahn, Kwangseog; Jun, Youngsoo

2013-01-01

Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation.

[Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian gerbil (Meriones unguiculatus) after 12-day spaceflight].

PubMed

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.

Pollution status of Pakistan: a retrospective review on heavy metal contamination of water, soil, and vegetables.

PubMed

Waseem, Amir; Arshad, Jahanzaib; Iqbal, Farhat; Sajjad, Ashif; Mehmood, Zahid; Murtaza, Ghulam

2014-01-01

Trace heavy metals, such as arsenic, cadmium, lead, chromium, nickel, and mercury, are important environmental pollutants, particularly in areas with high anthropogenic pressure. In addition to these metals, copper, manganese, iron, and zinc are also important trace micronutrients. The presence of trace heavy metals in the atmosphere, soil, and water can cause serious problems to all organisms, and the ubiquitous bioavailability of these heavy metal can result in bioaccumulation in the food chain which especially can be highly dangerous to human health. This study reviews the heavy metal contamination in several areas of Pakistan over the past few years, particularly to assess the heavy metal contamination in water (ground water, surface water, and waste water), soil, sediments, particulate matter, and vegetables. The listed contaminations affect the drinking water quality, ecological environment, and food chain. Moreover, the toxicity induced by contaminated water, soil, and vegetables poses serious threat to human health.

High-throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination.

PubMed

Tan, Yann-Chong; Blum, Lisa K; Kongpachith, Sarah; Ju, Chia-Hsin; Cai, Xiaoyong; Lindstrom, Tamsin M; Sokolove, Jeremy; Robinson, William H

2014-03-01

We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination. Published by Elsevier Inc.

Electrostatic complementarity between proteins and ligands. 1. Charge disposition, dielectric and interface effects.

PubMed

Chau, P L; Dean, P M

1994-10-01

Electrostatic interactions have always been considered an important factor governing ligand-receptor interactions. Previous work in this field has established the existence of electrostatic complementarity between the ligand and its receptor site. However, this property has not been treated rigorously, and the description remains largely qualitative. In this work, 34 data sets of high quality were chosen from the Brookhaven Protein Databank. The electrostatic complementary has been calculated between the surface potentials; complementarity is absent between adjacent or neighbouring atoms of the ligand and the receptor. There is little difference between complementarities on the total ligand surface and the interfacial region. Altering the homogeneous dielectric to distance-dependent dielectrics reduces the complementarity slightly, but does not affect the pattern of complementarity.

Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction

PubMed Central

Hinz, Andreas; Lutje Hulsik, David; Forsman, Anna; Koh, Willie Wee-Lee; Belrhali, Hassan; Gorlani, Andrea; de Haard, Hans; Weiss, Robin A.; Verrips, Theo; Weissenhorn, Winfried

2010-01-01

HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment VHH D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 VHH at 1.5 Å resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site. PMID:20463957

Structural insights into humanization of anti-tissue factor antibody 10H10.

PubMed

Teplyakov, Alexey; Obmolova, Galina; Malia, Thomas J; Raghunathan, Gopalan; Martinez, Christian; Fransson, Johan; Edwards, Wilson; Connor, Judith; Husovsky, Matthew; Beck, Heena; Chi, Ellen; Fenton, Sandra; Zhou, Hong; Almagro, Juan Carlos; Gilliland, Gary L

Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.

Minimal Residual Disease Detection and Evolved IGH Clones Analysis in Acute B Lymphoblastic Leukemia Using IGH Deep Sequencing.

PubMed

Wu, Jinghua; Jia, Shan; Wang, Changxi; Zhang, Wei; Liu, Sixi; Zeng, Xiaojing; Mai, Huirong; Yuan, Xiuli; Du, Yuanping; Wang, Xiaodong; Hong, Xueyu; Li, Xuemei; Wen, Feiqiu; Xu, Xun; Pan, Jianhua; Li, Changgang; Liu, Xiao

2016-01-01

Acute B lymphoblastic leukemia (B-ALL) is one of the most common types of childhood cancer worldwide and chemotherapy is the main treatment approach. Despite good response rates to chemotherapy regiments, many patients eventually relapse and minimal residual disease (MRD) is the leading risk factor for relapse. The evolution of leukemic clones during disease development and treatment may have clinical significance. In this study, we performed immunoglobulin heavy chain ( IGH ) repertoire high throughput sequencing (HTS) on the diagnostic and post-treatment samples of 51 pediatric B-ALL patients. We identified leukemic IGH clones in 92.2% of the diagnostic samples and nearly half of the patients were polyclonal. About one-third of the leukemic clones have correct open reading frame in the complementarity determining region 3 (CDR3) of IGH , which demonstrates that the leukemic B cells were in the early developmental stage. We also demonstrated the higher sensitivity of HTS in MRD detection and investigated the clinical value of using peripheral blood in MRD detection and monitoring the clonal IGH evolution. In addition, we found leukemic clones were extensively undergoing continuous clonal IGH evolution by variable gene replacement. Dynamic frequency change and newly emerged evolved IGH clones were identified upon the pressure of chemotherapy. In summary, we confirmed the high sensitivity and universal applicability of HTS in MRD detection. We also reported the ubiquitous evolved IGH clones in B-ALL samples and their response to chemotherapy during treatment.

Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis.

PubMed

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.

Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

PubMed Central

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715

Anti-tumor activities of peptides corresponding to conserved complementary determining regions from different immunoglobulins.

PubMed

Figueiredo, Carlos R; Matsuo, Alisson L; Massaoka, Mariana H; Polonelli, Luciano; Travassos, Luiz R

2014-09-01

Short synthetic peptides corresponding to sequences of complementarity-determining regions (CDRs) from different immunoglobulin families have been shown to induce antimicrobial, antiviral and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). Presently, we studied the in vitro and in vivo antitumor activity of synthetic peptides derived from conserved CDR sequences of different immunoglobulins against human tumor cell lines and murine B16F10-Nex2 melanoma aiming at the discovery of candidate molecules for cancer therapy. Four light- and heavy-chain CDR peptide sequences from different antibodies (C36-L1, HA9-H2, 1-H2 and Mg16-H2) showed cytotoxic activity against murine melanoma and a panel of human tumor cell lineages in vitro. Importantly, they also exerted anti-metastatic activity using a syngeneic melanoma model in mice. Other peptides (D07-H3, MN20v1, MS2-H3) were also protective against metastatic melanoma, without showing significant cytotoxicity against tumor cells in vitro. In this case, we suggest that these peptides may act as immune adjuvants in vivo. As observed, peptides induced nitric oxide production in bone-marrow macrophages showing that innate immune cells can also be modulated by these CDR peptides. The present screening supports the search in immunoglobulins of rather frequent CDR sequences that are endowed with specific antitumor properties and may be candidates to be developed as anti-cancer drugs. Copyright © 2014 Elsevier Inc. All rights reserved.

Biochemical and Structural Characterization of Lysophosphatidic Acid Binding by a Humanized Monoclonal Antibody

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

J Fleming; J Wojciak; M Campbell

Lysophosphatidic acid (LPA) is a common product of glycerophospholipid metabolism and an important mediator of signal transduction. Aberrantly high LPA concentrations accompany multiple disease states. One potential approach for treatment of these diseases, therefore, is the therapeutic application of antibodies that recognize and bind LPA as their antigen. We have determined the X-ray crystal structure of an anti-LPA antibody (LT3015) Fab fragment in its antigen-free form to 2.15 {angstrom} resolution and in complex with two LPA isotypes (14:0 and 18:2) to resolutions of 1.98 and 2.51 {angstrom}, respectively. The variable CDR (complementarity-determining region) loops at the antigen binding site adoptmore » nearly identical conformations in the free and antigen-bound crystal structures. The crystallographic models reveal that the LT3015 antibody employs both heavy- and light-chain CDR loops to create a network of eight hydrogen bonds with the glycerophosphate head group of its LPA antigen. The head group is almost completely excluded from contact with solvent, while the hydrocarbon tail is partially solvent-exposed. In general, mutation of amino acid residues at the antigen binding site disrupts LPA binding. However, the introduction of particular mutations chosen strategically on the basis of the structures can positively influence LPA binding affinity. Finally, these structures elucidate the exquisite specificity demonstrated by an anti-lipid antibody for binding a structurally simple and seemingly unconstrained target molecule.« less

Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

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

Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie

2009-11-23

We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragmentmore » revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.« less

Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

Folding and trimerization of clathrin subunits at the triskelion hub.

PubMed

Näthke, I S; Heuser, J; Lupas, A; Stock, J; Turck, C W; Brodsky, F M

1992-03-06

The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments.

Interaction between glycosaminoglycans and immunoglobulin light chains.

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

Jiang, X.; Myatt, E.; Lykos, P.

1997-01-01

Amyloidosis is a pathological process in which normally soluble proteins polymerize to form insoluble fibrils (amyloid). Amyloid formation is found in a number of diseases, including Alzheimer's disease, adult-onset diabetes, and light-chain-associated amyloidosis. No pharmaceutical methods currently exist to prevent this process or to remove the fibrils from tissue. The search for treatment and prevention methods is hampered by a limited understanding of the biophysical basis of amyloid formation. Glycosaminoglycans (GAGs) are long, unbranched heteropolysaccharides composed of repeating disaccharide subunits and are known to associate with amyloid fibrils. The interaction of amyloid-associated free light chains with GAGs was tested bymore » both size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. The results indicated that heparin 16 000 and chondroitin sulfate B and C precipitated both human intact light chains and recombinant light chain variable domains. Although all light chains interacted with heparin, the strongest interactions were obtained with proteins that had formed amyloid. Molecular modeling indicated the possibility of interaction between heparin and the conserved saddle like surface of the light chain dimer opposite the complementarity-determining segments that form part of the antigen-binding site of a functional antibody. This suggestion might offer a new path to block the aggregation of amyloid-associated light chain proteins, by design of antagonists based on properties of GAG binding. A hexasaccharide was modeled as the basis for a possible antagonist.« less

The D0 immunoglobulin-like domain plays a central role for the stronger binding of KIR3DL2 to B27 free heavy chain dimers

PubMed Central

Hatano, Hiroko; Shaw, Jacqueline; Marquardt, Kaitlin; Zhang, Zhiyong; Gauthier, Laurent; Chanteux, Stephanie; Rossi, Benjamin; Li, Demin; Mitchell, Julie; Kollnberger, Simon

2015-01-01

We have proposed that the killer cell immunoglobulin-like receptor KIR3DL2 binding more strongly to HLA-B27 (B27) β2m-free heavy chain (FHC) dimers regulates lymphocyte function in arthritis and infection. We compared the function of B27 FHC dimers with other class I heavy chains and identified contact residues in KIR3DL2. B27 FHC dimers interacted functionally with KIR3DL2 on NK and reporter cells more strongly than other class I FHC. Mutagenesis identified key residues in the D0 and other immunoglobulin-like domains which were shared and distinct from KIR3DL1, for KIR3DL2 binding to B27 and other class I FHC. We modeled B27 dimer binding to KIR3DL2 and compared experimental mutagenesis data with computational “hot spot” predictions. Modelling predicts the stronger binding of B27 dimers to KIR3DL2 is mediated by non-symmetrical complementary contacts of the D0 and D1 domains with the α1, α2 and α3 domains of both B27 heavy chains. By contrast, the D2 domain primarily contacts residues in the α2 domain of one B27 heavy chain. These findings both provide novel insights about the molecular basis of KIR3DL2 binding to HLA-B27 and other ligands and suggest an important role for KIR3DL2 HLA-B27 interactions in controlling the function of NK cells in HLA-B27+ individuals. PMID:25582852

Structural Characterization and Determinants of Specificity of Single-Chain Antibody Inhibitors of Membrane-Type Serine Protease 1

DTIC Science & Technology

2008-03-01

Colman, P. M. (1993). Shape complementarity at protein / protein interfaces . J Mol Biol 234, 946-50. 26. Huang, M., Syed, R., Stura, E. A., Stone, M. J...Å2 of surface area (Table 1). In the apo MT-SP1 structure20, Asp96 forms the bottom of the S4 pocket, allowing a positively charged substrate P4...of surface area that E2 buries on MT-SP1 is larger than the typical antibody/ protein antigen interaction, which averages about 875 Å2 26; 27. This

Sera of IgA nephropathy patients contain a heterogeneous population of relatively cationic alpha-heavy chains.

PubMed

Shuib, A S; Chua, C T; Hashim, O H

1998-01-01

Sera of IgA nephropathy (IgAN) patients and normal subjects were analysed by two-dimensional (2-D) gel electrophoresis. Densitometric analysis of the 2-D gels of IgAN patients and normal subjects revealed that their protein maps were comparable. There was no shift of pI values in the major alpha-heavy chain spots. However, the volume of the alpha-heavy chain bands were differently distributed. Distribution was significantly lower at the anionic region in IgAN patients (mean anionic:cationic ratio of 1.184 +/- 0.311) as compared to normal healthy controls (mean anionic:cationic ratio of 2.139 +/- 0.538). Our data are in support of the previously reported findings that IgA1 of IgAN patients were lacking in sialic acid residues.

Birth-Order Complementarity and Marital Adjustment.

ERIC Educational Resources Information Center

Vos, Cornelia J. Vanderkooy; Hayden, Delbert J.

1985-01-01

Tested the influence of birth-order complementarity on marital adjustment among 327 married women using the Spanier Dyadic Adjustment Scale (1976). Birth-order complementarity was found to be unassociated with marital adjustment. (Author/BL)

Ligand Binding Site Detection by Local Structure Alignment and Its Performance Complementarity

PubMed Central

Lee, Hui Sun; Im, Wonpil

2013-01-01

Accurate determination of potential ligand binding sites (BS) is a key step for protein function characterization and structure-based drug design. Despite promising results of template-based BS prediction methods using global structure alignment (GSA), there is a room to improve the performance by properly incorporating local structure alignment (LSA) because BS are local structures and often similar for proteins with dissimilar global folds. We present a template-based ligand BS prediction method using G-LoSA, our LSA tool. A large benchmark set validation shows that G-LoSA predicts drug-like ligands’ positions in single-chain protein targets more precisely than TM-align, a GSA-based method, while the overall success rate of TM-align is better. G-LoSA is particularly efficient for accurate detection of local structures conserved across proteins with diverse global topologies. Recognizing the performance complementarity of G-LoSA to TM-align and a non-template geometry-based method, fpocket, a robust consensus scoring method, CMCS-BSP (Complementary Methods and Consensus Scoring for ligand Binding Site Prediction), is developed and shows improvement on prediction accuracy. The G-LoSA source code is freely available at http://im.bioinformatics.ku.edu/GLoSA. PMID:23957286

Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment.

PubMed

Ladenson, Ruth C; Crimmins, Dan L; Landt, Yvonne; Ladenson, Jack H

2006-07-01

We have isolated and characterized a caffeine-specific, heavy-chain-only antibody fragment (V(HH)) from llama that is capable of being utilized to analyze caffeine in hot and cold beverages. Camelid species (llama and camel) were selected for immunization because of their potential to make heat-stable, heavy-chain-only antibodies. Llamas and camels were immunized with caffeine covalently linked to keyhole limpet hemocyanin, and recombinant antibody techniques were used to create phage displayed libraries of variable region fragments of the heavy-chain antibodies. Caffeine-specific V(HH) fragments were selected by their ability to bind to caffeine/bovine serum albumin (BSA) and confirmed by a positive reaction in a caffeine enzyme-linked immunosorbent assay (caffeine ELISA). One of these V(HH) fragments (VSA2) was expressed as a soluble protein and shown to recover its reactivity after exposure to temperatures up to 90 degrees C. In addition, VSA2 was able to bind caffeine at 70 degrees C. A competition caffeine ELISA was developed for the measurement of caffeine in beverages, and concentrations of caffeine obtained for coffee, Coca-Cola Classic, and Diet Coke agreed well with high performance liquid chromatography (HPLC) determination and literature values. VSA2 showed minimal cross reactivity with structurally related methylxanthines.

Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Monoclonal antibodies against loggerhead sea turtle, Caretta caretta, IgY isoforms reveal differential contributions to antibody titers and relatedness among other sea turtles.

PubMed

Rodgers, Maria L; Rice, Charles D

2018-05-19

Serum from loggerhead sea turtles, Caretta caretta, was collected from the southeast Atlantic Ocean during routine summer monitoring studies in 2017. Serum immunoglobulin IgY was purified and used to develop IgY isoform-specific monoclonal antibodies (mAb). mAb LH12 was developed against the 66 kDa heavy chain of IgY, mAb LH1 was developed against the truncated heavy chain of approximately 37 kDA, and mAb LH9 was developed against the 23 kDa light chains. mAb LH9 reacts with the light chains of all sea turtles, mAb LH12 reacts with the long heavy chain of all sea turtles within the family Cheloniidae, and mAb LH1 reacts with the truncated form of IgY in both olive and Kemp's ridley turtles. Circulating IgY antibodies against three different marine bacterial pathogens were determined in 16 loggerhead samples using these mAbs. mAb LH12 detects higher titers than mAb LH1, and mAb LH9 detects the highest titers. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pollution Status of Pakistan: A Retrospective Review on Heavy Metal Contamination of Water, Soil, and Vegetables

PubMed Central

Arshad, Jahanzaib; Iqbal, Farhat; Sajjad, Ashif; Mehmood, Zahid

2014-01-01

Trace heavy metals, such as arsenic, cadmium, lead, chromium, nickel, and mercury, are important environmental pollutants, particularly in areas with high anthropogenic pressure. In addition to these metals, copper, manganese, iron, and zinc are also important trace micronutrients. The presence of trace heavy metals in the atmosphere, soil, and water can cause serious problems to all organisms, and the ubiquitous bioavailability of these heavy metal can result in bioaccumulation in the food chain which especially can be highly dangerous to human health. This study reviews the heavy metal contamination in several areas of Pakistan over the past few years, particularly to assess the heavy metal contamination in water (ground water, surface water, and waste water), soil, sediments, particulate matter, and vegetables. The listed contaminations affect the drinking water quality, ecological environment, and food chain. Moreover, the toxicity induced by contaminated water, soil, and vegetables poses serious threat to human health. PMID:25276818

Evaluation of MMX1902 as an Oral Treatment for Duchenne Muscular Dystrophy

DTIC Science & Technology

2016-10-01

type control mice. Further, embryonic myosin heavy chain (eMHC) staining of the diaphragm showed a significant increase in eMHC positive muscle...resulting in cardiac functional measures comparable to exercised wild-type control mice (Table 1). Further, embryonic myosin heavy chain (eMHC) staining of...and cost-driver at scale-up, and the synthesis of 10.2 grams of MMX1902 at >98% purity. What opportunities for training and professional

A Motor-Driven Mechanism for Cell-Length Sensing

PubMed Central

Rishal, Ida; Kam, Naaman; Perry, Rotem Ben-Tov; Shinder, Vera; Fisher, Elizabeth M.C.; Schiavo, Giampietro; Fainzilber, Mike

2012-01-01

Summary Size homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing. Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development. Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1. Thus, molecular motors critically influence cell-length sensing and growth control. PMID:22773964

Simultaneous inhibition of multiple steps in the processing of N-linked oligosaccharides does not impair immunoglobulin secretion from rat hybridoma cells.

PubMed Central

Hashim, O H; Cushley, W

1988-01-01

The effects of inhibiting selected pairs of oligosaccharide-processing activities upon the secretion of IgM and IgG molecules have been investigated. In the presence of castanospermine (CSP) plus swainsonine (SW) or deoxynojirimycin (dNM) plus deoxymannojirimycin (dMM), secretion of IgM and IgG from rat hybridoma cells was unimpaired relative to control cultures. The structures of the N-linked oligosaccharides found on the Ig heavy chains isolated from treated cells or culture supernatants were shown to be qualitatively different from those associated with control Ig by persistent sensitivity to digestion by endo H. Furthermore, the electrophoretic mobilities of mu and gamma chains on SDS-PAGE derived from treated cells were consistently slower than those of control heavy chains. IgM and IgG were also efficiently secreted when all glucosidase and mannosidase activities were blocked, and the secreted heavy chains bore endo H-sensitive oligosaccharides. The data suggest that Ig secretion from hybridomas can proceed in the absence of N-linked oligosaccharide processing. Images Figure 1 Figure 2 Figure 3 PMID:3350578

Interpersonal complementarity in the mental health intake: a mixed-methods study.

PubMed

Rosen, Daniel C; Miller, Alisa B; Nakash, Ora; Halpern, Lucila; Halperin, Lucila; Alegría, Margarita

2012-04-01

The study examined which socio-demographic differences between clients and providers influenced interpersonal complementarity during an initial intake session; that is, behaviors that facilitate harmonious interactions between client and provider. Complementarity was assessed using blinded ratings of 114 videotaped intake sessions by trained observers. Hierarchical linear models were used to examine how match between client and provider in race/ethnicity, sex, and age were associated with levels of complementarity. A qualitative analysis investigated potential mechanisms that accounted for overall complementarity beyond match by examining client-provider dyads in the top and bottom quartiles of the complementarity measure. Results indicated significant interactions between client's race/ethnicity (Black) and provider's race/ethnicity (Latino) (p = .036) and client's age and provider's age (p = .044) on the Affiliation axis. The qualitative investigation revealed that client-provider interactions in the upper quartile of complementarity were characterized by consistent descriptions between the client and provider of concerns and expectations as well as depictions of what was important during the meeting. Results suggest that differences in social identities, although important, may be overcome by interpersonal variables early in the therapeutic relationship. Implications for both clinical practice and future research are discussed, as are factors relevant to working across cultures.

Complementarity and Area-Efficiency in the Prioritization of the Global Protected Area Network.

PubMed

Kullberg, Peter; Toivonen, Tuuli; Montesino Pouzols, Federico; Lehtomäki, Joona; Di Minin, Enrico; Moilanen, Atte

2015-01-01

Complementarity and cost-efficiency are widely used principles for protected area network design. Despite the wide use and robust theoretical underpinnings, their effects on the performance and patterns of priority areas are rarely studied in detail. Here we compare two approaches for identifying the management priority areas inside the global protected area network: 1) a scoring-based approach, used in recently published analysis and 2) a spatial prioritization method, which accounts for complementarity and area-efficiency. Using the same IUCN species distribution data the complementarity method found an equal-area set of priority areas with double the mean species ranges covered compared to the scoring-based approach. The complementarity set also had 72% more species with full ranges covered, and lacked any coverage only for half of the species compared to the scoring approach. Protected areas in our complementarity-based solution were on average smaller and geographically more scattered. The large difference between the two solutions highlights the need for critical thinking about the selected prioritization method. According to our analysis, accounting for complementarity and area-efficiency can lead to considerable improvements when setting management priorities for the global protected area network.

HLA-B27 Misfolding and Ankylosing Spondylitis

PubMed Central

Colbert, Robert A.; Tran, Tri M.; Layh-Schmitt, Gerlinde

2013-01-01

Understanding how HLA-B27 contributes to the pathogenesis of spondyloarthritis continues to be an important goal. Current efforts are aimed largely on three areas of investigation; peptide presentation to CD8 T cells, abnormal forms of the HLA-B27 heavy chain and their recognition by leukocyte immunoglobulin-like receptors on immune effector cells, and HLA-B27 heavy chain misfolding and intrinsic biological effects on affected cells. In this chapter we review our current understanding of the causes and consequences of HLA-B27 misfolding, which can be defined biochemically as a propensity to oligomerize and form complexes in the endoplasmic reticulum (ER) with the chaperone BiP (HSPA5/GRP78). HLA-B27 misfolding is linked to an unusual combination of polymorphisms that identify this allele, and cause the heavy chain to fold and load peptides inefficiently. Misfolding can result in ER-associated degradation (ERAD) of heavy chains, which is mediated in part by the E3 ubiquitin ligase HRD1 (SYVN1), and the ubiquitin conjugating enzyme UBE2JL. Upregulation of HLA-B27 and accumulation of misfolded heavy chains can activate ER stress signaling pathways that orchestrate the unfolded protein response. In transgenic rats where HLA-B27 is overexpressed, UPR activation is prominent. However, it is specific for heavy chain misfolding, since overexpression of HLA-B7, an allele that does not misfold, fails to generate ER stress. UPR activation has been linked to cytokine dysregulation, promoting lL-23, IFNβ, and lL-1α production, and may activate the IL-23/IL-17 axis in these rats. IL-1α and IFNβ are pro- and anti-osteoclastogenic cytokines, respectively, that modulate osteoclast development in HLA-B27-expressing transgenic rat monocytes. Translational studies of patient derived cells expressing HLA-B27 at physiologic levels have provided evidence that ER stress and UPR activation can occur in peripheral blood, but this has not been reported to date in isolated macrophages. Inflamed gastrointestinal tissue reveals evidence for HLA-B27 misfolding, ERAD, and autophagy, without acute UPR activation. A more complete picture of conditions that impact HLA-B27 folding and misfolding, the full spectrum and time course of consequences of ER stress, and critical cell types involved is needed to understand the role of HLA-B27 misfolding in spondyloarthritis pathogenesis. PMID:23993278

HLA-B27 misfolding and ankylosing spondylitis.

PubMed

Colbert, Robert A; Tran, Tri M; Layh-Schmitt, Gerlinde

2014-01-01

Understanding how HLA-B27 contributes to the pathogenesis of spondyloarthritis continues to be an important goal. Current efforts are aimed largely on three areas of investigation; peptide presentation to CD8T cells, abnormal forms of the HLA-B27 heavy chain and their recognition by leukocyte immunoglobulin-like receptors on immune effector cells, and HLA-B27 heavy chain misfolding and intrinsic biological effects on affected cells. In this chapter we review our current understanding of the causes and consequences of HLA-B27 misfolding, which can be defined biochemically as a propensity to oligomerize and form complexes in the endoplasmic reticulum (ER) with the chaperone BiP (HSPA5/GRP78). HLA-B27 misfolding is linked to an unusual combination of polymorphisms that identify this allele, and cause the heavy chain to fold and load peptides inefficiently. Misfolding can result in ER-associated degradation (ERAD) of heavy chains, which is mediated in part by the E3 ubiquitin ligase HRD1 (SYVN1), and the ubiquitin conjugating enzyme UBE2JL. Upregulation of HLA-B27 and accumulation of misfolded heavy chains can activate ER stress signaling pathways that orchestrate the unfolded protein response. In transgenic rats where HLA-B27 is overexpressed, UPR activation is prominent. However, it is specific for heavy chain misfolding, since overexpression of HLA-B7, an allele that does not misfold, fails to generate ER stress. UPR activation has been linked to cytokine dysregulation, promoting lL-23, IFNβ, and lL-1α production, and may activate the IL-23/IL-17 axis in these rats. IL-1α and IFNβ are pro- and anti-osteoclastogenic cytokines, respectively, that modulate osteoclast development in HLA-B27-expressing transgenic rat monocytes. Translational studies of patient derived cells expressing HLA-B27 at physiologic levels have provided evidence that ER stress and UPR activation can occur in peripheral blood, but this has not been reported to date in isolated macrophages. Inflamed gastrointestinal tissue reveals evidence for HLA-B27 misfolding, ERAD, and autophagy, without acute UPR activation. A more complete picture of conditions that impact HLA-B27 folding and misfolding, the full spectrum and time course of consequences of ER stress, and critical cell types involved is needed to understand the role of HLA-B27 misfolding in spondyloarthritis pathogenesis. Published by Elsevier Ltd.

Atoms-in-molecules study of the genetically encoded amino acids. III. Bond and atomic properties and their correlations with experiment including mutation-induced changes in protein stability and genetic coding.

PubMed

Matta, Chérif F; Bader, Richard F W

2003-08-15

This article presents a study of the molecular charge distributions of the genetically encoded amino acids (AA), one that builds on the previous determination of their equilibrium geometries and the demonstrated transferability of their common geometrical parameters. The properties of the charge distributions are characterized and given quantitative expression in terms of the bond and atomic properties determined within the quantum theory of atoms-in-molecules (QTAIM) that defines atoms and bonds in terms of the observable charge density. The properties so defined are demonstrated to be remarkably transferable, a reflection of the underlying transferability of the charge distributions of the main chain and other groups common to the AA. The use of the atomic properties in obtaining an understanding of the biological functions of the AA, whether free or bound in a polypeptide, is demonstrated by the excellent statistical correlations they yield with experimental physicochemical properties. A property of the AA side chains of particular importance is the charge separation index (CSI), a quantity previously defined as the sum of the magnitudes of the atomic charges and which measures the degree of separation of positive and negative charges in the side chain of interest. The CSI values provide a correlation with the measured free energies of transfer of capped side chain analogues, from the vapor phase to aqueous solution, yielding a linear regression equation with r2 = 0.94. The atomic volume is defined by the van der Waals isodensity surface and it, together with the CSI, which accounts for the electrostriction of the solvent, yield a linear regression (r2 = 0.98) with the measured partial molar volumes of the AAs. The changes in free energies of transfer from octanol to water upon interchanging 153 pairs of AAs and from cyclohexane to water upon interchanging 190 pairs of AAs, were modeled using only three calculated parameters (representing electrostatic and volume contributions) yielding linear regressions with r2 values of 0.78 and 0.89, respectively. These results are a prelude to the single-site mutation-induced changes in the stabilities of two typical proteins: ubiquitin and staphylococcal nuclease. Strong quadratic correlations (r2 approximately 0.9) were obtained between DeltaCSI upon mutation and each of the two terms DeltaDeltaH and TDeltaDeltaS taken from recent and accurate differential scanning calorimetry experiments on ubiquitin. When the two terms are summed to yield DeltaDeltaG, the quadratic terms nearly cancel, and the result is a simple linear fit between DeltaDeltaG and DeltaCSI with r2 = 0.88. As another example, the change in the stability of staphylococcal nuclease upon mutation has been fitted linearly (r2 = 0.83) to the sum of a DeltaCSI term and a term representing the change in the van der Waals volume of the side chains upon mutation. The suggested correlation of the polarity of the side chain with the second letter of the AA triplet genetic codon is given concrete expression in a classification of the side chains in terms of their CSI values and their group dipole moments. For example, all amino acids with a pyrimidine base as their second letter in mRNA possess side-chain CSI < or = 2.8 (with the exception of Cys), whereas all those with CSI > 2.8 possess an purine base. The article concludes with two proposals for measuring and predicting molecular complementarity: van der Waals complementarity expressed in terms of the van der Waals isodensity surface and Lewis complementarity expressed in terms of the local charge concentrations and depletions defined by the topology of the Laplacian of the electron density. A display of the experimentally accessible Laplacian distribution for a folded protein would offer a clear picture of the operation of the "stereochemical code" proposed as the determinant in the folding process. Copyright 2003 Wiley-Liss, Inc.

Affiliation and control in marital interaction: interpersonal complementarity is present but is not associated with affect or relationship quality.

PubMed

Cundiff, Jenny M; Smith, Timothy W; Butner, Jonathan; Critchfield, Kenneth L; Nealey-Moore, Jill

2015-01-01

The principle of complementarity in interpersonal theory states that an actor's behavior tends to "pull, elicit, invite, or evoke" responses from interaction partners who are similar in affiliation (i.e., warmth vs. hostility) and opposite in control (i.e., dominance vs. submissiveness). Furthermore, complementary interactions are proposed to evoke less negative affect and promote greater relationship satisfaction. These predictions were examined in two studies of married couples. Results suggest that complementarity in affiliation describes a robust general pattern of marital interaction, but complementarity in control varies across contexts. Consistent with behavioral models of marital interaction, greater levels of affiliation and lower control by partners-not complementarity in affiliation or control-were associated with less anger and anxiety and greater relationship quality. Partners' levels of affiliation and control combined in ways other than complementarity-mostly additively, but sometimes synergistically-to predict negative affect and relationship satisfaction. © 2014 by the Society for Personality and Social Psychology, Inc.

Light-induced Conversion of Trp to Gly and Gly Hydroperoxide in IgG1

PubMed Central

Haywood, Jessica; Mozziconacci, Olivier; Allegre, Kevin M.; Kerwin, Bruce A.; Schöneich, Christian

2013-01-01

The exposure of IgG1 in aqueous solution to light with λ = 254 nm or λ > 295 nm yields products consistent with Trp radical cation formation followed by αC-βC cleavage of the Trp side chain. The resulting glycyl radicals are either reduced to Gly, or add oxygen prior to reduction to Gly hydroperoxide. Photoirradiation at λ = 254 nm targets Trp at positions 191 (light chain), 309 and 377 (heavy chain) while photoirradiation at λ > 295 nm targets Trp at position 309 (heavy chain). Mechanistically, the formation of Trp radical cations likely proceeds via photo-induced electron- or hydrogen-transfer to disulfide bonds, yielding thiyl radicals and thiols, where thiols may serve as reductants for the intermediary glycyl or glycylperoxyl radicals. PMID:23363477

Biofuel supply chain, market, and policy analysis

NASA Astrophysics Data System (ADS)

Zhang, Leilei

Renewable fuel is receiving an increasing attention as a substitute for fossil based energy. The US Department of Energy (DOE) has employed increasing effort on promoting the advanced biofuel productions. Although the advanced biofuel remains at its early stage, it is expected to play an important role in climate policy in the future in the transportation sector. This dissertation studies the emerging biofuel supply chain and markets by analyzing the production cost, and the outcomes of the biofuel market, including blended fuel market price and quantity, biofuel contract price and quantity, profitability of each stakeholder (farmers, biofuel producers, biofuel blenders) in the market. I also address government policy impacts on the emerging biofuel market. The dissertation is composed with three parts, each in a paper format. The first part studies the supply chain of emerging biofuel industry. Two optimization-based models are built to determine the number of facilities to deploy, facility locations, facility capacities, and operational planning within facilities. Cost analyses have been conducted under a variety of biofuel demand scenarios. It is my intention that this model will shed light on biofuel supply chain design considering operational planning under uncertain demand situations. The second part of the dissertation work focuses on analyzing the interaction between the key stakeholders along the supply chain. A bottom-up equilibrium model is built for the emerging biofuel market to study the competition in the advanced biofuel market, explicitly formulating the interactions between farmers, biofuel producers, blenders, and consumers. The model simulates the profit maximization of multiple market entities by incorporating their competitive decisions in farmers' land allocation, biomass transportation, biofuel production, and biofuel blending. As such, the equilibrium model is capable of and appropriate for policy analysis, especially for those policies that have complex ramifications and result in sophisticate interactions among multiple stakeholders. The third part of the dissertation investigates the impacts of flexible fuel vehicles (FFVs) market penetration levels on the market outcomes, including cellulosic biofuel production and price, blended fuel market price, and profitability of each stakeholder in the biofuel supply chain for imperfectly competitive biofuel markets. In this paper, I investigate the penetration levels of FFVs by incorporating the substitution among different fuels in blended fuel demand functions through "cross price elasticity" in a bottom-up equilibrium model framework. The complementarity based problem is solved by a Taylor expansion-based iterative procedure. At each step of the iteration, the highly nonlinear complementarity problems with constant elasticity of demand functions are linearized into linear complimentarity problems and solved until it converges. This model can be applied to investigate the interaction between the stakeholders in the biofuel market, and to assist decision making for both cellulosic biofuel investors and government.

Interpersonal Complementarity in the Mental Health Intake: A Mixed-Methods Study

ERIC Educational Resources Information Center

Rosen, Daniel C.; Miller, Alisa B.; Nakash, Ora; Halperin, Lucila; Alegria, Margarita

2012-01-01

The study examined which socio-demographic differences between clients and providers influenced interpersonal complementarity during an initial intake session; that is, behaviors that facilitate harmonious interactions between client and provider. Complementarity was assessed using blinded ratings of 114 videotaped intake sessions by trained…

In vivo monitoring of neuronal loss in traumatic brain injury: a microdialysis study

PubMed Central

Tisdall, Martin M.; Girbes, Armand R.; Martinian, Lillian; Thom, Maria; Kitchen, Neil; Smith, Martin

2011-01-01

Traumatic brain injury causes diffuse axonal injury and loss of cortical neurons. These features are well recognized histologically, but their in vivo monitoring remains challenging. In vivo cortical microdialysis samples the extracellular fluid adjacent to neurons and axons. Here, we describe a novel neuronal proteolytic pathway and demonstrate the exclusive neuro-axonal expression of Pavlov’s enterokinase. Enterokinase is membrane bound and cleaves the neurofilament heavy chain at positions 476 and 986. Using a 100 kDa microdialysis cut-off membrane the two proteolytic breakdown products, extracellular fluid neurofilament heavy chains NfH476−986 and NfH476−1026, can be quantified with a relative recovery of 20%. In a prospective clinical in vivo study, we included 10 patients with traumatic brain injury with a median Glasgow Coma Score of 9, providing 640 cortical extracellular fluid samples for longitudinal data analysis. Following high-velocity impact traumatic brain injury, microdialysate extracellular fluid neurofilament heavy chain levels were significantly higher (6.18 ± 2.94 ng/ml) and detectable for longer (>4 days) compared with traumatic brain injury secondary to falls (0.84 ± 1.77 ng/ml, <2 days). During the initial 16 h following traumatic brain injury, strong correlations were found between extracellular fluid neurofilament heavy chain levels and physiological parameters (systemic blood pressure, anaerobic cerebral metabolism, excessive brain tissue oxygenation, elevated brain temperature). Finally, extracellular fluid neurofilament heavy chain levels were of prognostic value, predicting mortality with an odds ratio of 7.68 (confidence interval 2.15–27.46, P = 0.001). In conclusion, this study describes the discovery of Pavlov’s enterokinase in the human brain, a novel neuronal proteolytic pathway that gives rise to specific protein biomarkers (NfH476−986 and NfH476−1026) applicable to in vivo monitoring of diffuse axonal injury and neuronal loss in traumatic brain injury. PMID:21278408

Discrimination of germline V genes at different sequencing lengths and mutational burdens: A new tool for identifying and evaluating the reliability of V gene assignment.

PubMed

Zhang, Bochao; Meng, Wenzhao; Prak, Eline T Luning; Hershberg, Uri

2015-12-01

Immune repertoires are collections of lymphocytes that express diverse antigen receptor gene rearrangements consisting of Variable (V), (Diversity (D) in the case of heavy chains) and Joining (J) gene segments. Clonally related cells typically share the same germline gene segments and have highly similar junctional sequences within their third complementarity determining regions. Identifying clonal relatedness of sequences is a key step in the analysis of immune repertoires. The V gene is the most important for clone identification because it has the longest sequence and the greatest number of sequence variants. However, accurate identification of a clone's germline V gene source is challenging because there is a high degree of similarity between different germline V genes. This difficulty is compounded in antibodies, which can undergo somatic hypermutation. Furthermore, high-throughput sequencing experiments often generate partial sequences and have significant error rates. To address these issues, we describe a novel method to estimate which germline V genes (or alleles) cannot be discriminated under different conditions (read lengths, sequencing errors or somatic hypermutation frequencies). Starting with any set of germline V genes, this method measures their similarity using different sequencing lengths and calculates their likelihood of unambiguous assignment under different levels of mutation. Hence, one can identify, under different experimental and biological conditions, the germline V genes (or alleles) that cannot be uniquely identified and bundle them together into groups of specific V genes with highly similar sequences. Copyright © 2015 Elsevier B.V. All rights reserved.

Towards an understanding of the correlations in jet substructure

DOE PAGES

Adams, D.; Arce, A.; Asquith, L.; ...

2015-09-09

Over the past decade, a large number of jet substructure observables have been proposed in the literature, and explored at the LHC experiments. Such observables attempt to utilize the internal structure of jets in order to distinguish those initiated by quarks, gluons, or by boosted heavy objects, such as top quarks and W bosons. This report, originating from and motivated by the BOOST2013 workshop, presents original particle-level studies that aim to improve our understanding of the relationships between jet substructure observables, their complementarity, and their dependence on the underlying jet properties, particularly the jet radius and jet transverse momentum. Thismore » is explored in the context of quark/gluon discrimination, boosted W boson tagging and boosted top quark tagging.« less

Application of principal component analysis in the pollution assessment with heavy metals of vegetable food chain in the old mining areas

PubMed Central

2012-01-01

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. PMID:23234365

Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections.

PubMed Central

Duan, L; Pomerantz, R J

1994-01-01

The pooled degenerate-primer polymerase chain reaction (PCR) technology is now widely used in the amplification and cloning of murine hybridoma-specific immunoglobulin gene cDNAs. The design of primers is mainly based on the highly conserved 5' terminus of immunoglobulin gene variable regions and the constant region in the 3' terminus. Of note, most murine hybridoma cell lines are derived from the Sp2/0 cell line, which is demonstrated to express endogenous aberrant kappa chains (abV kappa). This high-level endogenous abV kappa mixes with specific kappa chains in the hybridomas and interferes with the efficiency of the reverse transcriptase (RT)-PCR cloning strategy. In this report, during the cloning of murine anti-human immunodeficiency virus type I (HIV-1) hybridoma immunoglobulin cDNAs, a specific primer-PCR screening system was developed, based on the abV kappa complementarity-defining region (CDR), to eliminate abV kappa-carrying plasmids. Furthermore, an abV kappa sequence-specific derived ribozyme was developed and packaged in a retroviral expression vector system. This abV kappa ribozyme can be transduced into different murine hybridomas, and expressed intracellularly to potently eliminate endogenous abV kappa RNA. Images PMID:7816635

Electrostatic complementarity at protein/protein interfaces.

PubMed

McCoy, A J; Chandana Epa, V; Colman, P M

1997-05-02

Calculation of the electrostatic potential of protein-protein complexes has led to the general assertion that protein-protein interfaces display "charge complementarity" and "electrostatic complementarity". In this study, quantitative measures for these two terms are developed and used to investigate protein-protein interfaces in a rigorous manner. Charge complementarity (CC) was defined using the correlation of charges on nearest neighbour atoms at the interface. All 12 protein-protein interfaces studied had insignificantly small CC values. Therefore, the term charge complementarity is not appropriate for the description of protein-protein interfaces when used in the sense measured by CC. Electrostatic complementarity (EC) was defined using the correlation of surface electrostatic potential at protein-protein interfaces. All twelve protein-protein interfaces studied had significant EC values, and thus the assertion that protein-protein association involves surfaces with complementary electrostatic potential was substantially confirmed. The term electrostatic complementarity can therefore be used to describe protein-protein interfaces when used in the sense measured by EC. Taken together, the results for CC and EC demonstrate the relevance of the long-range effects of charges, as described by the electrostatic potential at the binding interface. The EC value did not partition the complexes by type such as antigen-antibody and proteinase-inhibitor, as measures of the geometrical complementarity at protein-protein interfaces have done. The EC value was also not directly related to the number of salt bridges in the interface, and neutralisation of these salt bridges showed that other charges also contributed significantly to electrostatic complementarity and electrostatic interactions between the proteins. Electrostatic complementarity as defined by EC was extended to investigate the electrostatic similarity at the surface of influenza virus neuraminidase where the epitopes of two monoclonal antibodies, NC10 and NC41, overlap. Although NC10 and NC41 both have quite high values of EC for their interaction with neuraminidase, the similarity in electrostatic potential generated by the two on the overlapping region of the epitopes is insignificant. Thus, it is possible for two antibodies to recognise the electrostatic surface of a protein in dissimilar ways.

Manifestation of the structure of heavy nuclei in their alpha decays

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

Adamian, G. G., E-mail: adamian@theor.jinr.ru; Antonenko, N. V.; Bezbakh, A. N.

2016-11-15

Low-lying one- and two-quasiparticle states of heavy nuclei are predicted. Alpha-decay chains, including those that proceed through isomeric states, are examined on the basis of the predicted properties of superheavy nuclei.

Serum Free Light Chains in Neoplastic Monoclonal Gammopathies: Relative Under-Detection of Lambda Dominant Kappa/Lambda Ratio, and Underproduction of Free Lambda Light Chains, as Compared to Kappa Light Chains, in Patients With Neoplastic Monoclonal Gammopathies.

PubMed

Lee, Won Sok; Singh, Gurmukh

2018-07-01

Quantitative evaluation of serum free light chains is recommended for the work up of monoclonal gammopathies. Immunoglobulin light chains are generally produced in excess of heavy chains. In patients with monoclonal gammopathy, κ/λ ratio is abnormal less frequently with lambda chain lesions. This study was undertaken to ascertain if the levels of overproduction of the two light chain types and their detection rates are different in patients with neoplastic monoclonal gammopathies. Results of serum protein electrophoresis (SPEP), serum protein immunofixation electrophoresis (SIFE), urine protein electrophoresis (UPEP), urine protein immunofixation electrophoresis (UIFE), and serum free light chain assay (SFLCA) in patients with monoclonal gammopathies were examined retrospectively. The κ/λ ratios were appropriately abnormal more often in kappa chain lesions. Ratios of κ/λ were normal in about 25% of patients with lambda chain lesions in whom free homogenous lambda light chains were detectable in urine. An illustrative case suggests underproduction of free lambda light chains, in some instances. The lower prevalence of lambda dominant κ/λ ratio in lesions with lambda light chains is estimated to be due to relative under-detection of lambda dominant κ/λ ratio in about 25% of the patients and because lambda chains are not produced in as much excess of heavy chains as are kappa chains, in about 5% of the patients. The results question the medical necessity and clinical usefulness of the serum free light chain assay. UPEP/UIFE is under-utilized.

Ig heavy chain class switch recombination: mechanism and regulation

PubMed Central

Stavnezer, Janet; Schrader, Carol E.

2014-01-01

Ig heavy chain class switching occurs rapidly after activation of mature naïve B cells, resulting in a switch from expressing IgM and IgD to expression of IgG, IgE, or IgA; this switch improves the ability of antibodies to remove the pathogen that induces the humoral immune response. Class switching occurs by a deletional recombination between two different switch (S) regions, each of which is associated with a heavy chain constant (CH) region gene. Class switch recombination (CSR) is instigated by activation-induced cytidine deaminase (AID), which converts cytosines in S regions to uracils. The uracils are subsequently removed by two DNA repair pathways, resulting in mutations, single-strand DNA breaks, and the double-strand breaks required for CSR. We discuss several aspects of CSR, including how CSR is induced, CSR in B-cell progenitors, the roles for transcription and chromosomal looping in CSR, and the roles of certain DNA repair enzymes in CSR. PMID:25411432

Recombinant dissection of myosin heavy chain of Toxocara canis shows strong clustering of antigenic regions.

PubMed

Obwaller, A; Duchêne, M; Bruhn, H; Steipe, B; Tripp, C; Kraft, D; Wiedermann, G; Auer, H; Aspöck, H

2001-05-01

Myosins from nematode parasites elicit strong humoral and cellular immune responses and have been investigated as vaccine candidates. In this study we cloned and sequenced a cDNA coding for myosin heavy chain from Toxocara canis, a nematode parasite of canids which may also infect humans and cause various unspecific symptoms. To determine the major antigenic regions the myosin heavy chain was systematically dissected into ten overlapping recombinant fusion polypeptides which were purified by metal chelate chromatography. Single fragments were then tested for their IgG reactivity in sera from toxocarosis patients and healthy probands. Two regions, one region at the mid to carboxy-terminal end of the head domain and one region in the rod domain, were identified as major antigens, which in combination were positive with 86% of the sera. The other domains were less reactive. This shows that the patients' IgG reactivity was not directed evenly against all parts of the molecule, but was rather clustered in few regions.

Use of bortezomib in heavy-chain deposition disease: a report of 3 cases.

PubMed

Patel, Kinjal; Dillon, John J; Leung, Nelson; Bomback, Andrew S; Appel, Gerald B; D'Agati, Vivette; Canetta, Pietro A

2014-07-01

Heavy-chain deposition disease (HCDD) is a rare complication of plasma cell dyscrasia in which monoclonal heavy chains deposit in glomerular and tubular basement membranes of the kidney. Clinical and pathologic features of HCDD have been well described in case reports and series, but evidence supporting specific therapies is sparse. Historically, the disease has had a poor prognosis, intensifying the need to clarify optimal treatments. We describe 3 cases of HCDD with biopsy-proven glomerular involvement, severe nephrotic syndrome, and decline in kidney function that were treated successfully with bortezomib, a proteasome inhibitor. None of these patients had multiple myeloma. In all cases, bortezomib-based therapy resulted in sustained resolution of nephrotic syndrome and improvement in kidney function. All 3 patients developed peripheral neuropathy; otherwise, treatment was well tolerated. To our knowledge, this is the first description of the clinical effectiveness of bortezomib against HCDD. Copyright © 2014 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Human heavy chain disease protein WIS: implications for the organization of immunoglobulin genes.

PubMed Central

Franklin, E C; Prelli, F; Frangione, B

1979-01-01

Protein WIS is a human gamma3 heavy (H) chain disease immunoglobulin variant whose amino acid sequence is most readily interpreted by postulating that three residues of the amino terminus are followed by a deletion of most of the variable (VH) domain, which ends at the variable-constant (VC) joining region. Then there is a stretch of eight residues, three of which are unusual, while the other five have striking homology to the VC junction sequence. This is followed by a second deletion, which ends at the beginning of the quadruplicated hinge region. These findings are consistent with mutations resulting in deletions of most of the gene coding for the V region and CH1 domain followed by splicing at the VC joining region and at the hinge. These structural features fit well the notion of genetic discontinuity between V and C genes and also suggest similar mechanisms of excision and splicing in the interdomain regions of the C gene of the heavy chain. PMID:106391

A novel MYH7 mutation links congenital fiber type disproportion and myosin storage myopathy.

PubMed

Ortolano, Saida; Tarrío, Rosa; Blanco-Arias, Patricia; Teijeira, Susana; Rodríguez-Trelles, Francisco; García-Murias, María; Delague, Valerie; Lévy, Nicolas; Fernández, José M; Quintáns, Beatriz; Millán, Beatriz San; Carracedo, Angel; Navarro, Carmen; Sobrido, María-Jesús

2011-04-01

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patient's biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy. Copyright © 2011 Elsevier B.V. All rights reserved.

Computational sequence analysis of predicted long dsRNA transcriptomes of major crops reveals sequence complementarity with human genes.

PubMed

Jensen, Peter D; Zhang, Yuanji; Wiggins, B Elizabeth; Petrick, Jay S; Zhu, Jin; Kerstetter, Randall A; Heck, Gregory R; Ivashuta, Sergey I

2013-01-01

Long double-stranded RNAs (long dsRNAs) are precursors for the effector molecules of sequence-specific RNA-based gene silencing in eukaryotes. Plant cells can contain numerous endogenous long dsRNAs. This study demonstrates that such endogenous long dsRNAs in plants have sequence complementarity to human genes. Many of these complementary long dsRNAs have perfect sequence complementarity of at least 21 nucleotides to human genes; enough complementarity to potentially trigger gene silencing in targeted human cells if delivered in functional form. However, the number and diversity of long dsRNA molecules in plant tissue from crops such as lettuce, tomato, corn, soy and rice with complementarity to human genes that have a long history of safe consumption supports a conclusion that long dsRNAs do not present a significant dietary risk.

IgG-Paraoxonase-1 Fusion Protein for Targeted Drug Delivery Across the Human Blood-Brain Barrier

PubMed Central

Boado, Ruben J.; Zhang, Yun; Zhang, Yufeng; Wang, Yuntao; Pardridge, William M.

2009-01-01

Paraoxonase (PON)-1 is the most potent human protein with organophosphatase activity against organophosphate (OP) toxins. OP compounds readily cross the blood-brain barrier (BBB), and have lethal mechanisms of action within the brain. The production of a brain penetrating form of human PON1, which crosses the BBB, is possible with the re-engineering of the enzyme as a fusion protein with a monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry the PON1 into brain. The human PON1 was fused to the carboxyl terminus of the heavy chain of the chimeric HIRMAb. COS cells were dual transfected with the heavy chain gene and the light chain gene, and the HIRMAb-PON1 fusion protein was affinity purified with protein A chromatography. Western blotting with antibodies to human IgG or human PON1 showed the heavy chain of the HIRMAb-PON1 fusion protein was 40 kDa larger than the heavy chain of the chimeric HIRMAb. The ED50 of binding to the HIR extracellular domain was 0.55 ± 0.07 nM and 1.1 ±0.1 nM, respectively, for the chimeric HIRMAb and the HIRMAb-PON1 fusion protein. The PON1 enzyme activity of the fusion protein was approximately 25% of the enzyme activity in human plasma, based on a fluorometric enzymatic assay. In conclusion, human PON1 has been re-engineered as an IgG-organophosphatase fusion protein that penetrates the human BBB. PMID:19434854

Functional Expression of a Bacterial Heavy Metal Transporter in Arabidopsis Enhances Resistance to and Decreases Uptake of Heavy Metals1[w

PubMed Central

Lee, Joohyun; Bae, Hyunju; Jeong, Jeeyon; Lee, Jae-Yun; Yang, Young-Yell; Hwang, Inhwan; Martinoia, Enrico; Lee, Youngsook

2003-01-01

Large parts of agricultural soil are contaminated with lead (Pb) and cadmium (Cd). Although most environments are not heavily contaminated, the low levels observed nonetheless pose a high risk of heavy metal accumulation in the food chain. Therefore, approaches to develop plants with reduced heavy metal uptake are important. Recently, many transgenic plants with increased heavy metal resistance and uptake of heavy metals were developed for the purpose of phytoremediation. However, to reduce heavy metal in the food chain, plants that transfer less heavy metals to the shoot are required. We tested whether an Escherichia coli gene, ZntA, which encodes a Pb(II)/Cd(II)/Zn(II) pump, could be useful for developing plants with reduced heavy metal content. Yeast cells transformed with this gene had improved resistance to Pb(II) and Cd(II). In Arabidopsis plants transformed with ZntA, ZntA was localized at the plasma membrane and improved the resistance of the plants to Pb(II) and Cd(II). The shoots of the transgenic plants had decreased Pb and Cd content. Moreover, the transgenic protoplasts showed lower accumulation of Cd and faster release of preloaded Cd than wild-type protoplasts. These results show that a bacterial transporter gene, ZntA, can be functionally expressed in plant cells, and that that it may be useful for the development of crop plants that are safe from heavy metal contamination. PMID:14512517

Monoclonal antibodies for the measurement of class-specific antibody responses in the green turtle, Chelonia mydas.

PubMed

Herbst, L H; Klein, P A

1995-06-01

Monoclonal antibodies (Mabs) were developed against the known immunoglobulin classes of the green turtle, Chelonia mydas. Plasma protein fractions enriched for 5.7S IgY, 7S IgY, and IgM turtle immunoglobulins were used to immunize Balb/c mice for hybridoma production and for hybridoma screening. Fifteen hybridomas produced Mabs with specificity for turtle immunoglobulins and for affinity purified dinitrophenol (DNP) specific turtle antibodies. Three Mabs specific for either turtle 5.7S IgY heavy chain (HL814), 7S IgY heavy chain (HL857), or IgM heavy chain (HL846) were purified and used in an enzyme-linked immunosorbent assay (ELISA) to measure antibody responses in two turtles immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) over a 10 month period. In both turtles the 7S IgY antibody response developed within 5 weeks of the first inoculation and remained high over the following 9 months. The 5.7S IgY antibody response was detected in one turtle at 3-4 months and in the other at 8 months, and reached high levels in both individuals by 10 months. The IgM responses were difficult to interpret. One turtle had pre-inoculation anti-DNP IgM antibody in its plasma and the other developed only a weak, transient response at about 4 months. The class-specific antibody activity in immune turtle plasma could be strongly inhibited by soluble DNP or by rabbit anti-DNP specific antiserum, showing that these antibody responses were directed predominantly to the DNP hapten on the DNP-BSA antigen. Antibody responses to the BSA carrier could not be detected in either turtle over the course of the immunization. Mab HL814, specific for an epitope on the 5.7S green turtle immunoglobulin heavy chain, will be useful for characterizing the molecular relationships of 5.7S, 7S and IgM heavy chains and the role of 5.7S antibody in humoral immunity in this species. All anti-turtle Ig Mabs were screened against the plasma globulins of Loggerhead (Caretta caretta), Olive Ridley (Lepidochelys olivacea), Kemp's Ridley (Lepidochelys kempi), Hawksbill (Eretmochelys imbricata), and Leatherback (Dermochelys coriacea). While the Mabs specific for IgM and 5.7S IgY reacted only with the green turtle, two Mabs specific for light chain reacted with all species except the leatherback, and nine mabs specific for 7S IgY heavy chain reacted with all five species. Thus, these Mabs may be useful for immunodiagnostic applications in these endangered species as well.

Integrability in heavy quark effective theory

NASA Astrophysics Data System (ADS)

Braun, Vladimir M.; Ji, Yao; Manashov, Alexander N.

2018-06-01

It was found that renormalization group equations in the heavy-quark effective theory (HQET) for the operators involving one effective heavy quark and light degrees of freedom are completely integrable in some cases and are related to spin chain models with the Hamiltonian commuting with the nondiagonal entry C( u) of the monodromy matrix. In this work we provide a more complete mathematical treatment of such spin chains in the QISM framework. We also discuss the relation of integrable models that appear in the HQET context with the large-spin limit of integrable models in QCD with light quarks. We find that the conserved charges and the "ground state" wave functions in HQET models can be obtained from the light-quark counterparts in a certain scaling limit.

The fully actuated traffic control problem solved by global optimization and complementarity

NASA Astrophysics Data System (ADS)

Ribeiro, Isabel M.; de Lurdes de Oliveira Simões, Maria

2016-02-01

Global optimization and complementarity are used to determine the signal timing for fully actuated traffic control, regarding effective green and red times on each cycle. The average values of these parameters can be used to estimate the control delay of vehicles. In this article, a two-phase queuing system for a signalized intersection is outlined, based on the principle of minimization of the total waiting time for the vehicles. The underlying model results in a linear program with linear complementarity constraints, solved by a sequential complementarity algorithm. Departure rates of vehicles during green and yellow periods were treated as deterministic, while arrival rates of vehicles were assumed to follow a Poisson distribution. Several traffic scenarios were created and solved. The numerical results reveal that it is possible to use global optimization and complementarity over a reasonable number of cycles and determine with efficiency effective green and red times for a signalized intersection.

Immunoglobulin λ Gene Rearrangement Can Precede κ Gene Rearrangement

DOE PAGES

Berg, Jörg; Mcdowell, Mindy; Jäck, Hans-Martin; ...

1990-01-01

Imore » mmunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two lightchain loci: κ and λ .t has been reported that κ loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged λ -chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the κ locus, and that only upon failure to produce a functional κ chain is there an attempt to rearrange the λ locus; and (b) the stochastic theory, which postulates that rearrangement at the λ locus proceeds at a rate that is intrinsically much slower than that at the κ locus. We show here that λ -chain genes are generated whether or not the κ locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.« less

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions

PubMed Central

Bradbury, Andrew R. M.; Trinklein, Nathan D.; Wilkinson, Ian C.; Tandon, Atul K.; Anderson, Stephen; Bladen, Catherine L.; Jones, Brittany; Aldred, Shelley Force; Bestagno, Marco; Burrone, Oscar; Maynard, Jennifer; Ferrara, Fortunato; Görnemann, Janina; Glanville, Jacob; Wolf, Philipp; Frenzel, Andre; Wong, Julin; Koh, Xin Yu; Eng, Hui-Yan; Lane, David; Lefranc, Marie-Paule; Clark, Mike

2018-01-01

ABSTRACT Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use. PMID:29485921

The removal of pyroglutamic acid from monoclonal antibodies without denaturation of the protein chains.

PubMed

Werner, William E; Wu, Sylvia; Mulkerrin, Michael

2005-07-01

Typically, the removal of pyroglutamate from the protein chains of immunoglobulins with the enzyme pyroglutamate aminopeptidase requires the use of chaotropic and reducing agents, quite often with limited success. This article describes a series of optimization experiments using elevated temperatures and detergents to denature and stabilize the heavy chains of immunoglobulins such that the pyroglutamate at the amino terminal was accessible to enzymatic removal using the thermostable protease isolated from Pyrococcus furiosus. The detergent polysorbate 20 (Tween 20) was used successfully to facilitate the removal of pyroglutamate residues. A one-step digestion was developed using elevated temperatures and polysorbate 20, rather than chaotropic and reducing agents, with sample cleanup and preparation for Edman sequencing performed using a commercial cartridge containing the PVDF membrane. All of the immunoglobulins digested with this method yielded heavy chain sequence, but the extent of deblocking was immunglobulin dependent (typically>50%).

Poliomyelitis: immunoglobulin-containing cells in the central nervous system in acute and convalescent phases of the human disease.

PubMed Central

Esiri, M M

1980-01-01

The immunoperoxidase method has been used to demonstrate the presence of immunoglobulin-containing cells in the central nervous system in acute and convalescent phases of poliomyelitis. These cells were found in considerable numbers in the areas of damage during the acute phase, and persisted at the same sites, though in smaller numbers, during the convalescent phase for at least 8 months. Most of the positively stained cells were plasma cells. IgA was the commonest heavy chain type demonstrated, with lesser amounts also of IgG and, during the acute phase, IgM. In the acute phase more lambda than kappa light chain was demonstrated but in the convalescent phase this ratio was reversed. More light chain than heavy chain was demonstrable during the acute phase. The significance of these results is briefly discussed. Images Fig. 2 PMID:6771081

The generation and selection of single-domain, v region libraries from nurse sharks.

PubMed

Flajnik, Martin F; Dooley, Helen

2009-01-01

The cartilaginous fish (sharks, skates, and rays) are the oldest phylogenetic group in which a human-type adaptive immune system and immunoglobulins (Igs) have been found. In addition to their conventional (heavy-light chain heterodimeric) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy chain homodimer that does not associate with light chains. Instead, its variable (V) regions act as independent, soluble units in order to bind antigen. In this chapter, we detail our immunization protocol in order to raise a humoral IgNAR response in the nurse shark (Ginglymostoma cirratum) and the subsequent cloning of the single-domain V regions from this isotype in order to select antigen-specific binders by phage display.

Cyanobacterial megamolecule sacran efficiently forms LC gels with very heavy metal ions.

PubMed

Okajima, Maiko K; Miyazato, Shinji; Kaneko, Tatsuo

2009-08-04

We extracted the megamolecular polysaccharide sacran, which contains carboxylate and sulfate groups, from the jellylike extracellular matrix (ECM) of the cyanobacterium Aphanothece sacrum, which has mineral adsorption bioactivity. We investigated the gelation properties of sacran binding with various heavy metal ions. The sacran chain adsorbed heavier metal ions such as indium, rare earth metals, and lead ions more efficiently to form gel beads. In addition, trivalent metal ions adsorbed onto the sacran chains more efficiently than did divalent ions. The investigation of the metal ion binding ratio on sacran chains demonstrated that sacran adsorbed gadolinium trivalent ions more efficiently than indium trivalent ions. Gel bead formation may be closely correlated to the liquid-crystalline organization of sacran.

Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis.

PubMed

Stoops, Janelle; Byrd, Samantha; Hasegawa, Haruki

2012-10-01

Russell bodies are intracellular aggregates of immunoglobulins. Although the mechanism of Russell body biogenesis has been extensively studied by using truncated mutant heavy chains, the importance of the variable domain sequences in this process and in immunoglobulin biosynthesis remains largely unknown. Using a panel of structurally and functionally normal human immunoglobulin Gs, we show that individual immunoglobulin G clones possess distinctive Russell body inducing propensities that can surface differently under normal and abnormal cellular conditions. Russell body inducing predisposition unique to each immunoglobulin G clone was corroborated by the intrinsic physicochemical properties encoded in the heavy chain variable domain/light chain variable domain sequence combinations that define each immunoglobulin G clone. While the sequence based intrinsic factors predispose certain immunoglobulin G clones to be more prone to induce Russell bodies, extrinsic factors such as stressful cell culture conditions also play roles in unmasking Russell body propensity from immunoglobulin G clones that are normally refractory to developing Russell bodies. By taking advantage of heterologous expression systems, we dissected the roles of individual subunit chains in Russell body formation and examined the effect of non-cognate subunit chain pair co-expression on Russell body forming propensity. The results suggest that the properties embedded in the variable domain of individual light chain clones and their compatibility with the partnering heavy chain variable domain sequences underscore the efficiency of immunoglobulin G biosynthesis, the threshold for Russell body induction, and the level of immunoglobulin G secretion. We propose that an interplay between the unique properties encoded in variable domain sequences and the state of protein homeostasis determines whether an immunoglobulin G expressing cell will develop the Russell body phenotype in a dynamic cellular setting. Copyright © 2012 Elsevier B.V. All rights reserved.

Regulation of contractile protein gene expression in unloaded mouse skeletal muscle

NASA Technical Reports Server (NTRS)

Criswell, D. S.; Carson, J. A.; Booth, F. W.

1996-01-01

Hindlimb unloading was performed on mice in an effort to study the regulation of contractile protein genes. In particular, the regulation of myosin heavy chain IIb was examined. During unloading, muscle fibers undergo a type conversion. Preliminary data from this study does not support the hypothesis that the fiber type conversion is due to an increase in promoter activity of fast isoform genes, such as myosin heavy chain IIb. The consequences of this finding are examined, with particular focus on other factors controlling gene regulation.

Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus.

PubMed

Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F; Criscitiello, Michael F

2018-04-17

Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. © 2018, Ott et al.

Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus

PubMed Central

Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F

2018-01-01

Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. PMID:29664399

Serum Free Light Chains in Neoplastic Monoclonal Gammopathies: Relative Under-Detection of Lambda Dominant Kappa/Lambda Ratio, and Underproduction of Free Lambda Light Chains, as Compared to Kappa Light Chains, in Patients With Neoplastic Monoclonal Gammopathies

PubMed Central

Lee, Won Sok; Singh, Gurmukh

2018-01-01

Background Quantitative evaluation of serum free light chains is recommended for the work up of monoclonal gammopathies. Immunoglobulin light chains are generally produced in excess of heavy chains. In patients with monoclonal gammopathy, κ/λ ratio is abnormal less frequently with lambda chain lesions. This study was undertaken to ascertain if the levels of overproduction of the two light chain types and their detection rates are different in patients with neoplastic monoclonal gammopathies. Methods Results of serum protein electrophoresis (SPEP), serum protein immunofixation electrophoresis (SIFE), urine protein electrophoresis (UPEP), urine protein immunofixation electrophoresis (UIFE), and serum free light chain assay (SFLCA) in patients with monoclonal gammopathies were examined retrospectively. Results The κ/λ ratios were appropriately abnormal more often in kappa chain lesions. Ratios of κ/λ were normal in about 25% of patients with lambda chain lesions in whom free homogenous lambda light chains were detectable in urine. An illustrative case suggests underproduction of free lambda light chains, in some instances. Conclusions The lower prevalence of lambda dominant κ/λ ratio in lesions with lambda light chains is estimated to be due to relative under-detection of lambda dominant κ/λ ratio in about 25% of the patients and because lambda chains are not produced in as much excess of heavy chains as are kappa chains, in about 5% of the patients. The results question the medical necessity and clinical usefulness of the serum free light chain assay. UPEP/UIFE is under-utilized. PMID:29904440

Heavy Traffic Feasible Hybrid Intracycle and Cyclic Sleep for Power Saving in 10G-EPON

PubMed Central

Wang, Liqian; Zhang, Zhiguo; Chen, Xue

2014-01-01

Energy consumption in optical access networks costs carriers substantial operational expense (OPEX) every year and is one of contributing factors for the global warming. To reduce energy consumption in the 10-gigabit Ethernet passive optical network (10G-EPON), a hybrid intracycle and cyclic sleep mechanism is proposed in this paper. Under heavy traffic load, optical network units (ONUs) can utilize short idle slots within each scheduling cycle to enter intracycle sleep without postponing data transmission. In this way, energy conservation is achieved even under heavy traffic load with quality of service (QoS) guarantee. Under light traffic load, ONUs perform long cyclic sleep for several scheduling cycles. The adoption of cyclic sleep instead of intracycle sleep under light traffic load can reduce unnecessary frequent transitions between sleep and full active work caused by using intracycle sleep. Further, the Markov chain of the proposed mechanism is established. The performances of the proposed mechanism and existing approaches are analyzed quantitatively based on the chain. For the proposed mechanism, power saving ability with QoS guarantee even under heavy traffic and better power saving performance than existing approaches are verified by the quantitative analysis. Moreover, simulations validate the above conclusions based on the chain. PMID:25177727

STCRDab: the structural T-cell receptor database

PubMed Central

de Oliveira, Saulo H P; Krawczyk, Konrad

2018-01-01

Abstract The Structural T–cell Receptor Database (STCRDab; http://opig.stats.ox.ac.uk/webapps/stcrdab) is an online resource that automatically collects and curates TCR structural data from the Protein Data Bank. For each entry, the database provides annotations, such as the α/β or γ/δ chain pairings, major histocompatibility complex details, and where available, antigen binding affinities. In addition, the orientation between the variable domains and the canonical forms of the complementarity-determining region loops are also provided. Users can select, view, and download individual or bulk sets of structures based on these criteria. Where available, STCRDab also finds antibody structures that are similar to TCRs, helping users explore the relationship between TCRs and antibodies. PMID:29087479

Comparative higher-order structure analysis of antibody biosimilars using combined bottom-up and top-down hydrogen-deuterium exchange mass spectrometry.

PubMed

Pan, Jingxi; Zhang, Suping; Borchers, Christoph H

2016-12-01

Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (MS) is a powerful technique for higher-order structural characterization of antibodies. Although the peptide-based bottom-up HDX approach and the protein-based top-down HDX approach have complementary advantages, the work done so far on biosimilars has involved only one or the other approach. Herein we have characterized the structures of two bevacizumab (BEV) biosimilars and compared them to the reference BEV using both methods. A sequence coverage of 87% was obtained for the heavy chain and 74% for the light chain in the bottom-up approach. The deuterium incorporation behavior of the peptic peptides from the three BEVs were compared side by side and showed no differences at various HDX time points. Top-down experiments were carried out using subzero temperature LC-MS, and the deuterium incorporation of the intact light chain and heavy chain were obtained. Top-down ETD was also performed to obtain amino acid-level HDX information that covered 100% of the light chain, but only 50% coverage is possible for the heavy chain. Consistent with the intact subunit level data, no differences were observed in the amino acid level HDX data. All these results indicate that there are no differences between the three BEV samples with respect to their high-order structures. The peptide level information from the bottom-up approach, and the residue level and intact subunit level information from the top-down approach were complementary and covered the entire antibody. Copyright © 2016 Elsevier B.V. All rights reserved.

Irreversible Heavy Chain Transfer to Chondroitin*

PubMed Central

Lauer, Mark E.; Hascall, Vincent C.; Green, Dixy E.; DeAngelis, Paul L.; Calabro, Anthony

2014-01-01

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture. PMID:25135638

Sterile neutrino searches at future e-e+, pp and e-p colliders

NASA Astrophysics Data System (ADS)

Antusch, Stefan; Cazzato, Eros; Fischer, Oliver

2017-05-01

Sterile neutrinos are among the most attractive extensions of the SM to generate the light neutrino masses observed in neutrino oscillation experiments. When the sterile neutrinos are subject to a protective symmetry, they can have masses around the electroweak scale and potentially large neutrino Yukawa couplings, which makes them testable at planned future particle colliders. We systematically discuss the production and decay channels at electron-positron, proton-proton and electron-proton colliders and provide a complete list of the leading order signatures for sterile neutrino searches. Among other things, we discuss several novel search channels, and present a first look at the possible sensitivities for the active-sterile mixings and the heavy neutrino masses. We compare the performance of the different collider types and discuss their complementarity.

C P -violation in the two Higgs doublet model: From the LHC to EDMs

NASA Astrophysics Data System (ADS)

Chen, Chien-Yi; Li, Hao-Lin; Ramsey-Musolf, Michael

2018-01-01

We study the prospective sensitivity to C P -violating two Higgs doublet models from the 14 TeV LHC and future electric dipole moment (EDM) experiments. We concentrate on the search for a resonant heavy Higgs that decays to a Z boson and a SM-like Higgs h , leading to the Z (ℓℓ)h (b b ¯ ) final state. The prospective LHC reach is analyzed using the Boosted Decision Tree method. We illustrate the complementarity between the LHC and low energy EDM measurements and study the dependence of the physics reach on the degree of deviation from the alignment limit. In all cases, we find that there exists a large part of parameter space that is sensitive to both EDMs and LHC searches.

D{sub H} element reading frame selection is influenced by an Ig heavy chain transgene, but not by bcl-2

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

Tarlinton, D.; Strasser, A.; McLean, M.

1995-04-01

Mouse B cell precursors containing Ig D{sub H}J{sub H} junctions in one particular reading frame are selectively lost during B cell development. In this register, arbitrarily referred to as reading frame 2, D{sub H}J{sub H} junctions give rise to an open reading frame starting upstream of the D{sub H} element and including the D{sub H}J{sub H}-peptide fused to the constant region of IgM. Expression of this protein, called D{mu}, has been strongly implicated in the loss of B cell precursors containing reading frame 2 D{sub H}J{sub H} junctions. In an attempt to elucidate the means of D{mu} counterselection, we havemore » examined the reading frame distribution of D{sub H}J{sub H} junctions in peripheral B cells from mice transgenic for either the human bcl-2 oncogene or for a functionally rearranged Ig {mu} heavy chain. In bcl-2 transgenic mice, reading frame 2 accounted for < 5% of the D{sub H}J{sub H} junctions in peripheral B cells, a value not significantly different from controls. Reading frames 1 and 3 were equally represented among the remaining junctions. By contrast, the reading frame distribution of endogenous D{sub H}J{sub H} junctions in splenic B cells from Ig {mu} heavy chain transgenic mice showed no evidence of bias against D{mu} encoding D{sub H}J{sub H} junctions. Reading frames 2 and 3 accounted for 27% and 30% of the sequenced D{sub H}J{sub H} junctions, respectively, and the remaining 43% were reading frame 1. Thus although the presence of BCL-2 cannot prevent the selective loss of reading frame 2 D{sub H}J{sub H} B cells, a functional {mu} heavy chain can. These results suggest that D{mu}-expressing B cell precursors may be selectively lost because of the premature and inappropriate cessation of heavy chain gene rearrangement rather than because of the induction of an apoptotic process which can be blocked by BCL-2. 42 refs., 4 figs., 4 tabs.« less

Chromosomal locations of mouse immunoglobulin genes.

PubMed Central

Valbuena, O; Marcu, K B; Croce, C M; Huebner, K; Weigert, M; Perry, R P

1978-01-01

The chromosomal locations of the structural genes coding for the constant portions of mouse heavy (H) and light chain immunoglobulins were studied by molecular hybridization techniques. Complementary DNA probes containing the constant-region sequences of kappa and lambdaI light chain and alpha, gamma2b, and mu heavy chain mRNAs were annealed to a large excess of DNA from a series of eight mouse-human hybrid cell lines that are deficient for various mouse chromosomes. The lines were scored as positive when a high proportion of a probe annealed and negative when an insignificant proportion annealed. Some lines were clearly negative for H and lambda and clearly positive for kappa. Others were positive or intermediate for lambda, positive for kappa and negative for H. Still others, including a line that was selected for the absence of the mouse X chromosome, were positive for all immunoglobulin species. These results demonstrate that the Clambda, Ckappa, and CH genes are located on different autosomes in the mouse. In contrast, the three heavy-chain families exhibited consistently uniform hybridization results, suggesting that the genes for Calpha, Cgamma, and Cmu are located on the same chromosome. A comparison of karyotypic data with hybridization data has limited the possible locations of the Ig genes to only a few chromosomes. PMID:96442

Comparison of llama VH sequences from conventional and heavy chain antibodies.

PubMed

Vu, K B; Ghahroudi, M A; Wyns, L; Muyldermans, S

1997-01-01

Forty different PCR clones encoding a llama variable heavy chain domain were analysed. The majority of these clones are derived from heavy-chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the VHH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama VHH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the VL. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H2 antibodies. The repertoire of the llama VHHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human VHs. The variability plot of the amino acids in the VHH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama VHHs of the homodimeric heavy-chain H2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H2L2 immunoglobulins.

An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells.

PubMed

Yokota, Etsuo; Ueda, Shunpei; Tamura, Kentaro; Orii, Hidefumi; Uchi, Satoko; Sonobe, Seiji; Hara-Nishimura, Ikuko; Shimmen, Teruo

2009-01-01

The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.

Inferring High-Confidence Human Protein-Protein Interactions

DTIC Science & Technology

2012-01-01

comprised proteins that had the same specific func- tion or were subunits of the same protein complex, such as branched chain keto acid E1 alpha (BCKDHA...and branched chain keto acid E1 beta (BCKDHB) [3,29], and dynein cytoplasmic 2 intermediate chain 1 (D2LIC) and dynein cytoplasmic 2 heavy chain 1...474.3 28.0 1337.0 BCKDHA 5 Branched chain keto acid dehydro. E1, alpha BCKDHB 4 Branched chain keto acid dehydro. E1, beta 4 471.4 29.0 1337.5 ARTN 2

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Greaser, M. L.; Schultz, E.

1998-01-01

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.

Complementarity effects on tree growth are contingent on tree size and climatic conditions across Europe

PubMed Central

Madrigal-González, Jaime; Ruiz-Benito, Paloma; Ratcliffe, Sophia; Calatayud, Joaquín; Kändler, Gerald; Lehtonen, Aleksi; Dahlgren, Jonas; Wirth, Christian; Zavala, Miguel A.

2016-01-01

Neglecting tree size and stand structure dynamics might bias the interpretation of the diversity-productivity relationship in forests. Here we show evidence that complementarity is contingent on tree size across large-scale climatic gradients in Europe. We compiled growth data of the 14 most dominant tree species in 32,628 permanent plots covering boreal, temperate and Mediterranean forest biomes. Niche complementarity is expected to result in significant growth increments of trees surrounded by a larger proportion of functionally dissimilar neighbours. Functional dissimilarity at the tree level was assessed using four functional types: i.e. broad-leaved deciduous, broad-leaved evergreen, needle-leaved deciduous and needle-leaved evergreen. Using Linear Mixed Models we show that, complementarity effects depend on tree size along an energy availability gradient across Europe. Specifically: (i) complementarity effects at low and intermediate positions of the gradient (coldest-temperate areas) were stronger for small than for large trees; (ii) in contrast, at the upper end of the gradient (warmer regions), complementarity is more widespread in larger than smaller trees, which in turn showed negative growth responses to increased functional dissimilarity. Our findings suggest that the outcome of species mixing on stand productivity might critically depend on individual size distribution structure along gradients of environmental variation. PMID:27571971

Germline TRAV5D-4 T-Cell Receptor Sequence Targets a Primary Insulin Peptide of NOD Mice

PubMed Central

Nakayama, Maki; Castoe, Todd; Sosinowski, Tomasz; He, XiangLing; Johnson, Kelly; Haskins, Kathryn; Vignali, Dario A.A.; Gapin, Laurent; Pollock, David; Eisenbarth, George S.

2012-01-01

There is accumulating evidence that autoimmunity to insulin B chain peptide, amino acids 9–23 (insulin B:9–23), is central to development of autoimmune diabetes of the NOD mouse model. We hypothesized that enhanced susceptibility to autoimmune diabetes is the result of targeting of insulin by a T-cell receptor (TCR) sequence commonly encoded in the germline. In this study, we aimed to demonstrate that a particular Vα gene TRAV5D-4 with multiple junction sequences is sufficient to induce anti-islet autoimmunity by studying retrogenic mouse lines expressing α-chains with different Vα TRAV genes. Retrogenic NOD strains expressing Vα TRAV5D-4 α-chains with many different complementarity determining region (CDR) 3 sequences, even those derived from TCRs recognizing islet-irrelevant molecules, developed anti-insulin autoimmunity. Induction of insulin autoantibodies by TRAV5D-4 α-chains was abrogated by the mutation of insulin peptide B:9–23 or that of two amino acid residues in CDR1 and 2 of the TRAV5D-4. TRAV13–1, the human ortholog of murine TRAV5D-4, was also capable of inducing in vivo anti-insulin autoimmunity when combined with different murine CDR3 sequences. Targeting primary autoantigenic peptides by simple germline-encoded TCR motifs may underlie enhanced susceptibility to the development of autoimmune diabetes. PMID:22315318

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

USDA-ARS?s Scientific Manuscript database

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...

Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A detected by sodium dodecyl sulfate-capillary gel electrophoresis.

PubMed

Kubota, Kei; Kobayashi, Naoki; Yabuta, Masayuki; Ohara, Motomu; Naito, Toyohiro; Kubo, Takuya; Otsuka, Koji

2017-06-05

This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal antibody-A (mAb-A), detected by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-cGE). The fragment migrated close to the internal standard (10kDa marker) of SDS-cGE and increased about 0.5% under a 25°C condition for 6 months. Generally, identification of fragments observed in SDS-cGE is challenging to carry out due to the difficulty of collecting analytical amounts of fractionations from the capillary. In this study, in-gel digestion peptide mapping and reversed phase liquid chromatography-mass spectrometry (RPLC-MS) were employed to elucidate the structure of the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain HC1-104. The fragment contained complementarity determining regions (CDRs), which are significant to antigen binding, and thus would affect the efficacy of mAb-A. In addition, SDS-cGE without the 10kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment revealed that the fragment increases 0.2% per year in storage at 5°C. The combination of the three analytical methodologies successfully identified the impurity peak detected by SDS-cGE, providing information critical to assuring the quality and stability of the biotherapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers

PubMed Central

Morris, Charles D.; Azadnia, Parisa; de Val, Natalia; Vora, Nemil; Honda, Andrew; Giang, Erick; Saye-Francisco, Karen; Cheng, Yushao; Lin, Xiaohe; Mann, Colin J.; Tang, Jeffrey; Sok, Devin; Burton, Dennis R.; Law, Mansun; Ward, Andrew B.

2017-01-01

ABSTRACT Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches. PMID:28246356

Characterization and screening of IgG binding to the neonatal Fc receptor

PubMed Central

Neuber, Tobias; Frese, Katrin; Jaehrling, Jan; Jäger, Sebastian; Daubert, Daniela; Felderer, Karin; Linnemann, Mechthild; Höhne, Anne; Kaden, Stefan; Kölln, Johanna; Tiller, Thomas; Brocks, Bodo; Ostendorp, Ralf; Pabst, Stefan

2014-01-01

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding. PMID:24802048

Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

PubMed

Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

2013-02-01

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

Characterization of human monoclonal antibodies that neutralize multiple poliovirus serotypes.

PubMed

Puligedda, Rama Devudu; Kouiavskaia, Diana; Al-Saleem, Fetweh H; Kattala, Chandana Devi; Nabi, Usman; Yaqoob, Hamid; Bhagavathula, V Sandeep; Sharma, Rashmi; Chumakov, Konstantin; Dessain, Scott K

2017-10-04

Following the eradication of wild poliovirus (PV), achieving and maintaining a polio-free status will require eliminating potentially pathogenic PV strains derived from the oral attenuated vaccine. For this purpose, a combination of non-cross-resistant drugs, such as small molecules and neutralizing monoclonal antibodies (mAbs), may be ideal. We previously isolated chimpanzee and human mAbs capable of neutralizing multiple PV types (cross-neutralization). Here, we describe three additional human mAbs that neutralize types 1 and 2 PV and one mAb that neutralizes all three types. Most bind conformational epitopes and have unusually long heavy chain complementarity determining 3 domains (HC CDR3). We assessed the ability of the mAbs to neutralize A12 escape mutant PV strains, and found that the neutralizing activities of the mAbs were disrupted by different amino acid substitutions. Competitive binding studies further suggested that the specific mAb:PV interactions that enable cross-neutralization differ among mAbs and serotypes. All of the cloned mAbs bind PV in the vicinity of the "canyon", a circular depression around the 5-fold axis of symmetry through which PV recognizes its cellular receptor. We were unable to generate escape mutants to two of the mAbs, suggesting that their epitopes are important for the PV life cycle. These data indicate that PV cross-neutralization involves binding to highly conserved structures within the canyon that binds to the cellular receptor. These may be facilitated by the long HC CDR3 domains, which may adopt alternative binding configurations. We propose that the human and chimpanzee mAbs described here could have potential as anti-PV therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ro52 autoantibodies arise from self-reactive progenitors in a mother of a child with neonatal lupus.

PubMed

Reed, Joanne H; Gorny, Miroslaw K; Li, Liuzhe; Cardozo, Timothy; Buyon, Jill P; Clancy, Robert M

2017-05-01

The detection of cardiac conduction defects in an 18-24 week old foetus in the absence of structural abnormalities predicts with near certainty the presence of autoantibodies against 60kD and 52kD SSA/Ro in the mother regardless of her health status. Previous studies have emphasized these autoantibodies as key mediators of tissue injury. The aim of this study was to focus on the anti-Ro52 response to determine whether these autoantibodies originate from progenitors that are inherently self-reactive or from B-cells that acquire self-reactivity during an immune response. We traced the evolution of two anti-Ro52 autoantibodies isolated from circulating IgG1-switched B-cells from an asymptomatic mother of a child with third degree congenital heart block. The autoantibodies were expressed as their immune form and as pre-immune ancestors by reverting somatic mutations to germline sequence. The reactivity of pre-immune and immune antibodies for Ro52, Ro60, La and DNA was measured. Both anti-Ro52 autoantibodies exhibited a low frequency of somatic mutations (3-4%) and utilised the same heavy and light chain genes but represented distinct clones based on differing complementarity determining region sequences. Pre- and post-immune antibodies showed specific binding to Ro52 with no measurable reactivity for other autoantigens. Ro52 binding was higher for immune antibodies compared to pre-immune counterparts demonstrating that autoreactivity was enhanced by affinity maturation. These data indicate that Ro52 reactivity is an intrinsic property of the germline antibody repertoire in a mother with a pathogenic antibody defined by cardiac injury in her offspring, and implies defects in both central and peripheral tolerance mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation-exchange HPLC.

PubMed

Lau, Hollis; Pace, Danielle; Yan, Boxu; McGrath, Theresa; Smallwood, Scott; Patel, Ketaki; Park, Jihea; Park, Sungae S; Latypov, Ramil F

2010-04-01

A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab-Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs. 2010 Elsevier B.V. All rights reserved.

Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.

PubMed

Raffaï, R; Weisgraber, K H; MacKenzie, R; Rupp, B; Rassart, E; Hirama, T; Innerarity, T L; Milne, R

2000-03-10

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.

Reshaping Human Antibodies: Grafting an Antilysozyme Activity

NASA Astrophysics Data System (ADS)

Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

1988-03-01

The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

Mapping of Heavy Chain Genes for Mouse Immunoglobulins M and D

NASA Astrophysics Data System (ADS)

Liu, Chih-Ping; Tucker, Philip W.; Mushinski, J. Frederic; Blattner, Frederick R.

1980-09-01

A single DNA fragment containing both μ and δ immunoglobulin heavy chain genes has been cloned from normal BALB/c mouse liver DNA with a new λ phage vector Charon 28. The physical distance between the membrane terminal exon of μ and the first domain of δ is 2466 base pairs, with δ on the 3' side of μ . A single transcript could contain a variable region and both μ and δ constant regions. The dual expression of immunoglobulins M and D on spleen B cells may be due to alternate splicing of this transcript.

Cysteine Racemization on IgG Heavy and Light Chains

PubMed Central

Zhang, Qingchun; Flynn, Gregory C.

2013-01-01

Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of d-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible β-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide. PMID:24142697

A dual chain chimeric antigen receptor (CAR) in the native antibody format for targeting immune cells towards cancer cells without the need of an scFv.

PubMed

Faitschuk, E; Nagy, V; Hombach, A A; Abken, H

2016-10-01

Adoptive cell therapy with chimeric antigen receptor (CAR)-modified T cells showed remarkable therapeutic efficacy in the treatment of leukaemia/lymphoma. However, the application to a variety of cancer entities is often constricted by the non-availability of a single chain antibody (scFv), which is usually the targeting domain in a CAR, while antibodies in the natural format are often available. To overcome the limitation, we designed a CAR that uses an antibody in its natural configuration for binding. Such CAR consists of two chains, the immunoglobulin light and heavy chain with their constant regions, whereby the heavy chain is anchored to the membrane and linked to an intracellular signalling domain for T-cell activation. The two chains form a stable heterodimer, a so-called dual chain CAR (dcCAR), and bind with high affinity and in a specific manner to their cognate antigen. By specific binding, the dcCAR activates engineered T cells for the release of pro-inflammatory cytokines and for target cell lysis. We provide evidence by three examples that the dcCAR format is universally applicable and thereby broadens the CAR cell therapy towards a larger variety of targets for which an scFv antibody is not available.

Integration of molecular dynamics simulation and hotspot residues grafting for de novo scFv design against Salmonella Typhi TolC protein.

PubMed

Leong, Siew Wen; Lim, Theam Soon; Ismail, Asma; Choong, Yee Siew

2018-05-01

With the development of de novo binders for protein targets from non-related scaffolds, many possibilities for therapeutics and diagnostics have been created. In this study, we described the use of de novo design approach to create single-chain fragment variable (scFv) for Salmonella enterica subspecies enterica serovar Typhi TolC protein. Typhoid fever is a global health concern in developing and underdeveloped countries. Rapid typhoid diagnostics will improve disease management and therapy. In this work, molecular dynamics simulation was first performed on a homology model of TolC protein in POPE membrane bilayer to obtain the central structure that was subsequently used as the target for scFv design. Potential hotspot residues capable of anchoring the binders to the target were identified by docking "disembodied" amino acid residues against TolC surface. Next, scFv scaffolds were selected from Protein Data Bank to harbor the computed hotspot residues. The hotspot residues were then incorporated into the scFv scaffold complementarity determining regions. The designs recapitulated binding energy, shape complementarity, and interface surface area of natural protein-antibody interfaces. This approach has yielded 5 designs with high binding affinity against TolC that may be beneficial for the future development of antigen-based detection agents for typhoid diagnostics. Copyright © 2017 John Wiley & Sons, Ltd.

Complementarity of genuine multipartite Bell nonlocality

NASA Astrophysics Data System (ADS)

Sami, Sasha; Chakrabarty, Indranil; Chaturvedi, Anubhav

2017-08-01

We introduce a feature of no-signaling (Bell) nonlocal theories: namely, when a system of multiple parties manifests genuine nonlocal correlation, then there cannot be arbitrarily high nonlocal correlation among any subset of the parties. We call this feature complementarity of genuine multipartite nonlocality. We use Svetlichny's criterion for genuine multipartite nonlocality and nonlocal games to derive the complementarity relations under no-signaling constraints. We find that the complementarity relations are tightened for the much stricter quantum constraints. We compare this notion with the well-known notion of monogamy of nonlocality. As a consequence, we obtain tighter nontrivial monogamy relations that take into account genuine multipartite nonlocality. Furthermore, we provide numerical evidence showcasing this feature using a bipartite measure and several other well-known tripartite measures of nonlocality.

Centrocins: isolation and characterization of novel dimeric antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis.

PubMed

Li, Chun; Haug, Tor; Moe, Morten K; Styrvold, Olaf B; Stensvåg, Klara

2010-09-01

As immune effector molecules, antimicrobial peptides (AMPs) play an important role in the invertebrate immune system. Here, we present two novel AMPs, named centrocins 1 (4.5kDa) and 2 (4.4kDa), purified from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. The native peptides are cationic and show potent activities against Gram-positive and Gram-negative bacteria. The centrocins have an intramolecular heterodimeric structure, containing a heavy chain (30 amino acids) and a light chain (12 amino acids). The cDNA encoding the peptides and genomic sequences were cloned and sequenced. One putative isoform (centrocin 1b) was identified and one intron was found in the genes coding for the centrocins. The full length protein sequence of centrocin 1 consists of 119 amino acids, whereas centrocin 2 consists of 118 amino acids which both include a preprosequence of 51 or 50 amino acids for centrocins 1 and 2, respectively, and an interchain of 24 amino acids between the heavy and light chain. The difference of molecular mass between the native centrocins and the deduced sequences from cDNA indicates that the native centrocins contain a post-translational brominated tryptophan. In addition, two amino acids at the C-terminal, Gly-Arg, were removed from the light chains during the post-translational processing. The separate peptide chains of centrocin 1 were synthesized and the heavy chain alone was shown to be sufficient for antimicrobial activity. The genome of the closely related species, the purple sea urchin (S. purpuratus), was shown to contain two putative proteins with high similarity to the centrocins. Copyright 2010 Elsevier Ltd. All rights reserved.

FOOD CHAIN TRANSFER AND BIOAVAILABILITY OF CD AND OTHER ELEMENTS IN PLANTS GROWN ON BIOSOLIDS AMENDED SOILS

EPA Science Inventory

Application of biosolids, livestock manures, compost, and many byproducts add heavy metals to soil. Exposure of humans, livestock and wildlife to these added heavy metals continues to be a concern despite the research and risk assessments which suggest otherwise. Key concepts gov...

A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells.

PubMed

Gerster, Anja; Wodarczyk, Claas; Reichenbächer, Britta; Köhler, Janet; Schulze, Andreas; Krause, Felix; Müller, Dethardt

2016-12-01

To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios. Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

PubMed

Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

2010-08-06

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. Copyright 2010 Elsevier Inc. All rights reserved.

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

PubMed Central

Shaffer, Justin F.; Kier, William M.

2015-01-01

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. PMID:26997860

A Cytoplasmic Dynein Heavy Chain Is Required for Oscillatory Nuclear Movement of Meiotic Prophase and Efficient Meiotic Recombination in Fission Yeast

PubMed Central

Yamamoto, Ayumu; West, Robert R.; McIntosh, J. Richard; Hiraoka, Yasushi

1999-01-01

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination. PMID:10366596

Impact of biodiesel source material and chemical structure on emissions of criteria pollutants from a heavy-duty engine.

PubMed

McCormick, R L; Graboski, M S; Alleman, T L; Herring, A M; Tyson, K S

2001-05-01

Biodiesel is an oxygenated diesel fuel made from vegetable oils and animal fats by conversion of the triglyceride fats to esters via transesterification. In this study we examined biodiesels produced from a variety of real-world feedstocks as well as pure (technical grade) fatty acid methyl and ethyl esters for emissions performance in a heavy-duty truck engine. The objective was to understand the impact of biodiesel chemical structure, specifically fatty acid chain length and number of double bonds, on emissions of NOx and particulate matter (PM). A group of seven biodiesels produced from real-world feedstocks and 14 produced from pure fatty acids were tested in a heavy-duty truck engine using the U.S. heavy-duty federal test procedure (transient test). It was found that the molecular structure of biodiesel can have a substantial impact on emissions. The properties of density, cetane number, and iodine number were found to be highly correlated with one another. For neat biodiesels, PM emissions were essentially constant at about 0.07 g/bhp-h for all biodiesels as long as density was less than 0.89 g/cm3 or cetane number was greater than about 45. NOx emissions increased with increasing fuel density or decreasing fuel cetane number. Increasing the number of double bonds, quantified as iodine number, correlated with increasing emissions of NOx. Thus the increased NOx observed for some fuels cannot be explained by the NOx/PM tradeoff and is therefore not driven by thermal NO formation. For fully saturated fatty acid chains the NOx emission increased with decreasing chain length for tests using 18, 16, and 12 carbon chain molecules. Additionally, there was no significant difference in NOx or PM emissions for the methyl and ethyl esters of identical fatty acids.

Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

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

Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina

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 sitesmore » (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.« less

Private algebras in quantum information and infinite-dimensional complementarity

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

Crann, Jason, E-mail: jason-crann@carleton.ca; Laboratoire de Mathématiques Paul Painlevé–UMR CNRS 8524, UFR de Mathématiques, Université Lille 1–Sciences et Technologies, 59655 Villeneuve d’Ascq Cédex; Kribs, David W., E-mail: dkribs@uoguelph.ca

We introduce a generalized framework for private quantum codes using von Neumann algebras and the structure of commutants. This leads naturally to a more general notion of complementary channel, which we use to establish a generalized complementarity theorem between private and correctable subalgebras that applies to both the finite and infinite-dimensional settings. Linear bosonic channels are considered and specific examples of Gaussian quantum channels are given to illustrate the new framework together with the complementarity theorem.

Landscaping the structures of GAVI country vaccine supply chains and testing the effects of radical redesign.

PubMed

Lee, Bruce Y; Connor, Diana L; Wateska, Angela R; Norman, Bryan A; Rajgopal, Jayant; Cakouros, Brigid E; Chen, Sheng-I; Claypool, Erin G; Haidari, Leila A; Karir, Veena; Leonard, Jim; Mueller, Leslie E; Paul, Proma; Schmitz, Michelle M; Welling, Joel S; Weng, Yu-Ting; Brown, Shawn T

2015-08-26

Many of the world's vaccine supply chains do not adequately provide vaccines, prompting several questions: how are vaccine supply chains currently structured, are these structures closely tailored to individual countries, and should these supply chains be radically redesigned? We segmented the 57 GAVI-eligible countries' vaccine supply chains based on their structure/morphology, analyzed whether these segments correlated with differences in country characteristics, and then utilized HERMES to develop a detailed simulation model of three sample countries' supply chains and explore the cost and impact of various alternative structures. The majority of supply chains (34 of 57) consist of four levels, despite serving a wide diversity of geographical areas and population sizes. These four-level supply chains loosely fall into three clusters [(1) 18 countries relatively more bottom-heavy, i.e., many more storage locations lower in the supply chain, (2) seven with relatively more storage locations in both top and lower levels, and (3) nine comparatively more top-heavy] which do not correlate closely with any of the country characteristics considered. For all three cluster types, our HERMES modeling found that simplified systems (a central location shipping directly to immunization locations with a limited number of Hubs in between) resulted in lower operating costs. A standard four-tier design template may have been followed for most countries and raises the possibility that simpler and more tailored designs may be warranted. Copyright © 2015 Elsevier Ltd. All rights reserved.

Megakaryocyte and platelet abnormalities in a patient with a W33C mutation in the conserved SH3-like domain of myosin heavy chain IIA.

PubMed

Kahr, Walter H A; Savoia, Anna; Pluthero, Fred G; Li, Ling; Christensen, Hilary; De Rocco, Daniela; Traivaree, Chanchai; Butchart, Sheila E; Curtin, Julie; Stollar, Elliott J; Forman-Kay, Julie D; Blanchette, Victor S

2009-12-01

Heterozygous mutations in MYH9, which encodes non-muscle myosin heavy chain IIA (MHC-IIA), result in autosomal dominant inherited MYH9-related disorders characterised by macro-thrombocytopenia, granulocyte inclusions, variable sensorineural deafness, cataracts and nephritis. MHC-IIA is assembled into a complex consisting of two pairs of light chains and two heavy chains, where the latter contain a neck region, SH3-like, motor and rod domains. We describe a patient with a Trp33Cys missense mutation in the SH3-like domain of MHC-IIA. Abnormal platelet function was observed using platelet aggregometry with the agonists epinephrine and adenosine diphosphate (ADP). Patient granulocytes and megakaryocytes, but not platelets, contained abnormal MHC-IIA inclusions visualised by confocal immunofluorescence or electron microscopy. Megakaryocytes grown in culture were smaller and contained hypolobulated nuclei compared to controls. Bone marrow-derived megakaryocytes revealed a preponderance of immature forms, the presence of structurally diverse inclusion bodies, and frequent emperipolesis as assessed by electron microscopy. Platelets and leukocytes contained indistinguishable amounts of total MHC-IIA determined by immunoblotting. Molecular modelling studies indicated that mutation of Trp33 destabilises the interface between the SH3-like and motor domain of MHC-IIA, which is close to previously described motor domain mutations, implying an important structural and/or functional role for this region in MHC-IIA.

Generalized uncertainty principle: implications for black hole complementarity

NASA Astrophysics Data System (ADS)

Chen, Pisin; Ong, Yen Chin; Yeom, Dong-han

2014-12-01

At the heart of the black hole information loss paradox and the firewall controversy lies the conflict between quantum mechanics and general relativity. Much has been said about quantum corrections to general relativity, but much less in the opposite direction. It is therefore crucial to examine possible corrections to quantum mechanics due to gravity. Indeed, the Heisenberg Uncertainty Principle is one profound feature of quantum mechanics, which nevertheless may receive correction when gravitational effects become important. Such generalized uncertainty principle [GUP] has been motivated from not only quite general considerations of quantum mechanics and gravity, but also string theoretic arguments. We examine the role of GUP in the context of black hole complementarity. We find that while complementarity can be violated by large N rescaling if one assumes only the Heisenberg's Uncertainty Principle, the application of GUP may save complementarity, but only if certain N -dependence is also assumed. This raises two important questions beyond the scope of this work, i.e., whether GUP really has the proposed form of N -dependence, and whether black hole complementarity is indeed correct.

Application of Monoclonal Antibodies in Functional and Comparative Investigations of Heavy-Chain Immunoglobulins in New World Camelids

PubMed Central

Daley, L. P.; Gagliardo, L. F.; Duffy, M. S.; Smith, M. C.; Appleton, J. A.

2005-01-01

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the γ chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids. PMID:15753251

Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids.

PubMed

Daley, L P; Gagliardo, L F; Duffy, M S; Smith, M C; Appleton, J A

2005-03-01

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.

[Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody].

PubMed

Liu, Fang; Li, Li; Zhang, Wei; Wang, Qi

2013-04-01

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.

A mechanical comparison of linear and double-looped hung supplemental heavy chain resistance to the back squat: a case study.

PubMed

Neelly, Kurt R; Terry, Joseph G; Morris, Martin J

2010-01-01

A relatively new and scarcely researched technique to increase strength is the use of supplemental heavy chain resistance (SHCR) in conjunction with plate weights to provide variable resistance to free weight exercises. The purpose of this case study was to determine the actual resistance being provided by a double-looped versus a linear hung SHCR to the back squat exercise. The linear technique simply hangs the chain directly from the bar, whereas the double-looped technique uses a smaller chain to adjust the height of the looped chain. In both techniques, as the squat descends, chain weight is unloaded onto the floor, and as the squat ascends, chain weight is progressively loaded back as resistance. One experienced and trained male weight lifter (age = 33 yr; height = 1.83 m; weight = 111.4 kg) served as the subject. Plate weight was set at 84.1 kg, approximately 50% of the subject's 1 repetition maximum. The SHCR was affixed to load cells, sampling at a frequency of 500 Hz, which were affixed to the Olympic bar. Data were collected as the subject completed the back squat under the following conditions: double-looped 1 chain (9.6 kg), double-looped 2 chains (19.2 kg), linear 1 chain, and linear 2 chains. The double-looped SHCR resulted in a 78-89% unloading of the chain weight at the bottom of the squat, whereas the linear hanging SHCR resulted in only a 36-42% unloading. The double-looped technique provided nearly 2 times the variable resistance at the top of the squat compared with the linear hanging technique, showing that attention must be given to the technique used to hang SHCR.

Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses.

PubMed

Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Kurosawa, Yoshikazu; Yokoyama, Shigeyuki

2014-07-01

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

PubMed Central

Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Yokoyama, Shigeyuki

2014-01-01

ABSTRACT Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines. PMID:24719430

Same-Day Imaging Using Small Proteins: Clinical Experience and Translational Prospects in Oncology.

PubMed

Krasniqi, Ahmet; D'Huyvetter, Matthias; Devoogdt, Nick; Frejd, Fredrik Y; Sörensen, Jens; Orlova, Anna; Keyaerts, Marleen; Tolmachev, Vladimir

2018-06-01

Imaging of expression of therapeutic targets may enable stratification of patients for targeted treatments. The use of small radiolabeled probes based on the heavy-chain variable region of heavy-chain-only immunoglobulins or nonimmunoglobulin scaffolds permits rapid localization of radiotracers in tumors and rapid clearance from normal tissues. This makes high-contrast imaging possible on the day of injection. This mini review focuses on small proteins for radionuclide-based imaging that would allow same-day imaging, with the emphasis on clinical applications and promising preclinical developments within the field of oncology. © 2018 by the Society of Nuclear Medicine and Molecular Imaging.

Detection of a ventricular-specific myosin heavy chain in adult and developing chicken heart

PubMed Central

1986-01-01

In the present study, a monoclonal antibody (McAb), ALD19, generated against myosin of slow tonic muscle, was shown to react with the heavy chain of ventricular myosin in the adult chicken heart. With this antibody, it was possible to detect a ventricular-specific myosin during myocardial differentiation and to show that the epitope recognized by ALD19 was present from the earliest stages of ventricular differentiation and maintained throughout development only in the ventricle. A second McAb, specific for atrial myosin heavy chain (MHC) (Gonzalez-Sanchez, A., and D. Bader, 1984, Dev. Biol., 103:151-158), was used as a control to detect an atrial-specific myosin in the caudal portion of the developing heart at Hamburger-Hamilton stage 15. It was found that the appearance of ventricular MHC predated the expression of atrial MHC by approximately 1 d in ovo and that specific MHCs were always differentially distributed. While a common primordial MHC may be present in the early heart, this study showed the tissue-specific expression of a ventricular MHC during the initial stages of heart development and its differential accumulation throughout development. PMID:3514633

Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly

PubMed Central

Xing, Yi; Böcking, Till; Wolf, Matthias; Grigorieff, Nikolaus; Kirchhausen, Tomas; Harrison, Stephen C

2010-01-01

The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly. PMID:20033059

Immunoglobulin isotypes in Atlantic salmon, Salmo salar.

PubMed

Hordvik, Ivar

2015-02-27

There are three major immunoglobulin (Ig) isotypes in salmonid fish: IgM, IgD and IgT, defined by the heavy chains μ, δ and τ, respectively. As a result of whole genome duplication in the ancestor of the salmonid fish family, Atlantic salmon (Salmo salar) possess two highly similar Ig heavy chain gene complexes (A and B), comprising two μ genes, two δ genes, three intact τ genes and five τ pseudogenes. The μA and μB genes correspond to two distinct sub-populations of serum IgM. The IgM-B sub-variant has a characteristic extra cysteine near the C-terminal part of the heavy chain and exhibits a higher degree of polymer disulfide cross-linking compared to IgM-A. The IgM-B:IgM-A ratio in serum is typically 60:40, but skewed ratios are also observed. The IgT isotype appears to be specialized to mucosal immune responses in salmonid fish. The concentration of IgT in serum is 100 to 1000 times lower than IgM. Secreted forms of IgD have been detected in rainbow trout, but not yet in Atlantic salmon.

Presence of Trifolium repens Promotes Complementarity of Water Use and N Facilitation in Diverse Grass Mixtures.

PubMed

Hernandez, Pauline; Picon-Cochard, Catherine

2016-01-01

Legume species promote productivity and increase the digestibility of herbage in grasslands. Considerable experimental data also indicate that communities with legumes produce more above-ground biomass than is expected from monocultures. While it has been attributed to N facilitation, evidence to identify the mechanisms involved is still lacking and the role of complementarity in soil water acquisition by vertical root differentiation remains unclear. We used a 20-months mesocosm experiment to investigate the effects of species richness (single species, two- and five-species mixtures) and functional diversity (presence of the legume Trifolium repens) on a set of traits related to light, N and water use and measured at community level. We found a positive effect of Trifolium presence and abundance on biomass production and complementarity effects in the two-species mixtures from the second year. In addition the community traits related to water and N acquisition and use (leaf area, N, water-use efficiency, and deep root growth) were higher in the presence of Trifolium. With a multiple regression approach, we showed that the traits related to water acquisition and use were with N the main determinants of biomass production and complementarity effects in diverse mixtures. At shallow soil layers, lower root mass of Trifolium and higher soil moisture should increase soil water availability for the associated grass species. Conversely at deep soil layer, higher root growth and lower soil moisture mirror soil resource use increase of mixtures. Altogether, these results highlight N facilitation but almost soil vertical differentiation and thus complementarity for water acquisition and use in mixtures with Trifolium. Contrary to grass-Trifolium mixtures, no significant over-yielding was measured for grass mixtures even those having complementary traits (short and shallow vs. tall and deep). Thus, vertical complementarity for soil resources uptake in mixtures was not only dependant on the inherent root system architecture but also on root plasticity. We also observed a time-dependence for positive complementarity effects due to the slow development of Trifolium in mixtures, possibly induced by competition with grasses. Overall, our data underlined that soil water resource was an important driver of over-yielding and complementarity effects in Trifolium-grass mixtures.

Partial versus Productive Immunoglobulin Heavy Locus Rearrangements in Chronic Lymphocytic Leukemia: Implications for B-Cell Receptor Stereotypy

PubMed Central

Tsakou, Eugenia; Agathagelidis, Andreas; Boudjoghra, Myriam; Raff, Thorsten; Dagklis, Antonis; Chatzouli, Maria; Smilevska, Tatjana; Bourikas, George; Merle-Beral, Helene; Manioudaki-Kavallieratou, Eleni; Anagnostopoulos, Achilles; Brüggemann, Monika; Davi, Frederic; Stamatopoulos, Kostas; Belessi, Chrysoula

2012-01-01

The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level. PMID:21968789

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

PubMed Central

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.

2014-01-01

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 non-complementarity 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. PMID:24615866

The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein

PubMed Central

Daley, Margaret E.; Sykes, Brian D.

2003-01-01

Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the 3Jαβ 1H-1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand. PMID:12824479

Tailoring Thermal Conductivity of Single-stranded Carbon-chain Polymers through Atomic Mass Modification

PubMed Central

Liao, Quanwen; Zeng, Lingping; Liu, Zhichun; Liu, Wei

2016-01-01

Tailoring the thermal conductivity of polymers is central to enlarge their applications in the thermal management of flexible integrated circuits. Progress has been made over the past decade by fabricating materials with various nanostructures, but a clear relationship between various functional groups and thermal properties of polymers remains to be established. Here, we numerically study the thermal conductivity of single-stranded carbon-chain polymers with multiple substituents of hydrogen atoms through atomic mass modification. We find that their thermal conductivity can be tuned by atomic mass modifications as revealed through molecular dynamics simulations. The simulation results suggest that heavy homogeneous substituents do not assist heat transport and trace amounts of heavy substituents can in fact hinder heat transport substantially. Our analysis indicates that carbon chain has the biggest contribution (over 80%) to the thermal conduction in single-stranded carbon-chain polymers. We further demonstrate that atomic mass modifications influence the phonon bands of bonding carbon atoms, and the discrepancies of phonon bands between carbon atoms are responsible for the remarkable drops in thermal conductivity and large thermal resistances in carbon chains. Our study provides fundamental insight into how to tailor the thermal conductivity of polymers through variable substituents. PMID:27713563

Complementarity in false memory illusions.

PubMed

Brainerd, C J; Reyna, V F

2018-03-01

For some years, the DRM illusion has been the most widely studied form of false memory. The consensus theoretical interpretation is that the illusion is a reality reversal, in which certain new words (critical distractors) are remembered as though they are old list words rather than as what they are-new words that are similar to old ones. This reality-reversal interpretation is supported by compelling lines of evidence, but prior experiments are limited by the fact that their memory tests only asked whether test items were old. We removed that limitation by also asking whether test items were new-similar. This more comprehensive methodology revealed that list words and critical distractors are remembered quite differently. Memory for list words is compensatory: They are remembered as old at high rates and remembered as new-similar at very low rates. In contrast, memory for critical distractors is complementary: They are remembered as both old and new-similar at high rates, which means that the DRM procedure induces a complementarity illusion rather than a reality reversal. The conjoint recognition model explains complementarity as a function of three retrieval processes (semantic familiarity, target recollection, and context recollection), and it predicts that complementarity can be driven up or down by varying the mix of those processes. Our experiments generated data on that prediction and introduced a convenient statistic, the complementarity ratio, which measures (a) the level of complementarity in memory performance and (b) whether its direction is reality-consistent or reality-reversed. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

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

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

Wu, Xueling; Zhou, Tongqing; Zhu, Jiang

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 ofmore » 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.« less

Determination of heavy metals in the ambient atmosphere.

PubMed

Suvarapu, Lakshmi Narayana; Baek, Sung-Ok

2017-01-01

Heavy metal determination in ambient air is an important task for environmental researchers because of their toxicity to human beings. Some heavy metals (hexavalent chromium (Cr), arsenic (As), cadmium (Cd) and nickel (Ni)) have been listed as carcinogens. Furthermore, heavy metals in the atmosphere can accumulate in various plants and animals and enter humans through the food chain. This article reviews the determination of heavy metals in the atmosphere in different areas of the world since 2006. The results showed that most researchers concentrated on toxic metals, such as Cr, Cd, Ni, As and lead. A few studies used plant materials as bio-monitors for the atmospheric levels of heavy metals. Some researchers found higher concentrations of heavy metals surrounding industrial areas compared with residential and/or commercial areas. Most studies reported the major sources of the particulate matter and heavy metals in the atmosphere to be industrial emissions, vehicular emissions and secondary aerosols.

Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

1989-01-01

Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

Point Mutations in the Stem Region and the Fourth AAA Domain of Cytoplasmic Dynein Heavy Chain Partially Suppress the Phenotype of NUDF/LIS1 Loss in Aspergillus nidulans

PubMed Central

Zhuang, Lei; Zhang, Jun; Xiang, Xin

2007-01-01

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger ΔnudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the ΔnudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo. PMID:17237507

Phytoplankton Assemblage Characteristics in Recurrently Fluctuating Environments

PubMed Central

Roelke, Daniel L.; Spatharis, Sofie

2015-01-01

Annual variations in biogeochemical and physical processes can lead to nutrient variability and seasonal patterns in phytoplankton productivity and assemblage structure. In many coastal systems river inflow and water exchange with the ocean varies seasonally, and alternating periods can arise where the nutrient most limiting to phytoplankton growth switches. Transitions between these alternating periods can be sudden or gradual and this depends on human activities, such as reservoir construction and interbasin water transfers. How such activities might influence phytoplankton assemblages is largely unknown. Here, we employed a multispecies, multi-nutrient model to explore how nutrient loading switching mode might affect characteristics of phytoplankton assemblages. The model is based on the Monod-relationship, predicting an instantaneous growth rate from ambient inorganic nutrient concentrations whereas the limiting nutrient at any given time was determined by Liebig’s Law of the Minimum. Our simulated phytoplankton assemblages self-organized from species rich pools over a 15-year period, and only the surviving species were considered as assemblage members. Using the model, we explored the interactive effects of complementarity level in trait trade-offs within phytoplankton assemblages and the amount of noise in the resource supply concentrations. We found that the effect of shift from a sudden resource supply transition to a gradual one, as observed in systems impacted by watershed development, was dependent on the level of complementarity. In the extremes, phytoplankton species richness and relative overyielding increased when complementarity was lowest, and phytoplankton biomass increased greatly when complementarity was highest. For low-complementarity simulations, the persistence of poorer-performing phytoplankton species of intermediate R*s led to higher richness and relative overyielding. For high-complementarity simulations, the formation of phytoplankton species clusters and niche compression enabled higher biomass accumulation. Our findings suggest that an understanding of factors influencing the emergence of life history traits important to complementarity is necessary to predict the impact of watershed development on phytoplankton productivity and assemblage structure. PMID:25799563

Atwood's Heavy Chain

ERIC Educational Resources Information Center

Beeken, Paul

2011-01-01

While perusing various websites in search of a more challenging lab for my students, I came across a number of ideas where replacing the string in an Atwood's machine with a simple ball chain like the kind found in lamp pulls created an interesting system to investigate. The replacement of the string produced a nice nonuniform acceleration, but…

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

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

Mullinax, R.L.; Gross, E.A.; Amberg, J.R.

1990-10-01

The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These humanmore » antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.« less

Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

PubMed Central

Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

2012-01-01

The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

PubMed

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2016-09-01

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cloning of the IgM heavy chain of the bottlenose dolphin (Tursiops truncatus), and initial analysis of VH gene usage.

PubMed

Lundqvist, Mats L; Kohlberg, Kathleen E; Gefroh, Holly A; Arnaud, Philippe; Middleton, Darlene L; Romano, Tracy A; Warr, Gregory W

2002-07-01

Clones encoding the dolphin IgM heavy (micro) chain gene were isolated from a cDNA library of peripheral blood leukocytes. Genomic Southern blot analyses showed that the dolphin IGHM gene is most likely present in a single copy, and its sequence shows greatest similarity to those of the IGHM gene of the sheep, pig and cow, evolutionarily related artiodactyls. The transmembrane (TM) form of the IGHM chain was isolated by 3' RACE. While showing similarities to the TM regions of other mammalian IGHM chains, the highly conserved Ser residue of the CART motif is substituted with a Gly in the dolphin. In contrast to the pig and cow, which utilize only a single VH family, the dolphin expresses at least two distinct VH families, belonging to the mammalian VH clans I and III. At least two JH genes were identified in the dolphin. Some CDR3 regions of the dolphin VH are long (up to 21 amino acids), and contain multiple Cys residues, hypothesized to stabilize the CDR3 structure through disulfide bond formation.

Black hole complementarity with the generalized uncertainty principle in Gravity's Rainbow

NASA Astrophysics Data System (ADS)

Gim, Yongwan; Um, Hwajin; Kim, Wontae

2018-02-01

When gravitation is combined with quantum theory, the Heisenberg uncertainty principle could be extended to the generalized uncertainty principle accompanying a minimal length. To see how the generalized uncertainty principle works in the context of black hole complementarity, we calculate the required energy to duplicate information for the Schwarzschild black hole. It shows that the duplication of information is not allowed and black hole complementarity is still valid even assuming the generalized uncertainty principle. On the other hand, the generalized uncertainty principle with the minimal length could lead to a modification of the conventional dispersion relation in light of Gravity's Rainbow, where the minimal length is also invariant as well as the speed of light. Revisiting the gedanken experiment, we show that the no-cloning theorem for black hole complementarity can be made valid in the regime of Gravity's Rainbow on a certain combination of parameters.

Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands.

PubMed

Das, Dibyendu Kumar; Mallis, Robert J; Duke-Cohan, Jonathan S; Hussey, Rebecca E; Tetteh, Paul W; Hilton, Mark; Wagner, Gerhard; Lang, Matthew J; Reinherz, Ellis L

2016-12-02

The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands*♦

PubMed Central

Das, Dibyendu Kumar; Mallis, Robert J.; Duke-Cohan, Jonathan S.; Hussey, Rebecca E.; Tetteh, Paul W.; Hilton, Mark; Wagner, Gerhard; Lang, Matthew J.; Reinherz, Ellis L.

2016-01-01

The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR. PMID:27707880

Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

NASA Astrophysics Data System (ADS)

Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

1985-03-01

The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

The C-terminal heavy-chain domain of botulinum neurotoxin a is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions.

PubMed

Ayyar, B Vijayalakshmi; Aoki, K Roger; Atassi, M Zouhair

2015-04-01

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Polymer-Coated Nanoparticles for Reversible Emulsification and Recovery of Heavy Oil.

PubMed

Qi, Luqing; Song, Chen; Wang, Tianxiao; Li, Qilin; Hirasaki, George J; Verduzco, Rafael

2018-06-05

Heavy crude oil has poor solubility and a high density, making recovery and transport much more difficult and expensive than for light crude oil. Emulsifiers can be used to produce low viscosity oil-in-water emulsions for recovery and transport, but subsequent demulsification can be challenging. Here, we develop and implement interfacially active, pH-responsive polymer-coated nanoparticles (PNPs) to reversibly stabilize, recover, and break oil/water emulsions through variation of solution pH. Silica particles with poly(2-(dimethylamino)ethyl methacrylate) (DMAEMA) chains covalently grafted to the surface are prepared although a reversible addition fragmentation chain transfer grafting-through technique. The resulting DMAEMA PNPs can stabilize emulsions of high viscosity Canadian heavy oil at PNP concentrations as low as 0.1 wt % and at neutral pH. The performance of the DMAEMA PNPs exceeds that of DMAEMA homopolymer additives, which we attribute to the larger size and irreversible adsorption of DMAEMA PNPs to the oil/water interface. After recovery, the emulsion can be destabilized by the addition of acid to reduce pH, resulting in separation and settling of the heavy oil from the aqueous phase. Recovery of more than 10 wt % of the crude heavy oil-in-place is achieved by flooding with aqueous solution of 0.1 wt % DMAEMA PNPs without any additional surfactant or chemical. This work demonstrates the applicability of PNPs as surface active materials for enhanced oil recovery processes and for heavy oil transport.

[Study on the DNA vaccine against foot-and-mouth disease virus using the heavy chain constant region of swine IgG as the carrier for peptide epitopes].

PubMed

Li, G J; Yan, W Y; Xu, Q X; Sheng, Z T; Zheng, Z X

2001-05-01

The peptide of amino acids 141-160 of VP1 protein of foot-and-mouth disease virus (FMDV) is a major B cell epitope and the peptide of amino acids 21-40 is an important T cell epitope. In this study, the DNA fragments of 141-160 and 21-40 peptide epitopes of a strain of type O FMDV was chemically synthesized and arranged into a tandem repeat 141-160 (20AA)-21-40 (20AA)-141-160 (20AA). This tandem sequence was fused to the 3' end of the heavy chain constant region gene of swine immunoglobulin G and was then cloned into mammalian expression vector pCDM8 to form a recombinant plasmid pCDM8FZ3. After pCDM8FZ3 was inoculated intramuscularly into guinea pigs, it elicited a neutralizing antibody response and a specific spleen T cell proliferative response, and 66% of the vaccinated animals were protected from viral challenge. Our study indicated that the heavy chain constant region of swine IgG can act as the carrier protein for FMDV peptide epitopes, and pC-DM8FZ3 is a potential DNA vaccine candidate to prevent FMDV infection.

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

PubMed

Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

2012-04-20

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.

Camel heavy chain antibodies against prostate-specific membrane antigen.

PubMed

Evazalipour, Mehdi; Tehrani, Bahram Soltani; Abolhassani, Mohsen; Morovvati, Hamid; Omidfar, Kobra

2012-12-01

Prostate-specific membrane antigen (PSMA), a type II integral membrane glycoprotein, is highly overexpressed in all forms of prostate cancer tissues. It has also been demonstrated in a wide range of neovasculature of non-prostatic solid tumors, including bladder, pancreas, lung, kidney, colorectal, and gastric cancers. Given the unique expression of PSMA, it is considered an alluring target for antibody-based imaging and therapy of cancer. In the present study, the production and characterization of camel heavy chain antibodies (HCAbs) specific for the external domain of the PSMA are reported. Due to the absence of the CH1 domain, HCAbs are smaller than their counterparts in conventional antibodies. In this study, camel antibodies were generated through immunization of Camelus dromedarius with a synthetic 28 amino acid peptide corresponding to the external surface domain of antigen and PSMA-expressing cell lines. Different binding properties to protein A and protein G affinity columns were deployed to separate three subclasses of camel IgG. The affinity purified HCAbs bound selectively to the synthetic peptide in enzyme linked immunosorbent assay (ELISA) and reacted specifically with PSMA-expressing cell line through immunocytochemistry study. Currently, we are attempting to develop recombinant variable domain of these heavy chain antibodies (VHH or nanobody) for tumor imaging and cancer therapy.

The Complete Nucleotide Sequence of the Human Immunoglobulin Heavy Chain Variable Region Locus

PubMed Central

Matsuda, Fumihiko; Ishii, Kazuo; Bourvagnet, Patrice; Kuma, Kei-ichi; Hayashida, Hidenori; Miyata, Takashi; Honjo, Tasuku

1998-01-01

The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified. The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of VH region was calculated to be ∼6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes. Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family. However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region. PMID:9841928

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

PubMed

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.

Complementarity and Compensation: Bridging the Gap between Writing and Design.

ERIC Educational Resources Information Center

Killingsworth, M. Jimmie; Sanders, Scott P.

1990-01-01

Outlines two rhetorical principles for producing iconic-mosaic texts--the principle of complementarity and the principle of compensation. Shows how these principles can be applied to practical problems in coordinating the writing and design processes in student projects. (RS)

Educational Finance Policy: A Search for Complementarities.

ERIC Educational Resources Information Center

Geske, Terry G.

1983-01-01

An overview of recent state level policy developments and policy analysis research as related to equity and efficiency objectives in public school finance is presented. Emphasis is placed on identifying complementarities, rather than the tradeoffs, between equity and efficiency criteria. (Author/LC)

Is the firewall consistent? Gedanken experiments on black hole complementarity and firewall proposal

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

Hwang, Dong-il; Lee, Bum-Hoon; Yeom, Dong-han, E-mail: dongil.j.hwang@gmail.com, E-mail: bhl@sogang.ac.kr, E-mail: innocent.yeom@gmail.com

2013-01-01

In this paper, we discuss the black hole complementarity and the firewall proposal at length. Black hole complementarity is inevitable if we assume the following five things: unitarity, entropy-area formula, existence of an information observer, semi-classical quantum field theory for an asymptotic observer, and the general relativity for an in-falling observer. However, large N rescaling and the AMPS argument show that black hole complementarity is inconsistent. To salvage the basic philosophy of the black hole complementarity, AMPS introduced a firewall around the horizon. According to large N rescaling, the firewall should be located close to the apparent horizon. We investigatemore » the consistency of the firewall with the two critical conditions: the firewall should be near the time-like apparent horizon and it should not affect the future infinity. Concerning this, we have introduced a gravitational collapse with a false vacuum lump which can generate a spacetime structure with disconnected apparent horizons. This reveals a situation that there is a firewall outside of the event horizon, while the apparent horizon is absent. Therefore, the firewall, if it exists, not only does modify the general relativity for an in-falling observer, but also modify the semi-classical quantum field theory for an asymptotic observer.« less

Is the firewall consistent? Gedanken experiments on black hole complementarity and firewall proposal

NASA Astrophysics Data System (ADS)

Hwang, Dong-il; Lee, Bum-Hoon; Yeom, Dong-han

2013-01-01

In this paper, we discuss the black hole complementarity and the firewall proposal at length. Black hole complementarity is inevitable if we assume the following five things: unitarity, entropy-area formula, existence of an information observer, semi-classical quantum field theory for an asymptotic observer, and the general relativity for an in-falling observer. However, large N rescaling and the AMPS argument show that black hole complementarity is inconsistent. To salvage the basic philosophy of the black hole complementarity, AMPS introduced a firewall around the horizon. According to large N rescaling, the firewall should be located close to the apparent horizon. We investigate the consistency of the firewall with the two critical conditions: the firewall should be near the time-like apparent horizon and it should not affect the future infinity. Concerning this, we have introduced a gravitational collapse with a false vacuum lump which can generate a spacetime structure with disconnected apparent horizons. This reveals a situation that there is a firewall outside of the event horizon, while the apparent horizon is absent. Therefore, the firewall, if it exists, not only does modify the general relativity for an in-falling observer, but also modify the semi-classical quantum field theory for an asymptotic observer.

Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA

PubMed Central

Howe, Kenneth James; Ares, Manuel

1997-01-01

Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA–RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5′ splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5′ splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing. PMID:9356473

Polymorphisms in the F Pocket of HLA-B27 Subtypes Strongly Affect Assembly, Chaperone Interactions, and Heavy-Chain Misfolding.

PubMed

Guiliano, David B; North, Helen; Panayoitou, Eleni; Campbell, Elaine C; McHugh, Kirsty; Cooke, Fiona G M; Silvestre, Marine; Bowness, Paul; Powis, Simon J; Antoniou, Antony N

2017-03-01

HLA-B27 is associated with the inflammatory spondyloarthritides (SpA), although subtypes HLA-B*27:06 and HLA-B*27:09 are not. These subtypes differ from the HLA-B*27:05 disease-associated allele primarily at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen-binding groove. Dimerization of HLA-B27 during assembly has been implicated in disease onset. The purpose of this study was to investigate the factors that influence differences in dimerization between disease-associated and non-disease-associated HLA-B27 alleles. HLA-B*27:05 and mutants resembling the HLA-B*27:06 and 09 subtypes were expressed in the rat C58 T cell line, the human CEM T cell line and its calnexin-deficient variant CEM.NKR. Immunoprecipitation, pulse-chase experiments, flow cytometry, and immunoblotting were performed to study the assembly kinetics, heavy-chain dimerization, and chaperone associations. By expressing HLA-B*27:05, 06-like, and 09 alleles on a restrictive rat transporter associated with antigen processing background, we demonstrate that a tyrosine expressed at p116, either alone or together with an aspartic acid residue at p114, inhibited HLA-B27 dimerization and increased the assembly rate. F-pocket residues altered the associations with chaperones of the early major histocompatibility complex class I folding pathway. Calnexin was demonstrated to participate in endoplasmic reticulum (ER) stress-mediated degradation of dimers, whereas the oxidoreductase ERp57 does not appear to influence dimerization. Residues within the F pocket of the peptide-binding groove, which differ between disease-associated and non-disease-associated HLA-B27 subtypes, can influence the assembly process and heavy-chain dimerization, events which have been linked to the initiation of disease pathogenesis. © 2016, American College of Rheumatology.

ALLOTYPE EXCLUSION IN UNIFORM RABBIT ANTIBODY TO STREPTOCOCCAL CARBOHYDRATE

PubMed Central

Kindt, Thomas J.; Todd, Charles W.; Eichmann, Klaus; Krause, Richard M.

1970-01-01

Rabbit antibodies to streptococcal polysaccharide are described which show selectivity of expression of the allotypic specificities on both the heavy (H) and light (L) chains. One of these antibodies binds weakly to Sephadex. A purification method based on this binding has yielded antibody completely lacking any group a allotypic marker on its H chains. PMID:5419853

Analysis of immunoglobulin heavy and light chain variable genes in post-transplant lymphoproliferative disorders.

PubMed

Capello, Daniela; Cerri, Michaela; Muti, Giuliana; Lucioni, Marco; Oreste, Pierluigi; Gloghini, Annunziata; Berra, Eva; Deambrogi, Clara; Franceschetti, Silvia; Rossi, Davide; Alabiso, Oscar; Morra, Enrica; Rambaldi, Alessandro; Carbone, Antonino; Paulli, Marco; Gaidano, Gianluca

2006-12-01

Post-transplant lymphoproliferative disorders (PTLD) derive from antigen-experienced B-cells and represent a major complication of solid organ transplantation. We characterized usage, mutation frequency and mutation pattern of immunoglobulin variable (IGV) gene rearrangements in 50 PTLD (polymorphic PTLD, n=10; diffuse large B-cell lymphoma, n=35; and Burkitt/Burkitt-like lymphoma, n=5). Among PTLD yielding clonal IGV amplimers, a functional IGV heavy chain (IGHV) rearrangement was found in 40/50 (80.0%) cases, whereas a potentially functional IGV light chain rearrangement was identified in 36/46 (78.3%) PTLD. By combining IGHV and IGV light chain rearrangements, 10/50 (20.0%) PTLD carried crippling mutations, precluding expression of a functional B-cell receptor (BCR). Immunohistochemistry showed detectable expression of IG light chains in only 18/43 (41.9%) PTLD. Failure to detect a functional IGV rearrangement associated with lack of IGV expression. Our data suggest that a large fraction of PTLD arise from germinal centre (GC)-experienced B-cells that display impaired BCR. Since a functional BCR is required for normal B-cell survival during GC transit, PTLD development may implicate rescue from apoptosis and expansion of B-cells that have failed the GC reaction. The high frequency of IGV loci inactivation appears to be a peculiar feature of PTLD among immunodeficiency-associated lymphoproliferations.

Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1

PubMed Central

Mazor, Masha; Alkrinawi, Soliman; Chalifa-Caspi, Vered; Manor, Esther; Sheffield, Val C.; Aviram, Micha; Parvari, Ruti

2011-01-01

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. PMID:21496787

Strong contributions from vertical triads to helix-partner preferences in parallel coiled coils.

PubMed

Steinkruger, Jay D; Bartlett, Gail J; Woolfson, Derek N; Gellman, Samuel H

2012-09-26

Pairing preferences in heterodimeric coiled coils are determined by complementarities among side chains that pack against one another at the helix-helix interface. However, relationships between dimer stability and interfacial residue identity are not fully understood. In the context of the "knobs-into-holes" (KIH) packing pattern, one can identify two classes of interactions between side chains from different helices: "lateral", in which a line connecting the adjacent side chains is perpendicular to the helix axes, and "vertical", in which the connecting line is parallel to the helix axes. We have previously analyzed vertical interactions in antiparallel coiled coils and found that one type of triad constellation (a'-a-a') exerts a strong effect on pairing preferences, while the other type of triad (d'-d-d') has relatively little impact on pairing tendencies. Here, we ask whether vertical interactions (d'-a-d') influence pairing in parallel coiled-coil dimers. Our results indicate that vertical interactions can exert a substantial impact on pairing specificity, and that the influence of the d'-a-d' triad depends on the lateral a' contact within the local KIH motif. Structure-informed bioinformatic analyses of protein sequences reveal trends consistent with the thermodynamic data derived from our experimental model system in suggesting that heterotriads involving Leu and Ile are preferred over homotriads involving Leu and Ile.

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20.

PubMed

Lin, Su-Chang; Chung, Jee Y; Lamothe, Betty; Rajashankar, Kanagalaghatta; Lu, Miao; Lo, Yu-Chih; Lam, Amy Y; Darnay, Bryant G; Wu, Hao

2008-02-15

Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.

Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdol-Amir

2006-02-01

The methylotrophic yeast Pichia pastoris has become a highly popular expression host system for the recombinant production of a wide variety of proteins, such as antibody fragments. Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy chain antibody (VHH). To test the feasibility of expressing VHHs in the yeast, which on account of their small size and antigen recognition properties would have a major impact on antibody engineering strategies, we constructed two VHH genes encoding the single-domain antibody fragments with specificity for a cancer associated mucin, MUC1. The recombinant strains of the yeast P. pastoris were developed which secrete single-domain antibody fragment to the culture supernatant as a biologically active protein. Supplementation of medium with sorbitol (in pre-induction phase) and casamino acid or EDTA (in induction phase) provided ideal condition of increasing the yield of VHH production compared to culture condition devoid of above recipe. The secreted protein was purified following a 80% ammonium sulfate precipitation step, followed by a affinity chromatography column. The specific activity in enzyme-linked immunosorbant assay (ELISA) of the purified yeast VHH was higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level and correctly folded production of VHH antibody fragments with potential in vivo diagnostic and therapeutic applications. This is the first report of expression of VHH in P. pastoris.

Characterization of a Gene Encoding Clathrin Heavy Chain in Maize Up-Regulated by Salicylic Acid, Abscisic Acid and High Boron Supply

PubMed Central

Zeng, Mu-Heng; Liu, Sheng-Hong; Yang, Miao-Xian; Zhang, Ya-Jun; Liang, Jia-Yong; Wan, Xiao-Rong; Liang, Hong

2013-01-01

Clathrin, a three-legged triskelion composed of three clathrin heavy chains (CHCs) and three light chains (CLCs), plays a critical role in clathrin-mediated endocytosis (CME) in eukaryotic cells. In this study, the genes ZmCHC1 and ZmCHC2 encoding clathrin heavy chain in maize were cloned and characterized for the first time in monocots. ZmCHC1 encodes a 1693-amino acid-protein including 29 exons and 28 introns, and ZmCHC2 encodes a 1746-amino acid-protein including 28 exons and 27 introns. The high similarities of gene structure, protein sequences and 3D models among ZmCHC1, and Arabidopsis AtCHC1 and AtCHC2 suggest their similar functions in CME. ZmCHC1 gene is predominantly expressed in maize roots instead of ubiquitous expression of ZmCHC2. Consistent with a typical predicted salicylic acid (SA)-responsive element and four predicted ABA-responsive elements (ABREs) in the promoter sequence of ZmCHC1, the expression of ZmCHC1 instead of ZmCHC2 in maize roots is significantly up-regulated by SA or ABA, suggesting that ZmCHC1 gene may be involved in the SA signaling pathway in maize defense responses. The expressions of ZmCHC1 and ZmCHC2 genes in maize are down-regulated by azide or cold treatment, further revealing the energy requirement of CME and suggesting that CME in plants is sensitive to low temperatures. PMID:23880865

Chronic lymphocytic leukemia patients exposed to ionizing radiation due to the Chernobyl NPP accident--with focus on immunoglobulin heavy chain gene analysis.

PubMed

Abramenko, Iryna; Bilous, Nadia; Chumak, Anatoliy; Davidova, Ekaterina; Kryachok, Iryna; Martina, Zoya; Nechaev, Stanislav; Dyagil, Iryna; Bazyka, Dmytriy; Bebeshko, Vladimir

2008-04-01

Clinical data and immunoglobulin variable heavy chain (IgVH) gene configuration were analyzed in 47 CLL patients, exposed to ionizing radiation (IR) due to Chernobyl NPP accident, and 141 non-exposed patients. Clean-up workers of the second quarter of 1986 (n=19) were picked out as separate group with the highest number of unmutated cases (94.4%), increased usage of IgVH1-69 (33.3%) and IgVH3-21 (16.7%) genes, high frequency of secondary solid tumors (6 cases) and Richter transformation (4 cases). These preliminary data suggest that CLL in the most suffered contingent due to Chernobyl NPP accident might have some specific features.

Major histocompatibility complex class I molecules modulate embryonic neuritogenesis and neuronal polarization

PubMed Central

Bilousova, Tina; Dang, Hoa; Xu, Willem; Gustafson, Sarah; Jin, Yingli; Wickramasinghe, Lalinda; Won, Tony; Bobarnac, Gabriela; Middleton, Blake; Tian, Jide; Kaufman, Daniel L.

2012-01-01

We studied cultured hippocampal neurons from embryonic wildtype, major histocompatibility complex class I (MHCI) heavy chain-deficient (KbDb−/−) and NSE-Db (which have elevated neuronal MHCI expression) C57BL/6 mice. KbDb−/− neurons displayed slower neuritogenesis and establishment of polarity, while NSE-Db neurons had faster neurite outgrowth, more primary neurites, and tended to have accelerated polarization. Additional studies with ϐ2M−/− neurons, exogenous ϐ2M, and a self-MHCI monomer suggest that free heavy chain cis interactions with other surface molecules can promote neuritogenesis while tripartite MHCI interactions with classical MHCI receptors can inhibit axon outgrowth. Together with the results of others, MHCI appears to differentially modulate neuritogenesis and synaptogenesis. PMID:22503373

Atwood's Heavy Chain

NASA Astrophysics Data System (ADS)

Beeken, Paul

2011-11-01

While perusing various websites in search of a more challenging lab for my students, I came across a number of ideas where replacing the string in an Atwood's machine with a simple ball chain like the kind found in lamp pulls created an interesting system to investigate. The replacement of the string produced a nice nonuniform acceleration, but one that my AP® students found difficult to analyze given their current math background. As the year progressed, we began to explore the importance of work and its utility in making predictions on systems that did not lend themselves to easy analysis using Newtonian mechanics. The effort made it apparent that the heavy rope Atwood's machine would make a perfect system for investigation using the lessons gained from work and energy.

Functional traits explain ecosystem function through opposing mechanisms.

PubMed

Cadotte, Marc W

2017-08-01

The ability to explain why multispecies assemblages produce greater biomass compared to monocultures, has been a central goal in the quest to understand biodiversity effects on ecosystem function. Species contributions to ecosystem function can be driven by two processes: niche complementarity and a selection effect that is influenced by fitness (competitive) differences, and both can be approximated with measures of species' traits. It has been hypothesised that fitness differences are associated with few, singular traits while complementarity requires multidimensional trait measures. Here, using experimental data from plant assemblages, I show that the selection effect was strongest when trait dissimilarity was low, while complementarity was greatest with high trait dissimilarity. Selection effects were best explained by a single trait, plant height. Complementarity was correlated with dissimilarity across multiple traits, representing above and below ground processes. By identifying the relevant traits linked to ecosystem function, we obtain the ability to predict combinations of species that will maximise ecosystem function. © 2017 John Wiley & Sons Ltd/CNRS.

Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

PubMed

Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

2003-09-01

The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

Vaccines against Botulism.

PubMed

Sundeen, Grace; Barbieri, Joseph T

2017-09-02

Botulinum neurotoxins (BoNT) cause the flaccid paralysis of botulism by inhibiting the release of acetylcholine from motor neurons. There are seven serotypes of BoNT (A-G), with limited therapies, and no FDA approved vaccine for botulism. An investigational formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was used to vaccinate people who are at high risk of contracting botulism. However, this formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was losing potency and was discontinued. This article reviews the different vaccines being developed to replace the discontinued toxoid vaccine. These vaccines include DNA-based, viral vector-based, and recombinant protein-based vaccines. DNA-based vaccines include plasmids or viral vectors containing the gene encoding one of the BoNT heavy chain receptor binding domains (HC). Viral vectors reviewed are adenovirus, influenza virus, rabies virus, Semliki Forest virus, and Venezuelan Equine Encephalitis virus. Among the potential recombinant protein vaccines reviewed are HC, light chain-heavy chain translocation domain, and chemically or genetically inactivated holotoxin.

Vaccines against Botulism

PubMed Central

Sundeen, Grace; Barbieri, Joseph T.

2017-01-01

Botulinum neurotoxins (BoNT) cause the flaccid paralysis of botulism by inhibiting the release of acetylcholine from motor neurons. There are seven serotypes of BoNT (A-G), with limited therapies, and no FDA approved vaccine for botulism. An investigational formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was used to vaccinate people who are at high risk of contracting botulism. However, this formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was losing potency and was discontinued. This article reviews the different vaccines being developed to replace the discontinued toxoid vaccine. These vaccines include DNA-based, viral vector-based, and recombinant protein-based vaccines. DNA-based vaccines include plasmids or viral vectors containing the gene encoding one of the BoNT heavy chain receptor binding domains (HC). Viral vectors reviewed are adenovirus, influenza virus, rabies virus, Semliki Forest virus, and Venezuelan Equine Encephalitis virus. Among the potential recombinant protein vaccines reviewed are HC, light chain-heavy chain translocation domain, and chemically or genetically inactivated holotoxin. PMID:28869493

Molecular architecture of protein-RNA recognition sites.

PubMed

Barik, Amita; C, Nithin; Pilla, Smita P; Bahadur, Ranjit Prasad

2015-01-01

The molecular architecture of protein-RNA interfaces are analyzed using a non-redundant dataset of 152 protein-RNA complexes. We find that an average protein-RNA interface is smaller than an average protein-DNA interface but larger than an average protein-protein interface. Among the different classes of protein-RNA complexes, interfaces with tRNA are the largest, while the interfaces with the single-stranded RNA are the smallest. Significantly, RNA contributes more to the interface area than its partner protein. Moreover, unlike protein-protein interfaces where the side chain contributes less to the interface area compared to the main chain, the main chain and side chain contributions flipped in protein-RNA interfaces. We find that the protein surface in contact with the RNA in protein-RNA complexes is better packed than that in contact with the DNA in protein-DNA complexes, but loosely packed than that in contact with the protein in protein-protein complexes. Shape complementarity and electrostatic potential are the two major factors that determine the specificity of the protein-RNA interaction. We find that the H-bond density at the protein-RNA interfaces is similar with that of protein-DNA interfaces but higher than the protein-protein interfaces. Unlike protein-DNA interfaces where the deoxyribose has little role in intermolecular H-bonds, due to the presence of an oxygen atom at the 2' position, the ribose in RNA plays significant role in protein-RNA H-bonds. We find that besides H-bonds, salt bridges and stacking interactions also play significant role in stabilizing protein-nucleic acids interfaces; however, their contribution at the protein-protein interfaces is insignificant.

An overview of heavy-atom derivatization of protein crystals

PubMed Central

Pike, Ashley C. W.; Garman, Elspeth F.; Krojer, Tobias; von Delft, Frank; Carpenter, Elisabeth P.

2016-01-01

Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative. PMID:26960118

A molecular model for cocaine binding by the immunotherapeutic human/mouse chimeric monoclonal antibody 2E2.

PubMed

Lape, Michael; Paula, Stefan; Ball, William J

2010-06-01

Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (gamma1 heavy chain)/murine (lambda light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2's ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a homology model for the 3-D structure of its Fv fragment. Subsequent computational docking studies revealed the intermolecular interactions potentially responsible for mAb 2E2's cocaine binding properties. The driving force of cocaine binding was identified as a combination of hydrophobic interactions and a single hydrogen bond between a light chain tyrosine residue and a carbonyl oxygen atom of cocaine. The model also allowed for an in silico evaluation of single/double residue mutations in the heavy and light chain variable regions that might further enhance mAb 2E2's cocaine binding properties. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

A Molecular Model for Cocaine Binding by the Immunotherapeutic Human/Mouse Chimeric Monoclonal Antibody 2E2

PubMed Central

Lape, Michael; Paula, Stefan; Ball, William J.

2010-01-01

Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (γ1 heavy chain)/murine (λ light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2’s ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a homology model for the 3-D structure of its Fv fragment. Subsequent computational docking studies revealed the intermolecular interactions potentially responsible for mAb 2E2’s cocaine binding properties. The driving force of cocaine binding was identified as a combination of hydrophobic interactions and a single hydrogen bond between a light chain tyrosine residue and a carbonyl oxygen atom of cocaine. The model also allowed for an in silico evaluation of single/double residue mutations in the heavy and light chain variable regions that might further enhance mAb 2E2’s cocaine binding properties. PMID:20185210

Follow-up of IgD-κ multiple myeloma by monitoring free light chains and total heavy chain IgD: A case report

PubMed Central

De Santis, Elena; Masi, Serena; Cordone, Iole; Pisani, Francesco; Zuppi, Cecilia; Mattei, Fabrizio; Conti, Laura; Cigliana, Giovanni

2016-01-01

Immunoglobulin (Ig)D-κ multiple myeloma (MM) is a rare neoplastic disease characterized by an aggressive and rapidly progressing course, which constitutes only a very small proportion of all MM cases. In the present report, the clinical case of a 51-year-old Caucasian woman diagnosed with IgD-κ MM is described. The patient underwent different chemotherapeutic treatments subsequently to a single autologous stem cell transplantation. Despite the inherent difficulty of monitoring IgD levels and performing serum immunofixation electrophoresis, the clinical outcome of the patient was almost uniquely monitored by measuring the levels of κ and λ free light chains (FLCs) and total heavy chain IgD. The data suggest the non-invasive potential and usefulness of FLCs evaluation for early detection of stringent complete remission, follow-up and early detection of disease relapse. In addition, this diagnostic procedure has successfully been employed for the therapeutic monitoring of the present patient, and may represent a very helpful, non-invasive tool for the follow-up of IgD myeloma patients without the requirement of serial bone marrow aspirate. PMID:27588135

Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus.

PubMed

Sun, S; Mo, W; Ji, Y; Liu, S

2001-01-01

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Fab, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions. Copyright 2001 John Wiley & Sons, Ltd.

Climate change mitigation and adaptation in the land use sector: from complementarity to synergy.

PubMed

Duguma, Lalisa A; Minang, Peter A; van Noordwijk, Meine

2014-09-01

Currently, mitigation and adaptation measures are handled separately, due to differences in priorities for the measures and segregated planning and implementation policies at international and national levels. There is a growing argument that synergistic approaches to adaptation and mitigation could bring substantial benefits at multiple scales in the land use sector. Nonetheless, efforts to implement synergies between adaptation and mitigation measures are rare due to the weak conceptual framing of the approach and constraining policy issues. In this paper, we explore the attributes of synergy and the necessary enabling conditions and discuss, as an example, experience with the Ngitili system in Tanzania that serves both adaptation and mitigation functions. An in-depth look into the current practices suggests that more emphasis is laid on complementarity-i.e., mitigation projects providing adaptation co-benefits and vice versa rather than on synergy. Unlike complementarity, synergy should emphasize functionally sustainable landscape systems in which adaptation and mitigation are optimized as part of multiple functions. We argue that the current practice of seeking co-benefits (complementarity) is a necessary but insufficient step toward addressing synergy. Moving forward from complementarity will require a paradigm shift from current compartmentalization between mitigation and adaptation to systems thinking at landscape scale. However, enabling policy, institutional, and investment conditions need to be developed at global, national, and local levels to achieve synergistic goals.

Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat.

PubMed

Andruchov, Oleg; Galler, Stefan

2008-03-01

This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

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

PubMed Central

Rumfelt, Lynn L.; Avila, David; Diaz, Marilyn; Bartl, Simona; McKinney, E. Churchill; Flajnik, Martin F.

2001-01-01

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, IgM1gj, 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 H1gj 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. H1gj 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 IgM1gj. 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. PMID:11172027

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

PubMed Central

Housset, D; Mazza, G; Grégoire, C; Piras, C; Malissen, B; Fontecilla-Camps, J C

1997-01-01

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand. PMID:9250664

Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8+ T cell response.

PubMed

Culshaw, Abigail; Ladell, Kristin; Gras, Stephanie; McLaren, James E; Miners, Kelly L; Farenc, Carine; van den Heuvel, Heleen; Gostick, Emma; Dejnirattisai, Wanwisa; Wangteeraprasert, Apirath; Duangchinda, Thaneeya; Chotiyarnwong, Pojchong; Limpitikul, Wannee; Vasanawathana, Sirijitt; Malasit, Prida; Dong, Tao; Rossjohn, Jamie; Mongkolsapaya, Juthathip; Price, David A; Screaton, Gavin R

2017-11-01

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) β-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8 + T cell populations specific for variants of the nonstructural protein epitope NS3 133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3 133 -DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2 + TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second β-chain complementarity-determining region (CDR2β). Extensive mutagenesis studies of three distinct TRBV11-2 + TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2β loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.

Crystal structures of ricin toxin's enzymatic subunit (RTA) in complex with neutralizing and non-neutralizing single-chain antibodies.

PubMed

Rudolph, Michael J; Vance, David J; Cheung, Jonah; Franklin, Matthew C; Burshteyn, Fiana; Cassidy, Michael S; Gary, Ebony N; Herrera, Cristina; Shoemaker, Charles B; Mantis, Nicholas J

2014-08-26

Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Symmetry of Fv architecture is conducive to grafting a second antibody binding site in the Fv region.

PubMed Central

Keck, P C; Huston, J S

1996-01-01

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single-chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovered an architectural basis common to many Fv regions that permits grafting a chi-site onto the Fv surface that diametrically opposes the normal combining site. By using molecular graphics analysis, chimeric complementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an overall geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions for inserting chi CDRs are very highly conserved, as are the corresponding chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply this approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins. Images FIGURE 2 FIGURE 3 PMID:8889174

Response of rhizosphere microbial community structure and diversity to heavy metal co-pollution in arable soil.

PubMed

Deng, Linjing; Zeng, Guangming; Fan, Changzheng; Lu, Lunhui; Chen, Xunfeng; Chen, Ming; Wu, Haipeng; He, Xiaoxiao; He, Yan

2015-10-01

Due to the emerging environmental issues related to heavy metals, concern about the soil quality of farming lands near manufacturing district is increasing. Investigating the function of soil microorganisms exposed to long-term heavy metal contamination is meaningful and important for agricultural soil utilization. This article studied the potential influence of several heavy metals on microbial biomass, activity, abundance, and community composition in arable soil near industrial estate in Zhuzhou, Hunan province, China. The results showed that soil organic contents (SOC) were significantly positive correlated with heavy metals, whereas dehydrogenase activity (DHA) was greatly depressed by the heavy metal stress. Negative correlation was found between heavy metals and basal soil respiration (BSR), and no correlation was found between heavy metals and microbial biomass content (MBC). The quantitative PCR (QPCR) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis could suggest that heavy metal pollution has significantly decreased abundance of bacteria and fungi and also changed their community structure. The results could contribute to evaluate heavy metal pollution level in soil. By combining different environmental parameters, it would promote the better understanding of heavy metal effect on the size, structure, and activity of microbial community in arable soil.

Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

PubMed

Shinohara, Mayuka; Sumino, Yasuhiro; Sato, Fuminori; Kiyono, Tohru; Hashimoto, Naohiro; Mimata, Hiromitsu

2017-06-01

To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence. © 2017 The Japanese Urological Association.

Three IgH isotypes, IgM, IgA and IgY are expressed in Gentoo penguin and zebra finch.

PubMed

Han, Binyue; Li, Yan; Han, Haitang; Zhao, Yaofeng; Pan, Qingjie; Ren, Liming

2017-01-01

Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (Taeniopygia guttata) and Gentoo penguin (Pygoscelis papua), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.

A Motif in the Clathrin Heavy Chain Required for the Hsc70/Auxilin Uncoating Reaction

PubMed Central

Rapoport, Iris; Boll, Werner; Yu, Anan; Böcking, Till

2008-01-01

The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent “disassembly enzyme” for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631–1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70–facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone. PMID:17978091

Expression and biological effects of high levels of serum IgE in epsilon heavy chain transgenic mice.

PubMed

Adamczewski, M; Köhler, G; Lamers, M C

1991-03-01

We have generated and examined transgenic mice carrying a rearranged immunoglobulin transgene coding for the heavy chain of an IgE antibody. These mice produce the secreted form of the recombinant epsilon heavy chain. Serum IgE levels were increased at least 100-fold over control values. Transgenic epsilon mRNA was detected in spleen and thymus, not in liver and heart. Transgenic epsilon production in vitro was slightly up-regulated by T cells, but not affected by interleukin 4 in vitro or Nippostrongylus infestation in vivo. The B cell and T cell compartments and antigen-specific IgE, IgG1 and IgM responses as well as the increase in endogenous IgE after Nippostrongylus infestation in transgenic mice were normal. These data indicate that the presence of high levels of transgenic IgE did not induce class-specific suppressive mechanisms. Transgenic IgE bound to Fc epsilon receptor type I and Fc epsilon receptor type II and mediated histamine release from mast cells in vitro and an allergic skin reaction in vivo. It inhibited an ovalbumin-specific skin reaction in ovalbumin-immunized transgenic mice only during the initial phases of the immune response. This result has a bearing on the feasibility of immune therapy of allergic diseases with substances that block binding of IgE to its receptors.

Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed

PubMed Central

Shaffer, Justin F.; Kier, William M.

2012-01-01

SUMMARY The speed of muscle contraction is largely controlled at the sarcomere level by the ATPase activity of the motor protein myosin. Differences in amino acid sequence in catalytically important regions of myosin yield different myosin isoforms with varying ATPase activities and resulting differences in cross-bridge cycling rates and interfilamentary sliding velocities. Modulation of whole-muscle performance by changes in myosin isoform ATPase activity is regarded as a universal mechanism to tune contractile properties, especially in vertebrate muscles. Invertebrates such as squid, however, may exhibit an alternative mechanism to tune contractile properties that is based on differences in muscle ultrastructure, including variable myofilament and sarcomere lengths. To determine definitively whether contractile properties of squid muscles are regulated via different myosin isoforms (i.e. different ATPase activities), the nucleotide and amino acid sequences of the myosin heavy chain from the squid Doryteuthis pealeii were determined from the mantle, arm, tentacle, fin and funnel retractor musculature. We identified three myosin heavy chain isoforms in squid muscular tissues, with differences arising at surface loop 1 and the carboxy terminus. All three isoforms were detected in all five tissues studied. These results suggest that the muscular tissues of D. pealeii express identical myosin isoforms, and it is likely that differences in muscle ultrastructure, not myosin ATPase activity, represent the most important mechanism for tuning contractile speeds. PMID:22189767

Myosin heavy chain profile of equine gluteus medius muscle following prolonged draught-exercise training and detraining.

PubMed

Serrano, A L; Rivero, J L

2000-04-01

Fourteen 4-year old Andalusian mares were used to examine the plasticity of myosin heavy chain (MHC) composition in horse skeletal muscle with heavy draught-exercise training and detraining. Seven horses underwent a training programme based on carriage exercises for 8 months. Afterwards, they were kept in paddocks for 3 months. The remaining seven animals were used as control horses. Three gluteus medius muscle biopsies were removed at depths of 20, 40 and 60 mm from each horse before (month 0), during the training (months 3 and 8) and after detraining (month 11). Myosin heavy chain composition was analysed by electrophoresis and immunohistochemically with anti-MHC monoclonal antibodies. Fibre areas, oxidative capacity and capillaries were studied histochemically. After 8 months of training, MHC-IIX and IIX fibres decreased whereas MHC-I and type I and I + IIA fibres increased. Neither MHC-IIA nor the percentage of IIA fibres changed when the data were considered as a whole, but the proportion of MHC-IIA increased in the superficial region of the muscle after 8 months of training. Mean areas of type II fibres were not affected by training and detraining, but the cross-sectional of type I fibres increased after 3 month of training and not further increases were recorded afterward. The percentage of high-oxidative capacity fibres and the number of capillaries per mm2 increased with training. Most of these muscular adaptations reverted after detraining. These results indicate that long term draught-exercise training induces a reversible transition of MHC composition in equine muscle in the order IIX --> IIA --> I. The physiological implication of these changes is an impact on the velocity of shortening and fatigue resistance of muscle fibres.

Heavy metals in vegetables and respective soils irrigated by canal, municipal waste and tube well waters.

PubMed

Ismail, Amir; Riaz, Muhammad; Akhtar, Saeed; Ismail, Tariq; Amir, Mamoona; Zafar-ul-Hye, Muhammad

2014-01-01

Heavy metal contamination in the food chain is of serious concern due to the potential risks involved. The results of this study revealed the presence of maximum concentration of heavy metals in the canal followed by sewerage and tube well water. Similarly, the vegetables and respective soils irrigated with canal water were found to have higher heavy metal contamination followed by sewerage- and tube-well-watered samples. However, the heavy metal content of vegetables under study was below the limits as set by FAO/WHO, except for lead in canal-water-irrigated spinach (0.59 mg kg(-1)), radish pods (0.44 mg kg(-1)) and bitter gourd (0.33 mg kg(-1)). Estimated daily intakes of heavy metals by the consumption of selected vegetables were found to be well below the maximum limits. However, a complete estimation of daily intake requires the inclusion of other dietary and non-dietary exposure sources of heavy metals.

Structural Mimicry of the Dengue Virus Envelope Glycoprotein Revealed by the Crystallographic Study of an Idiotype–Anti-idiotype Fab Complex

PubMed Central

Wong, Yee Hwa; Goh, Boon Chong; Lim, She Yah; Teo, En Wei; Lim, Angeline P. C.; Dedon, Pete C.; Hanson, Brendon J.

2017-01-01

ABSTRACT A detailed understanding of the fine specificity of serotype-specific human antibodies is vital for the development and evaluation of new vaccines for pathogenic flaviviruses such as dengue virus (DENV) and Zika virus. In this study, we thoroughly characterize the structural footprint of an anti-idiotype antibody (E1) specific for a potent, fully human DENV serotype 1-specific antibody, termed HM14c10, derived from a recovered patient. The crystal structure at a resolution of 2.5 Å of a complex between the Fab fragments of E1 and HM14c10 provides the first detailed molecular comparison of an anti-idiotype paratope specific for a human antibody with its analogous epitope, a discontinuous quaternary structure located at the surface of the viral particle that spans adjacent envelope (E) proteins. This comparison reveals that the footprints left by E1 and E on HM14c10 largely overlap, explaining why the formation of binary complexes is mutually exclusive. Structural mimicry of the DENV E epitope by the E1 combining site is achieved via the formation of numerous interactions with heavy chain complementarity domain regions (CDRs) of HM14c10, while fewer interactions are observed with its light chain than for the E protein. We show that E1 can be utilized to detect HM14c10-like antibodies in sera from patients who recovered from DENV-1, infection suggesting that this is a public (common) idiotype. These data demonstrate the utility of employing an anti-idiotype antibody to monitor a patient's specific immune responses and suggest routes for the improvement of E “mimicry” by E1 by increasing its recognition of the Fab HM14c10 light chain CDRs. IMPORTANCE A chimeric yellow fever-dengue live-attenuated tetravalent vaccine is now being marketed. Dengue remains a significant public health problem, because protection conferred by this vaccine against the four circulating serotypes is uneven. Reliable tools must be developed to measure the immune responses of individuals exposed to DENV either via viral infection or through vaccination. Anti-idiotypic antibodies provide precision tools for analyzing the pharmacokinetics of antibodies in an immune response and also for measuring the amount of circulating anti-infective therapeutic antibodies. Here, we characterize how an anti-idiotypic antibody (E1) binds antibody HM14c10, which potently neutralizes DENV serotype 1. We report the crystal structure at a resolution of 2.5 Å of a complex between the Fab fragments of E1 and HM14c10 and provide the first detailed molecular comparison between the anti-idiotype surface and its analogous epitope located at the surface of the dengue virus particle. PMID:28637753

[The heavy ion irradiation influence on the thermodynamic parameters of liquids in human body].

PubMed

Vlasenko, T S; Bulavin, L A; Sysoev, V M

2014-01-01

In this manuscript a theoretical model describing the influence of the heavy ion radiotherapy on the liquid matter in the human body is suggested. Based on the fundamental equations of Bogoliubov chain the effective temperatures in the case of constant particles fluent are found in the context of single component model. An existence of such temperatures allows the use of equilibrium thermodynamics formalism to nonequilibrium stationary state. The obtained results provide the possibility of predicting the liquid matter structural changes in the biological systems in the area influenced by the heavy ion beams.

Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1.

PubMed

Mazor, Masha; Alkrinawi, Soliman; Chalifa-Caspi, Vered; Manor, Esther; Sheffield, Val C; Aviram, Micha; Parvari, Ruti

2011-05-13

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The eukaryotic cell originated in the integration and redistribution of hyperstructures from communities of prokaryotic cells based on molecular complementarity.

PubMed

Norris, Vic; Root-Bernstein, Robert

2009-06-04

In the "ecosystems-first" approach to the origins of life, networks of non-covalent assemblies of molecules (composomes), rather than individual protocells, evolved under the constraints of molecular complementarity. Composomes evolved into the hyperstructures of modern bacteria. We extend the ecosystems-first approach to explain the origin of eukaryotic cells through the integration of mixed populations of bacteria. We suggest that mutualism and symbiosis resulted in cellular mergers entailing the loss of redundant hyperstructures, the uncoupling of transcription and translation, and the emergence of introns and multiple chromosomes. Molecular complementarity also facilitated integration of bacterial hyperstructures to perform cytoskeletal and movement functions.

Computational studies of new potential antimalarial compounds Stereoelectronic complementarity with the receptor

NASA Astrophysics Data System (ADS)

Portela, César; Afonso, Carlos M. M.; Pinto, Madalena M. M.; João Ramos, Maria

2003-09-01

One of the most important pharmacological mechanisms of antimalarial action is the inhibition of the aggregation of hematin into hemozoin. We present a group of new potential antimalarial molecules for which we have performed a DFT study of their stereoelectronic properties. Additionally, the same calculations were carried out for the two putative drug receptors involved in the referred activity, i.e., hematin μ-oxo dimer and hemozoin. A complementarity between the structural and electronic profiles of the planned molecules and the receptors can be observed. A docking study of the new compounds in relation to the two putative receptors is also presented, providing a correlation with the defined electrostatic complementarity.

Myosin heavy chain composition in the rat diaphragm - Effect of age and exercise training

NASA Technical Reports Server (NTRS)

Gosselin, Luc E.; Betlach, Michael; Vailas, Arthur C.; Greaser, Marion L.; Thomas, D. P.

1992-01-01

The effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Treadmill running at 75 percent maximal oxygen consumption resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23mo) trained animals (P less than 0.05). It was found that the ratio of fast to slow MHC was significantly higher (P less than 0.005) in the crural compared with costal diaphragm region in both age groups. A significant age-related increase in persentage of slow MHC was observed in both diaphragm regions. The relative proportion of slow MHC in either costal or crural region was not changed by exercise training.

Adult fast myosin pattern and Ca2+-induced slow myosin pattern in primary skeletal muscle culture

PubMed Central

Kubis, Hans-Peter; Haller, Ernst-August; Wetzel, Petra; Gros, Gerolf

1997-01-01

A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca2+ ionophore is added to the culture medium on day 11, raising intracellular [Ca2+] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs. PMID:9108130

Characterization of a highly polymorphic region 5′ to JH in the human immunoglobulin heavy chain

PubMed Central

Silva, Alcino J.; Johnson, John P.; White, Raymond L.

1987-01-01

A cloned DNA segment 1.25 kilobases (kb) upstream from the joining segments of the human heavy chain immunoglobulin gene revealed extensive polymorphic variation at this locus, and the polymorphic pattern was stably transmitted to the next generation. Genomic restriction analysis showed that the polymorphism was caused by insertions/deletions within an MspI/BamHI fragment. Sequencing of one allele, 848 base pairs (bp) long, revealed eleven 50-base-pair tandem repeats. A second allele, 648 bp long, was cloned from a human genomic cosmid library, sequenced, and found to contain four fewer repeats than the first allele. A survey of 186 chromosomes from unrelated individuals of primarily northern European descent revealed at least six alleles. Images PMID:2884636

Evolution of the Iga Heavy Chain Gene in the Genus Mus

PubMed Central

Osborne, B. A.; Golde, T. E.; Schwartz, R. L.; Rudikoff, S.

1988-01-01

To examine questions of immunoglobulin gene evolution, the IgA α heavy chain gene from Mus pahari, an evolutionarily distant relative to Mus musculus domesticus, was cloned and sequenced. The sequence, when compared to the IgA gene of BALB/c or human, demonstrated that the IgA gene is evolving in a mosaic fashion with the hinge region accumulating mutations most rapidly and the third domain at a considerably lower frequency. In spite of this pronounced accumulation of mutations, the hinge region appears to maintain the conformation of a random coil. A marked propensity to accumulate replacement over silent site changes in the coding regions was noted, as was a definite codon bias. The possibility that these two phenomena are interrelated is discussed. PMID:2842228

Six National Chains: Six Approaches to Development. Twelfth Annual Status Report on For Profit Child Care.

ERIC Educational Resources Information Center

Neugebauer, Roger

1999-01-01

While the nation's six largest for-profit child care organizations followed the same path of rapid growth in the 1970s and 1980s, slower growth in the 1990s has led the chains in different directions. Two organizations plagued by heavy financial losses have greatly reduced their expansion, while others have focused on moderate growth or…

Antagonistic and synergistic toxic effects of Pb and Cd in a simple food chain: nematodes feeding on bacteria and fungi

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

Doelman, P.; Nieboer, G.; Schrooten, J.

1984-06-01

Soil pollution with heavy metals may affect the functioning of the soil biota by inhibiting the decomposition of organic matter and may also influence food chains. This paper presents the results of an investigation on how the reproduction of soil nematodes can be influenced by feeding on bacteria or fungi contaminated with lead and cadmium.

Cell surface engineering of microorganisms towards adsorption of heavy metals.

PubMed

Li, Peng-Song; Tao, Hu-Chun

2015-06-01

Heavy metal contamination has become a worldwide environmental concern due to its toxicity, non-degradability and food-chain bioaccumulation. Conventional physical and chemical treatment methods for heavy metal removal have disadvantages such as cost-intensiveness, incomplete removal, secondary pollution and the lack of metal specificity. Microbial biomass-based biosorption is one of the approaches gaining increasing attention because it is effective, cheap, and environmental friendly and can work well at low concentrations. To enhance the adsorption properties of microbial cells to heavy metal ions, the cell surface display of various metal-binding proteins/peptides have been performed using a cell surface engineering approach. The surface engineering of Gram-negative bacteria, Gram-positive bacteria and yeast towards the adsorption of heavy metals are reviewed in this article. The problems and future perspectives of this technology are discussed.

MCCE2: improving protein pKa calculations with extensive side chain rotamer sampling.

PubMed

Song, Yifan; Mao, Junjun; Gunner, M R

2009-11-15

Multiconformation continuum electrostatics (MCCE) explores different conformational degrees of freedom in Monte Carlo calculations of protein residue and ligand pK(a)s. Explicit changes in side chain conformations throughout a titration create a position dependent, heterogeneous dielectric response giving a more accurate picture of coupled ionization and position changes. The MCCE2 methods for choosing a group of input heavy atom and proton positions are described. The pK(a)s calculated with different isosteric conformers, heavy atom rotamers and proton positions, with different degrees of optimization are tested against a curated group of 305 experimental pK(a)s in 33 proteins. QUICK calculations, with rotation around Asn and Gln termini, sampling His tautomers and torsion minimum hydroxyls yield an RMSD of 1.34 with 84% of the errors being <1.5 pH units. FULL calculations adding heavy atom rotamers and side chain optimization yield an RMSD of 0.90 with 90% of the errors <1.5 pH unit. Good results are also found for pK(a)s in the membrane protein bacteriorhodopsin. The inclusion of extra side chain positions distorts the dielectric boundary and also biases the calculated pK(a)s by creating more neutral than ionized conformers. Methods for correcting these errors are introduced. Calculations are compared with multiple X-ray and NMR derived structures in 36 soluble proteins. Calculations with X-ray structures give significantly better pK(a)s. Results with the default protein dielectric constant of 4 are as good as those using a value of 8. The MCCE2 program can be downloaded from http://www.sci.ccny.cuny.edu/~mcce. 2009 Wiley Periodicals, Inc.

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.

PubMed

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.

Compatibility and Complementarity of Classroom Ecology and Didactique Research Perspectives in Physical Education

ERIC Educational Resources Information Center

Leriche, Jérôme; Desbiens, Jean-François; Amade-Escot, Chantal; Tinning, Richard

2016-01-01

A large diversity of theoretical frameworks exists in the physical education literature. This article focuses on two of those frameworks to examine their compatibility and their complementarity. The classroom ecology paradigm concentrates on the balance between three task systems, two vectors, and programs of actions proposed by the physical…

A Portfolio for Optimal Collaboration of Human and Cyber Physical Production Systems in Problem-Solving

ERIC Educational Resources Information Center

Ansari, Fazel; Seidenberg, Ulrich

2016-01-01

This paper discusses the complementarity of human and cyber physical production systems (CPPS). The discourse of complementarity is elaborated by defining five criteria for comparing the characteristics of human and CPPS. Finally, a management portfolio matrix is proposed for examining the feasibility of optimal collaboration between them. The…

Microbes in Heavy Metal Remediation: A Review on Current Trends and Patents.

PubMed

Mishra, Geetesh Kumar

2017-01-01

Heavy metal pollution in the environmental samples like soil, water and runoff water is a worldwide problem. Such contamination of environmental matrices by the heavy metals accumulates due to various activities involving human driven sources and industries, although agriculture and sewage disposal are the largest source for the heavy metal contamination. Disposal of heavy metals or waste products containing heavy metals in the environment postures a trivial threat to public safety and health. Heavy metals are persistence and they can also cause biomagnifications and accumulate in food chain. Microbial bioremediation of heavy metal is emerging as an effective technique. Microbial bioremediation is a highly efficient environmental friendly procedure which also reduces the cost of cleanup process associated with heavy metal contamination. New methods for removal of heavy metals from the environmental samples are under development and most recent advancements have been made in exploring the knowledge of metal-microbes interactions and its use for heavy metal remediation. This review paper will focus on the microbial bioremediation process and highlight some of the newly developed patented methods for microbial bioremediation of the heavy metals from the environmental samples using microbial populations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quantification of total dinutuximab concentrations in neuroblastoma patients with liquid chromatography tandem mass spectrometry.

PubMed

El Amrani, Mohsin; Szanto, Celina L; Hack, C Erik; Huitema, Alwin D R; Nierkens, Stefan; van Maarseveen, Erik M

2018-06-25

Neuroblastoma is one of the most commonly found solid tumors in children. The monoclonal antibody dinutuximab (DNX) targets the sialic acid-containing glycosphingolipid GD2 expressed on almost all neuroblastoma tumor cells and induces cell lysis. However, the expression of GD2 is not limited to tumor cells only, but is also present on central nerve tissue and peripheral nerve cells explaining dinutuximab toxicity. The most common adverse reactions are pain and discomfort, which may lead to discontinuation of the treatment. Furthermore, there is little to no data available on exposure and effect relationships of dinutuximab. We, therefore, developed an easy method in order to quantify dinutuximab levels in human plasma. Ammonium sulfate (AS) was used to precipitate all immunoglobulins (IgGs) in human plasma. After centrifugation, supernatant containing albumin was decanted and the precipitated IgG fraction was re-dissolved in a buffer containing 0.5% sodium dodecyl sulfate (SDS). Samples were then reduced, alkylated, and digested with trypsin. Finally, a signature peptide in complementarity determining region 1 of DNX heavy chain was quantified on LC-MS/MS using a stable isotopically labeled peptide as internal standard. AS purification efficiently removed 97.5% of the albumin fraction in the supernatant layer. The validation performed on DNX showed that within-run and between-run coefficients of variation (CV) for lower limit of quantification (LLOQ) were 5.5 and 1.4%, respectively. The overall CVs for quality control (QC) low, QC med, and QC high levels were < 5%. Linearity in the range 1-32 mg/L was excellent (r 2  > 0.999). Selectivity, stability, and matrix effect were in concordance with EMA guidelines. In conclusion, a method to quantify DNX in human plasma was successfully developed. In addition, the high and robust process efficiency enabled the utilization of a stable isotopically labeled (SIL) peptide instead of SIL DNX, which was commercially unavailable. Graphical abstract.

Exploration of factors affecting the onset and maturation course of follicular lymphoma through simulations of the germinal center.

PubMed

Fenwick, Michael K; Escobedo, Fernando A

2009-08-01

Genetic mutations frequently observed in human follicular lymphoma (FL) B-cells result in aberrant expression of the anti-apoptotic protein bcl-2 and surface immunoglobulins (Igs) which display one or more novel variable (V) region N-glycosylation motifs. In the present study, we develop a simulation model of the germinal center (GC) to explore how these mutations might influence the emergence and clonal expansion of key mutants which provoke FL development. The simulations employ a stochastic method for calculating the cellular dynamics, which incorporates actual IgV region sequences and a simplified hypermutation scheme. We first bring our simulations into agreement with experimental data for well-characterized normal and bcl-2(+) anti-hapten GC responses in mice to provide a model for understanding how bcl-2 expression leads to permissive selection and memory cell differentiation of weakly competitive B-cells. However, as bcl-2 expression in the GC alone is thought to be insufficient for FL development, we next monitor simulated IgV region mutations to determine the emergence times of key mutants displaying aberrant N-glycosylation motifs recurrently observed in human FL IgV regions. Simulations of 26 germline V(H) gene segments indicate that particular IgV regions have a dynamical selective advantage by virtue of the speed with which one or more of their key sites can generate N-glycosylation motifs upon hypermutation. Separate calculations attribute the high occurrence frequency of such IgV regions in FL to an ability to produce key mutants at a fast enough rate to overcome stochastic processes in the GC that hinder clonal expansion. Altogether, these simulations characterize three pathways for FL maturation through positively selected N-glycosylations, namely, via one of two key sites within germline V(H) region gene segments, or via a site in the third heavy chain complementarity-determining region (CDR-H3) that is generated from VDJ recombination.

Decreased mutation frequencies among immunoglobulin G variable region genes during viremic HIV-1 infection.

PubMed

Bowers, Elisabeth; Scamurra, Ronald W; Asrani, Anil; Beniguel, Lydie; MaWhinney, Samantha; Keays, Kathryne M; Thurn, Joseph R; Janoff, Edward N

2014-01-01

HIV-1 infection is complicated by high rates of opportunistic infections against which specific antibodies contribute to immune defense. Antibody function depends on somatic hypermutation (SHM) of variable regions of immunoglobulin heavy chain genes (VH-D-J). We characterized the frequency of SHM in expressed IgG mRNA immunoglobulin transcripts from control and HIV-1-infected patients. We compared utilization of genes in the most prominent VH family (VH3) and mutation frequencies and patterns of cDNA from VH3-IgG genes from 10 seronegative control subjects and 21 patients with HIV-1 infection (6 without and 15 patients with detectable plasma viremia). Unique IgG VH3 family cDNA sequences (n = 1,565) were PCR amplified, cloned, and sequenced from blood. Sequences were analyzed using online (Vbase) and in-house immunoglobulin alignment resources. Mutation frequencies in the antigen-binding hypervariable complementarity determining regions (CDR1/2) of IgG class-switched B cells were lower among viremic HIV-1-infected patients vs. controls for nucleotides (CDR1/2: 10±5% vs. 13.5±6%, p = 0.03) and amino acids (CDR: 20%±10 vs. 25%±12, p = 0.02) and in structural framework regions. Mutation patterns were similar among groups. The most common VH3 gene, VH3-23, was utilized less frequently among viremic HIV-1-infected patients (p = 0.03), and overall, mutation frequencies were decreased in nearly all VH3 genes compared with controls. B cells from HIV-1-infected patients show decreased mutation frequencies, especially in antigen-binding VH3 CDR genes, and selective defects in gene utilization. Similar mutation patterns suggest defects in the quantity, but not quality, of mutator activity. Lower levels of SHM in IgG class-switched B cells from HIV-1-infected patients may contribute to the increased risk of opportunistic infections and impaired humoral responses to preventative vaccines.

Whole-body vibration in heavy equipment operators of a front-end loader: role of task exposure and tire configuration with and without traction chains.

PubMed

Blood, Ryan P; Rynell, Patrik W; Johnson, Peter W

2012-12-01

This study measured whole-body vibration (WBV) exposures in front-end loader operators, and evaluated the effects of traction chains and work tasks on their WBV exposures. WBV exposures were measured and compared across three different front-end loader tire configurations: (a) stock rubber tires, (b) rubber tires with ladder chains, and (c) rubber tires with basket chains. The operators completed three distinct standardized tasks: driving on a city street, simulated plowing, and a simulated scooping and dumping task. A portable data acquisition system collected tri-axial time weighted and raw WBV data per ISO 2631-1 and 2631-5 standards. In addition, Global Positioning System (GPS) data were collected in order to compare loader speeds across tire conditions and the standardized tasks. Relative to the stock rubber tires, both types of tire chains significantly increased WBV exposures with the ladder chains having substantially higher WBV exposures compared to basket chains. Additionally, there were task dependent differences in WBV exposures. During the driving task, the z-axis (up and down) was the predominant exposure; the plowing task had a more even distribution of exposure across all three axes; while during scooping and dumping task, the x-axis (fore and aft) had the highest WBV exposures. The GPS data indicated that there were significant speed differences across tasks but not between the basket and ladder chain conditions. Tires with ladder chains increased the front-end loader operators' exposure to WBV above the ISO 2631-1 recommended eight hour action limit increasing risk for adverse health effects. Although more expensive, basket chains are recommended over ladder chains since they substantially lowered the front-end loader operator's exposures and may ultimately reduce vibration related wear and tear on the vehicle. In order to reduce a heavy equipment vehicle (HEV) operator's chances for developing low back pain, this study provides information that health and safety professionals can use to reduce whole-body vibration (WBV) exposures when operating front-end wheel loaders with traction chains. Copyright © 2012 National Safety Council and Elsevier Ltd. All rights reserved.

Evaluating complementary networks of restoration plantings for landscape-scale occurrence of temporally dynamic species.

PubMed

Ikin, Karen; Tulloch, Ayesha; Gibbons, Philip; Ansell, Dean; Seddon, Julian; Lindenmayer, David

2016-10-01

Multibillion dollar investments in land restoration make it critical that conservation goals are achieved cost-effectively. Approaches developed for systematic conservation planning offer opportunities to evaluate landscape-scale, temporally dynamic biodiversity outcomes from restoration and improve on traditional approaches that focus on the most species-rich plantings. We investigated whether it is possible to apply a complementarity-based approach to evaluate the extent to which an existing network of restoration plantings meets representation targets. Using a case study of woodland birds of conservation concern in southeastern Australia, we compared complementarity-based selections of plantings based on temporally dynamic species occurrences with selections based on static species occurrences and selections based on ranking plantings by species richness. The dynamic complementarity approach, which incorporated species occurrences over 5 years, resulted in higher species occurrences and proportion of targets met compared with the static complementarity approach, in which species occurrences were taken at a single point in time. For equivalent cost, the dynamic complementarity approach also always resulted in higher average minimum percent occurrence of species maintained through time and a higher proportion of the bird community meeting representation targets compared with the species-richness approach. Plantings selected under the complementarity approaches represented the full range of planting attributes, whereas those selected under the species-richness approach were larger in size. Our results suggest that future restoration policy should not attempt to achieve all conservation goals within individual plantings, but should instead capitalize on restoration opportunities as they arise to achieve collective value of multiple plantings across the landscape. Networks of restoration plantings with complementary attributes of age, size, vegetation structure, and landscape context lead to considerably better outcomes than conventional restoration objectives of site-scale species richness and are crucial for allocating restoration investment wisely to reach desired conservation goals. © 2016 Society for Conservation Biology.

Tuning the electronic hybridization in the heavy fermion cage compound YbFe2Zn20 with Cd doping

NASA Astrophysics Data System (ADS)

Cabrera-Baez, M.; Ribeiro, R. A.; Avila, M. A.

2016-09-01

The tuning of the electronic properties of heavy fermion compounds by chemical substitution provides excellent opportunities for further understanding the physics of hybridized ions in crystal lattices. Here we present an investigation on the effects of Cd doping in flux-grown single crystals of the complex intermetallic cage compound YbFe2Zn20, which has been described as a heavy fermion with a Sommerfeld coefficient of 535 mJ mol-1 · K-2. The substitution of Cd for Zn disturbs the system by expanding the unit cell and, in this case, the size of the Zn cages that surround the Yb and Fe. With an increasing amount of Cd, the hybridization between the Yb 4f electrons and the conduction electrons is weakened, as shown by a decrease in the Sommerfeld coefficient, which should be accompanied by a valence shift of the Yb3+ due to the negative chemical pressure effect. This scenario is also supported by the low temperature DC magnetic susceptibility, which is gradually suppressed and shows an increment of the Kondo temperature, based on a shift to higher temperatures of the characteristic broad susceptibility peak. Furthermore, the DC resistivity decreases with the isoelectronic substitution of Cd for Zn, contrary to expectations in an increasingly disordered system, and implying that the valence shift is not related to charge carrier doping. The combined results demonstrate the excellent complementarity between positive physical pressure and negative chemical pressure, and point to a rich playground for exploring the physics and chemistry of strongly correlated electron systems in the general family of Zn20 compounds, despite their structural complexity.

A Toxoplasma gondii Class XIV Myosin, Expressed in Sf9 Cells with a Parasite Co-chaperone, Requires Two Light Chains for Fast Motility*

PubMed Central

Bookwalter, Carol S.; Kelsen, Anne; Leung, Jacqueline M.; Ward, Gary E.; Trybus, Kathleen M.

2014-01-01

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors. PMID:25231988

Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

PubMed

Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

KIR3DL2 binds to HLA-B27 dimers and free heavy chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis

PubMed Central

Wong-Baeza, Isabel; Ridley, Anna; Shaw, Jackie; Hatano, Hiroko; Rysnik, Oliwia; McHugh, Kirsty; Piper, Christopher; Brackenbridge, Simon; Fernandes, Ricardo; Chan, Anthoni; Bowness, Paul; Kollnberger, Simon

2013-01-01

1Abstract The Human Leukocyte Antigen HLA-B27(B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of antigen presenting cells (APC) as both classical β2m-associated B27 and as B27 free heavy chain forms (FHC) including disulphide-bonded heavy chain homodimers (termed B272). B27 FHC forms but not classical B27 bind to KIR3DL2. HLA-A3 which is not associated with spondyloarthritis (SpA) is also a ligand for KIR3DL2. Here we show that B272 and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B272 tetramers bound KIR3DL2 transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric and monomeric free heavy chains from HLA-B27 expressing cell lines. Binding to B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3ε–transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFNγ secretion and promoted greater survival of KIR3DL2+CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+SpA patients proliferated more in response to antigen presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27+ SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC. PMID:23440420

Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2

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

Sindi, Ramya A., E-mail: ramya.sindi2010@my.ntu.ac

Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20 h before exposure to CPF or CPO for 2–8 h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length ofmore » axon-like processes compared to the control, respectively, retraction of neurites being observed within 2 h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2 h exposure and reduced levels of reactivity of the same antibody following 8 h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2 h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8 h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation. - Highlights: • Chlorpyrifos and chlorpyrifos oxon induced rapid neurite retraction in N2a cells. • This occurred following transient hyperphosphorylation of ERK 1/2. • It was concomitant with hyperphosphorylation of neurofilament heavy chain (NFH). • Subsequent NFH dephosphorylation was associated with increased phosphatase activity. • Thus, the above changes reflect key events in chlorpyrifos induced neurodegeneration.« less

Designing Optimal LNG Station Network for U.S. Heavy-Duty Freight Trucks using Temporally and Spatially Explicit Supply Chain Optimization

NASA Astrophysics Data System (ADS)

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 unless a significant station capital subsidy, upwards of 50 percent and even then it might not be enough. However, a doubling of LNG truck demand will initialize network commercialization in the modeling base year, 2012 (the same year Clean Energy Corp. launched their national LNG network) in California and then gradually establish in other hotspot regions in Mid-West and Mid-Atlantic throughout the time horizon. The model shows that trucking routes in California are highly commercial due to high traffic volume and regional advantages. The model can be used by industry to inform necessary policies and to plan future infrastructure deployment along trucking routes that are likely to provide the highest returns.

Ecotoxic heavy metals transformation by bacteria and fungi in aquatic ecosystem.

PubMed

Chaturvedi, Amiy Dutt; Pal, Dharm; Penta, Santhosh; Kumar, Awanish

2015-10-01

Water is the most important and vital molecule of our planet and covers 75% of earth surface. But it is getting polluted due to high industrial growth. The heavy metals produced by industrial activities are recurrently added to it and considered as dangerous pollutants. Increasing concentration of toxic heavy metals (Pb(2+), Cd(2+), Hg(2+), Ni(2+)) in water is a severe threat for human. Heavy metal contaminated water is highly carcinogenic and poisonous at even relatively low concentrations. When they discharged in water bodies, they dissolve in the water and are distributed in the food chain. Bacteria and fungi are efficient microbes that frequently transform heavy metals and remove toxicity. The application of bacteria and fungi may offer cost benefit in water treatment plants for heavy metal transformation and directly related to public health and environmental safety issues. The heavy metals transformation rate in water is also dependent on the enzymatic capability of microorganisms. By transforming toxic heavy metals microbes sustain aquatic and terrestrial life. Therefore the application of microbiological biomass for heavy metal transformation and removal from aquatic ecosystem is highly significant and striking. This paper reviews the microbial transformation of heavy metal, microbe metal interaction and different approaches for microbial heavy metal remediation from water bodies.

[Research of Human-mouse Chimeric Antibodies Against Ebola Virus Nucleoprotein].

PubMed

Zhou, Rongping; Sun, Lina; Liu, Yang; Wu, Wei; Li, Chuan; Liang, Mifang; Qiu, Peihong

2016-01-01

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.

The Impact of Electronic Commerce on the Publishing Industry: Towards a Business Value Complementarity Framework of Electronic Publishing.

ERIC Educational Resources Information Center

Scupola, Ada

1999-01-01

Discussion of the publishing industry and its use of information and communication technologies focuses on the way in which electronic-commerce technologies are changing and could change the publishing processes, and develops a business complementarity model of electronic publishing to maximize profitability and improve the competitive position.…

Immunoglobulin heavy and light chains and T-cell receptor beta and gamma chains PCR assessment on cytological samples. A study comparing FTA cards and cryopreserved lymph node fine-needle cytology.

PubMed

Peluso, A L; Cozzolino, I; Bottiglieri, A; Lucchese, L; Di Crescenzo, R M; Langella, M; Selleri, C; Zeppa, P

2017-06-01

To evaluate and compare the DNA yield and quality extracted from lymph node fine needle cytology (FNC) samples stored on FTA cards to those cryopreserved, and to assess the immunoglobulin heavy and light chains (IGHK) and T-Cell receptor beta and gamma chains (TCRBG) PCR tests. DNA extractions were performed on FNC of 80 non-Hodgkin lymphomas (NHL), four myelomas and 56 benign reactive hyperplasias (BRH) cryopreserved and stored on FTA cards. The JAK2 gene was amplified to assess the DNA integrity and the IGHK/TCRBG clonality status was tested. IGHK monoclonality was found in 99% of B-cell NHL and 100% of myeloma. TCRBG monoclonality was found in 100% of T-cell NHL. TCRBG polyclonality was detected in 97% of B-cell NHL, 100% of myeloma and 96% of BRH. IGHK/TCRBG PCR data were confirmed by histological and/or follow-up controls. No differences were found in the DNA quality between cryopreservation and FTA cards storage methods. IGHK/TCRBG PCR of the lymphoproliferative process on FTA cards is comparable to those cryopreserved. FTA cards can be used to store lymph node FNC for further molecular investigations. © 2016 John Wiley & Sons Ltd.

Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis

PubMed Central

Feng, Zhonghui; Okada, Satoshi; Cai, Guoping; Zhou, Bing; Bi, Erfei

2015-01-01

MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species. PMID:25631819

Visualizing Key Hinges and a Potential Major Source of Compliance in the Lever Arm of Myosin

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

J Brown; V Senthil Kumar; E ONeall-Hennessey

2011-12-31

We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during themore » contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.« less

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

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

Brown, J.H.; Robinson, H.; Senthil Kumar, V. S.

2011-01-04

We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during themore » contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.« less

Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs)

PubMed Central

Maass, David R.; Sepulveda, Jorge; Pernthaner, Anton; Shoemaker, Charles B.

2007-01-01

Recombinant single domain antibody fragments (VHHs) that derive from the unusual camelid heavy chain only IgG class (HCAbs) have many favourable properties compared with single-chain antibodies prepared from conventional IgG. As a result, VHHs have become widely used as binding reagents and are beginning to show potential as therapeutic agents. To date, the source of VHH genetic material has been camels and llamas despite their large size and limited availability. Here we demonstrate that the smaller, more tractable and widely available alpaca is an excellent source of VHH coding DNA. Alpaca sera IgG consists of about 50% HCAbs, mostly of the short-hinge variety. Sequencing of DNA encoding more than 50 random VHH and hinge domains permitted the design of PCR primers that will amplify virtually all alpaca VHH coding DNAs for phage display library construction. Alpacas were immunized with ovine tumour necrosis factor α (TNFα) and a VHH phage display library was prepared from a lymph node that drains the sites of immunizations and successfully employed in the isolation of VHHs that bind and neutralize ovine TNFα. PMID:17568607

Tracing heavy metals in 'swine manure - maggot - chicken' production chain.

PubMed

Wang, Wanqiang; Zhang, Wenjuan; Wang, Xiaoping; Lei, Chaoliang; Tang, Rui; Zhang, Feng; Yang, Qizhi; Zhu, Fen

2017-08-21

With the development of large-scale livestock farming, manure pollution has drawn much attention. Conversion by insects is a rapid and cost-effective new method for manure management. Swine manure conversion with maggots (Musca domestica larvae) has developed, and the harvested maggots are often used as animal feed. However, the flow of heavy metals from manure to downstream processes cannot be ignored, and therefore, heavy metal content was measured in untreated raw manure, maggot-treated manure, harvested maggots and maggot-eating chickens (chest muscle and liver) to evaluate potential heavy metal risks. The levels of zinc, copper, chromium, selenium, cadmium and lead had significant differences between untreated raw manure and maggot-treated manure. The concentrations of all detected heavy metals, except for cadmium and selenium, in maggots met the limits established by the feed or feed additive standards of many countries. The bioaccumulation factor (BAF) of heavy metals decreased with the increase of the maggot instar, indicating that heavy metals were discharged from the bodies of maggots with the growth of maggots. Also, the contents of overall heavy metals in chickens fed harvested maggots met the standards for food. In conclusion, regarding heavy metals, it is eco-safe to use maggots in manure management.

Mapping the distribution of packing topologies within protein interiors shows predominant preference for specific packing motifs

PubMed Central

2011-01-01

Background Mapping protein primary sequences to their three dimensional folds referred to as the 'second genetic code' remains an unsolved scientific problem. A crucial part of the problem concerns the geometrical specificity in side chain association leading to densely packed protein cores, a hallmark of correctly folded native structures. Thus, any model of packing within proteins should constitute an indispensable component of protein folding and design. Results In this study an attempt has been made to find, characterize and classify recurring patterns in the packing of side chain atoms within a protein which sustains its native fold. The interaction of side chain atoms within the protein core has been represented as a contact network based on the surface complementarity and overlap between associating side chain surfaces. Some network topologies definitely appear to be preferred and they have been termed 'packing motifs', analogous to super secondary structures in proteins. Study of the distribution of these motifs reveals the ubiquitous presence of typical smaller graphs, which appear to get linked or coalesce to give larger graphs, reminiscent of the nucleation-condensation model in protein folding. One such frequently occurring motif, also envisaged as the unit of clustering, the three residue clique was invariably found in regions of dense packing. Finally, topological measures based on surface contact networks appeared to be effective in discriminating sequences native to a specific fold amongst a set of decoys. Conclusions Out of innumerable topological possibilities, only a finite number of specific packing motifs are actually realized in proteins. This small number of motifs could serve as a basis set in the construction of larger networks. Of these, the triplet clique exhibits distinct preference both in terms of composition and geometry. PMID:21605466

Selective amplification of T-cell receptor variable region species is demonstrable but not essential in early lesions of psoriasis vulgaris: analysis by anchored polymerase chain reaction and hypervariable region size spectratyping.

PubMed

Vekony, M A; Holder, J E; Lee, A J; Horrocks, C; Eperon, I C; Camp, R D

1997-07-01

Several groups have investigated the role of T cells in the pathogenesis of psoriasis by determination of T-cell receptor (TCR) B-chain variable (V) region usage, both in chronic plaque (psoriasis vulgaris) and guttate forms, with various results. Because there are no data on TCR expression in early psoriasis vulgaris, when specific cellular immune events may be expected to be most pronounced, we have analyzed early lesions (less than 3 wk old) of ten patients, with highly reproducible results. We have developed a highly controlled anchored polymerase chain reaction (PCR) method in which TCR beta chain species are all amplified with the same primer pair and products are quantified by dot blot hybridization with BV family-specific oligonucleotide probes. Overexpression of certain TCR BV genes was observed in the majority of lesional biopsies, but in samples in which the expanded BV family formed more than 10% of total lesional BV (half of the samples analyzed), BV2 and BV6 predominated. The consistency of overexpression of these BV species between patients was much less than in previous studies of TCRBV usage in established chronic plaque psoriasis lesions. Complementarity-determining region 3 (CDR3) size spectratyping demonstrated evidence for selective clonal T cell accumulation in less than half of the lesional samples showing BV expansion. These results indicate that selective amplification of TCRBV species occurs in early psoriasis vulgaris but is not essential to the pathogenic process and may be more important in the maintenance or expansion of chronic lesions.

Supramolecular Organization of the α121-α565 Collagen IV Network*

PubMed Central

Robertson, Wesley E.; Rose, Kristie L.; Hudson, Billy G.; Vanacore, Roberto M.

2014-01-01

Collagen IV is a family of 6 chains (α1-α6), that form triple-helical protomers that assemble into supramolecular networks. Two distinct networks with chain compositions of α121 and α345 have been established. These oligomerize into separate α121 and α345 networks by a homotypic interaction through their trimeric noncollagenous (NC1) domains, forming α121 and α345 NC1 hexamers, respectively. These are stabilized by novel sulfilimine (SN) cross-links, a covalent cross-link that forms between Met93 and Hyl211 at the trimer-trimer interface. A third network with a composition of α1256 has been proposed, but its supramolecular organization has not been established. In this study we investigated the supramolecular organization of this network by determining the chain identity of sulfilimine-cross-linked NC1 domains derived from the α1256 NC1 hexamer. High resolution mass spectrometry analyses of peptides revealed that sulfilimine bonds specifically cross-link α1 to α5 and α2 to α6 NC1 domains, thus providing the spatial orientation between interacting α121 and α565 trimers. Using this information, we constructed a three-dimensional homology model in which the α565 trimer shows a good chemical and structural complementarity to the α121 trimer. Our studies provide the first chemical evidence for an α565 protomer and its heterotypic interaction with the α121 protomer. Moreover, our findings, in conjunction with our previous studies, establish that the six collagen IV chains are organized into three canonical protomers α121, α345, and α565 forming three distinct networks: α121, α345, and α121-α565, each of which is stabilized by sulfilimine bonds between their C-terminal NC1 domains. PMID:25006246

Assessing climate change impact on complementarity between solar and hydro power in areas affected by glacier shrinkage

NASA Astrophysics Data System (ADS)

Diah Puspitarini, Handriyanti; François, Baptiste; Zoccatelli, Davide; Brown, Casey; Creutin, Jean-Dominique; Zaramella, Mattia; Borga, Marco

2017-04-01

Variable Renewable Energy (VRE) sources such as wind, solar and runoff sources are variable in time and space, following their driving weather variables. In this work we aim to analyse optimal mixes of energy sources, i.e. mixes of sources which minimize the deviation between energy load and generation, for a region in the Upper Adige river basin (Eastern Italian Alps) affected by glacier shrinking. The study focuses on hydropower (run of the river - RoR) and solar energy, and analyses the current situation as well different climate change scenarios. Changes in glacier extent in response to climate warming and/or altered precipitation regimes have the potential to substantially alter the magnitude and timing, as well as the spatial variation of watershed-scale hydrologic fluxes. This may change the complementarity with solar power as well. In this study, we analyse the climate change impact on complementarity between RoR and solar using the Decision Scaling approach (Brown et al. 2012). With this approach, the system vulnerability is separated from the climatic hazard that can come from any set of past or future climate conditions. It departs from conventional top-down impact studies because it explores the sensitivity of the system response to a plausible range of climate variations rather than its sensitivity to the time-varying outcome of individual GCM projections. It mainly relies on the development of Climate Response Functions that bring together i) the sensitivity of some system success and/or failure indicators to key external drivers (i.e. mean features of regional climate) and ii) the future values of these drivers as simulated from climate simulation chains. The main VRE sources used in the study region are solar- and hydro-power (with an important fraction of run-of-the river hydropower). The considered indicator of success is the 'energy penetration' coefficient, defined as the long-run percentage of energy demand naturally met by the VRE on an hourly basis. Climate response functions, developed in a 2D climate change space (change in mean temperature and precipitation), are built from multiple hydro-climatic scenarios obtained by perturbing the observed weather time series with the change factor method, and considering given glacier storage states. Climate experiments are further used for assessing these change factors from different emission scenarios, climate models and future prediction lead times. Their positioning on the Climate Response Function allows discussing the risk/opportunities pertaining to changes in VRE penetration in the future. Results show i) the large impact of glacier shrinkage on the complementarity between solar and RoR energy sources and ii) that the impact is decreasing with time, with the main alterations to be expected in the coming 30 years. Brown, C., Ghile, Y., Laverty, M., Li, K., (2012). Decision scaling: Linking bottom up vulnerability analysis with climate projections in the water sector. Water Resour Res 48. 515 doi:10.1029/2011WR011212

Effect of paper mill effluents on accumulation of heavy metals in coconut trees near Nanjangud, mysore district, Karnataka, India

NASA Astrophysics Data System (ADS)

Fazeli, M. Sharif; Sathyanarayan, S.; Satish, P. N.; Muthanna, Lata

1991-01-01

Physicochemical characteristics of wastewater from one of the paper mills near Nanjangud and the differential accumulation of heavy metals in parts of coconut trees growing in the area irrigated directly by the wastewaters of a paper mill were investigated. The total dissolved and suspended solids of wastewater were 1,136.9 mg/l and 2,185.4 mg/l, respectively. Biological oxygen demand (BOD) expands and COD is beyond the tolerance limit proposed by Indian standards. The concentrations of heavy metals like Cu, Pb, Zn, Ni, Co, and Cd in coconut water, root, and leaf are higher than the limits suggested by World Health Organization. Survival of coconut trees irrigated by polluted waters indicates tolerance to toxic heavy metals. Since coconut forms part of human food chain, accumulation of toxic heavy metals may lead to organic disorders.

Sequencing the Unrearranged Human Immunoglobin

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

Warren, Rene

2010-06-03

Rene Warren from Canada's Michael Smith Genome Sciences Centre discusses sequencing and finishing the IgH heavy chain locus on June 3, 2010 at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM.

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

PubMed

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.

Genetic diversity of the human immunoglobulin heavy chain VH region.

PubMed

Li, Honghua; Cui, Xiangfeng; Pramanik, Sreemanta; Chimge, Nyam-Osor

2002-12-01

The human immunoglobulin heavy chain VH region is one of the most complex regions in the human genome. The high level of diversity of this region has been shown by a number of studies. However, because of the limitations of the conventional experimental methods, it has been difficult to learn the extent of the diversity and the underlying mechanisms. This review describes a number of new genetic approaches developed in the authors' laboratory. By using these approaches, significant progress has been made in assigning different VH sequences to their respective loci, in learning the diversity of gene segment number and composition among the VH haplotypes, and in learning VH gene segment organization in individual haplotypes. Information obtained toward this direction could help in understanding the mechanisms underlying VH region diversity and the biological impact of the VH region diversity.

Submolecular recognition of the C-terminal domain of the heavy chain of botulinum neurotoxin type A by T cells from toxin-treated cervical dystonia patients.

PubMed

Oshima, Minako; Deitiker, Philip; Jankovic, Joseph; Aoki, K Roger; Atassi, M Zouhair

2016-04-01

We determined the T-cell proliferative responses of the peripheral blood lymphocytes (PBL) from 25 botulinum neurotoxin (BoNT)-treated patients to 31 overlapping synthetic peptides encompassing the C-terminal half (residues 855-1296) of BoNT/A heavy chain. Responses of PBL to HC peptides varied among patients. Samples from 14 patients treated solely with BoNT/A recognized 2-13 (average 6.4) peptides/sample at Z>3.0 level. Six peptide regions representing residues 855-873, 1023-1041, 1051-1069, 1093-1111, 1135-1153 and 1247-1265 were frequently recognized by 36-57% of these PBLs. Influence of treatment parameters on T-cell recognition of the peptides was also investigated. Copyright © 2015 Elsevier GmbH. All rights reserved.

Fiber transformation and replacement in low-frequency stimulated rabbit fast-twitch muscles.

PubMed

Schuler, M; Pette, D

1996-08-01

The fast-to-slow conversion of rabbit skeletal muscles by chronic low-frequency (10 Hz, 12 h daily) stimulation involves (1) sequential fast-to-slow fiber-type transitions in the order of type IID-->type IIA-->type I, and (2) the replacement of deteriorating fast-twitch glycolytic fibers by new fibers derived from satellite cells and myotubes. These two processes were analyzed in 30- and 60-day stimulated extensor digitorum longus and tibialis anterior muscles. Fast-to-slow transforming fibers were identified by myofibrillar actomyosin histochemistry as type C fibers and immunohistochemically by their reaction with monoclonal antibodies specific to slow and fast myosin heavy chain isoforms. In situ hybridization of mRNA specific to the myosin heavy chain I isoform identified all fibers expressing slow myosin, i.e., type I and C fibers. The fraction of transforming fibers ranged between 35% and 50% in 30-day stimulated muscles. The percentage of type I fibers (20%) was threefold elevated in extensor digitorum longus muscle, but unaltered (3.5%) in tibialis anterior muscle, suggesting that fast-to-slow fiber conversion was more advanced in the former than in the latter. Fiber replacement was indicated by the finding that the fiber populations of both muscles contained 15% myotubes or small fibers with central nuclei. In situ hybridization revealed that myotubes and small regenerating fibers uniformly expressed myosin heavy chain I mRNA. Similarly, high percentages of slow-myosin-expressing myotubes and small fibers were found in 60-day stimulated muscles.

Immunoglobulin heavy chain diversity in Pteropid bats: evidence for a diverse and highly specific antigen binding repertoire

PubMed Central

Tachedjian, Mary; Wang, Lin-Fa

2010-01-01

Bats are the natural host reservoir for range of emerging and re-emerging viruses, many of which cause significant morbidity and mortality in other mammals, yet appear to result in no clinical consequences for bats. The ability of bats to coexist with a variety of viruses presents an interesting immunological problem that has not been examined in any detail but which could provide significant insights into the evolution of antiviral mechanisms in mammals. Towards a better understanding of the bat immune system, we analysed the expressed heavy chain variable (VH) regions of antibodies from the black flying fox, Pteropus alecto. The germline repertoire of the closely related Pteropid bat, Pteropus vampyrus, whose genome has been sequenced was also examined for comparative purposes. Representative VH genes were found in all three mammalian VH clans (I, II and III) in both the expressed P. alecto VH repertoire and the germline P. vampyrus VH repertoire. Evidence for the use of multiple heavy chain diversity (DH) and joining (JH) segments for the generation of diverse VDJ rearrangements was also present in the expressed antibody repertoire of P. alecto. The long period of co-evolutionary history of bats with viruses may have resulted in a variety of highly specific VH segments being hardwired into the genomes of bats and may have implications for their ability to successfully cope with a diversity of viral antigens. PMID:20162414

Engineering Upper Hinge Improves Stability and Effector Function of a Human IgG1

PubMed Central

Yan, Boxu; Boyd, Daniel; Kaschak, Timothy; Tsukuda, Joni; Shen, Amy; Lin, Yuwen; Chung, Shan; Gupta, Priyanka; Kamath, Amrita; Wong, Anne; Vernes, Jean-Michel; Meng, Gloria Y.; Totpal, Klara; Schaefer, Gabriele; Jiang, Guoying; Nogal, Bartek; Emery, Craig; Vanderlaan, Martin; Carter, Paul; Harris, Reed; Amanullah, Ashraf

2012-01-01

Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys231 directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His229-mediated hinge cleavage. On the other hand, the substitution of His229 with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2–3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed. PMID:22203673

Engineering upper hinge improves stability and effector function of a human IgG1.

PubMed

Yan, Boxu; Boyd, Daniel; Kaschak, Timothy; Tsukuda, Joni; Shen, Amy; Lin, Yuwen; Chung, Shan; Gupta, Priyanka; Kamath, Amrita; Wong, Anne; Vernes, Jean-Michel; Meng, Gloria Y; Totpal, Klara; Schaefer, Gabriele; Jiang, Guoying; Nogal, Bartek; Emery, Craig; Vanderlaan, Martin; Carter, Paul; Harris, Reed; Amanullah, Ashraf

2012-02-17

Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins.

PubMed

Feige, Matthias J; Gräwert, Melissa A; Marcinowski, Moritz; Hennig, Janosch; Behnke, Julia; Ausländer, David; Herold, Eva M; Peschek, Jirka; Castro, Caitlin D; Flajnik, Martin; Hendershot, Linda M; Sattler, Michael; Groll, Michael; Buchner, Johannes

2014-06-03

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules.

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

PubMed Central

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

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

PubMed Central

Sotelo, Pablo H.; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

2012-01-01

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles. PMID:22692130

β-Myosin heavy chain variant Val606Met causes very mild hypertrophic cardiomyopathy in mice, but exacerbates HCM phenotypes in mice carrying other HCM mutations.

PubMed

Blankenburg, Robert; Hackert, Katarzyna; Wurster, Sebastian; Deenen, René; Seidman, J G; Seidman, Christine E; Lohse, Martin J; Schmitt, Joachim P

2014-07-07

Approximately 40% of hypertrophic cardiomyopathy (HCM) is caused by heterozygous missense mutations in β-cardiac myosin heavy chain (β-MHC). Associating disease phenotype with mutation is confounded by extensive background genetic and lifestyle/environmental differences between subjects even from the same family. To characterize disease caused by β-cardiac myosin heavy chain Val606Met substitution (VM) that has been identified in several HCM families with wide variation of clinical outcomes, in mice. Unlike 2 mouse lines bearing the malignant myosin mutations Arg453Cys (RC/+) or Arg719Trp (RW/+), VM/+ mice with an identical inbred genetic background lacked hallmarks of HCM such as left ventricular hypertrophy, disarray of myofibers, and interstitial fibrosis. Even homozygous VM/VM mice were indistinguishable from wild-type animals, whereas RC/RC- and RW/RW-mutant mice died within 9 days after birth. However, hypertrophic effects of the VM mutation were observed both in mice treated with cyclosporine, a known stimulator of the HCM response, and compound VM/RC heterozygous mice, which developed a severe HCM phenotype. In contrast to all heterozygous mutants, both systolic and diastolic function of VM/RC hearts was severely impaired already before the onset of cardiac remodeling. The VM mutation per se causes mild HCM-related phenotypes; however, in combination with other HCM activators it exacerbates the HCM phenotype. Double-mutant mice are suitable for assessing the severity of benign mutations. © 2014 American Heart Association, Inc.

Forecast and analysis of the cosmological redshift drift.

PubMed

Lazkoz, Ruth; Leanizbarrutia, Iker; Salzano, Vincenzo

2018-01-01

The cosmological redshift drift could lead to the next step in high-precision cosmic geometric observations, becoming a direct and irrefutable test for cosmic acceleration. In order to test the viability and possible properties of this effect, also called Sandage-Loeb (SL) test, we generate a model-independent mock data set in order to compare its constraining power with that of the future mock data sets of Type Ia Supernovae (SNe) and Baryon Acoustic Oscillations (BAO). The performance of those data sets is analyzed by testing several cosmological models with the Markov chain Monte Carlo (MCMC) method, both independently as well as combining all data sets. Final results show that, in general, SL data sets allow for remarkable constraints on the matter density parameter today [Formula: see text] on every tested model, showing also a great complementarity with SNe and BAO data regarding dark energy parameters.

Connections between the dynamical symmetries in the microscopic shell model

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

Georgieva, A. I., E-mail: anageorg@issp.bas.bg; Drumev, K. P.

2016-03-25

The dynamical symmetries of the microscopic shell model appear as the limiting cases of a symmetry adapted Pairing-Plus-Quadrupole Model /PQM/, with a Hamiltonian containing isoscalar and isovector pairing and quadrupole interactions. We establish a correspondence between each of the three types of pairing bases and Elliott’s SU(3) basis, that describes collective rotation of nuclear systems with quadrupole deformation. It is derived from their complementarity to the same LS coupling chain of the shell model number conserving algebra. The probability distribution of the S U(3) basis states within the pairing eigenstates is also obtained through a numerical diagonalization of the PQMmore » Hamiltonian in each limit. We introduce control parameters, which define the phase diagram of the model and determine the role of each term of the Hamiltonian in the correct reproduction of the experimental data for the considered nuclei.« less

Abiotic ligation of DNA oligomers templated by their liquid crystal ordering

NASA Astrophysics Data System (ADS)

Fraccia, Tommaso P.; Smith, Gregory P.; Zanchetta, Giuliano; Paraboschi, Elvezia; Yi, Yougwooo; Walba, David M.; Dieci, Giorgio; Clark, Noel A.; Bellini, Tommaso

2015-03-01

It has been observed that concentrated solutions of short DNA oligomers develop liquid crystal ordering as the result of a hierarchically structured supramolecular self-assembly. In mixtures of oligomers with various degree of complementarity, liquid crystal microdomains are formed via the selective aggregation of those oligomers that have a sufficient degree of duplexing and propensity for physical polymerization. Here we show that such domains act as fluid and permeable microreactors in which the order-stabilized molecular contacts between duplex terminals serve as physical templates for their chemical ligation. In the presence of abiotic condensing agents, liquid crystal ordering markedly enhances ligation efficacy, thereby enhancing its own phase stability. The coupling between order-templated ligation and selectivity provided by supramolecular ordering enables an autocatalytic cycle favouring the growth of DNA chains, up to biologically relevant lengths, from few-base long oligomers. This finding suggests a novel scenario for the abiotic origin of nucleic acids.

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

PubMed Central

Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

2015-01-01

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

Particle size effects on uptake of heavy metals from sewage sludge compost using natural zeolite clinoptilolite.

PubMed

Zorpas, Antonis A; Vassilis, Inglezakis; Loizidou, Maria; Grigoropoulou, Helen

2002-06-01

Land application of sewage sludge may be the least energy consuming and the most cost-effective means of sludge disposal or utilization. However, the major technical problem with land application of sludge concerns the high concentrations of heavy metals. These metals may be leached and enter the ecosystem, the food chain, and eventually the human population. This paper deals with the removal of heavy metals from sewage sludge compost using natural zeolite clinoptilolite, in respect to the particle size. The final results indicate that heavy metals can be sufficiently removed by using 25% w/w of zeolite with particle size of 3.3-4.0 mm. Pore clogging and structural damage in smaller particle sizes is probably the reason for lower uptake of metals by the latter.

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

Fazeli, M.S.; Sathyanarayan, S.; Satish, P.N.

Physicochemical characteristics of wastewater from one of the paper mills near Nanjangud and the differential accumulation of heavy metals in parts of coconut trees growing in the area irrigated directly by the wastewaters of a paper mill were investigated. The total dissolved and suspended oils of wastewater were 1,136.9 mg/l and 2,185.4 mg/l, respectively. Biological oxygen demand (BOD) expands and COD is beyond the tolerance limit proposed by Indian standards. The concentrations of heavy meals like Cu, Pb, Zn, Ni, Coo, and Cd in coconut water, root, and leaf are higher than the limits suggested by World Health Organization. Survivalmore » of coconut trees irrigated by polluted waters indicates tolerance to toxic heavy metals. Since coconut forms part of human food chain, accumulation of toxic heavy metals may lead to organic disorders.« less

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

PubMed

Zhang, Wenwu; Gunst, Susan J

2017-07-01

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

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

NASA Astrophysics Data System (ADS)

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

2014-10-01

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

Chlamydomonas Outer Arm Dynein Alters Conformation in Response to Ca2+

PubMed Central

Sakato, Miho; Sakakibara, Hitoshi

2007-01-01

We have previously shown that Ca2+ directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the β and γ heavy chains (HCs). The γ HC–associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca2+ with KCa = 3 × 10−5 M in vitro, suggesting it may act as a Ca2+ sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and γ HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the γ heavy chain. In vitro experiments indicate that LC4 undergoes a Ca2+-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca2+. The sedimentation profile of the γ HC subunit changed subtly upon Ca2+ addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated γ subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca2+ addition. We propose that Ca2+-dependent conformational change of LC4 has a direct effect on the stem domain of the γ HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm. PMID:17634291

Complementarity among four highly productive grassland species depends on resource availability.

PubMed

Roscher, Christiane; Schmid, Bernhard; Kolle, Olaf; Schulze, Ernst-Detlef

2016-06-01

Positive species richness-productivity relationships are common in biodiversity experiments, but how resource availability modifies biodiversity effects in grass-legume mixtures composed of highly productive species is yet to be explicitly tested. We addressed this question by choosing two grasses (Arrhenatherum elatius and Dactylis glomerata) and two legumes (Medicago × varia and Onobrychis viciifolia) which are highly productive in monocultures and dominant in mixtures (the Jena Experiment). We established monocultures, all possible two- and three-species mixtures, and the four-species mixture under three different resource supply conditions (control, fertilization, and shading). Compared to the control, community biomass production decreased under shading (-56 %) and increased under fertilization (+12 %). Net diversity effects (i.e., mixture minus mean monoculture biomass) were positive in the control and under shading (on average +15 and +72 %, respectively) and negative under fertilization (-10 %). Positive complementarity effects in the control suggested resource partitioning and facilitation of growth through symbiotic N2 fixation by legumes. Positive complementarity effects under shading indicated that resource partitioning is also possible when growth is carbon-limited. Negative complementarity effects under fertilization suggested that external nutrient supply depressed facilitative grass-legume interactions due to increased competition for light. Selection effects, which quantify the dominance of species with particularly high monoculture biomasses in the mixture, were generally small compared to complementarity effects, and indicated that these species had comparable competitive strengths in the mixture. Our study shows that resource availability has a strong impact on the occurrence of positive diversity effects among tall and highly productive grass and legume species.

On the Embedded Complementarity of Agent-Based and Aggregate Reasoning in Students' Developing Understanding of Dynamic Systems

ERIC Educational Resources Information Center

Stroup, Walter M.; Wilensky, Uri

2014-01-01

Placed in the larger context of broadening the engagement with systems dynamics and complexity theory in school-aged learning and teaching, this paper is intended to introduce, situate, and illustrate--with results from the use of network supported participatory simulations in classrooms--a stance we call "embedded complementarity" as an…

Has Complementarity between Employer-Sponsored Training and Education in the U.S. Changed during the 2000s?

ERIC Educational Resources Information Center

Waddoups, C. Jeffrey

2018-01-01

The study reveals that the positive correlation between formal education and job training (complementarity) has weakened during the 2000s. Using U.S. Census Bureau data from the Survey of Income and Program Participation, the study finds that although workers in all categories of educational attainment felt the decline, the effects were strongest…

Multiple-algorithm parallel fusion of infrared polarization and intensity images based on algorithmic complementarity and synergy

NASA Astrophysics Data System (ADS)

Zhang, Lei; Yang, Fengbao; Ji, Linna; Lv, Sheng

2018-01-01

Diverse image fusion methods perform differently. Each method has advantages and disadvantages compared with others. One notion is that the advantages of different image methods can be effectively combined. A multiple-algorithm parallel fusion method based on algorithmic complementarity and synergy is proposed. First, in view of the characteristics of the different algorithms and difference-features among images, an index vector-based feature-similarity is proposed to define the degree of complementarity and synergy. This proposed index vector is a reliable evidence indicator for algorithm selection. Second, the algorithms with a high degree of complementarity and synergy are selected. Then, the different degrees of various features and infrared intensity images are used as the initial weights for the nonnegative matrix factorization (NMF). This avoids randomness of the NMF initialization parameter. Finally, the fused images of different algorithms are integrated using the NMF because of its excellent data fusing performance on independent features. Experimental results demonstrate that the visual effect and objective evaluation index of the fused images obtained using the proposed method are better than those obtained using traditional methods. The proposed method retains all the advantages that individual fusion algorithms have.

Leveraging genome-wide datasets to quantify the functional role of the anti-Shine-Dalgarno sequence in regulating translation efficiency.

PubMed

Hockenberry, Adam J; Pah, Adam R; Jewett, Michael C; Amaral, Luís A N

2017-01-01

Studies dating back to the 1970s established that sequence complementarity between the anti-Shine-Dalgarno (aSD) sequence on prokaryotic ribosomes and the 5' untranslated region of mRNAs helps to facilitate translation initiation. The optimal location of aSD sequence binding relative to the start codon, the full extents of the aSD sequence and the functional form of the relationship between aSD sequence complementarity and translation efficiency have not been fully resolved. Here, we investigate these relationships by leveraging the sequence diversity of endogenous genes and recently available genome-wide estimates of translation efficiency. We show that-after accounting for predicted mRNA structure-aSD sequence complementarity increases the translation of endogenous mRNAs by roughly 50%. Further, we observe that this relationship is nonlinear, with translation efficiency maximized for mRNAs with intermediate levels of aSD sequence complementarity. The mechanistic insights that we observe are highly robust: we find nearly identical results in multiple datasets spanning three distantly related bacteria. Further, we verify our main conclusions by re-analysing a controlled experimental dataset. © 2017 The Authors.

Plant diversity effects on grassland productivity are robust to both nutrient enrichment and drought

PubMed Central

Isbell, Forest; Manning, Pete; Connolly, John; Bruelheide, Helge; Ebeling, Anne; Roscher, Christiane; van Ruijven, Jasper; Weigelt, Alexandra; Wilsey, Brian; Beierkuhnlein, Carl; de Luca, Enrica; Griffin, John N.; Hautier, Yann; Hector, Andy; Jentsch, Anke; Kreyling, Jürgen; Lanta, Vojtech; Loreau, Michel; Meyer, Sebastian T.; Mori, Akira S.; Naeem, Shahid; Palmborg, Cecilia; Polley, H. Wayne; Reich, Peter B.; Schmid, Bernhard; Siebenkäs, Alrun; Seabloom, Eric; Thakur, Madhav P.; Tilman, David; Vogel, Anja; Eisenhauer, Nico

2016-01-01

Global change drivers are rapidly altering resource availability and biodiversity. While there is consensus that greater biodiversity increases the functioning of ecosystems, the extent to which biodiversity buffers ecosystem productivity in response to changes in resource availability remains unclear. We use data from 16 grassland experiments across North America and Europe that manipulated plant species richness and one of two essential resources—soil nutrients or water—to assess the direction and strength of the interaction between plant diversity and resource alteration on above-ground productivity and net biodiversity, complementarity, and selection effects. Despite strong increases in productivity with nutrient addition and decreases in productivity with drought, we found that resource alterations did not alter biodiversity–ecosystem functioning relationships. Our results suggest that these relationships are largely determined by increases in complementarity effects along plant species richness gradients. Although nutrient addition reduced complementarity effects at high diversity, this appears to be due to high biomass in monocultures under nutrient enrichment. Our results indicate that diversity and the complementarity of species are important regulators of grassland ecosystem productivity, regardless of changes in other drivers of ecosystem function. PMID:27114579

By-product mutualism with evolving common enemies.

PubMed

De Jaegher, Kris

2017-05-07

The common-enemy hypothesis of by-product mutualism states that organisms cooperate when it is in their individual interests to do so, with benefits for other organisms arising as a by-product; in particular, such cooperation is hypothesized to arise when organisms face the common enemy of a sufficiently adverse environment. In an evolutionary game where two defenders can cooperate to defend a common resource, this paper analyzes the common-enemy hypothesis when adversity is endogenous, in that an attacker sets the number of attacks. As a benchmark, we first consider exogenous adversity, where adversity is not subject to evolution. In this case, the common-enemy hypothesis is predicted when the degree of complementarity between defenders' defensive efforts is sufficiently low. When the degree of complementarity is high, the hypothesis is predicted only when cooperation costs are high; when cooperation costs are instead low, a competing hypothesis is predicted, where adversity discourages cooperation. Second, we consider the case of endogenous adversity. In this case, we continue to predict the competing hypothesis for a high degree of complementarity and low cooperation costs. The common-enemy hypothesis, however, only continues to be predicted for the lowest degrees of complementarity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Countermine Warfare Analysis

DTIC Science & Technology

1981-06-01

which were Matilda and Valentine tanks fitted with heavy chain flails. As a front-mounted drum rotated, the chains were thrust forward to strike the...Detector, truck-mounted (UAZ-69 or UAZ-469 trucks) mine detector. The mine detection device is fitted to the truck brakes to stop it automati- cally...employed by the Soviets. The plows are controlled separately from inside the tank, and rollers are made up-of multiple discs of various I configurations. A

Tertiary structure of human {Lambda}6 light chains.

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

Pokkuluri, P. R.; Solomon, A.; Weiss, D. T.

1999-01-01

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

Bacterial mediated alleviation of heavy metal stress and decreased accumulation of metals in plant tissues: Mechanisms and future prospects.

PubMed

Etesami, Hassan

2018-01-01

Heavy metal pollution of agricultural soils is one of main concerns causing some of the different ecological and environmental problems. Excess accumulation of these metals in soil has changed microbial community (e.g., structure, function, and diversity), deteriorated soil, decreased the growth and yield of plant, and entered into the food chain. Plants' tolerance to heavy metal stress needs to be improved in order to allow growth of crops with minimum or no accumulation of heavy metals in edible parts of plant that satisfy safe food demands for the world's rapidly increasing population. It is well known that PGPRs (plant growth-promoting rhizobacteria) enhance crop productivity and plant resistance to heavy metal stress. Many recent reports describe the application of heavy metal resistant-PGPRs to enhance agricultural yields without accumulation of metal in plant tissues. This review provides information about the mechanisms possessed by heavy metal resistant-PGPRs that ameliorate heavy metal stress to plants and decrease the accumulation of these metals in plant, and finally gives some perspectives for research on these bacteria in agriculture in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Substituent effects that control conjugated oligomer conformation through non-covalent interactions

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

Sharber, Seth A.; Baral, Rom Nath; Frausto, Fanny

Although understanding the conformations and arrangements of conjugated materials as solids is key to their prospective applications, predictive power over these structural factors remains elusive. In this work, substituent effects tune non-covalent interactions between side-chain fluorinated benzyl esters and main-chain terminal arenes, in turn controlling the conformations and interchromophore aggregation of three-ring phenylene-ethynylenes (PEs). Cofacial fluoroarene–arene (ArF–ArH) interactions cause twisting in the PE backbone, interrupting intramolecular conjugation as well as blocking chromophore aggregation, both of which prevent the typically observed bathochromic shift observed upon transitioning PEs from solution to solid. This work highlights two structural factors that determine whether themore » ArF–ArH interactions, and the resulting twisted, unaggregated chromophores, occur in these solids: (i) the electron-releasing characteristic of substituents on ArH, with more electron-releasing character favoring ArF–ArH interactions, and (ii) the fluorination pattern of the ArF ring, with 2,3,4,5,6-pentafluorophenyl favoring ArF–ArH interactions over 2,4,6-trifluorophenyl. Furthermore, these trends indicate that considerations of electrostatic complementarity, whether through a polar-π or substituent–substituent mechanism, can serve as an effective design principle in controlling the interaction strengths, and therefore the optoelectronic properties, of these molecules as solids.« less

Substituent effects that control conjugated oligomer conformation through non-covalent interactions

DOE PAGES

Sharber, Seth A.; Baral, Rom Nath; Frausto, Fanny; ...

2017-03-31

Although understanding the conformations and arrangements of conjugated materials as solids is key to their prospective applications, predictive power over these structural factors remains elusive. In this work, substituent effects tune non-covalent interactions between side-chain fluorinated benzyl esters and main-chain terminal arenes, in turn controlling the conformations and interchromophore aggregation of three-ring phenylene-ethynylenes (PEs). Cofacial fluoroarene–arene (ArF–ArH) interactions cause twisting in the PE backbone, interrupting intramolecular conjugation as well as blocking chromophore aggregation, both of which prevent the typically observed bathochromic shift observed upon transitioning PEs from solution to solid. This work highlights two structural factors that determine whether themore » ArF–ArH interactions, and the resulting twisted, unaggregated chromophores, occur in these solids: (i) the electron-releasing characteristic of substituents on ArH, with more electron-releasing character favoring ArF–ArH interactions, and (ii) the fluorination pattern of the ArF ring, with 2,3,4,5,6-pentafluorophenyl favoring ArF–ArH interactions over 2,4,6-trifluorophenyl. Furthermore, these trends indicate that considerations of electrostatic complementarity, whether through a polar-π or substituent–substituent mechanism, can serve as an effective design principle in controlling the interaction strengths, and therefore the optoelectronic properties, of these molecules as solids.« less

Heavy metals in agricultural soils of the European Union with implications for food safety.

PubMed

Tóth, G; Hermann, T; Da Silva, M R; Montanarella, L

2016-03-01

Soil plays a central role in food safety as it determines the possible composition of food and feed at the root of the food chain. However, the quality of soil resources as defined by their potential impact on human health by propagation of harmful elements through the food chain has been poorly studied in Europe due to the lack of data of adequate detail and reliability. The European Union's first harmonized topsoil sampling and coherent analytical procedure produced trace element measurements from approximately 22,000 locations. This unique collection of information enables a reliable overview of the concentration of heavy metals, also referred to as metal(loid)s including As, Cd, Cr, Cu, Hg, Pb, Zn, Sb. Co, and Ni. In this article we propose that in some cases (e.g. Hg and Cd) the high concentrations of soil heavy metal attributed to human activity can be detected at a regional level. While the immense majority of European agricultural land can be considered adequately safe for food production, an estimated 6.24% or 137,000km(2) needs local assessment and eventual remediation action. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Metasynthesis of the Complementarity of Culturally Responsive and Inquiry-Based Science Education in K-12 Settings: Implications for Advancing Equitable Science Teaching and Learning

ERIC Educational Resources Information Center

Brown, Julie C.

2017-01-01

Employing metasynthesis as a method, this study examined 52 empirical articles on culturally relevant and responsive science education in K-12 settings to determine the nature and scope of complementarity between culturally responsive and inquiry-based science practices (i.e., science and engineering practices identified in the National Research…

Convergence analysis of modulus-based matrix splitting iterative methods for implicit complementarity problems.

PubMed

Wang, An; Cao, Yang; Shi, Quan

2018-01-01

In this paper, we demonstrate a complete version of the convergence theory of the modulus-based matrix splitting iteration methods for solving a class of implicit complementarity problems proposed by Hong and Li (Numer. Linear Algebra Appl. 23:629-641, 2016). New convergence conditions are presented when the system matrix is a positive-definite matrix and an [Formula: see text]-matrix, respectively.

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1.

PubMed

De Nardis, Camilla; Hendriks, Linda J A; Poirier, Emilie; Arvinte, Tudor; Gros, Piet; Bakker, Alexander B H; de Kruif, John

2017-09-01

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G 1 We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1

PubMed Central

De Nardis, Camilla; Hendriks, Linda J. A.; Poirier, Emilie; Arvinte, Tudor; Gros, Piet; Bakker, Alexander B. H.; de Kruif, John

2017-01-01

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G1. We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed “DEKK” consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics. PMID:28655766

Individual-based analyses reveal limited functional overlap in a coral reef fish community.

PubMed

Brandl, Simon J; Bellwood, David R

2014-05-01

Detailed knowledge of a species' functional niche is crucial for the study of ecological communities and processes. The extent of niche overlap, functional redundancy and functional complementarity is of particular importance if we are to understand ecosystem processes and their vulnerability to disturbances. Coral reefs are among the most threatened marine systems, and anthropogenic activity is changing the functional composition of reefs. The loss of herbivorous fishes is particularly concerning as the removal of algae is crucial for the growth and survival of corals. Yet, the foraging patterns of the various herbivorous fish species are poorly understood. Using a multidimensional framework, we present novel individual-based analyses of species' realized functional niches, which we apply to a herbivorous coral reef fish community. In calculating niche volumes for 21 species, based on their microhabitat utilization patterns during foraging, and computing functional overlaps, we provide a measurement of functional redundancy or complementarity. Complementarity is the inverse of redundancy and is defined as less than 50% overlap in niche volumes. The analyses reveal extensive complementarity with an average functional overlap of just 15.2%. Furthermore, the analyses divide herbivorous reef fishes into two broad groups. The first group (predominantly surgeonfishes and parrotfishes) comprises species feeding on exposed surfaces and predominantly open reef matrix or sandy substrata, resulting in small niche volumes and extensive complementarity. In contrast, the second group consists of species (predominantly rabbitfishes) that feed over a wider range of microhabitats, penetrating the reef matrix to exploit concealed surfaces of various substratum types. These species show high variation among individuals, leading to large niche volumes, more overlap and less complementarity. These results may have crucial consequences for our understanding of herbivorous processes on coral reefs, as algal removal appears to depend strongly on species-specific microhabitat utilization patterns of herbivores. Furthermore, the results emphasize the capacity of the individual-based analyses to reveal variation in the functional niches of species, even in high-diversity systems such as coral reefs, demonstrating its potential applicability to other high-diversity ecosystems. © 2013 The Authors. Journal of Animal Ecology © 2013 British Ecological Society.

Horizons of description: Black holes and complementarity

NASA Astrophysics Data System (ADS)

Bokulich, Peter Joshua Martin

Niels Bohr famously argued that a consistent understanding of quantum mechanics requires a new epistemic framework, which he named complementarity . This position asserts that even in the context of quantum theory, classical concepts must be used to understand and communicate measurement results. The apparent conflict between certain classical descriptions is avoided by recognizing that their application now crucially depends on the measurement context. Recently it has been argued that a new form of complementarity can provide a solution to the so-called information loss paradox. Stephen Hawking argues that the evolution of black holes cannot be described by standard unitary quantum evolution, because such evolution always preserves information, while the evaporation of a black hole will imply that any information that fell into it is irrevocably lost---hence a "paradox." Some researchers in quantum gravity have argued that this paradox can be resolved if one interprets certain seemingly incompatible descriptions of events around black holes as instead being complementary. In this dissertation I assess the extent to which this black hole complementarity can be undergirded by Bohr's account of the limitations of classical concepts. I begin by offering an interpretation of Bohr's complementarity and the role that it plays in his philosophy of quantum theory. After clarifying the nature of classical concepts, I offer an account of the limitations these concepts face, and argue that Bohr's appeal to disturbance is best understood as referring to these conceptual limits. Following preparatory chapters on issues in quantum field theory and black hole mechanics, I offer an analysis of the information loss paradox and various responses to it. I consider the three most prominent accounts of black hole complementarity and argue that they fail to offer sufficient justification for the proposed incompatibility between descriptions. The lesson that emerges from this dissertation is that we have as much to learn from the limitations facing our scientific descriptions as we do from the successes they enjoy. Because all of our scientific theories offer at best limited, effective accounts of the world, an important part of our interpretive efforts will be assessing the borders of these domains of description.

Nonrotational states in isotonic chains of heavy nuclei

NASA Astrophysics Data System (ADS)

Adamian, G. G.; Malov, L. A.; Antonenko, N. V.; Jolos, R. V.

2018-03-01

The ground-state deformations of heavy nuclei are explored with the microscopic-macroscopic approach using the single-particle Woods-Saxon potential of the quasiparticle-phonon model. The calculations of the energies of low-lying nonrotational states take into account the residual pairing and phonon-quasiparticle interactions. The spectra of these states are presented for the N =147 -161 isotones. The sensitivity of the calculated results to the parameters of the model is studied. A rather good description of the available experimental data is demonstrated.

Heavy Chain Diseases

MedlinePlus

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

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf)

PubMed Central

Shin, Geewook; Lee, Hyungjun; Palaksha, K. J.; Kim, Youngrim; Lee, Eunyoung; Shin, Yongseung; Lee, Eunggoo; Park, Kyungdae

2006-01-01

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles. PMID:16871026

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

NASA Technical Reports Server (NTRS)

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.

Application of SEU imaging for analysis of device architecture using a 25 MeV/u 86Kr ion microbeam at HIRFL

NASA Astrophysics Data System (ADS)

Liu, Tianqi; Yang, Zhenlei; Guo, Jinlong; Du, Guanghua; Tong, Teng; Wang, Xiaohui; Su, Hong; Liu, Wenjing; Liu, Jiande; Wang, Bin; Ye, Bing; Liu, Jie

2017-08-01

The heavy-ion imaging of single event upset (SEU) in a flash-based field programmable gate array (FPGA) device was carried out for the first time at Heavy Ion Research Facility in Lanzhou (HIRFL). The three shift register chains with separated input and output configurations in device under test (DUT) were used to identify the corresponding logical area rapidly once an upset occurred. The logic units in DUT were partly configured in order to distinguish the registers in SEU images. Based on the above settings, the partial architecture of shift register chains in DUT was imaged by employing the microbeam of 86Kr ion with energy of 25 MeV/u in air. The results showed that the physical distribution of registers in DUT had a high consistency with its logical arrangement by comparing SEU image with logic configuration in scanned area.

[Mutation analysis of beta myosin heavy chain gene in hypertrophic cardiomyopathy families].

PubMed

Fan, Xin-ping; Yang, Zhong-wei; Feng, Xiu-li; Yang, Fu-hui; Xiao, Bai; Liang, Yan

2011-08-01

To detect the gene mutations of beta-myosin heavy chain gene (MYH7) in Chinese pedigrees with hypertrophic cardiomyopathy (HCM), and to analyze the correlation between the genotype and phenotype. Exons 3, 5, 7-9, 11-16 and 18-23 of the MYH7 gene were amplified with PCR in three Chinese pedigrees with HCM. The products were sequenced. Sequence alignment between the detected and the standard sequences was performed. A missense mutation of Thr441Met in exon 14 was identified in a pedigree, which was not detected in the controls. Several synonymous mutations of MYH7 gene were detected in the three pedigrees. The mutation of Thr441Met, located in the actin binding domain of the globular head, was first identified in Chinese. It probably caused HCM. HCM is a heterogeneous disease. Many factors are involved in the process of its occurrence and development.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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

1994-05-01

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

Discovery of element 117: Super-heavy elements and the “island of stability”

DOE PAGES

Roberto, James B.; Rykaczewski, Krzysztof Piotr

2017-04-12

Element 117 (tennessine) joined the periodic table in November 2016. Two tennessine isotopes were synthesized by bombarding 249Bk from Oak Ridge National Laboratory with 48Ca ions at the Joint Institute of Nuclear Research, Russia, and 11 new heaviest isotopes of odd-Z elements were observed in subsequent decay chains. These isotopes exhibit increasing lifetimes as the closed nuclear shell at neutron number N = 184 is approached, providing evidence for the “island of stability” for super-heavy elements. Here, this article summarizes recent super-heavy element research with a focus on element 117, the role of actinide targets, and opportunities to synthesize elementsmore » 119 and 120.« less

Discovery of element 117: Super-heavy elements and the “island of stability”

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

Roberto, James B.; Rykaczewski, Krzysztof Piotr

Element 117 (tennessine) joined the periodic table in November 2016. Two tennessine isotopes were synthesized by bombarding 249Bk from Oak Ridge National Laboratory with 48Ca ions at the Joint Institute of Nuclear Research, Russia, and 11 new heaviest isotopes of odd-Z elements were observed in subsequent decay chains. These isotopes exhibit increasing lifetimes as the closed nuclear shell at neutron number N = 184 is approached, providing evidence for the “island of stability” for super-heavy elements. Here, this article summarizes recent super-heavy element research with a focus on element 117, the role of actinide targets, and opportunities to synthesize elementsmore » 119 and 120.« less

Molecular design of high performance zwitterionic liquids for enhanced heavy-oil recovery processes.

PubMed

Martínez-Magadán, J M; Cartas-Rosado, A R; Oviedo-Roa, R; Cisneros-Dévora, R; Pons-Jiménez, M; Hernández-Altamirano, R; Zamudio-Rivera, L S

2018-03-01

Branched gemini zwitterionic liquids, which contain two zwitterionic moieties of linked quaternary-ammonium and carboxylate groups, are proposed as chemicals to be applied in the Enhanced Oil Recovery (EOR) from fractured carbonate reservoirs. The zwitterionic moieties are bridged between them through an alkyl chain containing 12 ether groups, and each zwitterionic moiety has attached a long alkyl tail including a CC double bond. A theoretical molecular mechanism over which EOR could rest, consisting on both the disaggregation of heavy oil and the reservoir-rock wettability alteration, was suggested. Results show that chemicals can both reduce the viscosity and remove heavy-oil molecules from the rock surface. Copyright © 2018. Published by Elsevier Inc.

Invasive carnivores alter ecological function and enhance complementarity in scavenger assemblages on ocean beaches.

PubMed

Brown, Marion B; Schlacher, Thomas A; Schoeman, David S; Weston, Michael A; Huijbers, Chantal M; Olds, Andrew D; Connolly, Rod M

2015-10-01

Species composition is expected to alter ecological function in assemblages if species traits differ strongly. Such effects are often large and persistent for nonnative carnivores invading islands. Alternatively, high similarity in traits within assemblages creates a degree of functional redundancy in ecosystems. Here we tested whether species turnover results in functional ecological equivalence or complementarity, and whether invasive carnivores on islands significantly alter such ecological function. The model system consisted of vertebrate scavengers (dominated by raptors) foraging on animal carcasses on ocean beaches on two Australian islands, one with and one without invasive red foxes (Vulpes vulpes). Partitioning of scavenging events among species, carcass removal rates, and detection speeds were quantified using camera traps baited with fish carcasses at the dune-beach interface. Complete segregation of temporal foraging niches between mammals (nocturnal) and birds (diurnal) reflects complementarity in carrion utilization. Conversely, functional redundancy exists within the bird guild where several species of raptors dominate carrion removal in a broadly similar way. As predicted, effects of red foxes were large. They substantially changed the nature and rate of the scavenging process in the system: (1) foxes consumed over half (55%) of all carrion available at night, compared with negligible mammalian foraging at night on the fox-free island, and (2) significant shifts in the composition of the scavenger assemblages consuming beach-cast carrion are the consequence of fox invasion at one island. Arguably, in the absence of other mammalian apex predators, the addition of red foxes creates a new dimension of functional complementarity in beach food webs. However, this functional complementarity added by foxes is neither benign nor neutral, as marine carrion subsidies to coastal red fox populations are likely to facilitate their persistence as exotic carnivores.

Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

PubMed Central

Kraut, Daniel A; Sigala, Paul A; Pybus, Brandon; Liu, Corey W; Ringe, Dagmar; Petsko, Gregory A

2006-01-01

A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution. PMID:16602823

Sensitivity of midge larvae of Chironomus tentans Fabricius (Diptera Chironomidae) to heavy metals

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

Khangarot, B.S.; Ray, P.K.

1989-03-01

The discharge of heavy metals into the natural waters has numerous obvious impacts on physical, chemical and biological parameters of aquatic ecosystem. Bioassay tests are important steps in establishing appropriate water quality criteria and standards for diverse use of ponds, lakes, streams and river waters. Therefore, the acute toxicities of various heavy metals to water flea Daphnia magna, and snail Lymnaea acuminata, and toad tadpoles Bufo mentanostictus have been reported from the authors' laboratory. Chironomid larvae might be particularly useful as indicators of water quality because they are widely distributed in freshwater systems and often from diverse communities within particularmore » habitat. The aim of this study was to determine the acute toxicity of ten heavy metals to the midge larvae Chironomus tentans Fabricius, which forms an important link in aquatic food chain(s).« less

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Increased myosin heavy chain-beta with atrial expression of ventricular light chain-2 in canine cardiomyopathy.

PubMed

Fuller, Geraldine A; Bicer, Sabahattin; Hamlin, Robert L; Yamaguchi, Mamoru; Reiser, Peter J

2007-10-01

Dilated cardiomyopathy is a naturally occurring disease in humans and dogs. Human studies have shown increased levels of myosin heavy chain (MHC)-beta in failing ventricles and the left atria (LA) and of ventricular light chain (VLC)-2 in the right atria in dilated cardiomyopathy. This study evaluates the levels of MHC-beta in all heart chambers in prolonged canine right ventricular pacing. In addition, we determined whether levels of VLC2 were altered in these hearts. Failing hearts demonstrated significantly increased levels of MHC-beta in the right atria, right atrial appendage, LA, left atrial appendage (LAA), and right ventricle compared with controls. Significant levels of VLC2 were detected in the right atria of paced hearts. Differences in MHC-beta expression were observed between the LA and the LAA of paced and control dogs. MHC-beta expression was significantly greater in the LA of paced and control dogs compared with their respective LAA. The cardiac myosin isoform shifts in this study were similar to those observed in end-stage human heart failure and more severe than those reported in less prolonged pacing models, supporting the use of this model for further study of end-stage human heart failure. The observation of consistent differences between sampling sites, especially LA versus LAA, indicates the need for rigorous sampling consistency in future studies.

Insect Ferritins: typical or atypical?

PubMed Central

Pham, Daphne Q. D.; Winzerling, Joy J.

2010-01-01

Insects transmit millions of cases of disease each year, and cost millions of dollars in agricultural losses. The control of insect-borne diseases is vital for numerous developing countries, and the management of agricultural insect pests is a very serious business for developed countries. Control methods should target insect-specific traits in order to avoid non-target effects, especially in mammals. Since insect cells have had a billion years of evolutionary divergence from those of vertebrates, they differ in many ways that might be promising for the insect control field—especially, in iron metabolism because current studies have indicated that significant differences exist between insect and mammalian systems. Insect iron metabolism differs from that of vertebrates in the following respects. Insect ferritins have a heavier mass than mammalian ferritins. Unlike their mammalian counterparts, the insect ferritin subunits are often glycosylated and are synthesized with a signal peptide. The crystal structure of insect ferritin also shows a tetrahedral symmetry consisting of 12 heavy chain and 12 light chain subunits in contrast to that of mammalian ferritin that exhibits an octahedral symmetry made of 24 heavy chain and 24 light chain subunits. Insect ferritins associate primarily with the vacuolar system and serve as iron transporters—quite the opposite of the mammalian ferritins, which are mainly cytoplasmic and serve as iron storage proteins. This review will discuss these differences. PMID:20230873

Skill complementarity enhances heterophily in collaboration networks

PubMed Central

Xie, Wen-Jie; Li, Ming-Xia; Jiang, Zhi-Qiang; Tan, Qun-Zhao; Podobnik, Boris; Zhou, Wei-Xing; Stanley, H. Eugene

2016-01-01

Much empirical evidence shows that individuals usually exhibit significant homophily in social networks. We demonstrate, however, skill complementarity enhances heterophily in the formation of collaboration networks, where people prefer to forge social ties with people who have professions different from their own. We construct a model to quantify the heterophily by assuming that individuals choose collaborators to maximize utility. Using a huge database of online societies, we find evidence of heterophily in collaboration networks. The results of model calibration confirm the presence of heterophily. Both empirical analysis and model calibration show that the heterophilous feature is persistent along the evolution of online societies. Furthermore, the degree of skill complementarity is positively correlated with their production output. Our work sheds new light on the scientific research utility of virtual worlds for studying human behaviors in complex socioeconomic systems. PMID:26743687

Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres.

PubMed

Rannou, F; Droguet, M; Giroux-Metges, M A; Pennec, Y; Gioux, M; Pennec, J P

2009-11-01

The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocity of muscle fibres. The functional properties of the muscle fibre are also conditioned by its membrane excitability through the electrophysiological properties of sodium voltage-gated channels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus (PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibres are identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slow type I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared (Student's t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms are identified in PL and only type I in Sol single fibres. In PL, maximal sodium current (I(max)), maximal sodium conductance (g(Na,max)) and time constants of activation and inactivation ((m) and (h)) increase according to the scheme I-->IIa-->IIx-->IIb (P < 0.05). (m) values related to sodium channel type and/or function, are similar in Sol I and PL IIb fibres (P = 0.97) despite different contractile properties. The voltage dependence of activation (V(a,1/2)) shows a shift towards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL (P < 0.05). These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle like PL, while slow fibres of postural muscle such as soleus could be recruited in the same ways as IIb fibres in a fast muscle.

The B2 Alternatively Spliced Isoform of Nonmuscle Myosin II-B Lacks Actin-activated MgATPase Activity and In Vitro Motility

PubMed Central

Kim, Kye-Young; Kawamoto, Sachiyo; Bao, Jianjun; Sellers, James R.; Adelstein, Robert S.

2008-01-01

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the non-spliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells (Ma et al., Mol. Biol. Cell 15 (2006) 2138-2149). In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acids II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific. PMID:18060863

Effects of prolonged strenuous endurance exercise on plasma myosin heavy chain fragments and other muscular proteins. Cycling vs running.

PubMed

Koller, A; Mair, J; Schobersberger, W; Wohlfarter, T; Haid, C; Mayr, M; Villiger, B; Frey, W; Puschendorf, B

1998-03-01

This study evaluates creatine kinase, myosin heavy chain, and cardiac troponin blood levels following three types of exercise: 1) short-distance uphill or downhill running; 2) alpine ultramarathon; and 3) alpine long-distance cycling. Comparative field study; follow-up up to 10 days. Department of Sports Medicine. All biochemical markers were analysed at the Department of Medical Chemistry and Biochemistry. Subjects included healthy, trained males (N = 53). All subjects were nonsmokers and free from medication prior to and during the study. Each volunteer was an experienced runner or cyclist, who had at least once successfully finished the Swiss Alpine Marathon of Davos or the Otztal-Radmarathon before. Running or cycling. Plasma concentrations of creatine kinase, myosin heavy chain fragments and cardiac troponins were measured to diagnose skeletal and cardiac muscle damage, respectively. Skeletal muscle protein release is markedly different between uphill and downhill running, with very little evidence for muscle damage in the uphill runners. There is considerable muscle protein leakage in the ultramarathoners (67 km distance; 30 km downhill running). In contrast, only modest amounts of skeletal muscle damage are found after alpine long-distance cycling (230 km distance). This study proves that there is slow-twitch skeletal muscle fiber damage after prolonged strenuous endurance exercise and short-distance downhill running. Exhaustive endurance exercise involving downhill running and short-distance downhill running lead to more pronounced injury than strenuous endurance exercise involving concentric actions. From our results there is no reason for suggesting that prolonged intense exercise may induce myocardial injury in symptom-less athletes without cardiac deseases.

A novel role of HLA class I in the pathology of medulloblastoma.

PubMed

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-07-12

MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. We investigated expression of four essential components of MHC class I (heavy chain, beta2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of beta2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or beta2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility.

A novel role of HLA class I in the pathology of medulloblastoma

PubMed Central

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-01-01

Background MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. Methods We investigated expression of four essential components of MHC class I (heavy chain, β2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. Results The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of β2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. Conclusion MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or β2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility. PMID:19594892

Misfolding of major histocompatibility complex class I molecules in activated T cells allows cis-interactions with receptors and signaling molecules and is associated with tyrosine phosphorylation.

PubMed

Santos, Susana G; Powis, Simon J; Arosa, Fernando A

2004-12-17

Knowledge of the origin and biochemical status of beta(2)-microglobulin-free or misfolded major histocompatibility complex (MHC)-I molecules is essential for understanding their pleiotropic properties. Here we show that in normal human T cells, misfolding of MHC-I molecules is turned on upon activation and cell division and is proportional to the level of proliferation. Immunoprecipitation showed that a number of proteins are associated with MHC-I heavy chains at the surface of activated T cells, including the CD8alphabeta receptor and the chaperone tandem calreticulin/ERp57, associations that rely upon the existence of a pool of HC-10-reactive molecules. Biochemical analysis showed that misfolded MHC-I molecules present at the cell surface are fully glycosylated mature molecules. Importantly, misfolded MHC-I molecules are tyrosine phosphorylated and are associated with kinase activity. In vitro kinase assays followed by reprecipitation indicated that tyrosine phosphorylation of the class I heavy chain is probably mediated by a Src tyrosine kinase because Lck was found associated with HC-10 immunocomplexes. Finally, we show that inhibition of tyrosine phosphorylation by using the Src-family tyrosine kinase inhibitor PP2 resulted in enhanced release of MHC-I heavy chains from the cell surface of activated T cells and a slight down-regulation of cell surface W6/32-reactive molecules. This study provides new insights into the biology of MHC-I molecules and suggests that tyrosine phosphorylation may be involved in the regulation of MHC-I misfolding and expression.

Matrix-assisted laser desorption/ionization mass spectrometry for the evaluation of the C-terminal lysine distribution of a recombinant monoclonal antibody.

PubMed

Lazar, Alexandru C; Kloczewiak, Marek A; Mazsaroff, Istvan

2004-01-01

Recombinant monoclonal antibodies produced using mammalian cell lines contain multiple chemical modifications. One specific modification resides on the C-terminus of the heavy chain. Enzymes inside the cell can cleave the C-terminal lysine from the heavy-chain molecules, and variants with and without C-terminal lysine can be produced. In order to fully characterize the protein, there is a need for analytical methods that are able to account for the different product variants. Conventional analytical methods used for the measurement of the distribution of the two different variants are based on chemical or enzymatic degradation of the protein followed by chromatographic separation of the degradation products. Chromatographic separations with gradient elution have long run times, and analyses of multiple samples are time-consuming. This paper reports development of a novel method for the determination of the relative amounts of the two C-terminal heavy-chain variants based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) measurements of the cyanogen bromide degraded recombinant monoclonal antibody products. The distribution of the variants is determined from the MALDI-TOF mass spectra by measuring the peak areas of the two C-terminal peptides. The assay was used for the assessment of the C-terminal lysine distribution in different development lots. The method was able to differentiate between the products obtained using the same cell line as well as between products obtained from different cell lines. Copyright 2004 John Wiley & Sons, Ltd.

Production and characterization of monoclonal antibodies to IgM of Pacific herring (Clupea pallasii)

USGS Publications Warehouse

Purcell, Maureen K.; Bromage, Erin S.; Silva, Jessica; Hansen, John D.; Badil, Samantha M.; Woodson, James C.; Hershberger, Paul K.

2012-01-01

Pacific herring (Clupea pallasii) have a central role in the North Pacific ecosystem as a forage fish species and are natural reservoirs of several important finfish pathogens, including Viral hemorrhagic septicemia virus (VHSV). Here, we report the identification of the gene encoding the immunoglobulin mu (IgM) heavy chain, as well as the development and characterization of monoclonal antibodies (MAbs) that specifically react with Pacific herring IgM. Pacific herring immunoglobulin was purified and consisted of heavy and light chains of approximately 80 and 25 kDa. Three hybridoma clones were initially identified by ELISA as reactive with purified immunoglobulin but only one clone was able to detect an 80 kDa protein in Pacific and Atlantic herring (Clupea harengus) whole plasma by denaturing western blot. However, all three MAbs were able to precipitate an 80 kDa protein from Pacific herring and LCMS sequencing of peptide fragments derived from this protein matched the predicted amino acid sequence of the cloned, heavy chain gene. In addition, two of the MAbs stained cells within the putative lymphocyte gates for the spleen, anterior kidney and posterior kidney but were not reactive for myeloid/granulocyte gates, which is consistent with these MAbs reacting with surface IgM+ B-cells. To our knowledge, this is the first report of IgM-related gene sequences and anti-IgM monoclonal antibodies from any member of the family Clupeidae. The antibodies produced in this study are critical for achieving our long-term goal of conducting serological surveillance to assess pathogen exposure in natural populations of Pacific herring.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment.

PubMed

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition "modules" to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo . Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, F; Rasaee, M J; Forouzandeh Moghadam, M; Allameh, A A; Sadroddiny, E

2004-06-01

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

PubMed Central

Geoghegan, James C.; Fleming, Ryan; Damschroder, Melissa; Bishop, Steven M.; Sathish, Hasige A.; Esfandiary, Reza

2016-01-01

ABSTRACT Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies. PMID:27050875

A comparison of rat myosin from fast and slow skeletal muscle and the effect of disuse

NASA Technical Reports Server (NTRS)

Unsworth, B. R.; Witzmann, F. A.; Fitts, R. H.

1981-01-01

Certain enzymatic and structural features of myosin, purified from rat skeletal muscles representative of the fast twitch glycolytic (type IIb), the fast twitch oxidative (type IIa), and the slow twitch oxidative (type I) fiber, were determined and the results were compared with the measured contractile properties. Good correlation was found between the shortening velocities and Ca(2+)-activated ATPase activity for each fiber type. Short term hind limb immobilization caused prolongation of contraction time and one-half relaxation time in the fast twitch muscles and a reduction of these contractile properties in slow twitch soleus. Furthermore, the increased maximum shortening velocity in the immobilized soleus could be correlated with increased Ca(2+)-ATPase, but no change was observed in the enzymatic activity of the fast twitch muscles. No alteration in light chain distribution with disuse was observed in any of the fiber types. The myosin from slow twitch soleus could be distinguished from fast twitch myosins on the basis of the pattern of peptides generated by proteolysis of the heavy chains. Six weeks of hind limb immobilization resulted in both an increased ATPase activity and an altered heavy chain primary structure in the slow twitch soleus muscle.

A mutagenesis study of a catalytic antibody

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

Jackson, D.Y.; Prudent, J.R.; Baldwin, E.P.

1991-01-01

The authors have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (V{sub H}) of the phosphocholine-binding antibody S107.S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and fourmore » Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the V{sub H} binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.« less

PIGSPro: prediction of immunoGlobulin structures v2.

PubMed

Lepore, Rosalba; Olimpieri, Pier P; Messih, Mario A; Tramontano, Anna

2017-07-03

PIGSpro is a significant upgrade of the popular PIGS server for the prediction of the structure of immunoglobulins. The software has been completely rewritten in python following a similar pipeline as in the original method, but including, at various steps, relevant modifications found to improve its prediction accuracy, as demonstrated here. The steps of the pipeline include the selection of the appropriate framework for predicting the conserved regions of the molecule by homology; the target template alignment for this portion of the molecule; the selection of the main chain conformation of the hypervariable loops according to the canonical structure model, the prediction of the third loop of the heavy chain (H3) for which complete canonical structures are not available and the packing of the light and heavy chain if derived from different templates. Each of these steps has been improved including updated methods developed along the years. Last but not least, the user interface has been completely redesigned and an automatic monthly update of the underlying database has been implemented. The method is available as a web server at http://biocomputing.it/pigspro. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

PubMed

Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

2017-06-01

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

Structural Determination of the Broadly Reactive Anti-IGHV1-69 Anti-idiotypic Antibody G6 and Its Idiotope.

PubMed

Avnir, Yuval; Prachanronarong, Kristina L; Zhang, Zhen; Hou, Shurong; Peterson, Eric C; Sui, Jianhua; Zayed, Hatem; Kurella, Vinodh B; McGuire, Andrew T; Stamatatos, Leonidas; Hilbert, Brendan J; Bohn, Markus-Frederik; Kowalik, Timothy F; Jensen, Jeffrey D; Finberg, Robert W; Wang, Jennifer P; Goodall, Margaret; Jefferis, Roy; Zhu, Quan; Kurt Yilmaz, Nese; Schiffer, Celia A; Marasco, Wayne A

2017-12-12

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Bioremediation of Toxic Heavy Metals: A Patent Review.

PubMed

Verma, Neelam; Sharma, Rajni

2017-01-01

The global industrialization is fulfilling the demands of modern population at the cost of environmental exposure to various contaminants including heavy metals. These heavy metals affect water and soil quality. Moreover, these enter into the food chain and exhibit their lethal effects on the human health even when present at slightly higher concentration than required for normal metabolism. To the worst of their part, the heavy metals may become carcinogenic. Henceforth, the efficient removal of heavy metals is the demand of sustainable development. Remedy: Bioremediation is the 'green' imperative technique for the heavy metal removal without creating secondary metabolites in the ecosystem. The metabolic potential of several bacterial, algal, fungal as well as plant species has the efficiency to exterminate the heavy metals from the contaminated sites. Different strategies like bioaccumulation, biosorption, biotransformation, rhizofilteration, bioextraction and volatilization are employed for removal of heavy metals by the biological species. Bioremediation approach is presenting a splendid alternate for conventional expensive and inefficient methods for the heavy metal removal. The patents granted on the bioremediation of toxic heavy metals are summarized in the present manuscript which supported the applicability of bioremediation technique at commercial scale. However, the implementation of the present information and advanced research are mandatory to further explore the concealed potential of biological species to resume the originality of the environment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A monoclonal antibody distinguishes between two IgM heavy chain isotypes in Atlantic salmon and brown trout: protein characterization, 3D modeling and epitope mapping.

PubMed

Kamil, Atif; Falk, Knut; Sharma, Animesh; Raae, Arnt; Berven, Frode; Koppang, Erling Olaf; Hordvik, Ivar

2011-09-01

Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) possess two distinct subpopulations of IgM which can be separated by anion exchange chromatography. Accordingly, there are two isotypic μ genes in these species, related to ancestral tetraploidy. In the present work it was verified by mass spectrometry that IgM of peak 1 (subpopulation 1) have heavy chains previously designated as μB type whereas IgM of peak 2 (subpopulation 2) have heavy chains of μA type. Two adjacent cysteine residues are present near the C-terminal part of μB, in contrast to one cysteine residue in μA. Salmon IgM of both peak 1 and peak 2 contain light chains of the two most common isotypes: IgL1 and IgL3. In contrast to salmon and brown trout, IgM of rainbow trout (Oncorhynchus mykiss) is eluted in a single peak when subjected to anion exchange chromatography. Surprisingly, a monoclonal antibody MAb4C10 against rainbow trout IgM, reacted with μA in salmon, whereas in brown trout it reacted with μB. It is plausible to assume that DNA has been exchanged between the paralogous A and B loci during evolution while maintaining the two sub-variants, with and without the extra cysteine. MAb4C10 was conjugated to magnetic beads and used to separate cells, demonstrating that μ transcripts residing from captured cells were primarily of A type in salmon and B type in brown trout. An analysis of amino acid substitutions in μA and μB of salmon and brown trout indicated that the third constant domain is essential for MAb4C10 binding. This was supported by 3D modeling and was finally verified by studies of MAb4C10 reactivity with a series of recombinant μ3 constructs. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

Hart, William E.; Siirola, John Daniel

We describe new capabilities for modeling MPEC problems within the Pyomo modeling software. These capabilities include new modeling components that represent complementar- ity conditions, modeling transformations for re-expressing models with complementarity con- ditions in other forms, and meta-solvers that apply transformations and numeric optimization solvers to optimize MPEC problems. We illustrate the breadth of Pyomo's modeling capabil- ities for MPEC problems, and we describe how Pyomo's meta-solvers can perform local and global optimization of MPEC problems.

Functional divergence in nitrogen uptake rates explains diversity-productivity relationship in microalgal communities

DOE PAGES

Mandal, Shovon; Shurin, Jonathan B.; Efroymson, Rebecca A.; ...

2018-05-23

The relationship between biodiversity and productivity has emerged as a central theme in ecology. Mechanistic explanations for this relationship suggest that the role organisms play in the ecosystem (i.e., niches or functional traits) is a better predictor of ecosystem stability and productivity than taxonomic richness. Here, we tested the capacity of functional diversity in nitrogen uptake in experimental microalgal communities to predict the complementarity effect (CE) and selection effect (SE) of biodiversity on productivity. We grew five algal species as monocultures and as polycultures in pairwise combinations in homogeneous (ammonium, nitrate, or urea alone) and heterogeneous nitrogen (mixed nitrogen) environmentsmore » to determine whether complementarity between species may be enhanced in heterogeneous environments. We show that the positive diversity effects on productivity in heterogeneous environments resulted from complementarity effects with no positive contribution by species–specific SEs. Positive biodiversity effects in homogeneous environments, when present (nitrate and urea treatments but not ammonium), were driven both by CE and SE. Our results suggest that functional diversity increases species complementarity and productivity mainly in heterogeneous resource environments. Furthermore, these results provide evidence that the positive effect of functional diversity on community productivity depends on the diversity of resources present in the environment.« less

Functional divergence in nitrogen uptake rates explains diversity-productivity relationship in microalgal communities

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

Mandal, Shovon; Shurin, Jonathan B.; Efroymson, Rebecca A.

The relationship between biodiversity and productivity has emerged as a central theme in ecology. Mechanistic explanations for this relationship suggest that the role organisms play in the ecosystem (i.e., niches or functional traits) is a better predictor of ecosystem stability and productivity than taxonomic richness. Here, we tested the capacity of functional diversity in nitrogen uptake in experimental microalgal communities to predict the complementarity effect (CE) and selection effect (SE) of biodiversity on productivity. We grew five algal species as monocultures and as polycultures in pairwise combinations in homogeneous (ammonium, nitrate, or urea alone) and heterogeneous nitrogen (mixed nitrogen) environmentsmore » to determine whether complementarity between species may be enhanced in heterogeneous environments. We show that the positive diversity effects on productivity in heterogeneous environments resulted from complementarity effects with no positive contribution by species–specific SEs. Positive biodiversity effects in homogeneous environments, when present (nitrate and urea treatments but not ammonium), were driven both by CE and SE. Our results suggest that functional diversity increases species complementarity and productivity mainly in heterogeneous resource environments. Furthermore, these results provide evidence that the positive effect of functional diversity on community productivity depends on the diversity of resources present in the environment.« less

Functional group diversity of bee pollinators increases crop yield

PubMed Central

Hoehn, Patrick; Tscharntke, Teja; Tylianakis, Jason M; Steffan-Dewenter, Ingolf

2008-01-01

Niche complementarity is a commonly invoked mechanism underlying the positive relationship between biodiversity and ecosystem functioning, but little empirical evidence exists for complementarity among pollinator species. This study related differences in three functional traits of pollinating bees (flower height preference, daily time of flower visitation and within-flower behaviour) to the seed set of the obligate cross-pollinated pumpkin Cucurbita moschata Duch. ex Poir. across a land-use intensity gradient from tropical rainforest and agroforests to grassland in Indonesia. Bee richness and abundance changed with habitat variables and we used this natural variation to test whether complementary resource use by the diverse pollinator community enhanced final yield. We found that pollinator diversity, but not abundance, was positively related to seed set of pumpkins. Bees showed species-specific spatial and temporal variation in flower visitation traits and within-flower behaviour, allowing for classification into functional guilds. Diversity of functional groups explained even more of the variance in seed set (r2=45%) than did species richness (r2=32%) highlighting the role of functional complementarity. Even though we do not provide experimental, but rather correlative evidence, we can link spatial and temporal complementarity in highly diverse pollinator communities to pollination success in the field, leading to enhanced crop yield without any managed honeybees. PMID:18595841

Low energy electron catalyst: the electronic origin of catalytic strategies.

PubMed

Davis, Daly; Sajeev, Y

2016-10-12

Using a low energy electron (LEE) as a catalyst, the electronic origin of the catalytic strategies corresponding to substrate selectivity, reaction specificity and reaction rate enhancement is investigated for a reversible unimolecular elementary reaction. An electronic energy complementarity between the catalyst and the substrate molecule is the origin of substrate selectivity and reaction specificity. The electronic energy complementarity is induced by tuning the electronic energy of the catalyst. The energy complementarity maximizes the binding forces between the catalyst and the molecule. Consequently, a new electronically metastable high-energy reactant state and a corresponding new low barrier reaction path are resonantly created for a specific reaction of the substrate through the formation of a catalyst-substrate transient adduct. The LEE catalysis also reveals a fundamental structure-energy correspondence in the formation of the catalyst-substrate transient adduct. Since the energy complementarities corresponding to the substrate molecules of the forward and the backward steps of the reversible reactions are not the same due to their structural differences, the LEE catalyst exhibits a unique one-way catalytic strategy, i.e., the LEE catalyst favors the reversible reaction more effectively in one direction. A characteristic stronger binding of the catalyst to the transition state of the reaction than in the initial reactant state and the final product state is the molecular origin of barrier lowering.

Antigen-binding thymus-derived lymphocytes

PubMed Central

Hogg, Nancy M.; Greaves, M. F.

1972-01-01

Thymus-derived `rosette'-forming lymphocytes which have been separated from other SRBC-sensitive cells by means of cotton wool columns were examined for the presence of immunoglobulin. This was carried out by inhibition of rosette formation by anti-immunoglobulin sera. Inhibition was effected by a number of anti-IgM sera shown to contain antibodies with specificities directed towards the `hinge' region of the μ chain. No other heavy chain specific antisera were inhibitory. The ratio of rosette inhibition by anti-κ and anti-λ light chain sera varied during the course of the response to SRBC, the latter inhibiting by 89 per cent 3 days post-immunization. PMID:4113387

Rapid switch-off of the human myosin heavy chain IIX gene after heavy load muscle contractions is sustained for at least four days.

PubMed

Andersen, J L; Gruschy-Knudsen, T

2018-02-01

Long-term heavy load contractions decrease the relative amount of the myosin heavy chain (MHC) IIX isoform in human skeletal muscle, but the timing of the down-regulation in the short term is unknown. Untrained subjects performed two resistance bouts, in two consecutive days, with one leg, the other leg serving as a control (age 24±1, n=5). Muscle biopsies were obtained in both legs before, immediately after, and 24, 54, and 96 hours after exercise. Serial cryosection analysis combined immunohistochemistry and ATPase histochemistry with In Situ hybridization to identify the distribution of MHC isoforms and their corresponding transcripts, enabling identification of transitional fibers. Fibers positive solely for MHC IIX mRNA decreased in the exercised leg throughout the study period. At 96 hours post-exercise, no fibers solely expressed MHC IIX mRNA. In contrast, the number of fibers expressing MHC IIA mRNA increased throughout the study period. The percentage of fibers expressing mRNA for MHC I was unchanged in both legs at all time points. Pronounced depletion of glycogen in the MHC IIX fibers of the exercised leg verifies that the type IIX fibers were active during the heavy load contractions. Major mismatch between MHC at the mRNA and protein levels was only found in the fibers of the exercised leg. These data provide unequivocal in situ evidence of an immediate shutdown of the MHC IIX gene after resistance exercise. A further novel finding was that the silencing of the MHC IIX gene is sustained at least 4 days after removal of the stimulus. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus.

PubMed

Parvari, R; Avivi, A; Lentner, F; Ziv, E; Tel-Or, S; Burstein, Y; Schechter, I

1988-03-01

cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma-chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross-hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H-chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H-chains, a mechanism which is not operative in the chicken L-chain locus. The most notable among the chicken Igs is the so-called 7S IgG because its H-chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H-chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8-10 H-chain genes.

Data-driven analysis for the temperature and momentum dependence of the heavy-quark diffusion coefficient in relativistic heavy-ion collisions

NASA Astrophysics Data System (ADS)

Xu, Yingru; Bernhard, Jonah E.; Bass, Steffen A.; Nahrgang, Marlene; Cao, Shanshan

2018-01-01

By applying a Bayesian model-to-data analysis, we estimate the temperature and momentum dependence of the heavy quark diffusion coefficient in an improved Langevin framework. The posterior range of the diffusion coefficient is obtained by performing a Markov chain Monte Carlo random walk and calibrating on the experimental data of D -meson RAA and v2 in three different collision systems at the Relativistic Heavy-Ion Collidaer (RHIC) and the Large Hadron Collider (LHC): Au-Au collisions at 200 GeV and Pb-Pb collisions at 2.76 and 5.02 TeV. The spatial diffusion coefficient is found to be consistent with lattice QCD calculations and comparable with other models' estimation. We demonstrate the capability of our improved Langevin model to simultaneously describe the RAA and v2 at both RHIC and the LHC energies, as well as the higher order flow coefficient such as D meson v3. We show that by applying a Bayesian analysis, we are able to quantitatively and systematically study the heavy flavor dynamics in heavy-ion collisions.

Health risk assessment of heavy metals in soils and vegetables from wastewater irrigated area, Beijing-Tianjin city cluster, China.

PubMed

Wang, Yanchun; Qiao, Min; Liu, Yunxia; Zhu, Yongguan

2012-01-01

The possible health risks of heavy metals contamination to local population through food chain were evaluated in Beijing and Tianjin city cluster, China, where have a long history of sewage irrigation. The transfer factors (TF) for heavy metals from soil to vegetables for six elements including Cu, Zn, Pb, Cr, As and Cd were calculated and the pollution load indexes (PLI) were also assessed. Results indicate that only Cd exceeded the maximum acceptable limit in these sites. So far, the heavy metal concentrations in soils and vegetables were all below the permissible limits set by the Ministry of Environmental Protection of China and World Health Organization. The transfer factors of six heavy metals showed the trend as Cd > Zn > Cu > Pb > As > Cr, which were dependent on the vegetable species. The estimated dietary intakes of Cu, Zn, Pb, Cr, As and Cd were far below the tolerable limits and the target hazard quotient (THQ) values were less than 1, which suggested that the health risks of heavy metals exposure through consuming vegetables were generally assumed to be safe.

Heavy metal content in tea soils and their distribution in different parts of tea plants, Camellia sinensis (L). O. Kuntze.

PubMed

Seenivasan, Subbiah; Anderson, Todd Alan; Muraleedharan, Narayanannair

2016-07-01

Soils contaminated with heavy metals may pose a threat to environment and human health if metals enter the food chain over and above threshold levels. In general, there is a lack of information on the presence of heavy metals in tea [Camellia sinensis (L). O. Kuntze] plants and the soils in which they are grown. Therefore, an attempt was made to establish a database on the important heavy metals: cadmium (Cd), chromium (Cr), nickel (Ni), and lead (Pb). For an initial survey on heavy metals, soil samples were collected randomly from tea-growing areas of Tamil Nadu, Kerala, and Karnataka, India. Parallel studies were conducted in the greenhouse on uptake of Pb, Cd, and Ni from soils supplemented with these metals at different concentrations. Finally, metal distribution in the tea plants under field conditions was also documented to assess the accumulation potential and critical limit of uptake by plants.

Plant-driven removal of heavy metals from soil: uptake, translocation, tolerance mechanism, challenges, and future perspectives.

PubMed

Thakur, Sveta; Singh, Lakhveer; Wahid, Zularisam Ab; Siddiqui, Muhammad Faisal; Atnaw, Samson Mekbib; Din, Mohd Fadhil Md

2016-04-01

Increasing heavy metal (HM) concentrations in the soil have become a significant problem in the modern industrialized world due to several anthropogenic activities. Heavy metals (HMs) are non-biodegradable and have long biological half lives; thus, once entered in food chain, their concentrations keep on increasing through biomagnification. The increased concentrations of heavy metals ultimately pose threat on human life also. The one captivating solution for this problem is to use green plants for HM removal from soil and render it harmless and reusable. Although this green technology called phytoremediation has many advantages over conventional methods of HM removal from soils, there are also many challenges that need to be addressed before making this technique practically feasible and useful on a large scale. In this review, we discuss the mechanisms of HM uptake, transport, and plant tolerance mechanisms to cope with increased HM concentrations. This review article also comprehensively discusses the advantages, major challenges, and future perspectives of phytoremediation of heavy metals from the soil.

Phytoremediation strategies for soils contaminated with heavy metals: Modifications and future perspectives.

PubMed

Sarwar, Nadeem; Imran, Muhammad; Shaheen, Muhammad Rashid; Ishaque, Wajid; Kamran, Muhammad Asif; Matloob, Amar; Rehim, Abdur; Hussain, Saddam

2017-03-01

Presence of heavy metals in agricultural soils is of major environmental concern and a great threat to life on the earth. A number of human health risks are associated with heavy metals regarding their entry into food chain. Various physical, chemical and biological techniques are being used to remove heavy metals and metalloids from soils. Among them, phytoremediation is a good strategy to harvest heavy metals from soils and have been proven as an effective and economical technique. In present review, we discussed various sources and harmful effects of some important heavy metals and metalloids, traditional phytoremediation strategies, mechanisms involved in phytoremediation of these metals, limitations and some recent advances in phytoremediation approaches. Since traditional phytoremediation approach poses some limitations regarding their applications at large scale, so there is a dire need to modify this strategy using modern chemical, biological and genetic engineering tools. In view of above, the present manuscript brings both traditional and advanced phytoremediation techniques together in order to compare, understand and apply these strategies effectively to exclude heavy metals from soil keeping in view the economics and effectiveness of phytoremediation strategies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Warm season heavy rainfall events over the Huaihe River Valley and their linkage with wintertime thermal condition of the tropical oceans

NASA Astrophysics Data System (ADS)

Li, Laifang; Li, Wenhong; Tang, Qiuhong; Zhang, Pengfei; Liu, Yimin

2016-01-01

Warm season heavy rainfall events over the Huaihe River Valley (HRV) of China are amongst the top causes of agriculture and economic loss in this region. Thus, there is a pressing need for accurate seasonal prediction of HRV heavy rainfall events. This study improves the seasonal prediction of HRV heavy rainfall by implementing a novel rainfall framework, which overcomes the limitation of traditional probability models and advances the statistical inference on HRV heavy rainfall events. The framework is built on a three-cluster Normal mixture model, whose distribution parameters are sampled using Bayesian inference and Markov Chain Monte Carlo algorithm. The three rainfall clusters reflect probability behaviors of light, moderate, and heavy rainfall, respectively. Our analysis indicates that heavy rainfall events make the largest contribution to the total amount of seasonal precipitation. Furthermore, the interannual variation of summer precipitation is attributable to the variation of heavy rainfall frequency over the HRV. The heavy rainfall frequency, in turn, is influenced by sea surface temperature anomalies (SSTAs) over the north Indian Ocean, equatorial western Pacific, and the tropical Atlantic. The tropical SSTAs modulate the HRV heavy rainfall events by influencing atmospheric circulation favorable for the onset and maintenance of heavy rainfall events. Occurring 5 months prior to the summer season, these tropical SSTAs provide potential sources of prediction skill for heavy rainfall events over the HRV. Using these preceding SSTA signals, we show that the support vector machine algorithm can predict HRV heavy rainfall satisfactorily. The improved prediction skill has important implication for the nation's disaster early warning system.

Concentrations of selected heavy metals in benthic diatoms and sediment in the Westerschelde Estuary

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

Absil, M.C.P.; Scheppingen, Y. van

1996-12-31

In recent years considerable data have been compiled on heavy metal levels in biota in marine and estuarine environments. With respect to the fauna, much information is available on accumulation and effects of heavy metals in birds, fish and benthic macrofauna. Accumulation of heavy metals in aquatic flora has been studied mostly in benthic macroalgae, in particular in relation to the use as a biological monitor. The response of planktonic algal species to heavy metals has been studied extensively in cultured populations. Also. heavy metal concentrations in natural plankton have been studied. As far as we know, very few datamore » are available on the concentrations of heavy metals in the lowest benthic trophic level, the benthic microflora. It is a major food supply for numerous intertidal species, so it is obvious that microflora might play an important role in the accumulation of contaminants through coastal food chains. The aim of this research was to adjust a recently developed collection technique for benthic diatoms so that it is suitable for large-scale field studies. The method was then used to assess the concentration of the heavy metals Cd, Cu, Pb and Zn in benthic diatoms and sediments along an estuarine gradient. 18 refs., 2 figs., 1 tab.« less

Determination of the molecular weight of human gamma-3 chains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate

PubMed Central

Virella, G.; Parkhouse, R. M. E.

1972-01-01

The molecular weights (mol. wt) for heavy chains of human IgG were estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Polyclonal IgG and monoclonal IgG proteins of different subclasses were extensively reduced with 50 mM dithioerythritol, in the presence of 2 per cent sodium dodecyl sulphate, at 100°. Four control proteins of known mol. wt (cytochrome C, chymotrypsinogen A, egg albumin, and serum albumin) were used to construct a linear plot of electrophoretic mobility versus log mol. wt. From this plot, the following mol. wts were calculated: 53,650±700 for polyclonal IgG; 54,200±1065 for γ1, γ2, and γ4 chains, and 60,950±585 for γ3 chains. Those results confirm the larger size of γ3 chains reported by Saluk and Clem (1971). PMID:4346255

An improved error bound for linear complementarity problems for B-matrices.

PubMed

Gao, Lei; Li, Chaoqian

2017-01-01

A new error bound for the linear complementarity problem when the matrix involved is a B -matrix is presented, which improves the corresponding result in (Li et al. in Electron. J. Linear Algebra 31(1):476-484, 2016). In addition some sufficient conditions such that the new bound is sharper than that in (García-Esnaola and Peña in Appl. Math. Lett. 22(7):1071-1075, 2009) are provided.

Correlation complementarity yields bell monogamy relations.

PubMed

Kurzyński, P; Paterek, T; Ramanathan, R; Laskowski, W; Kaszlikowski, D

2011-05-06

We present a method to derive Bell monogamy relations by connecting the complementarity principle with quantum nonlocality. The resulting monogamy relations are stronger than those obtained from the no-signaling principle alone. In many cases, they yield tight quantum bounds on the amount of violation of single and multiple qubit correlation Bell inequalities. In contrast with the two-qubit case, a rich structure of possible violation patterns is shown to exist in the multipartite scenario.

Reviving Complementarity: John Wheeler's efforts to apply complementarity toward a quantum description of gravitation

NASA Astrophysics Data System (ADS)

Halpern, Paul

2017-01-01

In 1978, John Wheeler proposed the delayed-choice thought experiment as a generalization of the classic double slit experiment intended to help elucidate the nature of decision making in quantum measurement. In particular, he wished to illustrate how a decision made after a quantum system was prepared might retrospectively affect the outcome. He extended his methods to the universe itself, raising the question of whether the universe is a ``self-excited circuit'' in which scientific measurements in the present affect the quantum dynamics in the past. In this talk we'll show how Wheeler's approach revived the notion of Bohr's complementarity, which had by then faded from the prevailing discourse of quantum measurement theory. Wheeler's advocacy reflected, in part, his wish to eliminate the divide in quantum theory between measurer and what was being measured, bringing greater consistency to the ideas of Bohr, a mentor whom he deeply respected.

Bohrian Complementarity in the Light of Kantian Teleology

NASA Astrophysics Data System (ADS)

Pringe, Hernán

2014-03-01

The Kantian influences on Bohr's thought and the relationship between the perspective of complementarity in physics and in biology seem at first sight completely unrelated issues. However, the goal of this work is to show their intimate connection. We shall see that Bohr's views on biology shed light on Kantian elements of his thought, which enables a better understanding of his complementary interpretation of quantum theory. For this purpose, we shall begin by discussing Bohr's views on the analogies concerning the epistemological situation in biology and in physics. Later, we shall compare the Bohrian and the Kantian approaches to the science of life in order to show their close connection. On this basis, we shall finally turn to the issue of complementarity in quantum theory in order to assess what we can learn about the epistemological problems in the quantum realm from a consideration of Kant's views on teleology.

Bohr, Heisenberg and the divergent views of complementarity

NASA Astrophysics Data System (ADS)

Camilleri, Kristian

The fractious discussions between Bohr and Heisenberg in Copenhagen in 1927 have been the subject of much historical scholarship. However, little attention has been given to Heisenberg's understanding of the notion of complementary space-time and causal descriptions, which was presented for the first time in Bohr's lecture at the 1927 Como conference. In this paper, I argue that Heisenberg's own interpretation of this notion differed substantially from Bohr's. Whereas Bohr had intended this form of complementarity to entail a choice between a space-time description of the electron in an atom, and defining the energy of a stationary state, Heisenberg interpreted the 'causal' description in terms of ψ -function in configuration space. In disentangling the two views of complementarity, this paper sheds new light on the hidden philosophical disagreements between the proponents of these two founders of the so-called 'Copenhagen interpretation' of quantum mechanics.

Pollution load index for heavy metals in Mian-Ab plain soil, Khuzestan, Iran.

PubMed

Jorfi, Sahand; Maleki, Rohangiz; Jaafarzadeh, Neemat; Ahmadi, Mehdi

2017-12-01

Soil pollution by heavy metals is a major concern in agricultural area. Potential impact of heavy metals in agricultural soil on human health by accumulating in food chain demonstrated elsewhere. In this regard Mian-Ab plain as a major agricultural site of Khuzestan province considered for Arsenic, cadmium and lead concentration as the main potential toxic pollutants in soil. 50 topsoil samples were collected and analyzed by inductively coupled plasma mass spectrometry (ICP-MS). Also Contamination level of selected heavy metals in Mian-Ab Plain, was assessed by single factor contaminant index (PI) and pollution load index (PLI). Results show mean concentration of arsenic, cadmium and lead were 2.52, 0.30 and 7.21 mg kg -1 . Base on PLI results 12 point (24%) of the studied area show moderately polluted and 38 point (76%) show unpolluted area.

Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin.

PubMed

Wright, J F; Pernollet, M; Reboul, A; Aude, C; Colomb, M G

1992-05-05

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.

Identification of nickel response genes in abnormal early developments of sea urchin by differential display polymerase chain reaction.

PubMed

Ryu, Tae Kwon; Lee, Gunsup; Rhee, Yong; Park, Heung-Sik; Chang, Man; Lee, Sukchan; Lee, Jaean; Lee, Taek-Kyun

2012-10-01

Bioassays and biomarkers have been previously developed to assess the effects of heavy metal contaminants on the early life stages of the sea urchin. In this study, malformation in the early developmental processes was observed in sea urchin (Strongylocentrotus intermedius) larvae exposed to 10 ppm Ni for over 30 h. The most critical stage at which the triggering of nickel effects takes place is thought to be the blastula stage, which occurs after fertilization in larval development. To investigate the molecular-level responses of sea urchin exposed to heavy metal stress and to explore the differentially expressed genes that are induced or repressed by nickel, differential display polymerase chain reaction (DD-PCR) was used with sea urchin mRNAs. The malformation-related genes expressed in the early life stages of the sea urchin were cloned from larvae exposed to 10 ppm of nickel for 15 h, and accessed via DD-PCR. Sequence analysis results revealed that each of the genes evidenced high homology with EGF2, PCSK9, serine/threonine protein kinase, apolipophorin precursor protein, and MGC80921 protein/transcript variant 2. This result may prove useful in the development of novel biomarkers for the assessment of heavy metal stresses on sea urchin embryos. Copyright © 2012 Elsevier Inc. All rights reserved.

Vibration-induced multifocal neuropathy in forestry workers: electrophysiological findings in relation to vibration exposure and finger circulation.

PubMed

Bovenzi, M; Giannini, F; Rossi, S

2000-11-01

To investigate neural conduction in the upper limbs of symptomatic forestry workers with and without exposure to hand-transmitted vibration. A further aim was to assess the possible relationships between vibration exposure, nerve conduction and finger circulation in the forestry workers who used chain saws. A detailed neurophysiological investigation was performed on the upper extremities of 20 chain saw workers, 20 forestry operators with heavy manual work but without vibration exposure, and 20 healthy male controls. All subjects were screened to exclude polyneuropathy. Measurements of sensory and motor nerve conduction (velocity and amplitude) were obtained bilaterally from the median, ulnar and radial nerves. To assess peripheral vascular function, the forestry workers underwent a cold test with plethysmographic measurement of finger systolic blood pressure (FSBP). In the chain saw operators, vibration exposure was evaluated according to the International Standard ISO 5349. Indices of daily vibration exposure and lifetime cumulative vibration dose were estimated for each chain saw operator. Sensory nerve conduction in several segments of the median and radial nerves was significantly reduced in the chain saw operators compared with that in the workers doing heavy manual work and the controls. The neurophysiological pattern more frequently observed in the chain saw operators was a multifocal nerve conduction impairment to several neural segments with predominant involvement of sensory rather than motor fibres. Sensory nerve conduction velocities in the hands of the chain saw operators were inversely related to both daily and lifetime cumulative vibration exposures. In the vibration-exposed forestry workers, neither were sensori-motor complaints associated with vascular symptoms (finger whiteness) nor were electrophysiological data related to cold-induced changes in FSBP. Exposure to hand-transmitted vibration, in addition to ergonomic stress factors, can contribute to peripheral nerve disorders occurring in forestry workers who operate chain saws. The findings of this study suggest the existence of an exposure-effect relationship for vibration-induced neuropathy. Different underlying mechanisms are likely to be involved in the pathogenesis of the neurological and vascular components of the hand-arm vibration syndrome.

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

PubMed Central

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. PMID:26347575

COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids

PubMed Central

2016-01-01

Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3’-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5’-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5’-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. Specificity was enhanced by using 3’-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered. PMID:27783658

High Inter-Individual Diversity of Point Mutations, Insertions, and Deletions in Human Influenza Virus Nucleoprotein-Specific Memory B Cells

PubMed Central

Bussmann, Bianca M.; Horn, Susanne; Sieg, Michael; Jassoy, Christian

2015-01-01

The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4 % in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses. PMID:26086076

IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde–Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope

PubMed Central

Amir, Shahzada; Hartvigsen, Karsten; Hansen, Lotte F.; Woelkers, Douglas; Tsimikas, Sotirios; Binder, Christoph J.; Kipps, Thomas J.; Witztum, Joseph L.

2013-01-01

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde–acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells. PMID:23840319

China's Rare Earth Supply Chain: Illegal Production, and Response to new Cerium Demand

NASA Astrophysics Data System (ADS)

Nguyen, Ruby Thuy; Imholte, D. Devin

2016-07-01

As the demand for personal electronic devices, wind turbines, and electric vehicles increases, the world becomes more dependent on rare earth elements. Given the volatile, Chinese-concentrated supply chain, global attempts have been made to diversify supply of these materials. However, the overall effect of supply diversification on the entire supply chain, including increasing low-value rare earth demand, is not fully understood. This paper is the first attempt to shed some light on China's supply chain from both demand and supply perspectives, taking into account different Chinese policies such as mining quotas, separation quotas, export quotas, and resource taxes. We constructed a simulation model using Powersim Studio that analyzes production (both legal and illegal), production costs, Chinese and rest-of-world demand, and market dynamics. We also simulated new demand of an automotive aluminum-cerium alloy in the US market starting from 2018. Results showed that market share of the illegal sector has grown since 2007-2015, ranging between 22% and 25% of China's rare earth supply, translating into 59-65% illegal heavy rare earths and 14-16% illegal light rare earths. There will be a shortage in certain light and heavy rare earths given three production quota scenarios and constant demand growth rate from 2015 to 2030. The new simulated Ce demand would require supply beyond that produced in China. Finally, we illustrate revenue streams for different ore compositions in China in 2015.

China’s rare earth supply chain: Illegal production, and response to new cerium demand

DOE PAGES

Nguyen, Ruby Thuy; Imholte, D. Devin

2016-03-29

As the demand for personal electronic devices, wind turbines, and electric vehicles increases, the world becomes more dependent on rare earth elements. Given the volatile, Chinese-concentrated supply chain, global attempts have been made to diversify supply of these materials. However, the overall effect of supply diversification on the entire supply chain, including increasing low-value rare earth demand, is not fully understood. This paper is the first attempt to shed some light on China’s supply chain from both demand and supply perspectives, taking into account different Chinese policies such as mining quotas, separation quotas, export quotas, and resource taxes. We constructedmore » a simulation model using Powersim Studio that analyzes production (both legal and illegal), production costs, Chinese and rest-of-world demand, and market dynamics. We also simulated new demand of an automotive aluminum-cerium alloy in the U.S. market starting from 2018. Results showed that market share of the illegal sector has grown since 2007 to 2015, ranging between 22% and 25% of China’s rare earth supply, translating into 59–65% illegal heavy rare earths and 14–16% illegal light rare earths. There would be a shortage in certain light and heavy rare earths given three production quota scenarios and constant demand growth rate from 2015 to 2030. The new simulated Ce demand would require supply beyond that produced in China. Lastly, we illustrated revenue streams for different ore compositions in China in 2015.« less

Quantitation of bovine immunoglobulin isotypes and allotypes using monoclonal antibodies.

PubMed

Williams, D J; Newson, J; Naessens, J

1990-03-01

A panel of 10 monoclonal antibodies specific for bovine immunoglobulins M, A, G1, G2 and light chains were produced and enzyme-linked immunosorbent assays developed to measure Ig levels in body fluids and culture supernatants using this panel of MAbs. An inhibition ELISA was accurate and sensitive for MAbs of high affinity, detecting levels as low as 10 ng ml-1 of IgM using a high-affinity MAb, IL-A50 (dissociation constant = 1.3 X 10(-11) M). For MAbs of lower affinity (KD of less than 0.25 X 10(-9) M) a sandwich ELISA was more sensitive, detecting 0.1-1.0 microgram ml-1 Ig, provided a conjugate of an anti-light chain MAb was used. Using these ELISA techniques, four pairs of MAbs specific for bovine IgM, IgA, IgG1 and IgG2 respectively, were screened on sera from over 100 cattle of different breeds to determine whether any detected a polymorphic epitope. MAbs IL-A30, IL-A60, IL-A66, IL-A71, IL-A72, IL-A73 and IL-A74 were shown to recognise monomorphic determinants on their respective heavy chains. In contrast, the epitope recognised on the mu-heavy chain by MAb IL-A50, which had previously been shown to be polymorphic, was found to be allelic and inherited under the control of a single gene, probably Cu.

China’s rare earth supply chain: Illegal production, and response to new cerium demand

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

Nguyen, Ruby Thuy; Imholte, D. Devin

As the demand for personal electronic devices, wind turbines, and electric vehicles increases, the world becomes more dependent on rare earth elements. Given the volatile, Chinese-concentrated supply chain, global attempts have been made to diversify supply of these materials. However, the overall effect of supply diversification on the entire supply chain, including increasing low-value rare earth demand, is not fully understood. This paper is the first attempt to shed some light on China’s supply chain from both demand and supply perspectives, taking into account different Chinese policies such as mining quotas, separation quotas, export quotas, and resource taxes. We constructedmore » a simulation model using Powersim Studio that analyzes production (both legal and illegal), production costs, Chinese and rest-of-world demand, and market dynamics. We also simulated new demand of an automotive aluminum-cerium alloy in the U.S. market starting from 2018. Results showed that market share of the illegal sector has grown since 2007 to 2015, ranging between 22% and 25% of China’s rare earth supply, translating into 59–65% illegal heavy rare earths and 14–16% illegal light rare earths. There would be a shortage in certain light and heavy rare earths given three production quota scenarios and constant demand growth rate from 2015 to 2030. The new simulated Ce demand would require supply beyond that produced in China. Lastly, we illustrated revenue streams for different ore compositions in China in 2015.« less

Evolution and Distribution of Teleost myomiRNAs: Functionally Diversified myomiRs in Teleosts.

PubMed

Siddique, Bhuiyan Sharmin; Kinoshita, Shigeharu; Wongkarangkana, Chaninya; Asakawa, Shuichi; Watabe, Shugo

2016-06-01

Myosin heavy chain (MYH) genes belong to a multigene family, and the regulated expression of each member determines the physiological and contractile muscle properties. Among these, MYH6, MYH7, and MYH14 occupy unique positions in the mammalian MYH gene family because of their specific expression in slow/cardiac muscles and the existence of intronic micro(mi) RNAs. MYH6, MYH7, and MYH14 encode miR-208a, miR-208b, and miR-499, respectively. These MYH encoded miRNAs are designated as myomiRs because of their muscle-specific expression and functions. In mammals, myomiRs and host MYHs form a transcription network involved in muscle fiber-type specification; thus, genomic positions and expression patterns of them are well conserved. However, our previous studies revealed divergent distribution and expression of MYH14/miR-499 among teleosts, suggesting the unique evolution of myomiRs and host MYHs in teleosts. Here, we examined distribution and expression of myomiRs and host MYHs in various teleost species. The major cardiac MYH isoforms in teleosts are an intronless gene, atrial myosin heavy chain (amhc), and ventricular myosin heavy chain (vmhc) gene that encodes an intronic miRNA, miR-736. Phylogenetic analysis revealed that vmhc/miR-736 is a teleost-specific myomiR that differed from tetrapoda MYH6/MYH7/miR-208s. Teleost genomes also contain species-specific orthologs in addition to vmhc and amhc, indicating complex gene duplication and gene loss events during teleost evolution. In medaka and torafugu, miR-499 was highly expressed in slow/cardiac muscles whereas the expression of miR-736 was quite low and not muscle specific. These results suggest functional diversification of myomiRs in teleost with the diversification of host MYHs.

Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation.

PubMed

Mendias, Christopher L; Kayupov, Erdan; Bradley, Joshua R; Brooks, Susan V; Claflin, Dennis R

2011-07-01

Myostatin (MSTN) is a member of the transforming growth factor-β superfamily of cytokines and is a negative regulator of skeletal muscle mass. Compared with MSTN(+/+) mice, the extensor digitorum longus muscles of MSTN(-/-) mice exhibit hypertrophy, hyperplasia, and greater maximum isometric force production (F(o)), but decreased specific maximum isometric force (sF(o); F(o) normalized by muscle cross-sectional area). The reason for the reduction in sF(o) was not known. Studies in myotubes indicate that inhibiting myostatin may increase muscle mass by decreasing the expression of the E3 ubiquitin ligase atrogin-1, which could impact the force-generating capacity and size of muscle fibers. To gain a greater understanding of the influence of myostatin on muscle contractility, we determined the impact of myostatin deficiency on the contractility of permeabilized muscle fibers and on the levels of atrogin-1 and ubiquitinated myosin heavy chain in whole muscle. We hypothesized that single fibers from MSTN(-/-) mice have a greater F(o), but no difference in sF(o), and a decrease in atrogin-1 and ubiquitin-tagged myosin heavy chain levels. The results indicated that fibers from MSTN(-/-) mice have a greater cross-sectional area, but do not have a greater F(o) and have a sF(o) that is significantly lower than fibers from MSTN(+/+) mice. The extensor digitorum longus muscles from MSTN(-/-) mice also have reduced levels of atrogin-1 and ubiquitinated myosin heavy chain. These findings suggest that myostatin inhibition in otherwise healthy muscle increases the size of muscle fibers and decreases atrogin-1 levels, but does not increase the force production of individual muscle fibers.

Altered phenotypic expression of immunoglobulin heavy-chain variable-region (VH) genes in Alicia rabbits probably reflects a small deletion in the VH genes closest to the joining region.

PubMed

Allegrucci, M; Newman, B A; Young-Cooper, G O; Alexander, C B; Meier, D; Kelus, A S; Mage, R G

1990-07-01

Rabbits of the Alicia strain have a mutation (ali) that segregates with the immunoglobulin heavy-chain (lgh) locus and has a cis effect upon the expression of heavy-chain variable-region (VH) genes encoding the a2 allotype. In heterozygous a1/ali or a3/ali rabbits, serum immunoglobulins are almost entirely the products of the normal a1 or a3 allele and only traces of a2 immunoglobulin are detectable. Adult homozygous ali/ali rabbits likewise have normal immunoglobulin levels resulting from increased production of a-negative immunoglobulins and some residual ability to produce the a2 allotype. By contrast, the majority of the immunoglobulins of wild-type a2 rabbits are a2-positive and only a small percentage are a-negative. Genomic DNAs from homozygous mutant and wild-type animals were indistinguishable by Southern analyses using a variety of restriction enzyme digests and lgh probes. However, when digests with infrequently cutting enzymes were analyzed by transverse alternating-field electrophoresis, the ali DNA fragments were 10-15 kilobases smaller than the wild type. These fragments hybridized to probes both for VH and for a region of DNA a few kilobases downstream of the VH genes nearest the joining region. We suggest that this relatively small deletion affects a segment containing 3' VH genes with important regulatory functions, the loss of which leads to the ali phenotype. These results, and the fact that the 3' VH genes rearrange early in B-cell development, indicate that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression.

Altered phenotypic expression of immunoglobulin heavy-chain variable-region (VH) genes in Alicia rabbits probably reflects a small deletion in the VH genes closest to the joining region.

PubMed Central

Allegrucci, M; Newman, B A; Young-Cooper, G O; Alexander, C B; Meier, D; Kelus, A S; Mage, R G

1990-01-01

Rabbits of the Alicia strain have a mutation (ali) that segregates with the immunoglobulin heavy-chain (lgh) locus and has a cis effect upon the expression of heavy-chain variable-region (VH) genes encoding the a2 allotype. In heterozygous a1/ali or a3/ali rabbits, serum immunoglobulins are almost entirely the products of the normal a1 or a3 allele and only traces of a2 immunoglobulin are detectable. Adult homozygous ali/ali rabbits likewise have normal immunoglobulin levels resulting from increased production of a-negative immunoglobulins and some residual ability to produce the a2 allotype. By contrast, the majority of the immunoglobulins of wild-type a2 rabbits are a2-positive and only a small percentage are a-negative. Genomic DNAs from homozygous mutant and wild-type animals were indistinguishable by Southern analyses using a variety of restriction enzyme digests and lgh probes. However, when digests with infrequently cutting enzymes were analyzed by transverse alternating-field electrophoresis, the ali DNA fragments were 10-15 kilobases smaller than the wild type. These fragments hybridized to probes both for VH and for a region of DNA a few kilobases downstream of the VH genes nearest the joining region. We suggest that this relatively small deletion affects a segment containing 3' VH genes with important regulatory functions, the loss of which leads to the ali phenotype. These results, and the fact that the 3' VH genes rearrange early in B-cell development, indicate that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression. Images PMID:2115171

Myocardial short-range force responses increase with age in F344 rats

PubMed Central

Mitov, Mihail I.; Holbrook, Anastasia M.; Campbell, Kenneth S.

2009-01-01

The mechanical properties of triton-permeabilized ventricular preparations isolated from 4, 18 and 24-month-old F344 rats were analyzed to provide information about the molecular mechanisms that lead to age-related increases in diastolic myocardial stiffness in these animals. Passive stiffness (measured in solutions with minimal free Ca2+) did not change with age. This implies that the aging-associated dysfunction is not due to changes in titin or collagen molecules. Ca2+-activated preparations exhibited a characteristic short-range force response: force rose rapidly until the muscle reached its elastic limit and less rapidly thereafter. The elastic limit increased from 0.43 ± 0.01 % l0 (where l0 is the initial muscle length) in preparations from 4-month-old animals to 0.49 ± 0.01 % l0 in preparations from 24-month-old rats (p<0.001, ANOVA). Relative short-range force was defined as the maximum force produced during the short-range response normalized to the prevailing tension. This parameter increased from 0.110 ± 0.002 to 0.142 ± 0.002 over the same age-span (p<0.001, ANOVA). Analytical gel electrophoresis showed that the maximum stiffness of the preparations during the short-range response and the relative short-range force increased (p=0.031 and p=0.005 respectively) with the relative content of slow β myosin heavy chain molecules. Elastic limit values did not correlate with myosin isoform content. Simulations based on these results suggest that attached β myosin heavy chain cross-bridges are stiffer than links formed by α myosin heads. In conclusion, elevated content of stiffer β myosin heavy chain molecules may contribute to aging-associated increases in myocardial stiffness. PMID:19007786

Genetic stability of gene targeted immunoglobulin loci. I. Heavy chain isotype exchange induced by a universal gene replacement vector.

PubMed Central

Kardinal, C; Selmayr, M; Mocikat, R

1996-01-01

Gene targeting at the immunoglobulin loci of B cells is an efficient tool for studying immunoglobulin expression or generating chimeric antibodies. We have shown that vector integration induced by human immunoglobulin G1 (IgG1) insertion vectors results in subsequent vector excision mediated by the duplicated target sequence, whereas replacement events which could be induced by the same constructs remain stable. We could demonstrate that the distribution of the vector homology strongly influences the genetic stability obtained. To this end we developed a novel type of a heavy chain replacement vector making use of the heavy chain class switch recombination sequence. Despite the presence of a two-sided homology this construct is universally applicable irrespective of the constant gene region utilized by the B cell. In comparison to an integration vector the frequency of stable incorporation was strongly increased, but we still observed vector excision, although at a markedly reduced rate. The latter events even occurred with circular constructs. Linearization of the construct at various sites and the comparison with an integration vector that carries the identical homology sequence, but differs in the distribution of homology, revealed the following features of homologous recombination of immunoglobulin genes: (i) the integration frequency is only determined by the length of the homology flank where the cross-over takes place; (ii) a 5' flank that does not meet the minimum requirement of homology length cannot be complemented by a sufficient 3' flank; (iii) free vector ends play a role for integration as well as for replacement targeting; (iv) truncating recombination events are suppressed in the presence of two flanks. Furthermore, we show that the switch region that was used as 3' flank is non-functional in an inverted orientation. Images Figure 2 PMID:8958041

Molecular characterization and immunological response analysis of a novel transferrin-like, pacifastin heavy chain protein in giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879).

PubMed

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.

Immunoglobulin Tau Heavy Chain (IgT) in Flounder, Paralichthys olivaceus: Molecular Cloning, Characterization, and Expression Analyses

PubMed Central

Du, Yang; Tang, Xiaoqian; Zhan, Wenbin; Xing, Jing; Sheng, Xiuzhen

2016-01-01

Immunoglobulin tau (IgT) is a new teleost immunoglobulin isotype, and its potential function in adaptive immunity is not very clear. In the present study, the membrane-bound and secreted IgT (mIgT and sIgT) heavy chain genes were cloned for the first time and characterized in flounder (Paralichthys olivaceus), and found the nucleic acid sequence were exactly same in the Cτ1–Cτ4 constant domains of mIgT and sIgT, but different in variable regions and the C-terminus. The amino acid sequence of mIgT shared higher similarity with Bovichtus diacanthus (51.2%) and Dicentrarchus labrax (45.0%). Amino acid of flounder IgT, IgM, and IgD heavy chain was compared and the highest similarity was found between IgT Cτ1 and IgM Cμ1 (38%). In healthy flounder, the transcript levels of IgT mRNA were the highest in gill, spleen, and liver, and higher in peripheral blood leucocytes, skin, and hindgut. After infection and vaccination with Edwardsiella tarda via intraperitoneal injection and immersion, the qRT-PCR analysis demonstrated that the IgT mRNA level was significantly upregulated in all tested tissues, with similar dynamic tendency that increased firstly and then decreased, and higher in gill, skin, hindgut, liver, and stomach in immersion than in the injection group, but no significant difference existed in spleen and head kidney between immersion and injection groups. These results revealed that IgT responses could be simultaneously induced in both mucosal and systemic tissues after infection/vaccination via injection and immersion route, but IgT might play a more important role in mucosal immunity than in systemic immunity. PMID:27649168

ERAP2 functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers.

PubMed

Robinson, Philip C; Lau, Eugene; Keith, Patricia; Lau, Max C; Thomas, Gethin P; Bradbury, Linda A; Brown, Matthew A; Kenna, Tony J

2015-11-01

Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. There was no significant difference in HLA-class I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. Large differences were not seen in HLA-B27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The biochemistry and immunology of non-canonical forms of HLA-B27.

PubMed

Shaw, Jacqueline; Hatano, Hiroko; Kollnberger, Simon

2014-01-01

HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 is expressed at the cell surface of antigen presenting cells (APC) both as canonical β2m-associated and non-canonical β2m-free heavy chain (FHC) forms which include B27 dimers (termed B272). B27 FHC forms arise in an endosomal compartment from recycling β2m-associated B27. Formation of cell surface FHC dimers is critically dependent on an unpaired reactive cysteine 67 in the α1 helix of the class I heavy chain. HLA-B27 also form redox-inducible β2m-associated dimers on exosomes and apoptosing cells. By contrast with cell surface expressed cysteine 67-dependent heavy chain dimers these dimers are dependent on a cytoplasmic cysteine 325 for their formation. HLA-B27 binds to immunoregulatory receptors including members of the Killer cell Immunoglobulin-like (KIR) and Leukocyte Immunoglobulin-like receptor family. B27 FHC bind to different but overlapping sets of these immunoreceptors compared to classical β2m-associated HLA-B27. B27 FHC bind more strongly to KIR3DL2 and LILRB2 immune receptor than other β2m-associated HLA-class I ligands. Genetic studies have implicated genes which control production of the important proinflammatory cytokine IL-17 in the pathogenesis of spondyloarthritis. Cell surface HLA-B27 FHC binding to these immune receptors or acting through other mechanisms could impact on the pathogenesis of spondyloarthritis by promoting immune cell production of IL-17. Here we review the literature on these non-canonical forms of HLA-B27 and the immune receptors they bind to and discuss the possible relevance of these interactions to the pathogenesis of spondyloarthropathy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Genetic stability of gene targeted immunoglobulin loci. I. Heavy chain isotype exchange induced by a universal gene replacement vector.

PubMed

Kardinal, C; Selmayr, M; Mocikat, R

1996-11-01

Gene targeting at the immunoglobulin loci of B cells is an efficient tool for studying immunoglobulin expression or generating chimeric antibodies. We have shown that vector integration induced by human immunoglobulin G1 (IgG1) insertion vectors results in subsequent vector excision mediated by the duplicated target sequence, whereas replacement events which could be induced by the same constructs remain stable. We could demonstrate that the distribution of the vector homology strongly influences the genetic stability obtained. To this end we developed a novel type of a heavy chain replacement vector making use of the heavy chain class switch recombination sequence. Despite the presence of a two-sided homology this construct is universally applicable irrespective of the constant gene region utilized by the B cell. In comparison to an integration vector the frequency of stable incorporation was strongly increased, but we still observed vector excision, although at a markedly reduced rate. The latter events even occurred with circular constructs. Linearization of the construct at various sites and the comparison with an integration vector that carries the identical homology sequence, but differs in the distribution of homology, revealed the following features of homologous recombination of immunoglobulin genes: (i) the integration frequency is only determined by the length of the homology flank where the cross-over takes place; (ii) a 5' flank that does not meet the minimum requirement of homology length cannot be complemented by a sufficient 3' flank; (iii) free vector ends play a role for integration as well as for replacement targeting; (iv) truncating recombination events are suppressed in the presence of two flanks. Furthermore, we show that the switch region that was used as 3' flank is non-functional in an inverted orientation.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment

PubMed Central

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads. PMID:29520274

Genetic variants in the immunoglobulin heavy chain locus are associated with the IgG index in multiple sclerosis.

PubMed

Buck, Dorothea; Albrecht, Eva; Aslam, Muhammad; Goris, An; Hauenstein, Natalie; Jochim, Angela; Cepok, Sabine; Grummel, Verena; Dubois, Bénédicte; Berthele, Achim; Lichtner, Peter; Gieger, Christian; Winkelmann, Juliane; Hemmer, Bernhard

2013-01-01

Intrathecal synthesis of immunoglobulin gamma (IgG) synthesis is frequently observed in patients with multiple sclerosis (MS). Whereas the extent of intrathecal IgG synthesis varies largely between patients, it remains rather constant in the individual patient over time. The aim of this study was to identify common genetic variants associated with the IgG index as a marker of intrathecal IgG synthesis in MS. We performed a genome-wide association study of the IgG index in a discovery series of 229 patients. For confirmation we performed a replication in 2 independent series comprising 256 and 153 patients, respectively. The impact of associated single nucleotide polymorphisms (SNPs) on MS susceptibility was analyzed in an additional 1,854 cases and 5,175 controls. Significant association between the IgG index and 5 SNPs was detected in the discovery and confirmed in both replication series reaching combined p values of p = 6.5 × 10(-11) to p = 7.5 × 10(-16) . All identified SNPs are clustered around the immunoglobulin heavy chain (IGHC) locus on chromosome 14q32.33 and are in linkage disequilibrium (r(2) range, 0.71-0.95). The best associated SNP is located in an intronic region of the immunoglobulin gamma3 heavy chain gene. Additional sequencing identified the GM21* haplotype to be associated with a high IgG index. Further evaluation of the IGHC SNPs revealed no association with susceptibility to MS in our data set. The extent of intrathecal IgG in MS is influenced by the IGHC locus. No association with susceptibility to MS was found. Therefore GM haplotypes might affect intrathecal IgG synthesis independently of the underlying disease. Copyright © 2012 American Neurological Association.

A novel missense mutation, Leu390Val, in the cardiac beta-myosin heavy chain associated with pronounced septal hypertrophy in two families with hypertrophic cardiomyopathy.

PubMed

Havndrup, O; Bundgaard, H; Andersen, P S; Larsen, L A; Vuust, J; Kjeldsen, K; Christiansen, M

2000-12-01

An examination of the genetic background and phenotypic presentation of familial hypertrophic cardiomyopathy (FHC) with respect to specific mutations in the MYH7-gene encoding the cardiac beta-myosin heavy chain. Two families (n = 22) from a cohort of 67 families with FHC were studied at the National University Hospital, Rigshospitalet, Copenhagen. Clinical, non-invasive examinations of all included family members followed by molecular genetic analysis including PCR-single strand conformation polymorphism/heteroduplex (SSCP/HD) analysis and sequencing of exon 3-23 of the MYH7-gene. We found FHC associated with a missense mutation in two families, i.e. a C > G transversion at position g10124 and a G > T transversion at position g10126 causing the change of a leucine residue at codon 390 to a valine residue. The mutation is located in the actin-binding region of the beta-myosin heavy chain. The leucine residue is evolutionarily conserved in vertebrate myosins. In the two families, the phenotypic presentations in the clinically affected were characterized by asymmetric septal hypertrophy (septum diameter 18.8 (5.0) mm (mean (SD)) with only minor involvement of the left ventricular free wall (posterior wall diameter 11.0 (2.2) mm). Furthermore, the left ventricular systolic and diastolic functions were well preserved, even at a high age. The symptomatic status of the clinically affected patients depended on the presence or absence of a concomitant left ventricular outflow tract gradient. We report a novel missense mutation associated with FHC caused by a double nucleotide transversion. The penetrance of the mutation was not complete, but in clinically affected patients the mutation gives rise to an echocardiographic phenotype, predominantly characterized by pronounced septal hypertrophy.