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.

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

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

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.

Increased myosin heavy chain-beta with atrial expression of ventricular light chain-2 in canine cardiomyopathy.

PubMed

Fuller, Geraldine A; Bicer, Sabahattin; Hamlin, Robert L; Yamaguchi, Mamoru; Reiser, Peter J

2007-10-01

Dilated cardiomyopathy is a naturally occurring disease in humans and dogs. Human studies have shown increased levels of myosin heavy chain (MHC)-beta in failing ventricles and the left atria (LA) and of ventricular light chain (VLC)-2 in the right atria in dilated cardiomyopathy. This study evaluates the levels of MHC-beta in all heart chambers in prolonged canine right ventricular pacing. In addition, we determined whether levels of VLC2 were altered in these hearts. Failing hearts demonstrated significantly increased levels of MHC-beta in the right atria, right atrial appendage, LA, left atrial appendage (LAA), and right ventricle compared with controls. Significant levels of VLC2 were detected in the right atria of paced hearts. Differences in MHC-beta expression were observed between the LA and the LAA of paced and control dogs. MHC-beta expression was significantly greater in the LA of paced and control dogs compared with their respective LAA. The cardiac myosin isoform shifts in this study were similar to those observed in end-stage human heart failure and more severe than those reported in less prolonged pacing models, supporting the use of this model for further study of end-stage human heart failure. The observation of consistent differences between sampling sites, especially LA versus LAA, indicates the need for rigorous sampling consistency in future studies.

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.

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.
.

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

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.

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.

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

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.

Serum levels of immunoglobulin free light chains in patients with chronic hepatitis C presenting cryoglobulinemia.

PubMed

Oliveira, Isabela S; Cabral, Milena S; Jesus, Larissa S; Paraná, Raymundo; Atta, Ajax M; Sousa Atta, Maria Luiza B

2014-01-01

Hepatitis C virus (HCV) infects B-lymphocytes, provokes cellular dysfunction and causes lymphoproliferative diseases such as cryoglobulinemia and non-Hodgkin's B-cell lymphoma. In the present study, we investigated the serum levels of kappa and lambda free light chains (FLC) of immunoglobulins and the kappa/lambda FLC ratio in Brazilian patients with chronic HCV infection and cryoglobulinemia. We also analyzed the immunochemical composition of the cryoglobulins in these patients. Twenty-eight cryoglobulinemic HCV patients composed the target group, while 37 HCV patients without cryoglobulinemia were included as controls. The median levels of kappa and lambda FLC were higher in patients with cryoglobulinemia compared to controls (p=0.001 and p=0.003, respectively), but the kappa/lambda FLC ratio was similar in patients with and without cryoglobulinemia (p>0.05). The median FLC ratio was higher in HCV patients presenting with advanced fibrosis of the liver compared to HCV patients without fibrosis (p=0.004). Kappa and lambda FLC levels were strongly correlated with the IgA, IgG and IgM levels in the patients with cryoglobulinemia. In patients without cryoglobulinemia, the kappa FLC level was only correlated with the IgG level, whereas the lambda FLC were weakly correlated with the IgA, IgG and IgM levels. An immunochemical pattern of mixed cryoglobulins (MC), predominantly IgM, IgG, IgA and kappa light chain, was verified in these immune complexes. We concluded that HCV-infected patients presenting cryoglobulinemia have vigorous polyclonal B-lymphocyte activation due to chronic HCV infection and persistent immune stimulation. Copyright © 2014 Elsevier Editora Ltda. All rights reserved.

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.

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.

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.

Chromophore absorbance change quantification in tissue during low-level light therapy

NASA Astrophysics Data System (ADS)

Huynh, Daniel; Chung, Christine; Qian, Li; Lilge, Lothar

2012-03-01

Low Level Light Therapy (LLLT) has been implicated to stimulate tissue, promoting healing and reducing pain. One of the potential pathways stimulated by LLLT relates to the electron transport chain, where photon quantum energy can induce a change in the biochemical reactions within the cell. The aim of this study is to assess the feasibility to exploit light additionally as a diagnostic tool to determine tissue physiological states, particularly in quantifying the changes in redox states of Cytochrome C as a result of induced LLLT biochemical reactions.

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.

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.

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.

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.

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions.

PubMed

Townsend, Catherine L; Laffy, Julie M J; Wu, Yu-Chang Bryan; Silva O'Hare, Joselli; Martin, Victoria; Kipling, David; Fraternali, Franca; Dunn-Walters, Deborah K

2016-01-01

Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions

PubMed Central

Townsend, Catherine L.; Laffy, Julie M. J.; Wu, Yu-Chang Bryan; Silva O’Hare, Joselli; Martin, Victoria; Kipling, David; Fraternali, Franca; Dunn-Walters, Deborah K.

2016-01-01

Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response. PMID:27729912

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

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.

Structure and energetic basis of overrepresented λ light chain in systemic light chain amyloidosis patients.

PubMed

Zhao, Jun; Zhang, Baohong; Zhu, Jianwei; Nussinov, Ruth; Ma, Buyong

2018-06-01

Amyloid formation and deposition of immunoglobulin light-chain proteins in systemic amyloidosis (AL) cause major organ failures. While the κ light-chain is dominant (λ/κ=1:2) in healthy individuals, λ is highly overrepresented (λ/κ=3:1) in AL patients. The structural basis of the amyloid formation and the sequence preference are unknown. We examined the correlation between sequence and structural stability of dimeric variable domains of immunoglobulin light chains using molecular dynamics simulations of 24 representative dimer interfaces, followed by energy evaluation of conformational ensembles for 20 AL patients' light chain sequences. We identified a stable interface with displaced N-terminal residues, provides the structural basis for AL protein fibrils formation. Proline isomerization may cause the N-terminus to adopt amyloid-prone conformations. We found that λ light-chains prefer misfolded dimer conformation, while κ chain structures are stabilized by a natively folded dimer. Our study may facilitate structure-based small molecule and antibody design to inhibit AL. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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.

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.

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

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.
.

Convergent mechanisms favor fast amyloid formation in two lambda 6a Ig light chain mutants.

PubMed

Valdés-García, Gilberto; Millán-Pacheco, César; Pastor, Nina

2017-08-01

Extracellular deposition as amyloids of immunoglobulin light chains causes light chain amyloidosis. Among the light chain families, lambda 6a is one of the most frequent in light chain amyloidosis patients. Its germline protein, 6aJL2, and point mutants, R24G and P7S, are good models to study fibrillogenesis, because their stability and fibril formation characteristics have been described. Both mutations make the germline protein unstable and speed up its ability to aggregate. To date, there is no molecular mechanism that explains how these differences in amyloidogenesis can arise from a single mutation. To look into the structural and dynamical differences in the native state of these proteins, we carried out molecular dynamics simulations at room temperature. Despite the structural similarity of the germline protein and the mutants, we found differences in their dynamical signatures that explain the mutants' increased tendency to form amyloids. The contact network alterations caused by the mutations, though different, converge in affecting two anti-aggregation motifs present in light chain variable domains, suggesting a different starting point for aggregation in lambda chains compared to kappa chains. © 2017 Wiley Periodicals, Inc.

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.

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

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

2016-01-01

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. 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. 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. 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.

Searching for neurodegeneration in multiple sclerosis at clinical onset: Diagnostic value of biomarkers.

PubMed

Novakova, Lenka; Axelsson, Markus; Malmeström, Clas; Imberg, Henrik; Elias, Olle; Zetterberg, Henrik; Nerman, Olle; Lycke, Jan

2018-01-01

Neurodegeneration occurs during the early stages of multiple sclerosis. It is an essential, devastating part of the pathophysiology. Tools for measuring the degree of neurodegeneration could improve diagnostics and patient characterization. This study aimed to determine the diagnostic value of biomarkers of degeneration in patients with recent clinical onset of suspected multiple sclerosis, and to evaluate these biomarkers for characterizing disease course. This cross-sectional study included 271 patients with clinical features of suspected multiple sclerosis onset and was the baseline of a prospective study. After diagnostic investigations, the patients were classified into the following disease groups: patients with clinically isolated syndrome (n = 4) or early relapsing remitting multiple sclerosis (early RRMS; n = 93); patients with relapsing remitting multiple sclerosis with disease durations ≥2 years (established RRMS; n = 39); patients without multiple sclerosis, but showing symptoms (symptomatic controls; n = 89); and patients diagnosed with other diseases (n = 46). In addition, we included healthy controls (n = 51) and patients with progressive multiple sclerosis (n = 23). We analyzed six biomarkers of neurodegeneration: cerebrospinal fluid neurofilament light chain levels; cerebral spinal fluid glial fibrillary acidic protein; cerebral spinal fluid tau; retinal nerve fiber layer thickness; macula volume; and the brain parenchymal fraction. Except for increased cerebral spinal fluid neurofilament light chain levels, median 670 ng/L (IQR 400-2110), we could not find signs of early degeneration in the early disease group with recent clinical onset. However, the intrathecal immunoglobin G production and cerebral spinal fluid neurofilament light chain levels showed diagnostic value. Moreover, elevated levels of cerebral spinal fluid glial fibrillary acidic protein, thin retinal nerve fiber layers, and low brain parenchymal fractions were associated with progressive disease, but not with the other phenotypes. Thin retinal nerve fiber layers and low brain parenchymal fractions, which indicated neurodegeneration, were associated with longer disease duration. In clinically suspected multiple sclerosis, intrathecal immunoglobin G production and neurofilament light chain levels had diagnostic value. Therefore, these biomarkers could be included in diagnostic work-ups for multiple sclerosis. We found that the thickness of the retinal nerve fiber layer and the brain parenchymal fraction were not different between individuals that were healthy, symptomatic, or newly diagnosed with multiple sclerosis. This finding suggested that neurodegeneration had not reached a significant magnitude in patients with a recent clinical onset of multiple sclerosis.

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

PubMed Central

Dul, J L; Argon, Y

1990-01-01

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

[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.

Interaction between glycosaminoglycans and immunoglobulin light chains.

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

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

1997-01-01

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

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

PubMed

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

2013-04-01

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

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

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

Multispectral simulation environment for modeling low-light-level sensor systems

NASA Astrophysics Data System (ADS)

Ientilucci, Emmett J.; Brown, Scott D.; Schott, John R.; Raqueno, Rolando V.

1998-11-01

Image intensifying cameras have been found to be extremely useful in low-light-level (LLL) scenarios including military night vision and civilian rescue operations. These sensors utilize the available visible region photons and an amplification process to produce high contrast imagery. It has been demonstrated that processing techniques can further enhance the quality of this imagery. For example, fusion with matching thermal IR imagery can improve image content when very little visible region contrast is available. To aid in the improvement of current algorithms and the development of new ones, a high fidelity simulation environment capable of producing radiometrically correct multi-band imagery for low- light-level conditions is desired. This paper describes a modeling environment attempting to meet these criteria by addressing the task as two individual components: (1) prediction of a low-light-level radiance field from an arbitrary scene, and (2) simulation of the output from a low- light-level sensor for a given radiance field. The radiance prediction engine utilized in this environment is the Digital Imaging and Remote Sensing Image Generation (DIRSIG) model which is a first principles based multi-spectral synthetic image generation model capable of producing an arbitrary number of bands in the 0.28 to 20 micrometer region. The DIRSIG model is utilized to produce high spatial and spectral resolution radiance field images. These images are then processed by a user configurable multi-stage low-light-level sensor model that applies the appropriate noise and modulation transfer function (MTF) at each stage in the image processing chain. This includes the ability to reproduce common intensifying sensor artifacts such as saturation and 'blooming.' Additionally, co-registered imagery in other spectral bands may be simultaneously generated for testing fusion and exploitation algorithms. This paper discusses specific aspects of the DIRSIG radiance prediction for low- light-level conditions including the incorporation of natural and man-made sources which emphasizes the importance of accurate BRDF. A description of the implementation of each stage in the image processing and capture chain for the LLL model is also presented. Finally, simulated images are presented and qualitatively compared to lab acquired imagery from a commercial system.

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.

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

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.

Tau elevations in the brain extracellular space correlate with reduced amyloid-β levels and predict adverse clinical outcomes after severe traumatic brain injury.

PubMed

Magnoni, Sandra; Esparza, Thomas J; Conte, Valeria; Carbonara, Marco; Carrabba, Giorgio; Holtzman, David M; Zipfel, Greg J; Stocchetti, Nino; Brody, David L

2012-04-01

Axonal injury is believed to be a major determinant of adverse outcomes following traumatic brain injury. However, it has been difficult to assess acutely the severity of axonal injury in human traumatic brain injury patients. We hypothesized that microdialysis-based measurements of the brain extracellular fluid levels of tau and neurofilament light chain, two low molecular weight axonal proteins, could be helpful in this regard. To test this hypothesis, 100 kDa cut-off microdialysis catheters were placed in 16 patients with severe traumatic brain injury at two neurological/neurosurgical intensive care units. Tau levels in the microdialysis samples were highest early and fell over time in all patients. Initial tau levels were >3-fold higher in patients with microdialysis catheters placed in pericontusional regions than in patients in whom catheters were placed in normal-appearing right frontal lobe tissue (P = 0.005). Tau levels and neurofilament light-chain levels were positively correlated (r = 0.6, P = 0.013). Neurofilament light-chain levels were also higher in patients with pericontusional catheters (P = 0.04). Interestingly, initial tau levels were inversely correlated with initial amyloid-β levels measured in the same samples (r = -0.87, P = 0.000023). This could be due to reduced synaptic activity in areas with substantial axonal injury, as amyloid-β release is closely coupled with synaptic activity. Importantly, high initial tau levels correlated with worse clinical outcomes, as assessed using the Glasgow Outcome Scale 6 months after injury (r = -0.6, P = 0.018). Taken together, our data add support for the hypothesis that axonal injury may be related to long-term impairments following traumatic brain injury. Microdialysis-based measurement of tau levels in the brain extracellular space may be a useful way to assess the severity of axonal injury acutely in the intensive care unit. Further studies with larger numbers of patients will be required to assess the reproducibility of these findings and to determine whether this approach provides added value when combined with clinical and radiological information.

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.

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

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.

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

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.

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.

Three-dimensional Speckle Tracking Echocardiography in Light Chain Cardiac Amyloidosis: Examination of Left and Right Ventricular Myocardial Mechanics Parameters.

PubMed

Urbano-Moral, Jose Angel; Gangadharamurthy, Dakshin; Comenzo, Raymond L; Pandian, Natesa G; Patel, Ayan R

2015-08-01

The study of myocardial mechanics has a potential role in the detection of cardiac involvement in patients with amyloidosis. This study aimed to characterize 3-dimensional-speckle tracking echocardiography-derived left and right ventricular myocardial mechanics in light chain amyloidosis and examine their relationship with brain natriuretic peptide. In patients with light chain amyloidosis, left ventricular longitudinal and circumferential strain (n=40), and right ventricular longitudinal strain and radial displacement (n=26) were obtained by 3-dimensional-speckle tracking echocardiography. Brain natriuretic peptide levels were determined. All myocardial mechanics measurements showed differences when compared by brain natriuretic peptide level tertiles. Left and right ventricular longitudinal strain were highly correlated (r=0.95, P<.001). Left ventricular longitudinal and circumferential strain were reduced in patients with cardiac involvement (-9±4 vs -16±2; P<.001, and -24±6 vs -29±4; P=.01, respectively), with the most prominent impairment at the basal segments. Right ventricular longitudinal strain and radial displacement were diminished in patients with cardiac involvement (-9±3 vs -17±3; P<.001, and 2.7±0.8 vs 3.8±0.3; P=.002). On multivariate analysis, left ventricular longitudinal strain was associated with the presence of cardiac involvement (odds ratio = 1.6; 95% confidence interval, 1.04 to 2.37; P=.03) independent of the presence of brain natriuretic peptide and troponin I criteria for cardiac amyloidosis. Three-dimensional-speckle tracking echocardiography-derived left and right ventricular myocardial mechanics are increasingly altered as brain natriuretic peptide increases in light chain amyloidosis. There appears to be a strong association between left ventricular longitudinal strain and cardiac involvement, beyond biomarkers such as brain natriuretic peptide and troponin I. Copyright © 2015 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

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.

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.

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.

[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.

Characterization of renal amyloid derived from the variable region of the lambda light chain subgroup II.

PubMed Central

Picken, M. M.; Gallo, G.; Buxbaum, J.; Frangione, B.

1986-01-01

Amyloid fibrils were extracted from the kidney of a patient (CHE) shown to have tetramers and dimers of a monoclonal lambda light chain in his serum, and whose bone marrow cells in short-term culture synthesized these forms and a smaller lambda fragment of approximately 10,000 to 12,000 daltons. Biochemical and serologic analysis of a fraction of a size (obtained from amyloid fibrils extracted from the kidney) similar to that synthesized by the bone marrow cells revealed a light chain fragment corresponding to the amino terminal end of the variable region of the lambda light chain subgroup II. The presence of similarly sized short fragments of lambda light chain in both the synthesized and deposited protein suggests that aberrant synthesis and/or proteolytic degradation may play a pathogenetic role in the process of amyloidogenesis. Images Figure 1 PMID:3089021

Amyloid fibril protein AA in Papua New Guinean amyloidosis.

PubMed Central

Anders, R F; Price, M A; Wilkey, I S; Husby, G; Takitaki, F; Natvig, J B; McAdam, K P

1976-01-01

In this study of protein composition of amyloid fibrils isolated from eight patients representative of the spectrum of amyloidosis found in Papua New Guinea has been investigated. All fibril preparations, including three from patients with amyloidosis secondary to lepromatous leprosy and one from an unusual juvenile case of primary amyloidosis, contained the non-immunogobulin amyloid protein, protein AA. However, only 44% of thirty-six amyloid patients had detectable levels of the protein AA-related serum component, protein SAA. Alkali-degraded material from each of the fibril preparations failed to react in double immunodiffusion test with antiserum to the amyloid-related light chain VgammaV, but evidence was found for this immunoglobulin light chain-specificity in the serum of one patient. Images FIG. 1 PMID:820500

Amyloid fibril protein AA in Papua New Guinean amyloidosis.

PubMed

Anders, R F; Price, M A; Wilkey, I S; Husby, G; Takitaki, F; Natvig, J B; McAdam, K P

1976-04-01

In this study of protein composition of amyloid fibrils isolated from eight patients representative of the spectrum of amyloidosis found in Papua New Guinea has been investigated. All fibril preparations, including three from patients with amyloidosis secondary to lepromatous leprosy and one from an unusual juvenile case of primary amyloidosis, contained the non-immunogobulin amyloid protein, protein AA. However, only 44% of thirty-six amyloid patients had detectable levels of the protein AA-related serum component, protein SAA. Alkali-degraded material from each of the fibril preparations failed to react in double immunodiffusion test with antiserum to the amyloid-related light chain VgammaV, but evidence was found for this immunoglobulin light chain-specificity in the serum of one patient.

Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule.

PubMed

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

2012-01-01

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs. Copyright © 2011. Published by Elsevier GmbH.

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

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.

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

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

PubMed

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

2018-01-01

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

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.

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

Quantitation of bovine immunoglobulin isotypes and allotypes using monoclonal antibodies.

PubMed

Williams, D J; Newson, J; Naessens, J

1990-03-01

A panel of 10 monoclonal antibodies specific for bovine immunoglobulins M, A, G1, G2 and light chains were produced and enzyme-linked immunosorbent assays developed to measure Ig levels in body fluids and culture supernatants using this panel of MAbs. An inhibition ELISA was accurate and sensitive for MAbs of high affinity, detecting levels as low as 10 ng ml-1 of IgM using a high-affinity MAb, IL-A50 (dissociation constant = 1.3 X 10(-11) M). For MAbs of lower affinity (KD of less than 0.25 X 10(-9) M) a sandwich ELISA was more sensitive, detecting 0.1-1.0 microgram ml-1 Ig, provided a conjugate of an anti-light chain MAb was used. Using these ELISA techniques, four pairs of MAbs specific for bovine IgM, IgA, IgG1 and IgG2 respectively, were screened on sera from over 100 cattle of different breeds to determine whether any detected a polymorphic epitope. MAbs IL-A30, IL-A60, IL-A66, IL-A71, IL-A72, IL-A73 and IL-A74 were shown to recognise monomorphic determinants on their respective heavy chains. In contrast, the epitope recognised on the mu-heavy chain by MAb IL-A50, which had previously been shown to be polymorphic, was found to be allelic and inherited under the control of a single gene, probably Cu.

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

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.

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.

Exploring the folding pattern of a polymer chain in a single crystal by combining single-molecule force spectroscopy and steered molecular dynamics simulations.

PubMed

Song, Yu; Feng, Wei; Liu, Kai; Yang, Peng; Zhang, Wenke; Zhang, Xi

2013-03-26

Understanding the folding pattern of a single polymer chain within its single crystal will shed light on the mechanism of crystallization. Here, we use the combined techniques of atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations to study the folding pattern of a polyethylene oxide (PEO) chain in its single crystal. Our results show that the folding pattern of a PEO chain in the crystal formed in dilute solution follows the adjacent re-entry folding model. While in the crystal obtained from the melt, the nonadjacent folding with large and irregular loops contributes to big force fluctuations in the force-extension curves. The method established here can offer a novel strategy to directly unravel the chain-folding pattern of polymer single crystals at single-molecule level.

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.

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

Comparison of gel contraction mediated by airway smooth muscle cells from patients with and without asthma.

PubMed

Matsumoto, Hisako; Moir, Lyn M; Oliver, Brian G G; Burgess, Janette K; Roth, Michael; Black, Judith L; McParland, Brent E

2007-10-01

Exaggerated bronchial constriction is the most significant and life threatening response of patients with asthma to inhaled stimuli. However, few studies have investigated the contractility of airway smooth muscle (ASM) from these patients. The purpose of this study was to establish a method to measure contraction of ASM cells by embedding them into a collagen gel, and to compare the contraction between subjects with and without asthma. Gel contraction to histamine was examined in floating gels containing cultured ASM cells from subjects with and without asthma following overnight incubation while unattached (method 1) or attached (method 2) to casting plates. Smooth muscle myosin light chain kinase protein levels were also examined. Collagen gels containing ASM cells reduced in size when stimulated with histamine in a concentration-dependent manner and reached a maximum at a mean (SE) of 15.7 (1.2) min. This gel contraction was decreased by inhibitors for phospholipase C (U73122), myosin light chain kinase (ML-7) and Rho kinase (Y27632). When comparing the two patient groups, the maximal decreased area of gels containing ASM cells from patients with asthma was 19 (2)% (n = 8) using method 1 and 22 (3)% (n = 6) using method 2, both of which were greater than that of cells from patients without asthma: 13 (2)% (n = 9, p = 0.05) and 10 (4)% (n = 5, p = 0.024), respectively. Smooth muscle myosin light chain kinase levels were not different between the two groups. The increased contraction of asthmatic ASM cells may be responsible for exaggerated bronchial constriction in asthma.

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.

Block-accelerated aggregation multigrid for Markov chains with application to PageRank problems

NASA Astrophysics Data System (ADS)

Shen, Zhao-Li; Huang, Ting-Zhu; Carpentieri, Bruno; Wen, Chun; Gu, Xian-Ming

2018-06-01

Recently, the adaptive algebraic aggregation multigrid method has been proposed for computing stationary distributions of Markov chains. This method updates aggregates on every iterative cycle to keep high accuracies of coarse-level corrections. Accordingly, its fast convergence rate is well guaranteed, but often a large proportion of time is cost by aggregation processes. In this paper, we show that the aggregates on each level in this method can be utilized to transfer the probability equation of that level into a block linear system. Then we propose a Block-Jacobi relaxation that deals with the block system on each level to smooth error. Some theoretical analysis of this technique is presented, meanwhile it is also adapted to solve PageRank problems. The purpose of this technique is to accelerate the adaptive aggregation multigrid method and its variants for solving Markov chains and PageRank problems. It also attempts to shed some light on new solutions for making aggregation processes more cost-effective for aggregation multigrid methods. Numerical experiments are presented to illustrate the effectiveness of this technique.

Computational Identification Of CDR3 Sequence Archetypes Among Immunoglobulin Sequences in Chronic Lymphocytic Leukemia

PubMed Central

Messmer, Bradley T; Raphael, Benjamin J; Aerni, Sarah J; Widhopf, George F; Rassenti, Laura Z; Gribben, John G; Kay, Neil E; Kipps, Thomas J

2009-01-01

The leukemia cells of unrelated patients with chronic lymphocytic leukemia (CLL) display a restricted repertoire of immunoglobulin (Ig) gene rearrangements with preferential usage of certain Ig gene segments. We developed a computational method to rigorously quantify biases in Ig sequence similarity in large patient databases and to identify groups of patients with unusual levels of sequence similarity. We applied our method to sequences from 1577 CLL patients through the CLL Research Consortium (CRC), and identified 67 similarity groups into which roughly 20% of all patients could be assigned. Immunoglobulin light chain class was highly correlated within all groups and light chain gene usage was similar within sets. Surprisingly, over 40% of the identified groups were composed of somatically mutated genes. This study significantly expands the evidence that antigen selection shapes the Ig repertoire in CLL. PMID:18640719

Computational identification of CDR3 sequence archetypes among immunoglobulin sequences in chronic lymphocytic leukemia.

PubMed

Messmer, Bradley T; Raphael, Benjamin J; Aerni, Sarah J; Widhopf, George F; Rassenti, Laura Z; Gribben, John G; Kay, Neil E; Kipps, Thomas J

2009-03-01

The leukemia cells of unrelated patients with chronic lymphocytic leukemia (CLL) display a restricted repertoire of immunoglobulin (Ig) gene rearrangements with preferential usage of certain Ig gene segments. We developed a computational method to rigorously quantify biases in Ig sequence similarity in large patient databases and to identify groups of patients with unusual levels of sequence similarity. We applied our method to sequences from 1577 CLL patients through the CLL Research Consortium (CRC), and identified 67 similarity groups into which roughly 20% of all patients could be assigned. Immunoglobulin light chain class was highly correlated within all groups and light chain gene usage was similar within sets. Surprisingly, over 40% of the identified groups were composed of somatically mutated genes. This study significantly expands the evidence that antigen selection shapes the Ig repertoire in CLL.

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

Progress in LED technology for solid-state lighting

NASA Astrophysics Data System (ADS)

Bhardwaj, Jy; Guth, Greg; Cesaratto, John M.; Shchekin, Oleg B.; Soer, Wouter A.; Götz, Werner; Bonné, Ron; Song, Zhihua F.; den Breejen, Jeroen

2017-02-01

As solid-state lighting adoption moves from bulb socket replacement to lighting system engineering, luminaire manufacturers are beginning to actualize far greater cost savings through luminaire optimization rather than the simplistic process of component cost pareto management. Indeed, there are an increasing number of applications in which we see major shifts in the value chain in terms of increasing the L1 (LED) and L2 (LED array on PCB) value. The L1 value increase stems from a number of factors ranging from simply higher performing LEDs reducing the LED count, to L1 innovation such as high voltage LEDs, optimizing driver efficiency or to the use of high luminance LEDs enabling compact optics, allowing not only more design freedom but also cost reduction through space and weight savings. The L2 value increase is realized predominantly through increasing L2 performance with the use of algorithms that optimize L1 selection and placement and/or through L2 integration of drivers, control electronics, sensors, secondary lens and/or environmental protection, which is also initiating level collapse in the value chain. In this paper we will present the L1 and L2 innovations that are enabling this disruption as well as provide examples of fixture/luminaire level benefits.

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

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

PubMed Central

Moy, A B; Van Engelenhoven, J; Bodmer, J; Kamath, J; Keese, C; Giaever, I; Shasby, S; Shasby, D M

1996-01-01

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces. PMID:8613524

Transcranial Red and Near Infrared Light Transmission in a Cadaveric Model

PubMed Central

Jagdeo, Jared R.; Adams, Lauren E.; Brody, Neil I.; Siegel, Daniel M.

2012-01-01

Background and Objective Low level light therapy has garnered significant interest within the past decade. The exact molecular mechanisms of how red and near infrared light result in physiologic modulation are not fully understood. Heme moieties and copper within cells are red and near infrared light photoreceptors that induce the mitochondrial respiratory chain component cytochrome C oxidase, resulting in a cascade linked to cytoprotection and cellular metabolism. The copper centers in cytochrome C oxidase have a broad absorption range that peaks around 830 nm. Several in vitro and in vivo animal and human models exist that have demonstrated the benefits of red light and near infrared light for various conditions. Clinical applications for low level light therapy are varied. One study in particular demonstrated improved durable functional outcomes status post-stroke in patients treated with near infrared low level light therapy compared to sham treatment [1]. Despite previous data suggesting the beneficial effect in treating multiple conditions, including stroke, with low level light therapy, limited data exists that measures transmission in a human model. Study Design/Materials and Methods To investigate this idea, we measured the transmission of near infrared light energy, using red light for purposes of comparison, through intact cadaver soft tissue, skull bones, and brain using a commercially available LED device at 830 nm and 633 nm. Results Our results demonstrate that near infrared measurably penetrates soft tissue, bone and brain parenchyma in the formalin preserved cadaveric model, in comparison to negligible red light transmission in the same conditions. Conclusion These findings indicate that near infrared light can penetrate formalin fixed soft tissue, bone and brain and implicate that benefits observed in clinical studies are potentially related to direct action of near infrared light on neural tissue. PMID:23077622

Immunoglobulin gene usage in the human anti-pathogen response.

PubMed

Newkirk, M M; Rioux, J D

1995-09-01

The human antibody response to foreign pathogens is generated to a relatively small number of target surface proteins and carbohydrates that nonetheless have an extensive array of epitopes. The study of human monoclonal antibodies to different pathogens shows that there are a diversity of mechanisms used to generate a sufficient repertoire of antibodies to combat the invading pathogens. Although many different immunoglobulin gene elements are used to construct the anti-pathogen response, some elements are used more often than would be expected if all elements were used randomly. For example, the immune response to Haemophilus influenzae polysaccharide appears to be quite narrow, being restricted primarily to a specific heavy-chain gene, 3-15, and a lambda light-chain family II member, 4A. In contrast, for the immune response to cytomegalovirus proteins, a wider group of gene elements is needed. It is also surprising that despite an investigator bias for IgG- rather than IgM-secreting immortal B cells (because of their high affinity and neutralizing abilities), 26% of light chains and 13% of heavy chains showed a very low level of somatic mutation, equivalent to an IgM molecule that has not undergone affinity maturation. Although some highly mutated IgG molecules are present in the anti-pathogen response, most of the monoclonal antibodies specific for viruses or bacteria have a level of somatic hypermutation similar to that of the adult IgM repertoire. A number of studies have shown that there are similarities in the antibody responses to pathogens and to self (autoantibodies).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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

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.

[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).

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.

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

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

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

PubMed

Stoops, Janelle; Byrd, Samantha; Hasegawa, Haruki

2012-10-01

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

Myosin light chain phosphorylation is critical for adaptation to cardiac stress.

PubMed

Warren, Sonisha A; Briggs, Laura E; Zeng, Huadong; Chuang, Joyce; Chang, Eileen I; Terada, Ryota; Li, Moyi; Swanson, Maurice S; Lecker, Stewart H; Willis, Monte S; Spinale, Francis G; Maupin-Furlowe, Julie; McMullen, Julie R; Moss, Richard L; Kasahara, Hideko

2012-11-27

Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined. We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems. Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

Myosin dephosphorylation during rapid relaxation of hog carotid artery smooth muscle.

PubMed

Driska, S P; Stein, P G; Porter, R

1989-02-01

Changes in myosin light chain phosphorylation were measured during histamine-induced rhythmic contractions of hog carotid artery smooth muscle strips. Histamine made the muscle strips contract spontaneously every 1-5 min, and this allowed measurement of the time course of phosphorylation in relation to force development under conditions where diffusion of the agonist through tissue would not complicate the interpretation of the data. In the absence of histamine, phosphorylation was low [0.12 +/- 0.04 mol P/mol of the 20,000-Da light chain (LC 20)]. Phosphorylation was slightly (but not significantly) higher in the presence of 10 microM histamine in the relaxed state between contractions (0.20 +/- 0.03 mol P/mol LC 20). In muscle strips frozen during force development, when force had reached half of its peak value, phosphorylation was 0.38 +/- 0.06 mol P/mol LC 20. The highest levels of phosphorylation (0.49 +/- 0.04 mol P/mol LC 20) were found in strips frozen at the peak of the rhythmic contractions. Strips frozen when force had declined to half of the peak force showed low levels of phosphorylation (0.17 +/- 0.07 mol P/mol LC 20), indicating that the myosin light chain phosphatase activity was quite high. Mathematical modeling of the kinase and phosphatase reactions suggested that the apparent first-order phosphatase rate constant was at least 0.08 s-1 under these conditions. To obtain a better estimate of this rate constant, a second series of phosphorylation measurements were made early in the relaxation phase of the rhythmic contractions. The highest phosphatase rate constant obtained from these measurements was 0.23 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Bauhinia championii flavone inhibits apoptosis and autophagy via the PI3K/Akt pathway in myocardial ischemia/reperfusion injury in rats

PubMed Central

Jian, Jie; Xuan, Feifei; Qin, Feizhang; Huang, Renbin

2015-01-01

This study aimed to determine the effects of Bauhinia championii flavone (BCF) on myocardial ischemia/reperfusion injury (MI/RI) in rats and to explore potential mechanisms. The MI/RI model in rats was established by ligating the left anterior descending coronary artery for 30 minutes, then reperfusing for 3 hours. BCF at 20 mg/kg was given 20 minutes prior to ischemia via sublingual intravenous injection, with 24 μg/kg phosphoinositide 3-kinase inhibitor (PI3K; wortmannin) as a control. The creatine kinase-MB and nitric oxide content were assessed by colorimetry. The levels of mitochondrial permeability transition pores and tumor necrosis factor alpha were determined by an enzyme-linked immunosorbent assay. Cardiomyocyte apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Additionally, the expression of PI3K, endothelial nitric oxide synthase, caspase-3, and Beclin1 was analyzed by fluorescence quantitative polymerase chain reaction and Western blotting, respectively. Akt and microtubule-associated protein 1 light chain 3-II protein levels were also evaluated. Pretreatment with BCF significantly decreased the levels of creatine kinase-MB, tumor necrosis factor alpha, and mitochondrial permeability transition pores, but increased the nitric oxide content. Furthermore, BCF inhibited apoptosis, downregulated caspase-3, Beclin1, and microtubule-associated protein 1 light chain 3-II, upregulated PI3K, and increased the protein levels of phosphorylated Akt and endothelial nitric oxide synthase. However, all of the previously mentioned effects of BCF were blocked when BCF was coadministered with wortmannin. In conclusion, these observations indicated that BCF has cardioprotective effects against MI/RI by reducing cell apoptosis and excessive autophagy, which might be related to the activation of the PI3K/Akt signaling pathway. PMID:26604691

Bauhinia championii flavone inhibits apoptosis and autophagy via the PI3K/Akt pathway in myocardial ischemia/reperfusion injury in rats.

PubMed

Jian, Jie; Xuan, Feifei; Qin, Feizhang; Huang, Renbin

2015-01-01

This study aimed to determine the effects of Bauhinia championii flavone (BCF) on myocardial ischemia/reperfusion injury (MI/RI) in rats and to explore potential mechanisms. The MI/RI model in rats was established by ligating the left anterior descending coronary artery for 30 minutes, then reperfusing for 3 hours. BCF at 20 mg/kg was given 20 minutes prior to ischemia via sublingual intravenous injection, with 24 μg/kg phosphoinositide 3-kinase inhibitor (PI3K; wortmannin) as a control. The creatine kinase-MB and nitric oxide content were assessed by colorimetry. The levels of mitochondrial permeability transition pores and tumor necrosis factor alpha were determined by an enzyme-linked immunosorbent assay. Cardiomyocyte apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Additionally, the expression of PI3K, endothelial nitric oxide synthase, caspase-3, and Beclin1 was analyzed by fluorescence quantitative polymerase chain reaction and Western blotting, respectively. Akt and microtubule-associated protein 1 light chain 3-II protein levels were also evaluated. Pretreatment with BCF significantly decreased the levels of creatine kinase-MB, tumor necrosis factor alpha, and mitochondrial permeability transition pores, but increased the nitric oxide content. Furthermore, BCF inhibited apoptosis, downregulated caspase-3, Beclin1, and microtubule-associated protein 1 light chain 3-II, upregulated PI3K, and increased the protein levels of phosphorylated Akt and endothelial nitric oxide synthase. However, all of the previously mentioned effects of BCF were blocked when BCF was coadministered with wortmannin. In conclusion, these observations indicated that BCF has cardioprotective effects against MI/RI by reducing cell apoptosis and excessive autophagy, which might be related to the activation of the PI3K/Akt signaling pathway.

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.

Ferritin light chain gene mutations in two Brazilian families with hereditary hyperferritinemia-cataract syndrome

PubMed Central

Petroni, Roberta Cardoso; da Rosa, Susana Elaine Alves; de Carvalho, Flavia Pereira; Santana, Rúbia Anita Ferraz; Hyppolito, Joyce Esteves; Nascimento, Claudia Mac Donald Bley; Hamerschlak, Nelson; Campregher, Paulo Vidal

2017-01-01

ABSTRACT Hereditary hyperferritinemia-cataract syndrome is an autosomal dominant genetic disorder associated with mutations in the 5’UTR region of the ferritin light chain gene. These mutations cause the ferritin levels to increase even in the absence of iron overload. Patients also develop bilateral cataract early due to accumulation of ferritin in the lens, and many are misdiagnosed as having hemochromatosis and thus not properly treated. The first cases were described in 1995 and several mutations have already been identified. However, this syndrome is still a poorly understood. We report two cases of unrelated Brazilian families with clinical suspicion of the syndrome, which were treated in our department. For the definitive diagnosis, the affected patients, their parents and siblings were submitted to Sanger sequencing of the 5’UTR region for detection of the ferritin light gene mutation. Single nucleotide polymorphism-like mutations were found in the affected patients, previously described. The test assisted in making the accurate diagnosis of the disease, and its description is important so that the test can be incorporated into clinical practice. PMID:28746593

Current anti-myeloma therapies in renal manifestations of monoclonal light chain-associated Fanconi syndrome: a retrospective series of 49 patients.

PubMed

Vignon, M; Javaugue, V; Alexander, M P; El-Karoui, K; Karras, A; Roos-Weil, D; Royer, B; Asli, B; Knebelmann, B; Touchard, G; Jaccard, A; Arnulf, B; Bridoux, F; Leung, N; Fermand, J P

2017-01-01

We retrospectively reviewed 49 patients with light chain (LC) Fanconi syndrome (FS). Patients presented with chronic kidney disease (median estimated glomerular filtration rate (eGFR) of 33 ml/min/1.73 m 2 ) and tubular proteinuria. All patients tested had elevated fractional excretion of phosphate, uric acid, generalized aminoaciduria and/or normoglycemic glycosuria. Thirty-eight patients had monoclonal gammopathy of renal significance and eleven patients had an overt hematological malignancy. The monoclonal LC isotype was kappa in 46/49 cases. Kidney biopsy in 39 patients showed various proximal tubular lesions and characteristic LC intracytoplasmic crystalline inclusions in 24 patients. Forty-two patients received chemotherapy. Patients with plasma cell proliferation (n=38) received bortezomib-based regimens (n=11), immunomodulatory agents (n=7) or alkylating agents (n=6). High-dose melphalan (HDM) followed by autologous stem cell transplantation was performed in 14 patients. Hematological response was obtained in 90% of evaluable patients, assessed on serum free light chains (FLC). GFR remained stable as long as hematological response was maintained and declined when serum FLC level rebounded. Improvement in proximal tubule function occurred in 13 patients. In patients with LC-associated FS, chemotherapy using HDM and/or new generation anti-myeloma agents can stabilize renal function and improve proximal tubule function. Serum FLC should be used to assess the hematological response, related to renal outcome.

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

Chain dynamics and nanoparticle motion in attractive polymer nanocomposites subjected to large deformations.

PubMed

Senses, Erkan; Tyagi, Madhusudan; Natarajan, Bharath; Narayanan, Suresh; Faraone, Antonio

2017-11-08

The effect of large deformation on the chain dynamics in attractive polymer nanocomposites was investigated using neutron scattering techniques. Quasi-elastic neutron backscattering measurements reveal a substantial reduction of polymer mobility in the presence of attractive, well-dispersed nanoparticles. In addition, large deformations are observed to cause a further slowing down of the Rouse rates at high particle loadings, where the interparticle spacings are slightly smaller than the chain dimensions, i.e. in the strongly confined state. No noticeable change, however, was observed for a lightly confined system. The reptation tube diameter, measured by neutron spin echo, remained unchanged after shear, suggesting that the level of chain-chain entanglements is not significantly affected. The shear-induced changes in the interparticle bridging reflect the slow nanoparticle motion measured by X-ray photon correlation spectroscopy. These results provide a first step for understanding how large shear can significantly affect the segmental motion in nanocomposites and open up new opportunities for designing mechanically responsive soft materials.

Chain dynamics and nanoparticle motion in attractive polymer nanocomposites subjected to large deformations

DOE PAGES

Senses, Erkan; Tyagi, Madhusudan; Natarajan, Bharath; ...

2017-09-28

The effect of large deformation on the chain dynamics in attractive polymer nanocomposites was investigated using neutron scattering techniques. Quasielastic neutron backscattering measurements reveal a substantial reduction of polymer mobility in the presence of attractive, well-dispersed nanoparticles. Additionally, large deformations are observed to cause a further slowing down of the Rouse rates at high particle loadings, where the interparticle spacings are slightly smaller than the chain dimensions, i.e. in the strongly confined state. No noticeable change, however, was observed for a lightly confined system. The reptation tube diameter, measured by neutron spin echo, remained unchanged after shear, suggesting that themore » level of chain-chain entanglements is not significantly affected. The shearinduced changes in the interparticle bridging reflects on the slow nanoparticle motion measured by X-ray photon correlation spectroscopy. These results provide a first step for understanding how large shear can significantly affect the segmental motion in nanocomposites and open up new opportunities for designing mechanically responsive soft materials.« less

Chain dynamics and nanoparticle motion in attractive polymer nanocomposites subjected to large deformations

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

Senses, Erkan; Tyagi, Madhusudan; Natarajan, Bharath

The effect of large deformation on the chain dynamics in attractive polymer nanocomposites was investigated using neutron scattering techniques. Quasielastic neutron backscattering measurements reveal a substantial reduction of polymer mobility in the presence of attractive, well-dispersed nanoparticles. Additionally, large deformations are observed to cause a further slowing down of the Rouse rates at high particle loadings, where the interparticle spacings are slightly smaller than the chain dimensions, i.e. in the strongly confined state. No noticeable change, however, was observed for a lightly confined system. The reptation tube diameter, measured by neutron spin echo, remained unchanged after shear, suggesting that themore » level of chain-chain entanglements is not significantly affected. The shearinduced changes in the interparticle bridging reflects on the slow nanoparticle motion measured by X-ray photon correlation spectroscopy. These results provide a first step for understanding how large shear can significantly affect the segmental motion in nanocomposites and open up new opportunities for designing mechanically responsive soft materials.« less

An ab initio molecular dynamics and density functional theory study of the formation of phosphate chains from metathiophosphates.

PubMed

Mosey, Nicholas J; Woo, Tom K

2006-09-04

The reactions that occur between metathiophosphate (MTP) molecules are identified and examined through ab initio molecular dynamics simulations and static quantum chemical calculations at the density functional level of theory. The simulations show that certain types of MTPs can react to yield phosphate chains, while others only dimerize. These differences are rationalized in terms of reaction energies and the electronic structures of these molecules. In the reaction leading to the formation of phosphate chains, the reactive center, a tri-coordinate phosphorus atom, is continually regenerated. A polymerization mechanism linking MTPs to phosphate chains is developed on the basis of these results. This information sheds light on the underlying processes that may be responsible for the formation of phosphates under high-temperature conditions and may prove useful in the development of protocols for the rational synthesis of complex phosphate structures.

Human L-ferritin deficiency is characterized by idiopathic generalized seizures and atypical restless leg syndrome

PubMed Central

Cozzi, Anna; Santambrogio, Paolo; Privitera, Daniela; Broccoli, Vania; Rotundo, Luisa Ida; Garavaglia, Barbara; Benz, Rudolf; Altamura, Sandro; Goede, Jeroen S.; Muckenthaler, Martina U.

2013-01-01

The ubiquitously expressed iron storage protein ferritin plays a central role in maintaining cellular iron homeostasis. Cytosolic ferritins are composed of heavy (H) and light (L) subunits that co-assemble into a hollow spherical shell with an internal cavity where iron is stored. The ferroxidase activity of the ferritin H chain is critical to store iron in its Fe3+ oxidation state, while the L chain shows iron nucleation properties. We describe a unique case of a 23-yr-old female patient affected by a homozygous loss of function mutation in the L-ferritin gene, idiopathic generalized seizures, and atypical restless leg syndrome (RLS). We show that L chain ferritin is undetectable in primary fibroblasts from the patient, and thus ferritin consists only of H chains. Increased iron incorporation into the FtH homopolymer leads to reduced cellular iron availability, diminished levels of cytosolic catalase, SOD1 protein levels, enhanced ROS production and higher levels of oxidized proteins. Importantly, key phenotypic features observed in fibroblasts are also mirrored in reprogrammed neurons from the patient’s fibroblasts. Our results demonstrate for the first time the pathophysiological consequences of L-ferritin deficiency in a human and help to define the concept for a new disease entity hallmarked by idiopathic generalized seizure and atypical RLS. PMID:23940258

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.

Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

PubMed

Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

2016-07-01

At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as a common pathway contributing to the activity-dependent regulation of vesicle endocytosis at synapses. © 2016 International Society for Neurochemistry.

Prognostic Value of Serum Free Light Chain in Multiple Myeloma.

PubMed

El Naggar, Amel A; El-Naggar, Mostafa; Mokhamer, El-Hassan; Avad, Mona W

2015-01-01

The measurement of serum free light chain (sFLC) has been shown to be valuable in screening for the presence of plasma cell dyscrasia as well as for baseline prognosis in newly diagnosed patients. The aim of the present work was to study the prognostic value of sFLC in multiple myeloma in relation to other serum biomarkers, response to therapy and survival. Forty five newly diagnosed patients with MM were included in the study. Patients were divided into responders and non-responders groups according to response to therapy. sFLC and serum Amyloid A (SAA) were measured by immunonephelometry. The non-responders group showed a statistically significant higher kappa/lambda or lambda/kappa ratio and higher β2 microglobulin level, but lower albumin level at presentation, as compared to the responders group (P < 0.001). However, no statistically significant difference was detected between the two groups regarding SA A or calcium levels. Comparison between sFLC ratio obtained before and after therapy revealed significant decrease after treatment in the responders group (P = 0.05). Survival was significantly inferior in patients with an FLC ratio of ≥ 2.6 or ≤ 0.56 compared with those with an FLC ratio that was between 0.56 and 2.6 (P = 0.002).

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 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.

Effect of light chain V region duplication on IgG oligomerization and in vivo efficacy.

PubMed

Shuford, W; Raff, H V; Finley, J W; Esselstyn, J; Harris, L J

1991-05-03

A human immunoglobulin G1 (IgG1) antibody oligomer was isolated from a transfected myeloma cell line that produced a monoclonal antibody to group B streptococci. Compared to the IgG1 monomer, the oligomer was significantly more effective at protecting neonatal rats from infection in vivo. The oligomer was also shown to cross the placenta and to be stable in neonatal rats. Immunochemical analysis and complementary DNA sequencing showed that the transfected cell line produced two distinct kappa light chains: a normal light chain (Ln) with a molecular mass of 25 kilodaltons and a 37-kilodalton species (L37), the domain composition of which was variable-variable-constant (V-V-C). Cotransfection of vectors encoding the heavy chain and L37 resulted in production of oligomeric IgG.

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

Phase 2 trial of daily, oral epigallocatechin gallate in patients with light-chain amyloidosis.

PubMed

Meshitsuka, Sohsuke; Shingaki, Sumito; Hotta, Masatoshi; Goto, Miku; Kobayashi, Makoto; Ukawa, Yuuichi; Sagesaka, Yuko M; Wada, Yasuyo; Nojima, Masanori; Suzuki, Kenshi

2017-03-01

Previous studies have suggested that an increase in mitochondrial reactive oxygen species may cause organ damage in patients with light-chain (AL) amyloidosis; however, this damage can be decreased by antioxidant-agent treatment. Epigallocatechin gallate (EGCG), the major natural catechin in green tea, has potent antioxidant activity. Because EGCG has recently been reported to have a favorable toxicity profile for treating amyloidosis, we sought to examine the clinical efficacy and toxicity of EGCG in patients with AL amyloidosis. Fifty-seven patients were randomly assigned to the EGCG and observation groups and observed for six months. There were no increases in grade 3-5 adverse events and EGCG therapy was well tolerated. Although a decrease in the urinary albumin level was found in the EGCG group in patients with obvious albuminuria after treatment initiation, its antioxidant activity may not be sufficient to clarify the potential effect of EGCG in patients with AL amyloidosis. Because some of the biological markers responsible for organ damage were well correlated to the level of antioxidant potential in patients' plasma, the status of oxidative stress in the blood may indicate the extent of organ damage in clinical situations.

Combination of low level light therapy and nitrosyl-cobinamide accelerates wound healing

PubMed Central

Spitler, Ryan; Ho, Hsiang; Norpetlian, Frederique; Kong, Xiangduo; Jiang, Jingjing; Yokomori, Kyoko; Andersen, Bogi; Boss, Gerry R.; Berns, Michael W.

2015-01-01

Abstract. Low level light therapy (LLLT) has numerous therapeutic benefits, including improving wound healing, but the precise mechanisms involved are not well established; in particular, the underlying role of cytochrome C oxidase (C-ox) as the primary photoacceptor and the associated biochemical mechanisms still require further investigation. We previously showed the nitric oxide (NO) donating drug nitrosyl-cobinamide (NO-Cbi) enhances wound healing through a cGMP/cGMP-dependent protein kinase/ERK1/2 mechanism. Here, we show that the combination of LLLT and NO-Cbi markedly improves wound healing compared to either treatment alone. LLLT-enhanced wound healing proceeded through an electron transport chain-C-ox-dependent mechanism with a reduction of reactive oxygen species and increased adenosine triphosphate production. C-ox was validated as the primary photoacceptor by three observations: increased oxygen consumption, reduced wound healing in the presence of sodium azide, and disassociation of cyanide, a known C-ox ligand, following LLLT. We conclude that LLLT and NO-Cbi accelerate wound healing through two independent mechanisms, the electron transport chain-C-ox pathway and cGMP signaling, respectively, with both resulting in ERK1/2 activation. PMID:25562608

Combination of low level light therapy and nitrosyl-cobinamide accelerates wound healing

NASA Astrophysics Data System (ADS)

Spitler, Ryan; Ho, Hsiang; Norpetlian, Frederique; Kong, Xiangduo; Jiang, Jingjing; Yokomori, Kyoko; Andersen, Bogi; Boss, Gerry R.; Berns, Michael W.

2015-05-01

Low level light therapy (LLLT) has numerous therapeutic benefits, including improving wound healing, but the precise mechanisms involved are not well established; in particular, the underlying role of cytochrome C oxidase (C-ox) as the primary photoacceptor and the associated biochemical mechanisms still require further investigation. We previously showed the nitric oxide (NO) donating drug nitrosyl-cobinamide (NO-Cbi) enhances wound healing through a cGMP/cGMP-dependent protein kinase/ERK1/2 mechanism. Here, we show that the combination of LLLT and NO-Cbi markedly improves wound healing compared to either treatment alone. LLLT-enhanced wound healing proceeded through an electron transport chain-C-ox-dependent mechanism with a reduction of reactive oxygen species and increased adenosine triphosphate production. C-ox was validated as the primary photoacceptor by three observations: increased oxygen consumption, reduced wound healing in the presence of sodium azide, and disassociation of cyanide, a known C-ox ligand, following LLLT. We conclude that LLLT and NO-Cbi accelerate wound healing through two independent mechanisms, the electron transport chain-C-ox pathway and cGMP signaling, respectively, with both resulting in ERK1/2 activation.

Combination of low level light therapy and nitrosyl-cobinamide accelerates wound healing.

PubMed

Spitler, Ryan; Ho, Hsiang; Norpetlian, Frederique; Kong, Xiangduo; Jiang, Jingjing; Yokomori, Kyoko; Andersen, Bogi; Boss, Gerry R; Berns, Michael W

2015-05-01

Low level light therapy (LLLT) has numerous therapeutic benefits, including improving wound healing, but the precise mechanisms involved are not well established; in particular, the underlying role of cytochrome C oxidase (C-ox) as the primary photoacceptor and the associated biochemical mechanisms still require further investigation. We previously showed the nitric oxide (NO) donating drug nitrosyl-cobinamide (NO-Cbi) enhances wound healing through a cGMP/cGMP-dependent protein kinase/ERK1/2 mechanism. Here, we show that the combination of LLLT and NO-Cbi markedly improves wound healing compared to either treatment alone. LLLT-enhanced wound healing proceeded through an electron transport chain-C-ox-dependent mechanism with a reduction of reactive oxygen species and increased adenosine triphosphate production. C-ox was validated as the primary photoacceptor by three observations: increased oxygen consumption, reduced wound healing in the presence of sodium azide, and disassociation of cyanide, a known C-ox ligand, following LLLT. We conclude that LLLT and NO-Cbi accelerate wound healing through two independent mechanisms, the electron transport chain-C-ox pathway and cGMP signaling, respectively, with both resulting in ERK1/2 activation.

Effects of 530 nm green light on refractive status, melatonin, MT1 receptor, and melanopsin in the guinea pig.

PubMed

Wang, Fei; Zhou, Jiaqi; Lu, Yi; Chu, Renyuan

2011-02-01

To investigate (i) the effect of monochromatic light on inhibiting induction of light-induced melatonin and (ii) the roles of melanopsin and MT1 receptor in light-induced myopia in the guinea pig. Forty-eight guinea pigs were randomly distributed into three treatment groups: white-light (control), green-light (530 nm), and blue-light (480 nm) groups. Levels of pineal gland melatonin were measured twice daily--10:00 a.m. and 10:00 p.m.--10 days after initial light treatment. Thirty additional guinea pigs were also assigned to these groups and treated similarly. For these latter animals, refractive status, ocular length, and vitreous depth were measured before and after light treatment. Eight weeks after light treatment, retinal and sceral levels of melanopsin, melatonin receptor type (MT) 1, and mRNA protein were determined by Western blotting, real-time polymerase chain reaction (RT-PCR), and immunohistochemistry. The level of pineal gland melatonin in the green-light group was significantly higher than that in the blue-light group. Biometric measurements showed that guinea pigs in the green-light group had a somewhat myopic refractive status. Expressions of retinal melanopsin mRNA and protein were significantly higher in the blue-light group and lower in the green-light group when compared to controls. Conversely, expressions of MT1 receptor mRNA and protein in retina and sclera were significantly higher in the green-light group and lower in the blue-light group when compared to controls. Green light appears to suppress induction of melatonin production. In addition, 530 nm of green light is involved in the development of myopia. In the guinea pig, MT1 receptor and melanopsin appear to play roles in the development of myopia induced by 530 nm of light.

High level expression and immunochemical characterization of botulinum neurotoxin type F light chain.

PubMed

Chauhan, Ritika; Chauhan, Vinita; Rao, Mula Kameshwar; Chaudhary, Dilip; Bhagyawant, Sameer; Dhaked, Ram Kumar

2018-06-01

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

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

PubMed

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

1994-12-01

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

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).

Four methods for determining the composition of trace radioactive surface contamination of low-radioactivity metal

NASA Astrophysics Data System (ADS)

O'Keeffe, H. M.; Burritt, T. H.; Cleveland, B. T.; Doucas, G.; Gagnon, N.; Jelley, N. A.; Kraus, C.; Lawson, I. T.; Majerus, S.; McGee, S. R.; Myers, A. W.; Poon, A. W. P.; Rielage, K.; Robertson, R. G. H.; Rosten, R. C.; Stonehill, L. C.; VanDevender, B. A.; Van Wechel, T. D.

2011-12-01

Four methods for determining the composition of low-level uranium- and thorium-chain surface contamination are presented. One method is the observation of Cherenkov light production in water. In two additional methods a position-sensitive proportional counter surrounding the surface is used to make both a measurement of the energy spectrum of alpha particle emissions and also coincidence measurements to derive the thorium-chain content based on the presence of short-lived isotopes in that decay chain. The fourth method is a radiochemical technique in which the surface is eluted with a weak acid, the eluate is concentrated, added to liquid scintillator and assayed by recording beta-alpha coincidences. These methods were used to characterize two 'hotspots' on the outer surface of one of the 3He proportional counters in the Neutral Current Detection array of the Sudbury Neutrino Observatory experiment. The methods have similar sensitivities, of order tens of ng, to both thorium- and uranium-chain contamination.

In vitro Fab display: a cell-free system for IgG discovery

PubMed Central

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Magnolol Inhibits Human Glioblastoma Cell Migration by Regulating N-Cadherin.

PubMed

Cheng, Yu-Chen; Tsao, Min-Jen; Chiu, Chen-Yang; Kan, Po-Chieh; Chen, Ying

2018-06-01

Glioblastoma is a primary malignant brain tumor with a poor prognosis. An effective treatment for glioblastoma is needed. Magnolol is a natural compound from Magnolia officinalis suggested to have antiproliferative activity. The aim of this research was to investigate the anticancer effects of magnolol in glioma, with an emphasis on migration and the underlying mechanism. Magnolol decreased the expression of focal adhesion-related proteins and inhibited LN229 and U87MG glioma cell migration. The levels of phosphorylated myosin light chain (p-MLC), phosphorylated myosin light chain kinase and myosin phosphatase target subunit 1 were reduced in response to magnolol treatment. In addition, immunostaining and membrane fractionation showed that the distribution of N-cadherin at the glioma cell membrane was decreased by magnolol. In an orthotropic xenograft animal model, magnolol treatment not only inhibited tumor progression but also reduced p-MLC and N-cadherin protein expression. In conclusion, magnolol reduces cell migration, potentially through regulating focal adhesions and N-cadherin in glioma cells. Magnolol is a potential candidate for glioma treatment.

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.

Regions of botulinum neurotoxin A light chain recognized by human anti-toxin antibodies from cervical dystonia patients immunoresistant to toxin treatment. The antigenic structure of the active toxin recognized by human antibodies.

PubMed

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

2011-07-01

This work was aimed at determining the BoNT/A L-chain antigenic regions recognized by blocking antibodies in human antisera from cervical dystonia patients who had become immunoresistant to BoNT/A treatment. Antisera from 28 immunoresistant patients were analyzed for binding to each of 32 overlapping synthetic peptides that spanned the entire L-chain. A mixture of the antisera showed that antibodies bound to three peptides, L11 (residues 141-159), L14 (183-201) and L18 (239-257). When mapped separately, the antibodies were bound only by a limited set of peptides. No peptide bound antibodies from all the patients and amounts of antibodies bound to a given peptide varied with the patient. Peptides L11, L14 and L18 were recognized predominantly. A small but significant number of patients had antibodies to peptides L27 (365-383) and L29 (379-397). Other peptides were recognized at very low and perhaps insignificant antibody levels by a minority (15% or less) of patients or had no detectable antibody with any of the sera. In the 3-dimensional structure, antibody-binding regions L11, L14 and L18 of the L-chain occupy surface areas and did not correlate with electrostatic potential, hydrophilicity/hydrophobicity, or temperature factor. These three antigenic regions reside in close proximity to the belt of the heavy chain. The regions L11 and L18 are accessible in both the free light chain and the holotoxin forms, while L14 appears to be less accessible in the holotoxin. Antibodies against these regions could prevent delivery of the L-chain into the neurons by inhibition of the translocation. Copyright © 2011 Elsevier GmbH. All rights reserved.

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

PubMed

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

2006-12-01

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

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

Free kappa light chains in cerebrospinal fluid as a biomarker to assess risk conversion to multiple sclerosis.

PubMed

Menéndez-Valladares, P; García-Sánchez, M I; Cuadri Benítez, P; Lucas, M; Adorna Martínez, M; Carranco Galán, V; García De Veas Silva, J L; Bermudo Guitarte, C; Izquierdo Ayuso, G

2015-01-01

Multiple sclerosis (MS) initiates with a first attack or clinically isolated syndrome (CIS). The importance of an early treatment in MS leads to the search, as soon as possible, for novel biomarkers which can predict conversion from CIS to MS. The purpose of this study was to assess the predictive value of the kappa index ([Formula: see text] index), using kappa free light light chains ([Formula: see text]FLCs) in cerebrospinal fluid (CSF), for the conversion of CIS patients to MS, and compare its accuracy with other parameters used in clinical practice. FLC levels were analysed in CSF from 176 patients: 70 as control group, 77 CIS, and 29 relapsing-remitting MS. FLC levels were quantified by nephelometry. [Formula: see text] Index sensitivity and specificity (93.1%; 95.7%) was higher than those from the immunoglobulin G (IgG) index (75.9%; 94.3%), and lower than those from oligoclonal IgG bands (OCGBs) (96.5%; 98.6%). The optimal cut-off for [Formula: see text] index was 10.62. Most of the CIS patients with [Formula: see text] index >10.62 presented OCGBs, IgG index >0.56 and fulfilled magnetic resonance imaging (MRI) criteria. CIS patients above [Formula: see text] index cut-off of 10.62 present 7.34-fold risk of conversion to MS than CIS below this value. The [Formula: see text] index correlated with positive OCGBs, IgG index above 0.56 and MRI criteria.

Free kappa light chains in cerebrospinal fluid as a biomarker to assess risk conversion to multiple sclerosis

PubMed Central

Menéndez-Valladares, P; García-Sánchez, MI; Cuadri Benítez, P; Lucas, M; Adorna Martínez, M; Carranco Galán, V; García De Veas Silva, JL; Bermudo Guitarte, C

2015-01-01

Background Multiple sclerosis (MS) initiates with a first attack or clinically isolated syndrome (CIS). The importance of an early treatment in MS leads to the search, as soon as possible, for novel biomarkers which can predict conversion from CIS to MS. Objective The purpose of this study was to assess the predictive value of the kappa index (κ index), using kappa free light light chains (κFLCs) in cerebrospinal fluid (CSF), for the conversion of CIS patients to MS, and compare its accuracy with other parameters used in clinical practice. Methods FLC levels were analysed in CSF from 176 patients: 70 as control group, 77 CIS, and 29 relapsing–remitting MS. FLC levels were quantified by nephelometry. Results κ Index sensitivity and specificity (93.1%; 95.7%) was higher than those from the immunoglobulin G (IgG) index (75.9%; 94.3%), and lower than those from oligoclonal IgG bands (OCGBs) (96.5%; 98.6%). The optimal cut-off for κ index was 10.62. Most of the CIS patients with κ index >10.62 presented OCGBs, IgG index >0.56 and fulfilled magnetic resonance imaging (MRI) criteria. Conclusion CIS patients above κ index cut-off of 10.62 present 7.34-fold risk of conversion to MS than CIS below this value. The κ index correlated with positive OCGBs, IgG index above 0.56 and MRI criteria. PMID:28607709

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.

Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

PubMed Central

Barbeito, Ana G.; Garringer, Holly J.; Baraibar, Martin A.; Gao, Xiaoying; Arredondo, Miguel; Núñez, Marco T.; Smith, Mark A.; Ghetti, Bernardino; Vidal, Ruben

2009-01-01

Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. PMID:19519778

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.

Light irradiation helps magnetotactic bacteria eliminate intracellular reactive oxygen species.

PubMed

Li, Kefeng; Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Lulu; Song, Tao

2017-09-01

Magnetotactic bacteria (MTB) demonstrate photoresponse. However, little is known about the biological significance of this behaviour. Magnetosomes exhibit peroxidase-like activity and can scavenge reactive oxygen species (ROS). Magnetosomes extracted from the Magnetospirillum magneticum strain AMB-1 show enhanced peroxidase-like activity under illumination. The present study investigated the effects of light irradiation on nonmagnetic (without magnetosomes) and magnetic (with magnetosomes) AMB-1 cells. Results showed that light irradiation did not affect the growth of nonmagnetic and magnetic cells but significantly increased magnetosome synthesis and reduced intracellular ROS level in magnetic cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyse the expression level of magnetosome formation-associated genes (mamA, mms6, mms13 and mmsF) and stress-related genes (recA, oxyR, SOD, amb0664 and amb2684). Results showed that light irradiation upregulated the expression of mms6, mms13 and mmsF. Furthermore, light irradiation upregulated the expression of stress-related genes in nonmagnetic cells but downregulated them in magnetic cells. Additionally, magnetic cells exhibited stronger phototactic behaviour than nonmagnetic ones. These results suggested that light irradiation could heighten the ability of MTB to eliminate intracellular ROS and help them adapt to lighted environments. This phenomenon may be related to the enhanced peroxidase-like activity of magnetosomes under light irradiation. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Ultrafast optical pulse convertor caused by oscillations of the energy level structure in the conjugated polymer poly(p-phenylenevinylene).

PubMed

Zhang, Yusong; Chen, Weikang; Lin, Zhe; Li, Sheng; George, Thomas F

2017-08-21

For a conjugated polymer irradiated by two optical pulses, the whole process of excitation, involving lattice oscillations, oscillations of the energy level structure, and evolution of the electron cloud, is investigated. Localization of the electron cloud appears in the first 100 fs of irradiation, which in turn induces vibrations of lattice of the polymer chain as well as oscillations of the band gap. These oscillations filter the absorption of the external optical field inversely and convert the original optical field to an ultrafast light field whose intensity varies with a certain period. Based on the mechanism, oscillations of the energy level structure, induced by the external excitation, can be designed as an ultrafast response optical convertor that is able to change the external optical pulse into a new effective light field with a certain oscillation period. This helps provide new insight into designing nanostructures for polymeric optoelectronics.

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…

Differential effects of ambient or diminished CO2 and O2 levels on thylakoid membrane structure in light-stressed plants.

PubMed

Tsabari, Onie; Nevo, Reinat; Meir, Sagit; Carrillo, L Ruby; Kramer, David M; Reich, Ziv

2015-03-01

Over-reduction of the photosynthetic electron transport chain may severely damage the photosynthetic apparatus as well as other constituents of the chloroplast and the cell. Here, we exposed Arabidopsis leaves to saturating light either under normal atmospheric conditions or under CO2--and O2 -limiting conditions, which greatly increase excitation and electron pressures by draining terminal electron acceptors. The two treatments were found to have very different, often opposing, effects on the structure of the thylakoid membranes, including the width of the granal lumenal compartment. Modulation of the latter is proposed to be related to movements of ions across the thylakoid membrane, which alter the relative osmolarity of the lumen and stroma and affect the partitioning of the proton motive force into its electrical and osmotic components. The resulting changes in thylakoid organization and lumenal width should facilitate the repair of photodamaged photosystem II complexes in response to light stress under ambient conditions, but are expected to inhibit the repair cycle when the light stress occurs concurrently with CO2 and O2 depletion. Under the latter conditions, the changes in thylakoid structure are predicted to complement other processes that restrict the flow of electrons into the high-potential chain, thus moderating the production of deleterious reactive oxygen species at photosystem I. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

[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.

Photoinduced Regeneration of an Aptamer-Based Electrochemical Sensor for Sensitively Detecting Adenosine Triphosphate.

PubMed

Zhang, Xiaoyu; Song, Chunxia; Yang, Ke; Hong, Wenwen; Lu, Ying; Yu, Ping; Mao, Lanqun

2018-04-17

Electrochemical aptasensors generally include three elements, that is, recognition element, signal-transformation element, and regeneration element. In this study, a new adenosine triphosphate (ATP) aptasensor is developed by combining three elements into one DNA oligonucleotide chain. In the DNA oligonucleotide chain, DNA aptamer is used as the recognition element, ferrocene group attached at the 3'-end of the aptamer is used as the signal-transformation element, and azobenzene moiety embedded into the DNA chain is used as the regeneration element. In addition to the similar analytical properties with the traditional ones, the aptasensor developed here is easily regenerated with UV-light irradiation. The current response recorded on the aptasensor increases with increasing the concentration of ATP in the incubation solution and is linear with the logarithm of ATP concentration in the range from 1 nM to 100 μM. The limit of detection is 0.5 nM (S/N = 3). The basal level of ATP in the rat brain cortex microdialysate is determined to be 21.33 ± 4.1 nM ( n = 3). After being challenged with ATP, the aptasensor could be readily regenerated by UV-light irradiation for more than seven cycles. The regeneration of the aptasensor is proposed to be regulated by conversing azobenzene from its trans to cis form under UV irradiation.

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

PubMed

Simpson, L L; Stiles, B G; Zepeda, H; Wilkins, T D

1989-01-01

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

PubMed Central

Simpson, L L; Stiles, B G; Zepeda, H; Wilkins, T D

1989-01-01

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins. Images PMID:2521214

Analysis of the gravitaxis signal transduction chain in Euglena gracilis

NASA Astrophysics Data System (ADS)

Nasir, Adeel

Abstract Euglena gracilis is a photosynthetic, eukaryotic flagellate. It can adapt autotrophic and heterotrophic mode of growth and respond to different stimuli, this makes it an organism of choice for different research disciplines. It swims to reach a suitable niche by employing different stimuli such as oxygen, light, gravity and different chemicals. Among these stimuli light and gravity are the most important. Phototaxis (locomotion under light stimulus) and gravitaxis (locomotion under gravity stimulus) synergistically help cells to attain an optimal niche in the environment. However, in the complete absence of light or under scarcity of detectable light, cells can totally depend on gravity to find its swimming path. Therefore gravity has certain advantages over other stimuli.Unlike phototatic signal transduction chain of Euglena gracilis no clear primary gravity receptor has been identified in Euglena cells so far. However, there are some convincing evidence that TRP like channels act as a primary gravity receptor in Euglena gracilis.Use of different inhibitors gave rise to the involvement of protein kinase and calmodulin proteins in signal transduction chain of Euglena gracilis. Recently, specific calmodulin (Calmodulin 2) and protein kinase (PKA) have been identified as potential candidates of gravitactic signal transduction chain. Further characterization and investigation of these candidates was required. Therefore a combination of biochemical and genetic techniques was employed to localize proteins in cells and also to find interacting partners. For localization studies, specific antibodies were raised and characterized. Specificity of antibodies was validated by knockdown mutants, Invitro-translated proteins and heterologously expressed proteins. Cell fractionation studies, involving separation of the cell body and flagella for western blot analysis and confocal immunofluorescence studies were performed for subcellular localization. In order to find interacting partners, yeast two hybrid screen was conducted by using commercially synthesized cDNA library for Euglena gracilis. For both protein kinase and calmodulin some putative interacting partners were found. These plausible candidates are subjected for further validation studies, to verify the protein-protein interaction. In addition, some differential expression studies are also performed for these proteins to evaluate their expression levels under conditions which are known to affect gravitaxis in Euglena gracilis. Taken together, these data are in good agreement with some of already predicted studies for protein localization, but at the same time provides new insights for further studies.

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.

Tacrolimus has immunosuppressive effects on heavy/light chain pairs and free light chains in patients after heart transplantation: A relationship with infection.

PubMed

Lavríková, Petra; Sečník, Peter; Kubíček, Zdenek; Jabor, Antonín; Hošková, Lenka; Franeková, Janka

2018-06-15

The aim of the study was to investigate the relationship between tacrolimus (TAC) immunosuppressive treatment and serum concentrations of immunoglobulin heavy/light chain pairs (sHLC) and free light chains (sFLC) in patients after heart transplantation (HTX) and to use these biomarkers to predict the risk of infection in these patients. A total of 88 patients with an immunosuppressive regimen involving tacrolimus who underwent HTX were analyzed over 24 months of follow-up. sFLC and sHLC levels were determined before and at three time points after HTX. TAC concentrations were determined at several time points after HTX, and mean TAC concentrations and areas under the curve (AUCs) of TAC concentration were calculated. Relevant clinical data were obtained from patients' medical records. A larger AUC of TAC was associated with decreases in the concentrations of IgG total (p < 0.05); similarly, cumulative AUC of TAC during 18 post-transplant months correlated inversely with sHLC IgG kappa (r = -0.228, p < 0.05) and IgG total (r = -0.352, p < 0.05). Concentrations of sFLC kappa, sFLC lambda, sHLC IgG kappa, and sHLC IgG total were significantly lower in infected patients (in the 9th month after HTX, all p < 0.05). Combined criteria for increased AUC (greater than the median of 12.9 mg·d/l) and decreased sFLC kappa (less than the median of 12.5 mg/l) correlated with the presence of infection (p < 0.03) in the 9th month after HTX. Ratio of concentration of TAC to sFLC kappa or lambda was significantly higher in infected patients (both p < 0.05). Intensive treatment with tacrolimus after HTX is possibly reflected by decreases in sFLC and sHLC (mainly sHLC IgG). Patients with decreased concentrations of these biomarkers are at increased risk for infection, primarily in the 9th month after HTX, when the concentrations of tacrolimus were the highest. Copyright © 2018 Elsevier B.V. All rights reserved.

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).

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.

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

PubMed

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

2011-09-01

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

Folding and trimerization of clathrin subunits at the triskelion hub.

PubMed

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

1992-03-06

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

Biomass supply chain optimisation for Organosolv-based biorefineries.

PubMed

Giarola, Sara; Patel, Mayank; Shah, Nilay

2014-05-01

This work aims at providing a Mixed Integer Linear Programming modelling framework to help define planning strategies for the development of sustainable biorefineries. The up-scaling of an Organosolv biorefinery was addressed via optimisation of the whole system economics. Three real world case studies were addressed to show the high-level flexibility and wide applicability of the tool to model different biomass typologies (i.e. forest fellings, cereal residues and energy crops) and supply strategies. Model outcomes have revealed how supply chain optimisation techniques could help shed light on the development of sustainable biorefineries. Feedstock quality, quantity, temporal and geographical availability are crucial to determine biorefinery location and the cost-efficient way to supply the feedstock to the plant. Storage costs are relevant for biorefineries based on cereal stubble, while wood supply chains present dominant pretreatment operations costs. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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

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

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

PubMed Central

Smith, Ian C.; Gittings, William; Huang, Jian; McMillan, Elliott M.; Quadrilatero, Joe; Tupling, A. Russell

2013-01-01

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca2+ concentration ([Ca2+]i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca2+-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca2+ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca2+]i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca2+ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca2+]i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation. PMID:23401574

[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.

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.

The effectiveness of riboflavin and ultraviolet light pathogen reduction technology in eliminating Trypanosoma cruzi from leukoreduced whole blood.

PubMed

Jimenez-Marco, Teresa; Cancino-Faure, Beatriz; Girona-Llobera, Enrique; Alcover, M Magdalena; Riera, Cristina; Fisa, Roser

2017-06-01

The parasitic Chagas disease is caused by the protozoan Trypanosoma cruzi, which is mainly transmitted by insect vectors. Other infection routes, both in endemic and in nonendemic areas, include organ and marrow transplantation, congenital transmission, and blood transfusion. Asymptomatic chronic chagasic individuals may have a low and transient parasitemia in peripheral blood and, consequently, they can unknowingly transmit the disease via blood transfusion. Riboflavin and ultraviolet (UV) light pathogen reduction is a method to reduce pathogen transfusion transmission risk based on damage to the pathogen nucleic acids. In this study, we tested the effectiveness of this technology for the elimination of T. cruzi parasites in artificially contaminated whole blood units (WBUs) and thus for decreasing the risk of T. cruzi transfusion transmission. The contaminated WBUs were leukoreduced by filtration and treated with riboflavin and UV light. The level of pathogen reduction was quantified by a real-time polymerase chain reaction (qPCR) and a real-time reverse transcription-polymerase chain reaction (RT-qPCR) as a viability assay. The RNA (cDNA) quantification of the parasites showed a more than 99% reduction of viable T. cruzi parasites after leukoreduction and a complete reduction (100%) after the riboflavin and UV light treatment. Riboflavin and UV light treatment and leukoreduction used in conjunction appears to eliminate significant amounts of viable T. cruzi in whole blood. Both strategies could complement other blood bank measures already implemented to prevent the transmission of T. cruzi via blood transfusion. © 2017 AABB.

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.

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.

ATM Substrate Chk2-interacting Zn2+ Finger (ASCIZ) Is a Bi-functional Transcriptional Activator and Feedback Sensor in the Regulation of Dynein Light Chain (DYNLL1) Expression*

PubMed Central

Jurado, Sabine; Conlan, Lindus A.; Baker, Emma K.; Ng, Jane-Lee; Tenis, Nora; Hoch, Nicolas C.; Gleeson, Kimberly; Smeets, Monique; Izon, David; Heierhorst, Jörg

2012-01-01

The highly conserved DYNLL1 (LC8) protein was originally discovered as a light chain of the dynein motor complex, but is increasingly emerging as a sequence-specific regulator of protein dimerization with hundreds of targets and wide-ranging cellular functions. Despite its important roles, DYNLL1's own regulation remains poorly understood. Here we identify ASCIZ (ATMIN/ZNF822), an essential Zn2+ finger protein with dual roles in the DNA base damage response and as a developmental transcription factor, as a conserved regulator of Dynll1 gene expression. DYNLL1 levels are reduced by ∼10-fold in the absence of ASCIZ in human, mouse and chicken cells. ASCIZ binds directly to the Dynll1 promoter and regulates its activity in a Zn2+ finger-dependent manner. DYNLL1 protein in turn interacts with ten binding sites in the ASCIZ transcription activation domain, and high DYNLL1 levels inhibit the transcriptional activity of ASCIZ. In addition, DYNLL1 was also required for DNA damage-induced ASCIZ focus formation. The dual ability of ASCIZ to activate Dynll1 gene expression and to sense free DYNLL1 protein levels enables a simple dynamic feedback loop to adjust DYNLL1 levels to cellular needs. The ASCIZ-DYNLL1 feedback loop represents a novel mechanism for auto-regulation of gene expression, where the gene product directly inhibits the transcriptional activator while bound at its own promoter. PMID:22167198

Optical Properties of a Single Carbon Chain-Doped Silicene Nanoribbon

NASA Astrophysics Data System (ADS)

Lu, Dao-Bang; Song, Yu-Ling; Huang, Xiao-yu; Wang, Chong

2018-05-01

Using first-principles spin polarization density function theory calculations, we have studied the electronic and optical properties of zigzag-edge silicene nanoribbons (ZSiNRs) doped with a single carbon chain. Because of the doped carbon chain, there are several defect states in the band structures of ZSiNRs across the Fermi level, and the ferromagnetic ground state is metallic. The dielectric functions in all three dimensions are completely different from each other, and thus the system exhibits strong optical anisotropism. The carbon chain influenced the dielectric functions most at low energy. The first peak in the E//x direction of the dielectric spectrum exhibits a significant blueshift, and its value has changed as well. The main absorption wavelength depends on the polarization direction of the incident light, but occurs within the UV region for all polarization directions. The peaks of the energy loss spectra correspond to the trailing edges in the reflectivity spectrum, and the highest peak corresponds to a plasmon frequency. Our results could be useful for investigating nanodevices based on silicene nanoribbons.

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

DISTINCT ANTIBODY SPECIES: STRUCTURAL DIFFERENCES CREATING THERAPEUTIC OPPORTUNITIES

PubMed Central

Muyldermans, Serge; Smider, Vaughn V.

2016-01-01

Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies. PMID:26922135

Illustrating the Molecular Origin of Mechanical Stress in Ductile Deformation of Polymer Glasses.

PubMed

Li, Xiaoxiao; Liu, Jianning; Liu, Zhuonan; Tsige, Mesfin; Wang, Shi-Qing

2018-02-16

New experiments show that tensile stress vanishes shortly after preyield deformation of polymer glasses while tensile stress after postyield deformation stays high and relaxes on much longer time scales, thus hinting at a specific molecular origin of stress in ductile cold drawing: chain tension rather than intersegmental interactions. Molecular dynamics simulation based on a coarse-grained model for polystyrene confirms the conclusion that the chain network plays an essential role, causing the glassy state to yield and to respond with a high level of intrachain retractive stress. This identification sheds light on the future development regarding an improved theoretical account for molecular mechanics of polymer glasses and the molecular design of stronger polymeric materials to enhance their mechanical performance.

Illustrating the Molecular Origin of Mechanical Stress in Ductile Deformation of Polymer Glasses

NASA Astrophysics Data System (ADS)

Li, Xiaoxiao; Liu, Jianning; Liu, Zhuonan; Tsige, Mesfin; Wang, Shi-Qing

2018-02-01

New experiments show that tensile stress vanishes shortly after preyield deformation of polymer glasses while tensile stress after postyield deformation stays high and relaxes on much longer time scales, thus hinting at a specific molecular origin of stress in ductile cold drawing: chain tension rather than intersegmental interactions. Molecular dynamics simulation based on a coarse-grained model for polystyrene confirms the conclusion that the chain network plays an essential role, causing the glassy state to yield and to respond with a high level of intrachain retractive stress. This identification sheds light on the future development regarding an improved theoretical account for molecular mechanics of polymer glasses and the molecular design of stronger polymeric materials to enhance their mechanical performance.

4-Amino-7-chloroquinolines: probing ligand efficiency provides botulinum neurotoxin serotype A light chain inhibitors with significant antiprotozoal activity

PubMed Central

Opsenica, Igor M.; Tot, Mikloš; Gomba, Laura; Nuss, Jonathan E.; Sciotti, Richard J.; Bavari, Sina; Burnett, James C.; Šolaja, Bogdan A.

2013-01-01

Structurally simplified analogs of dual antimalarial and botulinum neurotoxin serotype A light chain (BoNT/A LC) inhibitor bis-aminoquinoline (1) were prepared. New compounds were designed to improve ligand efficiency while maintaining or exceeding the inhibitory potency of 1. Three of the new compounds are more active than 1 against both indications. Metabolically, the new inhibitors are relatively stable and non-toxic. Twelve, 14, and 15 are more potent BoNT/A LC inhibitors than 1. Additionally, 15 has excellent in vitro antimalarial efficacy, with IC90 values ranging from 4.45-12.11 nM against five Plasmodium falciparum (P.f.) strains: W2, D6, C235, C2A, C2B. The results indicate that the same level of inhibitory efficacy provided by 1 can be retained/exceeded with less structural complexity. Twelve, 14, and 15 provide new platforms for the development of more potent dual BoNT/A LC and P.f. inhibitors adhering to generally accepted chemical properties associated with the druggability of synthetic molecules. PMID:23815186

Blebbistatin, a myosin II inhibitor, suppresses Ca(2+)-induced and "sensitized"-contraction of skinned tracheal muscles from guinea pig.

PubMed

Yumoto, Masatoshi; Watanabe, Masaru

2013-01-01

Blebbistatin, a potent inhibitor of myosin II, has inhibiting effects on Ca(2+)-induced contraction and contractile filament organization without affecting the Ca(2+)-sensitivity to the force and phosphorylation level of myosin regulatory light chain (MLC20) in skinned (cell membrane permeabilized) taenia cecum from the guinea pig (Watanabe et al., Am J Physiol Cell Physiol. 2010; 298: C1118-26). In the present study, we investigated blebbistatin effects on the contractile force of skinned tracheal muscle, in which myosin filaments organization is more labile than that in the taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration, but had little effects on the Ca(2+)- induced myosin light chain phosphorylation. Also blebbistatin at 10 μM and higher significantly suppressed GTP-γS-induced "sensitized" force development. Since the force inhibiting effects of blebbistatin on the skinned trachea were much stronger than those in skinned taenia cecum, blebbistatin might directly affect myosin filaments organization.

An overview of the legislation and light microscopy for detection of processed animal proteins in feeds.

PubMed

Liu, Xian; Han, Lujia; Veys, Pascal; Baeten, Vincent; Jiang, Xunpeng; Dardenne, Pierre

2011-08-01

From the first cases of bovine spongiform encephalopathy (BSE) among cattle in the United Kingdom in 1986, the route of infection of BSE is generally believed by means of feeds containing low level of processed animal proteins (PAPs). Therefore, many feed bans and alternative and complementary techniques were resulted for the BSE safeguards in the world. Now the feed bans are expected to develop into a "species to species" ban, which requires the corresponding species-specific identification methods. Currently, banned PAPs can be detected by various methods as light microscopy, polymerase chain reaction, enzyme-linked immunosorbent assay, near infrared spectroscopy, and near infrared microscopy. Light microscopy as described in the recent Commission Regulation EC/152/2009 is the only official method for the detection and characterization of PAPs in feed in the European Union. It is able to detect the presence of constituents of animal origin in feed at the level of 1 g/kg with hardly any false negative. Nevertheless, light microscopy has the limitation of lack of species specificity. This article presents a review of legislations on the use of PAPs in feedstuff, the detection details of animal proteins by light microscopy, and also presents and discusses the analysis procedure and expected development of the technique. Copyright © 2010 Wiley-Liss, Inc.

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

Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

PubMed Central

Yotova, Iveta; Quan, Ping; Gaba, Aulona; Leditznig, Nadja; Pateisky, Petra; Kurz, Christine; Tschugguel, Walter

2012-01-01

Endometriosis is a disease characterized by the localization of endometrial tissue outside the uterine cavity. The differences observed in migration of human endometrial stromal cells (hESC) obtained from patients with endometriosis versus healthy controls were proposed to correlate with the abnormal activation of Raf-1/ROCKII signalling pathway. To evaluate the mechanism by which Raf-1 regulates cytoskeleton reorganization and motility, we used primary eutopic (Eu-, n = 16) and ectopic (Ec-, n = 8; isolated from ovarian cysts) hESC of patients with endometriosis and endometriosis-free controls (Co-hESC, n = 14). Raf-1 siRNA knockdown in Co- and Eu-hESC resulted in contraction and decreased migration versus siRNA controls. This phenotype was reversed following the re-expression of Raf-1 in these cells. Lowest Raf-1 levels in Ec-hESC were associated with hyperactivated ROCKII and ezrin/radixin/moesin (E/R/M), impaired migration and a contracted phenotype similar to Raf-1 knockdown in Co- and Eu-hESC. We further show that the mechanism by which Raf-1 mediates migration in hESC includes direct myosin light chain phosphatase (MYPT1) phosphorylation and regulation of the levels of E/R/M, paxillin, MYPT1 and myosin light chain (MLC) phosphorylation indirectly via the hyperactivation of ROCKII kinase. Furthermore, we suggest that in contrast to Co-and Eu-hESC, where the cellular Raf-1 levels regulate the rate of migration, the low cellular Raf-1 content in Ec-hESC, might ensure their restricted migration by preserving the contracted cellular phenotype. In conclusion, our findings suggest that cellular levels of Raf-1 adjust the threshold of hESC migration in endometriosis. PMID:22225925

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.

The significance and predictive value of free light chains in the urine of patients with chronic inflammatory rheumatic disease.

PubMed

Bramlage, Carsten Paul; Froelich, Britta; Wallbach, Manuel; Minguet, Joan; Grupp, Clemens; Deutsch, Cornelia; Bramlage, Peter; Koziolek, Michael; Müller, Gerhard Anton

2016-12-01

In patients with rheumatic diseases, reliable markers for determining disease activity are scarce. One potential parameter is the level of immunoglobulin free light chains (FLCs), which is known to be elevated in the blood of patients with certain rheumatic diseases. Few studies have quantified FLCs in urine, a convenient source of test sample, in patients with different rheumatic diseases. We carried out a retrospective analysis of patients with rheumatic disease attending the University hospital of Goettingen, Germany. Subjects were included if they had urine levels of both κ and λ FLCs available and did not have myeloma. Data regarding systemic inflammation and kidney function were recorded, and FLC levels were correlated with inflammatory markers. Of the 382 patients with rheumatic disease, 40.1 % had chronic polyarthritis, 21.2 % connective tissue disease, 18.6 % spondyloarthritis and 15.7 % vasculitis. Elevated levels of κ FLCs were found for 84 % of patients and elevated λ for 52.7 %. For the patients with rheumatoid arthritis, FLCs correlated with C-reactive protein (κ, r = 0.368, p < 0.001; λ, r = 0.398, p < 0.001) and erythrocyte sedimentation rate (κ, r = 0.692, p < 0.001; λ, r = 0.612, p < 0.001). Patients being treated with rituximab displayed FLC levels similar to those of the reference group. There were clear elevations in both κ and λ FLCs in patients with rheumatic disease, but not in κ/λ ratio. The correlation between FLCs and inflammatory markers in patients with rheumatoid arthritis demonstrates their potential for predicting disease activity.

Proposed Mechanisms of Photobiomodulation or Low-Level Light Therapy

PubMed Central

de Freitas, Lucas Freitas; Hamblin, Michael R

2016-01-01

Photobiomodulation (PBM) also known as low-level laser (or light) therapy (LLLT), has been known for almost 50 years but still has not gained widespread acceptance, largely due to uncertainty about the molecular, cellular, and tissular mechanisms of action. However, in recent years, much knowledge has been gained in this area, which will be summarized in this review. One of the most important chromophores is cytochrome c oxidase (unit IV in the mitochondrial respiratory chain), which contains both heme and copper centers and absorbs light into the near-infra-red region. The leading hypothesis is that the photons dissociate inhibitory nitric oxide from the enzyme, leading to an increase in electron transport, mitochondrial membrane potential and ATP production. Another hypothesis concerns light-sensitive ion channels that can be activated allowing calcium to enter the cell. After the initial photon absorption events, numerous signaling pathways are activated via reactive oxygen species, cyclic AMP, NO and Ca2+, leading to activation of transcription factors. These transcription factors can lead to increased expression of genes related to protein synthesis, cell migration and proliferation, anti-inflammatory signaling, anti-apoptotic proteins, antioxidant enzymes. Stem cells and progenitor cells appear to be particularly susceptible to LLLT. PMID:28070154

Proposed Mechanisms of Photobiomodulation or Low-Level Light Therapy.

PubMed

de Freitas, Lucas Freitas; Hamblin, Michael R

2016-01-01

Photobiomodulation (PBM) also known as low-level laser (or light) therapy (LLLT), has been known for almost 50 years but still has not gained widespread acceptance, largely due to uncertainty about the molecular, cellular, and tissular mechanisms of action. However, in recent years, much knowledge has been gained in this area, which will be summarized in this review. One of the most important chromophores is cytochrome c oxidase (unit IV in the mitochondrial respiratory chain), which contains both heme and copper centers and absorbs light into the near-infra-red region. The leading hypothesis is that the photons dissociate inhibitory nitric oxide from the enzyme, leading to an increase in electron transport, mitochondrial membrane potential and ATP production. Another hypothesis concerns light-sensitive ion channels that can be activated allowing calcium to enter the cell. After the initial photon absorption events, numerous signaling pathways are activated via reactive oxygen species, cyclic AMP, NO and Ca2+, leading to activation of transcription factors. These transcription factors can lead to increased expression of genes related to protein synthesis, cell migration and proliferation, anti-inflammatory signaling, anti-apoptotic proteins, antioxidant enzymes. Stem cells and progenitor cells appear to be particularly susceptible to LLLT.

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.

Nitric oxide measurements in hTERT-RPE cells and subcellular fractions exposed to low levels of red light

NASA Astrophysics Data System (ADS)

Wigle, Jeffrey C.; Castellanos, Cherry C.; Denton, Michael L.; Holwitt, Eric A.

2014-02-01

Cells in a tissue culture model for laser eye injury exhibit increased resistance to a lethal pulse of 2.0-μm laser radiation if the cells are first exposed to 2.88 J/cm2 of red light 24 hr prior to the lethal laser exposure. Changes in expression of various genes associated with apoptosis have been observed, but the biochemical link between light absorption and gene expression remains unknown. Cytochome c oxidase (CCOX), in the electron transport chain, is the currentlyhypothesized absorber. Absorption of the red light by CCOX is thought to facilitate displacement of nitric oxide (NO) by O2 in the active site, increasing cellular respiration and intracellular ATP. However, NO is also an important regulator and mediator of numerous physiological processes in a variety of cell and tissue types that is synthesized from l-arginine by NO synthases. In an effort to determine the relative NO contributions from these competing pathways, we measured NO levels in whole cells and subcellular fractions, with and without exposure to red light, using DAF-FM, a fluorescent dye that stoichiometrically reacts with NO. Red light induced a small, but consistently reproducible, increase in fluorescence intensity in whole cells and some subcellular fractions. Whole cells exhibited the highest overall fluorescence intensity followed by (in order) cytosolic proteins, microsomes, then nuclei and mitochondria.

Early Application of High Cut-Off Haemodialysis for de-Novo Myeloma Nephropathy is Associated with Long-Term Dialysis-Independency and Renal Recovery

PubMed Central

Khalafallah, Alhossain A.; Loi, Sie Wuong; Love, Sarah; Mohamed, Muhajir; Mace, Rose; Khalil, Ramy; Girgs, Miriam; Raj, Rajesh; Mathew, Mathew

2013-01-01

Background Multiple myeloma (MM) is a haematological malignancy associated with kidney injury resulting from cast nephropathy, which can be caused by monoclonal free light chains (FLC). It has been demonstrated that early reduction of FLC can lead to a higher proportion of patients recovering renal function with a better outcome, especially if high cut-off haemodialysis (HCO-HD) combined with chemotherapy is used. Patients and Methods In this study, four cases with MM nephropathy were treated with HCO-HD and chemotherapy at a single institution during the period from August 2009 to August 2011. All of the patients presented with acute renal failure and high serum FLC. All patients underwent a bone marrow biopsy to confirm the diagnosis of MM, according to the WHO criteria. Three patients had de novo MM and one patient had relapsed light chain myeloma disease. All patients underwent HCO-HD concomitantly with specific myeloma therapy once the diagnosis or relapse of MM was established. Results After a medial follow up of 26 months, (range, 13–36) our data showed that all patients had a significant decrease in serum FLC through HCO-HD, proving the effectiveness of HCO-HD in managing MM. De-novo MM patients restored their renal function and achieved low-level FLC early in the treatment and became dialysis-independent. One patient with relapsed myeloma remained dialysis-dependent. Conclusion In summary, our study suggests that in myeloma nephropathy associated with light-chain MM, HCO-HD should be initiated as early as possible. At the same time a specific MM treatment should be initiated to gain control of the disease and salvage the kidneys in order to achieve dialysis-independency. Further randomized trials to confirm our results are warranted. PMID:23350020

Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

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

Dey, Sumit K.; Saha, Shekhar; Das, Provas

2014-08-01

3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reducedmore » in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.« less

Red light regulation of ethylene biosynthesis and gravitropism in etiolated pea stems

NASA Technical Reports Server (NTRS)

Steed, C. L.; Taylor, L. K.; Harrison, M. A.

2004-01-01

During gravitropism, the accumulation of auxin in the lower side of the stem causes increased growth and the subsequent curvature, while the gaseous hormone ethylene plays a modulating role in regulating the kinetics of growth asymmetries. Light also contributes to the control of gravitropic curvature, potentially through its interaction with ethylene biosynthesis. In this study, red-light pulse treatment of etiolated pea epicotyls was evaluated for its effect on ethylene biosynthesis during gravitropic curvature. Ethylene biosynthesis analysis included measurements of ethylene; the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC); malonyl-conjugated ACC (MACC); and expression levels of pea ACC oxidase (Ps-ACO1) and ACC synthase (Ps-ACS1, Ps-ACS2) genes by reverse transcriptase-polymerase chain reaction analysis. Red-pulsed seedlings were given a 6 min pulse of 11 micromoles m-2 s-1 red-light 15 h prior to horizontal reorientation for consistency with the timeline of red-light inhibition of ethylene production. Red-pulse treatment significantly reduced ethylene production and MACC levels in epicotyl tissue. However, there was no effect of red-pulse treatment on ACC level, or expression of ACS or ACO genes. During gravitropic curvature, ethylene production increased from 60 to 120 min after horizontal placement in both control and red-pulsed epicotyls. In red-pulsed tissues, ACC levels increased by 120 min after horizontal reorientation, accompanied by decreased MACC levels in the lower portion of the epicotyl. Overall, our results demonstrate that ethylene production in etiolated epicotyls increases after the initiation of curvature. This ethylene increase may inhibit cell growth in the lower portion of the epicotyl and contribute to tip straightening and reduced overall curvature observed after the initial 60 min of curvature in etiolated pea epicotyls.

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

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.

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

The phototoxicity of phenothiazinium-based photosensitizers to bacterial membranes.

PubMed

Hussain, Saimah; Harris, Frederick; Phoenix, David A

2006-02-01

The ability of phenothiazinium-based photosensitizers to induce photodamage to Escherichia coli membranes is investigated. Phenothiazinium-based photosensitizers were found to be somewhat lipophilic (log P>0.7) and to induce surface-pressure changes (3-12 mN m(-1)) in lipid monolayers mimetic of bacterial membranes, implying that these molecules are able to penetrate biological membranes. Under dark and light conditions (3.15 J cm(-1) for 30 min), phenothiazinium-based photosensitizers were incubated with E. coli cells. These cells showed levels of dark bacteriolysis that ranged between 6% and 13%, with light conditions leading to no significant increase in these levels. Gas chromatography-based analyses showed such incubations to produce no significant changes in the levels of C(16) and C(18) fatty acid chain saturation found in E. coli whole lipid-extracts. It is concluded that the phenothiazinium-based photosensitizers studied may not use E. coli membranes as their primary photodynamic target, but may inflict photodamage on cytoplasmic targets, possibly DNA.

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.

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains

PubMed Central

Hora, Manuel; Carballo-Pacheco, Martin; Weber, Benedikt; Morris, Vanessa K.; Wittkopf, Antje; Buchner, Johannes; Strodel, Birgit; Reif, Bernd

2017-01-01

Antibody light chain amyloidosis is a rare disease caused by fibril formation of secreted immunoglobulin light chains (LCs). The huge variety of antibody sequences puts a serious challenge to drug discovery. The green tea polyphenol epigallocatechin-3-gallate (EGCG) is known to interfere with fibril formation in general. Here we present solution- and solid-state NMR studies as well as MD simulations to characterise the interaction of EGCG with LC variable domains. We identified two distinct EGCG binding sites, both of which include a proline as an important recognition element. The binding sites were confirmed by site-directed mutagenesis and solid-state NMR analysis. The EGCG-induced protein complexes are unstructured. We propose a general mechanistic model for EGCG binding to a conserved site in LCs. We find that EGCG reacts selectively with amyloidogenic mutants. This makes this compound a promising lead structure, that can handle the immense sequence variability of antibody LCs. PMID:28128355

The force dependence of isometric and concentric potentiation in mouse muscle with and without skeletal myosin light chain kinase.

PubMed

Gittings, William; Aggarwal, Harish; Stull, James T; Vandenboom, Rene

2015-01-01

The isometric potentiation associated with myosin phosphorylation is force dependent. The purpose of this study was to assess the influence of a pre-existing period of isometric force on the concentric force potentiation displayed by mouse muscles with and without the ability to phosphorylate myosin. We tested isometric (ISO) and concentric (CON) potentiation, as well as concentric potentiation after isometric force (ISO-CON), in muscles from wild-type (WT) and skeletal myosin light chain kinase-deficient (skMLCK(-/-)) mice. A conditioning stimulus increased (i.e., potentiated) mean concentric force in the ISO-CON and CON conditions to 1.31 ± 0.02 and 1.35 ± 0.02 (WT) and to 1.19 ± 0.02 and 1.21 ± 0.01 (skMLCK(-/-)) of prestimulus levels, respectively (data n = 6-8, p < 0.05). No potentiation of mean isometric force was observed in either genotype. The potentiation of mean concentric force was inversely related to relative tetanic force level (P/Po) in both genotypes. Moreover, concentric potentiation varied greatly within each contraction type and was negatively correlated with unpotentiated force in both genotypes. Thus, although no effect of pre-existing force was observed, strong and inverse relationships between concentric force potentiation and unpotentiated concentric force may suggest an influence of attached and force-generating crossbridges on potentiation magnitude in both WT and skMLCK(-/-) muscles.

The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain1[OPEN

PubMed Central

2018-01-01

Phosphorus (P) is an essential macronutrient, and P deficiency limits plant productivity. Recent work showed that P deficiency affects electron transport to photosystem I (PSI), but the underlying mechanisms are unknown. Here, we present a comprehensive biological model describing how P deficiency disrupts the photosynthetic machinery and the electron transport chain through a series of sequential events in barley (Hordeum vulgare). P deficiency reduces the orthophosphate concentration in the chloroplast stroma to levels that inhibit ATP synthase activity. Consequently, protons accumulate in the thylakoids and cause lumen acidification, which inhibits linear electron flow. Limited plastoquinol oxidation retards electron transport to the cytochrome b6f complex, yet the electron transfer rate of PSI is increased under steady-state growth light and is limited under high-light conditions. Under P deficiency, the enhanced electron flow through PSI increases the levels of NADPH, whereas ATP production remains restricted and, hence, reduces CO2 fixation. In parallel, lumen acidification activates the energy-dependent quenching component of the nonphotochemical quenching mechanism and prevents the overexcitation of photosystem II and damage to the leaf tissue. Consequently, plants can be severely affected by P deficiency for weeks without displaying any visual leaf symptoms. All of the processes in the photosynthetic machinery influenced by P deficiency appear to be fully reversible and can be restored in less than 60 min after resupply of orthophosphate to the leaf tissue. PMID:29540590

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.

L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

PubMed

Maddala, Rupalatha; Nagendran, Tharkika; de Ridder, Gustaaf G; Schey, Kevin L; Rao, Ponugoti Vasantha

2013-01-01

Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial role for LTCCs in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the mechanical properties of lens, all of which have a vital role in maintaining lens function and cytoarchitecture.

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.

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.

Elevated propionate and butyrate in fecal ferments of hydrolysates generated by oxalic acid treatment of corn bran arabinoxylan.

PubMed

Rumpagaporn, Pinthip; Reuhs, Brad L; Cantu-Jungles, Thaisa M; Kaur, Amandeep; Patterson, John A; Keshavarzian, Ali; Hamaker, Bruce R

2016-12-07

Previous work in our laboratory showed that alkali-solubilized corn arabinoxylan (CAX) has a slow initial, but later complete, in vitro human fecal fermentation. CAX and a moderately high molecular weight hydrolysate (CH) were propiogenic, and produced low levels of butyrate. Here, we show that oxalic acid-generated hydrolysates from CAX, which include a large xylooligosaccharide, and free arabinose fractions, increased short chain fatty acid (SCFA) production, which included relatively high levels of both propionate and butyrate, an unusual SCFA combination. Hydrolytic degradation of CAX by acid hydrolysis (0.05 M oxalic acid at 100 °C for 2 h) and subsequent graded ethanol precipitations were used to obtain mixtures with different molecular weight ranges. Ethanol-precipitated fractions (F 0-65%, F 65-75%, F 75-85%) were mostly lower than 100 kDa and F > 85% was composed of monosaccharides and oligosaccharides of DP 2-8. Oxalic acid treatment caused the removal of all single arabinose unit branch chains and some di/trisaccharide branch chains, producing lightly substituted xylan backbone fragments, most of which were in the oligosaccharide (DP < 10) size range. In vitro human fecal fermentation analyses showed all oxalic acid-hydrolysate fractions were slower fermenting than fructooligosaccharides (FOS), but produced similar or higher amounts of total SCFAs. Butyrate production in two hydrolyzate fractions was double that of CH, while propionate levels remained relatively high.

Immunoglobulin λ Gene Rearrangement Can Precede κ Gene Rearrangement

DOE PAGES

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

1990-01-01

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

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

PubMed Central

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

2018-01-01

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

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.

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.

High-Performance Visible-Blind UV Phototransistors Based on n-Type Naphthalene Diimide Nanomaterials.

PubMed

Song, Inho; Lee, Seung-Chul; Shang, Xiaobo; Ahn, Jaeyong; Jung, Hoon-Joo; Jeong, Chan-Uk; Kim, Sang-Wook; Yoon, Woojin; Yun, Hoseop; Kwon, O-Pil; Oh, Joon Hak

2018-04-11

This study investigates the performance of single-crystalline nanomaterials of wide-band gap naphthalene diimide (NDI) derivatives with methylene-bridged aromatic side chains. Such materials are found to be easily used as high-performance, visible-blind near-UV light detectors. NDI single-crystalline nanoribbons are assembled using a simple solution-based process (without solvent-inclusion problems), which is then applied to organic phototransistors (OPTs). Such OPTs exhibit excellent n-channel transistor characteristics, including an average electron mobility of 1.7 cm 2 V -1 s -1 , sensitive UV detection properties with a detection limit of ∼1 μW cm -2 , millisecond-level responses, and detectivity as high as 10 15 Jones, demonstrating the highly sensitive organic visible-blind UV detectors. The high performance of our OPTs originates from the large face-to-face π-π stacking area between the NDI semiconducting cores, which is facilitated by methylene-bridged aromatic side chains. Interestingly, NDI-based nanoribbon OPTs exhibit a distinct visible-blind near-UV detection with an identical detection limit, even under intense visible light illumination (for example, 10 4 times higher intensity than UV light intensity). Our findings demonstrate that wide-band gap NDI-based nanomaterials are highly promising for developing high-performance visible-blind UV photodetectors. Such photodetectors could potentially be used for various applications including environmental and health-monitoring systems.

Validation of Roche LightCycler Epstein-Barr virus quantification reagents in a clinical laboratory setting.

PubMed

Gulley, Margaret L; Fan, Hongxin; Elmore, Sandra H

2006-11-01

Epstein-Barr virus (EBV) is associated with a wide range of benign and malignant diseases, including infectious mononucleosis, lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. Measurement of EBV viral load in plasma is increasingly used for rapid assessment of disease status. We evaluated the performance characteristics of an EBV polymerase chain reaction assay that uses commercial reagents and instruments from Roche Diagnostics (Indianapolis, IN). DNA was extracted from plasma using a MagNaPure instrument, and viral load was measured by real-time polymerase chain reaction on a LightCycler. Analyte-specific reagents included primers and hybridization probes targeting the EBV LMP2 gene and a spiked control sequence. Accuracy and reproducibility were established using DNA from three cell lines. The assay was sensitive to approximately 750 copies of EBV DNA per milliliter of plasma and was linear across at least four orders of magnitude. The assay detected EBV DNA in three of five samples from nasopharyngeal carcinoma patients, seven of nine infectious mononucleosis samples, and 34/34 samples from immunosuppressed patients with clinically significant EBV-related disease, whereas EBV DNA was undetectable in plasma from 21 individuals without EBV-related disease. In conclusion, this LightCycler EBV assay is rapid, sensitive, and linear for quantifying EBV viral load. The assay appears to be useful for measuring clinically significant EBV levels in immunodeficient patients.

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

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.

Factor VIII inhibitor in a patient with mild haemophilia A and an Asn618-->Ser mutation responsive to immune tolerance induction and cyclophosphamide.

PubMed

Vlot, André J; Wittebol, Shulamiet; Strengers, Paul F W; Turenhout, Ellen A M; Voorberg, Jan; van den Berg, H Marijke; Mauser-Bunschoten, Eveline P

2002-04-01

We describe a patient with mild haemophilia A (original value of factor VIII activity 0.30 U/ml) who developed an inhibitor (36.1 Bethesda U/ml) which cross-reacted with his endogenous factor VIII. This caused a decline in basal factor VIII level (< 0.01 U/ml) and severe haemorrhagic events. Treatment to induce immune tolerance was started with factor VIII/von Willebrand factor (VWF) concentrates, but inhibitor levels increased progressively and the patient suffered serious bleeding. Cyclophosphamide was administered and, after 8 months treatment, factor VIII levels increased to 0.20 U/ml and the inhibitor could no longer be detected. Screening of his factor VIII gene revealed a missense mutation in exon 13 that predicts substitution of Asn618-->Ser in the A2 domain of factor VIII. Immunoprecipitation analysis showed that the antibodies present in the patient's plasma reacted with metabolically labelled A2 domain and, to a lesser extent, with factor VIII light chain. Inhibitory antibodies were completely neutralized by recombinant A2 domain, whereas no neutralization was observed after the addition of factor VIII light chain (A3-C1-C2) and C2 domain. More detailed analysis showed that the majority of inhibitory antibodies were directed against residues Arg484-Ile508, a previously identified binding site for factor VIII inhibitors. Our findings suggest that immune tolerance therapy and cyclophosphamide were successful in eradicating inhibitory antibodies against a common epitope on factor VIII.

Serum free light chains are reduced in endurance trained older adults: Evidence that exercise training may reduce basal inflammation in older adults.

PubMed

Heaney, Jennifer L J; Phillips, Anna C; Drayson, Mark T; Campbell, John P

2016-05-01

Traditionally, free light chains (FLCs) are used as key serum biomarkers in the diagnosis and monitoring of plasma cell malignancies, but polyclonal FLCs can also be used as an accurate real-time indicator of immune-activation and inflammation. The primary aim of the present study was to assess the effects of exercise training status on serum FLCs in older adults, and secondly, to examine if training status moderated serum FLC responses to acute exercise. Kappa and lambda serum FLC levels were measured in 45 healthy older adults (aged ≥ 60 years) who were either sedentary, physically active or endurance trained. FLCs were measured at baseline and in response to an acute bout of submaximal exercise. The endurance trained group had significantly lower levels of kappa and lambda serum FLCs compared with physically active or sedentary elderly adults; these effects were independent of age, BMI and renal function. There was no significant difference in whole immunoglobulins between groups. Exercise training status had no effect on serum FLC responses to acute exercise, which were marginal. In conclusion, endurance training was associated with lower FLC levels compared with less physically active individuals. These findings suggest that long-term endurance training may be beneficial in reducing basal inflammation in older adults as well as elevated FLCs present in inflammatory and autoimmune conditions, often associated with ageing. FLCs may serve as a useful biomarker for monitoring the efficacy of exercise intervention studies in healthy and clinical populations. Copyright © 2016 Elsevier Inc. All rights reserved.

Large effect of irradiance on hydrogen isotope fractionation of alkenones in Emiliania huxleyi

NASA Astrophysics Data System (ADS)

van der Meer, Marcel T. J.; Benthien, Albert; French, Katherine L.; Epping, Eric; Zondervan, Ingrid; Reichart, Gert-Jan; Bijma, Jelle; Sinninghe Damsté, Jaap S.; Schouten, Stefan

2015-07-01

The hydrogen isotopic (δD) composition of long-chain alkenones produced by certain haptophyte algae has been suggested as a potential proxy for reconstructing paleo sea surface salinity. However, environmental parameters other than salinity may also affect the δD of alkenones. We investigated the impact of the level of irradiance on hydrogen isotopic fractionation of alkenones versus growth water by cultivating two strains of the cosmopolitan haptophyte Emiliania huxleyi at different light intensities. The hydrogen isotope fractionation decreased by approximately 40‰ when irradiance was increased from 15 to 200 μmol photons m-2 s-1 above which it was relatively constant. The response is likely a direct effect of photosystem I and II activity as the relationship of the fractionation factor α versus light intensity can be described by an Eilers-Peeters photosynthesis model. This irradiance effect is in agreement with published δD data of alkenones derived from suspended particulate matter collected from different depths in the photic zone of the Gulf of California and the eastern tropical North Pacific. However, haptophyte algae tend to bloom at relatively high light intensities (>500 μmol photons m-2 s-1) occurring at the sea surface, at which hydrogen isotope fractionation is relatively constant and not affected by changes in light intensity. Alkenones accumulating in the sediment are likely mostly derived from these surface water haptophyte blooms, when the largest amount of biomass is produced. Therefore, the observed irradiance effect is unlikely to affect the applicability of the hydrogen isotopic composition of sedimentary long chain alkenones as a proxy for paleosalinity.

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.

Sustainable Engineering and Improved Recycling of PET for High-Value Applications: Transforming Linear PET to Lightly Branched PET with a Novel, Scalable Process

NASA Astrophysics Data System (ADS)

Pierre, Cynthia; Torkelson, John

2009-03-01

A major challenge for the most effective recycling of poly(ethylene terephthalate) concerns the fact that initial melt processing of PET into a product leads to substantial degradation of molecular weight. Thus, recycled PET has insufficient melt viscosity for reuse in high-value applications such as melt-blowing of PET bottles. Academic and industrial research has tried to remedy this situation by synthesis and use of ``chain extenders'' that can lead to branched PET (with higher melt viscosity than the linear recycled PET) via condensation reactions with functional groups on the PET. Here we show that simple processing of PET via solid-state shear pulverization (SSSP) leads to enhanced PET melt viscosity without need for chemical additives. We hypothesize that this branching results from low levels of chain scission accompanying SSSP, leading to formation of polymeric radicals that participate in chain transfer and combination reactions with other PET chains and thereby to in situ branch formation. The pulverized PET exhibits vastly enhanced crystallization kinetics, eliminating the need to employ cold crystallization to achieve maximum PET crystallinity. Results of SSSP processing of PET will be compared to results obtained with poly(butylene terephthalate).

Biological effects of blocking blue and other visible light on the mouse retina.

PubMed

Narimatsu, Toshio; Ozawa, Yoko; Miyake, Seiji; Kubota, Shunsuke; Yuki, Kenya; Nagai, Norihiro; Tsubota, Kazuo

2014-08-01

To elucidate the biological effects of blocking fluorescent light on the retina using specific blocking materials. Seven- to 8-week-old BALB/c mice were divided into three groups and placed in one of the three boxes: one blocked ultraviolet and violet wavelengths of light (violet blockade), one blocked ultraviolet, violet, blue and some other visible wavelengths (blue-plus blockade), and one allowed most visible light to pass through (control). They were then exposed to a white fluorescent lamp for 1 h at 5.65E-05 mW/cm(2) /s. After treatment, the electroretinogram, retinal outer nuclear layer thickness and retinal outer segment length were measured. In addition, retinal apoptotic cells were quantified by TdT-mediated dUTP nick-end labelling assay and c-Fos messenger RNA, and protein levels were measured by real-time reverse-transcription polymerase chain reaction and immunoblot analyses, respectively. The blue-plus blockade group retained a significantly better electroretinogram response following light exposure than the control or violet blockade groups. The blue-plus blockade group also exhibited greater outer nuclear layer thickness and greater outer-segment length, and fewer apoptotic cells after light exposure than the other groups. The c-Fos messenger RNA and protein levels were substantially reduced in the blue-plus blockade group and reduced to a lesser extent in the violet blockade group. The blockade of blue plus additional visible wavelengths of light was most effective in protecting the retina from light-induced damage. The blockade of violet light alone was also effective in reducing intracellular molecular responses, but these effects were not sufficient for attenuating retinal degeneration. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

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.

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

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.

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.

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.

Diurnal Regulation of Cellular Processes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Insights from Transcriptomic, Fluxomic, and Physiological Analyses

PubMed Central

Saha, Rajib; Liu, Deng; Hoynes-O’Connor, Allison; Liberton, Michelle; Yu, Jingjie; Bhattacharyya-Pakrasi, Maitrayee; Balassy, Andrea; Zhang, Fuzhong; Maranas, Costas D.

2016-01-01

ABSTRACT Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper) were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H), and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP+ showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium. PMID:27143387

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

Proximal tubule H-ferritin mediates iron trafficking in acute kidney injury

PubMed Central

Zarjou, Abolfazl; Bolisetty, Subhashini; Joseph, Reny; Traylor, Amie; Apostolov, Eugene O.; Arosio, Paolo; Balla, Jozsef; Verlander, Jill; Darshan, Deepak; Kuhn, Lukas C.; Agarwal, Anupam

2013-01-01

Ferritin plays a central role in iron metabolism and is made of 24 subunits of 2 types: heavy chain and light chain. The ferritin heavy chain (FtH) has ferroxidase activity that is required for iron incorporation and limiting toxicity. The purpose of this study was to investigate the role of FtH in acute kidney injury (AKI) and renal iron handling by using proximal tubule–specific FtH-knockout mice (FtHPT–/– mice). FtHPT–/– mice had significant mortality, worse structural and functional renal injury, and increased levels of apoptosis in rhabdomyolysis and cisplatin-induced AKI, despite significantly higher expression of heme oxygenase-1, an antioxidant and cytoprotective enzyme. While expression of divalent metal transporter-1 was unaffected, expression of ferroportin (FPN) was significantly lower under both basal and rhabdomyolysis-induced AKI in FtHPT–/– mice. Apical localization of FPN was disrupted after AKI to a diffuse cytosolic and basolateral pattern. FtH, regardless of iron content and ferroxidase activity, induced FPN. Interestingly, urinary levels of the iron acceptor proteins neutrophil gelatinase–associated lipocalin, hemopexin, and transferrin were increased in FtHPT–/– mice after AKI. These results underscore the protective role of FtH and reveal the critical role of proximal tubule FtH in iron trafficking in AKI. PMID:24018561

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.

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.

Identification and Levels of 2′-Carboxyarabinitol in Leaves 1

PubMed Central

Moore, Brandon d.; Sharkey, Thomas D.; Kobza, John; Seemann, Jeffrey R.

1992-01-01

2′-Carboxyarabinitol 1-phosphate (CA1P) is a naturally occurring inhibitor of ribulose-1,5 bisphosphate carboxylase/oxygenase activity. A chloroplast phosphatase has previously been identified that degrades CA1P in vitro to carboxyarabinitol (CA) plus phosphate, but CA has not yet been detected in plants. Here, we detail procedures to isolate and assay CA from leaves and utilize mass spectrometry to demonstrate for the first time that CA is present in plants. CA was present in leaves of all 13 species examined, including those of C3, C4, and Crassulacean acid metabolism photosynthetic subgroups. CA was present both in species with high levels of CA1P (e.g. Phaseolus vulgaris, Lycopersicon esculentum, Beta vulgaris) as well as in species with low levels of CA1P (e.g. Spinacea oleracea, Triticum aestivum). CA levels in the light were sometimes greater than those in the dark. Bean leaves had the most CA of any species tested, with levels in the light approaching 1 micromole per milligram of chlorophyll. In illuminated bean leaves, about 63% of the CA is located outside the chloroplast. CA is one of only a few branched chain sugar acids to be identified from plants. PMID:16669072

Cloning and expression analysis of carboxyltransferase of acetyl-coA carboxylase from Jatropha curcas.

PubMed

Xie, Wu-Wei; Gao, Shun; Wang, Sheng-Hua; Zhu, Jin-Qiu; Xu, Ying; Tang, Lin; Chen, Fang

2010-01-01

A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl-CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fluorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased significantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a significant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fluctuations compared to those of the control. These findings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.

Low-Level Light Therapy with 410 nm Light Emitting Diode Suppresses Collagen Synthesis in Human Keloid Fibroblasts: An In Vitro Study

PubMed Central

Lee, Hyun Soo; Jung, Soo-Eun; Kim, Sue Kyung; Kim, You-Sun; Sohn, Seonghyang

2017-01-01

Background Keloids are characterized by excessive collagen deposition in the dermis, in which transforming growth factor β (TGF-β)/Smad signaling plays an important role. Low-level light therapy (LLLT) is reported as effective in preventing keloids in clinical reports, recently. To date, studies investigating the effect of LLLT on keloid fibroblasts are extremely rare. Objective We investigated the effect of LLLT with blue (410 nm), red (630 nm), and infrared (830 nm) light on the collagen synthesis in keloid fibroblasts. Methods Keloid fibroblasts were isolated from keloid-revision surgery samples and irradiated using 410-, 630-, 830-nm light emitting diode twice, with a 24-hour interval at 10 J/cm2. After irradiation, cells were incubated for 24 and 48 hours and real-time quantitative reverse transcription polymerase chain reaction was performed. Western blot analysis was also performed in 48 hours after last irradiation. The genes and proteins of collagen type I, TGF-β1, Smad3, and Smad7 were analyzed. Results We observed no statistically significant change in the viability of keloid fibroblasts after irradiation. Collagen type I was the only gene whose expression significantly decreased after irradiation at 410 nm when compared to the non-irradiated control. Western blot analysis showed that LLLT at 410 nm lowered the protein levels of collagen type I compared to the control. Conclusion LLLT at 410 nm decreased the expression of collagen type I in keloid fibroblasts and might be effective in preventing keloid formation in their initial stage. PMID:28392641

Low-Level Light Therapy with 410 nm Light Emitting Diode Suppresses Collagen Synthesis in Human Keloid Fibroblasts: An In Vitro Study.

PubMed

Lee, Hyun Soo; Jung, Soo-Eun; Kim, Sue Kyung; Kim, You-Sun; Sohn, Seonghyang; Kim, You Chan

2017-04-01

Keloids are characterized by excessive collagen deposition in the dermis, in which transforming growth factor β (TGF-β)/Smad signaling plays an important role. Low-level light therapy (LLLT) is reported as effective in preventing keloids in clinical reports, recently. To date, studies investigating the effect of LLLT on keloid fibroblasts are extremely rare. We investigated the effect of LLLT with blue (410 nm), red (630 nm), and infrared (830 nm) light on the collagen synthesis in keloid fibroblasts. Keloid fibroblasts were isolated from keloid-revision surgery samples and irradiated using 410-, 630-, 830-nm light emitting diode twice, with a 24-hour interval at 10 J/cm 2 . After irradiation, cells were incubated for 24 and 48 hours and real-time quantitative reverse transcription polymerase chain reaction was performed. Western blot analysis was also performed in 48 hours after last irradiation. The genes and proteins of collagen type I, TGF-β1, Smad3, and Smad7 were analyzed. We observed no statistically significant change in the viability of keloid fibroblasts after irradiation. Collagen type I was the only gene whose expression significantly decreased after irradiation at 410 nm when compared to the non-irradiated control. Western blot analysis showed that LLLT at 410 nm lowered the protein levels of collagen type I compared to the control. LLLT at 410 nm decreased the expression of collagen type I in keloid fibroblasts and might be effective in preventing keloid formation in their initial stage.

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.

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.

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

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

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.

Src Family Kinases (SFK) Mediate Angiotensin II-Induced Myosin Light Chain Phosphorylation and Hypertension.

PubMed

Qin, Bo; Zhou, Junlan

2015-01-01

Angiotensin (Ang) II is the major bioactive peptide of the renin-angiotensin system (RAS); it contributes to the pathogenesis of hypertension by inducing vascular contraction and adverse remodeling, thus elevated peripheral resistance. Ang II also activates Src family kinases (SFK) in the vascular system, which has been implicated in cell proliferation and migration. However, the role of SFK in Ang II-induced hypertension is largely unknown. In this study, we found that administration of a SFK inhibitor SU6656 markedly lowered the level of systemic BP in Ang II-treated mice, which was associated with an attenuated phosphorylation of the smooth-muscle myosin-light-chain (MLC) in the mesenteric resistant arteries. In the cultured human coronary artery smooth muscle cells (SMCs), pretreatment with SU6656 blocked Ang II-induced MLC phosphorylation and contraction. These results for the first time demonstrate that SFK directly regulate vascular contractile machinery to influence BP. Thus our study provides an additional mechanistic link between Ang II and vasoconstriction via SFK-enhanced MLC phosphorylation in SMCs, and suggests that targeted inhibition of Src may provide a new therapeutic opportunity in the treatment of hypertension.

Pre-Conditioning with Low-Level Laser (Light) Therapy: Light Before the Storm

PubMed Central

Agrawal, Tanupriya; Gupta, Gaurav K.; Rai, Vikrant; Carroll, James D.; Hamblin, Michael R.

2014-01-01

Pre-conditioning by ischemia, hyperthermia, hypothermia, hyperbaric oxygen (and numerous other modalities) is a rapidly growing area of investigation that is used in pathological conditions where tissue damage may be expected. The damage caused by surgery, heart attack, or stroke can be mitigated by pre-treating the local or distant tissue with low levels of a stress-inducing stimulus, that can induce a protective response against subsequent major damage. Low-level laser (light) therapy (LLLT) has been used for nearly 50 years to enhance tissue healing and to relieve pain, inflammation and swelling. The photons are absorbed in cytochrome(c) oxidase (unit four in the mitochondrial respiratory chain), and this enzyme activation increases electron transport, respiration, oxygen consumption and ATP production. A complex signaling cascade is initiated leading to activation of transcription factors and up- and down-regulation of numerous genes. Recently it has become apparent that LLLT can also be effective if delivered to normal cells or tissue before the actual insult or trauma, in a pre-conditioning mode. Muscles are protected, nerves feel less pain, and LLLT can protect against a subsequent heart attack. These examples point the way to wider use of LLLT as a pre-conditioning modality to prevent pain and increase healing after surgical/medical procedures and possibly to increase athletic performance. PMID:25552961

Pulsed flows, tributary inputs, and food web structure in a highly regulated river

USGS Publications Warehouse

Sabo, John; Caron, Melanie; Doucett, Richard R.; Dibble, Kimberly L.; Ruhi, Albert; Marks, Jane; Hungate, Bruce; Kennedy, Theodore A.

2018-01-01

1.Dams disrupt the river continuum, altering hydrology, biodiversity, and energy flow. Although research indicates that tributary inputs have the potential to dilute these effects, knowledge at the food web level is still scarce.2.Here we examined the riverine food web structure of the Colorado River below Glen Canyon Dam, focusing on organic matter sources, trophic diversity, and food chain length. We asked how these components respond to pulsed flows from tributaries following monsoon thunderstorms that seasonally increase streamflow in the American Southwest.3.Tributaries increased the relative importance of terrestrial organic matter, particularly during the wet season below junctures of key tributaries. This contrasted with the algal-based food web present immediately below Glen Canyon Dam.4.Tributary inputs during the monsoon also increased trophic diversity and food chain length: food chain length peaked below the confluence with the largest tributary (by discharge) in Grand Canyon, increasing by >1 trophic level over a 4-5 kilometre reach possibly due to aquatic prey being flushed into the mainstem during heavy rain events.5.Our results illustrate that large tributaries can create seasonal discontinuities, influencing riverine food web structure in terms of allochthony, food web diversity, and food chain length.6.Synthesis and applications. Pulsed flows from unregulated tributaries following seasonal monsoon rains increase the importance of terrestrially-derived organic matter in large, regulated river food webs, increasing food chain length and trophic diversity downstream of tributary inputs. Protecting unregulated tributaries within hydropower cascades may be important if we are to mitigate food web structure alteration due to flow regulation by large dams. This is critical in the light of global hydropower development, especially in megadiverse, developing countries where dam placement (including completed and planned structures) is in tributaries.

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.

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

USDA-ARS?s Scientific Manuscript database

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

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.

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.

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

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.

Method for altering antibody light chain interactions

DOEpatents

Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

2002-01-01

A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

Diagnostic and prognostic significance of neurofilament light chain NF-L, but not progranulin and S100B, in the course of amyotrophic lateral sclerosis: Data from the German MND-net.

PubMed

Steinacker, Petra; Huss, André; Mayer, Benjamin; Grehl, Torsten; Grosskreutz, Julian; Borck, Guntram; Kuhle, Jens; Lulé, Dorothée; Meyer, Thomas; Oeckl, Patrick; Petri, Susanne; Weishaupt, Jochen; Ludolph, Albert C; Otto, Markus

2017-02-01

There is a need for diagnostic, prognostic, and monitoring blood biomarkers for ALS. We aimed to analyse and compare proposed candidate markers for disease progression in the course of ALS. Blood samples were taken from 125 ALS patients, including nine patients with C9orf72 or SOD1 mutation, at regular intervals of six months. ALS patients were characterized by the ALS functional rating scale (ALSFRS-R) and the Edinburgh Cognitive and Behavioural ALS Screen (ECAS). We quantified neurofilament light chain (NF-L), S100B, and progranulin (PGRN) and analysed it in relation to disease progression. Results showed that, at baseline, serum concentrations of NF-L but not PGRN or S100B discriminated significantly between ALS and controls. Within 24 months follow-up the marker concentrations remained stable. Baseline serum NF-L levels correlated with survival time, which was confirmed in subgroups with fast, intermediate, and slow disease progression and there was a weak association with disease duration. For S100B and PGRN we found an association with ALSFRS-R score changes and a trend for decreased levels in the fast progressor subgroup. In conclusion, serum NF-L in any ALS disease stage is a promising marker to support diagnosis and predict outcome, while serum PGRN and S100B are only of minor prognostic value.

Light chains removal by extracorporeal techniques in acute kidney injury due to multiple myeloma: a position statement of the Onconephrology Work Group of the Italian Society of Nephrology.

PubMed

Fabbrini, P; Finkel, K; Gallieni, M; Capasso, G; Cavo, M; Santoro, A; Pasquali, S

2016-12-01

Acute kidney injury (AKI) is a frequent complication of multiple myeloma and is associated with increased short-term mortality. Additionally, even a single episode of AKI can eventually lead to end-stage renal disease (ESRD), significantly reducing quality of life and long-term survival. In the setting of multiple myeloma, severe AKI (requiring dialysis) is typically secondary to cast nephropathy (CN). Renal injury in CN is due to intratubular obstruction from precipitation of monoclonal serum free light chains (sFLC) as well as direct tubular toxicity of sFLC via stimulation of nuclear factor (NF)κB inflammatory pathways. Current mainstays of CN treatment are early removal of precipitating factors such as nephrotoxic drugs, acidosis and dehydration, together with rapid reduction of sFLC levels. Introduction of the proteasome inhibitor bortezomib has significantly improved the response rates in multiple myeloma due to its ability to rapidly reduce sFLC levels and has been referred to as "renoprotective" therapy. As an adjunct to chemotherapy, several new extracorporeal techniques have raised interest as a further means to reduce sFLC concentrations in the treatment of CN. Whether addition of extracorporeal therapies to renoprotective therapy can result in better renal recovery is still a matter of debate and there are currently no guidelines in this field. In this positon paper, we offer an overview of the available data and the authors' perspectives on extracorporeal treatments in CN.

The Impacts of Phosphorus Deficiency on the Photosynthetic Electron Transport Chain.

PubMed

Carstensen, Andreas; Herdean, Andrei; Schmidt, Sidsel Birkelund; Sharma, Anurag; Spetea, Cornelia; Pribil, Mathias; Husted, Søren

2018-05-01

Phosphorus (P) is an essential macronutrient, and P deficiency limits plant productivity. Recent work showed that P deficiency affects electron transport to photosystem I (PSI), but the underlying mechanisms are unknown. Here, we present a comprehensive biological model describing how P deficiency disrupts the photosynthetic machinery and the electron transport chain through a series of sequential events in barley ( Hordeum vulgare ). P deficiency reduces the orthophosphate concentration in the chloroplast stroma to levels that inhibit ATP synthase activity. Consequently, protons accumulate in the thylakoids and cause lumen acidification, which inhibits linear electron flow. Limited plastoquinol oxidation retards electron transport to the cytochrome b 6 f complex, yet the electron transfer rate of PSI is increased under steady-state growth light and is limited under high-light conditions. Under P deficiency, the enhanced electron flow through PSI increases the levels of NADPH, whereas ATP production remains restricted and, hence, reduces CO 2 fixation. In parallel, lumen acidification activates the energy-dependent quenching component of the nonphotochemical quenching mechanism and prevents the overexcitation of photosystem II and damage to the leaf tissue. Consequently, plants can be severely affected by P deficiency for weeks without displaying any visual leaf symptoms. All of the processes in the photosynthetic machinery influenced by P deficiency appear to be fully reversible and can be restored in less than 60 min after resupply of orthophosphate to the leaf tissue. © 2018 American Society of Plant Biologists. All Rights Reserved.

Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

PubMed

Mead, Emma J; Chiverton, Lesley M; Spurgeon, Sarah K; Martin, Elaine B; Montague, Gary A; Smales, C Mark; von der Haar, Tobias

2012-01-01

Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.

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.

Metal-Free Atom Transfer Radical Polymerization of Methyl Methacrylate with ppm Level of Organic Photocatalyst.

PubMed

Huang, Zhicheng; Gu, Yu; Liu, Xiaodong; Zhang, Lifen; Cheng, Zhenping; Zhu, Xiulin

2017-05-01

It is well known that the recently developed photoinduced metal-free atom transfer radical polymerization (ATRP) has been considered as a promising methodology to completely eliminate transition metal residue in polymers. However, a serious problem needs to be improved, namely, large amount of organic photocatalysts should be used to keep the controllability over molecular weights and molecular weight distributions. In this work, a novel photocatalyst 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN) with strong excited state reduction potential is successfully used to mediate a metal-free ATRP of methyl methacrylate just with parts per million (ppm) level usage under irradiation of blue light emitting diode at room temperature, using ethyl α-bromophenyl-acetate as a typical initiator with high initiator efficiency. The polymerization kinetic study, multiple controlled "on-off" light switching cycle regulation, and chain extension experiment confirm the "living"/controlled features of this promising photoinduced metal-free ATRP system with good molecular weight control in the presence of ppm level photocatalyst 4CzIPN. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Computerized temperature monitoring of the vaccine cold chain in a tropical climate, Chad].

PubMed

Schlumberger, M; Mireux, F; Tchang, S G; Mboutbogol, D; Cheikh, D O; Hissein, A A; Youssouf, B O; Brahimi, M M; Gamatié, Y

2011-06-01

Because new EPI liquid vaccines are highly sensitive to freezing and overheating, close monitoring of the cold chain is mandatory. The new Testostore 171-1 electronic thermometer (Testo) provides more reliable monitoring of cold chain temperature than freezer indicators, vaccine vial monitors and color strips that only indicate if vaccines are out-of-date. The Testo thermometer uses a probe placed in refrigeration units to periodically measure and store temperature readings. Temperature curves are displayed via a USB connection on a laptop computer running special software (Comfort software light). Testo temperature data can easily be communicated to all management levels by e-mail. The first experience using the Testo system in Africa involved regional EPI supervision in Mondou, Logone Occidental, Chad. After a preliminary mission in Chad in 2006 showed the feasibility of using this method to manage the national cold chain at all levels, a nurse was appointed as EPI supervisor and given a refresher course in Chad's capital Ndjamena in March 2009. In April-May 2009, the supervisor was sent back to the Logone Occidental Region to monitor, by himself, refrigeration units making up the regional and district cold chain for vaccine storage in five health centers (rural and urban). Temperature curve readings were performed on site in the presence of the medical staff and results were compared to those recorded twice a day on conventional temperature charts using lamellar thermometers installed in refrigerators doors. Testo curves showed that liquid vaccine storage temperatures fell below freezing too frequently and that temperatures readings of door thermometers were often inaccurate. Testo readings also detected power outages in refrigeration units used in urban settings and flame extinctions in kerosene lamp refrigerators due to refrigerator breakdown or windy weather conditions before the rainy season. The main advantage of this monitoring method is to provide reliable data as a basis not only for detection of possible freezing of liquid vaccines but also for discussion of cold chain management and improvement with medical staff.

3D printing scaffold coupled with low level light therapy for neural tissue regeneration.

PubMed

Zhu, Wei; George, Jonathan K; Sorger, Volker J; Grace Zhang, Lijie

2017-04-12

3D printing has shown promise for neural regeneration by providing customized nerve scaffolds to structurally support and bridge the defect gap as well as deliver cells or various bioactive substances. Low-level light therapy (LLLT) exhibits positive effects on rehabiliation of degenerative nerves and neural disorders. With this in mind, we postulate that 3D printed neural scaffold coupling with LLLT will generate a new strategy to repair neural degeneration. To achieve this goal, we applied red laser light to stimualte neural stem cells on 3D printed scaffolds and investigated the subsequent cell response with respect to cell proliferation and differentiation. Here we show that cell prolifeartion rate and intracellular reactive oxgen species synthesis were significantly increased after 15 s laser stimulation follwed by 1 d culture. Over culturing time of 14 d in vitro, the laser stimulation promoted neuronal differentiation of neural stem cells, while the glial differentiation was suppressed based on results of both immunocytochemistry studies and real-time quantitative reverse transcription polymerase chain reaction testing. These findings suggest that integration of 3D printing and LLLT might provide a powerful methodology for neural tissue engineering.

Comparative Confocal and Histopathological Study of Corneal Changes in Multiple Myeloma.

PubMed

Micali, Antonio; Roszkowska, Anna M; Postorino, Elisa I; Rania, Laura; Aragona, Emanuela; Wylegala, Edward; Nowinska, Anna; Ieni, Antonio; Calimeri, Sebastiano; Pisani, Antonina; Aragona, Pasquale; Puzzolo, Domenico

2017-01-01

Corneal opacities rarely occur in multiple myeloma (MM). Our study correlates the findings of in vivo confocal microscopy (IVCM), a useful diagnostic tool, with histopathological features of corneal opacities appearing in a patient with MM. Case report. A 53-year-old man developed corneal opacities in both eyes, more pronounced in the left eye. After IVCM examination, he underwent penetrating keratoplasty in the left eye, and the button was processed for light and electron microscopy and immunohistochemistry. The diagnosis of MM was made, as confirmed by the elevation of IgGk light chains. IVCM demonstrated hyperreflective areas at the epithelial level, hyperreflective keratocytes of dendritic and lamellar morphology in whole stroma, and hyperreflective endothelial cells. Histopathological examination disclosed many vacuoles in the epithelial cell cytoplasm and a homogenous granular material in the Bowman layer. In stroma, keratocytes of different shape and size, with vesicles laden with an abnormal material, were evident. In Descemet membrane, the posterior nonbanded zone had a honeycomb appearance because of the presence of many roundish spaces among wide-spaced collagen fibers. Endothelial cells demonstrated vesicles filled with a material of uneven electron density. Immunohistochemical analysis showed strong positivity for IgGk light chains in keratocytes and among stromal lamellae. This is the first study describing a correspondence between IVCM features and histopathological alterations observed in corneal opacities in MM. The results of this study improve the current understanding of the pictures obtained by IVCM studies.

Oxygen radicals shaping evolution: why fatty acid catabolism leads to peroxisomes while neurons do without it: FADH₂/NADH flux ratios determining mitochondrial radical formation were crucial for the eukaryotic invention of peroxisomes and catabolic tissue differentiation.

PubMed

Speijer, Dave

2011-02-01

Oxygen radical formation in mitochondria is a highly important, but incompletely understood, attribute of eukaryotic cells. I propose a kinetic model in which the ratio between electrons entering the respiratory chain via FADH₂ or NADH is a major determinant in radical formation. During the breakdown of glucose, this ratio is low; during fatty acid breakdown, this ratio is much higher. The longer the fatty acid, the higher the ratio and the higher the level of radical formation. This means that very long chain fatty acids should be broken down without generation of FADH₂ for mitochondria. This is accomplished in peroxisomes, thus explaining their role and evolution. The model explains many recent observations regarding radical formation by the respiratory chain. It also sheds light on the reasons for the lack of neuronal fatty acid (beta-) oxidation and for beneficial aspects of unsaturated fatty acids. Last but not least, it has very important implications for all models describing eukaryotic origins.

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.

Involvement of the chloroplast plastoquinone pool in the Mehler reaction.

PubMed

Vetoshkina, Daria V; Ivanov, Boris N; Khorobrykh, Sergey A; Proskuryakov, Ivan I; Borisova-Mubarakshina, Maria M

2017-09-01

Light-dependent oxygen reduction in the photosynthetic electron transfer chain, i.e. the Mehler reaction, has been studied using isolated pea thylakoids. The role of the plastoquinone pool in the Mehler reaction was investigated in the presence of dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-INT), the inhibitor of plastohydroquinone oxidation by cytochrome b6/f complex. Oxygen reduction rate in the presence of DNP-INT was higher than in the absence of the inhibitor in low light at pH 6.5 and 7.6, showing that the capacity of the plastoquinone pool to reduce molecular oxygen in this case exceeded that of the entire electron transfer chain. In the presence of DNP-INT, appearance of superoxide anion radicals outside thylakoid membrane represented approximately 60% of the total superoxide anion radicals produced. The remaining 40% of the produced superoxide anion radicals was suggested to be trapped by plastohydroquinone molecules within thylakoid membrane, leading to the formation of hydrogen peroxide (H 2 O 2 ). To validate the reaction of superoxide anion radical with plastohydroquinone, xanthine/xanthine oxidase system was integrated with thylakoid membrane in order to generate superoxide anion radical in close vicinity of plastohydroquinone. Addition of xanthine/xanthine oxidase to the thylakoid suspension resulted in a decrease in the reduction level of the plastoquinone pool in the light. The obtained data provide additional clarification of the aspects that the plastoquinone pool is involved in both reduction of oxygen to superoxide anion radicals and reduction of superoxide anion radicals to H 2 O 2 . Significance of the plastoquinone pool involvement in the Mehler reaction for the acclimation of plants to light conditions is discussed. © 2017 Scandinavian Plant Physiology Society.

Epigenetic Contribution of the Myosin Light Chain Kinase Gene to the Risk for Acute Respiratory Distress Syndrome

PubMed Central

Szilágyi, Keely L.; Liu, Cong; Zhang, Xu; Wang, Ting; Fortman, Jeffrey D.; Zhang, Wei; Garcia, Joe G.N.

2016-01-01

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with a considerable case fatality rate (~30-40%). Health disparities exist with African descent subjects (ADs) exhibiting greater mortality than European descent individuals (EDs). Myosin light chain kinase (MLCK) is encoded by MYLK whose genetic variants are implicated in ARDS pathogenesis and may influence ARDS mortality. As baseline population-specific epigenetic changes, i.e. cytosine modifications, have been observed between AD and ED individuals, epigenetic variations in MYLK may provide insights into ARDS disparities. We compared methylation levels of MYLK CpGs between ARDS patients and ICU controls overall and by ethnicity in a nested case control study of 39 ARDS cases and 75 non-ARDS intensive care unit controls. Two MYLK CpG sites (cg03892735, cg23344121) were differentially modified between ARDS subjects and controls (p<0.05; q<0.25) in a logistic regression model, where no effect modification from ethnicity or age was found. One CpG site was associated with ARDS in patients less than 58 years old, cg19611163 (intron 19,20). Two CpG sites were associated with ARDS in EDs only, gene body CpG (cg01894985, intron 2,3) and CpG (cg16212219, intron 31,32), with higher modification levels exhibited in ARDS subjects than controls. Cis-acting mQTL (modified cytosine quantitative trait loci) were identified using linear regression between local genetic variants and modification levels for two ARDS-associated CpGs (cg23344121, cg16212219). In summary, these ARDS-associated MYLK CpGs with effect modification by ethnicity and local mQTL, suggest that MYLK epigenetic variation and local genetic background may contribute to health disparities observed in ARDS. PMID:27543902

Influences of different dietary energy level on sheep testicular development associated with AMPK/ULK1/autophagy pathway.

PubMed

Pang, Jing; Li, Fengzhe; Feng, Xu; Yang, Hua; Han, Le; Fan, Yixuan; Nie, Haitao; Wang, Zhen; Wang, Feng; Zhang, Yanli

2018-03-01

Energy balance is an important feature for spermatozoa production in the testis. The 5'-AMP-activated protein kinase (AMPK) is a sensor of cell energy, has been implicated as a mediator between gonadal function and energy balance. Herein, we intended to determine the physiological effects of AMPK on testicular development in feed energy restricted and compensated pre-pubertal rams. Lambs had restricted feeding for 2 months and then provided compensatory feeding for another 3 months. Feed levels were 100%(control), 15% and 30% of energy restriction (ER) diets, respectively. The results showed that lambs fed the 30% ER diet had significantly lower testicular weight (P < .05) and spermatids number in the seminiferous tubules, but there were no differences between control and 15% ER groups. Meanwhile, 15% ER and 30% ER diets induced testis autophagy and apoptosis through activating AMPK-ULK1(ULK1, Unc-51 like autophagy activating kinase) signal pathway with characterization of increased Beclin-1 and Light chain 3-Ⅱ/Light chain 3-Ⅰ (LC3-II/LC3-I) ratio, up-regulated the ratio of pro-apoptotic Bcl-2-associated X protein (BAX) and anti-apoptotic B-cell lymphoma 2 (Bcl-2), as well as activated AMPK, phosphorylated AMPK(p-AMPK) and ULK1. Furthermore, a compensation of these parameters occurred when the lambs were re-fed with normal energy requirement after restriction. Taken together, dietary energy levels influence testicular development through autophagy and apoptosis interplay mediated by AMPK-ULK1 signal pathway, which also indicates the important role of the actions of AMPK in the testis homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Xiao-Yao-San, a Chinese Medicine Formula, Ameliorates Chronic Unpredictable Mild Stress Induced Polycystic Ovary in Rat

PubMed Central

Sun, Hao-Yu; Li, Quan; Liu, Yu-Ying; Wei, Xiao-Hong; Pan, Chun-Shui; Fan, Jing-Yu; Han, Jing-Yan

2017-01-01

Chronic stress induces endocrine disturbance, which contributes to the development of polycystic ovary syndrome (PCOS), a condition that remains a challenge for clinicians to cope with. The present study investigated the effect of Xiao-Yao-San (XYS), a traditional Chinese medicine formula used for treatment of gynecological disease, on the chronic stress-induced polycystic ovary and its underlying mechanism. Female Sprague-Dwaley rats underwent a 3 weeks chronic unpredictable mild stress (CUMS) procedure to establish the PCOS model, followed by 4 weeks treatment with XYS (0.505 g/kg or 1.01 g/kg) by gavage. Granulosa cells were exposed to noradrenaline (1 mM) in vitro for 24 h, followed by incubation with or without XYS-treated rat serum for 24 h. Post-treatment with XYS ameliorated CUMS-induced irregular estrous cycles and follicles development abnormalities, decrease of estradiol and progesterone level as well as increase of luteinizing hormone in serum, reduced cystic follicles formation and the apoptosis and autophagy of granulosa cells, attenuated the increase in dopamine beta hydroxylase and c-fos level in locus coeruleus, the noradrenaline level in serum and ovarian tissue, and the expression of beta 2 adrenergic receptor in ovarian tissue. Besides, XYS alleviated the reduction of phosphorylation of ribosomal protein S6 kinase polypeptide I and protein kinase B, as well as the increase of microtubule-associated protein light chain 3-I to microtubule-associated protein light chain 3-II conversion both in vivo and in vitro. This study demonstrated XYS as a potential strategy for CUMS induced polycystic ovary, and suggested that the beneficial role of XYS was correlated with the regulation of the sympathetic nerve activity. PMID:29018356

Gastroprotective effects of sulforaphane and thymoquinone against acetylsalicylic acid-induced gastric ulcer in rats.

PubMed

Zeren, Sezgin; Bayhan, Zulfu; Kocak, Fatma Emel; Kocak, Cengiz; Akcılar, Raziye; Bayat, Zeynep; Simsek, Hasan; Duzgun, Sukru Aydin

2016-06-15

Nonsteroidal anti-inflammatory drugs (NSAIDs) commonly cause gastric ulcers (GUs). We investigated the effects of sulforaphane (SF) and thymoquinone (TQ) in rats with acetylsalicylic acid (ASA)-induced GUs. Thirty-five male Wistar-Albino rats were divided into five groups: control; ASA; ASA with vehicle; ASA + SF; and ASA + TQ. Compounds were administered by oral gavage before GU induction. GUs were induced by intragastric administration of ASA. Four hours after GU induction, rats were killed and stomachs excised. Total oxidant status, total antioxidant status, total thiol, nitric oxide, asymmetric dimethylarginine, tumor necrosis factor-alpha levels, superoxide dismutase activity, and glutathione peroxidase activity in tissue were measured. Messenger RNA expression of dimethylarginine dimethylaminohydrolases, heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2, and nuclear factor kappa-light-chain-enhancer of activated B cells were analyzed. Renal tissues were evaluated by histopathologic and immunohistochemical means. SF and TQ reduced GU indices, apoptosis, total oxidant status, asymmetric dimethylarginine, and tumor necrosis factor-alpha levels, nuclear factor kappa-light-chain-enhancer of activated B cells, and inducible nitric oxide synthase expressions (P < 0.001, P = 0.001). Both examined compounds increased superoxide dismutase activity, glutathione peroxidase activity, total antioxidant status, total thiol, nitric oxide levels, endothelial nitric oxide synthase, dimethylarginine dimethylaminohydrolases, HO-1, nuclear factor erythroid 2-related factor 2, and HO-1 expressions (P < 0.001). These results suggest that pretreatment with SF or TQ can reduce ASA-induced GUs via anti-inflammatory, antioxidant, and antiapoptotic effects. These compounds may be useful therapeutic strategies to prevent the gastrointestinal adverse effects that limit nonsteroidal anti-inflammatory drugs use. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

[The prognostic value of baseline serum free light chain in cardiac amyloidosis].

PubMed

Zhao, Lei; Tian, Zhuang; Fang, Quan

2016-03-01

To analyze the prognostic value of baseline serum free light chain (sFLC) in light-chain (AL) cardiac amyloidosis. Twenty-seven patients with AL cardiac amyloidosis were retrospectively reviewed from January 2014 to January 2015. sFLC was measured by immuoturbidimetric assay. Baseline characteristics, echocardiographic parameters and electrocardiogram data were analyzed. According to the median baseline dFLC (involved sFLC minus uninvolved sFLC), patients were categorized into either the low dFLC(≤ 307 mg/L) or the high dFLC group (>307 mg/L). More subjects in the high dFLC group with early/late diastolic mitral velocity ratio (E/A ratio) over 2 (71.4% vs 30.8%, P=0.035), and subjects in this group had a shorter median survival time than those in the low dFLC group (3 months vs 17 months, P=0.004). A similar phenomenon for median survival time was observed when the subjects were redivided either by a new cut-off value of 180 mg/L for dFLC (low dFLC group: 17 months; high dFLC group: 4 months, P=0.014) or a κ/λ ratio, in which subjects with κ type sFLC-ratio ≤ 19.6 and λ type sFLC-ratio>0.065 were in the low sFLC-ratio group (17 months) and those with κ type sFLC-ratio > 19.6 and λ type sFLC-ratio ≤ 0.065 were in the high sFLC-ratio group (4 months, P=0.023). In multivariate analysis, dFLC and New York Heart Association (NYHA)classification of cardiac function were two risk factors associated with all-cause mortality in patients, among which the hazard ratio for higher dFLC was 4.28 (95%CI 1.55-11.8, P=0.005). The level of sFLC could be a marker for the prognosis of AL cardiac amyloidosis.

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.

Elevated levels of ferritin in the cerebrospinal fluid of amyotrophic lateral sclerosis patients.

PubMed

Zheng, Y; Gao, L; Wang, D; Zang, D

2017-08-01

The aim of the study was to detect changes in the levels of ferritin heavy chain (FHC), ferritin light chain (FLC), and transferrin in the cerebrospinal fluid (CSF) and serum of amyotrophic lateral sclerosis (ALS) patients and to analyze the correlations between the levels of these proteins and various clinical parameters. Cerebrospinal fluid and serum samples were obtained from 54 ALS patients and 46 non-inflammatory neurological disease control (non-INDC) patients. CSF and serum FHC, FLC, and transferring levels were measured via the enzyme-linked immunosorbent method using a commercial ELISA kit, and the times from onset (durations), ALS functional rating scale-revised (ALSFRS-r) scores, and disease progression rates (DPRs) were analyzed by registered neurologists. Statistical analysis was performed via Prism software. Compared with controls, ALS patients exhibited significantly increased FHC and FLC levels in CSF, which were positively correlated with DPR and negatively correlated with duration. Serum transferrin levels were significantly increased in ALS patients but were not correlated with disease progression. FHC and FLC in CSF rapidly increased as the disease worsened. This study demonstrated that the clinical measurement of FHC and FLC in CSF may be beneficial for disease differentiation and evaluating progression in patients with ALS. Compared with levels in serum, the levels of FHC and FLC in CSF might be more reliable for diagnosing and assessing the progression of ALS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Evidence for the Involvement of Lfc and Tctex-1 in Axon Formation

PubMed Central

Conde, Cecilia; Arias, Cristina; Robin, Maria; Li, Aiqun; Saito, Masaki; Chuang, Jen-Zen; Nairn, Angus C.; Sung, Ching-Hwa; Cáceres, Alfredo

2013-01-01

RhoA and Rac play key and opposite roles during neuronal polarization. We now show that Lfc, a guanosine nucleotide exchange factor (GEF), localizes to the Golgi apparatus and growth cones of developing neurons and negatively regulates neurite sprouting and axon formation through a Rho signaling pathway. Tctex-1, a dynein light chain implicated in axon outgrowth by modulating actin dynamics and Rac activity, colocalizes and physically interacts with Lfc, thus inhibiting its GEF activity, decreasing Rho-GTP levels, and functionally antagonizing Lfc during neurite formation. PMID:20463241

Devastating tsunami inspires efforts to reduce future tsunami destruction

USGS Publications Warehouse

Lockridge, P.A.

1987-01-01

The beacon from landmark Scotch Cap lighthouse pierced the moonless night of April 1, 1946, in Alaska's remote Aleutain Island chain. In the reinforced concrete lighthouse, five men were engaged in various support operations connected with the maintenance of the 80,000 candlepower beam. Perched atop a building constructed 5 years earlier on a bluff 32 feet above sea level, the proud new light rose a total of 92 feet above the swirling restless seas. On a cliff behind the lighthouse, a second building housed the Coast Guard radio-direction-finding station. 

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.

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.

The PedR transcriptional regulator interacts with thioredoxin to connect photosynthesis with gene expression in cyanobacteria.

PubMed

Horiuchi, Mayumi; Nakamura, Kinu; Kojima, Kouji; Nishiyama, Yoshitaka; Hatakeyama, Wakako; Hisabori, Toru; Hihara, Yukako

2010-10-01

The redox state of the photosynthetic electron transport chain acts as a critical sensing mechanism by regulating the transcription of key genes involved in the acclimation response to a change in the environment. In the present study we show that the small LuxR-type regulator PedR interacts with Trx (thioredoxin) to achieve photosynthetic electron-transport-dependent transcriptional regulation in the cyanobacterium Synechocystis sp. PCC 6803. TrxM, an isoform of Trx, was isolated as an interacting factor of PedR by pull-down assays. In vitro analysis revealed that the intermolecular disulfide bond formed between Cys80 residues of the PedR homodimer was reduced by both TrxM and TrxX. It has been shown previously that, although PedR is active under low-light conditions, it becomes transiently inactivated following a shift to high-light conditions, with a concomitant conformational change [Nakamura and Hihara (2006) J. Biol. Chem. 281, 36758-36766]. In the present study, we found that the conformational change of PedR and the change in the transcript level of its target gene were minimal when mutants of Synechocystis that lack ferredoxin-Trx reductase or NADPH-Trx reductase were exposed to high levels of light. These results indicate that the reduction of PedR by Trx causes transient inactivation of PedR upon the shift of cyanobacterial cells to high-light conditions.

Analysis of Class I Major Histocompatibility Complex Gene Transcription in Human Tumors Caused by Human Papillomavirus Infection

PubMed Central

Gameiro, Steven F.; Zhang, Ali; Ghasemi, Farhad; Barrett, John W.; Mymryk, Joe S.

2017-01-01

Oncoproteins from high-risk human papillomaviruses (HPV) downregulate the transcription of the class I major histocompatibility complex (MHC-I) antigen presentation apparatus in tissue culture model systems. This could allow infected or transformed cells to evade the adaptive immune response. Using data from over 800 human cervical and head & neck tumors from The Cancer Genome Atlas (TCGA), we determined the impact of HPV status on the mRNA expression of all six MHC-I heavy chain genes, and the β2 microglobulin light chain. Unexpectedly, these genes were all expressed at high levels in HPV positive (HPV+) cancers compared with normal control tissues. Indeed, many of these genes were expressed at significantly enhanced levels in HPV+ tumors. Similarly, the transcript levels of several other components of the MHC-I peptide-loading complex were also high in HPV+ cancers. The coordinated expression of high mRNA levels of the MHC-I antigen presentation apparatus could be a consequence of the higher intratumoral levels of interferon γ in HPV+ carcinomas, which correlate with signatures of increased infiltration by T- and NK-cells. These data, which were obtained from both cervical and oral tumors in large human cohorts, indicates that HPV oncoproteins do not efficiently suppress the transcription of the antigen presentation apparatus in human tumors. PMID:28891951

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.

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

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

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

Evidence for Ig Light Chain Isotype Exclusion in Shark B Lymphocytes Suggests Ordered Mechanisms.

PubMed

Iacoangeli, Anna; Lui, Anita; Haines, Ashley; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

2017-09-01

Unlike most vertebrates, the shark IgL gene organization precludes secondary rearrangements that delete self-reactive VJ rearranged genes. Nurse sharks express four L chain isotypes, κ, λ, σ, and σ-2, encoded by 35 functional minigenes or clusters. The sequence of gene activation/expression and receptor editing of these isotypes have not been studied. We therefore investigated the extent of isotypic exclusion in separated B cell subpopulations. Surface Ig (sIg)κ-expressing cells, isolated with mAb LK14 that recognizes Cκ, carry predominantly nonproductive rearrangements of other L chain isotypes. Conversely, after depletion with LK14, sIgM + cells contained largely nonproductive κ and enrichment for in-frame VJ of the others. Because some isotypic inclusion was observed at the mRNA level, expression in the BCR was examined. Functional λ mRNA was obtained, as expected, from the LK14-depleted population, but was also in sIgκ + splenocytes. Whereas λ somatic mutants from the depleted sample displayed evidence of positive selection, the λ genes in sIgκ + cells accumulated bystander mutations indicating a failure to express their products at the cell surface in association with the BCR H chain. In conclusion, a shark B cell expresses one L chain isotype at the surface and other isotypes as nonproductive VJ, sterile transcripts, or in-frame VJ whose products may not associate with the H chain. Based on the mRNA content found in the B cell subpopulations, an order of L chain gene activation is suggested as: σ-2 followed by κ, then σ and λ. Copyright © 2017 by The American Association of Immunologists, Inc.

Ablation of ceramide synthase 2 exacerbates dextran sodium sulphate-induced colitis in mice due to increased intestinal permeability.

PubMed

Kim, Ye-Ryung; Volpert, Giora; Shin, Kyong-Oh; Kim, So-Yeon; Shin, Sun-Hye; Lee, Younghay; Sung, Sun Hee; Lee, Yong-Moon; Ahn, Jung-Hyuck; Pewzner-Jung, Yael; Park, Woo-Jae; Futerman, Anthony H; Park, Joo-Won

2017-12-01

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long-chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very-long acyl chain ceramides with concomitant increase of long chain bases and C16-ceramides, were more susceptible to dextran sodium sulphate-induced colitis, and their survival rate was markedly decreased compared with that of wild-type littermates. Using mixed bone-marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule-A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco-2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2-knockdown via CRISPR-Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long-chain bases and C16-ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC-dextran levels, indicating that altered SLs including deficiency of very-long-chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Redox proteomic analysis of serum from aortic anerurysm patients: insights on oxidation of specific protein target.

PubMed

Spadaccio, Cristiano; Coccia, Raffaella; Perluigi, Marzia; Pupo, Gilda; Schininà, Maria Eugenia; Giorgi, Alessandra; Blarzino, Carla; Nappi, Francesco; Sutherland, Fraser W; Chello, Massimo; Di Domenico, Fabio

2016-06-21

oxidative stress is undoubtedly one of the main players in abdominal aortic aneurysm (AAA) pathophysiology. Recent studies in AAA patients reported an increase in the indices of oxidative damage at the tissue level and in biological fluids coupled with the loss of counter-regulatory mechanisms of protection from oxidative stress. We recently reported, in a proteomic analysis of AAA patient sera, changes in the expression of several proteins exerting important modulatory activities on cellular proliferation, differentiation and response to damage. This study aimed to explore the involvement of protein oxidation, at peripheral levels, in AAA. a redox proteomic approach was used to investigate total and specific protein carbonylation and protein-bound 4-hydroxy-2-nonenal (HNE) in the serum of AAA patients compared with age-matched controls. our results show increased oxidative damage to protein as indexed by the total carbonyl levels and total protein-bound HNE. By redox proteomics we identified specific carbonylation of three serum proteins: serum retinol-binding protein, vitamin D-binding protein and fibrinogen α-chain HNE. We also identified increased protein-bound HNE levels for hemopexin, IgK chain C region and IgK chain V-III region SIE. In addition we found a high correlation between specific protein carbonylation and protein-bound HNE and the aortic diameter. Moreover the analysis of serum proteins with antioxidant activity demonstrates the oxidation of albumin together with the overexpression of transferrin, haptoglobin and HSPs 90, 70, 60 and 32. this study support the involvement of oxidative stress in the pathogenesis of AAA and might provide a further degree of knowledge in the cause-effect role of oxidative stress shedding new light on the molecular candidates involved in the disease.

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.

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.

A novel option for reducing free light chains in myeloma kidney: supra-hemodiafiltration with endogenous reinfusion (HFR).

PubMed

Pasquali, Sonia; Iannuzzella, Francesco; Corradini, Mattia; Mattei, Silvia; Bovino, Achiropita; Stefani, Alfredo; Palladino, Giuseppe; Caiazzo, Marialuisa

2015-04-01

In myeloma cast nephropathy, fast reduction of serum free light chain (FLC) levels correlates with renal recovery. Recently, extracorporeal treatments using filters with a high-molecular weight cut-off have been successfully used for FLC removal. However, using these new filters, high cost and elevated albumin leakage are common drawbacks. We studied a new and cheaper therapeutic approach with adsorbent resins to evaluate its efficacy. We treated four patients, affected by dialysis-dependent acute kidney injury (AKI) due to biopsy proven de novo FLC myeloma cast nephropathy. Each patient underwent bortezomib chemotherapy and extracorporeal treatment with the supra-hemodiafiltration with endogenous reinfusion (HFR) technique (Supra-HFR, Bellco Mirandola, Modena, Italy). Supra-HFR is a kind of hemodiafiltration that utilizes separated convection, diffusion and adsorption. The sorbent cartridge has a high affinity for FLC (both κ and λ) but is able to re-infuse albumin, avoiding the need for albumin perfusions. Supra HFR treatments (4 h each) were carried out for eight consecutive days and then every other day. All patients showed a significant reduction of serum FLC, whereas serum albumin concentration remained unchanged. Renal function recovered in three out of four patients. FLC removal with adsorbent resins represents an effective therapeutic strategy that does not require replacement with albumin .

[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.

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

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.

Neurofilament light chain level is a weak risk factor for the development of MS

PubMed Central

Arrambide, Georgina; Eixarch, Herena; Villar, Luisa M.; Alvarez-Cermeño, José C.; Picón, Carmen; Kuhle, Jens; Disanto, Giulio; Kappos, Ludwig; Sastre-Garriga, Jaume; Pareto, Deborah; Simon, Eva; Comabella, Manuel; Río, Jordi; Nos, Carlos; Tur, Carmen; Castilló, Joaquín; Vidal-Jordana, Angela; Galán, Ingrid; Arévalo, Maria J.; Auger, Cristina; Rovira, Alex; Montalban, Xavier

2016-01-01

Objective: To determine the prognostic value of selected biomarkers in clinically isolated syndromes (CIS) for conversion to multiple sclerosis (MS) and disability accrual. Methods: Data were acquired from 2 CIS cohorts. The screening phase evaluated patients developing clinically definite MS (CIS-CDMS) and patients who remained as CIS during a 2-year minimum follow-up (CIS-CIS). We determined levels of neurofascin, semaphorin 3A, fetuin A, glial fibrillary acidic protein, and neurofilament light (NfL) and heavy chains in CSF (estimated mean [95% confidence interval; CI]). We evaluated associations between biomarker levels, conversion, disability, and magnetic resonance parameters. In the replication phase, we determined NfL levels (n = 155) using a 900 ng/L cutoff. Primary endpoints in uni- and multivariate analyses were CDMS and 2010 McDonald MS. Results: The only biomarker showing significant differences in the screening was NfL (CIS-CDMS 1,553.1 [1,208.7–1,897.5] ng/L and CIS-CIS 499.0 [168.8–829.2] ng/L, p < 0.0001). The strongest associations were with brain parenchymal fraction change (rs = −0.892) and percentage brain volume change (rs = −0.842) at 5 years. NfL did not correlate with disability. In the replication phase, more NfL-positive patients, according to the cutoff, evolved to MS. Every 100-ng/L increase in NfL predicted CDMS (hazard ratio [HR] = 1.009, 95% CI 1.005–1.014) and McDonald MS (HR = 1.009, 95% CI 1.005–1.013), remaining significant for CDMS in the multivariate analysis (adjusted HR = 1.005, 95% CI 1.000–1.011). This risk was lower than the presence of oligoclonal bands or T2 lesions. Conclusions: NfL is a weak independent risk factor for MS. Its role as an axonal damage biomarker may be more relevant as suggested by its association with medium-term brain volume changes. PMID:27521440

Serum free light chains, interferon-alpha, and interleukins in systemic lupus erythematosus.

PubMed

Jolly, M; Francis, S; Aggarwal, R; Mikolaitis, R A; Niewold, T B; Chubinskaya, S; Block, J A; Scanzello, C; Sequeira, W

2014-08-01

Interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-alpha (IFN-α), and free light chains (FLCs: lambda, kappa) have all been noted to be of importance in systemic lupus erythematosus (SLE). Herein, we quantified and explored the relationship between these inflammatory mediators and disease activity in SLE; and stratified by their current anti-dsDNA antibody status. Seventy-seven SLE patients underwent assessment of disease activity using the SLE disease activity index (SLEDAI). Serum FLC (lambda, kappa, and total), IL-6, IL-10, and IFN-α were quantified. Demographics of disease characteristics were determined by chart reviews. Statistical analyses included Mann-Whitney test, chi square, and linear regression analyses. Mean (SD) age of the patients was 44.9 ± 12.7 years; SLEDAI (mean ± SD) was 3.4 ± 4.0. Serum lambda FLC levels had a moderate correlation (r = 0.46 with physician global assessment, 0.44 with SLEDAI) and the strongest correlation with disease activity as compared with other inflammatory mediators including current dsDNA antibody status. After adjusting for prednisone use, the correlation of lambda FLC with PGA (r = 0.48) and SLEDAI (r = 0.52) was better than of current dsDNA antibody status with PGA (r = 0.33) and adjusted SLEDAI (r = 0.24), respectively. IL-10 and IFN-α activity did not correlate with disease activity. Serum FLC and IL-6 levels could differentiate between active and inactive SLE patients. Serum lambda FLC and IL-6 levels differed significantly among patients with and without current dsDNA antibodies. Serum lambda FLC levels accounted for 31% of variance in SLEDAI scores. Serum FLC and IL-6 are potentially useful biomarkers of disease activity in SLE. Further studies, with larger study sample and longitudinal design, are indicated. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

HIV induces production of IL-18 from intestinal epithelial cells that increases intestinal permeability and microbial translocation

PubMed Central

Allam, Ossama; Samarani, Suzanne; Mehraj, Vikram; Jenabian, Mohammad-Ali; Tremblay, Cecile; Routy, Jean-Pierre; Amre, Devendra

2018-01-01

Interleukin-18 (IL-18) is a pleiotropic cytokine of the IL-1 family with multiple context dependent functions. We and others have shown that HIV infection is accompanied by increased circulating levels of IL-18 along with decreased levels of its antagonist, Interleukin-18 Binding Protein (IL-18BP). The infection is also accompanied by intestinal inflammation and decreased intestinal integrity as measured by intestinal permeability, regeneration and repair. However, little is known concerning the relation between high level of IL-18 associated with the viral infection and intestinal permeability. Here we demonstrate that HIV treatment increases production of IL-18 and decreases that of IL-18BP production in human intestinal epithelial cell (IEC) lines. IL-18 causes apoptosis of the IEC by activating caspase-1 and caspase-3. It induces epithelial barrier hyperpermeability by decreasing and disrupting both tight and adherens junction proteins, occludin, claudin 2 and beta-catenin. Disorganization of F-actin was also observed in the IEC that were exposed to the cytokine. Moreover IL-18 decreases transepithelial electrical resistance (TEER) in Caco-2 and increases permeability in HT29 monolayers. The cells’ treatment with IL-18 causes an increase in the expression of phosphorylated myosin II regulatory light-chain (p-MLC) and myosin light-chain kinase (MLCK), and a decrease in phosphorylated Signal Transducer and Activator of Transcription (p-STAT)-5. This increase in p-MLC is suppressed by a Rho-kinase (ROCK)-specific inhibitor. Interestingly, the levels of the cytokine correlate with those of LPS in the circulation in three different categories of HIV infected patients (HAART-naïve and HAART-treated HIV-infected individuals, and Elite controls) as well as in healthy controls. Collectively, these results suggest that the HIV-induced IL-18 plays a role in increased intestinal permeability and microbial translocation observed in HIV-infected individuals. PMID:29601578

Hemodiafiltration improves free light chain removal and normalizes κ/λ ratio in hemodialysis patients.

PubMed

Bourguignon, Chloé; Chenine, Leïla; Bargnoux, Anne Sophie; Leray-Moragues, Hélène; Canaud, Bernard; Cristol, Jean-Paul; Morena, Marion

2016-04-01

Serum free light chain (FLC) levels are correlated with chronic kidney disease (CKD) stages and are highest in patients on hemodialysis (HD). Aim of this study was to assess the FLC removal efficiency of Elisio™-210H dialyzer using either high-flux HD or on line high efficiency hemodiafiltration (HDF) modalities in CKD-5D patients. In this prospective and comparative study, 20 CKD-5D patients free from multiple myeloma were randomized in two groups: HD versus on line HDF. All patients were dialyzed with Elisio™-210H dialyzer. Serum samples were collected before and after the midweek dialysis session, before randomization and at the end of the study to measure κ and λ FLC concentrations. Reduction ratios were corrected for net ultrafiltration. For both HD and HDF mode, κ and λ FLC concentrations were significantly lower after dialysis than before but median reductions in κ and λ FLC levels were significantly higher in HDF versus HD groups (κ 73.5 vs. 65.5 %, p = 0.04 and λ 51.0 vs. 36.6 %, p = 0.07). After dialysis, all κ/λ ratio values were between 0.26 and 1.65 which is the reference range described in subjects with normal kidney function, for both HD and HDF groups (median κ/λ ratios were 0.80 [0.47-1.22] and 0.67 [0.50-0.79] respectively). This study shows the superiority of on line HDF compared with HD to remove both κ and λ FLC. Moreover, all post-dialysis κ/λ ratios reached normal reference range.

Forskolin induces myosin light chain dephosphorylation in bovine trabecular meshwork cells.

PubMed

Ramachandran, Charanya; Satpathy, Minati; Mehta, Dolly; Srinivas, Sangly P

2008-02-01

Enhanced contractility of the actin cytoskeleton in trabecular meshwork (TM) cells is implicated in increased resistance to aqueous humor outflow. In this study, we have investigated effects of forskolin, which is known to elevate cAMP and also enhance aqueous humor outflow, on myosin light chain (MLC) phosphorylation, a biochemical marker of actin contractility. Experiments were performed using cultured bovine TM cells. Phosphorylated MLC (pMLC), expressed as the % of untreated cells, was assessed by urea-glycerol gel electrophoresis and Western blotting. RhoA activity was determined by affinity precipitation of RhoA-GTP to RhoA binding domain of an effector of RhoA. Intracellular cAMP levels were measured by ELISA. Exposure to LPA (lysophosphatidic acid) led to increased MLC phosphorylation (LPA: pMLC=133%) and activation of RhoA. These responses of LPA were suppressed by co-treatment with forskolin (LPA+forskolin: pMLC=88%). Similarly, ET-1 and nocodazole-induced MLC phosphorylation (ET-1: pMLC=145%; nocodazole: pMLC=145%) as well as RhoA activation were suppressed by co-treatment with forskolin (ET-1+forskolin: pMLC=99%; nocodazole+forskolin: pMLC=107%). Exposure to forskolin alone led to MLC dephosphorylation (pMLC=68%). Forskolin alone led to a 4-fold increase in cAMP levels. This increase was not affected when co-treated with LPA or ET-1. Forskolin prevents MLC phosphorylation induced by LPA, ET-1, and nocodazole through inhibition of RhoA-Rho kinase axis. MLC dephosphorylation and consequent relaxation of actin cytoskeleton in TM cells presumably underlies the increased outflow facility reported in response to forskolin.

Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury

PubMed Central

Zahs, Anita; Bird, Melanie D.; Ramirez, Luis; Turner, Jerrold R.; Choudhry, Mashkoor A.

2012-01-01

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1β and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury. PMID:22790598

Inverse correlation between microtubule-associated protein 1A/1B-light chain 3 and p62/sequestosome-1 expression in the progression of cutaneous squamous cell carcinoma.

PubMed

Yoshihara, Nagisa; Takagi, Atsushi; Ueno, Takashi; Ikeda, Shigaku

2014-04-01

The expression of autophagy-related markers has occasionally been reported to correlate with the clinical stage of disease in patients with solid cancer, indicating autophagy activation. However, there have been no such reports for cutaneous squamous cell carcinoma. In this study, we investigated the expression levels of two autophagy-related markers, microtubule-associated protein IA/IB light chain 3 (LC3) and p62/sequestosome-1 (p62), in cutaneous squamous cell carcinoma specimens and assessed their correlation to clinicopathological factors in patients with this type of cancer. As a marker of the autophagosome, LC3 expression increases with autophagosome formation/accumulation, whereas p62 expression decreases due to selective degradation via autophagy. We performed immunostaining for LC3 and p62 in 50 cutaneous squamous cell carcinoma specimens obtained from patients treated by surgical resection, counted the number of cells that showed positive staining, and calculated the percentage of positive cells per low-power microscopic field. We next investigated the correlations between the expression levels of these markers and various clinicopathological factors. The results indicated that LC3 expression increased significantly with advanced clinical stage (P < 0.001) and increased tumor diameter (P = 0.046). By contrast, the expression of p62 decreased significantly with advanced clinical stage (P < 0.001) and increased tumor diameter (P = 0.001). These results suggest that autophagy becomes activated during disease progression in patients with cutaneous squamous cell carcinoma. © 2014 Japanese Dermatological Association.

A post-transcriptional compensatory pathway in heterozygous ventricular myosin light chain 2-deficient mice results in lack of gene dosage effect during normal cardiac growth or hypertrophy.

PubMed

Minamisawa, S; Gu, Y; Ross, J; Chien, K R; Chen, J

1999-04-09

Our previous study of homozygous mutants of the ventricular specific isoform of myosin light chain 2 (mlc-2v) demonstrated that mlc-2v plays an essential role in murine heart development (Chen, J., Kubalak, S. W., Minamisawa, S., Price, R. L., Becker, K. D., Hickey, R., Ross, J., Jr., and Chien, K. R. (1998) J. Biol. Chem. 273, 1252-1256). As gene dosage of some myofibrillar proteins can affect muscle function, we have analyzed heterozygous mutants in depth. Ventricles of heterozygous mutants displayed a 50% reduction in mlc-2v mRNA, yet expressed normal levels of protein both under basal conditions and following induction of cardiac hypertrophy by aortic constriction. Heterozygous mutants exhibited cardiac function comparable to that of wild-type littermate controls both prior to and following aortic constriction. There were no significant differences in contractility and responses to calcium between wild-type and heterozygous unloaded cardiomyocytes. We conclude that heterozygous mutants show neither a molecular nor a physiological cardiac phenotype either at base line or following hypertrophic stimuli. These results suggest that post-transcriptional compensatory mechanisms play a major role in maintaining the level of MLC-2v protein in murine hearts. In addition, as our mlc-2v knockout mutants were created by a knock-in of Cre recombinase into the endogenous mlc-2v locus, this study demonstrates that heterozygous mlc-2v cre knock-in mice are appropriate for ventricular specific gene targeting.

Propofol Inhibits HeLa Cells by Impairing Autophagic Flux via AMP-Activated Protein Kinase (AMPK) Activation and Endoplasmic Reticulum Stress Regulated by Calcium.

PubMed

Chen, Xi; Li, Kai; Zhao, Guoqing

2018-04-18

BACKGROUND Propofol has antitumor effects against various cancers. However, the mechanism of action of propofol in HeLa human cervical cancer cells has not been elucidated. MATERIAL AND METHODS We treated HeLa human cervical cancer cells with different concentrations of propofol. Cell viability was evaluated with Cell Counting Kit-8 and apoptosis was analyzed by annexin V-fluorescein isothiocyanate and propidium iodide staining and flow cytometry. Autophagosome formation was evaluated based on microtubule-associated protein light chain (LC)3 conversion and light chain 3 puncta formation. Autophagosome clearance was assessed according to p62 protein level and autolysosome generation. RESULTS We found that propofol decreased cell viability and increased autophagosome generation in HeLa cells. Autophagosome formation was evaluated based on LC3 conversion and LC3 puncta formation. Autophagosome clearance was assessed according to p62 protein level. The AMPK/mTOR signaling pathway was found to be activated in propofol-induced autophagosome accumulation. Fluorescence analysis using LysoTracker dye revealed that propofol blocked autophagosome-lysosome fusion. Administration of rapamycin increased autophagosome clearance in propofol-treated HeLa cells. Additionally, propofol induced endoplasmic reticulum (ER) stress and disrupted intracellular Ca2+ balance, thereby enhancing autophagosome accumulation. Suppressing ER stress by treatment with tauroursodeoxycholic acid (TUDCA) enhanced these effects, suggesting that the cytotoxicity of propofol is related to induction of ER stress. CONCLUSIONS This study is the first to provide evidence that propofol-mediated autophagy regulation is an underlying part of the mechanism by which propofol regulates HeLa cells progression.

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.

The electrical response of turtle cones to flashes and steps of light.

PubMed

Baylor, D A; Hodgkin, A L; Lamb, T D

1974-11-01

1. The linear response of turtle cones to weak flashes or steps of light was usually well fitted by equations based on a chain of six or seven reactions with time constants varying over about a 6-fold range.2. The temperature coefficient (Q(10)) of the reciprocal of the time to peak of the response to a flash was 1.8 (15-25 degrees C), corresponding to an activation energy of 10 kcal/mole.3. Electrical measurements with one internal electrode and a balancing circuit gave the following results on red-sensitive cones of high resistance: resistance across cell surface in dark 50-170 MOmega; time constant in dark 4-6.5 msec. The effect of a bright light was to increase the resistance and time constant by 10-30%.4. If the cell time constant, resting potential and maximum hyperpolarization are known, the fraction of ionic channels blocked by light at any instant can be calculated from the hyperpolarization and its rate of change. At times less than 50 msec the shape of this relation is consistent with the idea that the concentration of a blocking molecule which varies linearly with light intensity is in equilibrium with the fraction of ionic channels blocked.5. The rising phase of the response to flashes and steps of light covering a 10(5)-fold range of intensities is well fitted by a theory in which the essential assumptions are that (i) light starts a linear chain of reactions leading to the production of a substance which blocks ionic channels in the outer segment, (ii) an equilibrium between the blocking molecules and unblocked channels is established rapidly, and (iii) the electrical properties of the cell can be represented by a simple circuit with a time constant in the dark of about 6 msec.6. Deviations from the simple theory which occur after 50 msec are attributed partly to a time-dependent desensitization mechanism and partly to a change in saturation potential resulting from a voltage-dependent change in conductance.7. The existence of several components in the relaxation of the potential to its resting level can be explained by supposing that the ;substance' which blocks light sensitive ionic channels is inactivated in a series of steps.

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.

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.

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

Effects of light, food availability and temperature stress on the function of photosystem II and photosystem I of coral symbionts.

PubMed

Hoogenboom, Mia O; Campbell, Douglas A; Beraud, Eric; Dezeeuw, Katrina; Ferrier-Pagès, Christine

2012-01-01

Reef corals are heterotrophic coelenterates that achieve high productivity through their photosynthetic dinoflagellate symbionts. Excessive seawater temperature destabilises this symbiosis and causes corals to "bleach," lowering their photosynthetic capacity. Bleaching poses a serious threat to the persistence of coral reefs on a global scale. Despite expanding research on the causes of bleaching, the mechanisms remain a subject of debate. This study determined how light and food availability modulate the effects of temperature stress on photosynthesis in two reef coral species. We quantified the activities of Photosystem II, Photosystem I and whole chain electron transport under combinations of normal and stressful growth temperatures, moderate and high light levels and the presence or absence of feeding of the coral hosts. Our results show that PS1 function is comparatively robust against temperature stress in both species, whereas PS2 and whole chain electron transport are susceptible to temperature stress. In the symbiotic dinoflagellates of Stylophora pistillata the contents of chlorophyll and major photosynthetic complexes were primarily affected by food availability. In Turbinaria reniformis growth temperature was the dominant influence on the contents of the photosynthetic complexes. In both species feeding the host significantly protected photosynthetic function from high temperature stress. Our findings support the photoinhibition model of coral bleaching and demonstrate that PS1 is not a major site for thermal damage during bleaching events. Feeding mitigates bleaching in two scleractinian corals, so that reef responses to temperature stresses will likely be influenced by the coinciding availabilities of prey for the host.

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.

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.

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.

Neurofilament light antibodies in serum reflect response to natalizumab treatment in multiple sclerosis.

PubMed

Amor, Sandra; van der Star, Baukje J; Bosca, Isabel; Raffel, Joel; Gnanapavan, Sharmilee; Watchorn, Jonathan; Kuhle, Jens; Giovannoni, Gavin; Baker, David; Malaspina, Andrea; Puentes, Fabiola

2014-09-01

Increased levels of antibodies to neurofilament light protein (NF-L) in biological fluids have been found to reflect neuroinflammatory responses and neurodegeneration in multiple sclerosis (MS). To evaluate whether levels of serum antibodies against NF-L correlate with clinical variants and treatment response in MS. The autoantibody reactivity to NF-L protein was tested in serum samples from patients with relapsing-remitting MS (RRMS) (n=22) and secondary progressive MS (SPMS) (n=26). Two other cohorts of RRMS patients under treatment with natalizumab were analysed cross-sectionally (n=16) and longitudinally (n=24). The follow-up samples were taken at 6, 12, 18 and 24 months after treatment, and the NF-L antibody levels were compared against baseline levels. NF-L antibodies were higher in MS clinical groups than healthy controls and in RRMS compared to SPMS patients (p<0.001). NF-L antibody levels were lower in natalizumab treated than in untreated patients (p<0.001). In the longitudinal series, NF-L antibody levels decreased over time and a significant difference was found following 24 months of treatment compared with baseline measurements (p=0.001). Drug efficacy in MS treatment indicates the potential use of monitoring the content of antibodies against the NF-L chain as a predictive biomarker of treatment response in MS. © The Author(s) 2014.

Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

PubMed Central

Rompolas, Panteleimon; Patel-King, Ramila S.; King, Stephen M.

2012-01-01

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a “high-load environment,” we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters. PMID:22855525

Interfacial welding of dynamic covalent network polymers

NASA Astrophysics Data System (ADS)

Yu, Kai; Shi, Qian; Li, Hao; Jabour, John; Yang, Hua; Dunn, Martin L.; Wang, Tiejun; Qi, H. Jerry

2016-09-01

Dynamic covalent network (or covalent adaptable network) polymers can rearrange their macromolecular chain network by bond exchange reactions (BERs) where an active unit replaces a unit in an existing bond to form a new bond. Such macromolecular events, when they occur in large amounts, can attribute to unusual properties that are not seen in conventional covalent network polymers, such as shape reforming and surface welding; the latter further enables the important attributes of material malleability and powder-based reprocessing. In this paper, a multiscale modeling framework is developed to study the surface welding of thermally induced dynamic covalent network polymers. At the macromolecular network level, a lattice model is developed to describe the chain density evolution across the interface and its connection to bulk stress relaxation due to BERs. The chain density evolution rule is then fed into a continuum level interfacial model that takes into account surface roughness and applied pressure to predict the effective elastic modulus and interfacial fracture energy of welded polymers. The model yields particularly accessible results where the moduli and interfacial strength of the welded samples as a function of temperature and pressure can be predicted with four parameters, three of which can be measured directly. The model identifies the dependency of surface welding efficiency on the applied thermal and mechanical fields: the pressure will affect the real contact area under the consideration of surface roughness of dynamic covalent network polymers; the chain density increment on the real contact area of interface is only dependent on the welding time and temperature. The modeling approach shows good agreement with experiments and can be extended to other types of dynamic covalent network polymers using different stimuli for BERs, such as light and moisture etc.

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

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

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

1990-10-01

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

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

PubMed Central

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

2012-01-01

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

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

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.

Photon transport in a dissipative chain of nonlinear cavities

NASA Astrophysics Data System (ADS)

Biella, Alberto; Mazza, Leonardo; Carusotto, Iacopo; Rossini, Davide; Fazio, Rosario

2015-05-01

By means of numerical simulations and the input-output formalism, we study photon transport through a chain of coupled nonlinear optical cavities subject to uniform dissipation. Photons are injected from one end of the chain by means of a coherent source. The propagation through the array of cavities is sensitive to the interplay between the photon hopping strength and the local nonlinearity in each cavity. We characterize photon transport by studying the populations and the photon correlations as a function of the cavity position. When complemented with input-output theory, these quantities provide direct information about photon transmission through the system. The position of single-photon and multiphoton resonances directly reflects the structure of the many-body energy levels. This shows how a study of transport along a coupled cavity array can provide rich information about the strongly correlated (many-body) states of light even in presence of dissipation. The numerical algorithm we use, based on the time-evolving block decimation scheme adapted to mixed states, allows us to simulate large arrays (up to 60 cavities). The scaling of photon transmission with the number of cavities does depend on the structure of the many-body photon states inside the array.

Structural Determination of the Broadly Reactive Anti-IGHV1-69 Anti-idiotypic Antibody G6 and Its Idiotope.

PubMed

Avnir, Yuval; Prachanronarong, Kristina L; Zhang, Zhen; Hou, Shurong; Peterson, Eric C; Sui, Jianhua; Zayed, Hatem; Kurella, Vinodh B; McGuire, Andrew T; Stamatatos, Leonidas; Hilbert, Brendan J; Bohn, Markus-Frederik; Kowalik, Timothy F; Jensen, Jeffrey D; Finberg, Robert W; Wang, Jennifer P; Goodall, Margaret; Jefferis, Roy; Zhu, Quan; Kurt Yilmaz, Nese; Schiffer, Celia A; Marasco, Wayne A

2017-12-12

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Protective Effect of Hericium erinaceus on Alcohol Induced Hepatotoxicity in Mice

PubMed Central

Hao, Lijun; Xie, Yuxi; Wu, Guikai; Cheng, Aibin; Liu, Xiaogang; Zheng, Rongjuan; Huo, Hong; Zhang, Junwei

2015-01-01

We investigated the effects of Hericium erinaceus (HEM) on liver injury induced by acute alcohol administration in mice. Mice received ethanol (5 g/kg BW) by gavage every 12 hrs for a total of 3 doses. HEM (200 mg/kg BW) was gavage before ethanol administration. Subsequent serum alanine aminotransferase (ALT) level, aspartate aminotransaminase (AST) level, Maleic dialdehyde (MDA) level, hepatic total antioxidant status (TAOS), and activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were determined by ELISA and immunohistochemistry, respectively. HEM administration markedly (P < 0.05) decreased serum ALT, AST, and MDA levels. The hepatic histopathological observations showed that HEM had a relatively significant role in mice model, which had alcoholic liver damage. In conclusion, we observed that HEM (200 mg/kg BW) supplementation could restrain the hepatic damage caused by acute alcohol exposure. PMID:25960751

Protective Effect of Hericium erinaceus on Alcohol Induced Hepatotoxicity in Mice.

PubMed

Hao, Lijun; Xie, Yuxi; Wu, Guikai; Cheng, Aibin; Liu, Xiaogang; Zheng, Rongjuan; Huo, Hong; Zhang, Junwei

2015-01-01

We investigated the effects of Hericium erinaceus (HEM) on liver injury induced by acute alcohol administration in mice. Mice received ethanol (5 g/kg BW) by gavage every 12 hrs for a total of 3 doses. HEM (200 mg/kg BW) was gavage before ethanol administration. Subsequent serum alanine aminotransferase (ALT) level, aspartate aminotransaminase (AST) level, Maleic dialdehyde (MDA) level, hepatic total antioxidant status (TAOS), and activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were determined by ELISA and immunohistochemistry, respectively. HEM administration markedly (P < 0.05) decreased serum ALT, AST, and MDA levels. The hepatic histopathological observations showed that HEM had a relatively significant role in mice model, which had alcoholic liver damage. In conclusion, we observed that HEM (200 mg/kg BW) supplementation could restrain the hepatic damage caused by acute alcohol exposure.

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age.

PubMed

Gannon, Joan; Doran, Philip; Kirwan, Anne; Ohlendieck, Kay

2009-11-01

The age-dependent decline in skeletal muscle mass and function is believed to be due to a multi-factorial pathology and represents a major factor that blocks healthy aging by increasing physical disability, frailty and loss of independence in the elderly. This study has focused on the comparative proteomic analysis of contractile elements and revealed that the most striking age-related changes seem to occur in the protein family representing myosin light chains (MLCs). Comparative screening of total muscle extracts suggests a fast-to-slow transition in the aged MLC population. The mass spectrometric analysis of the myofibril-enriched fraction identified the MLC2 isoform of the slow-type MLC as the contractile protein with the most drastically changed expression during aging. Immunoblotting confirmed an increased abundance of slow MLC2, concomitant with a switch in fast versus slow myosin heavy chains. Staining of two-dimensional gels of crude extracts with the phospho-specific fluorescent dye ProQ-Diamond identified the increased MLC2 spot as a muscle protein with a drastically enhanced phosphorylation level in aged fibres. Comparative immunofluorescence microscopy, using antibodies to fast and slow myosin isoforms, confirmed a fast-to-slow transformation process during muscle aging. Interestingly, the dramatic increase in slow MLC2 expression was restricted to individual senescent fibres. These findings agree with the idea that aged skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower-twitching fibre population and suggest the slow MLC2 isoform as a potential biomarker for fibre type shifting in sarcopenia of old age.

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.

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

PubMed Central

Bhoo, Seong Hee; Lai, Huafang; Ma, Julian; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.

2014-01-01

Summary Filoviruses (Ebola and Marburg viruses) cause severe and often fatal hemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identify Ebola and Marburg viruses as “category A” pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of Nicotiana benthamiana produced assembled immunoglobulin, which was purified by ammonium sulfate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine. PMID:21281425

Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness.

PubMed

Gil, Fulvio R; Zitouni, Nedjma B; Azoulay, Eric; Maghni, Karim; Lauzon, Anne-Marie

2006-11-01

Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana.

PubMed

Phoolcharoen, Waranyoo; Bhoo, Seong H; Lai, Huafang; Ma, Julian; Arntzen, Charles J; Chen, Qiang; Mason, Hugh S

2011-09-01

Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as 'category A' pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size-exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

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

Hua, Tian; Wu, Dong; Ding, Wei

2012-10-15

Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCRmore » refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the Km values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.« less

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

PubMed Central

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-01-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression. PMID:22434878

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

PubMed

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-07-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

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.

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.

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

Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia

PubMed Central

Ghiotto, Fabio; Fais, Franco; Valetto, Angelo; Albesiano, Emilia; Hashimoto, Shiori; Dono, Mariella; Ikematsu, Hideyuki; Allen, Steven L.; Kolitz, Jonathan; Rai, Kanti R.; Nardini, Marco; Tramontano, Anna; Ferrarini, Manlio; Chiorazzi, Nicholas

2004-01-01

Studies of B cell antigen receptors (BCRs) expressed by leukemic lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that B lymphocytes with some level of BCR structural restriction become transformed. While analyzing rearranged VHDJH and VLJL genes of 25 non–IgM-producing B-CLL cases, we found five IgG+ cases that display strikingly similar BCRs (use of the same H- and L-chain V gene segments with unique, shared heavy chain third complementarity-determining region [HCDR3] and light chain third complementarity-determining region [LCDR3] motifs). These H- and L-chain characteristics were not identified in other B-CLL cases or in normal B lymphocytes whose sequences are available in the public databases. Three-dimensional modeling studies suggest that these BCRs could bind the same antigenic epitope. The structural features of the B-CLL BCRs resemble those of mAb’s reactive with carbohydrate determinants of bacterial capsules or viral coats and with certain autoantigens. These findings suggest that the B lymphocytes that gave rise to these IgG+ B-CLL cells were selected for this unique BCR structure. This selection could have occurred because the precursors of the B-CLL cells were chosen for their antigen-binding capabilities by antigen(s) of restricted nature and structure, or because the precursors derived from a B cell subpopulation with limited BCR heterogeneity, or both. PMID:15057307

Effectiveness of Haemodiafiltration with Heat Sterilized High-Flux Polyphenylene HF Dialyzer in Reducing Free Light Chains in Patients with Myeloma Cast Nephropathy

PubMed Central

2015-01-01

Introduction In cases of myeloma cast nephropathy in need of haemodialysis (HD), reduction of free light chains using HD with High-Cut-Off filters (HCO-HD), in combination with chemotherapy, may be associated with better renal recovery. The aim of the present study is to evaluate the effectiveness of haemodiafiltration (HDF) in reducing free light chain levels using a less expensive heat sterilized high-flux polyphenylene HF dialyzer (HF-HDF). Methods In a single-centre prospective cohort study, 327 dialysis sessions were performed using a 2.2 m2 heat sterilized high-flux polyphenylene HF dialyzer (Phylther HF22SD), a small (1.1m2) or large (2.1 m2) high-cut-off (HCO) dialyzer (HCOS and HCOL) in a cohort of 16 patients presenting with dialysis-dependent acute cast nephropathy and elevated free light chains (10 kappa, 6 lambda). The outcomes of the study were the mean reduction ratio (RR) of kappa and lambda, the proportion of treatments with an RR of at least 0.65, albumin loss and the description of patient outcomes. Statistical analysis was performed using linear and logistic regression through generalized estimating equation analysis so as to take into account repeated observation within subjects and adjust for session duration. Results There were no significant differences in the estimated marginal mean of kappa RR, which were respectively 0.67, 0.69 and 0.70 with HCOL-HD, HCOS-HDF and HF-HDF (P = 0.950). The estimated marginal mean of the proportions of treatments with a kappa RR ≥0.65 were 68%, 63% and 71% with HCOL-HD, HCOS-HDF and HF-HDF, respectively (P = 0.913). The estimated marginal mean of lambda RR were higher with HCOL-HDF (0.78), compared to HCOL-HD and HF-HDF (0.62, and 0.61 respectively). The estimated marginal mean proportion of treatments with a lambda RR ≥0.65 were higher with HCOL-HDF (81%), compared to 57% in HF-HDF (P = 0.042). The median albumin loss were 7, 21 and 63 g/session with HF-HDF, HCOL-HD and HCOL-HDF respectively (P = 0.044). Among survivors, 9 out of 10 episodes of acute kidney injuries became dialysis-independent following a median time of renal replacement therapy of 40 days (range 7–181). Conclusion Therefore, in patients with acute dialysis-dependent myeloma cast nephropathy, in addition to chemotherapy, HDF with a heat sterilized high-flux polyphenylene HF dialyzer could offer an alternative to HCO dialysis for extracorporeal kappa reduction with lower albumin loss. PMID:26466100

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

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.

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...

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.

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

p38 mitogen-activated protein kinase-induced nuclear factor kappa-light-chain-enhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury.

PubMed

Jiang, Shao-Yun; Zou, Yuan-Yuan; Wang, Jian-Tao

2012-01-01

In our previous study, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) played a neuroprotective role in retinal ischemia/reperfusion (I/R) injury in rats. However, the mechanism of NF-κB neuroprotection is still unclear. We hypothesize that p38 mitogen-activated protein kinase (MAPK) is expressed and NF-κB activity induced by p38 MAPK plays a neuroprotective role through antiapoptotic genes (B-cell lymphoma [Bcl]-2 and Bcl-XL) in retinal cells in retinal I/R injury. Retinal ischemia was induced by elevating intraocular pressure in rats. After retinal I/R, the p38 MAPK, NF-κB p65, Bcl-2, and Bcl-XL mRNA levels were measured with real-time polymerase chain reaction. NF-κB p65 activity was assessed with NF-κB enzyme-linked immunosorbent assay in retinal I/R injury and after application of the p38 MAPK inhibitor (SB203580). Furthermore, SB203580 and NF-κB p65 short interfering RNA (siRNA) were used in retinal I/R injury to examine the effects on Bcl-2 and Bcl-XL levels and nucleosome release in the retina and cell survival in the ganglion cell layer. The mRNA levels of NF-κB p65 and p38 MAPK reached a peak at 6 h after retinal I/R and then decreased gradually. The mRNA levels of Bcl-2 and Bcl-XL significantly increased at 2, 4, and 6 h, peaked at 8 h, and decreased gradually, but remained at a higher level compared with the normal control, which was accompanied by an increase in NF-κB p65 in nuclear extracts. After application of SB203580, the increase in the NF-κB p65 levels in the nucleus induced with I/R was completely abolished, and the mRNA expression of Bcl-2 and Bcl-XL decreased significantly compared with the I/R controls. In addition, NF-κB p65 siRNA inhibited Bcl-2 and Bcl-XL expression. Inhibition of the p38 MAPK-NF-κB pathway (using SB203580 or NF-κB p65 siRNA) increased retinal nucleosome release and decreased the number of ganglion cells. These findings provide evidence of crosstalk between p38 MAPK and NF-κB p65 and demonstrate a possible neuroprotective role for the p38 MAPK-NF-κB pathway through Bcl-2 and Bcl-XL in retinal I/R injury in rats.

p38 mitogen-activated protein kinase–induced nuclear factor kappa-light-chain-enhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury

PubMed Central

Jiang, Shao-Yun; Zou, Yuan-Yuan

2012-01-01

Purpose In our previous study, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) played a neuroprotective role in retinal ischemia/reperfusion (I/R) injury in rats. However, the mechanism of NF-κB neuroprotection is still unclear. We hypothesize that p38 mitogen-activated protein kinase (MAPK) is expressed and NF-κB activity induced by p38 MAPK plays a neuroprotective role through antiapoptotic genes (B-cell lymphoma [Bcl]-2 and Bcl-XL) in retinal cells in retinal I/R injury. Methods Retinal ischemia was induced by elevating intraocular pressure in rats. After retinal I/R, the p38 MAPK, NF-κB p65, Bcl-2, and Bcl-XL mRNA levels were measured with real-time polymerase chain reaction. NF-κB p65 activity was assessed with NF-κB enzyme-linked immunosorbent assay in retinal I/R injury and after application of the p38 MAPK inhibitor (SB203580). Furthermore, SB203580 and NF-κB p65 short interfering RNA (siRNA) were used in retinal I/R injury to examine the effects on Bcl-2 and Bcl-XL levels and nucleosome release in the retina and cell survival in the ganglion cell layer. Results The mRNA levels of NF-κB p65 and p38 MAPK reached a peak at 6 h after retinal I/R and then decreased gradually. The mRNA levels of Bcl-2 and Bcl-XL significantly increased at 2, 4, and 6 h, peaked at 8 h, and decreased gradually, but remained at a higher level compared with the normal control, which was accompanied by an increase in NF-κB p65 in nuclear extracts. After application of SB203580, the increase in the NF-κB p65 levels in the nucleus induced with I/R was completely abolished, and the mRNA expression of Bcl-2 and Bcl-XL decreased significantly compared with the I/R controls. In addition, NF-κB p65 siRNA inhibited Bcl-2 and Bcl-XL expression. Inhibition of the p38 MAPK-NF-κB pathway (using SB203580 or NF-κB p65 siRNA) increased retinal nucleosome release and decreased the number of ganglion cells. Conclusions These findings provide evidence of crosstalk between p38 MAPK and NF-κB p65 and demonstrate a possible neuroprotective role for the p38 MAPK-NF-κB pathway through Bcl-2 and Bcl-XL in retinal I/R injury in rats. PMID:22876136

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

Fundamental and future prospects of printed ambipolar fluorene-type polymer light-emitting transistors for improved external quantum efficiency, mobility, and emission pattern

NASA Astrophysics Data System (ADS)

Kajii, Hirotake

2018-05-01

In this review, we focus on the improved external quantum efficiency, field-effect mobility, and emission pattern of top-gate-type polymer light-emitting transistors (PLETs) based on ambipolar fluorene-type polymers. A low-temperature, high-efficiency, printable red phosphorescent PLET based on poly(alkylfluorene) with modified alkyl side chains fabricated by a film transfer process is demonstrated. Device fabrication based on oriented films leads to an improved EL intensity owing to the increase in field-effect mobility. There are three factors that affect the transport of carriers, i.e., the energy level, threshold voltage, and mobility of each layer for heterostructure PLETs, which result in various emission patterns such as the line-shaped, multicolor and in-plane emission pattern in the full-channel area between source and drain electrodes. Fundamentals and future prospects in heterostructure devices are discussed and reviewed.

Correlation of Device Performance and Fermi Level Shift in the Emitting Layer of Organic Light-Emitting Diodes with Amine-Based Electron Injection Layers.

PubMed

Stolz, Sebastian; Lemmer, Uli; Hernandez-Sosa, Gerardo; Mankel, Eric

2018-03-14

We investigate three amine-based polymers, polyethylenimine and two amino-functionalized polyfluorenes, as electron injection layers (EILs) in organic light-emitting diodes (OLEDs) and find correlations between the molecular structure of the polymers, the electronic alignment at the emitter/EIL interface, and the resulting device performance. X-ray photoelectron spectroscopy measurements of the emitter/EIL interface indicate that all three EIL polymers induce an upward shift of the Fermi level in the emitting layer close to the interface similar to n-type doping. The absolute value of this Fermi level shift, which can be explained by an electron transfer from the EIL polymers into the emitting layer, correlates with the number of nitrogen-containing groups in the side chains of the polymers. Whereas polyethylenimine (PEI) and one of the investigated polyfluorenes (PFCON-C) have six such groups per monomer unit, the second investigated polyfluorene (PFN) only possesses two. Consequently, we measure Fermi level shifts of 0.5-0.7 eV for PEI and PFCON-C and only 0.2 eV for PFN. As a result of these Fermi level shifts, the energetic barrier for electron injection is significantly lowered and OLEDs which comprise PEI or PFCON-C as an EIL exhibit a more than twofold higher luminous efficacy than OLEDs with PFN.

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.

Comparative Indole-3-Acetic Acid Levels in the Slender Pea and Other Pea Phenotypes 1

PubMed Central

Law, David M.; Davies, Peter J.

1990-01-01

Free indole-3-acetic acid levels were measured by gas chromatography-mass spectrometry in three ultra-tall `slender' Pisum sativum L. lines differing in gibberellin content. Measurements were made for apices and stem elongation zones of light-grown plants and values were compared with wild-type, dwarf, and nana phenotypes in which internode length is genetically regulated, purportedly via the gibberellin level. Indole-3-acetic acid levels of growing stems paralleled growth rates in all lines, and were high in all three slender genotypes. Growth was inhibited by p-chlorophenoxyisobutyric acid, demonstrating the requirement of auxin activity for stem elongation, and also by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. It is concluded that the slender phenotype may arise from constant activation of a gibberellin receptor or transduction chain event leading directly or indirectly to elevated levels of indole-3-acetic acid, and that increased indole-3-acetic acid levels are a significant factor in the promotion of stem elongation. PMID:16667653

The systemic iron-regulatory proteins hepcidin and ferroportin are reduced in the brain in Alzheimer’s disease

PubMed Central

2013-01-01

Background The pathological features of the common neurodegenerative conditions, Alzheimer’s disease (AD), Parkinson’s disease and multiple sclerosis are all known to be associated with iron dysregulation in regions of the brain where the specific pathology is most highly expressed. Iron accumulates in cortical plaques and neurofibrillary tangles in AD where it participates in redox cycling and causes oxidative damage to neurons. To understand these abnormalities in the distribution of iron the expression of proteins that maintain systemic iron balance was investigated in human AD brains and in the APP-transgenic (APP-tg) mouse. Results Protein levels of hepcidin, the iron-homeostatic peptide, and ferroportin, the iron exporter, were significantly reduced in hippocampal lysates from AD brains. By histochemistry, hepcidin and ferroportin were widely distributed in the normal human brain and co-localised in neurons and astrocytes suggesting a role in regulating iron release. In AD brains, hepcidin expression was reduced and restricted to the neuropil, blood vessels and damaged neurons. In the APP-tg mouse immunoreactivity for ferritin light-chain, the iron storage isoform, was initially distributed throughout the brain and as the disease progressed accumulated in the core of amyloid plaques. In human and mouse tissues, extensive AD pathology with amyloid plaques and severe vascular damage with loss of pericytes and endothelial disruption was seen. In AD brains, hepcidin and ferroportin were associated with haem-positive granular deposits in the region of damaged blood vessels. Conclusion Our results suggest that the reduction in ferroportin levels are likely associated with cerebral ischaemia, inflammation, the loss of neurons due to the well-characterised protein misfolding, senile plaque formation and possibly the ageing process itself. The reasons for the reduction in hepcidin levels are less clear but future investigation could examine circulating levels of the peptide in AD and a possible reduction in the passage of hepcidin across damaged vascular endothelium. Imbalance in the levels and distribution of ferritin light-chain further indicate a failure to utilize and release iron by damaged and degenerating neurons. PMID:24252754

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.

C5H9N isomers: pointers to possible branched chain interstellar molecules

NASA Astrophysics Data System (ADS)

Etim, Emmanuel E.; Gorai, Prasanta; Das, Ankan; Arunan, Elangannan

2017-04-01

The astronomical observation of isopropyl cyanide further stresses the link between the chemical composition of the interstellar medium (ISM) and molecular composition of the meteorites in which there is a dominance of branched chain amino acids as compared to the straight. However, observations of more branched chain molecules in ISM will firmly establish this link. In the light of this, we have considered C5H9N isomeric group in which the next higher member of the alkyl cyanide and other branched chain isomers belong. High-level quantum chemical calculations have been employed in estimating accurate energies of these isomers. From the results, the only isomer of the group that has been astronomically searched, n-butyl cyanide is not the most stable isomer and therefore, which might explain why its search could only yield upper limits of its column density without a successful detection. Rather, the two most stable isomers of the group are the branched chain isomers; tert-butylnitrile and isobutyl cyanide. Based on the rotational constants of these isomers, it is found that the expected intensity of tert-butylnitrile is the maximum among this isomeric group. Thus, this is proposed as the most probable candidate for astronomical observation. A simple LTE (local thermodynamic equilibrium) modelling has also been carried out to check the possibility of detecting tert-butyl cyanide in the millimetre-wave region. Contribution to the Topical Issue "Low-Energy Interactions related to Atmospheric and Extreme Conditions", edited by S. Ptasinska, M. Smialek-Telega, A. Milosavljevic and B. Sivaraman.

Human Immunodeficiency Virus Type 1 Employs the Cellular Dynein Light Chain 1 Protein for Reverse Transcription through Interaction with Its Integrase Protein

PubMed Central

Jayappa, Kallesh Danappa; Ao, Zhujun; Wang, Xiaoxia; Mouland, Andrew J.; Shekhar, Sudhanshu; Yang, Xi

2015-01-01

ABSTRACT In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) of DYNLL1, but not DYNLT1 or p150Glued, resulted in significantly lower levels of HIV-1 reverse transcription in cells. Following an attempt to determine how DYNLL1 could impact HIV-1 reverse transcription, we detected the DYNLL1 interaction with HIV-1 integrase (IN) but not with capsid (CA), matrix (MA), or reverse transcriptase (RT) protein. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs 52GQVD and 250VIQD in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1INQ53A/Q252A) exhibited impaired reverse transcription. Through further investigations, we have also detected relatively smaller amounts of particulate CA in DYNLL1-KD cells or in infections with HIV-1INQ53A/Q252A mutant virus. Overall, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and suggests the requirement of this virus-cell interaction for proper uncoating and efficient reverse transcription of HIV-1. IMPORTANCE Host cellular DYNLL1, DYNLT1, and p150Glued proteins have been implicated in the replication of several viruses. However, their roles in HIV-1 replication have not been investigated. For the first time, we demonstrated that during viral infection, HIV-1 IN interacts with DYNLL1, and their interaction was found to have a role in proper uncoating and efficient reverse transcription of HIV-1. Thus, interaction of IN and DYNLL1 may be a potential target for future anti-HIV therapy. Moreover, while our study has evaluated the involvement of IN in HIV-1 uncoating and reverse transcription, it also predicts a possible mechanism by which IN contributes to these early viral replication steps. PMID:25568209

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.

Tailorable Exciton Transport in Doped Peptide–Amphiphile Assemblies

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

Solomon, Lee A.; Sykes, Matthew E.; Wu, Yimin A.

Light-harvesting biomaterials are an attractive target in photovoltaics, photocatalysis, and artificial photosynthesis. Through peptide self-assembly, complex nanostructures can be engineered to study the role of chromophore organization during light absorption and energy transport. To this end, we demonstrate the one-dimensional transport of excitons along naturally occurring, light-harvesting, Zn-protoporphyrin IX chromophores within self-assembled peptide-amphiphile nanofibers. The internal structure of the nanofibers induces packing of the porphyrins into linear chains. We find that this peptide assembly can enable long-range exciton diffusion, yet it also induces the formation of excimers between adjacent molecules, which serve as exciton traps. Electronic coupling between neighboring porphyrinmore » molecules is confirmed by various spectroscopic methods. The exciton diffusion process is then probed through transient photoluminescence and absorption measurements and fit to a model for one-dimensional hopping. Because excimer formation impedes exciton hopping, increasing the interchromophore spacing allows for improved diffusivity, which we control through porphyrin doping levels. We show that diffusion lengths of over 60 nm are possible at low porphyrin doping, representing an order of magnitude improvement over the highest doping fractions.« less

Tailorable Exciton Transport in Doped Peptide-Amphiphile Assemblies.

PubMed

Solomon, Lee A; Sykes, Matthew E; Wu, Yimin A; Schaller, Richard D; Wiederrecht, Gary P; Fry, H Christopher

2017-09-26

Light-harvesting biomaterials are an attractive target in photovoltaics, photocatalysis, and artificial photosynthesis. Through peptide self-assembly, complex nanostructures can be engineered to study the role of chromophore organization during light absorption and energy transport. To this end, we demonstrate the one-dimensional transport of excitons along naturally occurring, light-harvesting, Zn-protoporphyrin IX chromophores within self-assembled peptide-amphiphile nanofibers. The internal structure of the nanofibers induces packing of the porphyrins into linear chains. We find that this peptide assembly can enable long-range exciton diffusion, yet it also induces the formation of excimers between adjacent molecules, which serve as exciton traps. Electronic coupling between neighboring porphyrin molecules is confirmed by various spectroscopic methods. The exciton diffusion process is then probed through transient photoluminescence and absorption measurements and fit to a model for one-dimensional hopping. Because excimer formation impedes exciton hopping, increasing the interchromophore spacing allows for improved diffusivity, which we control through porphyrin doping levels. We show that diffusion lengths of over 60 nm are possible at low porphyrin doping, representing an order of magnitude improvement over the highest doping fractions.

Serum free light chains in myeloma patients with an intact M-protein by immunofixation: potential roles for response assessment and prognosis during induction therapy with novel agents

PubMed Central

Mori, Sherry; Crawford, Brooke S; Roddy, Julianna VF; Phillips, Gary; Elder, Pat; Hofmeister, Craig C; Efebera, Yvonne; Benson, Don M

2013-01-01

The ascertainment of serum free light chain levels (sFLC) has been shown to be valuable in screening for the presence of plasma cell dyscrasia as well as for baseline prognosis in newly diagnosed patients. For patients with amyloidosis and those with oligo- or non-secretory multiple myeloma (MM), serial measurement of sFLC has also been shown to be valuable in monitoring disease status. However, in patients with a measureable, intact monoclonal protein by immunofixation (M protein), the serial measurement of sFLC remains undefined and is currently not recommended in professional guidelines. Herein, we provide data comparing sFLC to M protein as biomarkers of response in newly-diagnosed patients with MM undergoing induction therapy with the novel agents thalidomide, lenalidomide and/or bortezomib. We show that while M protein appears to outperform sFLC comparatively over the course of induction therapy, the addition of FLC to M-protein further informs the characterization of residual disease status post-induction. Moreover, sFLC at the time of stem cell mobilization appears to hold prognostic power for survival endpoints following HDC/SCT. These findings suggest potentially novel roles for sFLC in patients with MM with an intact M-protein receiving novel agent-based induction strategies followed by HDC/SCT. PMID:22028144

GATA-6 and NF-κB Activate CPI-17 Gene Transcription and Regulate Ca2+ Sensitization of Smooth Muscle Contraction

PubMed Central

Boopathi, Ettickan; Hypolite, Joseph A.; Zderic, Stephen A.; Gomes, Cristiano Mendes; Malkowicz, Bruce; Liou, Hsiou-Chi; Wein, Alan J.

2013-01-01

Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca2+ sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH. PMID:23275439

The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

PubMed Central

Reck, Jaimee; Schauer, Alexandria M.; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A.; Porter, Mary E.

2016-01-01

The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

Counterion adsorption theory of dilute polyelectrolyte solutions: Apparent molecular weight, second virial coefficient, and intermolecular structure factor

PubMed Central

Muthukumar, M.

2012-01-01

Polyelectrolyte chains are well known to be strongly correlated even in extremely dilute solutions in the absence of additional strong electrolytes. Such correlations result in severe difficulties in interpreting light scattering measurements in the determination of the molecular weight, radius of gyration, and the second virial coefficient of charged macromolecules at lower ionic strengths from added strong electrolytes. By accounting for charge-regularization of the polyelectrolyte by the counterions, we present a theory of the apparent molecular weight, second virial coefficient, and the intermolecular structure factor in dilute polyelectrolyte solutions in terms of concentrations of the polymer and the added strong electrolyte. The counterion adsorption of the polyelectrolyte chains to differing levels at different concentrations of the strong electrolyte can lead to even an order of magnitude discrepancy in the molecular weight inferred from light scattering measurements. Based on counterion-mediated charge regularization, the second virial coefficient of the polyelectrolyte and the interchain structure factor are derived self-consistently. The effect of the interchain correlations, dominating at lower salt concentrations, on the inference of the radius of gyration and on molecular weight is derived. Conditions for the onset of nonmonotonic scattering wave vector dependence of scattered intensity upon lowering the electrolyte concentration and interpretation of the apparent radius of gyration are derived in terms of the counterion adsorption mechanism. PMID:22830728

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

The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

PubMed

Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

2015-11-01

Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. © 2015 Wiley Periodicals, Inc.

Protein expression of pectoralis major muscle in chickens in response to dietary methionine status.

PubMed

Corzo, A; Kidd, M T; Dozier, W A; Shack, L A; Burgess, S C

2006-04-01

The present study evaluated the effect of dietary methionine on breast-meat accretion and protein expression in skeletal muscle of broiler chickens in vivo. All broilers received a common pre-test diet up to 21 d of age, and were subsequently fed either a methionine-deficient (MD) or -adequate (MA) diet (3.1 v. 4.5 g/kg diet) from age 21 to 42 d. Dietary cystine levels were 3.7 v. 3.6 g/kg diet for the MD and MA diet, respectively. Detrimental effects on carcass yield (P=0.004), abdominal fat percentage (P=0.001), and breast-meat weight (P=0.001), yield (P=0.001), and uniformity (P=0.002) were observed and validated in birds fed MD diets. Via tandem MS, a total of 190 individual proteins were identified from pectoralis major (PM) muscle tissue. From the former composite, peptides from three proteins were observed to be present exclusively in breast muscle from those chickens fed the MD diet (pyruvate kinase, myosin alkali light chain-1, ribosomal-protein-L-29). No proteins were observed to be uniquely expressed in chickens fed MA diets. Research is warranted to further explore the possibility of the proteins pyruate kinase, myosin alkali light chain-1, or ribosomal protein L-29, as potential biological indicators of differences in protein expression of PM of chickens in response to a dietary methionine deficiency.

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.

An Analysis of United States Marine Corps Enlisted Entry-Level Training Using Supply Chain and Operations Management

DTIC Science & Technology

2010-12-01

An Analysis of United States Marine Corps Enlisted Entry-Level Training Using Supply Chain and Operations Management ______________________________________ By...Report 4. TITLE AND SUBTITLE: An Analysis of United States Marine Corps Enlisted Entry-Level Training Using Supply Chain and Operations Management 6...Level Training; United States Marine Corps; Operations Management ; Supply Chain Management; Process Analysis 16. PRICE CODE 17. SECURITY

Gender differences in the regulation of MLC20 phosphorylation and smooth muscle contraction in rat stomach

PubMed Central

Al-Shboul, Othman A.; Al-Dwairi, Ahmed N.; Alqudah, Mohammad A.; Mustafa, Ayman G.

2018-01-01

Evidence of sex-related differences in gastrointestinal (GI) functions has been reported in the literature. In addition, various GI disorders have disproportionate prevalence between the sexes. An essential step in the initiation of smooth muscle contraction is the phosphorylation of the 20-kDa regulatory myosin light chain (MLC20) by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK). However, whether male stomach smooth muscle inherits different contractile signaling mechanisms for the regulation of MLC20 phosphorylation from that in females has not been established. The present study was designed to investigate sex-associated differences in the regulation of MLC20 phosphorylation and thus muscle contraction in gastric smooth muscle cells (GSMCs). Experiments were performed on GSMCs freshly isolated from male and female rats. Contraction of the GSMCs in response to acetylcholine (ACh), a muscarinic agonist, was measured via scanning micrometry in the presence or absence of the MLCK inhibitor, ML-7. Additionally, the protein levels of MLC20, MLCK and phosphorylated MLC20 were measured by ELISA. The protein levels of MLC20 and MLCK were indifferent between the sexes. ACh induced greater contraction (P<0.05) as well as greater MLC20 phosphorylation (P<0.05) in male GSMCs compared with female. Pretreatment of GSMCs with ML-7 significantly reduced the ACh-induced contraction (P<0.05) and MLC20 phosphorylation (P<0.05) in the male and female cells, and notably, abolished the contractile differences between the sexes. In conclusion, MLC20 phosphorylation and thus muscle contraction may be activated to a greater extent in male rat stomach compared with that in females. PMID:29599980

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.

Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds1

PubMed Central

Toyomasu, Tomonobu; Kawaide, Hiroshi; Mitsuhashi, Wataru; Inoue, Yasunori; Kamiya, Yuji

1998-01-01

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3β-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3β-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3β-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action. PMID:9847128

CO2 utilization: an enabling element to move to a resource- and energy-efficient chemical and fuel production.

PubMed

Ampelli, Claudio; Perathoner, Siglinda; Centi, Gabriele

2015-03-13

CO(2) conversion will be at the core of the future of low-carbon chemical and energy industry. This review gives a glimpse into the possibilities in this field by discussing (i) CO(2) circular economy and its impact on the chemical and energy value chain, (ii) the role of CO(2) in a future scenario of chemical industry, (iii) new routes for CO(2) utilization, including emerging biotechnology routes, (iv) the technology roadmap for CO(2) chemical utilization, (v) the introduction of renewable energy in the chemical production chain through CO(2) utilization, and (vi) CO(2) as a suitable C-source to move to a low-carbon chemical industry, discussing in particular syngas and light olefin production from CO(2). There are thus many stimulating possibilities offered by using CO(2) and this review shows this new perspective on CO(2) at the industrial, societal and scientific levels. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Epigenetic contribution of the myosin light chain kinase gene to the risk for acute respiratory distress syndrome.

PubMed

Szilágyi, Keely L; Liu, Cong; Zhang, Xu; Wang, Ting; Fortman, Jeffrey D; Zhang, Wei; Garcia, Joe G N

2017-02-01

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with a considerable case fatality rate (∼30%-40%). Health disparities exist with African descent (AD) subjects exhibiting greater mortality than European descent (ED) individuals. Myosin light chain kinase is encoded by MYLK, whose genetic variants are implicated in ARDS pathogenesis and may influence ARDS mortality. As baseline population-specific epigenetic changes, that is, cytosine modifications, have been observed between AD and ED individuals, epigenetic variations in MYLK may provide insights into ARDS disparities. We compared methylation levels of MYLK cytosine-guanine dinucleotides (CpGs) between ARDS patients and intensive care unit (ICU) controls overall and by ethnicity in a nested case-control study of 39 ARDS cases and 75 non-ARDS ICU controls. Two MYLK CpG sites (cg03892735 and cg23344121) were differentially modified between ARDS subjects and controls (P < 0.05; q < 0.25) in a logistic regression model, where no effect modification by ethnicity or age was found. One CpG site was associated with ARDS in patients aged <58 years, cg19611163 (intron 19, 20). Two CpG sites were associated with ARDS in EDs only, gene body CpG (cg01894985, intron 2, 3) and CpG (cg16212219, intron 31, 32), with higher modification levels exhibited in ARDS subjects than controls. Cis-acting modified cytosine quantitative trait loci (mQTL) were identified using linear regression between local genetic variants and modification levels for 2 ARDS-associated CpGs (cg23344121 and cg16212219). In summary, these ARDS-associated MYLK CpGs with effect modification by ethnicity and local mQTL suggest that MYLK epigenetic variation and local genetic background may contribute to health disparities observed in ARDS. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Outsourcing vaccine logistics to the private sector: The evidence and lessons learned from the Western Cape Province in South-Africa.

PubMed

Lydon, Patrick; Raubenheimer, Ticky; Arnot-Krüger, Michelle; Zaffran, Michel

2015-06-26

With few exceptions, immunization supply chains in developing countries continue to face chronic difficulties in providing uninterrupted availability of potent vaccines up to service delivery levels, and in the most efficient manner possible. As these countries struggle to keep pace with an ever growing number of vaccines, more and more Ministries of Health are considering options of engaging the private sector to manage vaccine storage, handling and distribution on their behalf. Despite this emerging trend, there is limited evidence on the benefits or challenges of this option to improve public supply chain performance for national immunization programmes. To bridge this knowledge gap, this study aims to shed light on the value proposition of outsourcing by documenting the specific experience of the Western Cape Province of South Africa. The methodology for this review rested on conducting two key supply chain assessments which allowed juxtaposing the performance of the government managed segments of the vaccine supply chain against those managed by the private sector. In particular, measures of effective vaccine management best practice and temperature control in the cold chain were analysed. In addition, the costs of engaging the private sector were analysed to get a better understanding of the economics underpinning outsourcing vaccine logistics. The results from this analysis confirmed some of the theoretical benefits of outsourcing to the private sector. Yet, if the experience in the Western Cape can be deemed a successful one, there are several policy and practice implications that developing countries should be mindful of when considering engaging the private sector. While outsourcing can help improve the performance of the vaccine supply chain, it has the potential to do the reverse if done incorrectly. The findings and lessons learnt from the Western Cape experience can serve as a step towards understanding the role of the private sector in immunization supply chain and logistics systems for developing countries. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

Poly(vinyl Chloride) Photostabilization in the Presence of Schiff Bases Containing a Thiadiazole Moiety.

PubMed

Shaalan, Naser; Laftah, Nawres; El-Hiti, Gamal A; Alotaibi, Mohammad Hayal; Muslih, Raad; Ahmed, Dina S; Yousif, Emad

2018-04-15

Five Schiff bases containing a thiadiazole moiety have been used as poly(vinyl chloride) photostabilizers at low concentrations. The efficiency of Schiff bases as photostabilizers was investigated using various techniques, for example, the changes in poly(vinyl chloride) infrared spectra, molecular weight, chain scission quantum yield, and surface morphology were monitored upon irradiation with an ultraviolet light. Evidently, all the additives used inhibited poly(vinyl chloride) photodegradation at a significant level. The most efficient Schiff base exhibited a high level of aromaticity and contained a hydroxyl group. It seems possible that such photostabilization could be due to the direct absorption of ultraviolet radiation by the additives. In addition, Schiff bases could act as radical scavengers and proton transfer facilitators to stabilize the polymeric materials.

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.

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.

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

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.

Role of the constant region domain in the structural diversity of human antibody light chains.

PubMed

Hifumi, Emi; Taguchi, Hiroaki; Kato, Ryuichi; Uda, Taizo

2017-04-01

Issues regarding the structural diversity (heterogeneity) of an antibody molecule have been the subject of discussion along with the development of antibody drugs. Research on heterogeneity has been extensive in recent years, but no clear solution has been reached. Heterogeneity is also observed in catalytic antibody κ light chains (CLs). In this study, we investigated how the constant region domain of CLs concerns structural diversity because it is a simple and good example for elucidating heterogeneity. By means of cation-exchange chromatography, SDS-PAGE, and 2-dimensional electrophoresis for the CL, multimolecular forms consisting of different electrical charges and molecular sizes coexisted in the solution, resulting in the similar heterogeneity of the full length of CLs. The addition of copper ion could cause the multimolecular forms to change to monomolecular forms. Copper ion contributed greatly to the enrichment of the dimer form of CL and the homogenization of the differently charged CLs. Two molecules of the CL protein bound one copper ion. The binding affinity of the ion was 48.0 μM -1 Several divalent metal ions were examined, but only zinc showed a similar effect.-Hifumi, E., Taguchi, H., Kato, R., Uda, T. Role of the constant region domain in the structural diversity of human antibody light chains. © FASEB.

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.

Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

PubMed Central

Barfod, Elisabeth T.; Moore, Ann L.; Van de Graaf, Benjamin G.; Lidofsky, Steven D.

2011-01-01

 The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress. PMID:21209319

Retinal pigment epithelial detachments and tears, and progressive retinal degeneration in light chain deposition disease.

PubMed

Spielberg, Leigh H; Heckenlively, John R; Leys, Anita M

2013-05-01

Light-chain deposition disease (LCDD) is a rare condition characterised by deposition of monoclonal immunoglobulin light chains (LCs) in tissues, resulting in varying degrees of organ dysfunction. This study reports the characteristic clinical ocular findings seen in advanced LCDD upon development of ocular fundus changes. This is the first report to describe this entity in vivo in a series of patients. A case series of ocular fundus changes in three patients with kidney biopsy-proven LCDD. All patients underwent best corrected visual acuity (BCVA) exam, perimetry, colour fundus photography and fluorescein angiography; two patients underwent indocyanine green angiography, optical coherence tomography, ultrasound and electroretinography; and one patient underwent fundus autofluorescence. Three patients, 53-60 years old at initial presentation, were studied. All three presented with night blindness, poor dark adaptation, metamorphopsia and visual loss. Examination revealed serous and serohaemorrhagic detachments, multiple retinal pigment epithelial (RPE) tears, diffuse RPE degeneration and progressive fibrotic changes. Neither choroidal neovascularisation nor other vascular abnormalities were present. Final best corrected visual acuity (BCVA) ranged from 20/40 to 20/300. Progressive LC deposition in the fundus seems to damage RPE pump function with flow disturbance between choroid and retina. This pathogenesis can explain the evolution to RPE detachments and subsequent rips and progressive retinal malfunction.

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

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

PubMed

Guzman-Villanueva, Diana; Migrino, Raymond Q; Truran, Seth; Karamanova, Nina; Franco, Daniel A; Burciu, Camelia; Senapati, Subhadip; Nedelkov, Dobrin; Hari, Parameswaran; Weissig, Volkmar

2018-06-01

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach.

PubMed

Chang, Tzuu-Wang; Janardhanan, Pavithra; Mello, Charlene M; Singh, Bal Ram; Cai, Shuowei

2016-09-01

Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2'-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2'-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance.

Selection of RNA aptamers against botulinum neurotoxin type A light chain through a non-radioactive approach

PubMed Central

Chang, Tzuu-Wang; Janardhanan, Pavithra; Mello, Charlene; Singh, Bal Ram; Cai, Shuowei

2016-01-01

Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive based SELEX process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nM range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that 2′-fluorine-pyrimidines modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for Systematic Evolution of Ligands by EXponential enrichment (SELEX), by using regular nucleotide during SELEX, and 2′-fluorine-pyrimidines modified nucleotide for final application to enhance their RNase-resistance. PMID:27085355

Nonmuscle myosin is regulated during smooth muscle contraction.

PubMed

Yuen, Samantha L; Ogut, Ozgur; Brozovich, Frank V

2009-07-01

The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca(2+)-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

Operating organic light-emitting diodes imaged by super-resolution spectroscopy

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

King, John T.; Granick, Steve

Super-resolution stimulated emission depletion (STED) microscopy is adapted here for materials characterization that would not otherwise be possible. With the example of organic light-emitting diodes (OLEDs), spectral imaging with pixel-by-pixel wavelength discrimination allows us to resolve local-chain environment encoded in the spectral response of the semi-conducting polymer, and correlate chain packing with local electroluminescence by using externally applied current as the excitation source. We observe nanoscopic defects that would be unresolvable by traditional microscopy. They are revealed in electroluminescence maps in operating OLEDs with 50 nm spatial resolution. We find that brightest emission comes from regions with more densely packedmore » chains. Conventional microscopy of an operating OLED would lack the resolution needed to discriminate these features, while traditional methods to resolve nanoscale features generally cannot be performed when the device is operating. As a result, this points the way towards real-time analysis of materials design principles in devices as they actually operate.« less

Operating organic light-emitting diodes imaged by super-resolution spectroscopy

DOE PAGES

King, John T.; Granick, Steve

2016-06-21

Super-resolution stimulated emission depletion (STED) microscopy is adapted here for materials characterization that would not otherwise be possible. With the example of organic light-emitting diodes (OLEDs), spectral imaging with pixel-by-pixel wavelength discrimination allows us to resolve local-chain environment encoded in the spectral response of the semi-conducting polymer, and correlate chain packing with local electroluminescence by using externally applied current as the excitation source. We observe nanoscopic defects that would be unresolvable by traditional microscopy. They are revealed in electroluminescence maps in operating OLEDs with 50 nm spatial resolution. We find that brightest emission comes from regions with more densely packedmore » chains. Conventional microscopy of an operating OLED would lack the resolution needed to discriminate these features, while traditional methods to resolve nanoscale features generally cannot be performed when the device is operating. As a result, this points the way towards real-time analysis of materials design principles in devices as they actually operate.« less

Molecule-assisted ferromagnetic atomic chain formation

NASA Astrophysics Data System (ADS)

Kumar, Manohar; Sethu, Kiran Kumar Vidya; van Ruitenbeek, Jan M.

2015-06-01

One dimensional systems strongly enhance the quantum character of electron transport. Such systems can be realized in 5 d transition metals Au, Pt, and Ir, in the form of suspended monatomic chains between bulk leads. Atomic chains between ferromagnetic leads would open up many perspectives in the context of spin-dependent transport and spintronics, but the evidence suggests that for pure metals only the mentioned three 5 d metals are susceptible to chain formation. It has been argued that the stability of atomic chains made up from ferromagnetic metals is compromised by the same exchange interaction that produces the local moments. Here we demonstrate that magnetic atomic chains can be induced to form in break junctions under the influence of light molecules. Explicitly, we find deuterium assisted chain formation in the 3 d ferromagnetic transition metals Fe and Ni. Chain lengths up to eight atoms are formed upon stretching the ferromagnetic atomic contact in deuterium atmosphere at cryogenic temperatures. From differential conductance spectra vibronic states of D2 can be identified, confirming the presence of deuterium in the atomic chains. Shot noise spectroscopy indicates the presence of weakly spin polarized transmission channels.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

PubMed Central

Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A.; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

2016-01-01

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. PMID:27869673

A redox-mediated modulation of stem bolting in transgenic Nicotiana sylvestris differentially expressing the external mitochondrial NADPH dehydrogenase.

PubMed

Liu, Yun-Jun; Nunes-Nesi, Adriano; Wallström, Sabá V; Lager, Ida; Michalecka, Agnieszka M; Norberg, Fredrik E B; Widell, Susanne; Fredlund, Kenneth M; Fernie, Alisdair R; Rasmusson, Allan G

2009-07-01

Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+) ratio was unaffected, the stem NADPH/NADP(+) ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+) ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.

Light Scattering Study of Mixed Micelles Made from Elastin-Like Polypeptide Linear Chains and Trimers

NASA Astrophysics Data System (ADS)

Terrano, Daniel; Tsuper, Ilona; Maraschky, Adam; Holland, Nolan; Streletzky, Kiril

Temperature sensitive nanoparticles were generated from a construct (H20F) of three chains of elastin-like polypeptides (ELP) linked to a negatively charged foldon domain. This ELP system was mixed at different ratios with linear chains of ELP (H40L) which lacks the foldon domain. The mixed system is soluble at room temperature and at a transition temperature (Tt) will form swollen micelles with the hydrophobic linear chains hidden inside. This system was studied using depolarized dynamic light scattering (DDLS) and static light scattering (SLS) to determine the size, shape, and internal structure of the mixed micelles. The mixed micelle in equal parts of H20F and H40L show a constant apparent hydrodynamic radius of 40-45 nm at the concentration window from 25:25 to 60:60 uM (1:1 ratio). At a fixed 50 uM concentration of the H20F, varying H40L concentration from 5 to 80 uM resulted in a linear growth in the hydrodynamic radius from about 11 to about 62 nm, along with a 1000-fold increase in VH signal. A possible simple model explaining the growth of the swollen micelles is considered. Lastly, the VH signal can indicate elongation in the geometry of the particle or could possibly be a result from anisotropic properties from the core of the micelle. SLS was used to study the molecular weight, and the radius of gyration of the micelle to help identify the structure and morphology of mixed micelles and the tangible cause of the VH signal.

Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway

PubMed Central

2016-01-01

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. PMID:27285777

Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody

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

Ponomarenko, Natalia; Chatziefthimiou, Spyros D.; Kurkova, Inna

2014-03-01

Catalytic antibody variants with κ and λ light-chain constant domains show differences in their crystal structures which lead to subtle changes in catalytic efficiency and thermodynamic parameters as well as in their affinity for peptide substrates. The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was foundmore » that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the V{sub L} and V{sub H} domains. These V{sub L}/V{sub H} domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.« less

Single photon detection imaging of Cherenkov light emitted during radiation therapy

NASA Astrophysics Data System (ADS)

Adamson, Philip M.; Andreozzi, Jacqueline M.; LaRochelle, Ethan; Gladstone, David J.; Pogue, Brian W.

2018-03-01

Cherenkov imaging during radiation therapy has been developed as a tool for dosimetry, which could have applications in patient delivery verification or in regular quality audit. The cameras used are intensified imaging sensors, either ICCD or ICMOS cameras, which allow important features of imaging, including: (1) nanosecond time gating, (2) amplification by 103-104, which together allow for imaging which has (1) real time capture at 10-30 frames per second, (2) sensitivity at the level of single photon event level, and (3) ability to suppress background light from the ambient room. However, the capability to achieve single photon imaging has not been fully analyzed to date, and as such was the focus of this study. The ability to quantitatively characterize how a single photon event appears in amplified camera imaging from the Cherenkov images was analyzed with image processing. The signal seen at normal gain levels appears to be a blur of about 90 counts in the CCD detector, after going through the chain of photocathode detection, amplification through a microchannel plate PMT, excitation onto a phosphor screen and then imaged on the CCD. The analysis of single photon events requires careful interpretation of the fixed pattern noise, statistical quantum noise distributions, and the spatial spread of each pulse through the ICCD.

[The value of serum free light chain in differential diagnosis of monoclonal gammopathy of renal significance].

PubMed

Li, C; Wen, Y B; Li, H; Su, W; Li, J; Cai, J F; Chen, L M; Li, X M; Li, X W

2017-08-08

Objective: To investigate the value of serum free light chain (FLC) in differential diagnosis of monoclonal gammopathy of renal significance (MGRS). Methods: Forty-nine hospitalized patients who underwent renal biopsy in Peking Union Medical College Hospital between January 2013 and December 2015 were included. Monoclonal gammopathy was detected by serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE), urine IFE and serum FLC. All patients were classified as MGRS ( n =32) and monoclonal gammopathy of undetermined significance (MGUS) ( n =17). Results: Renal lesions in MGRS subgroup included light chain amyloidosis ( n =24, 75.0%), light chain deposition disease ( n =7, 21.9%), and fibrillary glomerulopathy ( n =1, 3.1%). Renal diseases in MGUS subgroup included membranous nephropathy ( n =10), focal segmental glomerulosclerosi (FSGS) ( n =3), diabetic glomerulopathy ( n =1), Henoch-Schonlein purpura nephritis ( n =1), anti-GBM disease concurrent with membranous nephropathy ( n =1) and glomerulomegaly ( n =1). Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGRS subgroup were 12, 16, 23 and 30, respectively. Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGUS subgroup were 11, 17, 6 and 3, respectively. MGRS and MGUS subgroups differed significantly in positive rate of serum IFE ( P <0.001), as well as positive rate of urine IFE ( P =0.02) and abnormal rate of serum FLC ratio ( P <0.001). The sensitivity, specificity, total consistent rate of serum FLC ratio for diagnosis of MGRS were 93.8%, 82.4%, and 89.8% respectively. The sensitivity for diagnosing MGRS could be increased to 100% by combining serum FLC ratio and urine IFE. Conclusions: The significance of monoclonal gammopathy in patients with renal disease should be evaluated by renal pathology.On the premise of excluding lymphoplasmacytic malignancy, serum FLC ratio had promising diagnostic value for MGRS, which was helpful for differential diagnosis of patients who had contraindication to renal biopsy.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

PubMed

Guo, Ling; Wang, Zhen; Anderson, Courtney M; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V; Ondrejka, Sarah L; Ma, Xiao-Jun; Cook, James R

2018-03-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.