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

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.

Preferential use of lambda light chains is associated with defective mouse antibody responses to the capsular polysaccharide of Neisseria meningitidis group B.

PubMed

Colino, Jesus; Outschoorn, Ingrid

2004-01-01

The capsular polysaccharide of Neisseria meningitidis group B (CpsB) is a very poor immunogen in mammals; this has been considered to be due to the induction of tolerance to cross-reactive host glycoconjugates. It has hampered the development of an effective vaccine against this meningococcal group for many years. Syngeneic populations have a similar tolerogenic background. Thus, we used the variability in ability to mount CpsB-specific immunoglobulin (Ig) responses of individuals from these populations to reveal underlying mechanisms to tolerance contributing to the poor immunogenicity of CpsB. Here we analyze by ELISA, the individual CpsB-specific Ig response of BALB/c and other syngeneic mice to immunization with intact bacteria, using the distribution of light chains as a direct indicator of the repertoire dynamics of the response. Although approximately 96% of anti-CpsB Ig bear kappa-light chains, BALB/c mouse populations were heterogeneous in the light chain composition of their individual anti-CpsB Ig responses. The proportion of kappa and lambda-light chains used for anti-CpsB Ig was a private characteristic that remained relatively constant, for each individual, through repetitive immunizations regardless of the bacterial stimuli size. Despite the prevalence of individual use of kappa-light chains, 5% of BALB/c mice showed restricted usage of lambda-light chains in their CpsB-specific Ig responses, and an additional 11% use them significantly. The preferential use of lambda-light chains in these mice was strongly associated with defective IgM, and absent or barely detectable IgG anti-CpsB responses even after repetitive bacterial immunization. We conclude that differences in the private repertoire of specific Ig also contribute to mouse unresponsiveness to CpsB.

Complex of a Protective Antibody with its Ebola Virus GP Peptide Epitope: Unusual Features of a Vlambdalx Light Chain

DTIC Science & Technology

2008-01-01

Bioinformatics, 19, ii246–ii255. 52. Lawrence, M. C. & Colman, P. M. (1993). Shape complementarity at protein / protein interfaces . J. Mol. Biol. 234, 946...envelope spike, which is the sole protein expressed on the surface of the Ebola virus and is involved in receptor binding, tropism, and viral entry.6–9 It...variable light chain/heavy chain (VL/VH) interface of 13F6-1-2, ∼1025 Å2 surface area is buried on VL Fig. 1. Nucleotide and translated amino acid

Complex of a Protective Antibody with its Ebola Virus GP Peptide Epitope: Unusual Features of a V lambda x Light Chain

DTIC Science & Technology

2007-10-01

twists. Bioinformatics, 19, ii246–ii255. 52. Lawrence, M. C. & Colman, P. M. (1993). Shape complementarity at protein / protein interfaces . J. Mol. Biol...envelope spike, which is the sole protein expressed on the surface of the Ebola virus and is involved in receptor binding, tropism, and viral entry.6–9 It...26 At the variable light chain/heavy chain (VL/VH) interface of 13F6-1-2, ∼1025 Å2 surface area is buried on VL Fig. 1. Nucleotide and translated amino

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.

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.

Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting

PubMed Central

Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

2016-01-01

Objectives We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. Methods We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Results Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Conclusions Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance. PMID:27846293

A molecular model for self-assembly of amyloid fibrils: Immunoglobulin light chains

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

Stevens, F.J.; Myatt, E.A.; Westholm, F.A.

1995-08-29

The formation and pathological deposition of amyloid fibrils are defining features of many acquired and inherited disorders, including primary or light-chain-associated amyloidosis, Alzheimer`s disease, and adult-onset diabetes. No pharmacological methods exist to block this process or to effect the removal of fibrils from tissue, and thus, little can be done to prevent organ failure and ultimate death that result from deposition of amyloid. Knowledge of the pathogenesis, treatment, or prevention of these presently incurable diseases is limited due to the relative paucity of information regarding the biophysical basis of amyloid formation. Antibody light chains of different amino acid sequence showmore » differential amyloid-forming tendencies and, as such, can provide insight into the structural organization of amyloid fibrils as well as into basic mechanisms of protein self-assembly. We have compared primary structures of 180 human monoclonal light chains and have identified particular residues and positions within the variable domain that differentiate amyloid-from nonamyloid-associated proteins. We propose a molecular model that accounts for amyloid formation by antibody light chains and might also have implications for other forms of amyloidosis. 24 refs., 2 figs., 1 tab.« less

Antibody Light-Chain-Restricted Recognition of the Site of Immune Pressure in the RV144 HIV-1 Vaccine Trial Is Phylogenetically Conserved

DOE PAGES

Wiehe, Kevin; Easterhoff, David; Luo, Kan; ...

2014-11-29

In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognitionmore » prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. In conclusion, these data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.« less

Antibody Light-Chain-Restricted Recognition of the Site of Immune Pressure in the RV144 HIV-1 Vaccine Trial Is Phylogenetically Conserved

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

Wiehe, Kevin; Easterhoff, David; Luo, Kan

In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognitionmore » prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. In conclusion, these data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.« less

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

PubMed Central

Goodhardt, M; Babinet, C; Lutfalla, G; Kallenbach, S; Cavelier, P; Rougeon, F

1989-01-01

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo. Images PMID:2508061

INDUCTION OF RABBIT ANTIBODY WITH MOLECULAR UNIFORMITY AFTER IMMUNIZATION WITH GROUP C STREPTOCOCCI

PubMed Central

Eichmann, Klaus; Lackland, Henry; Hood, Leroy; Krause, Richard M.

1970-01-01

Antibodies with uniform properties may occur in rabbits after immunization with Group C streptococci. These precipitating antibodies possess specificity for the group-specific carbohydrate. Not uncommonly, their concentration is between 20 and 40 mg/ml of antiserum. Evidence for molecular uniformity in the case of one of these antibodies, described in detail here, includes: individual antigenic specificity; monodisperse distribution of the light chains by alkaline urea polyacrylamide disc electrophoresis; and a single amino acid in each of the first three N-terminal positions of the light chains. When the amino acid sequence of rabbit antibody b+ light chains (κ type) are aligned against their human κ counterparts, a definite homology is observed between the N-terminus of the human and the rabbit variable region. PMID:5409946

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

DOE PAGES

McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; ...

2015-09-22

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

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

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.

Diversity of immunoglobulin lambda light chain gene usage over developmental stages in the horse.

PubMed

Tallmadge, Rebecca L; Tseng, Chia T; Felippe, M Julia B

2014-10-01

To further studies of neonatal immune responses to pathogens and vaccination, we investigated the dynamics of B lymphocyte development and immunoglobulin (Ig) gene diversity. Previously we demonstrated that equine fetal Ig VDJ sequences exhibit combinatorial and junctional diversity levels comparable to those of adult Ig VDJ sequences. Herein, RACE clones from fetal, neonatal, foal, and adult lymphoid tissue were assessed for Ig lambda light chain combinatorial, junctional, and sequence diversity. Remarkably, more lambda variable genes (IGLV) were used during fetal life than later stages and IGLV gene usage differed significantly with time, in contrast to the Ig heavy chain. Junctional diversity measured by CDR3L length was constant over time. Comparison of Ig lambda transcripts to germline revealed significant increases in nucleotide diversity over time, even during fetal life. These results suggest that the Ig lambda light chain provides an additional dimension of diversity to the equine Ig repertoire. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

[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).

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.

Neutralisation and binding of VHS virus by monovalent antibody fragments.

PubMed

Cupit, P M; Lorenzen, N; Strachan, G; Kemp, G J; Secombes, C J; Cunningham, C

2001-12-04

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2 appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using the 3F1H10 variable heavy (VH) chain and variable light (Vkappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured by BIAcore analysis and found to correlate with the capacity of each molecule to neutralise DK-F1. These investigations, together with computer assisted molecular analysis of the theoretical influence of each mutation on antigen binding, led to the identification of a single mutation at position 35a in the VH domain as having the most marked impact on viral neutralisation.

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

PubMed Central

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

2016-01-01

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

Colour analysis and verification of CCTV images under different lighting conditions

NASA Astrophysics Data System (ADS)

Smith, R. A.; MacLennan-Brown, K.; Tighe, J. F.; Cohen, N.; Triantaphillidou, S.; MacDonald, L. W.

2008-01-01

Colour information is not faithfully maintained by a CCTV imaging chain. Since colour can play an important role in identifying objects it is beneficial to be able to account accurately for changes to colour introduced by components in the chain. With this information it will be possible for law enforcement agencies and others to work back along the imaging chain to extract accurate colour information from CCTV recordings. A typical CCTV system has an imaging chain that may consist of scene, camera, compression, recording media and display. The response of each of these stages to colour scene information was characterised by measuring its response to a known input. The main variables that affect colour within a scene are illumination and the colour, orientation and texture of objects. The effects of illumination on the appearance of colour of a variety of test targets were tested using laboratory-based lighting, street lighting, car headlights and artificial daylight. A range of typical cameras used in CCTV applications, common compression schemes and representative displays were also characterised.

Cargo selection by specific kinesin light chain 1 isoforms.

PubMed

Woźniak, Marcin J; Allan, Victoria J

2006-11-29

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.

Cargo selection by specific kinesin light chain 1 isoforms

PubMed Central

Woźniak, Marcin J; Allan, Victoria J

2006-01-01

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures. PMID:17093494

Characterization of antibodies directed against the immunoglobulin light kappa chain variable chain region (VK) of hepatitis C virus-related type-II mixed cryoglobulinemia and B-cell proliferations.

PubMed

de Re, Valli; Simula, Maria Paola; Pavan, Alessandro; Garziera, Marica; Marin, Dolores; Dolcetti, Riccardo; de Vita, Salvatore; Sansonno, Domenico; Geremia, Silvano; Toffoli, Giuseppe

2009-09-01

Autoimmune type-II cryoglobulinemia (II-MC) is sustained by hepatitis C virus (HCV) infection and B-cell (oligo)clones. This is the reason why the disease may be considered an "indolent B-cell lymphoma (NHL)." B clones show a restricted use of immunoglobulin variable genes (BCR), in particular in the use of the variable kappa (VK)3-20/15 light chain, and show a homology between their BCR functional regions and those of autoimmune rheumatoid factors. We underlined the BCR unique repertoire with frequent rheumatoid factor activity also observed in other autoimmune disorders associated with NHL. The immunoglobulin idiotype is a clonal B-cell marker and an ideal target for immunotherapy. Five monoclonal antibodies were produced in our laboratory toward the VK3-20 of a subject with HCV infection and a II-MC-associated NHL. Epitope determination was performed using the epitope excision approach. Monoclonal antibody reactivity was tested in vitro in ELISA, Western blot, and cytofluorimetry. Data confirmed that a panel of antibodies, reactive against shared idiotypes, can be produced from patients with HCV-associated B-cell lymphoproliferative diseases, thus obviating the need to produce an anti-idiotype antibody for each patient.

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

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.

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

Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics

PubMed Central

Vrana, Julie A.; Theis, Jason D.; Dasari, Surendra; Mereuta, Oana M.; Dispenzieri, Angela; Zeldenrust, Steven R.; Gertz, Morie A.; Kurtin, Paul J.; Grogg, Karen L.; Dogan, Ahmet

2014-01-01

Examination of abdominal subcutaneous fat aspirates is a practical, sensitive and specific method for the diagnosis of systemic amyloidosis. Here we describe the development and implementation of a clinical assay using mass spectrometry-based proteomics to type amyloidosis in subcutaneous fat aspirates. First, we validated the assay comparing amyloid-positive (n=43) and -negative (n=26) subcutaneous fat aspirates. The assay classified amyloidosis with 88% sensitivity and 96% specificity. We then implemented the assay as a clinical test, and analyzed 366 amyloid-positive subcutaneous fat aspirates in a 4-year period as part of routine clinical care. The assay had a sensitivity of 90%, and diverse amyloid types, including immunoglobulin light chain (74%), transthyretin (13%), serum amyloid A (%1), gelsolin (1%), and lysozyme (1%), were identified. Using bioinformatics, we identified a universal amyloid proteome signature, which has high sensitivity and specificity for amyloidosis similar to that of Congo red staining. We curated proteome databases which included variant proteins associated with systemic amyloidosis, and identified clonotypic immunoglobulin variable gene usage in immunoglobulin light chain amyloidosis, and the variant peptides in hereditary transthyretin amyloidosis. In conclusion, mass spectrometry-based proteomic analysis of subcutaneous fat aspirates offers a powerful tool for the diagnosis and typing of systemic amyloidosis. The assay reveals the underlying pathogenesis by identifying variable gene usage in immunoglobulin light chains and the variant peptides in hereditary amyloidosis. PMID:24747948

[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.

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

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

Oxidative Post-Translational Modifications of an Amyloidogenic Immunoglobulin Light Chain Protein.

PubMed

Lu, Yanyan; Jiang, Yan; Prokaeva, Tatiana; Connors, Lawreen H; Costello, Catherine E

2017-05-01

Immunoglobulin light chain amyloidosis (AL) is a plasma cell disorder characterized by overproduction and deposition of monoclonal immunoglobulin (Ig) light chains (LC) or variable region fragments as amyloid fibrils in various organs and tissues. Much clinical evidence indicates that patients with AL amyloidosis sustain cardiomyocyte impairment and suffer from oxidative stress. We seek to understand the underlying biochemical pathways whose disruption or amplification during sporadic or sustained disease states leads to harmful physiological consequences and to determine the detailed structures of intermediates and products that serve as signposts for the biochemical changes and represent potential biomarkers. In this study, matrix-assisted laser desorption/ionization mass spectrometry provided extensive evidence for oxidative post-translational modifications (PTMs) of an amyloidogenic Ig LC protein from a patient with AL amyloidosis. Some of the tyrosine residues were heavily mono- or di-chlorinated. In addition, a novel oxidative conversion to a nitrile moiety was observed for many of the terminal aminomethyl groups on lysine side chains. In vitro experiments using model peptides, in-solution oxidation, and click chemistry demonstrated that hypochlorous acid produced by the myeloperoxidase - hydrogen peroxide - chloride system could be responsible for these and other, more commonly observed modifications.

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.

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.

Quantum information processing with a travelling wave of light

NASA Astrophysics Data System (ADS)

Serikawa, Takahiro; Shiozawa, Yu; Ogawa, Hisashi; Takanashi, Naoto; Takeda, Shuntaro; Yoshikawa, Jun-ichi; Furusawa, Akira

2018-02-01

We exploit quantum information processing on a traveling wave of light, expecting emancipation from thermal noise, easy coupling to fiber communication, and potentially high operation speed. Although optical memories are technically challenging, we have an alternative approach to apply multi-step operations on traveling light, that is, continuous-variable one-way computation. So far our achievement includes generation of a one-million-mode entangled chain in time-domain, mode engineering of nonlinear resource states, and real-time nonlinear feedforward. Although they are implemented with free space optics, we are also investigating photonic integration and performed quantum teleportation with a passive liner waveguide chip as a demonstration of entangling, measurement, and feedforward. We also suggest a loop-based architecture as another model of continuous-variable computing.

Association of plasma cell subsets in the bone marrow and free light chain concentrations in the serum of monoclonal gammopathy patients.

PubMed

Ayliffe, Michael John; Behrens, Judith; Stern, Simon; Sumar, Nazira

2012-08-01

This study investigated bone marrow plasma cell subsets and monoclonal free light chain concentrations in blood of monoclonal gammopathy patients. 54 bone marrow samples were stained by double immunofluorescence to enumerate cellular subsets making either intact monoclonal immunoglobulin or free light chains only. Blood taken at the same time was assayed for free light chains by an automated immunoassay. Patients were assigned to three cellular population categories: single intact monoclonal immunoglobulin (59%), dual monoclonal immunoglobulin and free light chain only (31%), or single free light chain only (9%). The median affected free light chain concentration of each group was 75 mg/l, 903 mg/l and 3320 mg/l, respectively, but with substantial overlap. In myeloma patients the difference in serum free light chain concentrations between patients with free light chain only marrow cells and those without was statistically significant. Serum free light chain levels >600 mg/l result mostly from marrow cells restricted to free light chain production.

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

McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole

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

Anxiety and depression among amyloid light-chain cardiac amyloidosis patients: The role of life satisfaction.

PubMed

Smorti, Martina; Guarnieri, Silvia; Bergesio, Franco; Perfetto, Federico; Cappelli, Francesco

2016-06-01

The present study aimed to provide a contribution to the study of a rare disease, amyloid light-chain (AL) cardiac amyloidosis, which is the most common type of systemic amyloidosis. In AL amyloidosis prognosis is determined by cardiac involvement. Although the association between psychological distress (e.g. anxiety and depression) and AL cardiac amyloidosis is documented, very little is known about the psychosocial variables that may mediate the association. The aim of the study is therefore to examine the potential mediating role of life satisfaction in the relationship between cardiac symptom severity (independent variable) and anxious and depressive symptoms (dependent variables) in AL patients. Forty-three AL amyloidosis patients (57.1% males) with cardiac amyloidosis were administered the Satisfaction with Life Scale, the State-Trait Anxiety Inventory and the Centre for Epidemiological Study-Depression Scale. Clinical variables such as months since cardiac symptom onset and cardiac symptom severity were collected. Findings showed significant relationships between symptom severity and psychological disorders (e.g. anxiety and depression) and these were mediated by life satisfaction. Overall, findings highlight the importance of subjective well-being (e.g. life satisfaction) to reduce anxious and depressive symptoms and to improve general health in AL patients. © The European Society of Cardiology 2015.

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

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.

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

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.

Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four vλ6 proteins

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

Wall, J.S.; Gupta, V.; Wilkerson, M.

2004-04-01

Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (V{sub L}) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the V{sub L} domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable V{sub {lambda}}6 protein (Jto) with a long range electrostatic interaction between Asp and Arg side chains at position 29 and 68, respectively, and an unstable, highly fibrillogenic V{sub {lambda}}6 protein (Wil) that had neutral amino acidsmore » at these locations. To test this hypothesis, we have generated two Jto-related mutants designed to disrupt the interaction between Asp 29 and Arg 68 (JtoD29A and JtoR68S). Although the thermodynamic stabilities of unfolding for these two molecules were identical, they exhibited very different kinetics of fibril formation: the rate of JtoD29A fibrillogenesis was slow and comparable to the parent molecule, whereas that of JtoR68S was significantly faster. High-resolution X-ray diffraction analyses of crystals prepared from the two mutants having the same space group and unit cell dimensions revealed no significant main-chain conformational changes. However, several notable side-chain alterations were observed in JtoR68S, as compared with JtoD29A, that resulted in the solvent exposure of a greater hydrophobic surface and modifications in the electrostatic potential surface. We posit that these differences contributed to the enhanced fibrillogenic potential of the Arg 68 mutant, since both Jto mutants lacked the intrachain ionic interaction and were equivalently unstable. The information gleaned from our studies has provided insight into structural parameters that in addition to overall thermodynamic stability, contribute to the fibril forming propensity of immunoglobulin light chains.« less

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

A residue-specific shift in stability and amyloidogenicity of antibody variable domains.

PubMed

Nokwe, Cardine N; Zacharias, Martin; Yagi, Hisashi; Hora, Manuel; Reif, Bernd; Goto, Yuji; Buchner, Johannes

2014-09-26

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (VL) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of VL domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the VL domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol(-1), which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in VL domains of the κ family and not in VLλ or in VH domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Grasping the nettle: A bacterial invasin that targets immunoglobulin variable domains.

PubMed

Barlow, Paul

2018-06-01

In a new paper, the protein InvD from Yersinia pseudotuberculosis , a zoonotic pathogen, is shown to assist late-stage invasion of intestinal epithelia. Remarkably, InvD acts by binding the Fab region of IgG or IgA. It straddles adjacent light-chain and heavy-chain variable domains, but its binding is different from that of antigens in that complementarity-determining regions do not participate. Structure determination revealed that its Fab-interacting domain adopts an immunoglobulin-like fold, fused to the preceding immunoglobulin-like domain and carried on a long stalk anchored to the bacterial outer membrane. Possible roles of this unusual host-pathogen interaction include avoidance of clearance from the intestine by secretory IgA. © 2018 Barlow.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

PubMed

Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

2018-04-01

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

PubMed Central

Adler, Adam S.; Bedinger, Daniel; Adams, Matthew S.; Asensio, Michael A.; Edgar, Robert C.; Leong, Renee; Leong, Jackson; Mizrahi, Rena A.; Spindler, Matthew J.; Bandi, Srinivasa Rao; Huang, Haichun; Brams, Peter; Johnson, David S.

2018-01-01

ABSTRACT Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs. PMID:29376776

Transfer factor - hypotheses for its structure and function.

PubMed

Shifrine, M; Scibienski, R

1975-01-01

Transfer factor (TF) is a dialyzable extract from primed lymphocytes that is able to transfer specific delayed hypersensitivity from one animal to another. On the basis of available data we suggest that TF is a polypeptide with a molecular weight below 15,000 daltons. We hypothesize that TF is the variable light or heavy chain domain of immunoglobulin: such a molecule conforms with the accepted properties of TF and also has the necessary specificity requirements. We also hypothesize that TF is part of a receptor site. beta-2-microglobulin, a molecule that is an integral part of cell surfaces, could be the anchor for TF. beta-2-microglobulin has homologies with the constant portion of immunoglobulin light or heavy chain and thus would combine with the variable domain (TF) to form a complete receptor site for a specific antigen. The properties of TF suggest its mode of action, which is discussed in detail in the text. The biologic advantages of TF is its ability to confer immediate (immunologie specific) protection while the 'normal' immune response develops.

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

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

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.

Somatic diversification of chicken immunoglobulin light chains by point mutations.

PubMed

Parvari, R; Ziv, E; Lantner, F; Heller, D; Schechter, I

1990-04-01

The light-chain locus of chicken has 1 functional V lambda 1 gene, 1 J gene, and 25 pseudo-V lambda-genes (where V = variable and J = joining). A major problem is which somatic mechanisms expand this extremely limited germ-line information to generate many different antibodies. Weill's group [Reynaud, C. A., Anquez, V., Grimal, H. & Weill, J. C. (1987) Cell 48, 379-388] has shown that the pseudo-V lambda-genes diversify the rearranged V lambda 1 by gene conversion. Here we demonstrate that chicken light chains are further diversified by somatic point mutations and by V lambda 1-J flexible joining. Somatic point mutations were identified in the J and 3' noncoding DNA of rearranged light-chain genes of chicken. These regions were analyzed because point mutations in V lambda 1 are obscured by gene conversion; the J and 3' noncoding DNA are presented in one copy per haploid genome and are not subject to gene conversion. In rodents point mutations occur as frequently in the V-J coding regions as in the adjacent flanking DNA. Therefore, we conclude that somatic point mutations diversify the V lambda 1 of chicken. The frequency (0-1%) and distribution of the mutations (decreasing in number with increased distance from the V lambda 1 segment) in chicken were as observed in rodents. Sequence variability at the V lambda 1-J junctions could be attributed to imprecise joining of the V lambda 1 and J genes. The modification by gene conversion of rearranged V lambda 1 genes in the bursa was similar in chicken aged 3 months (9.5%) or 3 weeks (9.1%)--i.e., gene conversion that generates the preimmune repertoire in the bursa seems to level off around 3 weeks of age. This preimmune repertoire can be further diversified by somatic point mutations that presumably lead to the formation of antibodies with increased affinity. A segment with structural features of a matrix association region [(A + T)-rich and four topoisomerase II binding sites] was identified in the middle of the J-C lambda intron (where C = constant).

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.

Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease

PubMed Central

Sridevi, N. V.; Shukra, A. M.; Neelakantam, B.; Anilkumar, J.; Madhanmohan, M.; Rajan, S.; Dev Chandran

2014-01-01

Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals. PMID:24678404

Genome complexity in the coelacanth is reflected in its adaptive immune system

USGS Publications Warehouse

Saha, Nil Ratan; Ota, Tatsuya; Litman, Gary W.; Hansen, John; Parra, Zuly; Hsu, Ellen; Buonocore, Francesco; Canapa, Adriana; Cheng, Jan-Fang; Amemiya, Chris T.

2014-01-01

We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/β and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations.

Acquired Fanconi syndrome with proximal tubular cytoplasmic fibrillary inclusions of λ light chain restriction.

PubMed

Yao, Ying; Wang, Su-Xia; Zhang, You-Kang; Wang, Yan; Liu, Li; Liu, Gang

2014-01-01

Light chain proximal tubulopathy is a rarely reported entity associated with plasma cell dyscrasia that classically manifests as acquired Fanconi syndrome and is characterized by the presence of κ-restricted crystals in the proximal tubular cytoplasm. We herein present a case of multiple myeloma with Fanconi syndrome and acute kidney injury due to light chain proximal tubulopathy with light chain cast nephropathy. Prominent phagolysosomes and numerous irregularly shaped inclusions with a fibrillary matrix in the cytoplasm of the proximal tubules were identified on electron microscopy. A monotypic light chain of the λ type was detected in the distal tubular casts, proximal tubular cytoplasmic lysosomes and fibrillary inclusions on immunofluorescence and immune electron microscopy. This case underscores the importance of conducting careful ultrastructural investigations and immunocytologic examinations of light chains for detecting and diagnosing light chain proximal tubulopathy.

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.

[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 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

Involvement of myosin light-chain kinase in endothelial cell retraction

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

Wysolmerski, R.B.; Lagunoff, D.

Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca{sup 2+}. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-(gamma-({sup 35}S)thio)triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca{sup 2+}. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca{sup 2+}, and no thiophosphorylationmore » of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.« less

Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosins

NASA Astrophysics Data System (ADS)

Reinach, Fernando C.; Nagai, Kiyoshi; Kendrick-Jones, John

1986-07-01

The regulatory light chains, small polypeptides located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation. The demonstration that the regulatory light chains on scallop myosin can be replaced by light chains from other myosins has allowed us to compare the functional capabilities of different light chains1, but has not enabled us to probe the role of features, such as the Ca2+/Mg2+ binding site, that are common to all of them. Here, we describe the use of site-directed mutagenesis to study the function of that site. We synthesized the chicken skeletal myosin light chain in Escherichia coli and constructed mutants with substitutions within the Ca2+/Mg2+ binding site. When the aspartate residues at the first and sixth Ca2+ coordination positions are replaced by uncharged alanines, the light chains have a reduced Ca2+ binding capacity but still bind to scallop myosin with high affinity. Unlike the wild-type skeletal light chain which inhibits myosin interaction with actin, the mutants activate it. Thus, an intact Ca2+/Mg2+ binding site in the N-terminal region of the light chain is essential for regulating the interaction of myosin with actin.

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.

Mediation of acetylcholine and substance P induced contractions by myosin light chain phosphorylation in feline colonic smooth muscle.

PubMed

Washabau, Robert J; Holt, David E; Brockman, Daniel J

2002-05-01

To determine the role of myosin light chain phosphorylation in feline colonic smooth muscle contraction. Colonic tissue was obtained from eight 12- to 24-month-old cats. Colonic longitudinal smooth muscle strips were attached to isometric force transducers for measurements of isometric stress. Myosin light chain phosphorylation was determined by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stress and phosphorylation were determined following stimulation with ACh or SP, in the absence or presence of a calmodulin antagonist (W-7; 0.1 to 1.0 mM), myosin light chain kinase inhibitor (ML-9; 1 to 10 microM), or extracellular calcium free solutions. Unstimulated longitudinal colonic smooth muscle contained low amounts (6.9+/-3.2%) of phosphorylated myosin light chain. Phosphorylation of the myosin light chains was dose and time dependent with maximal values of 58.5% at 30 seconds of stimulation with 100 microM Ach and 60.2% at 45 seconds of stimulation with 100 nM SP Active isometric stress development closely paralleled phosphorylation of the myosin light chains in ACh- or SP-stimulated muscle. W-7 and ML-9 dose dependently inhibited myosin light chain phosphorylation and isometric stress development associated with ACh or SP stimulation. Removal of extracellular calcium inhibited myosin light chain phosphorylation and isometric stress development in ACh-stimulated smooth muscle. Feline longitudinal colonic smooth muscle contraction is calcium-, calmodulin-, and myosin light chain kinase-dependent. Myosin light chain phosphorylation is necessary for the initiation of contraction in feline longitudinal colonic smooth muscle. These findings may prove useful in determining the biochemical and molecular defects that accompany feline colonic motility disorders.

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis.

PubMed

del Pozo-Yauner, Luis; Wall, Jonathan S; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L; Pérez Carreón, Julio I; Ochoa-Leyva, Adrián; Fernández-Velasco, D Alejandro

2014-01-10

It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein. Copyright © 2013 Elsevier Inc. All rights reserved.

Four structural risk factors identify most fibril-forming kappa light chains.

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

Stevens, F. J.; Biosciences Division

2000-09-01

Antibody light chains (LCs) comprise the most structurally diverse family of proteins involved in amyloidosis. Many antibody LCs incorporate structural features that impair their stability and solubility, leading to their assembly into fibrils and to their subsequent pathological deposition when produced in excess during multiple myeloma and primary amyloidosis. The particular amino acid variations in antibody LCs that account for fibril formation and amyloidogenesis have not been identified. This study focuses on amyloidogenesis within the Kl family of human LCs. Reanalysis of the current database of primary structures of proteins from more than 100 patients who produced Kl LCS, 37more » of which were amyloidogenic, reveals apparent structural features that may contribute to amyloidosis. These features include loss of conserved residues or the gain of particular residues through mutation at sites involving a repertoire of approximately 20% of the amino acid positions in the light chain variable domain (V{sub L}). Moreover, 80% of all K1 amyloidogenic V{sub L}s are identifiable by the presence of at least one of three single-site substitutions or the acquisition of an N-linked glycosylation site through mutations. These findings suggest that it is feasible to predict fibril propensity by analysis of primary structure.« less

Immunoglobulin Light-Chain Amyloidosis: From Basics to New Developments in Diagnosis, Prognosis and Therapy.

PubMed

Muchtar, Eli; Buadi, Francis K; Dispenzieri, Angela; Gertz, Morie A

2016-01-01

Immunoglobulin amyloid light-chain (AL) amyloidosis is the most common form of systemic amyloidosis, where the culprit amyloidogenic protein is immunoglobulin light chains produced by marrow clonal plasma cells. AL amyloidosis is an infrequent disease, and since presentation is variable and often nonspecific, diagnosis is often delayed. This results in cumulative organ damage and has a negative prognostic effect. AL amyloidosis can also be challenging on the diagnostic level, especially when demonstration of Congo red-positive tissue is not readily obtained. Since as many as 31 known amyloidogenic proteins have been identified to date, determination of the amyloid type is required. While several typing methods are available, mass spectrometry has become the gold standard for amyloid typing. Upon confirming the diagnosis of amyloidosis, a pursuit for organ involvement is essential, with a focus on heart involvement, even in the absence of suggestive symptoms for involvement, as this has both prognostic and treatment implications. Details regarding initial treatment options, including stem cell transplantation, are provided in this review. AL amyloidosis management requires a multidisciplinary approach with careful patient monitoring, as organ impairment has a major effect on morbidity and treatment tolerability until a response to treatment is achieved and recovery emerges. © 2016 S. Karger AG, Basel.

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.

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires.

PubMed

DeKosky, Brandon J; Lungu, Oana I; Park, Daechan; Johnson, Erik L; Charab, Wissam; Chrysostomou, Constantine; Kuroda, Daisuke; Ellington, Andrew D; Ippolito, Gregory C; Gray, Jeffrey J; Georgiou, George

2016-05-10

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.

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

PubMed

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

2016-10-01

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

Characterization of myosin light chain in shrimp hemocytic phagocytosis.

PubMed

Han, Fang; Wang, Zhiyong; Wang, Xiaoqing

2010-11-01

Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp. Copyright 2010 Elsevier Ltd. All rights reserved.

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

PubMed

Velázquez-López, Isabel; Valdés-García, Gilberto; Romero Romero, Sergio; Maya Martínez, Roberto; Leal-Cervantes, Ana I; Costas, Miguel; Sánchez-López, Rosana; Amero, Carlos; Pastor, Nina; Fernández Velasco, D Alejandro

2018-07-01

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0 pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0 pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

Vasotropic light-chain amyloidosis and ischaemic cholangiopathy.

PubMed

Johnston, Emma L; Wilkinson, Mark; Knisely, A S

2015-06-25

A 75-year-old woman was incidentally found to have deranged liver function tests (LFTs). She was well, apart from 2 years of dyspnoea. Investigations had revealed atrial fibrillation and a right pleural effusion, without identified aetiology. On examination, the only finding was a palpable liver edge. Initial blood and ultrasound screening suggested no cause. The patient underwent liver biopsy. Microscopy showed κ-immunoglobulin light chains deposited exclusively in portal tracts, within blood vessel and bile duct walls. This pattern, although unusual, raised the possibility of κ-light chain disease. Serum electrophoresis was normal, as were serum immunoglobulin values. Serum concentrations of κ-light chains were elevated and microscopy of aspirated bone marrow found light-chain deposits with 10% plasmacytosis. Serum amyloid P (SAP) scintigraphy demonstrated splenic uptake. Myeloma, κ-light chain, with light-chain amyloidosis was diagnosed. The patient has responded well to cyclophosphamide, bortazomib and dexamethasone chemotherapy, and her LFTs are now nearly normal. 2015 BMJ Publishing Group Ltd.

Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of Fabricius

PubMed Central

Sayegh, Camil E.; Demaries, Sandra L.; Iacampo, Sandra; Ratcliffe, Michael J. H.

1999-01-01

Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor. PMID:10485907

Early Prognostic Value of Monitoring Serum Free Light Chain in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation.

PubMed

Özkurt, Zübeyde Nur; Sucak, Gülsan Türköz; Akı, Şahika Zeynep; Yağcı, Münci; Haznedar, Rauf

2017-03-16

We hypothesized the levels of free light chains obtained before and after autologous stem cell transplantation can be useful in predicting transplantation outcome. We analyzed 70 multiple myeloma patients. Abnormal free light chain ratios before stem cell transplantation were found to be associated early progression, although without any impact on overall survival. At day +30, the normalization of levels of involved free light chain related with early progression. According to these results almost one-third reduction of free light chain levels can predict favorable prognosis after autologous stem cell transplantation.

An investigation into UV light exposure as an experimental model for artificial aging on tensile strength and force delivery of elastomeric chain.

PubMed

Wahab, Siti Waznah; Bister, Dirk; Sherriff, Martyn

2014-02-01

This study investigated the effect of ultraviolet type A light (UVA) exposure on the tensile properties of elastomeric chain. UVA light exposure was used as model for artificial aging, simulating prolonged storage of elastomeric chain. Tensile strength (n = 60) was measured after exposing Ormco, Forestadent and 3M chains to UVA light for 0, 2, 3, and 4 weeks. Force decay was measured (n = 60) using chain exposed for 5, 10, and 14 days. The chains were subsequently stretched at a constant distance and the resulting forces measured at 0, 1, 24 hours and 7, 14, 21, and 28 days. This test simulated a clinical scenario of pre-stretching and subsequent shortening of elastomeric chain. Tensile strength had statistically significant difference and was directly related to the duration of ultraviolet (UV) light exposure. Forestadent chain, which had the second highest value for the 'as received' product, showed the most consistent values over time with the lowest degradation. Ormco showed the lowest values for 'as received' as well as after UV exposure; 3M chain had the highest loss of tensile strength. Force decay was also significantly different. UV light exposure of 10 days or more appears to mark a 'watershed' between products: 3M had most survivors, Forestadent chain had some survivors, depending on the time the chain was stretched for. None of the Ormco product survived UV light exposure for more than 5 days. UVA light exposure may be used as a model for artificial aging as it reduces force delivery and tensile strength of exposed chains.

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

Engineered Recombinant Single-Chain Fragment Variable Antibody for Immunosensors

PubMed Central

Shen, Zhihong; Mernaugh, Raymond L.; Yan, Heping; Yu, Lei; Zhang, Ying; Zeng, Xiangqun

2008-01-01

A recombinant single-chain fragment variable (scFv) antibody (designated A10B) was engineered to contain two histidines within the linker peptide used to join the scFv heavy and light chains. A piezoimmunosensor using the scFv was successfully developed. A10B scFv bound to the gold piezoimmunosensor surface were correctly oriented, retained antigen-binding activity, and coupled at high surface concentration. These results, and results obtained from an earlier study using an scFv containing a linker cysteine, suggest that the location on the linker sequence in which the amino acids were incorporated was well tolerated by the scFv and did not interfere with scFv antigen-binding activity. The scFv-modified QCM sensor was thoroughly characterized and used to specifically detect antigen in crude serum sample and had a sensitivity of 2.3 ± 0.15 nM (n = 4) with a linear range over 2.3 × 10−9–3.3 × 10−8 M. The piezoimmunosensor was also used to study the kinetics and thermodynamics of antigen/scFv antibody binding. PMID:16255580

The role of exoproteases in governing intraneuronal metabolism of botulinum toxin.

PubMed

Simpson, Lance L; Maksymowych, Andrew B; Kouguchi, Hirokazu; Dubois, Garrett; Bora, Roop S; Joshi, Suresh

2005-04-01

Botulinum toxin type A has a long duration of action, and thus it can block transmitter release for several weeks to several months. However, little is known about the precise mechanism that accounts for termination of toxin action. Therefore, experiments were done to gauge the effects of aminopeptidases and carboxypeptidases on the structure and function of the toxin. Exoproteases were added to the holotoxin, the native light chain, and a recombinant light chain. Treated toxin and light chain were examined for their effects on neuromuscular transmission and on isolated substrate. The data showed that aminopeptidase attack did not alter the N-terminus of the toxin/light chain, nor did it produce losses in biological activity. Carboxypeptidase attack did alter the C-terminus of the light chain, but not sufficiently to alter biological activity. The data suggest that the tertiary structure of the light chain confers upon the molecule substantial resistance to exoproteases.

Size, shape, and diffusivity of a single Debye-Hückel polyelectrolyte chain in solution.

PubMed

Soysa, W Chamath; Dünweg, B; Prakash, J Ravi

2015-08-14

Brownian dynamics simulations of a coarse-grained bead-spring chain model, with Debye-Hückel electrostatic interactions between the beads, are used to determine the root-mean-square end-to-end vector, the radius of gyration, and various shape functions (defined in terms of eigenvalues of the radius of gyration tensor) of a weakly charged polyelectrolyte chain in solution, in the limit of low polymer concentration. The long-time diffusivity is calculated from the mean square displacement of the centre of mass of the chain, with hydrodynamic interactions taken into account through the incorporation of the Rotne-Prager-Yamakawa tensor. Simulation results are interpreted in the light of the Odjik, Skolnick, Fixman, Khokhlov, and Khachaturian blob scaling theory (Everaers et al., Eur. Phys. J. E 8, 3 (2002)) which predicts that all solution properties are determined by just two scaling variables-the number of electrostatic blobs X and the reduced Debye screening length, Y. We identify three broad regimes, the ideal chain regime at small values of Y, the blob-pole regime at large values of Y, and the crossover regime at intermediate values of Y, within which the mean size, shape, and diffusivity exhibit characteristic behaviours. In particular, when simulation results are recast in terms of blob scaling variables, universal behaviour independent of the choice of bead-spring chain parameters, and the number of blobs X, is observed in the ideal chain regime and in much of the crossover regime, while the existence of logarithmic corrections to scaling in the blob-pole regime leads to non-universal behaviour.

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.

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires

PubMed Central

DeKosky, Brandon J.; Lungu, Oana I.; Park, Daechan; Johnson, Erik L.; Charab, Wissam; Chrysostomou, Constantine; Kuroda, Daisuke; Ellington, Andrew D.; Ippolito, Gregory C.; Gray, Jeffrey J.; Georgiou, George

2016-01-01

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19+CD20+CD27− IgM-naive B cells, >120,000 antibody clusters from CD19+CD20+CD27+ antigen–experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ–Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease. PMID:27114511

Clinicopathologic characteristics of light chain proximal tubulopathy with light chain inclusions involving multiple renal cell types
.

PubMed

Li, Xiaomei; Xu, Feng; Liang, Dandan; Liang, Shaoshan; Zhu, Xiaodong; Zhang, Mingchao; Huang, Xianghua; Liu, Zhihong; Zeng, Caihong

2018-02-01

Light chain proximal tubulopathy (LCPT) associated with plasma cell dyscrasias is a rare abnormality, especially cases involving multiple cell types. The aim of this study is to explore the characteristics and outcomes of these diseases. We comprehensively evaluated the clinical-pathological data, treatment, and outcomes of 6 LCPT patients with involvement of multiple cell types. In 3 cases, we found that the inclusions largely existed in tubular cells, while in 2 cases they coexisted in podocytes and tubular cells, and in 1 case they coexisted in histiocytes and tubular cells. The stain features and appearances of inclusions were specific and varied. Five patients displayed κ-light chains with crystal formation, while 1 patient displayed a λ subtype with increased lysosomes instead of crystals. Six patients presented with proteinuria, 4 with renal insufficiency, and 4 with complete or partial Fanconi syndrome. Our findings indicate that tubular cells are the most common location of cytoplasmic inclusions. Cases with κ-light chain storage are more common than λ, and the formation of crystals may be associated with the subtype of light chains. Immunoelectron microscopy could be used to increase sensitivity for the detection and location of monoclonal light chains. Therefore, these patients have some common clinical features with varied pathologic characteristics and prognoses but the same subtype of light chains.
.

Immunoglobulin Light Chains Form an Extensive and Highly Ordered Fibril Involving the N- and C-Termini

PubMed Central

2017-01-01

Light-chain (AL)-associated amyloidosis is a systemic disorder involving the formation and deposition of immunoglobulin AL fibrils in various bodily organs. One severe instance of AL disease is exhibited by the patient-derived variable domain (VL) of the light chain AL-09, a 108 amino acid residue protein containing seven mutations relative to the corresponding germline protein, κI O18/O8 VL. Previous work has demonstrated that the thermodynamic stability of native AL-09 VL is greatly lowered by two of these mutations, Y87H and N34I, whereas a third mutation, K42Q, further increases the kinetics of fibril formation. However, detailed knowledge regarding the residues that are responsible for stabilizing the misfolded fibril structure is lacking. In this study, using solid-state NMR spectroscopy, we show that the majority of the AL-09 VL sequence is immobilized in the fibrils and that the N- and C-terminal portions of the sequence are particularly well-structured. Thus, AL-09 VL forms an extensively ordered and β-strand-rich fibril structure. Furthermore, we demonstrate that the predominant β-sheet secondary structure and rigidity observed for in vitro prepared AL-09 VL fibrils are qualitatively similar to those observed for AL fibrils extracted from postmortem human spleen tissue, suggesting that this conformation may be representative of a common feature of AL fibrils. PMID:28261692

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.

Comparison of Free Light Chain Assays:  Freelite and N Latex in Diagnosis, Monitoring, and Predicting Survival in Light Chain Amyloidosis.

PubMed

Mahmood, Shameem; Wassef, Nancy L; Salter, Simon J; Sachchithanantham, Sajitha; Lane, T; Foard, D; Whelan, Carol J; Lachmann, Helen J; Gillmore, Julian D; Hawkins, Philip N; Wechalekar, Ashutosh D

2016-07-01

Measurement of serum free light chains (FLCs) is critical in diagnosis, prognosis, and monitoring treatment responses in light chain (AL) amyloidosis. We compare the Freelite assay (polyclonal antibodies to hidden light chain epitopes), which is the current gold standard, with a new assay: a mixture of monoclonal antibodies to light chain epitopes (N Latex). We collected 240 serum samples from 94 consecutive patients with newly diagnosed AL amyloidosis (at least three serial serum samples during the first 6 months) analyzed at the National Amyloidosis Centre, London, from January 2011 to April 2012. Concordance in detecting abnormal light chain components and hematologic response was assessed at 2, 4, and 6 months. The κ and λ clonal light chain involvement was 21% and 79%, respectively, with an abnormal κ/λ ratio or detectable protein in 78.7%. Median κ, λ, and difference in involved and uninvolved FLCs by Freelite and N Latex assays were 17.3 vs 16 mg/L (R(2 ) = 0.91), 48.8 vs 52.6 mg/L (R(2) = 0.52), and 43.2 vs 39.1 mg/L, respectively. Discordant κ/λ ratios at presentation were as follows: 10 of 90 abnormal by Freelite/normal by N Latex and 11 of 90 abnormal by N Latex/normal by Freelite. Both FLC assays show good correlation in detecting the abnormal light chain subtype with discordance in absolute values and thus are not interchangeable. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Inhibition of the protease activity of the light chain of type A botulinum neurotoxin by aqueous extract from stinging nettle (Urtica dioica) leaf.

PubMed

Gul, Nizamettin; Ahmed, S Ashraf; Smith, Leonard A

2004-11-01

We investigated the inhibitory effect of stinging nettle leaf extract on the protease activity of botulinum neurotoxin type A and B light chains. The nettle leaf infusion was fractionated and HPLC-based enzymatic assays were performed to determine the capacity of each fraction to inhibit the protease activity of botulinum neurotoxin type A and B light chains. Assay results demonstrated that a water-soluble fraction obtained from the nettle leaf infusion inhibited type A, but did not inhibit type B light chain protease activity. The inhibition mode of water soluble fraction against protease activity of type A light chain was analyzed and found to be a non-competitive.

[A wrong move in an amateur football player reveals a light chain myeloma].

PubMed

Peyneau, Marine; Nassiri, Shiva; Myara, Anne; Ohana, Salomon; Laplanche, Sophie

2016-01-01

Light chain multiple myeloma is a hematologic malignancy characterized by an excess of tumor plasma cells in the bone marrow and a monoclonal light chain in blood. It is generally diagnosed in patients aged 60-75 years old. Hypercalcemia, anemia, kidney failure, and bone pains are the main clinical and biological signs. Here is an atypical case report about a 30 year-old man who was diagnosed a light chain multiple myeloma. This patient had been suffering from back pain for 5 months. Osteolytic lesions were discovered on X-rays prescribed by the family practitioner. Admitted to the Emergency department, all blood tests showed results within the normal range. The serum protein electrophoresis was also normal. Only the urine analysis showed proteinuria. The urine immunofixation electrophoresis showed a massive κ light chain. The bone marrow aspiration cell count confirmed the myeloma diagnosis with an infiltration of dystrophic plasma cells. The patient was transferred to the hematology ward of Necker Hospital for treatment of light chain myeloma.

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

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

Serum neurofilament as a predictor of disease worsening and brain and spinal cord atrophy in multiple sclerosis.

PubMed

Barro, Christian; Benkert, Pascal; Disanto, Giulio; Tsagkas, Charidimos; Amann, Michael; Naegelin, Yvonne; Leppert, David; Gobbi, Claudio; Granziera, Cristina; Yaldizli, Özgür; Michalak, Zuzanna; Wuerfel, Jens; Kappos, Ludwig; Parmar, Katrin; Kuhle, Jens

2018-05-30

Neuro-axonal injury is a key factor in the development of permanent disability in multiple sclerosis. Neurofilament light chain in peripheral blood has recently emerged as a biofluid marker reflecting neuro-axonal damage in this disease. We aimed at comparing serum neurofilament light chain levels in multiple sclerosis and healthy controls, to determine their association with measures of disease activity and their ability to predict future clinical worsening as well as brain and spinal cord volume loss. Neurofilament light chain was measured by single molecule array assay in 2183 serum samples collected as part of an ongoing cohort study from 259 patients with multiple sclerosis (189 relapsing and 70 progressive) and 259 healthy control subjects. Clinical assessment, serum sampling and MRI were done annually; median follow-up time was 6.5 years. Brain volumes were quantified by structural image evaluation using normalization of atrophy, and structural image evaluation using normalization of atrophy, cross-sectional, cervical spinal cord volumes using spinal cord image analyser (cordial). Results were analysed using ordinary linear regression models and generalized estimating equation modelling. Serum neurofilament light chain was higher in patients with a clinically isolated syndrome or relapsing remitting multiple sclerosis as well as in patients with secondary or primary progressive multiple sclerosis than in healthy controls (age adjusted P < 0.001 for both). Serum neurofilament light chain above the 90th percentile of healthy controls values was an independent predictor of Expanded Disability Status Scale worsening in the subsequent year (P < 0.001). The probability of Expanded Disability Status Scale worsening gradually increased by higher serum neurofilament light chain percentile category. Contrast enhancing and new/enlarging lesions were independently associated with increased serum neurofilament light chain (17.8% and 4.9% increase per lesion respectively; P < 0.001). The higher the serum neurofilament light chain percentile level, the more pronounced was future brain and cervical spinal volume loss: serum neurofilament light chain above the 97.5th percentile was associated with an additional average loss in brain volume of 1.5% (P < 0.001) and spinal cord volume of 2.5% over 5 years (P = 0.009). Serum neurofilament light chain correlated with concurrent and future clinical and MRI measures of disease activity and severity. High serum neurofilament light chain levels were associated with both brain and spinal cord volume loss. Neurofilament light chain levels are a real-time, easy to measure marker of neuro-axonal injury that is conceptually more comprehensive than brain MRI.

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.

Immunoglobulin light chains, glycosaminoglycans and amyloid.

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

Stevens, F. J.; Kisilevsky, R.; Biosciences Division

2000-03-01

Immunoglobulin light chains are the precursor proteins for fibrils that are formed during primary amyloidosis and in amyloidosis associated with multiple myeloma. As found for the approximately 20 currently described forms of focal, localized, or systemic amyloidoses, light chain-related fibrils extracted from physiological deposits are invariably associated with glycosaminoglycans, predominantly heparan sulfate. Other amyloid-related proteins are either structurally normal, such as g2-microglobulin and islet amyloid polypeptide, fragments of normal proteins such as serum amyloid A protein or the precursor protein of the g peptide involved in Alzheimer's disease, or are inherited forms of single amino acid variants of a normalmore » protein such as found in the familial forms of amyloid associated with transthyretin. In contrast, the primary structures of light chains involved in fibril formation exhibit extensive mutational diversity rendering some proteins highly amyloidogenic and others non-pathological. The interactions between light chains and glycosaminoglycans are also affected by amino acid variation and may influence the clinical course of disease by enhancing fibril stability and contributing to resistance to protease degradation. Relatively little is currently known about the mechanisms by which glycosaminoglycans interact with light chains and light-chain fibrils. It is probable that future studies of this uniquely diverse family of proteins will continue o shed light on the processes of amyloidosis, and contribute as well to a greater understanding of the normal physiological roles of glycosaminoglycans.« less

Actin binding by Hip1 (huntingtin-interacting protein 1) and Hip1R (Hip1-related protein) is regulated by clathrin light chain.

PubMed

Wilbur, Jeremy D; Chen, Chih-Ying; Manalo, Venus; Hwang, Peter K; Fletterick, Robert J; Brodsky, Frances M

2008-11-21

The huntingtin-interacting protein family members (Hip1 and Hip1R in mammals and Sla2p in yeast) link clathrin-mediated membrane traffic to actin cytoskeleton dynamics. Genetic data in yeast have implicated the light chain subunit of clathrin in regulating this link. To test this hypothesis, the biophysical properties of mammalian Hip1 and Hip1R and their interaction with clathrin light chain and actin were analyzed. The coiled-coil domains (clathrin light chain-binding) of Hip1 and Hip1R were found to be stable homodimers with no propensity to heterodimerize in vitro. Homodimers were also predominant in vivo, accounting for cellular segregation of Hip1 and Hip1R functions. Coiled-coil domains of Hip1 and Hip1R differed in their stability and flexibility, correlating with slightly different affinities for clathrin light chain and more markedly with effects of clathrin light chain binding on Hip protein-actin interactions. Clathrin light chain binding induced a compact conformation of both Hip1 and Hip1R and significantly reduced actin binding by their THATCH domains. Thus, clathrin is a negative regulator of Hip-actin interactions. These observations necessarily change models proposed for Hip protein function.

Actin Binding by Hip1 (Huntingtin-interacting Protein 1) and Hip1R (Hip1-related Protein) Is Regulated by Clathrin Light Chain*S⃞

PubMed Central

Wilbur, Jeremy D.; Chen, Chih-Ying; Manalo, Venus; Hwang, Peter K.; Fletterick, Robert J.; Brodsky, Frances M.

2008-01-01

The huntingtin-interacting protein family members (Hip1 and Hip1R in mammals and Sla2p in yeast) link clathrin-mediated membrane traffic to actin cytoskeleton dynamics. Genetic data in yeast have implicated the light chain subunit of clathrin in regulating this link. To test this hypothesis, the biophysical properties of mammalian Hip1 and Hip1R and their interaction with clathrin light chain and actin were analyzed. The coiled-coil domains (clathrin light chain-binding) of Hip1 and Hip1R were found to be stable homodimers with no propensity to heterodimerize in vitro. Homodimers were also predominant in vivo, accounting for cellular segregation of Hip1 and Hip1R functions. Coiled-coil domains of Hip1 and Hip1R differed in their stability and flexibility, correlating with slightly different affinities for clathrin light chain and more markedly with effects of clathrin light chain binding on Hip protein-actin interactions. Clathrin light chain binding induced a compact conformation of both Hip1 and Hip1R and significantly reduced actin binding by their THATCH domains. Thus, clathrin is a negative regulator of Hip-actin interactions. These observations necessarily change models proposed for Hip protein function. PMID:18790740

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

PubMed

Battersby, J E; Snedecor, B; Chen, C; Champion, K M; Riddle, L; Vanderlaan, M

2001-08-24

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

PubMed Central

Klein, Christian; Sustmann, Claudio; Thomas, Markus; Stubenrauch, Kay; Croasdale, Rebecca; Schanzer, Jürgen; Brinkmann, Ulrich; Kettenberger, Hubert; Regula, Jörg T.; Schaefer, Wolfgang

2012-01-01

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress. PMID:22925968

Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

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

Wilbur, Jeremy D., E-mail: jwilbur@msg.ucsf.edu; Hwang, Peter K.; Brodsky, Frances M.

2010-03-01

Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coilmore » domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.« 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

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

PubMed Central

Smirnov, Ivan; Carletti, Eugénie; Kurkova, Inna; Nachon, Florian; Nicolet, Yvain; Mitkevich, Vladimir A.; Débat, Hélène; Avalle, Bérangère; Belogurov, Alexey A.; Kuznetsov, Nikita; Reshetnyak, Andrey; Masson, Patrick; Tonevitsky, Alexander G.; Ponomarenko, Natalia; Makarov, Alexander A.; Friboulet, Alain; Tramontano, Alfonso; Gabibov, Alexander

2011-01-01

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (VL) and variable heavy (VH) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes. PMID:21896761

Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

PubMed

Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

2011-07-01

A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

Adaptation of pelage color and pigment variations in Israeli subterranean blind mole rats, Spalax ehrenbergi [corrected].

PubMed

Singaravelan, Natarajan; Raz, Shmuel; Tzur, Shay; Belifante, Shirli; Pavlicek, Tomas; Beiles, Avigdor; Ito, Shosuke; Wakamatsu, Kazumasa; Nevo, Eviatar

2013-01-01

Concealing coloration in rodents is well established. However, only a few studies examined how soil color, pelage color, hair-melanin content, and genetics (i.e., the causal chain) synergize to configure it. This study investigates the causal chain of dorsal coloration in Israeli subterranean blind mole rats, Spalax ehrenbergi. We examined pelage coloration of 128 adult animals from 11 populations belonging to four species of Spalax ehrenbergi superspecies (Spalax galili, Spalax golani, Spalax carmeli, and Spalax judaei) and the corresponding coloration of soil samples from the collection sites using a digital colorimeter. Additionally, we quantified hair-melanin contents of 67 animals using HPLC and sequenced the MC1R gene in 68 individuals from all four mole rat species. Due to high variability of soil colors, the correlation between soil and pelage color coordinates was weak and significant only between soil hue and pelage lightness. Multiple stepwise forward regression revealed that soil lightness was significantly associated with all pelage color variables. Pelage color lightness among the four species increased with the higher southward aridity in accordance to Gloger's rule (darker in humid habitats and lighter in arid habitats). Darker and lighter pelage colors are associated with darker basalt and terra rossa, and lighter rendzina soils, respectively. Despite soil lightness varying significantly, pelage lightness and eumelanin converged among populations living in similar soil types. Partial sequencing of the MC1R gene identified three allelic variants, two of which were predominant in northern species (S. galili and S. golani), and the third was exclusive to southern species (S. carmeli and S. judaei), which might have caused the differences found in pheomelanin/eumelanin ratio. Darker dorsal pelage in darker basalt and terra rossa soils in the north and lighter pelage in rendzina and loess soils in the south reflect the combined results of crypsis and thermoregulatory function following Gloger's rule.

Crystal structure of a 3B3 variant - A broadly neutralizing HIV-1 scFv antibody

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

Clark, K. Reed; Walsh, Scott T.R.; NCH)

2009-12-10

We present the crystal structure determination of an anti-HIV-1 gp120 single-chain variable fragment antibody variant, 3B3, at 2.5 {angstrom} resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site-directed mutagenesis of the variable heavy chain (V{sub H}) complementary-determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross-clade primary isolates of HIV-1 by interaction with the recessed CD4-binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, theremore » is a reorientation of the CDR-H3 of the V{sub H} domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR-H3 of 3B3, in light of the b12-gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR-L3 of the variable light (VL) domain triggered by a point mutation in CDR-H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the VL and VH domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3-gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the VL and VH domains possibly through more favorable entropic effect through the expulsion of water.« less

A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring 6 Light-Chain Fibrillogenesis

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

Hernández-Santoyo, A.; Del Pozo Yauner, L; Fuentes-Silva, D

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although {lambda} chains, particularly those belonging to the {lambda}6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the {lambda}6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced itsmore » capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the VL (variable region of the light chain)-VL interface. This mutant crystallized in two orthorhombic polymorphs, P2{sub 1}2{sub 1}2{sub 1} and C222{sub 1}. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222{sub 1} lattice showed the establishment of intermolecular {beta}-{beta} interactions that involved the N-terminus and {beta}-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the VL interface in {lambda}6 LCs.« less

Pseudo-Peritoneal Carcinomatosis Presentation of a Crystal-Storing Histiocytosis With an Unmutated Monoclonal κ Light Chain

PubMed Central

Aline-Fardin, Aude; Bender, Sebastien; Fabiani, Bettina; Buob, David; Brahimi, Said; Verpont, Marie Christine; Mothy, Mohamad; Ronco, Pierre; Boffa, Jean Jacques; Aucouturier, Pierre; Garderet, Laurent

2015-01-01

Abstract Crystal-storing histiocytosis (CSH) is a rare complication of monoclonal gammopathies caused by accumulation of crystalline material inside macrophages, and it may result in a variety of clinical manifestations depending on the involved organs. Although immunoglobulin κ light chains (LCs) seem to be the most frequent pathogenic component, very few molecular data are currently available. A 69-year-old man presented with a very poor performance status. Remarkable features were mesenteric lymph node enlargement and proteinuria, including a monoclonal κ LC. Light and electron microscopy studies revealed the presence of crystals within macrophages in the lymph nodes, bone marrow, and kidney, leading to the diagnosis of CSH. The pathogenic κ LC variable domain sequence was identical to the germline Vk3-20∗01/Jk2∗01 gene segments, without any somatic mutation, suggesting an extra-follicular B cell proliferation. The patient was successfully treated with 4 cycles of bortezomib and dexamethasone. After a 12-month follow-up, he remains in hematological and renal remission. CSH may present as pseudo-peritoneal carcinomatosis and relate to a monoclonal κ LC encoded by an unmutated gene. Bortezomib-based therapy proved efficacious in this case. PMID:26266355

Ferritin light-chain subunits: key elements for the electron transfer across the protein cage.

PubMed

Carmona, Unai; Li, Le; Zhang, Lianbing; Knez, Mato

2014-12-18

The first specific functionality of the light-chain (L-chain) subunit of the universal iron storage protein ferritin was identified. The electrons released during iron-oxidation were transported across the ferritin cage specifically through the L-chains and the inverted electron transport through the L-chains also accelerated the demineralization of ferritin.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

PubMed

Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

2015-07-01

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab. © 2015 Wiley Periodicals, Inc.

Classifying Noisy Protein Sequence Data: A Case Study of Immunoglobulin Light Chains

DTIC Science & Technology

2005-01-01

collected from patients with and without amyloidosis , and indicates that the proposed modified classifi- ers are more robust to sequence variability than...piled from patients with and without amyloidosis provides unique features to serve as a model system, not only for conformational disease studies but...produced by patients with amyloidosis . SVMs have been used recently in a wide variety of applica- tions in computational biology (Noble, 2004). Most

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.

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.

Immunoglobulin Light Chains Form an Extensive and Highly Ordered Fibril Involving the N- and C-Termini.

PubMed

Piehl, Dennis W; Blancas-Mejía, Luis M; Wall, Jonathan S; Kennel, Stephen J; Ramirez-Alvarado, Marina; Rienstra, Chad M

2017-02-28

Light-chain (AL)-associated amyloidosis is a systemic disorder involving the formation and deposition of immunoglobulin AL fibrils in various bodily organs. One severe instance of AL disease is exhibited by the patient-derived variable domain (V L ) of the light chain AL-09, a 108 amino acid residue protein containing seven mutations relative to the corresponding germline protein, κI O18/O8 V L . Previous work has demonstrated that the thermodynamic stability of native AL-09 V L is greatly lowered by two of these mutations, Y87H and N34I, whereas a third mutation, K42Q, further increases the kinetics of fibril formation. However, detailed knowledge regarding the residues that are responsible for stabilizing the misfolded fibril structure is lacking. In this study, using solid-state NMR spectroscopy, we show that the majority of the AL-09 V L sequence is immobilized in the fibrils and that the N- and C-terminal portions of the sequence are particularly well-structured. Thus, AL-09 V L forms an extensively ordered and β-strand-rich fibril structure. Furthermore, we demonstrate that the predominant β-sheet secondary structure and rigidity observed for in vitro prepared AL-09 V L fibrils are qualitatively similar to those observed for AL fibrils extracted from postmortem human spleen tissue, suggesting that this conformation may be representative of a common feature of AL fibrils.

Serum-free light-chain assay: clinical utility and limitations.

PubMed

Bhole, Malini V; Sadler, Ross; Ramasamy, Karthik

2014-09-01

In the last decade, the introduction of the serum-free light-chain (sFLC) assay has been an important advance in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The immunoassay was developed to detect free light chains in serum by using anti-FLC antibodies which specifically recognised epitopes on light chains that were 'hidden' in intact immunoglobulins. Since its introduction in 2001, there have been several publications in the English language literature discussing the clinical utility as well as analytical limitations of the sFLC assay. These studies have highlighted both positive and negative aspects of the assay particularly with regard to its sensitivity and specificity and the technical challenges that can affect its performance. The contribution and significance of the sFLC assay in the management of light-chain myeloma, primary amyloid light-chain (AL) amyloidosis and non-secretory myeloma are well recognised and will be addressed in this review. The aim of this article is to also review the published literature with a view to providing a clear understanding of its utility and limitations in the diagnosis, prognosis and monitoring of plasma dyscrasias including intact immunoglobulin multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS). The increasing interest in using this assay in other haematological conditions will also be briefly discussed. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

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.

Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis

PubMed Central

1985-01-01

Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full- length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain- specific expression of V kappa Ser-associated phenotypic markers are discussed. PMID:3926938

[Plasma cell dyscrasias and renal damage].

PubMed

Pasquali, Sonia; Iannuzzella, Francesco; Somenzi, Danio; Mattei, Silvia; Bovino, Achiropita; Corradini, Mattia

2012-01-01

Kidney damage caused by immunoglobulin free light chains in the setting of plasma cell dyscrasias is common and may involve all renal compartments, from the glomerulus to the tubulointerstitium, in a wide variety of histomorphological and clinical patterns. The knowledge of how free light chains can promote kidney injury is growing: they can cause functional changes, be processed and deposited, mediate inflammation, apoptosis and fibrosis, and obstruct nephrons. Each clone of the free light chain is unique and its primary structure and post-translation modification can determine the type of renal disease. Measurement of serum free light chain concentrations and calculation of the serum kappa/lambda ratio, together with renal biopsy, represent essential diagnostic tools. An early and correct diagnosis of renal lesions due to plasma cell dyscrasias will allow early initiation of disease-specific treatment strategies. The treatment of free light chain nephropathies is evolving and knowledge of the pathways that promote renal damage should lead to further therapeutic developments.

Light Chain Cast Nephropathy: Practical Considerations in the Management of Myeloma Kidney-What We Know and What the Future May Hold.

PubMed

Manohar, Sandhya; Nasr, Samih H; Leung, Nelson

2018-05-03

To update and evaluate the current knowledge on pathogenesis and management of light chain cast nephropathy. Light chain cast nephropathy (LCCN) is the leading cause of acute renal failure in patients with multiple myeloma and is currently recognized as a myeloma defining event. The immunoglobulin free light chain plays an integral role in the pathogenesis of LCCN. The level of free light chain (FLC) in the blood and urine is directly associated with the risk of developing LCCN. Recovery of renal function is related to the speed and degree of the serum FLC reduction. Recently, two randomized trials using high cutoff dialyzer for the removal of serum FLC produced different results in terms of renal recovery. FLC plays a key role in the development and resolution of LCCN. Future therapies will aim to rapidly reduce its concentration or interrupt its interaction with Tamm-Horsfall protein.

Systematic Characterization and Comparative Analysis of the Rabbit Immunoglobulin Repertoire

PubMed Central

Lavinder, Jason J.; Hoi, Kam Hon; Reddy, Sai T.; Wine, Yariv; Georgiou, George

2014-01-01

Rabbits have been used extensively as a model system for the elucidation of the mechanism of immunoglobulin diversification and for the production of antibodies. We employed Next Generation Sequencing to analyze Ig germline V and J gene usage, CDR3 length and amino acid composition, and gene conversion frequencies within the functional (transcribed) IgG repertoire of the New Zealand white rabbit (Oryctolagus cuniculus). Several previously unannotated rabbit heavy chain variable (VH) and light chain variable (VL) germline elements were deduced bioinformatically using multidimensional scaling and k-means clustering methods. We estimated the gene conversion frequency in the rabbit at 23% of IgG sequences with a mean gene conversion tract length of 59±36 bp. Sequencing and gene conversion analysis of the chicken, human, and mouse repertoires revealed that gene conversion occurs much more extensively in the chicken (frequency 70%, tract length 79±57 bp), was observed to a small, yet statistically significant extent in humans, but was virtually absent in mice. PMID:24978027

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.

Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries.

PubMed

Tsurushita, N; Fu, H; Warren, C

1996-06-12

New phage display vectors for in vivo recombination of immunoglobulin (Ig) heavy (VH) and light (VL) chain variable genes, to make single-chain Fv fragments (scFv), were constructed. The VH and VL genes of monoclonal antibody (mAb) EP-5C7, which binds to both human E- and P-selectin, were cloned into a pUC19-derived plasmid vector, pCW93, and a pACYC184-derived phagemid vector, pCW99, respectively. Upon induction of Cre recombinase (phage P1 recombinase), the VH and VL genes were efficiently recombined into the same plasmid via the two loxP sites (phage P1 recombination sites), one located downstream from a VH gene in pCW93 and another upstream from a VL gene in pCW99. In the resulting phagemid, the loxP sequence also encodes a polypeptide linker connecting the VH and VL domains to form a scFv of EP-5C7. Whether expressed on the phage surface or as a soluble form, the EP-5C7 scFv showed specific binding to human E- and P-selectin. This phagemid vector system provides a way to recombine VH and VL gene libraries efficiently in vivo to make extremely large Ig combinatorial libraries.

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.

Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system.

PubMed

Sun, Dongbo; Shi, Hongyan; Chen, Jianfei; Shi, Da; Zhu, Qinghe; Zhang, Hong; Liu, Shengwang; Wang, Yunfeng; Qiu, Huaji; Feng, Li

2012-06-01

Porcine aminopeptidase N (pAPN) is a common cellular receptor for swine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). To investigate single-chain fragment variable (scFv) repertoire against pAPN, the genes encoding the immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) were amplified by reverse transcript polymerase chain reaction (RT-PCR) using a series of degenerate primers from the spleen of BABL/c mice immunized with native pAPN. The VL and VH amplicons were combined randomly by a 12 amino acid flexible linker by splicing by overlap extension PCR (SOE-PCR), which produced the scFv gene repertoire. After ligation of the scFv gene repertoire into the T7Select10-3b vector, a mouse scFv phage library specific for pAPN was produced through in vitro packaging. The primary scFv library against pAPN contained 2.0×10(7) recombinant phage clones, and the titer of the amplified library was 3.6×10(9)pfu/mL. BstNI restriction analysis and DNA sequencing revealed that 28 phage clones from the primary pAPN scFv library showed excellent diversity. The effectiveness of the scFv library against pAPN was verified further by phage ELISA using the recombinant protein of the pAPN C subunit as coating antigen. The construction and evaluation of a murine scFv library against the common receptor pAPN of porcine coronaviruses TGEV and PEDV using the T7 phage display system are described. Copyright © 2012 Elsevier B.V. All rights reserved.

Interactions of monochromatic visible light and near-IR radiation with cells: currently discussed mechanisms

NASA Astrophysics Data System (ADS)

Karu, Tiina I.

1995-05-01

Biological responses of cells to visible and near IR (laser) radiation occur due to physical and/or chemical changes in photoacceptor molecules, components of respiratory chains (cyt a/a3 in mitochondria, and cyt d in E. coli). As a result of the photoexcitation of electronic states, the following physical and/or chemical changes can occur: alteration of redox properties and acceleration of electron transfer, changes in biochemical activity due to local transient heating of chromophores, one-electron auto-oxidation and O2- production, and photodynamic action and 1O2 production. Different reaction channels can be activated to achieve the photobiological macroeffect. The primary physical and/or chemical changes induced by light in photoacceptor molecules are followed by a cascade of biochemical reactions in the cell that do not need further light activation and occur in the dark (photosignal transduction and amplification chains). These reactions are connected with changes in cellular homeostasis parameters. The crucial step here is thought to be an alteration of the cellular redox state: a shift towards oxidation is associated with stimulation of cellular vitality, and a shift towards reduction is linked to inhibition. Cells with a lower than normal pH, where the redox state is shifted in the reduced direction, are considered to be more sensitive to the stimulative action of light than those with the respective parameters being optimal or near optimal. This circumstance explains the possible variations in observed magnitudes of low-power laser effects. Light action on the redox state of a cell via the respiratory chain also explains the diversity of low-power laser effects. Beside explaining many controversies in the field of low-power laser effects (i.e., the diversity of effects, the variable magnitude or absence of effects in certain studies), the proposed redox-regulation mechanism may be a fundamental explanation of some clinical effects of irradiation, for example the positive results achieved in treating wounds, chronic inflammation, and ischemia, all characterized by acidosis and hypoxia.

Mechanisms of interaction of monochromatic visible light with cells

NASA Astrophysics Data System (ADS)

Karu, Tiina I.

1996-01-01

Biological responses of cells to visible and near IR (laser) radiation occur due to physical and/or chemical changes in photoacceptor molecules, components of respiratory chains (cyt a/a3 in mitochondria). As a result of the photoexcitation of electronic states, the following physical and/or chemical changes can occur: alteration of redox properties and acceleration of electron transfer, changes in biochemical activity due to local transient heating of chromophores, one-electron auto-oxidation and O'2- production, and photodynamic action and 1O2 production. Different reaction channels can be activated to achieve the photobiological macroeffect. The primary physical and/or chemical changes induced by light in photoacceptor molecules are followed by a cascade of biochemical reactions in the cell that do not need further light activation and occur in the dark (photosignal transduction and amplification chains). These reactions are connected with changes in cellular homeostasis parameters. The crucial step here is thought to be an alteration of the cellular redox state: a shift towards oxidation is associated with stimulation of cellular vitality, and a shift towards reduction is linked to inhibition. Cells with a lower than normal pH, where the redox state is shifted in the reduced direction, are considered to be more sensitive to the stimulative action of light than those with the respective parameters being optimal or near optimal. This circumstance explains the possible variations in observed magnitudes of low- power laser effects. Light action on the redox state of a cell via the respiratory chain also explains the diversity of low-power laser effects. Besides explaining many controversies in the field of low-power laser effects (i.e., the diversity of effects, the variable magnitude or absence of effects in certain studies), the proposed redox-regulation mechanism may be a fundamental explanation for some clinical effects of irradiation, for example the positive results achieved in treating wounds, chronic inflammation, and ischemia, all characterized by acidosis and hypoxia.

An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

PubMed

Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

2017-10-01

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

Light chain typing of immunoglobulins in small samples of biological material

PubMed Central

Rádl, J.

1970-01-01

A method is described for the typing of the light chains of immunoglobulins in small samples of sera or external secretions and without their previous isolation. It consists of immunoelectrophoresis in agar plates which contain specific antisera against one of the light chain types. All immunoglobulins of this type are thus selected by precipitation in the central area during the electrophoretic phase. Immunoglobulins of the opposite light chain type diffuse through the agar and react with the class specific antisera from the troughs. This results in the precipitin lines as in conventional immunoelectrophoresis. This technique has proved most useful for typing heterogenous or homogeneous immunoglobulins in normal and low concentration. The antisera used for incorporation in the agar should fulfil special requirements. They should contain a high level of antibodies against common surface determinants of the immunoglobulin light chains. The further possibilities of this immunoselection technique for typing different protein mixtures is discussed. ImagesFIG. 1FIG. 2FIG. 3FIG. 4FIG. 5FIG. 6 PMID:4098592

Serum-free light-chain analysis in diagnosis and management of multiple myeloma and related conditions.

PubMed

Milani, Paolo; Palladini, Giovanni; Merlini, Giampaolo

2016-01-01

The introduction of the serum-free light-chain (S-FLC) assay has been a breakthrough in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The first method, proposed in 2001, quantifies serum-free light-chains using polyclonal antibodies. More recently, assays based on monoclonal antibodies have entered into clinical practice. S-FLC measurement plays a central role in the screening for multiple myeloma and related conditions, in association with electrophoretic techniques. Analysis of S-FLC is essential in assessing the risk of progression of precursor diseases to overt plasma cell dyscrasias. It is also useful for risk stratification in solitary plasmacytoma and AL amyloidosis. The S-FLC measurement is part of the new diagnostic criteria for multiple myeloma, and provides a marker to follow changes in clonal substructure over time. Finally, the evaluation of S-FLC is fundamental for assessing the response to treatment in monoclonal light chain diseases.

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction*

PubMed Central

Zhang, Wen-Cheng; Peng, Ya-Jing; Zhang, Gen-Sheng; He, Wei-Qi; Qiao, Yan-Ning; Dong, Ying-Ying; Gao, Yun-Qian; Chen, Chen; Zhang, Cheng-Hai; Li, Wen; Shen, Hua-Hao; Ning, Wen; Kamm, Kristine E.; Stull, James T.; Gao, Xiang; Zhu, Min-Sheng

2010-01-01

Different interacting signaling modules involving Ca2+/calmodulin-dependent myosin light chain kinase, Ca2+-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K+-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance. PMID:20018858

Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-accessible surface area for 121 clinical stage monoclonal antibodies.

PubMed

Yang, Rong; Jain, Tushar; Lynaugh, Heather; Nobrega, R Paul; Lu, Xiaojun; Boland, Todd; Burnina, Irina; Sun, Tingwan; Caffry, Isabelle; Brown, Michael; Zhi, Xiaoyong; Lilov, Asparouh; Xu, Yingda

Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% H 2 O 2 for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.

Multiple Myeloma and Its Precursor Disease Among Firefighters Exposed to the World Trade Center Disaster.

PubMed

Landgren, Ola; Zeig-Owens, Rachel; Giricz, Orsolya; Goldfarb, David; Murata, Kaznouri; Thoren, Katie; Ramanathan, Lakshmi; Hultcrantz, Malin; Dogan, Ahmet; Nwankwo, George; Steidl, Ulrich; Pradhan, Kith; Hall, Charles B; Cohen, Hillel W; Jaber, Nadia; Schwartz, Theresa; Crowley, Laura; Crane, Michael; Irby, Shani; Webber, Mayris P; Verma, Amit; Prezant, David J

2018-06-01

The World Trade Center (WTC) attacks on September 11, 2001, created an unprecedented environmental exposure to known and suspected carcinogens suggested to increase the risk of multiple myeloma. Multiple myeloma is consistently preceded by the precursor states of monoclonal gammopathy of undetermined significance (MGUS) and light-chain MGUS, detectable in peripheral blood. To characterize WTC-exposed firefighters with a diagnosis of multiple myeloma and to conduct a screening study for MGUS and light-chain MGUS. Case series of multiple myeloma in firefighters diagnosed between September 11, 2001, and July 1, 2017, together with a seroprevalence study of MGUS in serum samples collected from Fire Department of the City of New York (FDNY) firefighters between December 2013 and October 2015. Participants included all WTC-exposed FDNY white, male firefighters with a confirmed physician diagnosis of multiple myeloma (n = 16) and WTC-exposed FDNY white male firefighters older than 50 years with available serum samples (n = 781). WTC exposure defined as rescue and/or recovery work at the WTC site between September 11, 2001, and July 25, 2002. Multiple myeloma case information, and age-adjusted and age-specific prevalence rates for overall MGUS (ie, MGUS and light-chain MGUS), MGUS, and light-chain MGUS. Sixteen WTC-exposed white male firefighters received a diagnosis of multiple myeloma after September 11, 2001; median age at diagnosis was 57 years (interquartile range, 50-68 years). Serum/urine monoclonal protein isotype/free light-chain data were available for 14 cases; 7 (50%) had light-chain multiple myeloma. In a subset of 7 patients, myeloma cells were assessed for CD20 expression; 5 (71%) were CD20 positive. In the screening study, we assayed peripheral blood from 781 WTC-exposed firefighters. The age-standardized prevalence rate of MGUS and light-chain MGUS combined was 7.63 per 100 persons (95% CI, 5.45-9.81), 1.8-fold higher than rates from the Olmsted County, Minnesota, white male reference population (relative rate, 1.76; 95% CI, 1.34-2.29). The age-standardized prevalence rate of light-chain MGUS was more than 3-fold higher than in the same reference population (relative rate, 3.13; 95% CI, 1.99-4.93). Environmental exposure to the WTC disaster site is associated with myeloma precursor disease (MGUS and light-chain MGUS) and may be a risk factor for the development of multiple myeloma at an earlier age, particularly the light-chain subtype.

Aging and the Immune Response to the Haemophilus influenzae Type b Capsular Polysaccharide: Retention of the Dominant Idiotype and Antibody Function in the Elderly

PubMed Central

Lucas, Alexander H.; Reason, Donald C.

1998-01-01

Anti-Haemophilus influenzae b polysaccharide (Hib PS) antibodies elicited in elderly subjects following conjugate vaccination expressed a light-chain variable-region (VL)-associated idiotype and had functional activities similar to those previously observed in children and younger adults. These findings indicate that advanced age is not accompanied by shifts in the major VL component of the Hib PS-specific repertoire or by diminution of the protective function of antibodies. PMID:9529108

The Clinical Presentation and Management of Systemic Light-Chain Amyloidosis in China.

PubMed

Huang, Xiang-Hua; Liu, Zhi-Hong

2016-04-01

Amyloidosis includes a group of diseases characterized by the extracellular deposition of various fibrillary proteins that can autoaggregate in a highly abnormal fibrillary conformation. The amyloid precursor protein of systemic light-chain (AL) amyloidosis is comprised of monoclonal light chains that are due to plasma cell dyscrasia. The clinical presentation of patients with AL amyloidosis varies from patient to patient. Current treatment strategies target the clone in order to decrease the production of the pathologic light chains. Recent advances in therapy have helped many patients with AL amyloidosis achieve hematologic and organ responses. AL amyloidosis is the most common type of systemic amyloidosis in China with increasing morbidity and a high mortality rate. The clinical presentation of AL amyloidosis is variable, and the median overall survival was found to be 36.3 months. The disease prognosis and risk stratification are linked to serialized measurement of cardiac biomarkers and free light chains. The treatment of AL amyloidosis is mainly based on chemotherapy and autologous hematopoietic stem cell transplantation (ASCT). The use of novel agents (thalidomide, lenalidomide, and bortezomib) alone and in combination with steroids and alkylating agents has shown efficacy and continues to be explored. AL amyloidosis is the most common type of systemic amyloidosis in China with increasing morbidity and a high mortality rate. The lack of prospective clinical trials using the current therapies is a challenge for evidence-based decision making concerning the treatment of AL amyloidosis. (1) AL amyloidosis is the most prevalent type of amyloidosis accounting for 65% of the amyloidosis-diagnosed patients in the UK and for 93% of the amyloidosis-diagnosed patients in China. The predisposition of men over women to develop AL amyloidosis might be higher in China than in Western countries (2:1 vs. 1.3:1). Both in the East and West, incidence increases with age. At the time of diagnosis, edema is twice as frequent and the proportion of renal involvement is higher in Chinese compared to Western patients. (2) Melphalan followed by ASCT is the current standard therapy but is restricted to eligible patients. The efficacy and safety of bortezomib combined with dexamethasone were proven in Western patients and recently confirmed in a Chinese cohort. Recent studies in China and the US indicate that bortezomib induction prior to ASCT increases the response rate. Thalidomide and lenalidomide have shown benefit, but toxicity and lack of clinical evidence exclude these agents from first-line therapy. The green tea extract epigallocatechin-3-gallate is under investigation as an inhibitor of AL amyloid formation and a compound that might dissolve amyloid.

Characterizing Chain Processes in Visible Light Photoredox Catalysis

PubMed Central

Cismesia, Megan A.

2015-01-01

The recognition that Ru(bpy)32+ andsimilar visible light absorbing transition metal complexes can be photocatalysts for a variety of synthetically useful organic reactions has resulted in a recent resurgence of interest in photoredox catalysis. However, many of the critical mechanistic aspects of this class of reactions remain poorly understood. In particular, the degree to which visible light photoredox reactions involve radical chain processes has been a point of some disagreement that has not been subjected to systematic analysis. We have now performed quantum yield measurements to demonstrate that threerepresentative, mechanistically distinct photoredox processes involve product-forming chain reactions. Moreover, we show that the combination of quantum yield and luminescence quenching experiments provides a rapid method to estimate the length of these chains. Together, these measurements constitute a robust, operationally facile strategy for characterizing chain processes in a wide range of visible light photoredox reactions. PMID:26668708

Therapeutic Approaches for Botulinum Intoxication Targeting Degradation of the Light Chain

DTIC Science & Technology

2013-04-01

SUBJECT TERMS Botulinum toxin , ubiquitin, chimeric toxin light chains, LcA, LcE, Yeast 2 hybrid, intracellular therapy. 16. SECURITY...Synaptic Research will develop dichain hybrids consisting of Clostridium botulinum toxin light chains (LCs) from serotypes A (long-lived) and E...stability to LCs of botulinum toxin can be assessed by mutation of dileucine residues and systematic deletion of residues from LcA-LcE chimeras to provide a

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

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

Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.

2011-12-16

Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chainmore » kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.« less

High-frequency expression of a conserved kappa light-chain variable-region gene in chronic lymphocytic leukemia

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

Kipps, T.J.; Fong, S.; Tomhave, E.

Malignant B lymphocytes from several patients with chronic lymphocytic leukemia (CLL) were examined for reactivity with murine monoclonal antibody 17.109. This antibody, prepared against the rheumatoid factor (RF) paraprotein Sie, recognizes a cross reactive idiotype on 48% of human IgM RF paraproteins, but does not react with IgM paraproteins without RF activity or substantially with normal pooled immunoglobulin. The 17.109-reactive idiotype is a marker for a kappa III variable-region gene, designated V/sub kappa/RF, that is conserved in outbred human populations. In a limited study of 31 CLL patients, the leukemic cells from 5 of 20 patients with kappa light chain-expressingmore » CLL were recognized by the 17.109 monoclonal antibody. Despite having malignant cells specifically reactive with this antibody, patients with 17.109-positive CLL did not have elevated serum levels of circulating antibody bearing 17.109-reactive determinants. Total RNAs isolated from the CLL B lymphocytes, or from hybridomas produced by fusing the CLL cells with the WI-L2-729-HF/sub 2/ cell line, were fractionated electrophoretically and examined by blot hybridization. Under stringent hybridization conditions capable of discerning a single base-pair mismatch, RNA from the 17.109-idiotype-positive CLL cells hybridized to synthetic oligonucleotide probes corresponding to framework and complementary-determining regions in the V/sub kappa/RF gene. The high frequency of the 17.109-associated idiotype and the V/sub kappa/RF gene in CLL suggests that the disease may arise from B lymphocytes that express a restricted set of inherited immunoglobulin variable-region genes with little or no somatic mutation.« less

Neurofilament light chain and oligoclonal bands are prognostic biomarkers in radiologically isolated syndrome.

PubMed

Matute-Blanch, Clara; Villar, Luisa M; Álvarez-Cermeño, José C; Rejdak, Konrad; Evdoshenko, Evgeniy; Makshakov, Gleb; Nazarov, Vladimir; Lapin, Sergey; Midaglia, Luciana; Vidal-Jordana, Angela; Drulovic, Jelena; García-Merino, Antonio; Sánchez-López, Antonio J; Havrdova, Eva; Saiz, Albert; Llufriu, Sara; Alvarez-Lafuente, Roberto; Schroeder, Ina; Zettl, Uwe K; Galimberti, Daniela; Ramió-Torrentà, Lluís; Robles, René; Quintana, Ester; Hegen, Harald; Deisenhammer, Florian; Río, Jordi; Tintoré, Mar; Sánchez, Alex; Montalban, Xavier; Comabella, Manuel

2018-04-01

The prognostic role of cerebrospinal fluid molecular biomarkers determined in early pathogenic stages of multiple sclerosis has yet to be defined. In the present study, we aimed to investigate the prognostic value of chitinase 3 like 1 (CHI3L1), neurofilament light chain, and oligoclonal bands for conversion to clinically isolated syndrome and to multiple sclerosis in 75 patients with radiologically isolated syndrome. Cerebrospinal fluid levels of CHI3L1 and neurofilament light chain were measured by enzyme-linked immunosorbent assay. Uni- and multivariable Cox regression models including as covariates age at diagnosis of radiologically isolated syndrome, number of brain lesions, sex and treatment were used to investigate associations between cerebrospinal fluid CHI3L1 and neurofilament light chain levels and time to conversion to clinically isolated syndrome and multiple sclerosis. Neurofilament light chain levels and oligoclonal bands were independent risk factors for the development of clinically isolated syndrome (hazard ratio = 1.02, P = 0.019, and hazard ratio = 14.7, P = 0.012, respectively) and multiple sclerosis (hazard ratio = 1.03, P = 0.003, and hazard ratio = 8.9, P = 0.046, respectively). The best cut-off to classify cerebrospinal fluid neurofilament light chain levels into high and low was 619 ng/l, and high neurofilament light chain levels were associated with a trend to shorter time to clinically isolated syndrome (P = 0.079) and significant shorter time to multiple sclerosis (P = 0.017). Similarly, patients with radiologically isolated syndrome presenting positive oligoclonal bands converted faster to clinically isolated syndrome and multiple sclerosis (P = 0.005 and P = 0.008, respectively). The effects of high neurofilament light chain levels shortening time to clinically isolated syndrome and multiple sclerosis were more pronounced in radiologically isolated syndrome patients with ≥37 years compared to younger patients. Cerebrospinal fluid CHI3L1 levels did not influence conversion to clinically isolated syndrome and multiple sclerosis in radiologically isolated syndrome patients. Overall, these findings suggest that cerebrospinal neurofilament light chain levels and oligoclonal bands are independent predictors of clinical conversion in patients with radiologically isolated syndrome. The association with a faster development of multiple sclerosis reinforces the importance of cerebrospinal fluid analysis in patients with radiologically isolated syndrome.

Microscopic theory of light-induced deformation in amorphous side-chain azobenzene polymers.

PubMed

Toshchevikov, V; Saphiannikova, M; Heinrich, G

2009-04-16

We propose a microscopic theory of light-induced deformation of side-chain azobenzene polymers taking into account the internal structure of polymer chains. Our theory is based on the fact that interaction of chromophores with the polarized light leads to the orientation anisotropy of azobenzene macromolecules which is accompanied by the appearance of mechanical stress. It is the first microscopic theory which provides the value of the light-induced stress larger than the yield stress. This result explains a possibility for the inscription of surface relief gratings in glassy side-chain azobenzene polymers. For some chemical architectures, elongation of a sample demonstrates a nonmonotonic behavior with the light intensity and can change its sign (a stretched sample starts to be uniaxially compressed), in agreement with experiments. Using a viscoplastic approach, we show that the irreversible strain of a sample, which remains after the light is switched off, decreases with increasing temperature and can disappear at certain temperature below the glass transition temperature. This theoretical prediction is also confirmed by recent experiments.

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility

PubMed Central

Green, Judith L.; Wall, Richard J.; Vahokoski, Juha; Yusuf, Noor A.; Ridzuan, Mohd A. Mohd; Stanway, Rebecca R.; Stock, Jessica; Knuepfer, Ellen; Brady, Declan; Martin, Stephen R.; Howell, Steven A.; Pires, Isa P.; Moon, Robert W.; Molloy, Justin E.; Kursula, Inari; Tewari, Rita

2017-01-01

Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain–interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion. PMID:28893907

Diagnostic reference range of κ/λ free light chain ratio to screen for Bence Jones proteinuria is not significantly influenced by GFR.

PubMed

Schmidt-Hieltjes, Yvonne; Elshof, Clemens; Roovers, Lian; Ruinemans-Koerts, Janneke

2016-05-01

The aim of our study was to analyse whether the κ/λ free light chain ratio reference range for screening for Bence Jones proteinuria should be dependent on the estimated glomerular filtration rate (eGFR). The serum κ/λ free light chain ratio, eGFR, serum M-protein and Bence Jones protein were measured in 544 patients for whom Bence Jones protein analysis was ordered. In the population of patients without Bence Jones proteinuria or a M-protein (n = 402), there is no gradual increase in κ/λ free light chain ratio with diminishing eGFR. The κ/λ free light chain ratio in this group was 0.56-1.86 (95% interval). With this diagnostic reference range of the κ/λ ratio, 105 of the 110 patients with Bence Jones protein could be identified correctly. Only five patients with Bence Jones proteinuria (<0.17 g/L) were missed, without diagnostic or therapeutic consequences. In 36 patients (6.6%), an abnormal κ/λ free light chain ratio was measured without the presence of Bence Jones proteinuria. A κ/λ free light chain ratio in serum can be used safely and efficiently to select urine samples which should be analysed for Bence Jones proteinuria with an electrophoresis/immunofixation technique. Using this diagnostic reference range, the number of urine samples which should be analysed by electrophoresis/immunofixation could be reduced by 74%. The diagnostic reference interval can be determined best in a group of patients for whom Bence Jones analysis is indicated. For calculation of this reference range, the eGFR value does not need to be taken into account. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Surface IgM λ light chain is involved in the binding and infection of infectious bursal disease virus (IBDV) to DT40 cells.

PubMed

Chi, Jiaqi; You, Leiming; Li, Peipei; Teng, Man; Zhang, Gaiping; Luo, Jun; Wang, Aiping

2018-04-01

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus in chickens. Surface immunoglobulin M (sIgM)-bearing B lymphocytes act as the major targets of IBDV in the bursa of Fabricius, and sIgM may function as one of the membrane binding sites responsible for IBDV infection. Recently, using the virus overlay protein binding assay, the chicken λ light chain of sIgM was identified to specifically interact with IBDV in a virulence-independent manner in vitro. To further investigate sIgM λ light chain-mediated IBDV binding and infection in pre-B cells, the cell line DT40, which is susceptible to both pathogenic and attenuated IBDV, was used. Based on the RNA interference strategy, the DT40 cell line whose λ light chain of sIgM was stably knocked down, herein termed DT40LKD, was generated by the genomic integration of a specific small hairpin RNA and a green fluorescence protein co-expression construct. Flow cytometry analysis indicated that the binding of IBDV to DT40LKD cells was significantly reduced due to the loss of sIgM λ light chain. In particular, reduced viral replication was observed in IBDV-incubated DT40LKD cells, and no viral release into cell culture medium was detected by the IBDV rapid diagnostic strips. In addition, the rescue of sIgM λ light chain expression restored viral binding and replication in DT40LKD cells. These results show that sIgM λ light chain appears to be beneficial for IBDV attachment and infection, suggesting that sIgM acts as a binding site involved in IBDV infection.

Primary CNS Nonamyloidogenic Light Chain Deposition Disease: Case Report and Brief Review.

PubMed

Mercado, Juan Jose; Markert, James M; Meador, William; Chapman, Philip; Perry, Arie; Hackney, James R

2017-12-01

The true incidence of light chain deposition disease (LCDD) restricted to the central nervous system (CNS) is unknown. To our knowledge only 7 cases of LCDD restricted to the brain have been previously reported. We herein describe an unusual example. A 44-year-old man presented with a history of ischemic retinopathy in 2004 and left lower extremity hypoesthesia in 2007 that progressed gradually to left-sided weakness and numbness in the 2 years prior to his hospitalization in 2015. A stereotactic brain biopsy was performed, displaying nonspecific hyaline deposits of amorphous "amyloid-like" material involving deep brain white matter and vessels. These were Congo red negative and were accompanied by a sparse lymphoplasmacytic infiltrate. Plasma cells demonstrated kappa light chain class restriction by chromogenic in situ hybridization (CISH). There was patchy reactivity with kappa immunohistochemistry in the amorphous deposits. A diagnosis of light chain deposition disease was made. Subsequent systemic myeloma and lymphoma workups were negative. Previously reported cases have included men and women, spanning the ages of 19 and 72 years, often presenting with hemiparesis, hypoesthesia, or seizures. Deposits have been reported in the cerebrum and cerebellum. T2/FLAIR (fluid attenuation inversion recovery) changes are usual, but lesions may or may not produce contrast enhancement. The light chain deposition may be of kappa or lambda class. Most lesions have been accompanied by local lymphoid and/or plasma cell infiltrates exhibiting light chain restriction of the same class as the deposits. In summary, LCDD limited to the CNS is a rare lesion consisting of deposition of amyloid-like, but Congo red-negative monotypic light chain usually produced by local lymphoplasmacytic infiltrates.

Pituitary adenylate cyclase-activating polypeptide is a potent inhibitor of the growth of light chain-secreting human multiple myeloma cells.

PubMed

Li, Min; Cortez, Shirley; Nakamachi, Tomoya; Batuman, Vecihi; Arimura, Akira

2006-09-01

Multiple myeloma represents a malignant proliferation of plasma cells in the bone marrow, which often overproduces immunoglobulin light chains. We have shown previously that pituitary adenylate cyclase-activating polypeptide (PACAP) markedly suppresses the release of proinflammatory cytokines from light chain-stimulated human renal proximal tubule epithelial cells and prevents the resulting tubule cell injury. In this study, we have shown that PACAP suppresses the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. The addition of PACAP suppressed light chain-producing myeloma cell-stimulated interleukin 6 (IL-6) secretion by the bone marrow stromal cells (BMSCs). A specific antagonist to either the human PACAP-specific receptor or the vasoactive intestinal peptide receptor attenuated the suppressive effect of PACAP on IL-6 production in the adhesion of human multiple myeloma cells to BMSCs. The secretion of IL-6 by BMSCs was completely inhibited by 10(-9) mol/L PACAP, which also attenuated the phosphorylation of both p42/44 and p38 mitogen-activated protein kinases (MAPK) as well as nuclear factor-kappaB (NF-kappaB) activation in response to the adhesion of multiple myeloma cells to BMSCs, whereas the inhibition of p42/44 MAPK signaling attenuated PACAP action. The signaling cascades involved in the inhibitory effect of PACAP on IL-6-mediated paracrine stimulation of light chain-secreting myeloma cell growth was mediated through the suppression of p38 MAPK as well as modulation of activation of transcription factor NF-kappaB. These findings suggest that PACAP may be a new antitumor agent that directly suppresses light chain-secreting myeloma cell growth and indirectly affects tumor cell growth by modifying the bone marrow milieu of the multiple myeloma.

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.

PubMed

Green, Judith L; Wall, Richard J; Vahokoski, Juha; Yusuf, Noor A; Ridzuan, Mohd A Mohd; Stanway, Rebecca R; Stock, Jessica; Knuepfer, Ellen; Brady, Declan; Martin, Stephen R; Howell, Steven A; Pires, Isa P; Moon, Robert W; Molloy, Justin E; Kursula, Inari; Tewari, Rita; Holder, Anthony A

2017-10-27

Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of Variable Resistance Using Chains on Bench Throw Performance in Trained Rugby Players.

PubMed

Godwin, Mark S; Fernandes, John F T; Twist, Craig

2018-04-01

Godwin, MS, Fernandes, JFT, and Twist, C. Effects of variable resistance using chains on bench throw performance in trained rugby players. J Strength Cond Res 32(4): 950-954, 2018-This study sought to determine the effects of variable resistance using chain resistance on bench throw performance. Eight male rugby union players (19.4 ± 2.3 years, 88.8 ± 6.0 kg, 1RM 105.6 ± 17.0 kg) were recruited from a national league team. In a randomized crossover design, participant's performed 3 bench throws at 45% one repetition maximum (1RM) at a constant load (no chains) or a variable load (30% 1RM constant load and 15% 1RM variable load; chains) with 7 days between conditions. For each repetition, the peak and mean velocity, peak power, peak acceleration, and time to peak velocity were recorded. Differences in peak and mean power were very likely trivial and unclear between the chain and no chain conditions, respectively. Possibly greater peak and likely greater mean bar velocity were accompanied by likely to most likely greater bar velocity between 50 and 400 ms from initiation of bench press in the chain condition compared with the no chain condition. Accordingly, bar acceleration was very likely greater in the chain condition compared with the no chain condition. In conclusion, these results show that the inclusion of chain resistance can acutely enhance several variables in the bench press throw and gives support to this type of training.

Treatment of Acute Renal Failure Secondary to Multiple Myeloma with Chemotherapy and Extended High Cut-Off Hemodialysis

PubMed Central

Hutchison, Colin A.; Bradwell, Arthur R.; Cook, Mark; Basnayake, Kolitha; Basu, Supratik; Harding, Stephen; Hattersley, John; Evans, Neil D.; Chappel, Mike J.; Sampson, Paul; Foggensteiner, Lukas; Adu, Dwomoa; Cockwell, Paul

2009-01-01

Background and objectives: Extended hemodialysis using a high cut-off dialyzer (HCO-HD) removes large quantities of free light chains in patients with multiple myeloma. However, the clinical utility of this method is uncertain. This study assessed the combination of chemotherapy and HCO-HD on serum free light chain concentrations and renal recovery in patients with myeloma kidney (cast nephropathy) and dialysis-dependent acute renal failure. Design, setting, participants, & measurements: An open-label study of the relationship between free light chain levels and clinical outcomes in 19 patients treated with standard chemotherapy regimens and HCO-HD. Results: There were sustained early reductions in serum free light chain concentrations (median 85% [range 50 to 97]) in 13 patients. These 13 patients became dialysis independent at a median of 27 d (range 13 to 120). Six patients had chemotherapy interrupted because of early infections and did not achieve sustained early free light chain reductions; one of these patients recovered renal function (at 105 d) the remaining 5 patients did not recover renal function. Patients who recovered renal function had a significantly improved survival (P < 0.012). Conclusion: In dialysis-dependent acute renal failure secondary to myeloma kidney, patients who received uninterrupted chemotherapy and extended HCO-HD had sustained reductions in serum free light chain concentrations and recovered independent renal function. PMID:19339414

Immunoglobulin light chain allelic inclusion in systemic lupus erythematosus

PubMed Central

Fraser, Louise D.; Zhao, Yuan; Lutalo, Pamela M. K.; D'Cruz, David P.; Cason, John; Silva, Joselli S.; Dunn‐Walters, Deborah K.; Nayar, Saba; Cope, Andrew P.

2015-01-01

The principles of allelic exclusion state that each B cell expresses a single light and heavy chain pair. Here, we show that B cells with both kappa and lambda light chains (Igκ and Igλ) are enriched in some patients with the systemic autoimmune disease systemic lupus erythematosus (SLE), but not in the systemic autoimmune disease control granulomatosis with polyangiitis. Detection of dual Igκ and Igλ expression by flow cytometry could not be abolished by acid washing or by DNAse treatment to remove any bound polyclonal antibody or complexes, and was retained after two days in culture. Both surface and intracytoplasmic dual light chain expression was evident by flow cytometry and confocal microscopy. We observed reduced frequency of rearrangements of the kappa‐deleting element (KDE) in SLE and an inverse correlation between the frequency of KDE rearrangement and the frequency of dual light chain expressing B cells. We propose that dual expression of Igκ and Igλ by a single B cell may occur in some patients with SLE when this may be a consequence of reduced activity of the KDE. PMID:26036683

Myosin conformational states determined by single fluorophore polarization

PubMed Central

Warshaw, David M.; Hayes, Eric; Gaffney, Donald; Lauzon, Anne-Marie; Wu, Junru; Kennedy, Guy; Trybus, Kathleen; Lowey, Susan; Berger, Christopher

1998-01-01

Muscle contraction is powered by the interaction of the molecular motor myosin with actin. With new techniques for single molecule manipulation and fluorescence detection, it is now possible to correlate, within the same molecule and in real time, conformational states and mechanical function of myosin. A spot-confocal microscope, capable of detecting single fluorophore polarization, was developed to measure orientational states in the smooth muscle myosin light chain domain during the process of motion generation. Fluorescently labeled turkey gizzard smooth muscle myosin was prepared by removal of endogenous regulatory light chain and re-addition of the light chain labeled at cysteine-108 with the 6-isomer of iodoacetamidotetramethylrhodamine (6-IATR). Single myosin molecule fluorescence polarization data, obtained in a motility assay, provide direct evidence that the myosin light chain domain adopts at least two orientational states during the cyclic interaction of myosin with actin, a randomly disordered state, most likely associated with myosin whereas weakly bound to actin, and an ordered state in which the light chain domain adopts a finite angular orientation whereas strongly bound after the powerstroke. PMID:9653135

Fab is the most efficient format to express functional antibodies by yeast surface display.

PubMed

Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

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.

Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

PubMed

Kirby, Karen A; Ong, Yee Tsuey; Hachiya, Atsuko; Laughlin, Thomas G; Chiang, Leslie A; Pan, Yun; Moran, Jennifer L; Marchand, Bruno; Singh, Kamalendra; Gallazzi, Fabio; Quinn, Thomas P; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G

2015-01-01

Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target. © FASEB.

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Walters, Benjamin T.; Janakiraman, Vasantharajan; Stinson, Jeremy; Patapoff, Thomas W.; Fuh, Germaine

2017-01-01

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. PMID:28057863

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

PubMed

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

2015-10-01

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

Light, nutrients, and food-chain length constrain planktonic energy transfer efficiency across multiple trophic levels

PubMed Central

Dickman, Elizabeth M.; Newell, Jennifer M.; González, María J.; Vanni, Michael J.

2008-01-01

The efficiency of energy transfer through food chains [food chain efficiency (FCE)] is an important ecosystem function. It has been hypothesized that FCE across multiple trophic levels is constrained by the efficiency at which herbivores use plant energy, which depends on plant nutritional quality. Furthermore, the number of trophic levels may also constrain FCE, because herbivores are less efficient in using plant production when they are constrained by carnivores. These hypotheses have not been tested experimentally in food chains with 3 or more trophic levels. In a field experiment manipulating light, nutrients, and food-chain length, we show that FCE is constrained by algal food quality and food-chain length. FCE across 3 trophic levels (phytoplankton to carnivorous fish) was highest under low light and high nutrients, where algal quality was best as indicated by taxonomic composition and nutrient stoichiometry. In 3-level systems, FCE was constrained by the efficiency at which both herbivores and carnivores converted food into production; a strong nutrient effect on carnivore efficiency suggests a carryover effect of algal quality across 3 trophic levels. Energy transfer efficiency from algae to herbivores was also higher in 2-level systems (without carnivores) than in 3-level systems. Our results support the hypothesis that FCE is strongly constrained by light, nutrients, and food-chain length and suggest that carryover effects across multiple trophic levels are important. Because many environmental perturbations affect light, nutrients, and food-chain length, and many ecological services are mediated by FCE, it will be important to apply these findings to various ecosystem types. PMID:19011082

Low-Power Light Guiding and Localization in Optoplasmonic Chains Obtained by Directed Self-Assembly

PubMed Central

Ahn, Wonmi; Zhao, Xin; Hong, Yan; Reinhard, Björn M.

2016-01-01

Optoplasmonic structures contain plasmonic components embedded in a defined photonic environment to create synergistic interactions between photonic and plasmonic components. Here, we show that chains of optical microspheres containing gold nanoparticles in their evanescent field combine the light guiding properties of a microsphere chain with the light localizing properties of a plasmonic nanoantenna. We implement these materials through template guided self-assembly and investigate their fundamental electromagnetic working principles through combination of electromagnetic simulations and experimental characterization. We demonstrate that optoplasmonic chains implemented by directed self-assembly achieve a significant reduction in guiding losses when compared with conventional plasmonic waveguides and, at the same time, retain the light localizing properties of plasmonic antennas at pre-defined locations. The results reinforce the potential of optoplasmonic structures for realizing low-loss optical interconnects with high bandwidth. PMID:26931149

Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

PubMed Central

Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S

1989-01-01

Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029

Biophysical basis of low-power-laser effects

NASA Astrophysics Data System (ADS)

Karu, Tiina I.

1996-06-01

Biological responses of cells to visible and near IR (laser) radiation occur due to physical and/or chemical changes in photoacceptor molecules, components of respiratory chains (cyt a/a3 in mitochondria). As a result of the photoexcitation of electronic states, the following physical and/or chemical changes can occur: alteration of redox properties and acceleration of electron transfer, changes in biochemical activity due to local transient heating of chromophores, one-electron auto-oxidation and O2- production, and photodynamic action and 1O2 production. Different reaction channels can be activated to achieve the photobiological macroeffect. The primary physical and/or chemical changes induced by light in photoacceptor molecules are followed by a cascade of biochemical reactions in the cell that do not need further light activation and occur in the dark (photosignal transduction and amplification chains). These actions are connected with changes in cellular homeostasis parameters. The crucial step here is thought to be an alteration of the cellular redox state: a shift towards oxidation is associated with stimulation of cellular vitality, and a shift towards reduction is linked to inhibition. Cells with a lower than normal pH, where the redox state is shifted in the reduced direction, are considered to be more sensitive to the stimulative action of light than those with the respective parameters being optimal or near optimal. This circumstance explains the possible variations in observed magnitudes of low-power laser effects. Light action on the redox state of a cell via the respiratory chain also explains the diversity of low-power laser effects. Beside explaining many controversies in the field of low-power laser effects (i.e., the diversity of effects, the variable magnitude or absence of effects in certain studies), the proposed redox-regulation mechanism may be a fundamental explanation for some clinical effects of irradiation, for example the positive results achieved in treating wounds, chronic inflammation, and ischemia, all characterized by acidosis and hypoxia.

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

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

Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx; Wall, Jonathan S.; González Andrade, Martín

2014-01-10

Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stabilitymore » and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.« less

Identification of Early Nuclear Target Genes of Plastidial Redox Signals that Trigger the Long-Term Response of Arabidopsis to Light Quality Shifts.

PubMed

Dietzel, Lars; Gläßer, Christine; Liebers, Monique; Hiekel, Stefan; Courtois, Florence; Czarnecki, Olaf; Schlicke, Hagen; Zubo, Yan; Börner, Thomas; Mayer, Klaus; Grimm, Bernhard; Pfannschmidt, Thomas

2015-08-01

Natural illumination conditions are highly variable and because of their sessile life style, plants are forced to acclimate to them at the cellular and molecular level. Changes in light intensity or quality induce changes in the reduction/oxidation (redox) state of the photosynthetic electron chain that acts as a trigger for compensatory acclimation responses comprising functional and structural adjustments of photosynthesis and metabolism. Such responses include redox-controlled changes in plant gene expression in the nucleus and organelles. Here we describe a strategy for the identification of early redox-regulated genes (ERGs) in the nucleus of the model organism Arabidopsis thaliana that respond significantly 30 or 60 min after the generation of a reduction signal in the photosynthetic electron transport chain. By comparing the response of wild-type plants with that of the acclimation mutant stn7, we could specifically identify ERGs. The results reveal a significant impact of chloroplast redox signals on distinct nuclear gene groups including genes for the mitochondrial electron transport chain, tetrapyrrole biosynthesis, carbohydrate metabolism, and signaling lipid synthesis. These expression profiles are clearly different from those observed in response to the reduction of photosynthetic electron transport by high light treatments. Thus, the ERGs identified are unique to redox imbalances in photosynthetic electron transport and were then used for analyzing potential redox-responsive cis-elements, trans-factors, and chromosomal regulatory hot spots. The data identify a novel redox-responsive element and indicate extensive redox control at transcriptional and chromosomal levels that point to an unprecedented impact of redox signals on epigenetic processes. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

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.

Statistical theory for protein combinatorial libraries. Packing interactions, backbone flexibility, and the sequence variability of a main-chain structure.

PubMed

Kono, H; Saven, J G

2001-02-23

Combinatorial experiments provide new ways to probe the determinants of protein folding and to identify novel folding amino acid sequences. These types of experiments, however, are complicated both by enormous conformational complexity and by large numbers of possible sequences. Therefore, a quantitative computational theory would be helpful in designing and interpreting these types of experiment. Here, we present and apply a statistically based, computational approach for identifying the properties of sequences compatible with a given main-chain structure. Protein side-chain conformations are included in an atom-based fashion. Calculations are performed for a variety of similar backbone structures to identify sequence properties that are robust with respect to minor changes in main-chain structure. Rather than specific sequences, the method yields the likelihood of each of the amino acids at preselected positions in a given protein structure. The theory may be used to quantify the characteristics of sequence space for a chosen structure without explicitly tabulating sequences. To account for hydrophobic effects, we introduce an environmental energy that it is consistent with other simple hydrophobicity scales and show that it is effective for side-chain modeling. We apply the method to calculate the identity probabilities of selected positions of the immunoglobulin light chain-binding domain of protein L, for which many variant folding sequences are available. The calculations compare favorably with the experimentally observed identity probabilities.

Estimation of polyclonal IgG4 hybrids in normal human serum.

PubMed

Young, Elizabeth; Lock, Emma; Ward, Douglas G; Cook, Alexander; Harding, Stephen; Wallis, Gregg L F

2014-07-01

The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains. Polyclonal IgG4 was purified from pooled or individual donor human sera and sequentially fractionated using light-chain affinity and size exclusion chromatography. Fractions were analysed by SDS-PAGE, immunoblotting, ELISA, immunodiffusion and matrix-assisted laser-desorption mass spectrometry. Polyclonal IgG4 purified from normal serum contained IgG4κ, IgG4λ and IgG4κ/λ molecules. Size exclusion chromatography showed that IgG4 was principally present in monomeric form (150 000 MW). SDS-PAGE, immunoblotting and ELISA showed the purity of the three IgG4 samples. Immunodiffusion, light-chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the IgG4κ/λ molecules. The amounts of IgG4κ/λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two IgG4 heavy chains plus one κ and one λ light chain. Polyclonal IgG (IgG4-depleted) was similarly fractionated according to light-chain specificity. No evidence of hybrid IgG κ/λ antibodies was observed. These results indicate that hybrid IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal human serum. © 2014 John Wiley & Sons Ltd.

Immunoglobulin light chain allelic inclusion in systemic lupus erythematosus.

PubMed

Fraser, Louise D; Zhao, Yuan; Lutalo, Pamela M K; D'Cruz, David P; Cason, John; Silva, Joselli S; Dunn-Walters, Deborah K; Nayar, Saba; Cope, Andrew P; Spencer, Jo

2015-08-01

The principles of allelic exclusion state that each B cell expresses a single light and heavy chain pair. Here, we show that B cells with both kappa and lambda light chains (Igκ and Igλ) are enriched in some patients with the systemic autoimmune disease systemic lupus erythematosus (SLE), but not in the systemic autoimmune disease control granulomatosis with polyangiitis. Detection of dual Igκ and Igλ expression by flow cytometry could not be abolished by acid washing or by DNAse treatment to remove any bound polyclonal antibody or complexes, and was retained after two days in culture. Both surface and intracytoplasmic dual light chain expression was evident by flow cytometry and confocal microscopy. We observed reduced frequency of rearrangements of the kappa-deleting element (KDE) in SLE and an inverse correlation between the frequency of KDE rearrangement and the frequency of dual light chain expressing B cells. We propose that dual expression of Igκ and Igλ by a single B cell may occur in some patients with SLE when this may be a consequence of reduced activity of the KDE. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

2016-06-15

Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection. Copyright © 2016. Published by Elsevier Inc.

SAbDab: the structural antibody database

PubMed Central

Dunbar, James; Krawczyk, Konrad; Leem, Jinwoo; Baker, Terry; Fuchs, Angelika; Georges, Guy; Shi, Jiye; Deane, Charlotte M.

2014-01-01

Structural antibody database (SAbDab; http://opig.stats.ox.ac.uk/webapps/sabdab) is an online resource containing all the publicly available antibody structures annotated and presented in a consistent fashion. The data are annotated with several properties including experimental information, gene details, correct heavy and light chain pairings, antigen details and, where available, antibody–antigen binding affinity. The user can select structures, according to these attributes as well as structural properties such as complementarity determining region loop conformation and variable domain orientation. Individual structures, datasets and the complete database can be downloaded. PMID:24214988

A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin.

PubMed

Berstein, R M; Schluter, S F; Shen, S; Marchalonis, J J

1996-04-16

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.

Isotype analysis of the anti-CENP-B anticentromere autoantibody: evidence for restricted clonality.

PubMed

Eisenberg, R A; Earnshaw, W C; Bordwell, B J; Craven, S Y; Cheek, R; Rothfield, N F

1989-10-01

Utilizing the centromere B fusion protein (CENP-B) and specific, matched monoclonal antiisotype reagents in an enzyme-linked immunosorbent assay, we found that anti-CENP-B autoantibodies were skewed to the IgG1 isotype. The overall kappa:lambda light chain ratio was 2:1, although several individual sera showed a strong predominance of one of the light chains. Isoelectric focusing of light chain-skewed sera showed polyclonal patterns. Our findings are consistent with the anti-CENP-B autoantibody response being a chronic, antigen-driven response.

Validation of serum free light chain reference ranges in primary care.

PubMed

Galvani, Luca; Flanagan, Jane; Sargazi, Mansour; Neithercut, William D

2016-05-01

The demand for measurement of serum immunoglobulin free kappa (κ) and lambda (λ) light chains has increased. The κ:λ ratio is used to assist in diagnosis/monitoring of plasma cell disorders. The binding site reference range for serum-free light chain κ:λ ratios of 0.26-1.65 was derived from healthy volunteers. Subsequently, a reference range of 0.37-3.1 for patients with chronic kidney disease has been proposed. Elevated free light chain concentrations and borderline raised free light chain ratios also may be found in polyclonal gammopathies and with other non-renal illnesses. This assessment was conducted to validate the established free light chain reference ranges in individuals from primary care. A total of 130 samples were identified from routine blood samples collected in primary care for routine biochemistry testing and estimated glomerular filtration rate calculation. The median and range of κ:λ ratios found in each estimated glomerular filtration rate group used for chronic kidney disease classification were higher than previously described. This was the case for individuals with normal or essentially normal renal function with estimated glomerular filtration rates>90, (0.58-1.76) and estimated glomerular filtration rate of 60-90 mL/min/1.73 m(2), (0.71-1.93). Individuals with estimated glomerular filtration rate 15-30, (0.72-4.50) and estimated glomerular filtration rate <15 ml/min/1.73 m(2) (0.71-4.95) also had higher values when compared to the current renal reference range of 0.37-3.10. Elevation of free light chain-κ:λ ratios may occur in the absence of a reduced renal function shown by a normal estimated glomerular filtration rate and in the presence of reduced renal function by estimated glomerular filtration rate when comparing results with the established reference ranges. Explanations include choice of analytical systems or the presence of other concurrent non-plasma cell illness. © The Author(s) 2016.

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

Loss of tubular creatinine secretion as the only sign of tubular proximal cell dysfunction in light chain proximal tubulopathy: A case report.

PubMed

Stehlé, Thomas; Vignon, Marguerite; Flamant, Martin; Figueres, Marie-Lucile; Rabant, Marion; Rodenas, Anita; Noël, Laure-Hélène; Arnulf, Bertrand; Vidal-Petiot, Emmanuelle

2016-06-01

Light chain proximal tubulopathy (LCPT) is a rare disease, characterized by cytoplasmic inclusions of light chain (usually kappa) immunoglobulins. Clinical presentation is usually a Fanconi syndrome. The proximal tubular dysfunction can be incomplete, and exceptional cases of LCPT without any tubular dysfunction have even been described. Here, we report a case of LCPT in which the only sign of proximal tubulopathy is the absence of secretion of creatinine, as assessed by the simultaneous measurement of renal clearance of creatinine and CrEDTA. The loss of tubular creatinine secretion as a sign of tubular proximal cell dysfunction ought to be identified in patients with light chain proximal tubulopathy as it leads to a clinically relevant underestimation of GFR by the creatinine-derived equations. The prevalence and prognostic significance of this particular proximal tubular damage in LCPT remain to be determined.

Low-power light guiding and localization in optoplasmonic chains obtained by directed self-assembly

DOE PAGES

Ahn, Wonmi; Zhao, Xin; Hong, Yan; ...

2016-03-02

Here, optoplasmonic structures contain plasmonic components embedded in a defined photonic environment to create synergistic interactions between photonic and plasmonic components. Here, we show that chains of optical microspheres containing gold nanoparticles in their evanescent field combine the light guiding properties of a microsphere chain with the light localizing properties of a plasmonic nanoantenna. We implement these materials through template guided self-assembly and investigate their fundamental electromagnetic working principles through combination of electromagnetic simulations and experimental characterization. We demonstrate that optoplasmonic chains implemented by directed self-assembly achieve a significant reduction in guiding losses when compared with conventional plasmonic waveguides and,more » at the same time, retain the light localizing properties of plasmonic antennas at pre-defined locations. The results reinforce the potential of optoplasmonic structures for realizing low-loss optical interconnects with high bandwidth.« less

Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.

PubMed

Barnidge, David R; Dasari, Surendra; Ramirez-Alvarado, Marina; Fontan, Adrian; Willrich, Maria A V; Tschumper, Renee C; Jelinek, Diane F; Snyder, Melissa R; Dispenzieri, Angela; Katzmann, Jerry A; Murray, David L

2014-11-07

We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

Detection of the value of consecutive serum total light chain (sTLC) in patients diagnosed with diffuse large B cell lymphoma.

PubMed

Zhai, Linzhu; Zhao, Yuanyuan; Peng, Songguo; Zhu, Ke; Yu, Rongjian; Chen, Hailong; Lin, Tongyu; Lin, Lizhu

2016-12-01

There are limited data on serum total light chain (sTLC) in lymphoma and its relative role on the outcome of diffuse large B cell lymphoma (DLBCL) patients. Blood samples from 46 cases newly diagnosed with DLBCL were collected consecutively during chemotherapy to detect sTLC, IgG, IgA, and IgM levels. Clinical data and survival outcomes were analyzed according to the results of sTLC measurements. In summary, 22 patients (47.8 %) had abnormal k or λ light chain, respectively, and 6 patients (13.0 %) had both abnormal k and λ light chains before chemotherapy. Patients with elevated k light chain more frequently displayed multiple extra-nodal organ involvement (P = 0.01) and had an inferior overall survival (OS) (P = 0.041) and progression-free survival (PFS) (P = 0.044) compared to patients with normal level of k light chain. Furthermore, patients with elevated level of both k and λ also exhibited significant association with shorter OS (P = 0.002) and PFS (P = 0.009). Both elevated k alone and concurrent elevated k and λ had independent adverse effects on PFS (P = 0.031 and P = 0.019, respectively). sTLC level was reduced gradually by treatment in this study and reached the lowest point after the fourth cycle of chemotherapy, which was consistent with the disease behavior during chemotherapy. Considering the small sample size of this study, these results should be confirmed in a larger prospective study.

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 traffic signal for heterodimeric amino acid transporters to transfer from the ER to the Golgi.

PubMed

Ganapathy, Vadivel

2009-01-15

Heterodimeric amino acid transporters represent a unique class of transport systems that consist of a light chain that serves as the 'transporter proper' and a heavy chain that is necessary for targeting the complex to the plasma membrane. The currently prevailing paradigm assigns no role for the light chains in the cellular processing of these transporters. In this issue of the Biochemical Journal, Sakamoto et al. provide evidence contrary to this paradigm. Their studies with the rBAT -b(0,+)AT (related to b(0,+) amino acid transporter-b(0,+)-type amino acid transporter) heterodimeric amino acid transporter show that the C-terminus of the light chain b(0,+)AT contains a sequence motif that serves as the traffic signal for the transfer of the heterodimeric complex from the endoplasmic reticulum to the Golgi. This is a novel function for the light chain in addition to its already established role as the subunit responsible for the transport activity. These new findings also seem to be applicable to other heterodimeric amino acid transporters as well.

Light-responsive expansion-contraction of spherical nanoparticle grafted with azopolymers

NASA Astrophysics Data System (ADS)

Fu, Jie; Zhang, Xinghua; Miao, Bing; Yan, Dadong

2017-04-01

Due to the very importance for both fundamental research and technological applications, smart materials with stimuli-responsive properties have been studied intensively. Theoretical investigation contributes to this endeavor through constructing and analyzing a model system which captures main features of the corresponding complex material, wherefrom useful insight can be provided to the trial-and-error experiments. We here report a theoretical study on the smart spherical nanoparticle grafted with light-responsive azobenzene-containing polymers. Utilizing the photoisomerization ability of the azobenzene group, nanoparticles can undergo a light-induced expansion-contraction transition. The wormlike chain based single chain in mean field theory, which has been developed by us recently, is used to investigate this transition in detail. Exploring a large parameter space, our results definitely determine the parameters, including the chain length and effective Kuhn length of grafted chain, nanoparticle radius, grafting density, and position of the azobenzene group along the chain contour, to admit optimum light-responsive behavior of the smart nanoparticle, which provides a guide for experimentalists to design this type of material in a rational manner.

Immunoturbidimetric assay for estimating free light chains of immunoglobulins in urine and serum.

PubMed Central

Tillyer, C R; Iqbal, J; Raymond, J; Gore, M; McIlwain, T J

1991-01-01

An immunoturbidimetric assay for the assessment of free kappa and lambda light chains of immunoglobulins was developed using a commercial polyclonal antiserum with reactivity towards epitopes on the light chains, which are not expressed when they are bound to heavy chains. The assay, on a centrifugal analyser, is simple and rapid. The limit of detection is 5 mg/l of free light chain, with an assay range of 5-120 mg/l, intrabatch precisions from 1.5-6.4%, and interbatch precisions from 6.5-8.9%. The assay was only slightly less sensitive than colloidal gold staining of cellulose acetate electrophoreses for the detection of Bence-Jones protein in urine. For the serial monitoring of response to chemotherapy in patients with myeloma, the assay correlated well with serum paraprotein estimates obtained by densitometric scanning of Ponceau stained cellulose acetate electrophoreses, but not with serum beta-2 microglobulin measurements, even after correction for the effects of creatinine. These assays may prove to be of use for the monitoring of tumour response in the treatment of Bence-Jones myeloma. PMID:1906071

Construction, expression and in vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia.

PubMed

Zhu, Lijuan; Liao, Wenjun; Zhu, Huifen; Lei, Ping; Wang, Zhihua; Shao, Jingfang; Zhang, Yue; Shen, Guanxin

2006-01-01

The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.

Imaging Variable Stars with HST

NASA Astrophysics Data System (ADS)

Karovska, Margarita

2011-05-01

The Hubble Space Telescope (HST) observations of astronomical sources, ranging from objects in our solar system to objects in the early Universe, have revolutionized our knowledge of the Universe its origins and contents.I will highlight results from HST observations of variable stars obtained during the past twenty or so years. Multiwavelength observations of numerous variable stars and stellar systems were obtained using the superb HST imaging capabilities and its unprecedented angular resolution, especially in the UV and optical. The HST provided the first detailed images probing the structure of variable stars including their atmospheres and circumstellar environments. AAVSO observations and light curves have been critical for scheduling of many of these observations and provided important information and context for understanding of the imaging results of many variable sources. I will describe the scientific results from the imaging observations of variable stars including AGBs, Miras, Cepheids, semi-regular variables (including supergiants and giants), YSOs and interacting stellar systems with a variable stellar components. These results have led to an unprecedented understanding of the spatial and temporal characteristics of these objects and their place in the stellar evolutionary chains, and in the larger context of the dynamic evolving Universe.

Imaging Variable Stars with HST

NASA Astrophysics Data System (ADS)

Karovska, M.

2012-06-01

(Abstract only) The Hubble Space Telescope (HST) observations of astronomical sources, ranging from objects in our solar system to objects in the early Universe, have revolutionized our knowledge of the Universe its origins and contents. I highlight results from HST observations of variable stars obtained during the past twenty or so years. Multiwavelength observations of numerous variable stars and stellar systems were obtained using the superb HST imaging capabilities and its unprecedented angular resolution, especially in the UV and optical. The HST provided the first detailed images probing the structure of variable stars including their atmospheres and circumstellar environments. AAVSO observations and light curves have been critical for scheduling of many of these observations and provided important information and context for understanding of the imaging results of many variable sources. I describe the scientific results from the imaging observations of variable stars including AGBs, Miras, Cepheids, semiregular variables (including supergiants and giants), YSOs and interacting stellar systems with a variable stellar components. These results have led to an unprecedented understanding of the spatial and temporal characteristics of these objects and their place in the stellar evolutionary chains, and in the larger context of the dynamic evolving Universe.

Spatially offset Raman spectroscopy for explosives detection through difficult (opaque) containers

NASA Astrophysics Data System (ADS)

Maskall, Guy T.; Bonthron, Stuart; Crawford, David

2013-10-01

With the continuing threat to aviation security from homemade explosive devices, the restrictions on taking a volume of liquid greater than 100 ml onto an aircraft remain in place. From January 2014, these restrictions will gradually be reduced via a phased implementation of technological screening of Liquids, Aerosols and Gels (LAGs). Raman spectroscopy offers a highly sensitive, and specific, technique for the detection and identification of chemicals. Spatially Offset Raman Spectroscopy (SORS), in particular, offers significant advantages over conventional Raman spectroscopy for detecting and recognizing contents within optically challenging (Raman active) containers. Containers vary enormously in their composition; glass type, plastic type, thickness, reflectance, and pigmentation are all variable and cause an infinite range of absorbances, fluorescence backgrounds, Rayleigh backscattered laser light, and container Raman bands. In this paper we show that the data processing chain for Cobalt Light Systems' INSIGHT100 bottlescanner is robust to such variability. We discuss issues of model selection for the detection stage and demonstrate an overall detection rate across a wide range of threats and containers of 97% with an associated false alarm rate of 0.1% or lower.

Surface immunoglobulins on blood lymphocytes of normal and immunodeficient persons studied by the mixed antiglobulin method

PubMed Central

Litwin, S. D.; Ochs, H.; Pollara, B.

1973-01-01

Surface immunoglobulins on human peripheral blood lymphocytes were investigated by the mixed antiglobulin technique—using the single layer mixed antiglobulin method as originally described (SLMA), and a modification employing a double layer of antibody (DLMA). Lymphocytes isolated from the blood of normal individuals had a mean of 7.8 and 18.4 per cent Ig + cells by the SLMA and DLMA techniques respectively. The DLMA data are similar to results obtained by other methods of detecting membrane Igs indicating that the mixed antiglobulin method is comparable in sensitivity. When the total numbers of Ig + cells, obtained by separate κ and λ testing, were compared with results obtained using single anti-light chain antisera, there was no significant difference, suggesting that most positive lymphocytes carry a single variety of light chain. Lymphocytes from the blood of seventeen patients with primary immunodeficiency were analysed. Four patients with variable immunodeficiency and four others with absent serum IgA all had normal surface Igs including α chains. All members of a family having an X-linked immunodeficiency had normal surface Igs including the affected members and a presumed carrier. Four cases of immunodeficiency associated with thymoma proved to have disparate findings. One patient exhibited a selective absence of μ antigens on the membranes of blood lymphocytes of over 2800 tested cells. Two other cases had normal surface Igs while a fourth patient, previously reported, lacked all surface Igs. PMID:4796276

Effect of diurnal photosynthetic activity on the fine structure of amylopectin from normal and waxy barley starch.

PubMed

Goldstein, Avi; Annor, George; Blennow, Andreas; Bertoft, Eric

2017-09-01

The impact of diurnal photosynthetic activity on the fine structure of the amylopectin fraction of starch synthesized by normal barley (NBS) and waxy barley (WBS), the latter completely devoid of amylose biosynthesis, was determined following the cultivation under normal diurnal or constant light growing conditions. The amylopectin fine structures were analysed by characterizing its unit chain length profiles after enzymatic debranching as well as its φ,β-limit dextrins and its clusters and building blocks after their partial and complete hydrolysis with α-amylase from Bacillus amyloliquefaciens, respectively. Regardless of lighting conditions, no structural effects were found when comparing both the amylopectin side-chain distribution and the internal chain fragments of these amylopectins. However, the diurnally grown NBS and WBS both showed larger amylopectin clusters and these had lower branching density and longer average chain lengths than clusters derived from plants grown under constant light conditions. Amylopectin clusters from diurnally grown plants also consisted of a greater number of building blocks, and shorter inter-block chain lengths compared to clusters derived from plants grown under constant light. Our data demonstrate that the diurnal light regime influences the fine structure of the amylopectin component both in amylose and non-amylose starch granules. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of light intensity on components and topographical structures of extracellular polysaccharides from the cyanobacteria Nostoc sp.

PubMed

Ge, Hongmei; Xia, Ling; Zhou, Xuping; Zhang, Delu; Hu, Chunxiang

2014-02-01

A study on the effects of light intensity (40 and 80 μE/m(2)/sec) on the components and topographical structures of extracellular polysaccharides (EPS) was carried out in cyanobacteria Nostoc sp.. EPS yield increased with light intensity. However, light intensity did not significantly affect the EPS fractions and monosaccharide composition. Higher light intensity generally resulted in higher protein content of EPS in similar fractions. The topographical structure of EPS, investigated by atomic force microscopy, appeared as spherical lumps, chains and networks. The long chains were observed at higher light intensity. Thus, light intensity affected the yield and nature of EPS.

[The H+/e- ratio in the photosynthetic electron transport chain].

PubMed

Ivanov, B N; Shmeleva, V L; Ovchinnikova, V I

1983-06-01

The number of protons adsorbed by tylakoids during one electron passage along the photosynthetic electron transport chain (i.e. the H+/e- ratio) was measured in isolated pea chloroplasts upon continuous illumination. Methylviologen was used as electron acceptor on the reducing side of PS I. It was found that at pH 6.0 upon illumination with red light (lambda greater than 620 nm) at an intensity of 2 . 10(5) erg/cm2 . s ("intensive" light) the H+/e- ratio is equal to 3. Upon illumination of dark-adapted chloroplasts with a "weak" light (900 erg/cm2 . s) the H+/e- ratio is equal to 2. Upon illumination of the chloroplasts with a "weak" after "intensive" light the value of this ratio is close to 3. Azide when added to the reaction mixture may interfere with the accuracy of measurements of the value of the H+/e- ratio by affecting proton exchange. Based on the changes in the H+/e- ratio induced by illumination it was assumed that at saturating intensity of the illuminating light the electron transport chain passes into a so-called "light" state when the mechanisms of proton-electron coupling differing from those of rare electron transfer ("weak" light, flashes) are triggered on. At pH 6.0 the "light" state of the electron transport chain is maintained for some time in the dark.

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

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

PubMed Central

Honey, Denise M.; Best, Annie; Qiu, Huawei

2018-01-01

ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

Longitudinal left ventricular function for prediction of survival in systemic light-chain amyloidosis: incremental value compared with clinical and biochemical markers.

PubMed

Buss, Sebastian J; Emami, Mostafa; Mereles, Derliz; Korosoglou, Grigorios; Kristen, Arnt V; Voss, Andreas; Schellberg, Dieter; Zugck, Christian; Galuschky, Christian; Giannitsis, Evangelos; Hegenbart, Ute; Ho, Anthony D; Katus, Hugo A; Schonland, Stefan O; Hardt, Stefan E

2012-09-18

The aim of the study was to determine whether longitudinal left ventricular (LV) function provides prognostic information in a large cohort of patients with systemic light-chain (AL) amyloidosis. AL amyloidosis is associated with a high incidence of cardiovascular events. Reduced myocardial longitudinal function is one of the hallmarks of myocardial involvement in this rare disease. Two hundred six consecutive patients with biopsy-proven AL amyloidosis were investigated in this prospective observational study. Echocardiographic imaging parameters, mean tissue Doppler-derived longitudinal strain (LS), and two-dimensional global longitudinal strain (2D-GLS) of the LV, cardiac serological biomarkers, and comprehensive clinical disease characteristics were assessed. The primary endpoint was all-cause mortality or heart transplantation. After a median follow-up of 1207 days, LS and 2D-GLS were significant predictors of survival in AL amyloidosis. The cutoff values discriminating survivors from nonsurvivors were -10.65% for LS and -11.78% for 2D-GLS. In a multivariable echocardiographic Cox model, only diastolic dysfunction and 2D-GLS remained as independent predictors of survival. In comprehensive clinical models, 2D-GLS (p < 0.0001), diastolic dysfunction (p < 0.01), the pathologic free light chains (p < 0.05), cardiac troponin-T (cTnT) (p < 0.01), and the Karnofsky index (p < 0.001) remained as independent predictors. 2D-GLS delineated a superior prognostic value compared with that derived from pathologic free light chains or cTnT in patients evaluated before firstline chemotherapy (n = 113; p < 0.0001), and remained the only independent predictor besides the Karnofsky index in subjects with preserved LV ejection fraction (≥50%; n = 127; p < 0.01). LS and 2D-GLS both offered significant incremental information (p < 0.001) for the assessment of outcome compared with clinical variables (age, Karnofsky index, and New York Heart Association functional class) and serological biomarkers. In the largest serial investigation reported so far, reduced LV longitudinal function served as an independent predictor of survival in AL amyloidosis and offered incremental information beyond standard clinical and serological parameters. Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Single Domain Antibodies as New Biomarker Detectors

PubMed Central

Fischer, Katja; Leow, Chiuan Yee; Chuah, Candy; McCarthy, James

2017-01-01

Biomarkers are defined as indicators of biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers have been widely used for early detection, prediction of response after treatment, and for monitoring the progression of diseases. Antibodies represent promising tools for recognition of biomarkers, and are widely deployed as analytical tools in clinical settings. For immunodiagnostics, antibodies are now exploited as binders for antigens of interest across a range of platforms. More recently, the discovery of antibody surface display and combinatorial chemistry techniques has allowed the exploration of new binders from a range of animals, for instance variable domains of new antigen receptors (VNAR) from shark and variable heavy chain domains (VHH) or nanobodies from camelids. These single domain antibodies (sdAbs) have some advantages over conventional murine immunoglobulin owing to the lack of a light chain, making them the smallest natural biomarker binders thus far identified. In this review, we will discuss several biomarkers used as a means to validate diseases progress. The potential functionality of modern singe domain antigen binders derived from phylogenetically early animals as new biomarker detectors for current diagnostic and research platforms development will be described. PMID:29039819

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

Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils-A metric for predicting amyloid propensity.

PubMed

Martin, Emily B; Williams, Angela; Wooliver, Craig; Heidel, R Eric; Adams, Sarah; Dunlap, John; Ramirez-Alvarado, Marina; Blancas-Mejia, Luis M; Lands, Ronald H; Kennel, Stephen J; Wall, Jonathan S

2017-01-01

Monoclonal free light chain (LC) proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL) amyloidosis and multiple myeloma (MM). Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10-30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM. We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05) than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains. The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies-this approach may improve the prognosis for these patients.

Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

PubMed

Barnidge, David R; Lundström, Susanna L; Zhang, Bo; Dasari, Surendra; Murray, David L; Zubarev, Roman A

2015-12-04

In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

Prognostic value of depressed midwall systolic function in cardiac light-chain amyloidosis.

PubMed

Perlini, Stefano; Salinaro, Francesco; Musca, Francesco; Mussinelli, Roberta; Boldrini, Michele; Raimondi, Ambra; Milani, Paolo; Foli, Andrea; Cappelli, Francesco; Perfetto, Federico; Palladini, Giovanni; Rapezzi, Claudio; Merlini, Giampaolo

2014-05-01

Cardiac amyloidosis represents an archetypal form of restrictive heart disease, characterized by profound diastolic dysfunction. As ejection fraction is preserved until the late stage of the disease, the majority of patients do fulfill the definition of diastolic heart failure, that is, heart failure with preserved ejection fraction (HFpEF). In another clinical model of HFpEF, that is, pressure-overload hypertrophy, depressed midwall fractional shortening (mFS) has been shown to be a powerful prognostic factor. To assess the potential prognostic role of mFS in cardiac light-chain amyloidosis with preserved ejection fraction, we enrolled 221 consecutive untreated patients, in whom a first diagnosis of cardiac light-chain amyloidosis was concluded between 2008 and 2010. HFpEF was present in 181 patients. Patients in whom cardiac involvement was excluded served as controls (n = 121). Prognosis was assessed after a median follow-up of 561 days. When compared with light-chain amyloidosis patients without myocardial involvement, cardiac light-chain amyloidosis was characterized by increased wall thickness (P <0.001), reduced end-diastolic left ventricular volumes (P <0.001), and diastolic dysfunction (P <0.001). In patients with preserved ejection fraction, mFS was markedly depressed [10.6% (8.7-13.5) vs. 17.8% (15.9-19.5) P <0.001]. At multivariable analysis, mFS, troponin I, and NT-pro-brain natriuretic peptide were the only significant prognostic determinants (P <0.001), whereas other indices of diastolic (E/E' ratio, transmitral and pulmonary vein flow velocities) and systolic function (tissue Doppler systolic indices, ejection fraction), or the presence/absence of congestive heart failure did not enter the model. In cardiac light-chain amyloidosis with normal ejection fraction, depressed circumferential mFS, a marker of myocardial contractile dysfunction, is a powerful predictor of survival.

Spectroscopic Studies of the Super Relaxed State of Skeletal Muscle

PubMed Central

Naber, Nariman; Pate, Edward; Canton, Marcella; Reggiani, Carlo; Cooke, Roger

2016-01-01

In the super-relaxed state of myosin, ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament in a structure known as the interacting-heads motif. In the disordered relaxed state myosin heads are not bound to the core of the thick filament and have an ATPase rate that is 10 fold greater. In the interacting-heads motif the two regulatory light chains appear to bind to each other. We have made single cysteine mutants of the regulatory light chain, placed both paramagnetic and fluorescent probes on them, and exchanged them into skinned skeletal muscle fibers. Many of the labeled light chains tended to disrupt the stability of the super-relaxed state, and showed spectral changes in the transition from the disordered relaxed state to the super-relaxed state. These data support the putative interface between the two regulatory light chains identified by cryo electron microscopy and show that both the divalent cation bound to the regulatory light chain and the N-terminus of the regulatory light chain play a role in the stability of the super-relaxed state. One probe showed a shift to shorter wavelengths in the super-relaxed state such that a ratio of intensities at 440nm to that at 520nm provided a measure of the population of the super-relaxed state amenable for high throughput screens for finding potential pharmaceuticals. The results provide a proof of concept that small molecules that bind to this region can destabilize the super-relaxed state and provide a method to search for small molecules that do so leading to a potentially effective treatment for Type 2 diabetes and obesity. PMID:27479128

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

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.

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

Undiagnosed light chain systemic amyloidosis: does it matter to anesthesiologists? -a case report-

PubMed Central

Kim, Gwan Ho; Lee, Woo Kyung; Na, Se Hee

2013-01-01

Light chain systemic amyloidosis is rare but may accompany laryngeal or pulmonary involvement, which may increase the risk in airway management. We present a case of a patient planned for resection of cervical epidural mass. The patient had face and neck ecchymoses and purpuras with an unknown cause. Mask ventilation and intubation were successful, but the operation was cancelled to evaluate bleeding from facial skin lesions. A diagnosis of light chain systemic amyloidosis prompted evaluation of involvement of other organs and treatment. This case shows the importance of preoperative evaluation and careful airway management in patients with systemic amyloidosis. PMID:24363850

Light-assisted, templated self-assembly of gold nanoparticle chains.

PubMed

Jaquay, Eric; Martínez, Luis Javier; Huang, Ningfeng; Mejia, Camilo A; Sarkar, Debarghya; Povinelli, Michelle L

2014-09-10

We experimentally demonstrate the technique of light-assisted, templated self-assembly (LATS) to trap and assemble 200 nm diameter gold nanoparticles. We excite a guided-resonance mode of a photonic-crystal slab with 1.55 μm laser light to create an array of optical traps. Unlike our previous demonstration of LATS with polystyrene particles, we find that the interparticle interactions play a significant role in the resulting particle patterns. Despite a two-dimensionally periodic intensity profile in the slab, the particles form one-dimensional chains whose orientations can be controlled by the incident polarization of the light. The formation of chains can be understood in terms of a competition between the gradient force due to the excitation of the mode in the slab and optical binding between particles.

Paraprotein-Related Kidney Disease: Evaluation and Treatment of Myeloma Cast Nephropathy.

PubMed

Finkel, Kevin W; Cohen, Eric P; Shirali, Anushree; Abudayyeh, Ala

2016-12-07

Nearly 50% of patients with multiple myeloma develop renal disease, most commonly from AKI caused by cast nephropathy. Development of AKI is associated with poor 1-year survival and reduces the therapeutic options available to patients. There is a great need for more effective therapies. Cast nephropathy is caused by the interaction and aggregation of filtered free light chains and Tamm-Horsfall protein causing intratubular obstruction and damage. The key to treating cast nephropathy is rapid lowering of free light chains, because this correlates with renal recovery. Newer chemotherapy agents rapidly lower free light chains and have been referred to as renoprotective. There is additional great interest in using extracorporeal therapies to remove serum free light chains. Small trials initially showed benefit of therapeutic plasma exchange to improve renal outcomes in cast nephropathy, but a large randomized trial of therapeutic plasma exchange failed to show benefit. A newer technique is extended high-cutoff hemodialysis. This modality uses a high molecular weight cutoff filter to remove free light chains. To date, trials of high-cutoff hemodialysis use in patients with cast nephropathy have been encouraging. However, there are no randomized trials showing the benefit of high-cutoff hemodialysis when used in addition to newer chemotherapeutic regimens. Until these studies are available, high-cutoff hemodialysis cannot be recommended as standard of care. Copyright © 2016 by the American Society of Nephrology.

Crystal structure at 2.8 A of the DLLRKN-containing coiled-coil domain of huntingtin-interacting protein 1 (HIP1) reveals a surface suitable for clathrin light chain binding.

PubMed

Ybe, Joel A; Mishra, Sanjay; Helms, Stephen; Nix, Jay

2007-03-16

Huntingtin interacting protein 1 (HIP1) is a member of a family of proteins whose interaction with Huntingtin is critical to prevent cells from initiating apoptosis. HIP1, and related protein HIP12/1R, can also bind to clathrin and membrane phospholipids, and HIP12/1R links the CCV to the actin cytoskeleton. HIP1 and HIP12/1R interact with the clathrin light chain EED regulatory site and stimulate clathrin lattice assembly. Here, we report the X-ray structure of the coiled-coil domain of HIP1 (residues 482-586) that includes residues crucial for binding clathrin light chain. The dimeric HIP1 crystal structure is partially splayed open. The comparison of the HIP1 model with coiled-coil predictions revealed the heptad repeat in the dimeric trunk (S2 path) is offset relative to the register of the heptad repeat from the N-terminal portion (S1 path) of the molecule. Furthermore, surface analysis showed there is a third hydrophobic path (S3) running parallel with S1 and S2. We present structural evidence supporting a role for the S3 path as an interaction surface for clathrin light chain. Finally, comparative analysis suggests the mode of binding between sla2p and clathrin light chain may be different in yeast.

Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

PubMed Central

Catera, Rosa; Hatzi, Katerina; Yan, Xiao-Jie; Zhang, Lu; Wang, Xiao Bo; Fales, Henry M.; Allen, Steven L.; Kolitz, Jonathan E.; Rai, Kanti R.; Chiorazzi, Nicholas

2008-01-01

Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells. PMID:18812466

The Ultrasensitivity of Living Polymers

NASA Astrophysics Data System (ADS)

O'Shaughnessy, Ben; Vavylonis, Dimitrios

2003-03-01

Synthetic and biological living polymers are self-assembling chains whose chain length distributions (CLDs) are dynamic. We show these dynamics are ultrasensitive: Even a small perturbation (e.g., temperature jump) nonlinearly distorts the CLD, eliminating or massively augmenting short chains. The origin is fast relaxation of mass variables (mean chain length, monomer concentration) which perturbs CLD shape variables before these can relax via slow chain growth rate fluctuations. Viscosity relaxation predictions agree with experiments on the best-studied synthetic system, α-methylstyrene.

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

Probing light chain mutation effects on thrombin via molecular dynamics simulations and machine learning.

PubMed

Xiao, Jiajie; Melvin, Ryan L; Salsbury, Freddie R

2018-03-02

Thrombin is a key component for chemotherapeutic and antithrombotic therapy development. As the physiologic and pathologic roles of the light chain still remain vague, here, we continue previous efforts to understand the impacts of the disease-associated single deletion of LYS9 in the light chain. By combining supervised and unsupervised machine learning methodologies and more traditional structural analyses on data from 10 μs molecular dynamics simulations, we show that the conformational ensemble of the ΔK9 mutant is significantly perturbed. Our analyses consistently indicate that LYS9 deletion destabilizes both the catalytic cleft and regulatory functional regions and result in some conformational changes that occur in tens to hundreds of nanosecond scaled motions. We also reveal that the two forms of thrombin each prefer a distinct binding mode of a Na + ion. We expand our understanding of previous experimental observations and shed light on the mechanisms of the LYS9 deletion associated bleeding disorder by providing consistent but more quantitative and detailed structural analyses than early studies in literature. With a novel application of supervised learning, i.e. the decision tree learning on the hydrogen bonding features in the wild-type and ΔK9 mutant forms of thrombin, we predict that seven pairs of critical hydrogen bonding interactions are significant for establishing distinct behaviors of wild-type thrombin and its ΔK9 mutant form. Our calculations indicate the LYS9 in the light chain has both localized and long-range allosteric effects on thrombin, supporting the opinion that light chain has an important role as an allosteric effector.

Successful treatment of nephrotic syndrome induced by lambda light chain deposition disease using lenalidomide: A case report and review of the literature
.

PubMed

Mima, Akira; Nagahara, Dai; Tansho, Kosuke

2018-06-01

Light chain deposition disease (LCDD) is a monoclonal immunoglobulin deposition disease (MIDD) that is characterized by the deposition of monoclonal light chains in multiple organs, including the kidney. It is a rare disorder caused by an underlying monoclonal plasma cell dyscrasia. LCDD with renal involvement causes proteinuria, which sometimes can lead to nephrotic syndrome. The monoclonal light chains are mostly in the κ form. Treatment of LCDD is the same as that for multiple myeloma (MM); however, some conventional anticancer drugs show substantial toxicity and therefore cannot be administered to older patients or those with renal impairment. An 80-year-old woman was referred to our department with severe nephrotic syndrome (13.6 g/gCr) and anemia. A renal biopsy showed mesangial proliferation and mesangial matrix expansion, and immunohistochemistry showed positive staining for λ chains along the glomerular basement membrane, but was negative for κ chains or amyloid deposition. A bone marrow biopsy revealed 64% plasma cells. Immunoglobulin G (IgG)-λ type M protein was detected, and the levels of free λ chain was significantly increased. We concluded that her nephrotic syndrome was caused by LCDD, which resulted from IgG-λ MM. The induction of a BCD (bortezomib, cyclophosphamide, and dexamethasone) treatment regimen did not lead to a hematological response or decrease in proteinuria. The administration of combination therapy of lenalidomide and prednisolone led to the successful reduction of proteinuria and hematuria. We presented a very rare case report describing the successful treatment of LCDD (λ chain)-induced nephrotic syndrome with lenalidomide.
.

The Analysis of Orders of Perishable Goods in Relation to the Bullwhip Effect in the Logistic Supply Chain of the Food Industry: a Case Study

NASA Astrophysics Data System (ADS)

Chocholáč, Jan; Průša, Petr

2016-12-01

The bullwhip effect generally refers to the phenomenon where order variability increases as the orders move upstream in the supply chain. It is serious problem for every member of the supply chain. This effect begins at customers and passes through the chain to producers, which are at the end of the logistic chain. Especially food supply chains are affected by this issue. These chains are unique for problems of expiration of goods (particularly perishable goods), variable demand, orders with quantity discounts and effort to maximize the customer satisfaction. This paper will present the problem of the bullwhip effect in the real supply chain in the food industry. This supply chain consists of approximately 350 stores, four central warehouses and more than 1000 suppliers, but the case study will examine 87 stores, one central warehouse and one supplier in 2015. The aim of this paper is the analysis of the order variability between the various links in this chain and confirmation of the bullwhip effect in this chain. The subject of the analysis will be perishable goods.

Cell origin of pyothorax-associated lymphoma: a lymphoma strongly associated with Epstein-Barr virus infection.

PubMed

Takakuwa, T; Tresnasari, K; Rahadiani, N; Miwa, H; Daibata, M; Aozasa, K

2008-03-01

Pyothorax-associated lymphoma (PAL) is an Epstein-Barr virus (EBV)-associated B cell lymphoma developing in the pleural cavity affected by chronic pyothorax. To clarify the cell origin of PAL, the expression of immunoglobulin heavy (IgH) and light chains in relation to somatic hypermutations (SHMs) of rearranged Ig heavy- and light-chain variable (IgV(H), IgV(L)) genes was examined using cell lines as well as clinical samples. SHMs without ongoing mutations of the IgV(H) gene were found in all PAL cell lines and clinical samples available for sequencing, indicating PAL to be derived from B cells at the postgerminal center (GC) stage of the differentiation process. They could be subdivided into post-GC cells with potentially productive IgV(H) genotypes (Group 1) and with sterile IgV(H) genotypes (Group 2). IgH expression was abrogated in Group 2 as expected and also in two cell lines in Group 1. DNA demethylation experiments with 5-aza-dC induced expression of IgH mRNA and protein in these cell lines. Most PAL cells were derived from crippled post-GC cells, which usually could not survive. Transformation of such B cells through EBV infection might provide a basis for the development of PAL with additional genetic changes.

Catalysts for synthesizing various short chain hydrocarbons

DOEpatents

Colmenares, Carlos

1991-01-01

Method and apparatus (10), including novel photocatalysts, are disclosed for the synthesis of various short chain hydrocarbons. Light-transparent SiO.sub.2 aerogels doped with photochemically active uranyl ions (18) are fluidized in a fluidized-bed reactor (12) having a transparent window (16), by hydrogen and CO, C.sub.2 H.sub.4 or C.sub.2 H.sub.6 gas mixtures (20), and exposed to radiation (34) from a light source (32) external to the reactor (12), to produce the short chain hydrocarbons (36).

Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.

PubMed

Henry, Kevin A

2018-01-01

Immunogenetic analyses of expressed antibody repertoires are becoming increasingly common experimental investigations and are critical to furthering our understanding of autoimmunity, infectious disease, and cancer. Next-generation DNA sequencing (NGS) technologies have now made it possible to interrogate antibody repertoires to unprecedented depths, typically by sequencing of cDNAs encoding immunoglobulin variable domains. In this chapter, we describe simple, fast, and reliable methods for producing and sequencing multiplex PCR amplicons derived from the variable regions (V H , V H H or V L ) of rearranged immunoglobulin heavy and light chain genes using the Illumina MiSeq platform. We include complete protocols and primer sets for amplicon sequencing of V H /V H H/V L repertoires directly from human, mouse, and llama lymphocytes as well as from phage-displayed V H /V H H/V L libraries; these can be easily be adapted to other types of amplicons with little modification. The resulting amplicons are diverse and representative, even using as few as 10 3 input B cells, and their generation is relatively inexpensive, requiring no special equipment and only a limited set of primers. In the absence of heavy-light chain pairing, single-domain antibodies are uniquely amenable to NGS analyses. We present a number of applications of NGS technology useful in discovery of single-domain antibodies from phage display libraries, including: (i) assessment of library functionality; (ii) confirmation of desired library randomization; (iii) estimation of library diversity; and (iv) monitoring the progress of panning experiments. While the case studies presented here are of phage-displayed single-domain antibody libraries, the principles extend to other types of in vitro display libraries.

Proposal and validation of prognostic scoring systems for IgG and IgA monoclonal gammopathies of undetermined significance.

PubMed

Rossi, Francesca; Petrucci, Maria Teresa; Guffanti, Andrea; Marcheselli, Luigi; Rossi, Davide; Callea, Vincenzo; Vincenzo, Federico; De Muro, Marianna; Baraldi, Alessandra; Villani, Oreste; Musto, Pellegrino; Bacigalupo, Andrea; Gaidano, Gianluca; Avvisati, Giuseppe; Goldaniga, Maria; Depaoli, Lorenzo; Baldini, Luca

2009-07-01

The presenting clinico-hematologic features of 1,283 patients with IgG and IgA monoclonal gammopathies of undetermined significance (MGUS) were correlated with the frequency of evolution into multiple myeloma (MM). Two IgG MGUS populations were evaluated: a training sample (553 patients) and a test sample (378 patients); the IgA MGUS population consisted of 352 patients. Forty-seven of the 553 training group patients and 22 of 378 test group IgG patients developed MM after a median follow-up of 6.7 and 3.6 years, respectively. Multivariate analysis showed that serum monoclonal component (MC) levels of < or =1.5 g/dL, the absence of light-chain proteinuria and normal serum polyclonal immunoglobulin levels defined a prognostically favorable subset of patients, and could be used to stratify the patients into three groups at different 10-year risk of evolution (hazard ratio, 1.0, 5.04, 11.2; P < 0.001). This scoring system was validated in the test sample. Thirty of the 352 IgA patients developed MM after a median follow-up of 4.8 years, and multivariate analysis showed that hemoglobin levels of <12.5 g/dL and reduced serum polyclonal immunoglobulin correlated with progression. A pooled statistical analysis of all of the patients confirmed the validity of Mayo Clinic risk model showing that IgA class, serum MC levels, and light-chain proteinuria are the most important variables correlated with disease progression. Using simple variables, we validated a prognostic model for IgG MGUS. Among the IgA cases, the possible prognostic role of hemoglobin emerged in addition to a decrease in normal immunoglobulin levels.

Serum free light chain reference values: a critical approach.

PubMed

Altinier, Sara; Seguso, Mara; Zaninotto, Martina; Varagnolo, Mariacristina; Adami, Fausto; Angeli, Paolo; Plebani, Mario

2013-05-01

The clinical usefulness of serum free light chain (FLC) measurement in the management of patients with plasma cell proliferative disorders has been reported in several papers, and most clinical studies use the reference ranges declared by the manufacturer. The aim of the present study was to evaluate the reproducibility of FLCs immunoassay and to validate the reference range, before introducing it in routine setting. Internal quality control materials and a pool of fresh serum samples were used to evaluate imprecision; 162 fresh sera from healthy blood donors were analyzed to evaluate the reference range for FLCs. In order to verify the κ/λ FLC ratio, 43 sera from patients with polyclonal hypergammaglobulinemia were tested. The FLC immunoassay was performed using a nephelometer with the Freelite reagents. The imprecision studies performed using a serum pool tested with two different lots of reagents showed a mean CV of 16.09% for κFLC and of 16.72% for λFLC. Lower CV%s and different mean values were found by calculating the results from each specific lot separately, while different results were obtained using the control materials provided by the manufacturer. In reference subjects, the 2.5-97.5th percentiles were found to be 4.52-22.33 and 4.84-21.88mg/L for κFLC and λFLC, respectively. The range for κ/λ ratio (0.65-2.36) was validated with the values obtained from subjects with polyclonal hypergammaglobulinemia. In retesting 15 samples from blood donor subjects with a different lot of reagents, mean bias percentages of 17.60 for κFLC and 15.26 for λFLC were obtained. These findings confirm the lot-to-lot variability of the FLC assays also in the measurement of polyclonal light chains, as well as the need to carefully validate the reference values. Published by Elsevier Inc.

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

Baden, Elizabeth M.; Owen, Barbara A.L.; Peterson, Francis C.

Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the kl O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90 degrees from the kl O18/O8 dimer interface. The three non-conservativemore » mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than kl O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.« less

Histopathologic and immunohistochemical features of Hashimoto thyroiditis.

PubMed

Amani, H Kazem

2011-01-01

Intrathyroid lymphoid tissue is accrued in Hashimoto thyroiditis (HT). Histologically, this acquired lymphoid tissue bears a close resemblance to mucosa-associated lymphoid tissue (MALT) and can evolve to lymphoma. To demonstrate the morphological, and immunohistochemical profiles of Hashimoto thyroiditis and to ascertain the importance of light chain restriction in distinguishing HT with extensive lymphoplasmacytoid infiltrate from MALT lymphoma. We studied histopathologically and immunohistochemically (CD20, CD3, Igk, Igl and cytokeratin) 30 cases of HT for evaluation of the lymphoid infiltrate and the presence of lymphoepithelial lesions (LELs). Distinguishing between early thyroid lymphoma and HT was evaluated by light chain restriction. These findings were compared with two cases of primary thyroid lymphoma. The histopathological findings were characteristic of HT. Immunohistochemistry confirmed inconspicuous, rare B-cell LELs as well as a prominent T-lymphocyte population. Testing for light chain restriction showed polyclonal population of plasma cells. The cases of MALT lymphoma had distinct destructive lymphoepithelial lesions, B-cell immunophenotyping and showed kappa light chain restriction in the plasmacytoid population. Hashimoto thyroiditis differs both histopathologically and immunohistochemically from thyroid lymphoma. In suspicious cases, immunohistochemistry could be helpful in reaching a definitive diagnosis.

A prospective study of nutritional status in immunoglobulin light chain amyloidosis

PubMed Central

Sattianayagam, Prayman T.; Lane, Thirusha; Fox, Zoe; Petrie, Aviva; Gibbs, Simon D.J.; Pinney, Jennifer H.; Risom, Signe S.; Rowczenio, Dorota M.; Wechalekar, Ashutosh D.; Lachmann, Helen J.; Gilbertson, Janet A.; Hawkins, Philip N.; Gillmore, Julian D.

2013-01-01

Weight loss is common in systemic immunoglobulin light chain amyloidosis but there are limited data on the impact of nutritional status on outcome. Using the Patient-Generated Subjective Global Assessment (PG-SGA) score, we prospectively examined nutritional status in 110 consecutive newly-diagnosed, treatment-naïve patients with immunoglobulin light chain amyloidosis attending the UK National Amyloidosis Centre. At study entry, 72 of 110 (66%) patients had a PG-SGA score of 4 or over, indicating malnutrition requiring specialist nutritional intervention. Number of amyloidotic organs, elevated alkaline phosphatase, presence of autonomic neuropathy and advanced Mayo disease stage were independently associated with poor nutritional status (P<0.05). Quality of life was substantially poorer among those with higher PG-SGA scores (P<0.001). Furthermore, PG-SGA score was a powerful independent predictor of patient survival (P=0.02). Malnutrition is prevalent and is associated with poor quality of life and reduced survival among patients with systemic immunoglobulin light chain amyloidosis. The PG-SGA score would be an appropriate tool to evaluate whether nutritional intervention could improve patient outcomes. PMID:22983575

Dynamics of a linear system coupled to a chain of light nonlinear oscillators analyzed through a continuous approximation

NASA Astrophysics Data System (ADS)

Charlemagne, S.; Ture Savadkoohi, A.; Lamarque, C.-H.

2018-07-01

The continuous approximation is used in this work to describe the dynamics of a nonlinear chain of light oscillators coupled to a linear main system. A general methodology is applied to an example where the chain has local nonlinear restoring forces. The slow invariant manifold is detected at fast time scale. At slow time scale, equilibrium and singular points are sought around this manifold in order to predict periodic regimes and strongly modulated responses of the system. Analytical predictions are in good accordance with numerical results and represent a potent tool for designing nonlinear chains for passive control purposes.

Plasmonic nanoparticle chain in a light field: a resonant optical sail.

PubMed

Albaladejo, Silvia; Sáenz, Juan José; Marqués, Manuel I

2011-11-09

Optical trapping and driving of small objects has become a topic of increasing interest in multidisciplinary sciences. We propose to use a chain made of metallic nanoparticles as a resonant light sail, attached by one end point to a transparent object and propelling it by the use of electromagnetic radiation. Driving forces exerted on the chain are theoretically studied as a function of radiation's wavelength and chain's alignments with respect to the direction of radiation. Interestingly, there is a window in the frequency spectrum in which null-torque equilibrium configuration, with minimum geometric cross section, corresponds to a maximum in the driving force.

Audit of Use and Overuse of Serum Protein Immunofixation Electrophoresis and Serum Free Light Chain Assay in Tertiary Health Care: A Case for Algorithmic Testing to Optimize Laboratory Utilization.

PubMed

Heaton, Christopher; Vyas, Shikhar G; Singh, Gurmukh

2016-04-01

Overuse of laboratory tests is a persistent issue. We examined the use and overuse of serum immunofixation electrophoresis and serum free light chain assays to develop an algorithm for optimizing utilization. A retrospective review of all tests, for investigation of monoclonal gammopathies, for all patients who had any of these tests done from April 24, 2014, through July 25, 2014, was carried out. The test orders were categorized as warranted or not warranted according to criteria presented in the article. A total of 237 patients were tested, and their historical records included 1,503 episodes of testing for one or more of serum protein electrophoresis, serum immunofixation electrophoresis, and serum free light chain assays. Only 46% of the serum immunofixation and 42% serum free light chain assays were warranted. Proper utilization, at our institution alone, would have obviated $64,182.95/year in health care costs, reduced laboratory cost of reagent alone by $26,436.04/year, and put $21,904.92/year of part B reimbursement at risk. Fewer than half of the serum immunofixation and serum free light chain assays added value. The proposed algorithm for testing should improve utilization. Risk to part B billing may be a disincentive to reducing test utilization. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Crystal structure at 2.8 Å of the DLLRKN-containing coiled-coil domain of Huntingtin-interacting protein 1 (HIP1) reveals a surface suitable for clathrin light chain binding

PubMed Central

Ybe, Joel A.; Mishra, Sanjay; Helms, Stephen; Nix, Jay

2007-01-01

Summary Huntingtin interacting protein 1 (HIP1) is a member of a family of proteins whose interaction with Huntingtin is critical to prevent cells from initiating apoptosis. HIP1, and related protein HIP12/1R, can also bind to clathrin and membrane phospholipids and HIP12/1R links the CCV to the actin cytoskeleton. HIP1 and HIP12/1R interact with the clathrin light chain EED regulatory site and stimulate clathrin lattice assembly. Here we report the X-ray structure of the coiled-coil domain of HIP1 from 482–586 that includes residues crucial for binding clathrin light chain. The dimeric HIP1 crystal structure is partially splayed open. The comparison of the HIP1 model with coiled-coil predictions revealed the heptad repeat in the dimeric trunk (S2 path) is offset relative to the register of the heptad repeat from the N-terminal portion (S1 path) of the molecule. Furthermore, surface analysis showed there is a third hydrophobic path (S3) running parallel to S1 and S2. We present structural evidence supporting a role for S3 path as an interaction surface for clathrin light chain. Finally, comparative analysis suggests the mode of binding between sla2p and clathrin light chain may be different in yeast. PMID:17257618

Coordinate changes of myosin light and heavy chain isoforms during forced fiber type transitions in rabbit muscle.

PubMed

Leeuw, T; Pette, D

1996-01-01

Skeletal muscle fibers are versatile entities, capable of changing their phenotype in response to altered functional demands. In the present study, fast-to-slow fiber type transitions were induced in rabbit tibialis anterior (fA) muscles by chronic low-frequency stimulation (CLFS). The time course of changes in relative protein concentrations of fast and slow myosin light chain (MLC) isoforms and changes in their relative synthesis rates by in vivo labeling with [35S]methionine were followed during stimulation periods of up to 60 days. Generally, relative synthesis rates and protein concentrations changed in parallel; i.e., fast isoforms decreased and slow isoforms increased. MLC3f, however, which turns over at a higher rate than the other light chains, exhibited a conspicuous discrepancy between a markedly reduced relative synthesis but only a moderate decrease in protein amount during the initial 2 weeks of CLFS. Apparently, MLC3f is regulated independent of MLC1f, with protein degradation playing an important role in its regulation. The exchange of fast MLC isoforms with their slow counterparts seemed to correspond to the ultimate fast-to-slow (MHCIIa-->MHCI) transition at the MHC level. However, due to an earlier onset of the fast-to-slow transition of the regulatory light chain and the delayed fast-to-slow exchange of the alkali light chains, a spectrum of hybrid isomyosins composed of fast and slow light and heavy chains must have existed transiently in transforming fibers. Such hybrid isomyosins appeared to be restricted to MHCIIa- and MHCI-based combinations. In conclusion, fiber type specific programs that normally coordinate the expression of myofibrillar protein isoforms seem to be maintained during fiber type transitions. Possible differences in post-transcriptional regulation may result in the transient accumulation of atypical combinations of fast and slow MLC and MHC isoforms, giving rise to the appearance of hybrid fibers under the conditions of forced fiber type conversion.

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP.

PubMed

Belov, Sergey; Buneva, Valentina N; Nevinsky, Georgy A

2017-10-01

Myelin basic protein (MBP) is a major protein of myelin-proteolipid shell of axons, and it plays an important role in pathogenesis of multiple sclerosis. In the literature, there are no data on how antibodies recognize different protein antigens including MBP. A stepwise increase in ligand complexity was used to estimate the relative contributions of virtually every amino acid residue (AA) of a specific 12-mer LSRFSWGAEGQK oligopeptide corresponding to immunodominant sequence of MBP to the light chains and to intact anti-MBP IgGs from sera of patients with multiple sclerosis. It was shown that the minimal ligands of the light chains of IgGs are many different free AAs (K d  = 0.51-0.016 M), and each free AA interacts with the specific subsite of the light chain intended for recognition of this AA in specific LSRFSW oligopeptide. A gradual transition from Leu to LSRFSWGAEGQK leads to an increase in the affinity from 10 -1 to 2.3 × 10 -4  M because of additive interactions of the light chain with 6 AAs of this oligopeptide and then the affinity reaches plateau. The contributions of 6 various AAs to the affinity of the oligopeptide are different (K d , M): 0.71 (S), 0.44 (R), 0.14 (F), 0.17 (S), and 0.62 (W). Affinity of nonspecific oligopeptides to the light chains of IgGs is significantly lower. Intact MBP interacts with both light and heavy chains of IgGs demonstrating 192-fold higher affinity than the specific oligopeptide. It is a first quantitative analysis of the mechanism of proteins recognition by antibodies. The thermodynamic model was constructed to describe the interactions of IgGs with MBP. The data obtained can be very useful for understanding how antibodies against many different proteins can recognize these proteins. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Hsc70-induced Changes in Clathrin-Auxilin Cage Structure Suggest a Role for Clathrin Light Chains in Cage Disassembly

PubMed Central

Young, Anna; Stoilova-McPhie, Svetla; Rothnie, Alice; Vallis, Yvonne; Harvey-Smith, Phillip; Ranson, Neil; Kent, Helen; Brodsky, Frances M; Pearse, Barbara M F; Roseman, Alan; Smith, Corinne J

2013-01-01

The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly. PMID:23710728

Managing Distance Education Institutions through Value Chain Analysis: the Nigerian Experience.

ERIC Educational Resources Information Center

Aderinto, J. A.; Akintayo, M. O.

Value chain analysis can gauge, analyze, and predict organization effects to control cost in light of achieving strategic organization objectives of distance education. Value chain analysis enables organizations to accomplish their goal or mission through cost effectiveness or differentiation. The value chain activity structure in a distance…

Molecular engineering to improve carrier lifetimes for organic photovoltaic devices with thick active layers

DOE PAGES

Oosterhout, Stefan D.; Braunecker, Wade A.; Owczarczyk, Zbyslaw R.; ...

2017-04-27

The morphology of the bulk heterojunction absorber layer in an organic photovoltaic (OPV) device has a profound effect on the electrical properties and efficiency of the device. Previous work has consistently demonstrated that the solubilizing side-chains of the donor material affect these properties and device performance in a non-trivial way. Here, using Time-Resolved Microwave Conductivity (TRMC), we show by direct measurements of carrier lifetimes that the choice of side chains can also make a substantial difference in photocarrier dynamics. We have previously demonstrated a correlation between peak photoconductance measured by TRMC and device efficiencies; here, we demonstrate that TRMC photocarriermore » dynamics have an important bearing on device performance in a case study of devices made from donor materials with linear vs. branched side-chains and with variable active layer thicknesses. We use Grazing-Incidence Wide Angle X-ray Scattering to elucidate the cause of the different carrier lifetimes as a function of different aggregation behavior in the polymers. Consequently, the results help establish TRMC as a technique for screening OPV donor materials whose devices maintain performance in thick active layers (>250 nm) designed to improve light harvesting, film reproducibility, and ease of processing.« less

[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.

Purification, Characterization and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkia.

USDA-ARS?s Scientific Manuscript database

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in close species. In this study, MLC with the molecular mass of 18kDa was purified from crayfish (Procambarus clarkii) muscle fibrils. Its physicochemical characterization showed that the...

Sequential cyclophosphamide-bortezomib-dexamethasone unmasks the harmful cardiac effect of dexamethasone in primary light-chain cardiac amyloidosis.

PubMed

Le Bras, Fabien; Molinier-Frenkel, Valerie; Guellich, Aziz; Dupuis, Jehan; Belhadj, Karim; Guendouz, Soulef; Ayad, Karima; Colombat, Magali; Benhaiem, Nicole; Tissot, Claire Marie; Hulin, Anne; Jaccard, Arnaud; Damy, Thibaud

2017-05-01

Chemotherapy combining cyclophosphamide, bortezomib and dexamethasone is widely used in light-chain amyloidosis. The benefit is limited in patients with cardiac amyloidosis mainly because of adverse cardiac events. Retrospective analysis of our cohort showed that 39 patients died with 42% during the first month. A new escalation-sequential regimen was set to improve the outcomes. Nine newly-diagnosed patients were prospectively treated with close monitoring of serum N-terminal pro-brain natriuretic peptide, troponin-T and free light chains. The results show that corticoids may destabilise the heart through fluid retention. Thus, a sequential protocol may be a promising approach to treat these patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination

PubMed Central

HWANG, JOYCE K.; ALT, FREDERICK W.; YEAP, LENG-SIEW

2015-01-01

The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination. PMID:26104555

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.

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.

A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

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

Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng

2010-08-13

Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody)more » antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.« less

Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

PubMed

Hayhurst, Andrew; Happe, Scott; Mabry, Robert; Koch, Zephyr; Iverson, Brent L; Georgiou, George

2003-05-01

Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

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.

Fluorescent signatures for variable DNA sequences

PubMed Central

Rice, John E.; Reis, Arthur H.; Rice, Lisa M.; Carver-Brown, Rachel K.; Wangh, Lawrence J.

2012-01-01

Life abounds with genetic variations writ in sequences that are often only a few hundred nucleotides long. Rapid detection of these variations for identification of genetic diseases, pathogens and organisms has become the mainstay of molecular science and medicine. This report describes a new, highly informative closed-tube polymerase chain reaction (PCR) strategy for analysis of both known and unknown sequence variations. It combines efficient quantitative amplification of single-stranded DNA targets through LATE-PCR with sets of Lights-On/Lights-Off probes that hybridize to their target sequences over a broad temperature range. Contiguous pairs of Lights-On/Lights-Off probes of the same fluorescent color are used to scan hundreds of nucleotides for the presence of mutations. Sets of probes in different colors can be combined in the same tube to analyze even longer single-stranded targets. Each set of hybridized Lights-On/Lights-Off probes generates a composite fluorescent contour, which is mathematically converted to a sequence-specific fluorescent signature. The versatility and broad utility of this new technology is illustrated in this report by characterization of variant sequences in three different DNA targets: the rpoB gene of Mycobacterium tuberculosis, a sequence in the mitochondrial cytochrome C oxidase subunit 1 gene of nematodes and the V3 hypervariable region of the bacterial 16 s ribosomal RNA gene. We anticipate widespread use of these technologies for diagnostics, species identification and basic research. PMID:22879378

A semi high-throughput method for screening small bispecific antibodies with high cytotoxicity.

PubMed

Sugiyama, Aruto; Umetsu, Mitsuo; Nakazawa, Hikaru; Niide, Teppei; Onodera, Tomoko; Hosokawa, Katsuhiro; Hattori, Shuhei; Asano, Ryutaro; Kumagai, Izumi

2017-06-06

Small bispecific antibodies that induce T-cell-mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell-recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 10 3 -times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.

Biomarkers in Immunoglobulin Light Chain Amyloidosis.

PubMed

Kufová, Z; Sevcikova, T; Growkova, K; Vojta, P; Filipová, J; Adam, Z; Pour, L; Penka, M; Rysava, R; Němec, P; Brozova, L; Vychytilova, P; Jurczyszyn, A; Grosicki, S; Barchnicka, A; Hajdúch, M; Simicek, M; Hájek, R

2017-01-01

Immunoglobulin light chain amyloidosis (AL amyloidosis - ALA) is a monoclonal gammopathy characterized by presence of aberrant plasma cells producing amyloidogenic immunoglobulin light chains. This leads to formation of amyloid fibrils in various organs and tissues, mainly in heart and kidney, and causes their dysfunction. As amyloid depositing in target organs is irreversible, there is a big effort to identify biomarker that could help to distinguish ALA from other monoclonal gammopathies in the early stages of disease, when amyloid deposits are not fatal yet. High throughput technologies bring new opportunities to modern cancer research as they enable to study disease within its complexity. Sophisticated methods such as next generation sequencing, gene expression profiling and circulating microRNA profiling are new approaches to study aberrant plasma cells from patients with light chain amyloidosis and related diseases. While generally known mutation in multiple myeloma patients (KRAS, NRAS, MYC, TP53) were not found in ALA, number of mutated genes is comparable. Transcriptome of ALA patients proves to be more similar to monoclonal gammopathy of undetermined significance patients, moreover level of circulating microRNA, that are known to correlate with heart damage, is increased in ALA patients, where heart damage in ALA typical symptom.Key words: amyloidosis - plasma cell - genome - transcriptome - microRNA.

High Performance Variable Emittance Devices for Spacecraft Application Based on Conducting Polymers Coupled with Ionic Liquids

NASA Astrophysics Data System (ADS)

Chandrasekhar, Prasanna; Zay, Brian J.; Barbolt, Scott; Werner, Robert; Birur, Gajanana C.; Paris, Anthony

2009-03-01

This contribution describes the fabrication, function and performance of thin-film variable emittance electrochromic skins fabricated using poly(aniline) as the conducting polymer (CP), a long-chain polymeric dopant, and an ionic liquid as electrolyte. The ionic electrolyte allows operation in space vacuum without any seals. A unique, space-durable coating applied to the external surface of the skins drastically lowers the solar absorptance of the skins, such that in their dark (highly emissive) electrochromic state, it is no more than 0.44, whilst in their light electrochromic state, it is ca. 0.3. Data presented show tailorable, variations from 0.19 to 0.90, ∀(s)<0.3, and nearly indefinite cyclability. Extended thermal vacuum, atomic-O, micrometeoroid, VUV and other studies show excellent space durability. Performance of a doughnut-shaped skin designed for a specific micro-spacecraft is also described.

Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.

PubMed

Bagheri, Salman; Yousefi, Mehdi; Safaie Qamsari, Elmira; Riazi-Rad, Farhad; Abolhassani, Mohsen; Younesi, Vahid; Dorostkar, Ruhollah; Movassaghpour, Ali Akbar; Sharifzadeh, Zahra

2017-03-01

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

PubMed Central

Yeow, Jonathan; Xu, Jiangtao; Boyer, Cyrille

2016-01-01

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (λ = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology. PMID:27340940

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst.

PubMed

Yeow, Jonathan; Xu, Jiangtao; Boyer, Cyrille

2016-06-08

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (λ = 460 nm, 0.7 mW/cm(2)), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor.

PubMed

Meixlsperger, Sonja; Köhler, Fabian; Wossning, Thomas; Reppel, Michael; Müschen, Markus; Jumaa, Hassan

2007-03-01

Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.

Falling Chains as Variable-Mass Systems: Theoretical Model and Experimental Analysis

ERIC Educational Resources Information Center

de Sousa, Celia A.; Gordo, Paulo M.; Costa, Pedro

2012-01-01

In this paper, we revisit, theoretically and experimentally, the fall of a folded U-chain and of a pile-chain. The model calculation implies the division of the whole system into two subsystems of variable mass, allowing us to explore the role of tensional contact forces at the boundary of the subsystems. This justifies, for instance, that the…

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

Ahn, Wonmi; Zhao, Xin; Hong, Yan

Here, optoplasmonic structures contain plasmonic components embedded in a defined photonic environment to create synergistic interactions between photonic and plasmonic components. Here, we show that chains of optical microspheres containing gold nanoparticles in their evanescent field combine the light guiding properties of a microsphere chain with the light localizing properties of a plasmonic nanoantenna. We implement these materials through template guided self-assembly and investigate their fundamental electromagnetic working principles through combination of electromagnetic simulations and experimental characterization. We demonstrate that optoplasmonic chains implemented by directed self-assembly achieve a significant reduction in guiding losses when compared with conventional plasmonic waveguides and,more » at the same time, retain the light localizing properties of plasmonic antennas at pre-defined locations. The results reinforce the potential of optoplasmonic structures for realizing low-loss optical interconnects with high bandwidth.« less

Difunctional bacteriophage conjugated with photosensitizers for Candida albicans-targeting photodynamic inactivation.

PubMed

Dong, Shuai; Shi, Hongxi; Zhang, Xintong; Chen, Xi; Cao, Donghui; Mao, Chuanbin; Gao, Xiang; Wang, Li

2018-01-01

Candida albicans is the most prevalent fungal pathogen of the human microbiota, causing infections ranging from superficial infections of the skin to life-threatening systemic infections. Due to the increasing occurrence of antibiotic-resistant C. albicans strains, new approaches to control this pathogen are needed. Photodynamic inactivation is an emerging alternative to treat infections based on the interactions between visible light and photosensitisers, in which pheophorbide a (PPA) is a chlorophyll-based photosensitizer that could induce cell death after light irradiation. Due to PPA's phototoxicity and low efficiency, the main challenge is to implement photosensitizer cell targeting and attacking. In this study, PPA was conjugated with JM-phage by EDC/NHS crosslinking. UV-Vis spectra was used to determine the optimum conjugation percentages of PPA and JM-phage complex for photodynamic inactivation. After photodynamic inactivation, the efficacy of PPA-JM-phage was assessed by performing in vitro experiments, such as MTS assay, scanning electron microscopy, measurement of dysfunctional mitochondria, ROS accumulation, S cell arrest and apoptotic pathway. A single-chain variable-fragment phage (JM) with high affinity to MP65 was screened from human single-fold single-chain variable-fragment libraries and designed as a binding target for C. albicans cells. Subsequently, PPa was integrated into JM phage to generate a combined nanoscale material, which was called PPA-JM-phage. After photodynamic inactivation, the growth of C. albicans was inhibited by PPA-JM-phage and apoptosis was observed. Scanning electron microscopy analysis revealed shrinking and rupturing of C. albicans . We also found that depolarization of mitochondrial membrane potential was decreased and intracellular reactive oxygen species levels were elevated significantly in C. albicans inhibited by PPA-JM-phage. Additionally, PPA-JM-phage also lead to S-phase arrest, and metacaspase activation resulting from mitochondrial dysfunction was also found to be involved in C. albicans apoptosis. PPa-JM-phage may induce C. albicans apoptosis through a caspase-dependent pathway and the results herein shed light on the potential application of phtototherapeutic nanostructures in fungal inactivation.

Difunctional bacteriophage conjugated with photosensitizers for Candida albicans-targeting photodynamic inactivation

PubMed Central

Dong, Shuai; Shi, Hongxi; Zhang, Xintong; Chen, Xi; Cao, Donghui; Mao, Chuanbin; Gao, Xiang; Wang, Li

2018-01-01

Background Candida albicans is the most prevalent fungal pathogen of the human microbiota, causing infections ranging from superficial infections of the skin to life-threatening systemic infections. Due to the increasing occurrence of antibiotic-resistant C. albicans strains, new approaches to control this pathogen are needed. Photodynamic inactivation is an emerging alternative to treat infections based on the interactions between visible light and photosensitisers, in which pheophorbide a (PPA) is a chlorophyll-based photosensitizer that could induce cell death after light irradiation. Due to PPA’s phototoxicity and low efficiency, the main challenge is to implement photosensitizer cell targeting and attacking. Methods In this study, PPA was conjugated with JM-phage by EDC/NHS crosslinking. UV-Vis spectra was used to determine the optimum conjugation percentages of PPA and JM-phage complex for photodynamic inactivation. After photodynamic inactivation, the efficacy of PPA-JM-phage was assessed by performing in vitro experiments, such as MTS assay, scanning electron microscopy, measurement of dysfunctional mitochondria, ROS accumulation, S cell arrest and apoptotic pathway. Results A single-chain variable-fragment phage (JM) with high affinity to MP65 was screened from human single-fold single-chain variable-fragment libraries and designed as a binding target for C. albicans cells. Subsequently, PPa was integrated into JM phage to generate a combined nanoscale material, which was called PPA-JM-phage. After photodynamic inactivation, the growth of C. albicans was inhibited by PPA-JM-phage and apoptosis was observed. Scanning electron microscopy analysis revealed shrinking and rupturing of C. albicans. We also found that depolarization of mitochondrial membrane potential was decreased and intracellular reactive oxygen species levels were elevated significantly in C. albicans inhibited by PPA-JM-phage. Additionally, PPA-JM-phage also lead to S-phase arrest, and metacaspase activation resulting from mitochondrial dysfunction was also found to be involved in C. albicans apoptosis. Conclusion PPa-JM-phage may induce C. albicans apoptosis through a caspase-dependent pathway and the results herein shed light on the potential application of phtototherapeutic nanostructures in fungal inactivation. PMID:29692614

[Histopathological diagnosis of amyloidosis].

PubMed

Hoshii, Yoshinobu

2006-05-01

For the diagnosis of amyloidosis, histological evidence of amyloid deposition is essential. Histologically, an amyloid deposit is stained orange red with Congo red and shows green birefringence under polarized light. When amyloidosis is clinically suspected, endoscopic biopsy of the stomach, duodenum or colon, or aspiration biopsy of abdominal fat is usually performed. If clinicians suspect amyloidosis, they should advise pathologists. Identification of the chemical type of amyloid is necessary with respect to treatment and prognosis. Immunohistochemical examination of amyloid in formalin-fixed, paraffin-embedded sections is simple to perform in most pathological laboratories. In Japan, almost all cases of systemic amyloidosis are classified as AL, AA, ATTR or Abeta2M amyloidosis, so the use of anti-immunoglobulin light chain, anti-amyloid A, anti-transthyretin and anti-beta2 microglobulin antibody is recommended for the classification of systemic amyloidosis. Formic acid pretreatment, which is often used for immunohistochemical detection of amyloidosis, is useful and easy for antigen retrieval. Amyloid deposits of AL amyloidosis are sometimes not immunostained well with commercial anti-immunoglobulin light chain antibody. Previously, we generated polyclonal antibodies against synthetic peptides corresponding to positions 118-134 of immunoglobulin lambda light chain and positions 116-133 of immunoglobulin kappa light chain. These antibodies are very useful for detecting AL amyloidosis because they react with amyloid deposits on formalin-fixed, paraffin-embedded specimens in almost all AL amyloidosis cases. Exact diagnosis and typing of amyloidosis are necessary for therapy.

Closed loop supply chain network design with fuzzy tactical decisions

NASA Astrophysics Data System (ADS)

Sherafati, Mahtab; Bashiri, Mahdi

2016-09-01

One of the most strategic and the most significant decisions in supply chain management is reconfiguration of the structure and design of the supply chain network. In this paper, a closed loop supply chain network design model is presented to select the best tactical and strategic decision levels simultaneously considering the appropriate transportation mode in activated links. The strategic decisions are made for a long term; thus, it is more satisfactory and more appropriate when the decision variables are considered uncertain and fuzzy, because it is more flexible and near to the real world. This paper is the first research which considers fuzzy decision variables in the supply chain network design model. Moreover, in this study a new fuzzy optimization approach is proposed to solve a supply chain network design problem with fuzzy tactical decision variables. Finally, the proposed approach and model are verified using several numerical examples. The comparison of the results with other existing approaches confirms efficiency of the proposed approach. Moreover the results confirms that by considering the vagueness of tactical decisions some properties of the supply chain network will be improved.

Measuring the influence of industry sector membership on supply chain disruption reporting.

PubMed

Alcantara, Patrick

2015-01-01

The global Supply Chain Resilience Survey by the Business Continuity Institute and Zurich Insurance is a comprehensive study on the state of supply chains in different organisations worldwide. As a benchmarking tool, it also contains data about business continuity arrangements in place to ensure supply chain resilience. Given this study's historically qualitative approach to reporting, this paper aims to introduce quantitative analysis. In this paper, responses that report membership in Standard Industrial Classification 2007 industry sectors from the 2013 Supply Chain Resilience Survey were disaggregated and related to supply chain disruption reporting. A chi-square test of independence reveals that membership in a particular industry sector influences reporting of supply chain disruption. Nonetheless, the relationship between these variables is weak. This study demonstrates interesting differences between industry sectors in terms of supply chain resilience. Further research is required in terms of other variables in order to provide granularity and relevant findings to supply chain planners.

Odd-even chain packing, molecular and thermal models for some long chain sodium(I) n-alkanoates

NASA Astrophysics Data System (ADS)

Nelson, Peter N.; Ellis, Henry A.

2014-10-01

A homologous series of sodium(I) n-alkanoates, NaCnH2n-1O2, with chain lengths n = 8-18, inclusive, have been synthesized and their structural and thermal properties investigated via Fourier Transform Infrared and Solid State 13C NMR spectroscopies, X-ray powder diffraction, Thermogravimetry, Differential Scanning Calorimetry, Polarizing light microscopy and variable temperature Infrared spectroscopy. The measurements show that metal-carboxylate coordination is via asymmetric chelating bidentate bonding with extensive carboxyl group inter-molecular interactions in which four oxygen atoms are bonded tetrahedrally to a sodium atom. Furthermore, the compounds crystallize in a monoclinic crystal system with the hydrocarbon chains in the fully extended all-trans conformation, advancing along the c-axis. Moreover, the chains are packed as tilted (θ ∼ 63°), non-overlapping, tail-to-tail lamellar bilayers that are not in the same plane, within a lamellar. Though these compounds are nearly isostructural, there are subtle differences in the packing of the hydrocarbon chains in the crystal lattice, resulting in odd-even alternation in the terminal methyl group asymmetric stretching vibration and chemical shift. These differences arise from the relative vertical distances between hydrocarbon planes within the lamellar; such that, for odd-chain compounds, larger inter-planar distances result in less efficient packing in the crystal lattice and hence, lower inter-planar van der Waals interactions between hydrocarbon chains. Thermal traces, for all compounds, show several partially reversible solid-solid pre-melting transitions associated with different degrees of gauche conformers in the alkyl chains. The reversible gauche-trans isomerism, of the methylene groups, is kinetically controlled; hence, super-cooling of the melt and other transitions, are observed for all compounds. The kinetics of chain reversion follow the exponential law of nucleation, though complicated by competing processes. Thermogravimetric data show that all compounds decompose at temperatures in excess of 690 K; therefore, free radical thermal cracking of the hydrocarbon chains, in conjunction with decarboxylation is proposed for their non-oxidative degradation mechanism.

Studies conducted as a search for physical mechanisms relating solar variability and the troposphere

NASA Technical Reports Server (NTRS)

Wu, S. T. (Editor)

1981-01-01

Chains of causative mechanisms that are hypothesized to relate solar variability to the behavior of the Earth's lower atmosphere were assessed. Solar variations believed most likely to constitute the forcing functions in hypothesized solar terrestrial atmosphere chain, changes in the Earth's atmospheric electrical characteristics due to solar variability, and the observed variations in atmospheric behavior that are influenced by solar variability were also examined.

Blood-based cerebral biomarkers in preeclampsia: Plasma concentrations of NfL, tau, S100B and NSE during pregnancy in women who later develop preeclampsia - A nested case control study.

PubMed

Bergman, Lina; Zetterberg, Henrik; Kaihola, Helena; Hagberg, Henrik; Blennow, Kaj; Åkerud, Helena

2018-01-01

To evaluate if concentrations of the neuronal proteins neurofilament light chain and tau are changed in women developing preeclampsia and to evaluate the ability of a combination of neurofilament light chain, tau, S100B and neuron specific enolase in identifying neurologic impairment before diagnosis of preeclampsia. A nested case-control study within a longitudinal study cohort was performed. 469 healthy pregnant women were enrolled between 2004-2007 and plasma samples were collected at gestational weeks 10, 25, 28, 33 and 37. Plasma concentrations of tau and neurofilament light chain were analyzed in 16 women who eventually developed preeclampsia and 36 controls throughout pregnancy with single molecule array (Simoa) method and compared within and between groups. S100B and NSE had been analyzed previously in the same study population. A statistical model with receiving characteristic operation curve was constructed with the four biomarkers combined. Plasma concentrations of neurofilament light chain were significantly increased in women who developed preeclampsia in gestational week 33 (11.85 ng/L, IQR 7.48-39.93 vs 6.80 ng/L, IQR 5.65-11.40) and 37 (22.15 ng/L, IQR 10.93-35.30 vs 8.40 ng/L, IQR 6.40-14.30) and for tau in gestational week 37 (4.33 ng/L, IQR 3.97-12.83 vs 3.77 ng/L, IQR 1.91-5.25) in contrast to healthy controls. A combined model for preeclampsia with tau, neurofilament light chain, S100B and neuron specific enolase in gestational week 25 displayed an area under the curve of 0.77, in week 28 it was 0.75, in week 33 it was 0.89 and in week 37 it was 0.83. Median week for diagnosis of preeclampsia was at 38 weeks of gestation. Concentrations of both tau and neurofilament light chain are increased in the end of pregnancy in women developing preeclampsia in contrast to healthy pregnancies. Cerebral biomarkers might reflect cerebral involvement before onset of disease.

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display.

PubMed

Schröter, Christian; Günther, Ralf; Rhiel, Laura; Becker, Stefan; Toleikis, Lars; Doerner, Achim; Becker, Janine; Schönemann, Andreas; Nasu, Daichi; Neuteboom, Berend; Kolmar, Harald; Hock, Björn

2015-01-01

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.

Cardiomyogenic Differentiation in Cardiac Myxoma Expressing Lineage-Specific Transcription Factors

PubMed Central

Kodama, Hiroaki; Hirotani, Takashi; Suzuki, Yusuke; Ogawa, Satoshi; Yamazaki, Kazuto

2002-01-01

We investigated five cases of cardiac myxoma and one case of cardiac undifferentiated sarcoma by light and electron microscopy, in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction for cardiomyocyte-specific transcription factors, Nkx2.5/Csx, GATA-4, MEF2, and eHAND. Conventional light microscopy revealed that cardiac myxoma and sarcoma cells presented variable cellular arrangements and different histological characteristics. Ultrastructurally, some of the myxoma cells exhibited endothelium-like or immature mesenchymal cell differentiation. Immunohistochemistry for Nkx2.5/Csx, GATA-4, and eHAND was slightly to intensely positive in all myxoma cases. MEF2 immunoreactivity was observed in all cases including the case of sarcoma, thus suggesting myogenic differentiation of myxoma or sarcoma cells. In situ hybridization for Nkx2.5/Csx also revealed that all myxoma cells, but not sarcoma cells, expressed mRNA of the cardiac homeobox gene, Nkx2.5/Csx. Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively. In conclusion, cardiac myxoma cells were found to express various amounts of cardiomyocyte-specific transcription factor gene products at the mRNA and protein levels, thus suggesting cardiomyogenic differentiation. These results support the concept that cardiac myxoma might arise from mesenchymal cardiomyocyte progenitor cells. PMID:12163362

Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671.

PubMed

Karageorgos, Ioannis; Gallagher, Elyssia S; Galvin, Connor; Gallagher, D Travis; Hudgens, Jeffrey W

2017-11-01

Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies. Published by Elsevier Ltd.

An exploration of counterfeit medicine surveillance strategies guided by geospatial analysis: lessons learned from counterfeit Avastin detection in the US drug supply chain.

PubMed

Cuomo, Raphael E; Mackey, Tim K

2014-12-02

To explore healthcare policy and system improvements that would more proactively respond to future penetration of counterfeit cancer medications in the USA drug supply chain using geospatial analysis. A statistical and geospatial analysis of areas that received notices from the Food and Drug Administration (FDA) about the possibility of counterfeit Avastin penetrating the US drug supply chain. Data from FDA warning notices were compared to data from 44 demographic variables available from the US Census Bureau via correlation, means testing and geospatial visualisation. Results were interpreted in light of existing literature in order to recommend improvements to surveillance of counterfeit medicines. This study analysed 791 distinct healthcare provider addresses that received FDA warning notices across 30,431 zip codes in the USA. Statistical outputs were Pearson's correlation coefficients and t values. Geospatial outputs were cartographic visualisations. These data were used to generate the overarching study outcome, which was a recommendation for a strategy for drug safety surveillance congruent with existing literature on counterfeit medication. Zip codes with greater numbers of individuals age 65+ and greater numbers of ethnic white individuals were most correlated with receipt of a counterfeit Avastin notice. Geospatial visualisations designed in conjunction with statistical analysis of demographic variables appeared more capable of suggesting areas and populations that may be at risk for undetected counterfeit Avastin penetration. This study suggests that dual incorporation of statistical and geospatial analysis in surveillance of counterfeit medicine may be helpful in guiding efforts to prevent, detect and visualise counterfeit medicines penetrations in the US drug supply chain and other settings. Importantly, the information generated by these analyses could be utilised to identify at-risk populations associated with demographic characteristics. Stakeholders should explore these results as another tool to improve on counterfeit medicine surveillance. 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.

Regions of recognition by blocking antibodies on the light chain of botulinum neurotoxin A: antigenic structure of the entire toxin.

PubMed

Dolimbek, Behzod Z; Steward, Lance E; Aoki, K Roger; Atassi, M Zouhair

2011-06-01

The continuous regions on botulinum neurotoxin A (BoNT/A) light (L) chain recognized by anti-toxin antibodies (Abs) from mouse, horse and chicken have been mapped. We synthesized a panel of thirty-two 19-residue peptides that overlapped consecutively by 5 residues and encompassed the entire L chain (residues 1-453). Mouse Abs recognized 5 major antigenic regions on the L chain, horse Abs recognized 9 while chicken Abs recognized 8 major antigenic regions. Overall, however, the three host species recognized, to some extent, similar, but not identical, peptides and the levels of Abs directed against a given region varied with the immunized host. Differences in the MHC of the host caused variation in levels of Ab recognition and some epitopes showed right or left frame-shifts among the species. Selected region(s) were also uniquely recognized by one species (e.g., peptide L1 by horse Abs). Mapping of the L chain antigenic regions and the previous localization of the regions on the H chain with the same antisera, has permitted description of the complete antigenic structure of BoNT/A. The locations in the 3-dimensional structure of the antigenic regions of the entire toxin are shown for mouse Abs. In the 3-D structure, the antigenic regions are on the surface of the toxin and when antibodies are bound the enzymatic activity of the light chain is obstructed. Copyright © 2010 Elsevier GmbH. All rights reserved.

Joint modelling rationale for chained equations

PubMed Central

2014-01-01

Background Chained equations imputation is widely used in medical research. It uses a set of conditional models, so is more flexible than joint modelling imputation for the imputation of different types of variables (e.g. binary, ordinal or unordered categorical). However, chained equations imputation does not correspond to drawing from a joint distribution when the conditional models are incompatible. Concurrently with our work, other authors have shown the equivalence of the two imputation methods in finite samples. Methods Taking a different approach, we prove, in finite samples, sufficient conditions for chained equations and joint modelling to yield imputations from the same predictive distribution. Further, we apply this proof in four specific cases and conduct a simulation study which explores the consequences when the conditional models are compatible but the conditions otherwise are not satisfied. Results We provide an additional “non-informative margins” condition which, together with compatibility, is sufficient. We show that the non-informative margins condition is not satisfied, despite compatible conditional models, in a situation as simple as two continuous variables and one binary variable. Our simulation study demonstrates that as a consequence of this violation order effects can occur; that is, systematic differences depending upon the ordering of the variables in the chained equations algorithm. However, the order effects appear to be small, especially when associations between variables are weak. Conclusions Since chained equations is typically used in medical research for datasets with different types of variables, researchers must be aware that order effects are likely to be ubiquitous, but our results suggest they may be small enough to be negligible. PMID:24559129

Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

PubMed Central

Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

2011-01-01

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

The role of free kappa and lambda light chains in the pathogenesis and treatment of inflammatory diseases.

PubMed

Esparvarinha, Mojgan; Nickho, Hamid; Mohammadi, Hamed; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Majidi, Jafar

2017-07-01

Kappa (κ) or lambda (λ) free light chains (FLCs) are produced from B cells during immunoglobulin synthesis. FLCs have been shown to participate in several key processes of immune responses. They are necessary to adjust PMN functions and assist PMN pre-stimulation. Moreover, they cause mast cell degranulation which releases pro-inflammatory mediators and stimulates local inflammatory responses in some conditions such as inflammatory bowel disease (IBD). Having low molecular weights which may straightly be toxic to proximal tubule cells (PTCs), FLCs can also have an important role in renal diseases. In this review we have highlighted the involvement of light chains in the pathogenesis of some inflammatory diseases and discussed their potential to be the targets of therapeutic purposes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Hypophosphatemic osteomalacia: an unusual clinical presentation of multiple myeloma.

PubMed

Reyskens, M; Sleurs, K; Verresen, L; Janssen, M; van den Bergh, J; van den Berg, J; Geusens, P

2015-07-01

An unusual case of a 75-year-old man is presented who had multiple stress fractures due to adult onset hypophosphatemic osteomalacia, which was the result of Fanconi syndrome, with light chain cast proximal tubulopathy due to multiple myeloma. A 75-year-old man presented with diffuse pain and muscle weakness. He had multiple stress fractures, low serum phosphate, decreased renal tubular reabsorption of phosphate, and normal PTH and FGF23, indicating adult onset hypophosphatemic osteomalacia. Phosphate supplements with calcitriol resulted in clinical recovery and healing of stress fractures. Because of proteinuria, a renal biopsy was performed that revealed Fanconi syndrome with light chain cast proximal tubulopathy and light kappa chains were found in serum and urine. A bone biopsy confirmed the diagnosis of multiple myeloma, and treatment with chemotherapy resulted in cytological and clinical recovery.

Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

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

Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.

2011-11-02

We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This resultmore » provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.« less

Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

PubMed

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity

PubMed Central

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity. PMID:25774965

Case for diagnosis. Systemic light chain amyloidosis with cutaneous involvement*

PubMed Central

Gontijo, João Renato Vianna; Pinto, Jackson Machado; de Paula, Maysa Carla

2017-01-01

Systemic light chain amiloydosis is a rare disease. Due to its typical cutaneous lesions, dermatologists play an essential role in its diagnosis. Clinical manifestations vary according to the affected organ and are often unspecific. Definitive diagnosis is achieved through biopsy. We report a patient with palpebral amyloidosis, typical bilateral ecchymoses and cardiac involvement, without plasma cell dyscrasia or lymphomas. The patient died shortly after the diagnosis. PMID:29166521

Double phosphorylation of the myosin regulatory light chain during rigor mortis of bovine Longissimus muscle.

PubMed

Muroya, Susumu; Ohnishi-Kameyama, Mayumi; Oe, Mika; Nakajima, Ikuyo; Shibata, Masahiro; Chikuni, Koichi

2007-05-16

To investigate changes in myosin light chains (MyLCs) during postmortem aging of the bovine longissimus muscle, we performed two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results of fluorescent differential gel electrophoresis showed that two spots of the myosin regulatory light chain (MyLC2) at pI values of 4.6 and 4.7 shifted toward those at pI values of 4.5 and 4.6, respectively, by 24 h postmortem when rigor mortis was completed. Meanwhile, the MyLC1 and MyLC3 spots did not change during the 14 days postmortem. Phosphoprotein-specific staining of the gels demonstrated that the MyLC2 proteins at pI values of 4.5 and 4.6 were phosphorylated. Furthermore, possible N-terminal region peptides containing one and two phosphoserine residues were detected in each mass spectrum of the MyLC2 spots at pI values of 4.5 and 4.6, respectively. These results demonstrated that MyLC2 became doubly phosphorylated during rigor formation of the bovine longissimus, suggesting involvement of the MyLC2 phosphorylation in the progress of beef rigor mortis. Bovine; myosin regulatory light chain (RLC, MyLC2); phosphorylation; rigor mortis; skeletal muscle.

Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins

NASA Technical Reports Server (NTRS)

Kretsinger, R. H.; Nakayama, S.

1993-01-01

In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

Recovery and fine structure variability of RGII sub-domains in wine (Vitis vinifera Merlot)

PubMed Central

Buffetto, F.; Ropartz, D.; Zhang, X. J.; Gilbert, H. J.; Guillon, F.; Ralet, M.-C.

2014-01-01

Background and Aims Rhamnogalacturonan II (RGII) is a structurally complex pectic sub-domain composed of more than 12 different sugars and 20 different linkages distributed in five side chains along a homogalacturonan backbone. Although RGII has long been described as highly conserved over plant evolution, recent studies have revealed variations in the structure of the polysaccharide. This study examines the fine structure variability of RGII in wine, focusing on the side chains A and B obtained after sequential mild acid hydrolysis. Specifically, this study aims to differentiate intrinsic structural variations in these RGII side chains from structural variations due to acid hydrolysis. Methods RGII from wine (Vitis vinifera Merlot) was sequentially hydrolysed with trifluoroacetic acid (TFA) and the hydrolysis products were separated by anion-exchange chromatography (AEC). AEC fractions or total hydrolysates were analysed by MALDI-TOF mass spectrometry. Key Results The optimal conditions to recover non-degraded side chain B, side chain A and RGII backbone were 0·1 m TFA at 40 °C for 16 h, 0·48 m TFA at 40 °C for 16 h (or 0·1 m TFA at 60 °C for 8 h) and 0·1 m TFA at 60 °C for 16 h, respectively. Side chain B was particularly prone to acid degradation. Side chain A and the RGII GalA backbone were partly degraded by 0·1 m TFA at 80 °C for 1–4 h. AEC allowed separation of side chain B, methyl-esterified side chain A and non-methyl-esterified side chain A. The structure of side chain A and the GalA backbone were highly variable. Conclusions Several modifications to the RGII structure of wine were identified. The observed dearabinosylation and deacetylation were primarily the consequence of acidic treatment, while variation in methyl-esterification, methyl-ether linkages and oxidation reflect natural diversity. The physiological significance of this variability, however, remains to be determined. PMID:24908680

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

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.

Hematopoiesis In The Equine Fetal Liver Suggests Immune Preparedness

PubMed Central

Battista, JM; Tallmadge, RL; Stokol, T; Felippe, MJB

2014-01-01

We investigated how the equine fetus prepares its pre-immune humoral repertoire for an imminent exposure to pathogens in the neonatal period, particularly how the primary hematopoietic organs are equipped to support B cell hematopoiesis and immunoglobulin (Ig) diversity. We demonstrated that the liver and the bone marrow at approximately 100 days of gestation (DG) are active sites of hematopoiesis based on the expression of signature mRNA (c-KIT, CD34, IL7R, CXCL12, IRF8, PU.1, PAX5, NOTCH1, GATA1, CEBPA) and protein markers (CD34, CD19, IgM, CD3, CD4, CD5, CD8, CD11b, CD172A) of hematopoietic development and leukocyte differentiation molecules, respectively. To verify Ig diversity achieved during the production of B cells, V(D)J segments were sequenced in primary lymphoid organs of the equine fetus and adult horse, revealing that similar heavy chain VDJ segments and CDR3 lengths were most frequently used independent of life stage. In contrast, different lambda light chain segments were predominant in equine fetal compared to adult stage and, surprisingly, the fetus had less restricted use of variable gene segments to construct the lambda chain. Fetal Igs also contained elements of sequence diversity, albeit to a smaller degree than that of the adult horse. Our data suggest that the B cells produced in the liver and bone marrow of the equine fetus generate a wide repertoire of pre-immune Igs for protection, and the more diverse use of different lambda variable gene segments in fetal life may provide the neonate an opportunity to respond to a wider range of antigens at birth. PMID:25179685

Light Regulation of the Arabidopsis Respiratory Chain. Multiple Discrete Photoreceptor Responses Contribute to Induction of Type II NAD(P)H Dehydrogenase Genes1

PubMed Central

Escobar, Matthew A.; Franklin, Keara A.; Svensson, Å. Staffan; Salter, Michael G.; Whitelam, Garry C.; Rasmusson, Allan G.

2004-01-01

Controlled oxidation reactions catalyzed by the large, proton-pumping complexes of the respiratory chain generate an electrochemical gradient across the mitochondrial inner membrane that is harnessed for ATP production. However, several alternative respiratory pathways in plants allow the maintenance of substrate oxidation while minimizing the production of ATP. We have investigated the role of light in the regulation of these energy-dissipating pathways by transcriptional profiling of the alternative oxidase, uncoupling protein, and type II NAD(P)H dehydrogenase gene families in etiolated Arabidopsis seedlings. Expression of the nda1 and ndc1 NAD(P)H dehydrogenase genes was rapidly up-regulated by a broad range of light intensities and qualities. For both genes, light induction appears to be a direct transcriptional effect that is independent of carbon status. Mutant analyses demonstrated the involvement of two separate photoreceptor families in nda1 and ndc1 light regulation: the phytochromes (phyA and phyB) and an undetermined blue light photoreceptor. In the case of the nda1 gene, the different photoreceptor systems generate distinct kinetic induction profiles that are integrated in white light response. Primary transcriptional control of light response was localized to a 99-bp region of the nda1 promoter, which contains an I-box flanked by two GT-1 elements, an arrangement prevalent in the promoters of photosynthesis-associated genes. Light induction was specific to nda1 and ndc1. The only other substantial light effect observed was a decrease in aox2 expression. Overall, these results suggest that light directly influences the respiratory electron transport chain via photoreceptor-mediated transcriptional control, likely for supporting photosynthetic metabolism. PMID:15333756

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

Engineering botulinum neurotoxin domains for activation by toxin light chain.

PubMed

Stancombe, Patrick R; Masuyer, Geoffrey; Birch-Machin, Ian; Beard, Matthew; Foster, Keith A; Chaddock, John A; Acharya, K Ravi

2012-02-01

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions. © 2011 Syntaxin Ltd. Journal compilation © 2011 FEBS.

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

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.

Atom Transfer Radical Polymerization of Functionalized Vinyl Monomers Using Perylene as a Visible Light Photocatalyst

PubMed Central

Theriot, Jordan C.; Ryan, Matthew D.; French, Tracy A.; Pearson, Ryan M.; Miyake, Garret M.

2016-01-01

A standardized technique for atom transfer radical polymerization of vinyl monomers using perylene as a visible-light photocatalyst is presented. The procedure is performed under an inert atmosphere using air- and water-exclusion techniques. The outcome of the polymerization is affected by the ratios of monomer, initiator, and catalyst used as well as the reaction concentration, solvent, and nature of the light source. Temporal control over the polymerization can be exercised by turning the visible light source off and on. Low dispersities of the resultant polymers as well as the ability to chain-extend to form block copolymers suggest control over the polymerization, while chain end-group analysis provides evidence supporting an atom-transfer radical polymerization mechanism. PMID:27166728

Opinion: the red-light response of stomatal movement is sensed by the redox state of the photosynthetic electron transport chain.

PubMed

Busch, Florian A

2014-02-01

Guard cells regulate CO2 uptake and water loss of a leaf by controlling stomatal movement in response to environmental factors such as CO2, humidity, and light. The mechanisms by which stomata respond to red light are actively debated in the literature, and even after decades of research it is still controversial whether stomatal movement is related to photosynthesis or not. This review summarizes the current knowledge of the red-light response of stomata. A comparison of published evidence suggests that stomatal movement is controlled by the redox state of photosynthetic electron transport chain components, in particular the redox state of plastoquinone. Potential consequences for the modeling of stomatal conductance are discussed.

Continuously Variable Transmission

NASA Technical Reports Server (NTRS)

Grana, D. C.

1985-01-01

Chain slides along two cones, in novel transmission concept. Transmission includes chain drive between two splined shafts. Chain sprockets follow surfaces of two cones. As one chain sprocket moves toward smaller diameter other chain sprocket moves toward larger diameter, thereby changing "gear" ratio. Movement initiated by tension applied to chain by planetary gear mechanism. Device positive, simple, and efficient over wide range of speed ratios.

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

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.

Structural considerations for functional anti-EGFR × anti-CD3 bispecific diabodies in light of domain order and binding affinity.

PubMed

Asano, Ryutaro; Nagai, Keisuke; Makabe, Koki; Takahashi, Kento; Kumagai, Takashi; Kawaguchi, Hiroko; Ogata, Hiromi; Arai, Kyoko; Umetsu, Mitsuo; Kumagai, Izumi

2018-03-02

We previously reported a functional humanized bispecific diabody (bsDb) that targeted EGFR and CD3 (hEx3-Db) and enhancement of its cytotoxicity by rearranging the domain order in the V domain. Here, we further dissected the effect of domain order in bsDbs on their cross-linking ability and binding kinetics to elucidate general rules regarding the design of functional bsDbs. Using Ex3-Db as a model system, we first classified the four possible domain orders as anti-parallel (where both chimeric single-chain components are variable heavy domain (VH)-variable light domain (VL) or VL-VH order) and parallel types (both chimeric single-chain components are mixed with VH-VL and VL-VH order). Although anti-parallel Ex3-Dbs could cross-link the soluble target antigens, their cross-linking ability between soluble targets had no correlation with their growth inhibitory effects. In contrast, the binding affinity of one of the two constructs with a parallel-arrangement V domain was particularly low, and structural modeling supported this phenomenon. Similar results were observed with E2x3-Dbs, in which the V region of the anti-EGFR antibody clone in hEx3 was replaced with that of another anti-EGFR clone. Only anti-parallel types showed affinity-dependent cancer inhibitory effects in each molecule, and E2x3-LH (both components in VL-VH order) showed the most intense anti-tumor activity in vitro and in vivo . Our results showed that, in addition to rearranging the domain order of bsDbs, increasing their binding affinity may be an ideal strategy for enhancing the cytotoxicity of anti-parallel constructs and that E2x3-LH is particularly attractive as a candidate next-generation anti-cancer drug.

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

PubMed Central

Chen, Cai-Ping; Chen, Xin; Qiao, Yan-Ning; Wang, Pei; He, Wei-Qi; Zhang, Cheng-Hai; Zhao, Wei; Gao, Yun-Qian; Chen, Chen; Tao, Tao; Sun, Jie; Wang, Ye; Gao, Ning; Kamm, Kristine E; Stull, James T; Zhu, Min-Sheng

2015-01-01

Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle. Key points Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. PMID:25433069

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction.

PubMed

Chen, Cai-Ping; Chen, Xin; Qiao, Yan-Ning; Wang, Pei; He, Wei-Qi; Zhang, Cheng-Hai; Zhao, Wei; Gao, Yun-Qian; Chen, Chen; Tao, Tao; Sun, Jie; Wang, Ye; Gao, Ning; Kamm, Kristine E; Stull, James T; Zhu, Min-Sheng

2015-02-01

Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

The effect of green supply chain management implementation to marketing performance through company competitiveness (study on paper industry in West Java)

NASA Astrophysics Data System (ADS)

Hijuzaman, O.; Rahayu, A.; Kusnendi

2018-05-01

The purpose of this study was to determine and analyze the effect of direct and indirect implementation of Green Supply-Chain Management (GSCM) on the Performance Marketing and Competitiveness Mediation Paper Company in West Java province. Object This research is the paper company that existed in West Java Province, which amounts to 30 companies as well as a sample of research, while variables studied are Variable Green Supply- Chain Management, Company Competitiveness Variable and Variable of Paper Marketing Industry Performance in West Java Province. The method used SEM with Variance Based or Component Based with Software used is SmartPLS 3.0. On this research are identified that no effect of Competitiveness on Marketing Performance in Paper Companies in West Java Province.

Light-induced yellowing of selectively 13C-enriched dehydrogenation polymers (DHPs). Part 1, Side-chain 13C-enriched DHP ([alpha], [beta], and [gamma]-13C)

Treesearch

Jim Parkas; Magnus Paulsson; Terashima Noritsugu; Ulla Westermark; Sally Ralph

2004-01-01

Light-induced yellowing has been studied using side-chain ([alpha], [beta], and [gamma]) 13C-enriched DHP (dehydrogenation polymer) and quantitative solution state 13C NMR spectroscopy. The DHP was formed from 13C-enriched coniferin using an enzymatic system consisting of [beta]-glucosidase, glucose oxidase, and peroxidase in a pH 6 buffer solution. The DHP was applied...

TCTEX1D4 Interactome in Human Testis: Unraveling the Function of Dynein Light Chain in Spermatozoa

PubMed Central

Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; da Cruz e Silva, Edgar

2014-01-01

Abstract Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function. PMID:24606217

Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle

PubMed Central

Cobley, James N.; Ab. Malik, Zulezwan; Morton, James P.; Close, Graeme L.; Edwards, Ben J.; Burniston, Jatin G.

2016-01-01

Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM). We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping. PMID:28248225

Genome-wide association study of immunoglobulin light chain amyloidosis in three patient cohorts: comparison with myeloma.

PubMed

da Silva Filho, M I; Försti, A; Weinhold, N; Meziane, I; Campo, C; Huhn, S; Nickel, J; Hoffmann, P; Nöthen, M M; Jöckel, K-H; Landi, S; Mitchell, J S; Johnson, D; Morgan, G J; Houlston, R; Goldschmidt, H; Jauch, A; Milani, P; Merlini, G; Rowcieno, D; Hawkins, P; Hegenbart, U; Palladini, G; Wechalekar, A; Schönland, S O; Hemminki, K

2017-08-01

Immunoglobulin light chain (AL) amyloidosis is characterized by tissue deposition of amyloid fibers derived from immunoglobulin light chain. AL amyloidosis and multiple myeloma (MM) originate from monoclonal gammopathy of undetermined significance. We wanted to characterize germline susceptibility to AL amyloidosis using a genome-wide association study (GWAS) on 1229 AL amyloidosis patients from Germany, UK and Italy, and 7526 healthy local controls. For comparison with MM, recent GWAS data on 3790 cases were used. For AL amyloidosis, single nucleotide polymorphisms (SNPs) at 10 loci showed evidence of an association at P<10 -5 with homogeneity of results from the 3 sample sets; some of these were previously documented to influence MM risk, including the SNP at the IRF4 binding site. In AL amyloidosis, rs9344 at the splice site of cyclin D1, promoting translocation (11;14), reached the highest significance, P=7.80 × 10 -11 ; the SNP was only marginally significant in MM. SNP rs79419269 close to gene SMARCD3 involved in chromatin remodeling was also significant (P=5.2 × 10 -8 ). These data provide evidence for common genetic susceptibility to AL amyloidosis and MM. Cyclin D1 is a more prominent driver in AL amyloidosis than in MM, but the links to aggregation of light chains need to be demonstrated.

A complementarity-determining region synthetic peptide acts as a miniantibody and neutralizes human immunodeficiency virus type 1 in vitro.

PubMed Central

Levi, M; Sällberg, M; Rudén, U; Herlyn, D; Maruyama, H; Wigzell, H; Marks, J; Wahren, B

1993-01-01

A complementarity-determining region (CDR) of the mouse monoclonal antibody (mAb) F58 was constructed with specificity to a neutralization-inducing region of human immunodeficiency virus type 1 (HIV-1). The mAb has its major reactivity to the amino acid sequence I--GPGRA in the V3 viral envelope region. All CDRs including several framework amino acids were synthesized from the sequence deduced by cloning and sequencing mAb F58 heavy- and light-chain variable domains. Peptides derived from the third heavy-chain domain (CDR-H3) alone or in combination with the other CDR sequences competed with F58 mAb for the V3 region. The CDR-H3 peptide was chemically modified by cyclization and then inhibited HIV-1 replication as well as syncytium formation by infected cells. Both the homologous IIIB viral strain to which the F58 mAb was induced and the heterologous SF2 strain were inhibited. This synthetic peptide had unexpectedly potent antiviral activity and may be a potential tool for treatment of HIV-infected persons. PMID:7685100

Synthesis of pore-variable mesoporous CdS and evaluation of its photocatalytic activity in degrading methylene blue

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

Zhang, Wei-Min, E-mail: chm_zhangwm@ujn.edu.cn; Jiang, Yao-Quan; Cao, Xiao-Yan

2013-10-15

Graphical abstract: - Highlights: • Self-templated synthesis of tubular CdS. • Cadmium complexes of aliphatic acids sustain the network of mesoporous structures. • Aliphatic acids affect the phase composition and particle size. • Pore size and volume vary with aliphatic acids having different hydrocarbonyl. - Abstract: In this study, mesoporous CdS polycrystallites have been synthesized using aliphatic acids of hexanoic acid, octanoic acid, and oleic acid as coordinating and capping agents, respectively. The fibrous Cd–fatty acid salts act as a template to form the tubular CdS. The organic species are found to be necessary for maintaining the network of mesoporousmore » CdS. The characterization results indicate that the shorter carbon chain length in aliphatic acids favors the wurtzite phase and particle size growth the specific surface area, pore diameter and pore volume show a monotonic raise with increasing carbon chain. The photocatalytic activities of mesoporous CdS tubes exhibit much higher efficiency than those of nanosized CdS powders in decolorizing methylene blue under simulated visible light.« less

Light-activated phenalen-1-one bactericides: efficacy, toxicity and mechanism compared with benzalkonium chloride.

PubMed

Muehler, Denise; Sommer, Kerstin; Wennige, Sara; Hiller, Karl-Anton; Cieplik, Fabian; Maisch, Tim; Späth, Andreas

2017-11-01

Five photoactive compounds with variable elongated alkyl-substituents in a phenalen-1-one structure were examined in view of structural similarity to the antimicrobial agent benzalkonium chloride (BAC). All phenalen-1-ones and BAC were evaluated for their antimicrobial properties against Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli, Pseudomonas aeruginosa and for their eukaryotic toxicity against normal human epidermal keratinocyte (NHEK) cells to narrow down the BAC-like effect and the photodynamic effect depending on the chemical structure. All compounds were investigated for effective concentration ranges, where a bacterial reduction of 5 log 10 is achieved, while an NHEK survival of 80% is ensured. Effective concentration ranges were found for four out of five photoactive compounds, but not for BAC and the compound with BAC-like alkyl chain length. Chain length size and polar area of the respective head-groups of phenalen-1-one compounds or BAC showed an influence on the incorporation inside lipid membranes and thus, head-groups may have an impact on the toxicity of antimicrobials.

The complementarity-determining region sequences in IgY antivenom hypervariable regions.

PubMed

da Rocha, David Gitirana; Fernandez, Jorge Hernandez; de Almeida, Claudia Maria Costa; da Silva, Claudia Letícia; Magnoli, Fabio Carlos; da Silva, Osmair Élder; da Silva, Wilmar Dias

2017-08-01

The data presented in this article are related to the research article entitled "Development of IgY antibodies against anti-snake toxins endowed with highly lethal neutralizing activity" (da Rocha et al., 2017) [1]. Complementarity-determining region (CDR) sequences are variable antibody (Ab) sequences that respond with specificity, duration and strength to identify and bind to antigen (Ag) epitopes. B lymphocytes isolated from hens immunized with Bitis arietans (Ba) and anti- Crotalus durissus terrificus (Cdt) venoms and expressing high specificity, affinity and toxicity neutralizing antibody titers were used as DNA sources. The VLF1, CDR1, CDR2, VLR1 and CDR3 sequences were validated by BLASTp, and values corresponding to IgY V L and V H anti-Ba or anti-Cdt venoms were identified, registered [ Gallus gallus IgY Fv Light chain (GU815099)/ Gallus gallus IgY Fv Heavy chain (GU815098)] and used for molecular modeling of IgY scFv anti-Ba. The resulting CDR1, CDR2 and CDR3 sequences were combined to construct the three - dimensional structure of the Ab paratope.

Highly efficient conductivity modulation of cinnamate-based light-responsive ionic liquids in aqueous solutions.

PubMed

Yang, Jie; Wang, Huiyong; Wang, Jianji; Zhang, Yue; Guo, Zhongjia

2014-12-11

A new class of cinnamate-based light-responsive ionic liquids was synthesized and characterized, and these ionic liquids with longer alkyl chains showed a remarkable increase in ionic conductivity under UV light irradiation in aqueous solutions.

A System-Oriented Approach for the Optimal Control of Process Chains under Stochastic Influences

NASA Astrophysics Data System (ADS)

Senn, Melanie; Schäfer, Julian; Pollak, Jürgen; Link, Norbert

2011-09-01

Process chains in manufacturing consist of multiple connected processes in terms of dynamic systems. The properties of a product passing through such a process chain are influenced by the transformation of each single process. There exist various methods for the control of individual processes, such as classical state controllers from cybernetics or function mapping approaches realized by statistical learning. These controllers ensure that a desired state is obtained at process end despite of variations in the input and disturbances. The interactions between the single processes are thereby neglected, but play an important role in the optimization of the entire process chain. We divide the overall optimization into two phases: (1) the solution of the optimization problem by Dynamic Programming to find the optimal control variable values for each process for any encountered end state of its predecessor and (2) the application of the optimal control variables at runtime for the detected initial process state. The optimization problem is solved by selecting adequate control variables for each process in the chain backwards based on predefined quality requirements for the final product. For the demonstration of the proposed concept, we have chosen a process chain from sheet metal manufacturing with simplified transformation functions.

Novel Strategy for Photopatterning Emissive Polymer Brushes for Organic Light Emitting Diode Applications

PubMed Central

2017-01-01

A light-mediated methodology to grow patterned, emissive polymer brushes with micron feature resolution is reported and applied to organic light emitting diode (OLED) displays. Light is used for both initiator functionalization of indium tin oxide and subsequent atom transfer radical polymerization of methacrylate-based fluorescent and phosphorescent iridium monomers. The iridium centers play key roles in photocatalyzing and mediating polymer growth while also emitting light in the final OLED structure. The scope of the presented procedure enables the synthesis of a library of polymers with emissive colors spanning the visible spectrum where the dopant incorporation, position of brush growth, and brush thickness are readily controlled. The chain-ends of the polymer brushes remain intact, affording subsequent chain extension and formation of well-defined diblock architectures. This high level of structure and function control allows for the facile preparation of random ternary copolymers and red–green–blue arrays to yield white emission. PMID:28691078

Novel Strategy for Photopatterning Emissive Polymer Brushes for Organic Light Emitting Diode Applications.

PubMed

Page, Zachariah A; Narupai, Benjaporn; Pester, Christian W; Bou Zerdan, Raghida; Sokolov, Anatoliy; Laitar, David S; Mukhopadhyay, Sukrit; Sprague, Scott; McGrath, Alaina J; Kramer, John W; Trefonas, Peter; Hawker, Craig J

2017-06-28

A light-mediated methodology to grow patterned, emissive polymer brushes with micron feature resolution is reported and applied to organic light emitting diode (OLED) displays. Light is used for both initiator functionalization of indium tin oxide and subsequent atom transfer radical polymerization of methacrylate-based fluorescent and phosphorescent iridium monomers. The iridium centers play key roles in photocatalyzing and mediating polymer growth while also emitting light in the final OLED structure. The scope of the presented procedure enables the synthesis of a library of polymers with emissive colors spanning the visible spectrum where the dopant incorporation, position of brush growth, and brush thickness are readily controlled. The chain-ends of the polymer brushes remain intact, affording subsequent chain extension and formation of well-defined diblock architectures. This high level of structure and function control allows for the facile preparation of random ternary copolymers and red-green-blue arrays to yield white emission.

Association of immunoglobulin G4 and free light chain with idiopathic pleural effusion.

PubMed

Murata, Y; Aoe, K; Mimura-Kimura, Y; Murakami, T; Oishi, K; Matsumoto, T; Ueoka, H; Matsunaga, K; Yano, M; Mimura, Y

2017-10-01

The cause of pleural effusion remains uncertain in approximately 15% of patients despite exhaustive evaluation. As recently described immunoglobulin (Ig)G4-related disease is a fibroinflammatory disorder that can affect various organs, including the lungs, we investigate whether idiopathic pleural effusion includes IgG4-associated etiology. Between 2000 and 2012, we collected 830 pleural fluid samples and reviewed 35 patients with pleural effusions undiagnosed after pleural biopsy at Yamaguchi-Ube Medical Center. Importantly, IgG4 immunostaining revealed infiltration of IgG4-positive plasma cells in the pleura of 12 patients (34%, IgG4 + group). The median effusion IgG4 level was 41 mg/dl in the IgG4 + group and 27 mg/dl in the IgG4 - group (P < 0·01). The light and heavy chains of effusion IgG4 antibodies of patients in the IgG4 + group were heterogeneous by two-dimensional electrophoresis, indicating the absence of clonality of the IgG4 antibodies. Interestingly, the κ light chains were more heterogeneous than the λ light chains. The measurement of the κ and λ free light chain (FLC) levels in the pleural fluids showed significantly different κ FLC levels (median: 28·0 versus 9·1 mg/dl, P < 0·01) and κ/λ ratios (median: 2·0 versus 1·2, P < 0·001) between the IgG4 + and IgG4 - groups. Furthermore, the κ/λ ratios were correlated with the IgG4 + /IgG + plasma cell ratios in the pleura of the IgG4 + group. Taken together, these results demonstrate the involvement of IgG4 in certain idiopathic pleural effusions and provide insights into the diagnosis, pathogenesis and therapeutic opportunities of IgG4-associated pleural effusion. © 2017 British Society for Immunology.

Matrix Metalloproteinases and their Tissue Inhibitors in Cardiac Amyloidosis: Relationship to Structural, Functional Myocardial Changes and to Light Chain Amyloid Deposition

PubMed Central

Biolo, Andreia; Ramamurthy, Sujata; Connors, Lawreen H.; O'Hara, Carl J.; Meier-Ewert, Hans K.; Hoo, Pamela T. Soo; Sawyer, Douglas B.; Seldin, David S.; Sam, Flora

2009-01-01

Background Cardiac amyloidosis is characterized by amyloid infiltration resulting in extracellular matrix (ECM) disruption. Amyloid cardiomyopathy due to immunoglobulin light chain protein (AL-CMP) deposition, has an accelerated clinical course and a worse prognosis compared to non-light chain cardiac amyloidoses i.e., forms associated with wild-type or mutated transthyretin (TTR). We therefore tested the hypothesis that determinants of proteolytic activity of the ECM, the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), would have distinct patterns and contribute to the pathogenesis of AL-CMP vs. TTR. Methods / Results We studied 40 patients with systemic amyloidosis: 10 AL-CMP patients, 20 patients with TTR-associated forms of cardiac amyloidosis, i.e. senile systemic amyloidois (SSA, involving wild-type TTR) or mutant TTR (ATTR), and 10 patients with AL amyloidosis without cardiac involvement. Serum MMP-2 and −9, TIMP-1, −2 and −4, brain natriuretic peptide (BNP) values and echocardiography were determined. AL-CMP and SSA-ATTR groups had similar degrees of increased left ventricular wall thickness (LVWT). However, BNP, MMP-9 and TIMP-1 levels were distinctly elevated accompanied by marked diastolic dysfunction in the AL-CMP group vs. no or minimal increases in the SSA-ATTR group. BNP, MMPs and TIMPs were not correlated with the degree of LVWT but were correlated to each other and to measures of diastolic dysfunction. Immunostaining of human endomyocardial biopsies showed diffuse expression of MMP-9 and TIMP-1 in AL-CMP and limited expression in SSA or ATTR hearts. Conclusions Despite comparable LVWT with TTR-related cardiac amyloidosis, AL-CMP patients have higher BNP, MMPs and TIMPs, which correlated with diastolic dysfunction. These findings suggest a relationship between light chains and ECM proteolytic activation that may play an important role in the functional and clinical manifestations of AL-CMP, distinct from the other non-light chain cardiac amyloidoses. PMID:19808299

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

DOE PAGES

Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; ...

2014-12-11

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

Immunoglobulin derived depositions in the nervous system: novel mass spectrometry application for protein characterization in formalin-fixed tissues.

PubMed

Rodriguez, Fausto J; Gamez, Jeffrey D; Vrana, Julie A; Theis, Jason D; Giannini, Caterina; Scheithauer, Bernd W; Parisi, Joseph E; Lucchinetti, Claudia F; Pendlebury, William W; Bergen, H Robert; Dogan, Ahmet

2008-10-01

Proteinaceous deposits are occasionally encountered in surgically obtained biopsies of the nervous system. Some of these are amyloidomas, although the precise nature of other cases remains uncertain. We studied 13 cases of proteinaceous aggregates in clinical specimens of the nervous system. Proteins contained within laser microdissected areas of interest were identified from tryptic peptide sequences by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). Immunohistochemical studies for immunoglobulin heavy and light chains and amyloidogenic proteins were performed in all cases. Histologically, the cases were classified into three groups: 'proteinaceous deposit not otherwise specified' (PDNOS) (n=6), amyloidoma (n=5), or 'intracellular crystals' (n=2). LC-MS/MS demonstrated the presence of lambda, but not kappa, light chain as well as serum amyloid P in all amyloidomas. lambda-Light-chain immunostaining was noted in amyloid (n=5), although demonstrable monotypic lymphoplasmacytic cells were seen in only one case. Conversely, in PDNOS kappa, but not lambda, was evident in five cases, both light chains being present in a single case. In three cases of PDNOS, a low-grade B-cell lymphoma consistent with marginal zone lymphoma was present in the brain specimen (n=2) or spleen (n=1). Lastly, in the 'intracellular crystals' group, the crystals were present within CD68+ macrophages in one case wherein kappa-light chain was found by LC-MS/MS only; the pathology was consistent with crystal-storing histiocytosis. In the second case, the crystals contained immunoglobulin G within CD138+ plasma cells. Our results show that proteinaceous deposits in the nervous system contain immunoglobulin components and LC-MS/MS accurately identifies the content of these deposits in clinical biopsy specimens. LC-MS/MS represents a novel application for characterization of these deposits and is of diagnostic utility in addition to standard immunohistochemical analyses.

Diffuse Peritoneal and Bowel Wall Infiltration by Light Chain-AL Amyloidosis with Omental Calcification Mimicking Abdominal Carcinomatosis - An Elderly Female with Incidental Finding of Light Chain Monoclonal Gammopathy of Undetermined Significance (LC-MGUS).

PubMed

Junejo, Shoaib; Ali, Yasir; Singh Lubana, Sandeep; Tuli, Sandeep S

2017-11-25

BACKGROUND Amyloidosis is the extracellular tissue deposition of plasma proteins, which after conformational changes, forms antiparallel beta pleated sheets of fibrils. Amyloid light-chain (AL) is a type of amyloidosis that is due to deposition of proteins derived from immunoglobulin (Ig) light chains. Gastrointestinal tract (GIT) involvement most often found in amyloid A (AA) amyloidosis type. There have been no reports of obstructive GIT AL amyloid patients having monoclonal gammopathy of undetermined significance (MGUS). Our case is the first case to show two coinciding conditions; one is the association of GIT AL amyloidosis with the incidental finding of a rare type of MGUS (LC-MGUS) and the other is the radiologic presentation of GIT amyloidosis with omental calcification mimicking the GIT malignancy. CASE REPORT A 68-year-old female presented with symptoms of partial bowel obstruction, including intermittent diffuse abdominal pain and constipation. After computed tomography (CT) abdomen and pelvis, an exploratory laparotomy was needed because of suspicion of abdominal carcinomatosis due to diffuse omental calcification. The tissue sent for biopsy surprisingly showed AL amyloidosis. The patient did not report any systemic symptoms. Further workup was advised to inquire about the plasma cell dyscrasia which eventually turned into a very rare version of MGUS knows as light chain MGUS (LC-MGUS). Following adequate resection of the involved structures, the patient was then placed on chemotherapy and successfully went into remission. CONCLUSIONS This case report illustrates that in an era of evidence based medicine, it is important to show through case reports the association of GIT AL amyloidosis with LC-MGUS, as the literature on this topic is lacking. It also points to the importance of timely intervention that can greatly enhance, not only the only the chances of remission but also prevention of further complications such as malignant transformation.

Heterogeneous and monoclonal helper T cells induce similar anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody populations in the primary adoptive response. II. Lambda light chain dominance and idiotope expression.

PubMed

Smith, F I; Tesch, H; Rajewsky, K

1984-02-01

When the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) is presented on different carrier molecules, different anti-NP antibody responses are stimulated. On stimulation with NP-lipopolysaccharide (LPS) [T-independent type 1 (TI-1) antigen] kappa + antibodies are the major population, whereas on stimulation with NP-Ficoll [T-independent type 2 (TI-2) antigen], NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin (CG) [T-dependent (TD) antigens], lambda 1+ antibodies dominate. The relative contribution of idiotopes Ac38 or Ac146 to the lambda 1+ anti-NP response was also different on comparison of TI-1 with TI-2 or TD anti-NP responses. We investigated whether light chain- or idiotype-specific T cells are responsible for these differences. Analysis of the anti-NP response of nude mice after immunization with NP-Ficoll showed lambda 1 dominance. Likewise primary adoptive transfer experiments using carrier-specific T cell lines to reconstitute the TD anti-NP response to NP-KLH or NP-CG, showed that help from carrier-specific T cells alone is capable of stimulating the characteristic lambda 1 dominant response. No significant difference could be found in the levels of Ac38 and Ac146 idiotope expression between mice reconstituted with splenic T cells and those reconstituted with T cell lines. These results suggest that light chain- or idiotype-specific T cells are required neither for the production of lambda 1 light chain dominance, nor for the appearance of idiotopes characteristic of the primary anti-NP response. The possible reasons for differences seen in both light chain and idiotope expression between primary anti-NP responses to the TI-1 antigen NP-LPS and those to TD or TI-2 antigens are discussed.

Variable context Markov chains for HIV protease cleavage site prediction.

PubMed

Oğul, Hasan

2009-06-01

Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA.

PubMed

Piette, J; Calberg-Bacq, C M; Lopez, M; van de Vorst, A

1984-04-05

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.

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.

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

A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain*

PubMed Central

Scruggs, Sarah B.; Reisdorph, Rick; Armstrong, Mike L.; Warren, Chad M.; Reisdorph, Nichole; Solaro, R. John; Buttrick, Peter M.

2010-01-01

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method. PMID:20445002

Therapeutic Approaches for Botulinum Intoxication Targeting Degradation of the Light Chain

DTIC Science & Technology

2014-06-01

protein and producing adequate amounts for in vitro testing. 15. SUBJECT TERMS- Botulinum toxin , ubiquitin, chimeric toxin light chains, LcA, LcE...that confer stability to LCs of botulinum toxin can be assessed by mutation of dileucine residues and systematic deletion of residues from LcA-LcE...cells. So What? Currently, there is no cure for botulinum poisoning once the toxin has entered a neuron. Moreover, the half-life of BoNT/A is very

Virtual medicinal chemistry: in silico pre-docking functional group transformation for discovery of novel inhibitors of botulinum toxin serotype A light chain.

PubMed

O'Malley, Sean; Sareth, Sina; Jiao, Guan-Sheng; Kim, Seongjin; Thai, April; Cregar-Hernandez, Lynne; McKasson, Linda; Margosiak, Stephen A; Johnson, Alan T

2013-05-01

A novel method for applying high-throughput docking to challenging metalloenzyme targets is described. The method utilizes information-based virtual transformation of library carboxylates to hydroxamic acids prior to docking, followed by compound acquisition, one-pot (two steps) chemical synthesis and in vitro screening. In two experiments targeting the botulinum neurotoxin serotype A metalloprotease light chain, hit rates of 32% and 18% were observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

Disseminated Mycobacterium intracellulare infection in a broad-snouted caiman Caiman latirostris.

PubMed

Kik, Marja J L

2013-11-25

A 10 yr old broad-snouted caiman Caiman latirostris from a small Dutch animal park was presented with long-term variable periods of anorexia and weight loss. Blood chemistry showed slightly elevated uric acid levels and low ionised calcium concentration. Ultrasonographical thickening of the intestinal wall in the region of the duodenum was evident. Pathological changes were a thickening of the wall of 90% of the small intestines, enlarged spleen with multifocal white foci and an enlarged light-brown liver. Histopathological lesions consisted of disseminated granulomas in the intestinal wall, the liver and the spleen. Multinucleated giant cells and epitheloid macrophages were abundant. Ziehl-Neelsen staining showed numerous intralesional acid-fast bacteria. Polymerase chain reaction for Mycobacterium intracellulare was positive.

Initiator and Photocatalyst-Free Visible Light Induced One-Pot Reaction: Concurrent RAFT Polymerization and CuAAC Click Reaction.

PubMed

Wang, Jie; Wang, Xinbo; Xue, Wentao; Chen, Gaojian; Zhang, Weidong; Zhu, Xiulin

2016-05-01

A new, visible light-catalyzed, one-pot and one-step reaction is successfully employed to design well-controlled side-chain functionalized polymers, by the combination of ambient temperature revisible addtion-fragmentation chain transfer (RAFT) polymerization and click chemistry. Polymerizations are well controlled in a living way under the irradiation of visible light-emitting diode (LED) light without photocatalyst and initiator, using the trithiocarbonate agent as iniferter (initiator-transfer agent-terminator) agent at ambient temperature. Fourier transfer infrared spectroscopy (FT-IR), NMR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) data confirm the successful one-pot reaction. Compared to the reported zero-valent metal-catalyzed one-pot reaction, the polymerization rate is much faster than that of the click reaction, and the visible light-catalyzed one-pot reaction can be freely and easily regulated by turning on and off the light. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling of an Adjustable Beam Solid State Light Project

NASA Technical Reports Server (NTRS)

Clark, Toni

2015-01-01

This proposal is for the development of a computational model of a prototype variable beam light source using optical modeling software, Zemax Optics Studio. The variable beam light source would be designed to generate flood, spot, and directional beam patterns, while maintaining the same average power usage. The optical model would demonstrate the possibility of such a light source and its ability to address several issues: commonality of design, human task variability, and light source design process improvements. An adaptive lighting solution that utilizes the same electronics footprint and power constraints while addressing variability of lighting needed for the range of exploration tasks can save costs and allow for the development of common avionics for lighting controls.

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

USDA-ARS?s Scientific Manuscript database

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

Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography.

PubMed

Kumaran, Desigan; Adler, Michael; Levit, Matthew; Krebs, Michael; Sweeney, Richard; Swaminathan, Subramanyam

2015-11-15

The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a ∼ 100 kDa heavy chain and a ∼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter. Published by Elsevier Ltd.

Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography

DOE PAGES

Kumaran, D.; Adler, M.; Levit, M.; ...

2015-10-17

The seven antigenically distinct serotypes (A to G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins: the heavy chain is responsible for neurospecific binding, internalization and translocation, and the light chain is responsible for substrate cleavage. Because of their extreme toxicity and prior history of weaponization, the BoNTs are considered to be potential bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than critical care;more » therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was synthesized and found to inhibit BoNT/A light chain (Balc) with high affinity. When tested in a cell-free Förster resonance excitation transfer (FRET) assay, CPI-1 was found to have a K i of 13.9 nM using full-length Balc448 and 42.1 nM using a truncated crystallizable form of light chain (Balc424). Co-crystallization of CPI-1 with Balc424 revealed that in the Balc-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn 2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter« less

Formation of pyroglutamic acid from N-terminal glutamic acid in immunoglobulin gamma antibodies.

PubMed

Chelius, Dirk; Jing, Kay; Lueras, Alexis; Rehder, Douglas S; Dillon, Thomas M; Vizel, Alona; Rajan, Rahul S; Li, Tiansheng; Treuheit, Michael J; Bondarenko, Pavel V

2006-04-01

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

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.

Discrete-Choice Modeling Of Non-Working Women’s Trip-Chaining Activity Based

NASA Astrophysics Data System (ADS)

Hayati, Amelia; Pradono; Purboyo, Heru; Maryati, Sri

2018-05-01

Start The urban developments of technology and economics are now changing the lifestyles of the urban societies. It is also changing their travel demand to meet their movement needs. Nowadays, urban women, especially in Bandung, West Java, have a high demand for their daily travel and tend to increase. They have the ease of accessibility to personal modes of transportation and freedom to go anywhere to meet their personal and family needs. This also happens to non-working women or as housewives in the city of Bandung. More than 50% of women’s mobility is outside the home, in the term of trip-chaining, from leaving to returning home in one day. It is based on their complex activities in order to meet the needs of family and home care. While less than 60% of male’s mobility is outdoors, it is a simple trip-chaining or only has a single trip. The trip-chaining has significant differences between non-working women and working-men. This illustrates the pattern of Mom and Dad’s mobility in a family with an activity-based approach for the same purpose, i.e. family welfare. This study explains how complex the trip-chaining of non-working urban women and as housewives, with an activity-based approach done outdoors in a week. Socio-economic and household demographic variables serve as the basis for measuring the independent variables affecting family welfare, as well as the variables of type, time and duration of activities performed by unemployed housewives. This study aims to examine the interrelationships between activity variables, especially the time of activity and travel, and socio-economic of household variables that can generate the complexity of women’s daily travel. Discrete Choice Modeling developed by Ben-Akiva, Chandra Bhat, etc., is used in this study to illustrate the relationship between activity and socio-economic demographic variables based on primary survey data in Bandung, West Java for 466 unemployed housewives. The results of the regression, by Seemingly Unrelated Regression approach methods, showed the interrelationship between all variables, including the complexity of trip chaining of housewives based on their daily activities. The type of mandatory and discretionary activities, and the duration of activities performed during the dismissal in the series of trip chains conducted are intended for the fulfillment of the welfare of all family member.

Serum Free Light Chain Assay and κ/λ Ratio: Performance in Patients With Monoclonal Gammopathy-High False Negative Rate for κ/λ Ratio

PubMed Central

Singh, Gurmukh

2017-01-01

Background Serum free light chain assay (SFLCA) and κ/λ ratio, and protein electrophoretic methods are used in the diagnosis and monitoring of monoclonal gammopathies. Methods Results for serum free light chains, serum and urine protein electrophoreses and immunofixation electrophoreses in 468 patients with a diagnosis of monoclonal gammopathy were compared. The results of the two methods were graded as concordant, non-concordant or discordant with the established diagnoses to assess the relative performance of the methods. Results of κ/λ ratio in samples with monoclonal protein detectable by electrophoretic methods were also analyzed. Results Protein electrophoreses results were concordant with the established diagnoses significantly more often than κ/λ ratio. The false negative rate for κ/λ ratio was higher than that for electrophoretic methods. κ/λ ratio was falsely negative in about 27% of the 1,860 samples with detectable monoclonal immunoglobulin. The false negative rate was higher in lesions with lambda chains (32%) than those with kappa chains (24%). The false negative rate for κ/λ ratio was over 55% in samples with monoclonal gammopathy of undetermined significance. Even at first encounter, the false negative rates for κ/λ ratios for monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma were 66.98%, 23.08%, and 30.15%, respectively, with false negative rate for lambda chain lesions being higher. Conclusions Electrophoretic studies of serum and urine are superior to SFLCA and κ/λ ratio. Abnormal κ/λ ratio, per se, is not diagnostic of monoclonal gammopathy. A normal κ/λ ratio does not exclude monoclonal gammopathy. False negative rates for lesions with lambda chain are higher than those for lesions with kappa chains. Electrophoretic studies of urine are underutilized. Clinical usefulness and medical necessity of SFLCA and κ/λ ratio is of questionable value in routine clinical testing. PMID:27924175

Serum Free Light Chain Assay and κ/λ Ratio: Performance in Patients With Monoclonal Gammopathy-High False Negative Rate for κ/λ Ratio.

PubMed

Singh, Gurmukh

2017-01-01

Serum free light chain assay (SFLCA) and κ/λ ratio, and protein electrophoretic methods are used in the diagnosis and monitoring of monoclonal gammopathies. Results for serum free light chains, serum and urine protein electrophoreses and immunofixation electrophoreses in 468 patients with a diagnosis of monoclonal gammopathy were compared. The results of the two methods were graded as concordant, non-concordant or discordant with the established diagnoses to assess the relative performance of the methods. Results of κ/λ ratio in samples with monoclonal protein detectable by electrophoretic methods were also analyzed. Protein electrophoreses results were concordant with the established diagnoses significantly more often than κ/λ ratio. The false negative rate for κ/λ ratio was higher than that for electrophoretic methods. κ/λ ratio was falsely negative in about 27% of the 1,860 samples with detectable monoclonal immunoglobulin. The false negative rate was higher in lesions with lambda chains (32%) than those with kappa chains (24%). The false negative rate for κ/λ ratio was over 55% in samples with monoclonal gammopathy of undetermined significance. Even at first encounter, the false negative rates for κ/λ ratios for monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma were 66.98%, 23.08%, and 30.15%, respectively, with false negative rate for lambda chain lesions being higher. Electrophoretic studies of serum and urine are superior to SFLCA and κ/λ ratio. Abnormal κ/λ ratio, per se , is not diagnostic of monoclonal gammopathy. A normal κ/λ ratio does not exclude monoclonal gammopathy. False negative rates for lesions with lambda chain are higher than those for lesions with kappa chains. Electrophoretic studies of urine are underutilized. Clinical usefulness and medical necessity of SFLCA and κ/λ ratio is of questionable value in routine clinical testing.

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

PubMed

Zhou, Xin X; Zou, Xinzhi; Chung, Hokyung K; Gao, Yuchen; Liu, Yanxia; Qi, Lei S; Lin, Michael Z

2018-02-16

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Planarian myosin essential light chain is involved in the formation of brain lateral branches during regeneration.

PubMed

Yu, Shuying; Chen, Xuhui; Yuan, Zuoqing; Zhou, Luming; Pang, Qiuxiang; Mao, Bingyu; Zhao, Bosheng

2015-08-01

The myosin essential light chain (ELC) is a structure component of the actomyosin cross-bridge, however, the functions in the central nervous system (CNS) development and regeneration remain poorly understood. Planarian Dugesia japonica has revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. In this study, the cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. It encodes a deduced protein with highly conserved functionally domains EF-Hand and Ca(2+) binding sites that shares significant similarity with other members of ELC. Whole mount in situ hybridization studies show that DjElc expressed in CNS during embryonic development and regeneration of adult planarians. Loss of function of DjElc by RNA interference during planarian regeneration inhibits brain lateral branches regeneration completely. In conclusion, these results demonstrated that DjElc is required for maintenance of neurons and neurite outgrowth, particularly for involving the brain later branch regeneration.

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

PubMed

Maire, E; Lelièvre, E; Brau, D; Lyons, A; Woodward, M; Fafeur, V; Vandenbunder, B

2000-04-10

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

Customer involvement in greening the supply chain: an interpretive structural modeling methodology

NASA Astrophysics Data System (ADS)

Kumar, Sanjay; Luthra, Sunil; Haleem, Abid

2013-04-01

The role of customers in green supply chain management needs to be identified and recognized as an important research area. This paper is an attempt to explore the involvement aspect of customers towards greening of the supply chain (SC). An empirical research approach has been used to collect primary data to rank different variables for effective customer involvement in green concept implementation in SC. An interpretive structural-based model has been presented, and variables have been classified using matrice d' impacts croises- multiplication appliqué a un classement analysis. Contextual relationships among variables have been established using experts' opinions. The research may help practicing managers to understand the interaction among variables affecting customer involvement. Further, this understanding may be helpful in framing the policies and strategies to green SC. Analyzing interaction among variables for effective customer involvement in greening SC to develop the structural model in the Indian perspective is an effort towards promoting environment consciousness.

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.

Integration of environmental aspects in modelling and optimisation of water supply chains.

PubMed

Koleva, Mariya N; Calderón, Andrés J; Zhang, Di; Styan, Craig A; Papageorgiou, Lazaros G

2018-04-26

Climate change becomes increasingly more relevant in the context of water systems planning. Tools are necessary to provide the most economic investment option considering the reliability of the infrastructure from technical and environmental perspectives. Accordingly, in this work, an optimisation approach, formulated as a spatially-explicit multi-period Mixed Integer Linear Programming (MILP) model, is proposed for the design of water supply chains at regional and national scales. The optimisation framework encompasses decisions such as installation of new purification plants, capacity expansion, and raw water trading schemes. The objective is to minimise the total cost incurring from capital and operating expenditures. Assessment of available resources for withdrawal is performed based on hydrological balances, governmental rules and sustainable limits. In the light of the increasing importance of reliability of water supply, a second objective, seeking to maximise the reliability of the supply chains, is introduced. The epsilon-constraint method is used as a solution procedure for the multi-objective formulation. Nash bargaining approach is applied to investigate the fair trade-offs between the two objectives and find the Pareto optimality. The models' capability is addressed through a case study based on Australia. The impact of variability in key input parameters is tackled through the implementation of a rigorous global sensitivity analysis (GSA). The findings suggest that variations in water demand can be more disruptive for the water supply chain than scenarios in which rainfalls are reduced. The frameworks can facilitate governmental multi-aspect decision making processes for the adequate and strategic investments of regional water supply infrastructure. Copyright © 2018. Published by Elsevier B.V.

Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.

PubMed

Seal, S N; Hoet, R M; Raats, J M; Radic, M Z

2000-09-01

To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

PubMed

Tesar, Devin; Hötzel, Isidro

2013-10-01

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

Spreaders and Sponges define metastasis in lung cancer: A Markov chain Monte Carlo Mathematical Model

PubMed Central

Newton, Paul K.; Mason, Jeremy; Bethel, Kelly; Bazhenova, Lyudmila; Nieva, Jorge; Norton, Larry; Kuhn, Peter

2013-01-01

The classic view of metastatic cancer progression is that it is a unidirectional process initiated at the primary tumor site, progressing to variably distant metastatic sites in a fairly predictable, though not perfectly understood, fashion. A Markov chain Monte Carlo mathematical approach can determine a pathway diagram that classifies metastatic tumors as ‘spreaders’ or ‘sponges’ and orders the timescales of progression from site to site. In light of recent experimental evidence highlighting the potential significance of self-seeding of primary tumors, we use a Markov chain Monte Carlo (MCMC) approach, based on large autopsy data sets, to quantify the stochastic, systemic, and often multi-directional aspects of cancer progression. We quantify three types of multi-directional mechanisms of progression: (i) self-seeding of the primary tumor; (ii) re-seeding of the primary tumor from a metastatic site (primary re-seeding); and (iii) re-seeding of metastatic tumors (metastasis re-seeding). The model shows that the combined characteristics of the primary and the first metastatic site to which it spreads largely determine the future pathways and timescales of systemic disease. For lung cancer, the main ‘spreaders’ of systemic disease are the adrenal gland and kidney, whereas the main ‘sponges’ are regional lymph nodes, liver, and bone. Lung is a significant self-seeder, although it is a ‘sponge’ site with respect to progression characteristics. PMID:23447576

Treatment With Bortezomib-based Therapy, Followed by Autologous Stem Cell Transplantation, Improves Outcomes in Light Chain Amyloidosis: A Retrospective Study.

PubMed

Jain, Tania; Kosiorek, Heidi E; Kung, Shu T; Shah, Vishal S; Dueck, Amylou C; Gonzalez-Calle, Veronica; Luft, Susan; Reeder, Craig B; Adams, Roberta; Noel, Pierre; Larsen, Jeremy T; Mikhael, Joseph; Bergsagel, Leif; Stewart, A Keith; Fonseca, Rafael

2018-05-04

The hematologic response is critical in patients with light chain amyloidosis because a good response is known to improve organ response and overall survival. We present a retrospective analysis to compare the hematologic and organ response in patients who received bortezomib-based therapy before autologous stem cell transplantation (ASCT) versus those who received non-bortezomib-based therapy before ASCT and those who underwent ASCT at diagnosis. Of a total of 63 patients who underwent ASCT for light chain amyloidosis, 34 received bortezomib-based therapy before ASCT (Bor-ASCT) and 29 did not receive bortezomib therapy (non-Bor-ASCT). A greater number of patients had involvement of ≥ 3 organs and cardiac involvement in the Bor-ASCT group, suggesting a greater risk at baseline in the Bor-ASCT group. At 3, 6, and 12 months after ASCT, the hematologic response was better in the Bor-ASCT group, with a statistically significance difference at 6 months (partial response or better in 82% vs. 20%; P = .002) and 12 months (partial response or better in 76% vs. 33%; P = .02). Organ responses (66% vs. 21%; P < .001) and median overall survival (not reached vs. 53 months; P = .001) were also greater in the Bor-ASCT group. Our study has shown that bortezomib-based therapy before ASCT improves the hematologic response, organ response and overall survival, potentially by decreasing the light chain load before ASCT. Copyright © 2018 Elsevier Inc. All rights reserved.

Covalent Binding Antibodies Suppress Advanced Glycation: On the Innate Tier of Adaptive Immunity

PubMed Central

Shcheglova, T.; Makker, S. P.

2009-01-01

Non-enzymatic protein glycation is a source of metabolic stress that contributes to cytotoxicity and tissue damage. Hyperglycemia has been linked to elevation of advanced glycation endproducts, which mediate much of the vascular pathology leading to diabetic complications. Enhanced glycation of immunoglobulins and their accelerated vascular clearance is proposed as a natural mechanism to intercept alternative advanced glycation endproducts, thereby mitigating microvascular disease. We reported that antibodies against the glycoprotein KLH have elevated reactivity for glycopeptides from diabetic serum. These reactions are mediated by covalent binding between antibody light chains and carbonyl groups of glycated peptides. Diabetic animals that were immunized to induce reactive antibodies had attenuated diabetic nephropathy, which correlated with reduced levels of circulating and kidney-bound glycation products. Molecular analysis of antibody glycation revealed the preferential modification of light chains bearing germline-encoded lambda V regions. We previously noted that antibody fragments carrying V regions in the germline configuration are selected from a human Fv library by covalent binding to a reactive organophosphorus ester. These Fv fragments were specifically modified at light chain V region residues, which map to the combining site at the interface between light and heavy chains. These findings suggest that covalent binding is an innate property of antibodies, which may be encoded in the genome for specific physiological purposes. This hypothesis is discussed in context with current knowledge of the natural antibodies that recognize altered self molecules and the catalytic autoantibodies found in autoimmune disease. PMID:22649604

Production and characterization of recombinant scFv against digoxin by phage display technology.

PubMed

Alirezapour, Behruz; Rajabibazl, Masoumeh; Rasaee, Mohhamad Javad; Omidfar, Kobra

2013-06-01

The cardiac glycoside digoxin is widely used for the treatment of congestive heart failure and cardiac arrhythmias. Digoxin is a highly toxic drug and consequently is routinely measured in sera of treated patients. In such cases, antibodies are required against digoxin for detection as well as detoxification purposes. To obtain recombinant single chain antibody against digoxin, RNA was extracted from spleen of BALB/c mice immunized with digoxin-BSA and converted to cDNA. The gene fragment corresponding to the variable regions of the repertoire of antibody genes were amplified by PCR. ScFv construct was generated by randomly joining individual heavy- and light-chain variable domains through gene splicing by overlapping extension PCR. Recombinant phage library expressing scFv polypeptides were produced. Phages with higher affinity toward digoxin were selected in the biopanning process. Sensitivity of produced recombinant MAb (AR85) was determined to be about 100 pg/well, while intact MAb (BBA) produced by hybridoma technology (data not shown) was reported to be around 100 pg/well too. The saturation value for recombinant scFv MAb was found to be 1000 ng/well while that for hybridoma MAb was reported to be 10 ng/well. The affinity constant of recombinant MAb (AR85) towards digoxin was also found to be around ka=3.8×10(7) M(-1) while that for hybridoma MAb (BBA) was reported to be ka=2.6×10(8) M(-1).

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

USDA-ARS?s Scientific Manuscript database

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

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

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.

Enhanced photocatalytic activity of degrading short chain chlorinated paraffins over reduced graphene oxide/CoFe2O4/Ag nanocomposite.

PubMed

Chen, Xin; Zhao, Qidong; Li, Xinyong; Wang, Dong

2016-10-01

Short chain chlorinated paraffins have recently attracted great attention because of their environmental persistence and biological toxicity as an important organic pollutant. In this work, reduced graphene oxide/CoFe2O4/Ag (RGO/CoFe2O4/Ag) nanocomposite was prepared and employed for photocatalytic degradation of short chain chlorinated paraffins. The process of photocatalytic degradation of short chain chlorinated paraffins over RGO/CoFe2O4/Ag under visible light (λ>400nm) was investigated by in situ Fourier transform infrared spectroscopy and the related mechanisms were proposed. An apparent degradation ratio of 91.9% over RGO/CoFe2O4/Ag could be obtained under visible light illumination of 12h, while only about 21.7% was obtained with commercial P25 TiO2 under the same experimental conditions, which demonstrates that the RGO/CoFe2O4/Ag nanocomposite is a potential candidate for effective photocatalytic removal of short chain chlorinated paraffins. Copyright © 2016 Elsevier Inc. All rights reserved.

Successful application of the dual-vector system II in creating a reliable phage-displayed combinatorial Fab library.

PubMed

Song, Suk-yoon; Hur, Byung-ung; Lee, Kyung-woo; Choi, Hyo-jung; Kim, Sung-soo; Kang, Goo; Cha, Sang-hoon

2009-03-31

The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously, but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted. Biopanning of one antibody library, termed DVFAB-1L library, which has a 1.3 x 10(7) combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II, an antibody library of a larger size, named DVFAB-131L, which has a 1.5 x 10(9) combinatorial antibody complexity, was also generated in a rapid manner by combining 1.3 x 10(7) heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort, and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecule.

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.

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.

Local recurrence as immunoglobulin G4 (IgG4)-related disease 10 years after radiotherapy to ocular adnexal extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue.

PubMed

Matsuo, Toshihiko; Ichimura, Kouichi; Yoshino, Tadashi

2011-01-01

In 2000, a 48-year-old woman developed a left orbital mass with lacrimal gland involvement and then, in 2003, a right orbital mass with lacrimal gland involvement, both of which were diagnosed as extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). She underwent 30 Gy external beam radiation to bilateral orbital lesions. The lymphoma cells in both lesions did not share the same clonality, as shown by amplification by polymerase chain reaction of the immunoglobulin heavy chain gene. Immunoglobulin light chain analysis by immunohistochemistry and messenger RNA in situ hybridization showed λ chain monotype in the left orbital lesion but κ chain monotype in the right orbital lesion. She developed recurrent left orbital mass with high uptake on fluorodeoxyglucose positron emission tomography fused with computed tomography in 2010, and excisional biopsy disclosed the formation of follicles and infiltration with immunoglobulin G4 (IgG4)-positive plasma cells mainly in interfollicular areas. The immunoglobulin light chain analysis showed the λ chain and κ chain bitype. With the immunohistopathological diagnosis of IgG4-related disease, the serum IgG4 level was found to show elevation at 376 mg/dL, and the patient chose observation. This is the first reported case of development of IgG4-related disease after bilataral orbital MALT lymphoma with external beam radiotherapy.

CSI 2264: CHARACTERIZING YOUNG STARS IN NGC 2264 WITH STOCHASTICALLY VARYING LIGHT CURVES

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

Stauffer, John; Rebull, Luisa; Carey, Sean

2016-03-15

We provide CoRoT and Spitzer light curves and other supporting data for 17 classical T Tauri stars in NGC 2264 whose CoRoT light curves exemplify the “stochastic” light curve class as defined in 2014 by Cody et al. The most probable physical mechanism to explain the optical variability within this light curve class is time-dependent mass accretion onto the stellar photosphere, producing transient hot spots. Where we have appropriate spectral data, we show that the veiling variability in these stars is consistent in both amplitude and timescale with the optical light curve morphology. The veiling variability is also well-correlated with the strengthmore » of the He i 6678 Å emission line, predicted by models to arise in accretion shocks on or near the stellar photosphere. Stars with accretion burst light curve morphology also have variable mass accretion. The stochastic and accretion burst light curves can both be explained by a simple model of randomly occurring flux bursts, with the stochastic light curve class having a higher frequency of lower amplitude events. Members of the stochastic light curve class have only moderate mass accretion rates. Their Hα profiles usually have blueshifted absorption features, probably originating in a disk wind. The lack of periodic signatures in the light curves suggests that little of the variability is due to long-lived hot spots rotating into or out of our line of sight; instead, the primary driver of the observed photometric variability is likely to be instabilities in the inner disk that lead to variable mass accretion.« less

Effect of low electric fields on alpha scintillation light yield in liquid argon

NASA Astrophysics Data System (ADS)

Agnes, P.; Albuquerque, I. F. M.; Alexander, T.; Alton, A. K.; Asner, D. M.; Back, H. O.; Baldin, B.; Biery, K.; Bocci, V.; Bonfini, G.; Bonivento, W.; Bossa, M.; Bottino, B.; Brigatti, A.; Brodsky, J.; Budano, F.; Bussino, S.; Cadeddu, M.; Cadoni, M.; Calaprice, F.; Canci, N.; Candela, A.; Caravati, M.; Cariello, M.; Carlini, M.; Catalanotti, S.; Cavalcante, P.; Chepurnov, A.; Cicalò, C.; Cocco, A. G.; Covone, G.; D'Angelo, D.; D'Incecco, M.; Davini, S.; De Cecco, S.; De Deo, M.; De Vincenzi, M.; Derbin, A.; Devoto, A.; Di Eusanio, F.; Di Pietro, G.; Dionisi, C.; Edkins, E.; Empl, A.; Fan, A.; Fiorillo, G.; Fomenko, K.; Forster, G.; Franco, D.; Gabriele, F.; Galbiati, C.; Giagu, S.; Giganti, C.; Giovanetti, G. K.; Goretti, A. M.; Granato, F.; Gromov, M.; Guan, M.; Guardincerri, Y.; Hackett, B. R.; Herner, K.; Hughes, D.; Humble, P.; Hungerford, E. V.; Ianni, A.; James, I.; Johnson, T. N.; Jollet, C.; Keeter, K.; Kendziora, C. L.; Koh, G.; Korablev, D.; Korga, G.; Kubankin, A.; Li, X.; Lissia, M.; Loer, B.; Lombardi, P.; Longo, G.; Ma, Y.; Machulin, I. N.; Mandarano, A.; Mari, S. M.; Maricic, J.; Marini, L.; Martoff, C. J.; Meregaglia, A.; Meyers, P. D.; Milincic, R.; Miller, J. D.; Montanari, D.; Monte, A.; Mount, B. J.; Muratova, V. N.; Musico, P.; Napolitano, J.; Navrer Agasson, A.; Odrowski, S.; Oleinik, A.; Orsini, M.; Ortica, F.; Pagani, L.; Pallavicini, M.; Pantic, E.; Parmeggiano, S.; Pelczar, K.; Pelliccia, N.; Pocar, A.; Pordes, S.; Pugachev, D. A.; Qian, H.; Randle, K.; Ranucci, G.; Razeti, M.; Razeto, A.; Reinhold, B.; Renshaw, A. L.; Rescigno, M.; Riffard, Q.; Romani, A.; Rossi, B.; Rossi, N.; Rountree, D.; Sablone, D.; Saggese, P.; Sands, W.; Savarese, C.; Schlitzer, B.; Segreto, E.; Semenov, D. A.; Shields, E.; Singh, P. N.; Skorokhvatov, M. D.; Smirnov, O.; Sotnikov, A.; Stanford, C.; Suvorov, Y.; Tartaglia, R.; Tatarowicz, J.; Testera, G.; Tonazzo, A.; Trinchese, P.; Unzhakov, E. V.; Verducci, M.; Vishneva, A.; Vogelaar, B.; Wada, M.; Walker, S.; Wang, H.; Wang, Y.; Watson, A. W.; Westerdale, S.; Wilhelmi, J.; Wojcik, M. M.; Xiang, X.; Xiao, X.; Xu, J.; Yang, C.; Zhong, W.; Zhu, C.; Zuzel, G.

2017-01-01

Measurements were made of scintillation light yield of alpha particles from the 222Rn decay chain within the DarkSide-50 liquid argon time projection chamber. The light yield was found to increase as the applied electric field increased, with alphas in a 200 V/cm electric field exhibiting a ~2% increase in light yield compared to alphas in no field.

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.

Control of in vivo disposition and immunogenicity of polymeric micelles by adjusting poly(sarcosine) chain lengths on surface

NASA Astrophysics Data System (ADS)

Kurihara, Kensuke; Ueda, Motoki; Hara, Isao; Ozeki, Eiichi; Togashi, Kaori; Kimura, Shunsaku

2017-07-01

Four kinds of A3B-type amphiphilic polydepsipeptides, (poly(sarcosine))3- b-poly( l-lactic acid) (the degree of polymerization of poly(sarcosine) are 10, 33, 55, and 85; S10 3 , S33 3 , S55 3 , and S85 3 ) were synthesized to prepare core-shell type polymeric micelles. Their in vivo dispositions and stimulations to trigger immune system to produce IgM upon multiple administrations to mice were examined. With increasing poly(sarcosine) chain lengths, the hydrophilic shell became thicker and the surface density at the most outer surface decreased on the basis of dynamic and static light scattering measurements. These two physical elements of polymeric micelles elicited opposite effects on the immune response in light of the chain length therefore to show an optimized poly(sarcosine) chain length existing between 33mer and 55mer to suppress the accelerated blood clearance phenomenon associated with polymeric micelles.

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.

Kepler Uniform Modeling of KOIs: MCMC Notes for Data Release 25

NASA Technical Reports Server (NTRS)

Hoffman, Kelsey L.; Rowe, Jason F.

2017-01-01

This document describes data products related to the reported planetary parameters and uncertainties for the Kepler Objects of Interest (KOIs) based on a Markov-Chain-Monte-Carlo (MCMC) analysis. Reported parameters, uncertainties and data products can be found at the NASA Exoplanet Archive . The codes used for this data analysis are available on the Github website (Rowe 2016). The relevant paper for details of the calculations is Rowe et al. (2015). The main differences between the model fits discussed here and those in the DR24 catalogue are that the DR25 light curves were used in the analysis, our processing of the MAST light curves took into account different data flags, the number of chains calculated was doubled to 200 000, and the parameters which are reported are based on a damped least-squares fit, instead of the median value from the Markov chain or the chain with the lowest 2 as reported in the past.

Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell gamma chain.

PubMed Central

Dialynas, D P; Murre, C; Quertermous, T; Boss, J M; Leiden, J M; Seidman, J G; Strominger, J L

1986-01-01

Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation. Images PMID:3458221

Is Congo red an amyloid-specific dye?

PubMed

Khurana, R; Uversky, V N; Nielsen, L; Fink, A L

2001-06-22

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.

Somatic hypermutation and antigen-driven selection of B cells are altered in autoimmune diseases.

PubMed

Zuckerman, Neta S; Hazanov, Helena; Barak, Michal; Edelman, Hanna; Hess, Shira; Shcolnik, Hadas; Dunn-Walters, Deborah; Mehr, Ramit

2010-12-01

B cells have been found to play a critical role in the pathogenesis of several autoimmune (AI) diseases. A common feature amongst many AI diseases is the formation of ectopic germinal centers (GC) within the afflicted tissue or organ, in which activated B cells expand and undergo somatic hypermutation (SHM) and antigen-driven selection on their immunoglobulin variable region (IgV) genes. However, it is not yet clear whether these processes occurring in ectopic GCs are identical to those in normal GCs. The analysis of IgV mutations has aided in revealing many aspects concerning B cell expansion, mutation and selection in GC reactions. We have applied several mutation analysis methods, based on lineage tree construction, to a large set of data, containing IgV productive and non-productive heavy and light chain sequences from several different tissues, to examine three of the most profoundly studied AI diseases - Rheumatoid Arthritis (RA), Multiple Sclerosis (MS) and Sjögren's Syndrome (SS). We have found that RA and MS sequences exhibited normal mutation spectra and targeting motifs, but a stricter selection compared to normal controls, which was more apparent in RA. SS sequence analysis results deviated from normal controls in both mutation spectra and indications of selection, also showing differences between light and heavy chain IgV and between different tissues. The differences revealed between AI diseases and normal control mutation patterns may result from the different microenvironmental influences to which ectopic GCs are exposed, relative to those in normal secondary lymphoid tissues. Copyright © 2010 Elsevier Ltd. All rights reserved.

Prognostic value of free light chains lambda and kappa in early multiple sclerosis.

PubMed

Voortman, Margarete M; Stojakovic, Tatjana; Pirpamer, Lukas; Jehna, Margit; Langkammer, Christian; Scharnagl, Hubert; Reindl, Markus; Ropele, Stefan; Seifert-Held, Thomas; Archelos, Juan-Jose; Fuchs, Siegrid; Enzinger, Christian; Fazekas, Franz; Khalil, Michael

2017-10-01

Cerebrospinal fluid (CSF) immunoglobulin free light chains (FLC) have been suggested as quantitative alternative to oligoclonal bands (OCB) in the diagnosis of multiple sclerosis (MS). However, little is known on their role in predicting clinical and paraclinical disease progression, particularly in early stages. To assess the prognostic value of FLC in OCB-positive patients with clinically isolated syndrome (CIS) suggestive of MS and early MS. We determined FLC kappa (KFLC) and lambda (LFLC) in CSF and serum by nephelometry in 61 patients (CIS ( n = 48), relapsing-remitting multiple sclerosis ( n = 13)) and 60 non-inflammatory neurological controls. Median clinical follow-up time in CIS was 4.8 years (interquartile range (IQR), 1.5-6.5 years). Patients underwent 3T magnetic resonance imaging (MRI) at baseline and follow-up (median time interval, 2.2 years; IQR, 1.0-3.7 years) to determine T2 lesion load (T2LL) and percent brain volume change (PBVC). CSF FLC were significantly increased in CIS/MS compared to controls (all p < 0.001). A lower KFLC/LFLC CSF ratio was associated with CIS-clinically definite multiple sclerosis (CDMS) conversion (hazard ratio (HR) = 2.89; 95% confidence interval (CI) = 1.17-7.14; p < 0.05). No correlations were found for FLC variables with T2LL or PBVC. Our study confirms increased intrathecal synthesis of FLC in CIS/MS which supports their diagnostic contribution. The KFLC/LFLC CSF ratio appears to have a prognostic value in CIS beyond OCB.

Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein*

PubMed Central

Sawano, Yoriko; Hatano, Ken-ichi; Miyakawa, Takuya; Tanokura, Masaru

2008-01-01

Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed φ dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition. PMID:18948264

Smart management of sample dilution using an artificial neural network to achieve streamlined processes and saving resources: the automated nephelometric testing of serum free light chain as case study.

PubMed

Ialongo, Cristiano; Pieri, Massimo; Bernardini, Sergio

2017-02-01

Saving resources is a paramount issue for the modern laboratory, and new trainable as well as smart technologies can be used to allow the automated instrumentation to manage samples more efficiently in order to achieve streamlined processes. In this regard the serum free light chain (sFLC) testing represents an interesting challenge, as it usually causes using a number of assays before achieving an acceptable result within the analytical range. An artificial neural network based on the multi-layer perceptron (MLP-ANN) was used to infer the starting dilution status of sFLC samples based on the information available through the laboratory information system (LIS). After the learning phase, the MLP-ANN simulation was applied to the nephelometric testing routinely performed in our laboratory on a BN ProSpec® System analyzer (Siemens Helathcare) using the N Latex FLC kit. The MLP-ANN reduced the serum kappa free light chain (κ-FLC) and serum lambda free light chain (λ-FLC) wasted tests by 69.4% and 70.8% with respect to the naïve stepwise dilution scheme used by the automated analyzer, and by 64.9% and 66.9% compared to a "rational" dilution scheme based on a 4-step dilution. Although it was restricted to follow-up samples, the MLP-ANN showed good predictive performance, which alongside the possibility to implement it in any automated system, made it a suitable solution for achieving streamlined laboratory processes and saving resources.

Cerebrospinal fluid and neuroimaging biomarker abnormalities suggest early neurological injury in a subset of individuals during primary HIV infection.

PubMed

Peluso, Michael J; Meyerhoff, Dieter J; Price, Richard W; Peterson, Julia; Lee, Evelyn; Young, Andrew C; Walter, Rudy; Fuchs, Dietmar; Brew, Bruce J; Cinque, Paola; Robertson, Kevin; Hagberg, Lars; Zetterberg, Henrik; Gisslén, Magnus; Spudich, Serena

2013-06-01

Cerebrospinal fluid (CSF) and neuroimaging abnormalities demonstrate neuronal injury during chronic AIDS, but data on these biomarkers during primary human immunodeficiency virus (HIV) infection is limited. We compared CSF concentrations of neurofilament light chain, t-tau, p-tau, amyloid precursor proteins, and amyloid-beta 42 in 92 subjects with primary HIV infection and 25 controls. We examined relationships with disease progression and neuroinflammation, neuropsychological testing, and proton-magnetic resonance spectroscopy (MRS)-based metabolites. Neurofilament light chain was elevated in primary HIV infection compared with controls (P = .0004) and correlated with CSF neopterin (r = 0.38; P = .0005), interferon gamma-induced protein 10 (r = 0.39; P = .002), white blood cells (r = 0.32; P = .004), protein (r = 0.59; P < .0001), and CSF/plasma albumin ratio (r = 0.60; P < .0001). Neurofilament light chain correlated with decreased N-acteylaspartate/creatine and glutamate/creatine in the anterior cingulate (r = -0.35, P = .02; r = -0.40, P = .009, respectively), frontal white matter (r = -0.43, P = .003; r = -0.30, P = .048, respectively), and parietal gray matter (r = -0.43, P = .003; r = -0.47, P = .001, respectively). Beta-amyloid was elevated in the primary infection group (P = .0005) and correlated with time infected (r = 0.34; P = .003). Neither marker correlated with neuropsychological abnormalities. T-tau and soluble amyloid precursor proteins did not differ between groups. Elevated neurofilament light chain and its correlation with MRS-based metabolites suggest early neuronal injury in a subset of participants with primary HIV infection through mechanisms involving central nervous system inflammation.

The Novel Zinc Finger Protein dASCIZ Regulates Mitosis in Drosophila via an Essential Role in Dynein Light-Chain Expression

PubMed Central

Zaytseva, Olga; Tenis, Nora; Mitchell, Naomi; Kanno, Shin-ichiro; Yasui, Akira; Heierhorst, Jörg; Quinn, Leonie M

2014-01-01

The essential zinc finger protein ASCIZ (also known as ATMIN, ZNF822) plays critical roles during lung organogenesis and B cell development in mice, where it regulates the expression of dynein light chain (DYNLL1/LC8), but its functions in other species including invertebrates are largely unknown. Here we report the identification of the Drosophila ortholog of ASCIZ (dASCIZ) and show that loss of dASCIZ function leads to pronounced mitotic delays with centrosome and spindle positioning defects during development, reminiscent of impaired dynein motor functions. Interestingly, similar mitotic and developmental defects were observed upon knockdown of the DYNLL/LC8-type dynein light chain Cutup (Ctp), and dASCIZ loss-of-function phenotypes could be suppressed by ectopic Ctp expression. Consistent with a genetic function of dASCIZ upstream of Ctp, we show that loss of dASCIZ led to reduced endogenous Ctp mRNA and protein levels and dramatically reduced Ctp–LacZ reporter gene activity in vivo, indicating that dASCIZ regulates development and mitosis as a Ctp transcription factor. We speculate that the more severe mitotic defects in the absence of ASCIZ in flies compared to mice may be due to redundancy with a second, ASCIZ-independent, Dynll2 gene in mammals in contrast to a single Ctp gene in Drosophila. Altogether, our data demonstrate that ASCIZ is an evolutionary highly conserved transcriptional regulator of dynein light-chain levels and a novel regulator of mitosis in flies. PMID:24336747

White Polymer Light-Emitting Diodes Based on Exciplex Electroluminescence from Polymer Blends and a Single Polymer.

PubMed

Liang, Junfei; Zhao, Sen; Jiang, Xiao-Fang; Guo, Ting; Yip, Hin-Lap; Ying, Lei; Huang, Fei; Yang, Wei; Cao, Yong

2016-03-09

In this Article, we designed and synthesized a series of polyfluorene derivatives, which consist of the electron-rich 4,4'-(9-alkyl-carbazole-3,6-diyl)bis(N,N-diphenylaniline) (TPA-Cz) in the side chain and the electron-deficient dibenzothiophene-5,5-dioxide (SO) unit in the main chain. The resulting copolymer PF-T25 that did not comprise the SO unit exhibited blue light-emission with the Commission Internationale de L'Eclairage coordinates of (0.16, 0.10). However, by physically blending PF-T25 with a blue light-emitting SO-based oligomer, a novel low-energy emission correlated to exciplex emerged due to the appropriate energy level alignment of TPA-Cz and the SO-based oligomers, which showed extended exciton lifetime as confirmed by time-resolved photoluminescent spectroscopy. The low-energy emission was also identified in copolymers consisting of SO unit in the main chain, which can effectively compensate for the high-energy emission to produce binary white light-emission. Polymer light-emitting diodes based on the exciplex-type single greenish-white polymer exhibit the peak luminous efficiency of 2.34 cd A(-1) and the maximum brightness of 12 410 cd m(-2), with Commission Internationale de L'Eclairage color coordinates (0.27, 0.39). The device based on such polymer showed much better electroluminescent stability than those based on blending films. These observations indicated that developing a single polymer with the generated exciplex emission can be a novel and effective molecular design strategy toward highly stable and efficient white polymer light-emitting diodes.

Synthesis of α,β-unsaturated aldehydes as potential substrates for bacterial luciferases.

PubMed

Brodl, Eveline; Ivkovic, Jakov; Tabib, Chaitanya R; Breinbauer, Rolf; Macheroux, Peter

2017-02-15

Bacterial luciferase catalyzes the monooxygenation of long-chain aldehydes such as tetradecanal to the corresponding acid accompanied by light emission with a maximum at 490nm. In this study even numbered aldehydes with eight, ten, twelve and fourteen carbon atoms were compared with analogs having a double bond at the α,β-position. These α,β-unsaturated aldehydes were synthesized in three steps and were examined as potential substrates in vitro. The luciferase of Photobacterium leiognathi was found to convert these analogs and showed a reduced but significant bioluminescence activity compared to tetradecanal. This study showed the trend that aldehydes, both saturated and unsaturated, with longer chain lengths had higher activity in terms of bioluminescence than shorter chain lengths. The maximal light intensity of (E)-tetradec-2-enal was approximately half with luciferase of P. leiognathi, compared to tetradecanal. Luciferases of Vibrio harveyi and Aliivibrio fisheri accepted these newly synthesized substrates but light emission dropped drastically compared to saturated aldehydes. The onset and the decay rate of bioluminescence were much slower, when using unsaturated substrates, indicating a kinetic effect. As a result the duration of the light emission is doubled. These results suggest that the substrate scope of bacterial luciferases is broader than previously reported. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase

PubMed Central

Baumann, Fabian; Bauer, Magnus Sebastian; Rees, Martin; Alexandrovich, Alexander; Gautel, Mathias; Pippig, Diana Angela; Gaub, Hermann Eduard

2017-01-01

Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region. DOI: http://dx.doi.org/10.7554/eLife.26473.001 PMID:28696205

Precise Protein Photolithography (P3): High Performance Biopatterning Using Silk Fibroin Light Chain as the Resist

PubMed Central

Liu, Wanpeng; Zhou, Zhitao; Zhang, Shaoqing; Shi, Zhifeng; Tabarini, Justin; Lee, Woonsoo; Zhang, Yeshun; Gilbert Corder, S. N.; Li, Xinxin; Dong, Fei; Cheng, Liang; Liu, Mengkun; Kaplan, David L.; Omenetto, Fiorenzo G.

2017-01-01

Precise patterning of biomaterials has widespread applications, including drug release, degradable implants, tissue engineering, and regenerative medicine. Patterning of protein‐based microstructures using UV‐photolithography has been demonstrated using protein as the resist material. The Achilles heel of existing protein‐based biophotoresists is the inevitable wide molecular weight distribution during the protein extraction/regeneration process, hindering their practical uses in the semiconductor industry where reliability and repeatability are paramount. A wafer‐scale high resolution patterning of bio‐microstructures using well‐defined silk fibroin light chain as the resist material is presented showing unprecedent performances. The lithographic and etching performance of silk fibroin light chain resists are evaluated systematically and the underlying mechanisms are thoroughly discussed. The micropatterned silk structures are tested as cellular substrates for the successful spatial guidance of fetal neural stems cells seeded on the patterned substrates. The enhanced patterning resolution, the improved etch resistance, and the inherent biocompatibility of such protein‐based photoresist provide new opportunities in fabricating large scale biocompatible functional microstructures. PMID:28932678

The invasion mode of GH(3) cells is conditioned by collagen subtype, and its efficiency depends on cell-cell adhesion.

PubMed

Azorín, Erika; Solano-Agama, Carmen; Mendoza-Garrido, M Eugenia

2012-12-15

The adaptation of GH(3) cells to different microenvironments is a consequence of a partial compromise with the tumor phenotype. A collagen type IV enriched microenvironment favors an invasive phenotype and increases the substrate adhesion capacity, whereas it decreases the phosphorylation of the regulatory myosin light chain and the aggregation capacity. In contrast, the higher internal tension and increased aggregation capacity induced by collagen type I/III are factors that reduce the invasion rate. Our results show, for the first time, the importance of collagen subtypes in determining the migratory strategy: collagen I/III favors mesenchymal-like motility, whereas collagen type IV induces an ameboid-type displacement. The reciprocal modulation of the myosin light chain kinase and the Rho-kinase determines the invasive capacity through changes in tissue cohesion, extracellular matrix affinity, regulatory myosin light chain phosphorylation and spatial distribution. The collagen subtype determines which of the mechano-transduction signaling pathways will regulate the tensional homeostasis and affect the invasion ability as well as the preferred migration strategy of the cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo

PubMed Central

Fazal, Fabeha; Gu, Lianzhi; Ihnatovych, Ivanna; Han, YooJeong; Hu, WenYang; Antic, Nenad; Carreira, Fernando; Blomquist, James F.; Hope, Thomas J.; Ucker, David S.; de Lanerolle, Primal

2005-01-01

Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis. PMID:15988034

First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate.

PubMed

Dooley, Helen; Stanfield, Robyn L; Brady, Rebecca A; Flajnik, Martin F

2006-02-07

The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.

Rapid detection of hepatitis B virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis B virus surface antigen.

PubMed

Chen, Yu-Ping; Qiao, Yuan-Yuan; Zhao, Xiao-Hang; Chen, Hong-Song; Wang, Yan; Wang, Zhuozhi

2007-06-01

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.

Rapid Detection of Hepatitis B Virus Surface Antigen by an Agglutination Assay Mediated by a Bispecific Diabody against Both Human Erythrocytes and Hepatitis B Virus Surface Antigen▿

PubMed Central

Chen, Yu-Ping; Qiao, Yuan-Yuan; Zhao, Xiao-Hang; Chen, Hong-Song; Wang, Yan; Wang, Zhuozhi

2007-01-01

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens. PMID:17442848

Engineering of a novel zipFv using leucine zipper motif against rabies virus glycoprotein G with improved protection potency in vivo.

PubMed

Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Li, Zhuang; Cheng, Yue; Jiang, Chunlai; Kong, Wei; Wu, Yongge

2017-06-01

Rabies is an acute zoonotic infectious disease with a high fatality rate but is preventable with vaccination and rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv), a small engineered antigen-binding protein derived from antibody variable heavy (V H ) and light (V L ) chains connected by a peptide linker, can potentially be used to replace RIG. Here, we produced two peptides V H -JUN-HIS and V L -FOS-HA separately in Escherichia coli and assembled them to form zipFv successfully in vitro. The new zipFv utilizes FOS and JUN leucine zippers to form an antibody structure similar to the IgG counterpart with two free N-terminal ends of V H and V L . The zipFv protein showed notable improvement in binding ability and affinity over its corresponding scFv. The zipFv also demonstrated greater stability in serum and the same protective rate as RIG against challenge with a standard rabies virus (CVS-24) in mice. Our results indicated zipFv as a novel and efficient antibody form with enhanced neutralizing potency. Copyright © 2017. Published by Elsevier B.V.

Basic Properties of Strong Mixing Conditions.

DTIC Science & Technology

1985-06-01

H. Dehling and W. Philipp. Almost sure invariance principles for weakly dependent vector-valued random variables. Ann. Probab. 10 (1982) 689-701. 34...Harris chains will not be discussed here.) It is well known that every stationary Harris chain has a well defined " period " p E {1,2,3,... (the chain is...chain is absolutely regular. (ii) More generally, for any strictly stationary real Harris chain, lim n (o in) = 1 - 1/p where p is the period . A

Identification of the significant factors in food safety using global sensitivity analysis and the accept-and-reject algorithm: application to the cold chain of ham.

PubMed

Duret, Steven; Guillier, Laurent; Hoang, Hong-Minh; Flick, Denis; Laguerre, Onrawee

2014-06-16

Deterministic models describing heat transfer and microbial growth in the cold chain are widely studied. However, it is difficult to apply them in practice because of several variable parameters in the logistic supply chain (e.g., ambient temperature varying due to season and product residence time in refrigeration equipment), the product's characteristics (e.g., pH and water activity) and the microbial characteristics (e.g., initial microbial load and lag time). This variability can lead to different bacterial growth rates in food products and has to be considered to properly predict the consumer's exposure and identify the key parameters of the cold chain. This study proposes a new approach that combines deterministic (heat transfer) and stochastic (Monte Carlo) modeling to account for the variability in the logistic supply chain and the product's characteristics. The model generates a realistic time-temperature product history , contrary to existing modeling whose describe time-temperature profile Contrary to existing approaches that use directly a time-temperature profile, the proposed model predicts product temperature evolution from the thermostat setting and the ambient temperature. The developed methodology was applied to the cold chain of cooked ham including, the display cabinet, transport by the consumer and the domestic refrigerator, to predict the evolution of state variables, such as the temperature and the growth of Listeria monocytogenes. The impacts of the input factors were calculated and ranked. It was found that the product's time-temperature history and the initial contamination level are the main causes of consumers' exposure. Then, a refined analysis was applied, revealing the importance of consumer behaviors on Listeria monocytogenes exposure. Copyright © 2014. Published by Elsevier B.V.

Analysis of correlated domain motions in IgG light chain reveals possible mechanisms of immunological signal transduction.

PubMed

Król, Marcin; Roterman, Irena; Piekarska, Barbara; Konieczny, Leszek; Rybarska, Janina; Stopa, Barbara; Spólnik, Paweł

2005-05-15

It was shown experimentally that binding of a micelle composed of Congo red molecules to immunological complexes leads to the enhanced stability of the latter, and simultaneously prevents binding of a complement molecule (C1q). The dye binds in a cavity created by the removal of N-terminal polypeptide chain, as observed experimentally in a model system-immunoglobulin G (IgG) light chain dimer. Molecular Dynamics (MD) simulations of three forms of IgG light chain dimer, with and without the dye, were performed to investigate the role of N-terminal fragment and self-assembled ligand in coupling between V and C domains. Root-mean-square distance (RMSD) time profiles show that removal of N-terminal fragment leads to destabilization of V domain. A micelle composed of four self-assembled dye molecules stabilizes and fixes the domain. Analysis of root-mean-square fluctuation (RMSF) values and dynamic cross-correlation matrices (DCCM) reveals that removal of N-terminal fragment results in complete decoupling between V and C domains. Binding of self-assembled Congo red molecules improves the coupling, albeit slightly. The disruption of a small beta-sheet composed of N- and C-terminal fragments of the domain (NC sheet) is the most likely reason for the decoupling. Self-assembled ligand, bound in the place originally occupied by N-terminal fragment, is not able to take over the function of the beta-sheet. Lack of correlation of motions between residues in V and C domains denotes that light chain-Congo red complexes have hampered ability to transmit conformational changes between domains. This is a likely explanation of the lack of complement binding by immunological complexes, which bind Congo red, and supports the idea that the NC sheet is the key structural fragment taking part in immunological signal transduction. Copyright 2005 Wiley-Liss, Inc.

On-shell constrained M 2 variables with applications to mass measurements and topology disambiguation

NASA Astrophysics Data System (ADS)

Cho, Won Sang; Gainer, James S.; Kim, Doojin; Matchev, Konstantin T.; Moortgat, Filip; Pape, Luc; Park, Myeonghun

2014-08-01

We consider a class of on-shell constrained mass variables that are 3+1 dimensional generalizations of the Cambridge M T2 variable and that automatically incorporate various assumptions about the underlying event topology. The presence of additional on-shell constraints causes their kinematic distributions to exhibit sharper endpoints than the usual M T2 distribution. We study the mathematical properties of these new variables, e.g., the uniqueness of the solution selected by the minimization over the invisible particle 4-momenta. We then use this solution to reconstruct the masses of various particles along the decay chain. We propose several tests for validating the assumed event topology in missing energy events from new physics. The tests are able to determine: 1) whether the decays in the event are two-body or three-body, 2) if the decay is two-body, whether the intermediate resonances in the two decay chains are the same, and 3) the exact sequence in which the visible particles are emitted from each decay chain.

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.

Charge-transfer dynamics in one-dimensional C 60 chains

NASA Astrophysics Data System (ADS)

Pérez-Dieste, V.; Tamai, A.; Greber, T.; Chiuzbaˇian, S. G.; Patthey, L.

2008-06-01

Charge transfer in highly-ordered C 60 chains grown on a Cu(5 5 3) vicinal surface is studied by means of resonant photoemission. Tuning the light polarization, autoionization of the highest occupied molecular orbital (HOMO) was expected to detect anisotropy in this one-dimensional system. For one monolayer C 60 we found no signature of autoionization. This indicates that for an electron which is excited from the C 1s level of C 60 to the lowest unoccupied molecular orbital (LUMO), hybridization leads to delocalization on the femtosecond time-scale and no influence of the light polarization is observed.

Cavity-Enhanced Raman Spectroscopy for Food Chain Management

PubMed Central

Sandfort, Vincenz; Goldschmidt, Jens; Wöllenstein, Jürgen

2018-01-01

Comprehensive food chain management requires the monitoring of many parameters including temperature, humidity, and multiple gases. The latter is highly challenging because no low-cost technology for the simultaneous chemical analysis of multiple gaseous components currently exists. This contribution proposes the use of cavity enhanced Raman spectroscopy to enable online monitoring of all relevant components using a single laser source. A laboratory scale setup is presented and characterized in detail. Power enhancement of the pump light is achieved in an optical resonator with a Finesse exceeding 2500. A simulation for the light scattering behavior shows the influence of polarization on the spatial distribution of the Raman scattered light. The setup is also used to measure three relevant showcase gases to demonstrate the feasibility of the approach, including carbon dioxide, oxygen and ethene. PMID:29495501

Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

USDA-ARS?s Scientific Manuscript database

A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

Human NF-κB1 Haploinsufficiency and Epstein-Barr Virus-Induced Disease-Molecular Mechanisms and Consequences.

PubMed

Hoeger, Birgit; Serwas, Nina Kathrin; Boztug, Kaan

2017-01-01

Nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κB1)-related human primary immune deficiencies have initially been characterized as defining a subgroup of common variable immunodeficiencies (CVIDs), representing intrinsic B-cell disorders with antibody deficiency and recurrent infections of various kind. Recent evidence indicates that NF-κB1 haploinsufficiency underlies a variable type of combined immunodeficiency (CID) affecting both B and T lymphocyte compartments, with a broadened spectrum of disease manifestations, including Epstein-Barr virus (EBV)-induced lymphoproliferative disease and immediate life-threatening consequences. As part of this review series focused on EBV-related primary immunodeficiencies, we discuss the current clinical and molecular understanding of monoallelic NFKB1 germline mutations with special focus on the emerging context of EBV-associated disease. We outline mechanistic implications of dysfunctional NF-κB1 in B and T cells and discuss the fatal relation of impaired T-cell function with the inability to clear EBV infections. Finally, we compare common and suggested treatment angles in the context of this complex disease.

Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences

PubMed Central

Hoeger, Birgit; Serwas, Nina Kathrin; Boztug, Kaan

2018-01-01

Nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κB1)-related human primary immune deficiencies have initially been characterized as defining a subgroup of common variable immunodeficiencies (CVIDs), representing intrinsic B-cell disorders with antibody deficiency and recurrent infections of various kind. Recent evidence indicates that NF-κB1 haploinsufficiency underlies a variable type of combined immunodeficiency (CID) affecting both B and T lymphocyte compartments, with a broadened spectrum of disease manifestations, including Epstein–Barr virus (EBV)-induced lymphoproliferative disease and immediate life-threatening consequences. As part of this review series focused on EBV-related primary immunodeficiencies, we discuss the current clinical and molecular understanding of monoallelic NFKB1 germline mutations with special focus on the emerging context of EBV-associated disease. We outline mechanistic implications of dysfunctional NF-κB1 in B and T cells and discuss the fatal relation of impaired T-cell function with the inability to clear EBV infections. Finally, we compare common and suggested treatment angles in the context of this complex disease. PMID:29403474

Airway structural alterations selectively associated with severe asthma.

PubMed

Benayoun, Laurent; Druilhe, Anne; Dombret, Marie-Christine; Aubier, Michel; Pretolani, Marina

2003-05-15

To identify airway pathologic abnormalities selectively associated with severe asthma, we examined 10 control subjects, 10 patients with intermittent asthma, 15 patients with mild-to-moderate persistent asthma, 15 patients with severe persistent asthma, and 10 patients with chronic obstructive pulmonary disease. Bronchial biopsies were assessed for epithelial integrity; subepithelial basement membrane (SBM) thickness; collagen type III deposition; eosinophil, neutrophil, and fibroblast numbers; mucous gland and airway smooth muscle (ASM) areas; SBM-ASM distance; ASM hypertrophy (increased cell size); and the expression of the contractile proteins alpha-actin, smooth muscle myosin heavy-chain isoforms, myosin light-chain kinase, and the phosphorylated form of the regulatory light chain of myosin. Neither mucosal eosinophilia nor neutrophilia, epithelial damage, or SBM thickness reflected asthma severity. In contrast, higher numbers of fibroblasts (p < 0.001), an increase in collagen type III deposition (p < 0.020), larger mucous gland (p < 0.040) and ASM (p < 0.001) areas, augmented ASM cell size (p < 0.001), and myosin light-chain kinase expression (p < 0.005) distinguished patients with severe persistent asthma from patients with milder disease or with chronic obstructive pulmonary disease. Stepwise multivariate regression analysis established that fibroblast numbers and ASM cell size were negatively associated with prebronchodilator and postbronchodilator FEV1 values in patients with asthma. We conclude that fibroblast accumulation and ASM hypertrophy in proximal airways are selective determinants of severe persistent asthma.

Bruton's tyrosine kinase and SLP-65 regulate pre-B cell differentiation and the induction of Ig light chain gene rearrangement.

PubMed

Kersseboom, Rogier; Ta, Van B T; Zijlstra, A J Esther; Middendorp, Sabine; Jumaa, Hassan; van Loo, Pieter Fokko; Hendriks, Rudolf W

2006-04-15

Bruton's tyrosine kinase (Btk) and the adapter protein SLP-65 (Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa) transmit precursor BCR (pre-BCR) signals that are essential for efficient developmental progression of large cycling into small resting pre-B cells. We show that Btk- and SLP-65-deficient pre-B cells have a specific defect in Ig lambda L chain germline transcription. In Btk/SLP-65 double-deficient pre-B cells, both kappa and lambda germline transcripts are severely reduced. Although these observations point to an important role for Btk and SLP-65 in the initiation of L chain gene rearrangement, the possibility remained that these signaling molecules are only required for termination of pre-B cell proliferation or for pre-B cell survival, whereby differentiation and L chain rearrangement is subsequently initiated in a Btk/SLP-65-independent fashion. Because transgenic expression of the antiapoptotic protein Bcl-2 did not rescue the developmental arrest of Btk/SLP-65 double-deficient pre-B cells, we conclude that defective L chain opening in Btk/SLP-65-deficient small resting pre-B cells is not due to their reduced survival. Next, we analyzed transgenic mice expressing the constitutively active Btk mutant E41K. The expression of E41K-Btk in Ig H chain-negative pro-B cells induced 1) surface marker changes that signify cellular differentiation, including down-regulation of surrogate L chain and up-regulation of CD2, CD25, and MHC class II; and 2) premature rearrangement and expression of kappa and lambda light chains. These findings demonstrate that Btk and SLP-65 transmit signals that induce cellular maturation and Ig L chain rearrangement independently of their role in termination of pre-B cell expansion.

Exact relativistic Toda chain eigenfunctions from Separation of Variables and gauge theory

NASA Astrophysics Data System (ADS)

Sciarappa, Antonio

2017-10-01

We provide a proposal, motivated by Separation of Variables and gauge theory arguments, for constructing exact solutions to the quantum Baxter equation associated to the N-particle relativistic Toda chain and test our proposal against numerical results. Quantum Mechanical non-perturbative corrections, essential in order to obtain a sensible solution, are taken into account in our gauge theory approach by considering codimension two defects on curved backgrounds (squashed S 5 and degenerate limits) rather than flat space; this setting also naturally incorporates exact quantization conditions and energy spectrum of the relativistic Toda chain as well as its modular dual structure.

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

PubMed Central

2015-01-01

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

Monoclonal antibodies for the identification and purification of vNAR domains and IgNAR immunoglobulins from the horn shark Heterodontus francisci.

PubMed

Juarez, Karla; Dubberke, Gudrun; Lugo, Pavel; Koch-Nolte, Friedrich; Buck, Friedrich; Haag, Friedrich; Licea, Alexei

2011-08-01

In addition to conventional antibodies, cartilaginous fish have evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs). These, together with the VHH camel domains, constitute the smallest naturally occurring domains able to recognize an antigen. Their special features, such as small size, long extended finger-like CDR3, and thermal and chemical stability, make them suitable candidates for biotechnological purposes. Here we describe the generation of two mouse monoclonal antibodies (MAbs), MAb 370-12 and MAb 533-10, that both specifically react with vNAR domains of the horn shark Heterodontus francisci. While the former recognizes a broad spectrum of recombinant vNAR proteins, the latter is more restricted. MAb 370-12 precipitated a single band from whole shark serum, which was identified as IgNAR by mass spectrometry. Additionally, we used MAb 370-12 to follow the IgNAR-mediated immune response of sharks during immunization protocols with two different antigens (complete cells and a synthethic peptide), thus corroborating that MAb 370-12 recognizes both isolated vNAR domains and whole IgNAR molecules. Both MAbs represent an affordable molecular, biochemical, and biotechnological tool in the field of shark single-domain antibodies.

Designing Performance Measurement For Supply Chain's Actors And Regulator Using Scale Balanced Scorecard And Data Envelopment Analysis

NASA Astrophysics Data System (ADS)

Kusrini, Elisa; Subagyo; Aini Masruroh, Nur

2016-01-01

This research is a sequel of the author's earlier conducted researches in the fields of designing of integrated performance measurement between supply chain's actors and regulator. In the previous paper, the design of performance measurement is done by combining Balanced Scorecard - Supply Chain Operation Reference - Regulator Contribution model and Data Envelopment Analysis. This model referred as B-S-Rc-DEA model. The combination has the disadvantage that all the performance variables have the same weight. This paper investigates whether by giving weight to performance variables will produce more sensitive performance measurement in detecting performance improvement. Therefore, this paper discusses the development of the model B-S-Rc-DEA by giving weight to its performance'variables. This model referred as Scale B-S-Rc-DEA model. To illustrate the model of development, some samples from small medium enterprises of leather craft industry supply chain in province of Yogyakarta, Indonesia are used in this research. It is found that Scale B-S-Rc-DEA model is more sensitive to detecting performance improvement than B-S- Rc-DEA model.

Established a new double antibodies sandwich enzyme-linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naïve mouse phage displayed library.

PubMed

Zhang, Xiao; Xu, Chongxin; Zhang, Cunzheng; Liu, Yuan; Xie, Yajing; Liu, Xianjin

2014-04-01

ScFvs are composed of the variable regions of the heavy and light chains via a short linker that maintain the specific antigen binding abilities of antibodies. In this study, we constructed a naïve mouse phage displayed library to generate scFvs against Cry1Ab toxin. After affinity panning, positive phage-scFvs were isolated, sequenced and characterized by ELISA. The best binding ability scFv-G9 was expressed and purified. SDS-PAGE indicated that the relative molecular mass of scFv was estimated at 28 kDa. The purified scFv-G9 was used to develop a new DAS-ELISA for detecting Cry1Ab toxin, within minimum detection limit of 0.008 μg mL(-1), a working range 0.018-6.23 μg mL(-1), and the linear curve displayed an acceptable correlation coefficient of 0.98. The cross-reactivity showed that scFv-G9 had strongly binding ability to Cry1Ac toxin, but not to Cry1B, Cry1C and Cry1F toxin. The average recoveries of Cry1Ab toxin from spiked leaf and rice samples were in the range 92.1-94.8%, and 91.6-98.6%, respectively, with a coefficient of variation (C.V) less than 5.0%. These results showed promising applications of scfv-G9 for detecting Cry1Ab toxin with new DAS-ELISA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize

NASA Technical Reports Server (NTRS)

Feldman, L. J.; Hidaka, H.

1993-01-01

Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

Verifying the Hanging Chain Model

ERIC Educational Resources Information Center

Karls, Michael A.

2013-01-01

The wave equation with variable tension is a classic partial differential equation that can be used to describe the horizontal displacements of a vertical hanging chain with one end fixed and the other end free to move. Using a web camera and TRACKER software to record displacement data from a vibrating hanging chain, we verify a modified version…

Effect of low electric fields on alpha scintillation light yield in liquid argon

DOE PAGES

Agnes, P.; Albuquerque, I. F. M.; Alexander, T.; ...

2017-01-24

Measurements were made of scintillation light yield of alpha particles from themore » $$^{222}$$Rn decay chain within the DarkSide-50 liquid argon time projection chamber. Furthermore, the light yield was found to increase as the applied electric field increased, with alphas in a 200 V/cm electric field exhibiting a 2% increase in light yield compared to alphas in no field.« less

Anisotropic Light Scattering from Ferrofluids

NASA Astrophysics Data System (ADS)

Rablau, Corneliu; Vaishnava, Prem; Naik, Ratna; Lawes, Gavin; Tackett, Ron; Sudakar, C.

2008-03-01

We have investigated the light scattering in DC magnetic fields from aqueous suspensions of Fe3O4 nanoparticles coated with tetra methyl ammonium hydroxide and γ-Fe2O3 nanoparticles embedded in alginate hydrogel. For Fe3O4 ferrofluid, anomalous light scattering behavior was observed when light propagated both parallel and perpendicular to the magnetic fields. This behavior is attributed to the alignment and aggregation of the nanoparticles in chain-like structures. A very different light scattering behavior was observed for γ-Fe2O3 alginate sample where, under the similar conditions, the application of the magnetic field produced no structured change in scattering. We attribute this difference to the absence of chain-like structures and constrained mobility of iron nanoparticles in the alginate sample. The observation is in agreement with our relaxation and dissipative heating results^1 where both samples exhibited Neel relaxation but only the Fe3O4 ferrofluid showed Brownian relaxation. The results suggest that Brownian relaxation and nanoparticle mobility are important for producing non-linear light scattering in such systems. ^1P.P. Vaishnava, R. Tackett, A. Dixit, C. Sudakar, R. Naik, and G. Lawes, J. Appl. Phys. 102, 063914 (2007).

Ammonium Assimilation Requires Mitochondrial Respiration in the Light 1

PubMed Central

Weger, Harold G.; Birch, Douglas G.; Elrifi, Ivor R.; Turpin, David H.

1988-01-01

Mass spectrometric analysis of O2 and CO2 exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH4Cl resulted in nearly a 250% increase in the rate of TCA cycle CO2 efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O2 consumption in both light and dark. Anaerobiosis inhibited the CO2 release caused by NH4Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O2 consumption in the light. This implied that the Mehler reaction did not play a significant role in O2 consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions. PMID:16665971

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

Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.

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

Serum Free Light Chain Assay and κ/λ Ratio Performance in Patients Without Monoclonal Gammopathies:  High False-Positive Rate.

PubMed

Singh, Gurmukh

2016-08-01

Serum free light chain assay is a recommended screening test for monoclonal gammopathies. Anecdotal observations indicated a high rate of false-positive abnormal κ/λ ratios. This study was undertaken to ascertain the magnitude of the false-positive rate and factors contributing to the error rate. Results of serum protein electrophoresis, serum free light chains, and related tests, usually done for investigation of suspected monoclonal gammopathy, were reviewed retrospectively for 270 patients and 297 observations. Using the conventional κ/λ ratio, 36.4% of the ratios were abnormal, in the absence of monoclonal gammopathy. When the renal κ/λ ratio was used, the rate of abnormal κ/λ ratios was 30.1%. In patients with a γ-globulin concentration of 1.6 g/dL or more, the usual κ/λ ratio was abnormal in 54.8% of the patients. Urine protein electrophoresis was used in 53 (19.6%) instances, whereas bone marrow examination was done in 65 (24.1%) cases. Usual κ/λ ratio was abnormal in 36.4% of the observations in patients without evidence of monoclonal gammopathy, and an abnormal κ/λ ratio should not be used as the sole indicator for diagnosis of neoplastic proliferation of the lympho-plasmacytic system. Hypergammaglobulinemia is associated with a higher rate of false-positive abnormal κ/λ ratios. Examination of urine for monoclonal immunoglobulins may be underused, and recommendations by some to use serum free light chain assay in place of, rather than as an adjunct to, urine electrophoresis are not warranted. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cerebrospinal Fluid and Neuroimaging Biomarker Abnormalities Suggest Early Neurological Injury in a Subset of Individuals During Primary HIV Infection

PubMed Central

Peluso, Michael J.; Meyerhoff, Dieter J.; Price, Richard W.; Peterson, Julia; Lee, Evelyn; Young, Andrew C.; Walter, Rudy; Fuchs, Dietmar; Brew, Bruce J.; Cinque, Paola; Robertson, Kevin; Hagberg, Lars; Zetterberg, Henrik; Gisslén, Magnus; Spudich, Serena

2013-01-01

Background. Cerebrospinal fluid (CSF) and neuroimaging abnormalities demonstrate neuronal injury during chronic AIDS, but data on these biomarkers during primary human immunodeficiency virus (HIV) infection is limited. Methods. We compared CSF concentrations of neurofilament light chain, t-tau, p-tau, amyloid precursor proteins, and amyloid-beta 42 in 92 subjects with primary HIV infection and 25 controls. We examined relationships with disease progression and neuroinflammation, neuropsychological testing, and proton-magnetic resonance spectroscopy (MRS)–based metabolites. Results. Neurofilament light chain was elevated in primary HIV infection compared with controls (P = .0004) and correlated with CSF neopterin (r = 0.38; P = .0005), interferon gamma-induced protein 10 (r = 0.39; P = .002), white blood cells (r = 0.32; P = .004), protein (r = 0.59; P < .0001), and CSF/plasma albumin ratio (r = 0.60; P < .0001). Neurofilament light chain correlated with decreased N-acteylaspartate/creatine and glutamate/creatine in the anterior cingulate (r = −0.35, P = .02; r = −0.40, P = .009, respectively), frontal white matter (r = −0.43, P = .003; r = −0.30, P = .048, respectively), and parietal gray matter (r = −0.43, P = .003; r = −0.47, P = .001, respectively). Beta-amyloid was elevated in the primary infection group (P = .0005) and correlated with time infected (r = 0.34; P = .003). Neither marker correlated with neuropsychological abnormalities. T-tau and soluble amyloid precursor proteins did not differ between groups. Conclusions. Elevated neurofilament light chain and its correlation with MRS-based metabolites suggest early neuronal injury in a subset of participants with primary HIV infection through mechanisms involving central nervous system inflammation. PMID:23460748

Functional Cardiac Recovery and Hematologic Response to Chemotherapy in Patients With Light-Chain Amyloidosis (from the Stanford University Amyloidosis Registry).

PubMed

Tuzovic, Mirela; Kobayashi, Yukari; Wheeler, Matthew; Barrett, Christopher; Liedtke, Michaela; Lafayette, Richard; Schrier, Stanley; Haddad, Francois; Witteles, Ronald

2017-10-15

Cardiac involvement is common in patients with light-chain (AL) amyloidosis and portends a poor prognosis, although little is known about the changes in cardiac mechanics after chemotherapy. We sought to explore the relation between amyloidosis staging and baseline cardiac mechanics and to investigate short-term changes in cardiac mechanics after chemotherapy. We identified 41 consecutive patients from the Stanford Amyloid Center who had echocardiograms and free light-chain values before and after chemotherapy, along with 40 age- and gender-matched controls. Echocardiographic assessment included left ventricular global longitudinal strain, E/e' ratio, and left atrial (LA) stiffness. Hematologic response to chemotherapy was defined as ≥50% reduction in the difference between the involved and the uninvolved free light chain (dFLC). The mean age was 66.9 ± 8.4 years and 66% were men. Before chemotherapy, global longitudinal strain, E/e' ratio, and LA stiffness were impaired in patients with amyloidosis compared with controls, and the severity of impairment worsened with advanced staging. After chemotherapy, hematologic response was observed in 30 (73%) patients. There was a significant association between the change in dFLC and cardiac function (E/e' ratio: r = -0.43, p = 0.01; LA stiffness: r = -0.35, p = 0.05). There was no significant improvement in cardiac mechanics in patients without a hematologic response to chemotherapy. In conclusion, amyloidosis stage correlated with noninvasive measurements of cardiac mechanics, and improvement in dFLC correlated with cardiac improvement on short-term follow-up echocardiography. Copyright © 2017 Elsevier Inc. All rights reserved.

Photoswitching of ferroelectric liquid crystals using chiral thioindigo dopants: The development of a photochemical switch hitter.

PubMed

Lemieux, Robert P

2004-01-01

By virtue of its spontaneous polarization (PS), a ferroelectric SmC* liquid crystal can be switched between two states corresponding to opposite molecular tilt orientations using an electric field, thus producing an ON-OFF light shutter between crossed polarizers. Considerable efforts have been made over the past decade to develop photonic FLC light shutters because of their potential uses in dynamic holography and optical data storage. The ON-OFF switching of a FLC light shutter can be triggered by light via a photoinversion of PS using a photochromic dopant. The spontaneous polarization is a chiral bulk property that can be left-handed (negative) or right-handed (positive), depending on the absolute configuration of the chiral component of the SmC* phase. In the approach described herein, the magnitude of PS is modulated via the photoisomerization of a chiral thioindigo dopant that undergoes a large increase in transverse dipole moment upon trans-cis photoisomerization. The sign of PS is photoinverted using an "ambidextrous" thioindigo dopant containing a chiral 2-octyloxy side chain that is coupled to the thioindigo core and induces a positive PS, and a chiral 2,3-difluorooctyloxy side chain that is decoupled from the core and induces a negative PS. In the trans form, the 2,3-difluorooctyloxy side chain predominates and the net PS induced by the dopant is negative. However, upon trans-cis-photoisomerization, the increase in transverse dipole moment of the 2-octyloxy/thioindigo unit raises its induced PS over that of the decoupled 2,3-difluorooctyloxy side chain, and thus inverts the net sign of PS induced by the dopant from negative to positive. Copyright 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.

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

Modeling water clarity in oceans and coasts

EPA Science Inventory

In oceans and coastal waters, phytoplankton is the primary producer of organic compounds which form the base for the food chain. The concentration of phytoplankton is a major factor controlling water clarity and the depth to which light penetrates in the water column. The light i...

Tetanus toxin is labeled with photoactivatable phospholipids at low pH

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

Montecucco, C.; Schiavo, G.; Brunner, J.

1986-02-25

The mechanism of cell penetration by tetanus toxin is unknown; it has been suggested that the toxin may penetrate into the lipid bilayer from a low-pH vesicular compartment. In this work, the interaction of tetanus toxin with liposomal model membranes has been studied by following its photoinduced cross-linking with either a nitrene or a carbene photolytically generated from corresponding light-sensitive phosphatidylcholine analogues. The toxin was labeled only at pHs lower than 5.5. The low pH acquired hydrophobicity of tetanus toxin appears to be confined to its light chain and to the 45-kDa NH2-terminal fragment of the heavy chain. Negatively chargedmore » lipids promote the interaction of this toxin with the hydrocarbon chain of phospholipids. The relevance of the present findings to the possible mechanism of nerve cell penetration by tetanus toxin is discussed.« less

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

Toughening elastomers with sacrificial bonds and watching them break.

PubMed

Ducrot, Etienne; Chen, Yulan; Bulters, Markus; Sijbesma, Rint P; Creton, Costantino

2014-04-11

Elastomers are widely used because of their large-strain reversible deformability. Most unfilled elastomers suffer from a poor mechanical strength, which limits their use. Using sacrificial bonds, we show how brittle, unfilled elastomers can be strongly reinforced in stiffness and toughness (up to 4 megapascals and 9 kilojoules per square meter) by introducing a variable proportion of isotropically prestretched chains that can break and dissipate energy before the material fails. Chemoluminescent cross-linking molecules, which emit light as they break, map in real time where and when many of these internal bonds break ahead of a propagating crack. The simple methodology that we use to introduce sacrificial bonds, combined with the mapping of where bonds break, has the potential to stimulate the development of new classes of unfilled tough elastomers and better molecular models of the fracture of soft materials.

The Viscoelastic Properties of Nematic Monodomains Containing Liquid Crystal Polymers.

NASA Astrophysics Data System (ADS)

Gu, Dongfeng

The work presented here investigates the viscoelastic properties of nematic materials containing liquid crystal polymers (LCP). We focus on how the elastic constants and the viscosity coefficients of the mixture systems are influenced by polymer architectures. In dynamic light scattering studies of the relaxation of the director orientation fluctuations for the splay, twist, and bend deformation modes, decrease of the relaxation rates was observed when LCPs were dissolved into low molar mass nematics (LMMN). For the side-chain LCPs, the slowing down in the bend mode is comparable to or larger than those of the splay and twist modes. For main-chain LCPs, the relative changes in the relaxation rates for the twist and splay modes are about one order of magnitude larger than that for the bend mode. The results of light scattering under an electric field show that the decrease in the twist relaxation rate is due to a large increase in the twist viscosity and a minor decrease in the twist elastic constant. These changes were found to increase with decrease of the spacer length, with increase of molecular weight, and with decrease of the backbone flexibility. In Freedericksz transition measurements, the splay and bend elastic constants and the dielectric anisotropies of the nematic mixtures were determined and the values are 5~15% lower than those of the pure solvent. From the analysis of the results of Freedericksz transition and light scattering experiments, a complete set of the elastic constants and viscosity coefficients corresponding to the three director deformation modes were obtained for the LCP mixtures. The changes in the viscosity coefficients due to addition of LCPs were analysed to estimate the anisotropic shapes of the polymer backbone via a hydrodynamic model. The results suggest that an oblate backbone configuration is maintained by the side-chain LCPs and a prolate chain configuration appears for the main-chain LCPs. The rheological behavior of a side-chain and a main-chain LCP nematic solutions were investigated. The addition of the side-chain LCP into a flow-aligning LMMN (5CB) induces director tumbling in the mixture, and, the dissolution of the main-chain LCP into a director tumbling LMMN (8CB) makes the solution become a flow-aligning nematic. Based on the hydrodynamic theory, these observations are further confirmation of the chain anisotropies of the LCPs investigated. Ericksen's transversely isotropic fluid model was used to extract the various viscosity coefficients with good accuracy. In addition, we believe that this is the first time the bulk rheological consequences of director tumbling in LMMNs has been observed.

Nursing home financial performance: the role of ownership and chain affiliation.

PubMed

Weech-Maldonado, Robert; Laberge, Alex; Pradhan, Rohit; Johnson, Christopher E; Yang, Zhou; Hyer, Kathryn

2012-01-01

The nursing home industry serves one of the most vulnerable populations, and its financial sustainability is a matter of public concern. However, limited empirical evidence exists on the impact of ownership and chain affiliation on nursing home financial performance. The aim of this study was to examine the joint effects of ownership and chain affiliation on the financial performance of the nursing home industry for the study period 1999-2004 on a national sample of 11,236 nursing homes per year. Data included the Medicare Cost Reports; the Online Survey, Certification, and Reporting file; and the Area Resource File. Dependent variables included operating and total margins. Independent variables included four ownership/chain affiliation combinations: for-profit chain, for-profit independent, not-for-profit chain, and not-for-profit independent. Random effects generalized least square regressions were performed. Results show that for-profit nursing homes delivered better financial performance than not-for-profit facilities did across both operating and total margins. However, the relationship between chain affiliation and financial performance was more nuanced. In the case of operating margin, chain-affiliated facilities delivered superior financial performance irrespective of ownership type; however, in the case of total margin, independents outperformed chain-affiliated facilities among for-profits. Our findings show an interactive effect of ownership and chain affiliation on nursing home financial performance, suggesting the pursuit of different organizational strategies by different ownership/chain affiliation subgroups (for-profit chain, for-profit independent, not-for-profit chain, and not-for-profit independent), with implications for financial performance. For-profit independent nursing homes managed to be the top performing group in terms of overall financial despite the operating financial advantage of for-profit chain-affiliated nursing homes. Similarly, not-for-profit independent nursing homes and not-for-profit chain homes had comparable overall financial performance despite the operating financial advantage of chain homes.

Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Reymond, P.; Short, T. W.; Briggs, W. R.; Poff, K. L.

1992-01-01

Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.

Decorin causes autophagy in endothelial cells via Peg3

PubMed Central

Buraschi, Simone; Neill, Thomas; Goyal, Atul; Poluzzi, Chiara; Smythies, James; Owens, Rick T.; Schaefer, Liliana; Torres, Annabel; Iozzo, Renato V.

2013-01-01

Soluble decorin affects the biology of several receptor tyrosine kinases by triggering receptor internalization and degradation. We found that decorin induced paternally expressed gene 3 (Peg3), an imprinted tumor suppressor gene, and that Peg3 relocated into autophagosomes labeled by Beclin 1 and microtubule-associated light chain 3. Decorin evoked Peg3-dependent autophagy in both microvascular and macrovascular endothelial cells leading to suppression of angiogenesis. Peg3 coimmunoprecipitated with Beclin 1 and LC3 and was required for maintaining basal levels of Beclin 1. Decorin, via Peg3, induced transcription of Beclin 1 and microtubule-associated protein 1 light chain 3 alpha genes, thereby leading to a protracted autophagic program. Mechanistically, decorin interacted with VEGF receptor 2 (VEGFR2) in a region overlapping with its natural ligand VEGFA, and VEGFR2 was required for decorin-evoked Beclin 1 and microtubule-associated protein 1 light chain 3 alpha expression as well as for Peg3 induction in endothelial cells. Moreover, decorin induced VEGFR2-dependent mitochondrial fragmentation and loss of mitochondrial membrane potential. Thus, we have unveiled a mechanism for a secreted proteoglycan in inducing Peg3, a master regulator of macroautophagy in endothelial cells. PMID:23798385

Risk factors for venous thromboembolism in immunoglobulin light chain amyloidosis

PubMed Central

Bever, Katherine M.; Masha, Luke I.; Sun, Fangui; Stern, Lauren; Havasi, Andrea; Berk, John L.; Sanchorawala, Vaishali; Seldin, David C.; Sloan, J. Mark

2016-01-01

Patients with immunoglobulin light chain amyloidosis are at risk for both thrombotic and bleeding complications. While the hemostatic defects have been extensively studied, less is known about thrombotic complications in this disease. This retrospective study examined the frequency of venous thromboembolism in 929 patients with immunoglobulin light chain amyloidosis presenting to a single referral center, correlated risk of venous thromboembolism with clinical and laboratory factors, and examined complications of anticoagulation in this population. Sixty-five patients (7%) were documented as having at least one venous thromboembolic event. Eighty percent of these patients had events within one year prior to or following diagnosis. Lower serum albumin was associated with increased risk of VTE, with a hazard ratio of 4.30 (CI 1.60–11.55; P=0.0038) for serum albumin less than 3 g/dL compared to serum albumin greater than 4 g/dL. Severe bleeding complications were observed in 5 out of 57 patients with venous thromboembolism undergoing treatment with anticoagulation. Prospective investigation should be undertaken to better risk stratify these patients and to determine the optimal strategies for prophylaxis against and management of venous thromboembolism. PMID:26452981

Risk factors for venous thromboembolism in immunoglobulin light chain amyloidosis.

PubMed

Bever, Katherine M; Masha, Luke I; Sun, Fangui; Stern, Lauren; Havasi, Andrea; Berk, John L; Sanchorawala, Vaishali; Seldin, David C; Sloan, J Mark

2016-01-01

Patients with immunoglobulin light chain amyloidosis are at risk for both thrombotic and bleeding complications. While the hemostatic defects have been extensively studied, less is known about thrombotic complications in this disease. This retrospective study examined the frequency of venous thromboembolism in 929 patients with immunoglobulin light chain amyloidosis presenting to a single referral center, correlated risk of venous thromboembolism with clinical and laboratory factors, and examined complications of anticoagulation in this population. Sixty-five patients (7%) were documented as having at least one venous thromboembolic event. Eighty percent of these patients had events within one year prior to or following diagnosis. Lower serum albumin was associated with increased risk of VTE, with a hazard ratio of 4.30 (CI 1.60-11.55; P=0.0038) for serum albumin less than 3 g/dL compared to serum albumin greater than 4 g/dL. Severe bleeding complications were observed in 5 out of 57 patients with venous thromboembolism undergoing treatment with anticoagulation. Prospective investigation should be undertaken to better risk stratify these patients and to determine the optimal strategies for prophylaxis against and management of venous thromboembolism. Copyright© Ferrata Storti Foundation.

Renal complications in multiple myeloma and related disorders: survivorship care plan of the International Myeloma Foundation Nurse Leadership Board.

PubMed

Faiman, Beth M; Mangan, Patricia; Spong, Jacy; Tariman, Joseph D

2011-08-01

Kidney dysfunction is a common clinical feature of symptomatic multiple myeloma. Some degree of renal insufficiency or renal failure is present at diagnosis or will occur during the course of the disease and, if not reversed, will adversely affect overall survival and quality of life. Chronic insults to the kidneys from other illnesses, treatment, or multiple myeloma itself can further damage renal function and increase the risk for additional complications, such as anemia. Patients with multiple myeloma who have light chain (Bence Jones protein) proteinuria may experience renal failure or progress to end-stage renal disease (ESRD) and require dialysis because of light chain cast nephropathy. Kidney failure in patients with presumed multiple myeloma also may result from amyloidosis, light chain deposition disease, or acute tubular necrosis caused by nephrotoxic agents; therefore, identification of patients at risk for kidney damage is essential. The International Myeloma Foundation's Nurse Leadership Board has developed practice recommendations for screening renal function, identifying positive and negative contributing risk and environmental factors, selecting appropriate therapies and supportive care measures to decrease progression to ESRD, and enacting dialysis to reduce and manage renal complications in patients with multiple myeloma.

Update on treatment of light chain amyloidosis

PubMed Central

Mahmood, Shameem; Palladini, Giovanni; Sanchorawala, Vaishali; Wechalekar, Ashutosh

2014-01-01

Light chain amyloidosis is the most common type of amyloidosis as a consequence of protein misfolding of aggregates composed of amyloid fibrils. The clinical features are dependent on the organs involved, typically cardiac, renal, hepatic, peripheral and autonomic neuropathy and soft tissue. A tissue biopsy or fat aspirate is needed to confirm the presence/type of amyloid and prognostic tools are important in a risk stratified approach to treatment. Autologous stem cell transplant eligibility should be assessed at baseline, weighing the reversible or non-reversible contraindications, toxicity of treatment and chemotherapy alternatives available. Chemotherapy options include melphalan, thalidomide, bortezomib, lenalidomide, bendamustine in combination with dexamethasone. Many studies have explored these treatment modalities, with ongoing debate about the optimal first line and sequential treatment thereafter. Attaining a very good partial response or better is the treatment goal coupled with early assessment central to optimizing treatment. One major challenge remains increasing the awareness of this disease, frequently diagnosed late as the presenting symptoms mimic many other medical conditions. This review focuses on the treatments for light chain amyloidosis, how these treatments have evolved over the years, improved patient risk stratification, toxicities encountered and future directions. PMID:24497558

Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1) Are Essential for the Binding of Clathrin Light Chain.

PubMed

Ybe, Joel A; Clegg, Mary E; Illingworth, Melissa; Gonzalez, Claire; Niu, Qian

2009-01-01

The interaction between HIP family proteins (HIP1 and HIP12/1R) and clathrin is fundamental to endocytosis. We used circular dichroism (CD) to study the stability of an HIP1 subfragment (aa468-530) that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83°C. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494). The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae), HIP1 from D. melanogaster, and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.

Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1) Are Essential for the Binding of Clathrin Light Chain

PubMed Central

Ybe, Joel A.; Clegg, Mary E.; Illingworth, Melissa; Gonzalez, Claire; Niu, Qian

2009-01-01

The interaction between HIP family proteins (HIP1 and HIP12/1R) and clathrin is fundamental to endocytosis. We used circular dichroism (CD) to study the stability of an HIP1 subfragment (aa468-530) that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83°C. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494). The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae), HIP1 from D. melanogaster, and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms. PMID:22820750

Damaging effects of visible light. Comprehensive progress report

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

None

1981-01-01

Research progress is reported on studies of retinal light damage. A myriad of variables effect the production of light damage. These include age, prior light history, body temperature, vitamin A status, intensity, wavelength and duration of light. The intensity-duration function and the age function have been studied in detail in rats. Studies have been begun on the wavelength variable. (ACR)

An Integer Programming Model for Multi-Echelon Supply Chain Decision Problem Considering Inventories

NASA Astrophysics Data System (ADS)

Harahap, Amin; Mawengkang, Herman; Siswadi; Effendi, Syahril

2018-01-01

In this paper we address a problem that is of significance to the industry, namely the optimal decision of a multi-echelon supply chain and the associated inventory systems. By using the guaranteed service approach to model the multi-echelon inventory system, we develop a mixed integer; programming model to simultaneously optimize the transportation, inventory and network structure of a multi-echelon supply chain. To solve the model we develop a direct search approach using a strategy of releasing nonbasic variables from their bounds, combined with the “active constraint” method. This strategy is used to force the appropriate non-integer basic variables to move to their neighbourhood integer points.

New construction of eigenstates and separation of variables for SU( N) quantum spin chains

NASA Astrophysics Data System (ADS)

Gromov, Nikolay; Levkovich-Maslyuk, Fedor; Sizov, Grigory

2017-09-01

We conjecture a new way to construct eigenstates of integrable XXX quantum spin chains with SU( N) symmetry. The states are built by repeatedly acting on the vacuum with a single operator B good( u) evaluated at the Bethe roots. Our proposal serves as a compact alternative to the usual nested algebraic Bethe ansatz. Furthermore, the roots of this operator give the separated variables of the model, explicitly generalizing Sklyanin's approach to the SU( N) case. We present many tests of the conjecture and prove it in several special cases. We focus on rational spin chains with fundamental representation at each site, but expect many of the results to be valid more generally.

Fabrication of ZnS nanoparticle chains on a protein template

PubMed Central

Hulleman, J.; Kim, S. M.; Tumkur, T.; Rochet, J.-C.; Stach, E.; Stanciu, L.

2011-01-01

In the present study, we have exploited the properties of a fibrillar protein for the template synthesis of zinc sulfide (ZnS) nanoparticle chains. The diameter of the ZnS nanoparticle chains was tuned in range of ~30 to ~165 nm by varying the process variables. The nanoparticle chains were characterized by field emission scanning electron microscopy, UV–Visible spectroscopy, transmission electron microscopy, electron energy loss spectroscopy, and high-resolution transmission electron microscopy. The effect of incubation temperature on the morphology of the nanoparticle chains was also studied. PMID:21804765

Softening of the stiffness of bottle-brush polymers by mutual interaction

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

Bolisetty, S.; Airaud, C.; Rosenfeldt, S.

2007-04-15

We study bottle-brush macromolecules in a good solvent by small-angle neutron scattering (SANS), static light scattering (SLS), and dynamic light scattering (DLS). These polymers consist of a linear backbone to which long side chains are chemically grafted. The backbone contains about 1600 monomer units (weight average) and every second monomer unit carries side chains with approximately 60 monomer units. The SLS and SANS data extrapolated to infinite dilution lead to the form factor of the polymer that can be described in terms of a wormlike chain with a contour length of 380 nm and a persistence length of 17.5 nm.more » An analysis of the DLS data confirms these model parameters. The scattering intensities taken at finite concentration can be modeled using the polymer reference interaction site model. It reveals a softening of the bottle-brush polymers caused by their mutual interaction. We demonstrate that the persistence decreases from 17.5 nm down to 5 nm upon increasing the concentration from dilute solution to the highest concentration (40.59 g/l) under consideration. The observed softening of the chains is comparable to the theoretically predicted decrease of the electrostatic persistence length of linear polyelectrolyte chains at finite concentrations.« less

Metallocene-Containing Homopolymers and Heterobimetallic Block Copolymers via Photoinduced RAFT Polymerization

PubMed Central

Yang, Peng; Pageni, Parasmani; Kabir, Mohammad Pabel; Zhu, Tianyu; Tang, Chuanbing

2017-01-01

We report the synthesis of cationic cobaltocenium and neutral ferrocene containing homopolymers mediated by photoinduced reversible addition-fragmentation chain transfer (RAFT) polymerization with a photocatalyst fac-[Ir(ppy)3]. The homopolymers were further used as macromolecular chain transfer agents to synthesize diblock copolymers via chain extension. Controlled/“living” feature of photoinduced RAFT polymerization was confirmed by kinetic studies even without prior deoxygenation. A light switch between ON and OFF provided a spatiotemporal control of polymerization. PMID:29276651

Modeling Water Clarity and Light Quality in Oceans

EPA Science Inventory

Phytoplankton is a primary producer of organic compounds, and it forms the base of the food chain in ocean waters. The concentration of phytoplankton in the water column controls water clarity and the amount and quality of light that penetrates through it. The availability of ade...

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.

Impairment of extramitochondrial oxidative phosphorylation in mouse rod outer segments by blue light irradiation.

PubMed

Calzia, Daniela; Panfoli, Isabella; Heinig, Nora; Schumann, Ulrike; Ader, Marius; Traverso, Carlo Enrico; Funk, Richard H W; Roehlecke, Cora

2016-06-01

Exposure to short wavelength light causes increased reactive oxygen intermediates production in the outer retina, particularly in the rod Outer Segments (OS). Consistently, the OS were shown to conduct aerobic ATP production through the ectopic expression of the electron transfer chain complexes I-IV and F1Fo-ATP synthase. These facts prompted us to verify if the oxidative phosphorylation in the OS is implied in the oxidative damage of the blue-light (BL) treated OS, in an organotypic model of mouse retina. Whole mouse eyeball cultures were treated with short wavelength BL (peak at 405 nm, output power 1 mW/cm(2)) for 6 h. Immunogold transmission electron microscopy confirmed the expression of Complex I and F1Fo-ATP synthase in the OS. In situ histochemical assays on unfixed sections showed impairment of respiratory Complexes I and II after BL exposure, both in the OS and IS, utilized as a control. Basal O2 consumption and ATP synthesis were impaired in the OS purified from blue-light irradiated eyeball cultures. Electron transfer capacity between Complex I and II as well as activity of Complexes I and II was decreased in blue-light irradiated purified OS. The severe malfunctioning of the OS aerobic respiratory capacity after 6 h BL treatment may be the consequence of a self-induced damage. BL exposure would cause an initial over-functioning of both the phototransduction and respiratory chain, with reactive oxygen species production. In a self-renewal vicious cycle, membrane and protein oxidative damage, proton leakage and uncoupling, would impair redox chains, perpetuating the damage and causing hypo-metabolism with eventual apoptosis of the rod. Data may shed new light on the rod-driven retinopathies such as Age Related Macular Degeneration, of which blue-light irradiated retina represents a model. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

PubMed

Martinez, J M; Elfarissi, H; De Velasco, B; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

2000-01-01

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

Chemical variability along the value chains of turmeric (Curcuma longa): a comparison of nuclear magnetic resonance spectroscopy and high performance thin layer chromatography.

PubMed

Booker, Anthony; Frommenwiler, Debora; Johnston, Deborah; Umealajekwu, Chinenye; Reich, Eike; Heinrich, Michael

2014-03-14

Herbal medicine value chains have generally been overlooked compared with food commodities. Not surprisingly, revenue generation tends to be weighted towards the end of the chain and consequently the farmers and producers are the lowest paid beneficiaries. Value chains have an impact both on the livelihood of producers and on the composition and quality of products commonly sold locally and globally and consequently on the consumers. In order to understand the impact of value chains on the composition of products, we studied the production conditions for turmeric (Curcuma longa) and the metabolomic composition of products derived from it. We aimed at integrating these two components in order to gain a better understanding of the effect of different value chains on the livelihoods of some producers. This interdisciplinary project uses a mixed methods approach. Case studies were undertaken on two separate sites in India. Data was initially gathered on herbal medicine value chains by means of semi-structured interviews and non-participant observations. Samples were collected from locations in India, Europe and the USA and analysed using (1)H NMR spectroscopy coupled with multivariate analysis software and with high performance thin layer chromatography (HPTLC). We investigate medicinal plant value chains and interpret the impact different value chains have on some aspects of the livelihoods of producers in India and, for the first time, analytically assess the chemical variability and quality implications that different value chains may have on the products available to end users in Europe. There are benefits to farmers that belonged to an integrated chain and the resulting products were subject to a higher standard of processing and storage. By using analytical methods, including HPTLC and (1)H NMR spectroscopy, it has been possible to correlate some variations in product composition for selected producers and identify strengths and weaknesses of some types of value chains. The two analytical techniques provide different and complementary data and together they can be used to effectively differentiate between a wide variety of crude drug powders and herbal medicinal products. This project demonstrates that there is a need to study the links between producers and consumers of commodities produced in so-called 'provider countries' and that metabolomics offer a novel way of assessing the chemical variability along a value chain. This also has implications for understanding the impact this has on the livelihood of those along the value chain. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Role of different hematologic variables in defining the risk of malignant transformation in monoclonal gammopathy.

PubMed

Baldini, L; Guffanti, A; Cesana, B M; Colombi, M; Chiorboli, O; Damilano, I; Maiolo, A T

1996-02-01

The presenting clinico-hematologic features of 386 patients with nonmyelomatous monoclonal gammopathy (MG) were correlated with the frequency of malignant transformation to evaluate the most important variables conditioning its evolution into multiple myeloma (MM) or Waldenström macroglobulinemia (WM). Most of the patients (335) had monoclonal gammopathy of undetermined significance (MGUS: 39 IgA, 242 IgG, 54 IgM): the remaining 51 patients (12 IgA, 39 IgG) fulfilled all of the MGUS diagnostic criteria (according to Durie) except that bone marrow plasma cell (BMPC) content was 10% to 30%, and so they were defined as having monoclonal gammopathy of borderline significance (MGBS). There were no significant differences between the MGUS and MGBS groups in terms of age, sex, or median follow-up. After a median follow-up of 70 and 53 months, respectively, 23 of 335 MGUS and 19 of 51 MGBS patients had undergone a malignant evolution. Univariate analysis of the IgA and IgG patients showed that the cumulative probability of the disease evolving into MM correlated with diagnostic definition (MGBS v MGUS), BMPC content (> or = 10% v < 5% and < or = 5% v > 5%) and reduced serum polyclonal Ig. In the IgG cases, there was also a significant correlation with detectable Bence Jones proteinuria, serum monoclonal component (MC) levels and age at diagnosis (> 70 v < = or 55 years). In the IgG cases as a whole, the same variables remained in the Cox model where the BMPC percentage was considered after natural logarithmic transformation and the monoclonal component as g/dL value. The relative risks of developing MM are the following: 2.4 for each 1 g/dL increase of IgG, serum MC, 3.5 for detectable light chain proteinuria, 4.4 for the increase of 1 unit in log. BMPC percentage, 6.1 for age > 70, 3.6 and 13.1 for a reduction in one or two polyclonal Ig. In conclusion, our study allows the identification of a particular subset of MGUS patients (MC < = or 1.5 g/dL, BMPC < 5%, no reduction in polyclonal Ig and no detectable light chain proteinuria) at very low-risk of evolution, who can be considered as having benign monoclonal gammopathies. We also describe a previously undefined group of MG patients (with monoclonal gammopathy of borderline significance) who are at high-risk of malignant evolution. These findings could have a considerable impact on the cost/benefit ratio of monitoring programs in these patients.

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.

Topology of an intracellular transduction chain (phototropism of Phycomyces): 1. Joint review of functional, temporal, and spatial aspects.

PubMed

Wenzler, D; Reinhardt, M; Fukshansky, L

2001-08-21

Two light-induced growth reactions in a unicellular cylindrical sporangiophore of Phycomyces blakesleeanus-vertical growth acceleration under symmetrical irradiation (photomecism) and directional growth under unilateral irradiation (phototropism)-share common input light perception as well as common output growth mechanism but have strongly divergent dynamics and other distinctive features. This divergence culminates in the phototropic paradoxes the main of which states that photomecism shows total adaptation, while phototropism does not adapt. The basis for this contradiction is that the phototropic transduction chain, unlike that of photomecism, faces a spatially non-uniform stimulus and processes a series of spatial patterns (light and absorption profiles, adaptation profile, etc.). The only way to resolve the paradoxes and correlate features of both responses within a single transduction chain is to assume non-local signal transduction, e.g. a cross-talk between different azimuthal locations within the cylindrical cell. On the other hand, to establish the presence of an appropriate cross-talk is equivalent of gaining insight into the topology of the transduction chain. This series of two papers contains a review reconsidering the entire field from this viewpoint (Paper 1) and a mathematical model of pattern transduction which unifies features of phototropism and resolves the paradoxes (Paper 2). At the same time, this is the first "proof of concept" for the "activity/pooling (a/p) networks"-a specific mathematical apparatus designed to analyse systemic properties and control in metabolic pathways. Copyright 2001 Academic Press.

Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway.

PubMed

Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Hunter, C Neil; Bocian, David F; Holten, Dewey; Niedzwiedzki, Dariusz M

2016-06-23

Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids.

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

A Unique Role for Endothelial Cell Kinesin Light Chain 1, Variant 1 in Leukocyte Transendothelial Migration

PubMed Central

Cyrus, Bita F.; Muller, William A.

2017-01-01

A reservoir of parajunctional membrane in endothelial cells, the lateral border recycling compartment (LBRC), is critical for transendothelial migration (TEM). We have previously shown that targeted recycling of the LBRC to the site of TEM requires microtubules and a kinesin molecular motor. However, the identity of the kinesin and mechanism of cargo binding were not known. We show that microinjection of endothelial cells with a monoclonal antibody specific for kinesin-1 significantly blocked LBRC-targeted recycling and TEM. In complementary experiments, knocking down KIF5B, a ubiquitous kinesin-1 isoform, in endothelial cells significantly decreased targeted recycling of the LBRC and leukocyte TEM. Kinesin heavy chains move cargo along microtubules by one of many kinesin light chains (KLCs), which directly bind the cargo. Knocking down KLC 1 isoform variant 1 (KLC1C) significantly decreased LBRC-targeted recycling and TEM, whereas knocking down other isoforms of KLC1 had no effect. Re-expression of KLC1C resistant to the knockdown shRNA restored targeted recycling and TEM. Thus kinesin-1 and KLC1C are specifically required for targeted recycling and TEM. These data suggest that of the many potential combinations of the 45 kinesin family members and multiple associated light chains, KLC1C links the LBRC to kinesin-1 (KIF5B) during targeted recycling and TEM. Thus, KLC1C can potentially be used as a target for anti-inflammatory therapy. PMID:26994343

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

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

What are we missing? Scope 3 greenhouse gas emissions accounting in the metals and minerals industry

NASA Astrophysics Data System (ADS)

Greene, Suzanne E.

2018-05-01

Metal and mineral companies have significant greenhouse gas emissions in their upstream and downstream value chains due to outsourced extraction, beneficiation and transportation activities, depending on a firm's business model. While many companies move towards more transparent reporting of corporate greenhouse gas emissions, value chain emissions remain difficult to capture, particularly in the global supply chain. Incomplete reports make it difficult for companies to track emissions reductions goals or implement sustainable supply chain improvements, especially for commodity products that form the base of many other sector's value chains. Using voluntarily-reported CDP data, this paper sheds light on hotspots in value chain emissions for individual metal and mineral companies, and for the sector as a whole. The state of value chain emissions reporting for the industry is discussed in general, with a focus on where emissions could potentially be underestimated and how estimates could be improved.

Value of Conditioned Reinforcers as a Function of Temporal Context

ERIC Educational Resources Information Center

O'Daly, Matthew; Meyer, Steven; Fantino, Edmund

2005-01-01

In two experiments, pigeons were trained on a multiple-chain schedule, in which the initial link for one chain was a variable-interval (VI) 100s schedule and for the other chain a VI 10s schedule. The terminal links were both fixed-time 30s schedules signaled by differently colored stimuli. Following training, the pigeons had their preference for…

Basic properties and variability

NASA Technical Reports Server (NTRS)

Querci, Francois R.

1987-01-01

Giant and supergiant M, S, and C stars are discussed in this survey of research. Basic properties as determined by spectra, chemical composition, photometry, or variability type are discussed. Space motions and space distributions of cool giants are described. Distribution of these stars in our galaxy and those nearby is discussed. Mira variables in particular are surveyed with emphasis on the following topics: (1) phase lag phenomenon; (2) Mira light curves; (3) variations in color indices; (4) determination of multiple periods; (5) correlations between quantities such as period length, light-curve shape, infrared (IR) excess, and visible and IR color diagram; (6) semiregular (SR) variables and different time scales in SR light variations; (7) irregular variable Lb and Lc stars; (8) different time-scale light variations; (9) hydrogen-deficient carbon (HdC) stars, in particular RCB stars; and (10) irreversible changes and rapid evolution in red variable stars.

Line patterning of anisotropic spin chains by polarized laser for application in micro-thermal management

NASA Astrophysics Data System (ADS)

Terakado, Nobuaki; Takahashi, Ryosuke; Takahashi, Yoshihiro; Fujiwara, Takumi

2017-05-01

The control of heat flow has become increasingly important in energy saving and harvesting. Among various thermal management materials, spinon thermal conductivity materials are promising for heat flow control at microscales because they exhibit high, anisotropic thermal conductivity resulting from spin chains. However, there has been only little development of the materials for controlling heat flow. Here, we present the line patterning of the spin chain structure on a SrCuO2 nanocrystalline film by laser scanning. When the polarization direction of laser light was orthogonal to the scanning direction, we found that the spin-chain structure anisotropically grew on the patterned line.

Membranous nephropathy (bubbling appearance and spike formation) without immunoglobulin deposition in a patient with systemic lupus erythematosus.

PubMed

Miura, Naoto; Mori, Yuki; Yoshino, Masabumi; Suga, Norihiro; Kitagawa, Wataru; Yamada, Harutaka; Nishikawa, Kazuhiro; Imai, Hirokazu

2008-12-01

A 53-year-old Japanese man with systemic lupus erythematosus developed proteinuria and hematuria after a urinary stone episode. A light microscopic study of a kidney biopsy specimen demonstrated a bubbling appearance and spike formation of the basement membrane. Immunofluorescent studies revealed that there were no significant depositions of immunoglobulins, such as IgG (-), IgA (-), IgM (+/-), kappa light chain (+/-), lambda light chain (+/-), or C3 (-) in the glomerular capillary wall, though C1q was present as one-plus positive staining in mesangial areas. Electron microscopic studies showed that the thickness of the basement membrane varied from thin to thick without electron dense deposits, and that the cellular components of the podocyte were irregularly present in the basement membrane. Urinary protein decreased after the usage of prednisolone and mizoribine; however, proteinuria aggravated after an episode of urinary stone during the same treatment.

Modification of Antibody Function by Mutagenesis.

PubMed

Dasch, James R; Dasch, Amy L

2017-09-01

The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained. © 2017 Cold Spring Harbor Laboratory Press.

Clinical and veterinary isolates of Salmonella enterica serovar Enteritidis defective in lipopolysaccharide O-chain polymerization

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

Guard-Petter, J.; Parker, C.T.; Asokan, K.

1999-05-01

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and themore » O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, the authors analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.« less

Evaluation of the participation of ferredoxin in oxygen reduction in the photosynthetic electron transport chain of isolated pea thylakoids.

PubMed

Kozuleva, Marina A; Ivanov, Boris N

2010-07-01

The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP(+). The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 microM in the absence of NADP(+) increased the proportion of oxygen reduction by Fd from 25-35 to 40-60% in different experiments. This proportion decreased with increasing light intensity. When NADP(+) was added in the presence of 15 microM Fd, which was optimal for the NADP(+) reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP(+) and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.

Ammonium Assimilation Requires Mitochondrial Respiration in the Light : A Study with the Green Alga Selenastrum minutum.

PubMed

Weger, H G; Birch, D G; Elrifi, I R; Turpin, D H

1988-03-01

Mass spectrometric analysis of O(2) and CO(2) exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH(4)Cl resulted in nearly a 250% increase in the rate of TCA cycle CO(2) efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O(2) consumption in both light and dark. Anaerobiosis inhibited the CO(2) release caused by NH(4)Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O(2) consumption in the light. This implied that the Mehler reaction did not play a significant role in O(2) consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions.

The ASAS-SN Catalog of Variable Stars I: The Serendipitous Survey

NASA Astrophysics Data System (ADS)

Jayasinghe, T.; Kochanek, C. S.; Stanek, K. Z.; Shappee, B. J.; Holoien, T. W.-S.; Thompson, Todd A.; Prieto, J. L.; Dong, Subo; Pawlak, M.; Shields, J. V.; Pojmanski, G.; Otero, S.; Britt, C. A.; Will, D.

2018-04-01

The All-Sky Automated Survey for Supernovae (ASAS-SN) is the first optical survey to routinely monitor the whole sky with a cadence of ˜2 - 3 days down to V≲ 17 mag. ASAS-SN has monitored the whole sky since 2014, collecting ˜100 - 500 epochs of observations per field. The V-band light curves for candidate variables identified during the search for supernovae are classified using a random forest classifier and visually verified. We present a catalog of 66,533 bright, new variable stars discovered during our search for supernovae, including 27,753 periodic variables and 38,780 irregular variables. V-band light curves for the ASAS-SN variables are available through the ASAS-SN variable stars database (https://asas-sn.osu.edu/variables). The database will begin to include the light curves of known variable stars in the near future along with the results for a systematic, all-sky variability survey.

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

Berenstein, David; Kavli Institute for Theoretical Physics, University of California at Santa Barbara, California 93106; Correa, Diego H.

We study an XXX open spin chain with variable number of sites, where the variability is introduced only at the boundaries. This model arises naturally in the study of giant gravitons in the anti-de Sitter-space/conformal field-theory correspondence. We show how to quantize the spin chain by mapping its states to a bosonic lattice of finite length with sources and sinks of particles at the boundaries. Using coherent states, we show how the Hamiltonian for the bosonic lattice gives the correct description of semiclassical open strings ending on giant gravitons.

[Effects of Pseudomonas syringae pv. tabaci infection on tobacco photosynthetic apparatus under light or dark conditions.

PubMed

Cheng, Dan Dan; Sun, Jian Ping; Chai, Yuan; Zhu, Yi Yong; Zhao, Min; Sun, Guang Yu; Sun, Xing Bin

2016-08-01

Pseudomonas syringae pv. tabaci (Pst) is a hemi-biotrophic bacterial pathogen that causes the formation of brown spots named wildfire disease. Pst has received considerable attention in recent years. However, most of the studies focused on the tolerance and defense mechanisms of the host and non-host plants against Pst infection and a toxin originally described as being from Pst named tabtoxin, little information is available on the photosynthetic performance of tobacco leaves after Pst infection. Exploring the effects of Pst on the photosystem Ⅱ (PSⅡ) will not only help in clarifying tobacco-Pst interaction mechanisms, but also deepen the understanding of bacterial pathogen disease from a physiological perspective. By analyzing chlorophyll a fluorescence transient, performing western blot of thylakoid membrane and measuring the content of reactive oxygen species (ROS) and total chlorophyll, the effects of Pst on PS2 in tobacco were studied under light (200 μmol·m -2 ·s -1 ) or dark conditions. The results showed that chlorophyll content significantly decreased and significant chlorosis of the infiltrated zone was observed compared to the untreated ones, and tobacco leaves exhibited a visible and overt wildfire symptom at 3 days post Pst infection (dpi) under light and dark conditions. The H 2 O 2 content increased at 3 dpi compared to untreated ones in tobacco leaves under light and dark conditions, and was much higher under light than dark condition. Besides, markedly increase of the normalized relative variable fluorescence at the K step (W K ) and the relative variable fluorescence at the J step (V J ), significant decrease of maximal quantum yield of PS2 (F v /F m ) and density of Q A - reducing PS2 reaction centers per cross section (RC/CSm) were observed in tobacco leaves after Pst infection at 3 dpi under light and dark conditions. Moreover, inhibition of the K and J steps was more pronounced in the dark, as indicated by the greater increase of W K and V J under darkness compared with the light conditions during Pst inoculation. Dramatic (net) degradation of D1 protein and PsaO, the core protein of PS2 reaction center and oxygen evolving complex (OEC) respectively, at 3 dpi after Pst infection was observed in tobacco leaves under both light or dark conditions, and the decline was more exacerbated under dark than light condition. The results indicated that the electron transport from Q A to Q B of photosynthesis electron transport chain was severely blocked, OEC was damaged on both the donor and acceptor sides, and the reaction center of PS2 was severely damaged by Pst infection in tobacco lea-ves under either light or dark condition. Photoinhibition and photoinhibition-like damage of PSⅡ was observed after Pst infection, and the damage to PS2 under dark condition was much more severe than under light condition in tobacco leaves.

Investigation of Structure in the Light Curves of a Sample of Newly Discovered Long Period Variable Stars

NASA Astrophysics Data System (ADS)

Craine, E. R.; Culver, R. B.; Eykholt, R.; Flurchick, K. M.; Kraus, A. L.; Tucker, R. A.; Walker, D. K.

2015-09-01

Long period variable stars exhibit hump structures, and possibly flares, in their light curves. While the existence of humps is not controversial, the presence of flaring activity is less clear. Mining of a sky survey database of new variable star discoveries (the first MOTESS-GNAT Variable Star Catalog (MG1-VSC)) has led to identification of 47 such stars for which there are sufficient data to explore the presence of anomalous light curve features. We find a number of hump structures, and see one possible flare, suggesting that they are rare events. We present light curves and measured parameters for these stars, and a population statistical analysis.

A fusion-protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

PubMed

Hu, Jia; Chen, Xiang; Zhang, Xuhua; Yuan, Xiaopeng; Yang, Mingjuan; Dai, Hui; Yang, Wei; Zhou, Qinghua; Wen, Weihong; Wang, Qirui; Qin, Weijun; Zhao, Aizhi

2018-05-01

A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (V H ) and light (V L ) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development. © 2018 The Protein Society.

Nanobodies®: new ammunition to battle viruses.

PubMed

Vanlandschoot, Peter; Stortelers, Catelijne; Beirnaert, Els; Ibañez, Lorena Itatí; Schepens, Bert; Depla, Erik; Saelens, Xavier

2011-12-01

In 1989, a new type of antibody was identified, first in the sera of dromedaries and later also in all other species of the Camelidae family. These antibodies do not contain a light chain and also lack the first constant heavy domain. Today it is still unclear what the evolutionary advantage of such heavy chain-only antibodies could be. In sharp contrast, the broad applicability of the isolated variable antigen-binding domains (VHH) was rapidly recognized, especially for the development of therapeutic proteins, called Nanobodies(®). Here we summarize first some of the unique characteristics and features of VHHs. These will next be described in the context of different experimental therapeutic applications of Nanobodies against different viruses: HIV, Hepatitis B virus, influenza virus, Respiratory Syncytial virus, Rabies virus, FMDV, Poliovirus, Rotavirus, and PERVs. Next, the diagnostic application of VHHs (Vaccinia virus, Marburg virus and plant Tulip virus X), as well as an industrial application (lytic lactococcal 936 phage) will be described. In addition, the described data show that monovalent Nanobodies can possess unique characteristics not observed with conventional antibodies. The straightforward formatting into bivalent, multivalent, and/or multispecific Nanobodies allowed tailoring molecules for potency and cross-reactivity against viral targets with high sequence diversity. Copyright © 2011. Published by Elsevier B.V.

Fusion protein of CDR mimetic peptide with Fc inhibit TNF-alpha induced cytotoxicity.

PubMed

Qin, Weisong; Feng, Jiannan; Li, Yan; Lin, Zhou; Shen, Beifen

2006-02-01

The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-alpha (TNF-alpha) neutralizing monoclonal antibody (named as Z12) with TNF-alpha, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-alpha. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-alpha induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.

IgV gene intraclonal diversification and clonal evolution in B-cell chronic lymphocytic leukaemia.

PubMed

Bagnara, Davide; Callea, Vincenzo; Stelitano, Caterina; Morabito, Fortunato; Fabris, Sonia; Neri, Antonino; Zanardi, Sabrina; Ghiotto, Fabio; Ciccone, Ermanno; Grossi, Carlo Enrico; Fais, Franco

2006-04-01

Intraclonal diversification of immunoglobulin (Ig) variable (V) genes was evaluated in leukaemic cells from a B-cell chronic lymphocytic leukaemia (B-CLL) case over a 2-year period at four time points. Intraclonal heterogeneity was analysed by sequencing 305 molecular clones derived from polymerase chain reaction amplification of B-CLL cell IgV heavy (H) and light (C) chain gene rearrangements. Sequences were compared with evaluating intraclonal variation and the nature of somatic mutations. Although IgV intraclonal variation was detected at all time points, its level decreased with time and a parallel emergence of two more represented V(H)DJ(H) clones was observed. They differed by nine nucleotide substitutions one of which only caused a conservative replacement aminoacid change. In addition, one V(L)J(L) rearrangement became more represented over time. Analyses of somatic mutations suggest antigen selection and impairment of negative selection of neoplastic cells. In addition, a genealogical tree representing a model of clonal evolution of the neoplastic cells was created. It is of note that, during the period of study, the patient showed clinical progression of disease. We conclude that antigen stimulation and somatic hypermutation may participate in disease progression through the selection and expansion of neoplastic subclone(s).

Quantification of Peptides from Immunoglobulin Constant and Variable Regions by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry for Assessment of Multiple Myeloma Patients

PubMed Central

Remily-Wood, Elizabeth R.; Benson, Kaaron; Baz, Rachid C.; Chen, Y. Ann; Hussein, Mohamad; Hartley-Brown, Monique A.; Sprung, Robert W.; Perez, Brianna; Liu, Richard Z.; Yoder, Sean; Teer, Jamie; Eschrich, Steven A.; Koomen, John M.

2014-01-01

Purpose Quantitative mass spectrometry assays for immunoglobulins (Igs) are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, e.g. multiple myeloma. Experimental design Using LC-MS/MS data, Ig constant region peptides and transitions were selected for liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM). Quantitative assays were used to assess Igs in serum from 83 patients. Results LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1–4, IgA1–2, IgM, IgD, and IgE, as well as kappa(κ) and lambda(λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 multiple myeloma cell line and two MM patients. Conclusions and Clinical Relevance LC-MRM assays targeting constant region peptides determine the type and isoform of the involved immunoglobulin and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher interassay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques. PMID:24723328

Quantification of peptides from immunoglobulin constant and variable regions by LC-MRM MS for assessment of multiple myeloma patients.

PubMed

Remily-Wood, Elizabeth R; Benson, Kaaron; Baz, Rachid C; Chen, Y Ann; Hussein, Mohamad; Hartley-Brown, Monique A; Sprung, Robert W; Perez, Brianna; Liu, Richard Z; Yoder, Sean J; Teer, Jamie K; Eschrich, Steven A; Koomen, John M

2014-10-01

Quantitative MS assays for Igs are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, for example, multiple myeloma (MM). Using LC-MS/MS data, Ig constant region peptides, and transitions were selected for LC-MRM MS. Quantitative assays were used to assess Igs in serum from 83 patients. RNA sequencing and peptide-based LC-MRM are used to define peptides for quantification of the disease-specific Ig. LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1-4, IgA1-2, IgM, IgD, and IgE, as well as kappa (κ) and lambda (λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 MM cell line and two MM patients. LC-MRM assays targeting constant region peptides determine the type and isoform of the involved Ig and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher inter-assay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determinants of supply chain coordination of milk and dairy industries in Ethiopia: a case of Addis Ababa and its surroundings.

PubMed

Lemma, Habtamu Regassa; Singh, Rajwinder; Kaur, Navjot

2015-01-01

Coordination of different business activities among units becomes vital as organizations pay much attention to their core activities. Thus, their fruitfulness constantly relies on their capacity to coordinate their internal and external activities in the supply chain outside their own boundaries. Giving consideration to these obvious reasons, this paper aims to investigate the determinants of supply chain coordination of milk and dairy industries. The data were collected from 330 milk suppliers, processors, and retailers in the central part of Ethiopia. The structural equation modeling has been employed to develop the structural relationship between key constructs and measured variables. In total, 15 measured variables for coordination in the supply chain have been identified. These are further grouped into four factors namely, non-price coordination, price coordination, relationships and product development decision. It has been observed that the implementation of these factors could maximize the coordination linkage among supply chain members. Thus, dairy sectors should take the identified coordination factors into account in each of their business dealings.

The role of discharge variation in scaling of drainage area and food chain length in rivers

USGS Publications Warehouse

Sabo, John L.; Finlay, Jacques C.; Kennedy, Theodore A.; Post, David M.

2010-01-01

Food chain length (FCL) is a fundamental component of food web structure. Studies in a variety of ecosystems suggest that FCL is determined by energy supply, environmental stability, and/or ecosystem size, but the nature of the relationship between environmental stability and FCL, and the mechanism linking ecosystem size to FCL, remain unclear. Here we show that FCL increases with drainage area and decreases with hydrologic variability and intermittency across 36 North American rivers. Our analysis further suggests that hydrologic variability is the mechanism underlying the correlation between ecosystem size and FCL in rivers. Ecosystem size lengthens river food chains by integrating and attenuating discharge variation through stream networks, thereby enhancing environmental stability in larger river systems.

The role of discharge variation in scaling of drainage area and food chain length in rivers.

PubMed

Sabo, John L; Finlay, Jacques C; Kennedy, Theodore; Post, David M

2010-11-12

Food chain length (FCL) is a fundamental component of food web structure. Studies in a variety of ecosystems suggest that FCL is determined by energy supply, environmental stability, and/or ecosystem size, but the nature of the relationship between environmental stability and FCL, and the mechanism linking ecosystem size to FCL, remain unclear. Here we show that FCL increases with drainage area and decreases with hydrologic variability and intermittency across 36 North American rivers. Our analysis further suggests that hydrologic variability is the mechanism underlying the correlation between ecosystem size and FCL in rivers. Ecosystem size lengthens river food chains by integrating and attenuating discharge variation through stream networks, thereby enhancing environmental stability in larger river systems.

Epoxy-Based Organogels for Thermally Reversible Light Scattering Films and Form-Stable Phase Change Materials.

PubMed

Puig, Julieta; Dell' Erba, Ignacio E; Schroeder, Walter F; Hoppe, Cristina E; Williams, Roberto J J

2017-03-29

Alkyl chains of β-hydroxyesters synthesized by the capping of terminal epoxy groups of diglycidylether of bisphenol A (DGEBA) with palmitic (C16), stearic (C18), or behenic (C22) fatty acids self-assemble forming a crystalline phase. Above a particular concentration solutions of these esters in a variety of solvents led to supramolecular (physical) gels below the crystallization temperature of alkyl chains. A form-stable phase change material (FS-PCM) was obtained by blending the ester derived from behenic acid with eicosane. A blend containing 20 wt % ester was stable as a gel up to 53 °C and exhibited a heat storage capacity of 161 J/g, absorbed during the melting of eicosane at 37 °C. Thermally reversible light scattering (TRLS) films were obtained by visible-light photopolymerization of poly(ethylene glycol) dimethacrylate-ester blends (50 wt %) in the gel state at room temperature. The reaction was very fast and not inhibited by oxygen. TRLS films consisted of a cross-linked methacrylic network interpenetrated by the supramolecular network formed by the esters. Above the melting temperature of crystallites formed by alkyl chains, the film was transparent due to the matching between refractive indices of the methacrylic network and the amorphous ester. Below the crystallization temperature, the film was opaque because of light dispersion produced by the organic crystallites uniformly dispersed in the material. Of high significance for application was the fact that the contrast ratio did not depend on heating and cooling rates.

Quantifying side-chain conformational variations in protein structure

PubMed Central

Miao, Zhichao; Cao, Yang

2016-01-01

Protein side-chain conformation is closely related to their biological functions. The side-chain prediction is a key step in protein design, protein docking and structure optimization. However, side-chain polymorphism comprehensively exists in protein as various types and has been long overlooked by side-chain prediction. But such conformational variations have not been quantitatively studied and the correlations between these variations and residue features are vague. Here, we performed statistical analyses on large scale data sets and found that the side-chain conformational flexibility is closely related to the exposure to solvent, degree of freedom and hydrophilicity. These analyses allowed us to quantify different types of side-chain variabilities in PDB. The results underscore that protein side-chain conformation prediction is not a single-answer problem, leading us to reconsider the assessment approaches of side-chain prediction programs. PMID:27845406

Quantifying side-chain conformational variations in protein structure

NASA Astrophysics Data System (ADS)

Miao, Zhichao; Cao, Yang

2016-11-01

Protein side-chain conformation is closely related to their biological functions. The side-chain prediction is a key step in protein design, protein docking and structure optimization. However, side-chain polymorphism comprehensively exists in protein as various types and has been long overlooked by side-chain prediction. But such conformational variations have not been quantitatively studied and the correlations between these variations and residue features are vague. Here, we performed statistical analyses on large scale data sets and found that the side-chain conformational flexibility is closely related to the exposure to solvent, degree of freedom and hydrophilicity. These analyses allowed us to quantify different types of side-chain variabilities in PDB. The results underscore that protein side-chain conformation prediction is not a single-answer problem, leading us to reconsider the assessment approaches of side-chain prediction programs.

Quantifying side-chain conformational variations in protein structure.

PubMed

Miao, Zhichao; Cao, Yang

2016-11-15

Protein side-chain conformation is closely related to their biological functions. The side-chain prediction is a key step in protein design, protein docking and structure optimization. However, side-chain polymorphism comprehensively exists in protein as various types and has been long overlooked by side-chain prediction. But such conformational variations have not been quantitatively studied and the correlations between these variations and residue features are vague. Here, we performed statistical analyses on large scale data sets and found that the side-chain conformational flexibility is closely related to the exposure to solvent, degree of freedom and hydrophilicity. These analyses allowed us to quantify different types of side-chain variabilities in PDB. The results underscore that protein side-chain conformation prediction is not a single-answer problem, leading us to reconsider the assessment approaches of side-chain prediction programs.

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.

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

NASA Astrophysics Data System (ADS)

He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

2017-05-01

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Designing of an artificial light energy converter in the form of short-chain dyad when combined with core-shell gold/silver nanocomposites.

PubMed

Dutta Pal, Gopa; Paul, Somnath; Bardhan, Munmun; De, Asish; Ganguly, Tapan

2017-06-05

UV-vis absorption, steady state and time resolved fluorescence and absorption spectroscopic investigations demonstrate that the short chain dyad MNTMA when combined with gold-silver core-shell (Au@Ag) nanocomposite , forms elongated conformers in the excited state whereas for the dyad - Ag (spherical) system the majority of dyads remains in a folded conformation. In the dyad-core-shell nanocomposite system, energy wasting charge recombination rate slows down primarily due to elongated conformation and thus it may be anticipated that this hybrid nanocomposite system may serve as a better light energy conversion device. Copyright © 2017 Elsevier B.V. All rights reserved.

[18F]-NaF PET/CT imaging in cardiac amyloidosis.

PubMed

Van Der Gucht, Axel; Galat, Arnault; Rosso, Jean; Guellich, Aziz; Garot, Jérôme; Bodez, Diane; Plante-Bordeneuve, Violaine; Hittinger, Luc; Dubois-Randé, Jean-Luc; Evangelista, Eva; Sasanelli, Myriam; Chalaye, Julia; Meignan, Michel; Itti, Emmanuel; Damy, Thibaud

2016-08-01

Cardiac amyloidosis (CA) is recognized as a common cause of restrictive cardiomyopathy and heart failure due to the deposition of insoluble proteins in the myocardial interstitium. We emphasize the role of [18F]-sodium fluoride (NaF) PET/CT as a potential noninvasive tool to identify and differentiate the transthyretin-related cardiac amyloidosis from the light-chain cardiac amyloidosis. We report cases of a 73-year-old man and a 75-year-old woman followed in our center for congestive heart failure with marked alteration of the left ventricular ejection fraction due to familial transthyretin Val122Ile cardiac amyloidosis and light-chain cardiac amyloidosis, respectively, confirmed on endomyocardial biopsy.

Highly sensitive and unbiased approach for elucidating antibody repertoires

PubMed Central

Lin, Sherry G.; Ba, Zhaoqing; Du, Zhou; Zhang, Yu; Hu, Jiazhi; Alt, Frederick W.

2016-01-01

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. PMID:27354528

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer.

PubMed

Frigerio, B; Fracasso, G; Luison, E; Cingarlini, S; Mortarino, M; Coliva, A; Seregni, E; Bombardieri, E; Zuccolotto, G; Rosato, A; Colombatti, M; Canevari, S; Figini, M

2013-06-01

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy. To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers. Published by Elsevier Ltd.

From Chains for Mean Value Inequalities to Mitrinovic's Problem II

ERIC Educational Resources Information Center

Zhu, Ling

2005-01-01

In this note, an integrated form of some significant means with two variables is provided, and some chains for mean value inequalities are obtained. At the same time, a concise family of algebraic functions appears, which satisfy Mitrinovic's requirements.

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

Fast response dry-type artificial molecular muscles with [c2]daisy chains

NASA Astrophysics Data System (ADS)

Iwaso, Kazuhisa; Takashima, Yoshinori; Harada, Akira

2016-06-01

Hierarchically organized myosin and actin filaments found in biological systems exhibit contraction and expansion behaviours that produce work and force by consuming chemical energy. Inspired by these naturally occurring examples, we have developed photoresponsive wet- and dry-type molecular actuators built from rotaxane-based compounds known as [c2]daisy chains (specifically, [c2]AzoCD2 hydrogel and [c2]AzoCD2 xerogel). These actuators were prepared via polycondensation between four-armed poly(ethylene glycol) and a [c2]daisy chain based on α-cyclodextrin as the host component and azobenzene as a photoresponsive guest component. The light-induced actuation arises from the sliding motion of the [c2]daisy chain unit. Ultraviolet irradiation caused the gels to bend towards the light source. The response of the [c2]AzoCD2 xerogel, even under dry conditions, is very fast (7° every second), which is 10,800 times faster than the [c2]AzoCD2 hydrogel (7° every 3 h). In addition, the [c2]AzoCD2 xerogel was used as a crane arm to lift an object using ultraviolet irradiation to produce mechanical work.

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.

Minimally invasive surgery: only as good as the picture.

PubMed Central

Drury, Nigel E.; Pollard, Rebecca; Dyer, Jonathan P.

2004-01-01

BACKGROUND: In minimally invasive surgery, there is increased reliance on real-time 2-dimensional images. The fibre-optic light lead is one of the most frequently damaged elements of the 'imaging chain', leading to a poor quality picture. METHODS: Light leads with a honeycomb projection were connected to a light source and the resulting beam directed at a sheet of paper. Darkened sectors with diminished or absent light transmission were recorded. RESULTS: All suitable light leads in routine use were examined. A mean of 22.2% (SD 7.8%) of the projection had diminished or absent light transmission. CONCLUSION: Sub-optimal endoscopic equipment was in routine use. PMID:15005945

IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST.

PubMed

Giudicelli, Véronique; Duroux, Patrice; Kossida, Sofia; Lefranc, Marie-Paule

2017-06-26

IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain. The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation. For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino changes). The functionality is generic and can analyse any IG or TR single chain nucleotide sequence containing two V domains, provided that the corresponding species IMGT reference directory is available. The "Analysis of single chain Fragment variable (scFv)" implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST provides the identification and full characterization of the two V domains of full-length scFv (~850 bp) nucleotide sequences from combinatorial libraries. The analysis can also be performed on concatenated paired chains of expressed antigen receptor IG or TR repertoires.

A new era for homolytic aromatic substitution: replacing Bu3SnH with efficient light-induced chain reactions.

PubMed

Gurry, Michael; Aldabbagh, Fawaz

2016-04-28

Herein is a pertinent review of recent photochemical homolytic aromatic substitution (HAS) literature. Issues with using the reductant Bu3SnH in an oxidative process where the net loss of a hydrogen atom occurs is discussed. Nowadays more efficient light-induced chain reactions are used resulting in HAS becoming a synthetic mechanism of choice rivaling organometallic, transition-metal and electrophilic aromatic substitution protocols. The review includes aromatic substitution as part of a tandem or cascade reaction, Pschorr reaction, as well as HAS facilitated by ipso-substitution, and Smiles rearrangement. Recently visible-light photoredox catalysis, which is carried out at room temperature has become one of the most important means of aromatic substitution. The main photoredox catalysts used are polypyridine complexes of Ru(ii) and Ir(iii), although eosin Y is an alternative allowing metal-free HAS. Other radical initiator-free aromatic substitutions have used 9-mesityl-10-methylacridinium ion and N,N-bis(2,6-diisopropylphenyl)perylene-3,4,9,10-bis(dicarboximide) as the photoredox catalyst, UV-light, photoinduced electron-transfer, zwitterionic semiquinone radical anions, and Barton ester intermediates.

Integrating biomass quality variability in stochastic supply chain modeling and optimization for large-scale biofuel production

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

Castillo-Villar, Krystel K.; Eksioglu, Sandra; Taherkhorsandi, Milad

The production of biofuels using second-generation feedstocks has been recognized as an important alternative source of sustainable energy and its demand is expected to increase due to regulations such as the Renewable Fuel Standard. However, the pathway to biofuel industry maturity faces unique, unaddressed challenges. Here, to address this challenges, this article presents an optimization model which quantifies and controls the impact of biomass quality variability on supply chain related decisions and technology selection. We propose a two-stage stochastic programming model and associated efficient solution procedures for solving large-scale problems to (1) better represent the random nature of the biomassmore » quality (defined by moisture and ash contents) in supply chain modeling, and (2) assess the impact of these uncertainties on the supply chain design and planning. The proposed model is then applied to a case study in the state of Tennessee. Results show that high moisture and ash contents negatively impact the unit delivery cost since poor biomass quality requires the addition of quality control activities. Experimental results indicate that supply chain cost could increase as much as 27%–31% when biomass quality is poor. We assess the impact of the biomass quality on the topological supply chain. Our case study indicates that biomass quality impacts supply chain costs; thus, it is important to consider the impact of biomass quality in supply chain design and management decisions.« less

Integrating biomass quality variability in stochastic supply chain modeling and optimization for large-scale biofuel production

DOE PAGES

Castillo-Villar, Krystel K.; Eksioglu, Sandra; Taherkhorsandi, Milad

2017-02-20

The production of biofuels using second-generation feedstocks has been recognized as an important alternative source of sustainable energy and its demand is expected to increase due to regulations such as the Renewable Fuel Standard. However, the pathway to biofuel industry maturity faces unique, unaddressed challenges. Here, to address this challenges, this article presents an optimization model which quantifies and controls the impact of biomass quality variability on supply chain related decisions and technology selection. We propose a two-stage stochastic programming model and associated efficient solution procedures for solving large-scale problems to (1) better represent the random nature of the biomassmore » quality (defined by moisture and ash contents) in supply chain modeling, and (2) assess the impact of these uncertainties on the supply chain design and planning. The proposed model is then applied to a case study in the state of Tennessee. Results show that high moisture and ash contents negatively impact the unit delivery cost since poor biomass quality requires the addition of quality control activities. Experimental results indicate that supply chain cost could increase as much as 27%–31% when biomass quality is poor. We assess the impact of the biomass quality on the topological supply chain. Our case study indicates that biomass quality impacts supply chain costs; thus, it is important to consider the impact of biomass quality in supply chain design and management decisions.« less

Metal-Dependent Amyloid β-Degrading Catalytic Antibody Construct

PubMed Central

Nishiyama, Yasuhiro; Taguchi, Hiroaki; Hara, Mariko; Planque, Stephanie A.; Mitsuda, Yukie; Paul, Sudhir

2015-01-01

Catalytic antibodies (catabodies) that degrade target antigens rapidly are rare. We describe the metal-dependence of catabody construct 2E6, an engineered heterodimer of immunoglobulin light chain variable domains that hydrolyzes amyloid β peptides (Aβ) specifically. In addition to the electrophilic phosphonate inhibitor of serine proteases, the metal chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline completely inhibited the hydrolysis of Aβ by catabody 2E6. Formation of catabody-electrophilic phosphonate inhibitor adducts was unaffected by EDTA, suggesting that the metal exerts a favorable effect on a catalytic step after the initial catabody nucleophilic attack on Aβ. The EDTA inactivated catabody failed to disaggregate fibrillar Aβ, indicating the functional importance of the Aβ hydrolytic activity. Treating the EDTA-inactivated catabody with Zn2+ or Co2+ restored the Aβ hydrolytic activity, and Zn2+-induced catabody conformational transitions were evident by fluorescence emission spectroscopy. The studies reveal the absolute catabody dependence on a metal cofactor. PMID:24698848

Biased immunoglobulin light chain gene usage in the shark1

PubMed Central

Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

2015-01-01

This study of a large family of kappa light (L) chain clusters in nurse shark completes the characterization of its classical immunoglobulin (Ig) gene content (two heavy chain classes, mu and omega, and four L chain isotopes, kappa, lambda, sigma, and sigma-2). The shark kappa clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over a ~500 bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ca. 39 kappa clusters are pre-rearranged in the germline (GL-joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, non-productive, and sterile transcripts of the kappa clusters compared to the other three L chain isotypes. Kappa cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and non-productive rearrangements. These results show that the individual activation of the spatially distant kappa clusters is non-random. Although both split and GL-joined kappa genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing. PMID:26342033

Toughening elastomers with sacrificial bonds and watching them break

NASA Astrophysics Data System (ADS)

Creton, Costantino

2014-03-01

Most unfilled elastomers are relatively brittle, in particular when the average molecular weight between crosslinks is lower than the average molecular weight between entanglements. We created a new class of tough elastomers by introducing isotropically prestretched chains inside ordinary acrylic elastomers by successive swelling and polymerization steps. These new materials combine a high entanglement density with a densely crosslinked structure reaching elastic moduli of 4 MPa and fracture strength of 25 MPa. The highly prestretched chains are the minority in the material and can break in the bulk of the material before catastrophic failure occurs, increasing the toughness of the material by two orders of magnitude up to 5 kJ/m2. To investigate the details of the toughening mechanism we introduced specific sacrificial dioxetane bonds in the prestretched chains that emit light when they break. In uniaxial extension cyclic experiments, we checked that the light emission corresponded exactly and quantitatively to the energy dissipation in each cycle demonstrating that short chains break first and long chains later. We then watched crack propagation in notched samples and mapped spatially the location of bond breakage ahead of the crack tip before and during propagation. This new toughening mechanism for elastomers creates superentangled rubbers and is ideally suited to overcome the trade-off between toughness and stiffness of ordinary elastomers. We gratefully acknowledge funding from DSM Ahead

Survival condition for low-frequency quasi-one-dimensional breathers in a two-dimensional strongly anisotropic crystal

NASA Astrophysics Data System (ADS)

Savin, A. V.; Zubova, E. A.; Manevitch, L. I.

2005-06-01

We investigate a two-dimensional (2D) strongly anisotropic crystal (2D SAC) on substrate: 2D system of coupled linear chains of particles with strong intrachain and weak interchain interactions, each chain being subjected to the sine background potential. Nonlinear dynamics of one of these chains when the rest of them are fixed is reduced to the well known Frenkel-Kontorova (FK) model. Depending on strengh of the substrate, the 2D SAC models a variety of physical systems: polymer crystals with identical chains having light side groups, an array of inductively coupled long Josephson junctions, anisotropic crystals having light and heavy sublattices. Continuum limit of the FK model, the sine-Gordon (sG) equation, allows two types of soliton solutions: topological solitons and breathers. It is known that the quasi-one-dimensional topological solitons can propagate also in a chain of 2D system of coupled chains and even in a helix chain in a three-dimensional model of polymer crystal. In contrast to this, numerical simulation shows that the long-living breathers inherent to the FK model do not exist in the 2D SAC with weak background potential. The effect changes scenario of kink-antikink collision with small relative velocity: at weak background potential the collision always results only in intensive phonon radiation while kink-antikink recombination in the FK model results in long-living low-frequency sG breather creation. We found the survival condition for breathers in the 2D SAC on substrate depending on breather frequency and strength of the background potential. The survival condition bears no relation to resonances between breather frequency and frequencies of phonon band—contrary to the case of the FK model.

Probing AGN Accretion Physics through AGN Variability: Insights from Kepler

NASA Astrophysics Data System (ADS)

Kasliwal, Vishal Pramod

Active Galactic Nuclei (AGN) exhibit large luminosity variations over the entire electromagnetic spectrum on timescales ranging from hours to years. The variations in luminosity are devoid of any periodic character and appear stochastic. While complex correlations exist between the variability observed in different parts of the electromagnetic spectrum, no frequency band appears to be completely dominant, suggesting that the physical processes producing the variability are exceedingly rich and complex. In the absence of a clear theoretical explanation of the variability, phenomenological models are used to study AGN variability. The stochastic behavior of AGN variability makes formulating such models difficult and connecting them to the underlying physics exceedingly hard. We study AGN light curves serendipitously observed by the NASA Kepler planet-finding mission. Compared to previous ground-based observations, Kepler offers higher precision and a smaller sampling interval resulting in potentially higher quality light curves. Using structure functions, we demonstrate that (1) the simplest statistical model of AGN variability, the damped random walk (DRW), is insufficient to characterize the observed behavior of AGN light curves; and (2) variability begins to occur in AGN on time-scales as short as hours. Of the 20 light curves studied by us, only 3-8 may be consistent with the DRW. The structure functions of the AGN in our sample exhibit complex behavior with pronounced dips on time-scales of 10-100 d suggesting that AGN variability can be very complex and merits further analysis. We examine the accuracy of the Kepler pipeline-generated light curves and find that the publicly available light curves may require re-processing to reduce contamination from field sources. We show that while the re-processing changes the exact PSD power law slopes inferred by us, it is unlikely to change the conclusion of our structure function study-Kepler AGN light curves indicate that the DRW is insufficient to characterize AGN variability. We provide a new approach to probing accretion physics with variability by decomposing observed light curves into a set of impulses that drive diffusive processes using C-ARMA models. Applying our approach to Kepler data, we demonstrate how the time-scales reported in the literature can be interpreted in the context of the growth and decay time-scales for flux perturbations and tentatively identify the flux perturbation driving process with accretion disk turbulence on length-scales much longer than the characteristic eddy size. Our analysis technique is applicable to (1) studying the connection between AGN sub-type and variability properties; (2) probing the origins of variability by studying the multi-wavelength behavior of AGN; (3) testing numerical simulations of accretion flows with the goal of creating a library of the variability properties of different accretion mechanisms; (4) hunting for changes in the behavior of the accretion flow by block-analyzing observed light curves; and (5) constraining the sampling requirements of future surveys of AGN variability.

Chain-Growth Methods for the Synthesis of High Molecular Weight Conducting and Semiconducting Polymers

DTIC Science & Technology

2013-08-25

to produce the desired polymerization in analogy to the well-known “super glue ” anionic polymerization. Although there are abundant examples of...light (a) and UV light (b). 5 are further functionalized and block polymers formed with polynorborene have elastomeric properties. The...top) and UV (bottom) light show the evolution of the band gap of the polymer with increasing molecular weight. The plot on the right shows the

The characteristics of polyimide photoalignment layer with chalcone derivatives produced by linear polarized UV light

NASA Astrophysics Data System (ADS)

Jung, Kyoung Hoon; Hyun, Soon-Young; Song, Dong-Mee; Shin, Dong-Myung

2003-01-01

The photoalignment of liquid crystal (LC) molecules located onto polyimide films with chalcone derivatives using linearly polarized UV (LPUV) light is investigated. The LPUV light irradiation generated dimerization products of the chalcones followed by isomerization of the chalcone derivatives. The alignment directions of LC molecules were either homeotropic or planar with respect to plane of polyimide film, depending upon the alkyl chain length attached on the chalcones.