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Sample records for c1 light chain

  1. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    SciTech Connect

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.

    2009-06-01

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  2. Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity.

    PubMed

    Jin, Rongsheng; Sikorra, Stefan; Stegmann, Christian M; Pich, Andreas; Binz, Thomas; Brunger, Axel T

    2007-09-18

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity. PMID:17718519

  3. Light chain nephropathy.

    PubMed

    Darouich, Sihem; Bettaieb, Ilhem; Aouadia, Raja; Hedri, Hafedh; Abderrahim, Ezzeddine; Goucha, Rym; Khedher, Adel

    2015-01-01

    Light chain deposition disease (LCDD) is characterized by the tissue deposition of monotypic immunoglobulin light chains of either kappa or lambda isotype. It is the archetypal systemic disease that is most frequently diagnosed on a kidney biopsy, although the deposits may involve several other organs. This brief review focuses on the clinicopathological features of LCDD-associated nephropathy with an emphasis on the diagnostic and therapeutic difficulties related to this elusive condition. PMID:26022011

  4. Myosin light-chain phosphatase.

    PubMed Central

    Morgan, M; Perry, S V; Ottaway, J

    1976-01-01

    1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain. Images PLATE 1 PMID:186030

  5. Serum Free Light Chains

    MedlinePlus

    ... changes in the ratio of kappa and lambda production, which indicate an excess of one clone of ... test to detect abnormal monoclonal protein (M-protein) production and to calculate a kappa/lambda free light ...

  6. Interaction between glycosaminoglycans and immunoglobulin light chains.

    SciTech Connect

    Jiang, X.; Myatt, E.; Lykos, P.; Stevens, F. J.; Center for Mechanistic Biology and Biotechnology; Illinois Inst. of Tech.

    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 by 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.

  7. Immunoglobulin light chains, glycosaminoglycans and amyloid.

    SciTech Connect

    Stevens, F. J.; Kisilevsky, R.; Biosciences Division; Queen's Univ.

    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 normal 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.

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

    PubMed Central

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

    2016-01-01

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

  9. Radioimmunoassay of free light chains of immunoglobulins in urine

    SciTech Connect

    Robinson, E.L.; Gowland, E.; Ward, I.D.; Scarffe, J.H.

    1982-01-01

    Radioimmunoassays for kappa and lambda light chains of immunoglobulins in urine are described. The assays, which involve antisera to free light chains, were sufficiently sensitive to measure the light chains in unconcentrated urine from healthy subjects. The usefulness of the assays in clinical practice is illustrated by measurements of light chain excretion by patients, including serial studies on patients undergoing treatment for myeloma.

  10. Myosin light chains: Teaching old dogs new tricks

    PubMed Central

    Heissler, Sarah M; Sellers, James R

    2014-01-01

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

  11. Structure-activity relationships of C1 and C6 side chains of zaragozic acid A derivatives.

    PubMed

    Ponpipom, M M; Girotra, N N; Bugianesi, R L; Roberts, C D; Berger, G D; Burk, R M; Marquis, R W; Parsons, W H; Bartizal, K F; Bergstom, J D

    1994-11-11

    Systematic modification of the C6 acyl side chain of zaragozic acid A, a potent squalene synthase inhibitor, was undertaken to improve its biological activity. Simplification of the C6 side chain to the octanoyl ester has deleterious effects; increasing the linear chain length improves the in vitro activity up to the tetradecanoyl ester. An omega-phenoxy group is a better activity enhancer than an omega-phenyl group. A number of C6 carbamates, ethers, and carbonates were prepared and found to have similar activity profiles as the C6 esters. In the preparation of C6 ethers, C4 and C4,6 bisethers were also isolated; their relative activity is: C6 > C4 > C4,6. These C6 long-chain derivatives are subnanomolar squalene synthase inhibitors; they are, however, only weakly active in inhibiting hepatic cholesterol synthesis in mice. The C6 short-chain derivatives are much less active in vitro, but they all have improved oral activity in mice. Modification of the C1 alkyl side chain of the n-butanoyl analogue (ED50 4.5 mg/kg) did not improve the po activity further. A number of these C6 long-chain derivatives are also potent antifungal agents in vitro. PMID:7966163

  12. [Light Chain Amyloidosis: an Update for Treatment].

    PubMed

    Shen, Kai-Ni; Li, Jian

    2015-06-01

    Systemic light chain amyloidosis (AL amyloidosis) is the most common type of amyloidosis, in which deposition of misfolded monoclonal light chain secreted by underlying clonal plasma cells leads to organ dysfunction. Tissue biopsy of involved organ is needed to confirm the type of amyloid deposits, thus proper treatment could be applied. Laser microdissection followed by mass spectrometry, performed on formalin-fixed paraffin-embedded specimens, has been proven superior to traditional methods on accurate diagnosis of amyloidosis. Prognosis depends on the extent of cardiac involvement. The Mayo staging system using NT-ProBNP, cardiac troponin-T and free light chain, is the most robust method for risk stratification and treatment guidance. The introduction of autologous stem cell transplantation (auto-ASCT) resulted in long-term survival in responders, while treatment-related toxicity substantially limited the number of eligible candidates. Novel agents, especially bortezomib, thalidomide and lenalidomide hold promise to achieve comparable hematological responses with auto-ASCT, which might play significant role in treatment of recurrent or refractory AL amyloidosis. PMID:26117060

  13. Shared epitopes of avian immunoglobulin light chains.

    PubMed

    Benčina, Mateja; Cizelj, Ivanka; Berčič, Rebeka Lucijana; Narat, Mojca; Benčina, Dušan; Dovč, Peter

    2014-04-15

    Like all jawed vertebrates, birds (Aves) also produce antibodies i.e. immunoglobulins (Igs) as a defence mechanism against pathogens. Their Igs are composed of two identical heavy (H) and light (L) chains which are of lambda isotype. The L chain consists of variable (VL), joining (JL) and constant (CL) region. Using enzyme immunoassays (EIA) and two monoclonal antibodies (mAbs) (3C10 and CH31) to chicken L chain, we analysed their cross-reactivity with sera from 33 avian species belonging to nine different orders. Among Galliformes tested, mAbs 3C10 and CH31 reacted with L chains of chicken, turkey, four genera of pheasants, tragopan and peafowl, but not with sera of grey partridge, quail and Japanese quail. Immunoglobulins of guinea-fowl reacted only with mAb 3C10. Both mAbs reacted also with the L chain of Eurasian griffon (order Falconiformes) and domestic sparrow (order Passeriformes). Sera from six other orders of Aves did not react with either of the two mAbs. EIA using mAbs 3C10 and CH31 enabled detection of antibodies to major avian pathogens in sera of chickens, turkeys, pheasants, peafowl, Eurasian griffon and guinea-fowl (only with mAb 3C10). The N-terminal amino acid sequence of pheasant L chain (19 residues) was identical to that of chicken. Sequences of genes encoding the L chain constant regions of pheasants, turkey and partridge were determined and deposited in the public database (GenBank accession numbers: FJ 649651, FJ 649652 and FJ 649653, respectively). Among them, amino acid sequence of pheasants is the most similar to that of chicken (97% similarity), whereas those of turkey and partridge have greater similarity to each other (89%) than to any other avian L chain sequence. The characteristic deletion of two amino acids which is present in the L chain constant region in Galliformes has been most likely introduced to their L chain after their divergence from Anseriformes. PMID:24603015

  14. 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.

  15. 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

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

    PubMed

    Vincent, N D; Cummins, P

    1985-04-01

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

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

    SciTech Connect

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    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 chain 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

  18. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Léguillette, Renaud; Laviolette, Michel; Bergeron, Celine; Zitouni, Nedjma; Kogut, Paul; Solway, Julian; Kachmar, Linda; Hamid, Qutayba; Lauzon, Anne-Marie

    2009-01-01

    Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. PMID:19011151

  19. Comparison of serum free light chain and urine electrophoresis for the detection of the light chain component of monoclonal immunoglobulins in light chain and intact immunoglobulin multiple myeloma

    PubMed Central

    Dejoie, Thomas; Attal, Michel; Moreau, Philippe; Harousseau, Jean-Luc; Avet-Loiseau, Herve

    2016-01-01

    Response criteria for multiple myeloma are based upon changes in monoclonal protein levels quantified using serum and/or urine protein electrophoresis. The latter lacks sensitivity at low monoclonal protein levels and since 2001, the serum free light chain test has been available and its clinical utility proven, yet guidelines have not recommended it as a replacement for urine assessment. Herein we evaluated responses using serum free light chain measurements and serum and urine electrophoresis after 2 and 4 cycles of therapy and after stem cell transplantation in 25 light chain and 157 intact immunoglobulin myeloma patients enrolled in the IFM 2007-02 MM trial. All 25 light chain patients had measurable disease by serum free light chain and urine methods at presentation. By contrast 98 out of 157 intact immunoglobulin patients had measurable disease by serum free light chain compared to 55 out of 157 by urine electrophoresis. In all patients there was substantial agreement between predicate (serum/urine protein electrophoresis) and test (serum protein electrophoresis and serum free light chain) methods for response assessment (Weighted Kappa=0.83). Urine immunofixation became negative in 47% light chain and 43% intact immunoglobulin patients after 2 cycles of therapy. At this time the serum free light chain ratio normalised in only 11% and 27% patients, respectively. In summary we found good agreement between methods for response assessment, but the serum free light chain test provided greater sensitivity than urine electrophoresis for monitoring. To our knowledge this is the first report comparing both methods for response assignment based on the International Myeloma Working Group guidelines. PMID:26635032

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

    SciTech Connect

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

    2006-01-12

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

  1. Formation of Amyloid Fibers by Monomeric Light Chain Variable Domains*

    PubMed Central

    Brumshtein, Boris; Esswein, Shannon R.; Landau, Meytal; Ryan, Christopher M.; Whitelegge, Julian P.; Phillips, Martin L.; Cascio, Duilio; Sawaya, Michael R.; Eisenberg, David S.

    2014-01-01

    Systemic light chain amyloidosis is a lethal disease characterized by excess immunoglobulin light chains and light chain fragments composed of variable domains, which aggregate into amyloid fibers. These fibers accumulate and damage organs. Some light chains induce formation of amyloid fibers, whereas others do not, making it unclear what distinguishes amyloid formers from non-formers. One mechanism by which sequence variation may reduce propensity to form amyloid fibers is by shifting the equilibrium toward an amyloid-resistant quaternary structure. Here we identify the monomeric form of the Mcg immunoglobulin light chain variable domain as the quaternary unit required for amyloid fiber assembly. Dimers of Mcg variable domains remain stable and soluble, yet become prone to assemble into amyloid fibers upon disassociation into monomers. PMID:25138218

  2. Stretch activates myosin light chain kinase in arterial smooth muscle

    SciTech Connect

    Barany, K.; Rokolya, A.; Barany, M. )

    1990-11-30

    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol (32P)phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the (32P)phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in (32P)phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.

  3. Unusual Presentation of Light Chain Deposition Disease: A Case Report

    PubMed Central

    Uppal, Mayank; Amitabh, Vindu; Agrawal, Usha

    2016-01-01

    Light Chain Deposition Disease (LCDD) is a rare disease characterized by deposition of monoclonal non-amyloid light chains in multiple organs. We report an unusual histologic manifestation of LCDD in a 55-year-old female patient, who presented with nephrotic syndrome and an increased serum creatinine. This case of LCDD had features of cast nephropathy on biopsy which is diagnostic of myeloma kidney, when the patient was clinically asymptomatic. Serum electrophoresis showed no abnormal band. There was no other evidence of a B-cell clonal disorder or amyloidosis. Following chemotherapy, improvement in renal function correlated with a reduction in circulating light-chain levels. PMID:27437235

  4. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies.

    PubMed

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite(®) has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines. PMID:26969773

  5. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies

    PubMed Central

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite® has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines. PMID:26969773

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

    PubMed Central

    Buxbaum, J

    1986-01-01

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

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

    PubMed

    Rostkova, Elena; Gautel, Mathias; Pfuhl, Mark

    2015-04-01

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

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

    PubMed

    Stevenson, G T; Straus, D

    1968-07-01

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

  9. Optical Spectral Observations of a Flickering White-light Kernel in a C1 Solar Flare

    NASA Astrophysics Data System (ADS)

    Kowalski, Adam F.; Cauzzi, Gianna; Fletcher, Lyndsay

    2015-01-01

    We analyze optical spectra of a two-ribbon, long-duration C1.1 flare that occurred on 2011 August 18 within AR 11271 (SOL2011-08-18T15:15). The impulsive phase of the flare was observed with a comprehensive set of space-borne and ground-based instruments, which provide a range of unique diagnostics of the lower flaring atmosphere. Here we report the detection of enhanced continuum emission, observed in low-resolution spectra from 3600 Å to 4550 Å acquired with the Horizontal Spectrograph at the Dunn Solar Telescope. A small, <=0.''5 (1015 cm2) penumbral/umbral kernel brightens repeatedly in the optical continuum and chromospheric emission lines, similar to the temporal characteristics of the hard X-ray variation as detected by the Gamma-ray Burst Monitor on the Fermi spacecraft. Radiative-hydrodynamic flare models that employ a nonthermal electron beam energy flux high enough to produce the optical contrast in our flare spectra would predict a large Balmer jump in emission, indicative of hydrogen recombination radiation from the upper flare chromosphere. However, we find no evidence of such a Balmer jump in the bluemost spectral region of the continuum excess. Just redward of the expected Balmer jump, we find evidence of a "blue continuum bump" in the excess emission which may be indicative of the merging of the higher order Balmer lines. The large number of observational constraints provides a springboard for modeling the blue/optical emission for this particular flare with radiative-hydrodynamic codes, which are necessary to understand the opacity effects for the continuum and emission line radiation at these wavelengths.

  10. OPTICAL SPECTRAL OBSERVATIONS OF A FLICKERING WHITE-LIGHT KERNEL IN A C1 SOLAR FLARE

    SciTech Connect

    Kowalski, Adam F.; Cauzzi, Gianna; Fletcher, Lyndsay

    2015-01-10

    We analyze optical spectra of a two-ribbon, long-duration C1.1 flare that occurred on 2011 August 18 within AR 11271 (SOL2011-08-18T15:15). The impulsive phase of the flare was observed with a comprehensive set of space-borne and ground-based instruments, which provide a range of unique diagnostics of the lower flaring atmosphere. Here we report the detection of enhanced continuum emission, observed in low-resolution spectra from 3600 Å to 4550 Å acquired with the Horizontal Spectrograph at the Dunn Solar Telescope. A small, ≤0.''5 (10{sup 15} cm{sup 2}) penumbral/umbral kernel brightens repeatedly in the optical continuum and chromospheric emission lines, similar to the temporal characteristics of the hard X-ray variation as detected by the Gamma-ray Burst Monitor on the Fermi spacecraft. Radiative-hydrodynamic flare models that employ a nonthermal electron beam energy flux high enough to produce the optical contrast in our flare spectra would predict a large Balmer jump in emission, indicative of hydrogen recombination radiation from the upper flare chromosphere. However, we find no evidence of such a Balmer jump in the bluemost spectral region of the continuum excess. Just redward of the expected Balmer jump, we find evidence of a ''blue continuum bump'' in the excess emission which may be indicative of the merging of the higher order Balmer lines. The large number of observational constraints provides a springboard for modeling the blue/optical emission for this particular flare with radiative-hydrodynamic codes, which are necessary to understand the opacity effects for the continuum and emission line radiation at these wavelengths.

  11. Chronic myopathy due to immunoglobulin light chain amyloidosis

    PubMed Central

    Manoli, Irini; Kwan, Justin Y.; Wang, Qian; Rushing, Elisabeth J.; Tsokos, Maria; Arai, Andrew E.; Burch, Warner M.; Dispenzieri, Angela; McPherron, Alexandra C.; Gahl, William A.

    2013-01-01

    Amyloid myopathy associated with a plasma cell dyscrasia is a rare cause of muscle hypertrophy. It can be a challenging diagnosis, since pathological findings are often elusive. In addition, the mechanism by which immunoglobulin light-chain deposition stimulates muscle overgrowth remains poorly understood. We present a 53–year old female with a 10-year history of progressive generalized muscle overgrowth. Congo-red staining and immunohistochemistry revealed perivascular lambda light chain amyloid deposits, apparent only in a second muscle biopsy. The numbers of central nuclei and satellite cells were increased, suggesting enhanced muscle progenitor cell formation. Despite the chronicity of the light chain disease, the patient showed complete resolution of hematologic findings and significant improvement of her muscle symptoms following autologous bone marrow transplantation. This case highlights the importance of early diagnosis and therapy for this treatable cause of a chronic myopathy with muscle hypertrophy. PMID:23465863

  12. 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. PMID:26036683

  13. Mantle cell lymphoma cell lines show no evident immunoglobulin heavy chain stereotypy but frequent light chain stereotypy.

    PubMed

    Pighi, Chiara; Barbi, Stefano; Bertolaso, Anna; Zamò, Alberto

    2013-08-01

    Mantle cell lymphoma shows a peculiar immunogenetic profile, but the functional consequences of this fact are unknown. We have determined the precise sequences of rearranged heavy and light chain genes in several mantle cell lymphoma cell lines and investigated the presence of heavy and light chain stereotypy. These cell lines use IGHV and IGLV genes that are known to be preferentially rearranged in mantle cell lymphoma, but we found no evidence of heavy chain stereotypy. In contrast, one cell line (Mino) showed a nearly identical light chain complementarity-determining region 3 when compared to the only published light chain cluster. Two cell line couples (Jeko-1/UPN-2 and JVM-2/JVM-13) showed a highly similar light chain that satisfied the criteria for stereotypy. Our data show that mantle cell lymphoma cell lines resemble the IGHV and IGLV usage of mantle cell lymphoma, and foster the hypothesis that light chain stereotypy might be under-recognized. PMID:23245212

  14. Cysteine Racemization on IgG Heavy and Light Chains

    PubMed Central

    Zhang, Qingchun; Flynn, Gregory C.

    2013-01-01

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

  15. Structural and Thermodynamic Characterization of a Cytoplasmic Dynein Light Chain-Intermediate Chain Complex

    SciTech Connect

    Williams,J.; Roulhac, P.; Roy, A.; Vallee, R.; Fitzgerald, M.; Hendrickson, W.

    2007-01-01

    Cytoplasmic dynein is a microtubule-based motor protein complex that plays important roles in a wide range of fundamental cellular processes, including vesicular transport, mitosis, and cell migration. A single major form of cytoplasmic dynein associates with membranous organelles, mitotic kinetochores, the mitotic and migratory cell cortex, centrosomes, and mRNA complexes. The ability of cytoplasmic dynein to recognize such diverse forms of cargo is thought to be associated with its several accessory subunits, which reside at the base of the molecule. The dynein light chains (LCs) LC8 and TcTex1 form a subcomplex with dynein intermediate chains, and they also interact with numerous protein and ribonucleoprotein partners. This observation has led to the hypothesis that these subunits serve to tether cargo to the dynein motor. Here, we present the structure and a thermodynamic analysis of a complex of LC8 and TcTex1 associated with their intermediate chain scaffold. The intermediate chains effectively block the major putative cargo binding sites within the light chains. These data suggest that, in the dynein complex, the LCs do not bind cargo, in apparent disagreement with a role for LCs in dynein cargo binding interactions.

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

    PubMed

    Solomon, A; Weiss, D T

    1987-08-01

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

  17. Fast and slow light in zigzag microring resonator chains.

    PubMed

    Chamorro-Posada, P; Fraile-Pelaez, F J

    2009-03-01

    We analyze fast- and slow-light transmission in a zigzag microring resonator chain. In the superluminal case, a new light-transmission effect is found whereby the input optical pulse is reproduced in an almost-simultaneous manner at the various system outputs. When the input carrier is tuned to a different frequency, the system permits to slow down the propagating optical signal. Between these two extreme cases, the relative delay can be tuned within a broad range. We propose, and analyze numerically, a laser-array configuration for the stable operation of active devices. PMID:19252573

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

    SciTech Connect

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

    1999-01-01

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

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

    SciTech Connect

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

    2015-09-22

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

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

    DOE PAGESBeta

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

    2015-09-22

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

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

    PubMed Central

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

    2015-01-01

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

  2. Light chain amyloidosis - current findings and future prospects.

    PubMed

    Baden, Elizabeth M; Sikkink, Laura A; Ramirez-Alvarado, Marina

    2009-10-01

    Systemic light chain amyloidosis (AL) is one of several protein misfolding diseases and is characterized by extracellular deposition of immunoglobulin light chains in the form of amyloid fibrils [1]. Immunoglobulin (Ig) proteins consist of two light chains (LCs) and two heavy chains (HCs) that ordinarily form a heterotetramer which is secreted by a plasma cell. In AL, however, a monoclonal plasma cell population produces an abundance of a pathogenic LC protein. In this case, not all of the LCs pair with the HCs, and free LCs are secreted into circulation. The LC-HC dimer is very stable, and losing this interaction may result in an unstable LC protein [2]. Additionally, somatic mutations are thought to cause amyloidogenic proteins to be less stable compared to non-amyloidogenic proteins [3-5], leading to protein misfolding and amyloid fibril formation. The amyloid fibrils cause tissue damage and cell death, leading to patient death within 12-18 months if left untreated [6]. Current therapies are harsh and not curative, including chemotherapy and autologous stem cell transplants. Studies of protein pathogenesis and fibril formation mechanisms may lead to better therapies with an improved outlook for patient survival. Much has been done to determine the molecular factors that make a particular LC protein amyloidogenic and to elucidate the mechanism of amyloid fibril formation. Anthony Fink's work, particularly with discerning the role of intermediates in the fibril formation pathway, has made a remarkable impact in the field of amyloidosis research. This review provides a general overview of the current state of AL research and also attempts to capture the most recent ideas and knowledge generated from the Fink laboratory. PMID:19538145

  3. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity. PMID:25733667

  4. Russell body duodenitis with immunoglobulin kappa light chain restriction

    PubMed Central

    Munday, William R; Kapur, Lucy Harn; Xu, Mina; Zhang, Xuchen

    2015-01-01

    Russell bodies are eosinophilic intracytoplasmic globules which are likely the result of disturbed secretion of immunoglobulins that accumulate within the plasma cell. Russell body collections have been identified within the stomach, known as Russell body gastritis. Similar lesions within the duodenum are referred to as Russell body duodenitis, which is rare. Several Russell body gastritis case reports are associated with Helicobacter pylori. However, the etiology of Russell body duodenitis remains unclear. Here we report the first case of Russell body duodenitis with immunoglobulin light chain restriction in a background of peptic duodenitis. PMID:25610537

  5. Russell body duodenitis with immunoglobulin kappa light chain restriction.

    PubMed

    Munday, William R; Kapur, Lucy Harn; Xu, Mina; Zhang, Xuchen

    2015-01-16

    Russell bodies are eosinophilic intracytoplasmic globules which are likely the result of disturbed secretion of immunoglobulins that accumulate within the plasma cell. Russell body collections have been identified within the stomach, known as Russell body gastritis. Similar lesions within the duodenum are referred to as Russell body duodenitis, which is rare. Several Russell body gastritis case reports are associated with Helicobacter pylori. However, the etiology of Russell body duodenitis remains unclear. Here we report the first case of Russell body duodenitis with immunoglobulin light chain restriction in a background of peptic duodenitis. PMID:25610537

  6. Light-chain cardiac amyloidosis with neuropathy: a case report

    PubMed Central

    Xu, Zhan-Wen; Li, Ya-Qin; Liu, Li-xia; Zhou, Bing-Juan

    2015-01-01

    Light-chain amyloidosis is a relatively rare multisystem disorder. The disease often is normally difficult to diagnose due to its broad range of characters without specific symptoms. A 62-year-old male patient presented with heart failure after experiencing a long period of unexplained and untreated gastrointestinal symptoms. Clinical examination and laboratory findings indicated a systemic process with cardiac involvement. Echocardiography revealed concentric left ventricular hypertrophy with enhanced echogenicity and preserved ejection fraction. Rectum biopsy confirmed amyloid deposition. The side effect of delayed diagnosis on prognosis and the appropriate diagnostic strategy has been discussed. PMID:26257516

  7. Myosin Light Chain Kinase (MLCK) Regulates Cell Migration in a Myosin Regulatory Light Chain Phosphorylation-independent Mechanism*

    PubMed Central

    Chen, Chen; Tao, Tao; Wen, Cheng; He, Wei-Qi; Qiao, Yan-Ning; Gao, Yun-Qian; Chen, Xin; Wang, Pei; Chen, Cai-Ping; Zhao, Wei; Chen, Hua-Qun; Ye, An-Pei; Peng, Ya-Jing; Zhu, Min-Sheng

    2014-01-01

    Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration. PMID:25122766

  8. Skeletal muscle myosin light chains are essential for physiological speeds of shortening.

    PubMed

    Lowey, S; Waller, G S; Trybus, K M

    1993-09-30

    In muscle each myosin head contains a regulatory light chain (LC2) that is wrapped around the head/rod junction, and an alkali light chain that is distal to LC2 (ref. 1). The role of these light chains in vertebrate skeletal muscle myosin has remained obscure. Here we prepare heavy chains that are free of both light chains in order to determine by a motility assay whether the light chains are necessary for movement. We find that removal of light chains from myosin reduces the velocity of actin filaments from 8.8 microns s-1 to 0.8 microns s-1 without significantly decreasing the ATPase activity. Reconstitution of myosin with LC2 or alkali light chain increases filament velocity to intermediate rates, and readdition of both classes of light chains fully restores the original sliding velocity. We conclude that even though the light chains are not essential for enzymatic activity, light-chain/heavy-chain interactions play an important part in the conversion of chemical energy into movement. PMID:8413589

  9. 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. PMID:26452981

  10. 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

  11. Structure and diversity of Mexican axolotl lambda light chains.

    PubMed

    André, S; Guillet, F; Charlemagne, J; Fellah, J S

    2000-11-01

    We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cgamma domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus Cgamma, but the KATLVCL stretch, which is well conserved in the Cgamma and T-cell receptor Cbeta domains of many vertebrate species, is not well conserved. All axolotl Vgamma sequences closely match several human and Xenopus Vgamma-like sequences and, although the axolotl Cgamma and Vgamma sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vgamma CDR3 junctions seems to be of the same order as that of mammalian Vgamma chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the gamma type. This may explain in part the poor humoral response of the axolotl. PMID:11132150

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

    SciTech Connect

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

    1993-04-01

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

  13. The light chains of kinesin-1 are autoinhibited.

    PubMed

    Yip, Yan Y; Pernigo, Stefano; Sanger, Anneri; Xu, Mengjia; Parsons, Maddy; Steiner, Roberto A; Dodding, Mark P

    2016-03-01

    The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLC(TPR)), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2(TPR) domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme. PMID:26884162

  14. Normal pre-B cells express a receptor complex of mu heavy chains and surrogate light-chain proteins.

    PubMed

    Nishimoto, N; Kubagawa, H; Ohno, T; Gartland, G L; Stankovic, A K; Cooper, M D

    1991-07-15

    Precursors of B cells, which constitute a subpopulation of the lymphocytes in bone marrow, can be identified by their surface expression of nonimmunoglobulin markers and the absence of immunoglobulin kappa and lambda light chains. Most pre-B cells synthesize mu heavy chains but, without light-chain partners, these undergo rapid cytoplasmic degradation. In the present study, we demonstrate that late stage pre-B cells, like their neoplastic counterparts, express low levels of a surface receptor composed of mu chains paired with a surrogate light-chain complex formed by Vpre-B and lambda 5-like proteins. The data define a previously suspected but unrecognized stage in normal pre-B-cell differentiation. Expression of a clonally diverse receptor renders this population of immature B-lineage cells potentially vulnerable to clonal selection by antigens and idiotypic interactions. PMID:1906177

  15. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    SciTech Connect

    Pozo-Yauner, Luis del; Wall, Jonathan S.; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L.; Pérez Carreón, Julio I.; and others

    2014-01-10

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

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

    SciTech Connect

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

    1997-12-01

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

  17. Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

    PubMed Central

    Stuchell-Brereton, Melissa D.; Siglin, Amanda; Li, Jun; Moore, Jeffrey K.; Ahmed, Shubbir; Williams, John C.; Cooper, John A.

    2011-01-01

    Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function. PMID:21633107

  18. Light-chain binding sites on renal brush-border membranes

    SciTech Connect

    Batuman, V.; Dreisbach, A.W.; Cyran, J.

    1990-05-01

    Immunoglobulin light chains are low-molecular-weight proteins filtered at the renal glomerulus and catabolized within the proximal tubular epithelium. Excessive production and urinary excretion of light chains are associated with renal dysfunction. They also interfere with proximal renal tubule epithelial functions in vitro. We studied the binding of 125I-labeled kappa- and lambda-light chains, obtained from the urine of multiple myeloma patients, to rat and human renal proximal tubular brush-border membranes. Light-chain binding to brush borders was also demonstrated immunologically by flow cytometry. Computer analysis of binding data was consistent with presence of a single class of low-affinity, high-capacity, non-cooperative binding sites with relative selectivity for light chains on both rat and human kidney brush-border membranes. The dissociation constants of light chains ranged from 1.6 X 10(-5) to 1.2 X 10(-4) M, and maximum binding capacity ranged from 4.7 +/- 1.3 X 10(-8) to 8.0 +/- 0.9 X 10(-8) (SD) mol/mg protein at 25 degrees C. Kappa- and lambda-light chains competed with each other for binding with comparable affinity constants. Competition by albumin and beta-lactoglobulin, however, was much weaker, suggesting relative site selectivity for light chains. These binding sites probably function as endocytotic receptors for light chains and possibly other low-molecular-weight proteins.

  19. A second immunoglobulin light chain isotype in the rainbow trout.

    PubMed

    Partula, S; Schwager, J; Timmusk, S; Pilström, L; Charlemagne, J

    1996-01-01

    A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus. PMID:8881036

  20. Is accuracy of serum free light chain measurement achievable?

    PubMed

    Jacobs, Joannes F M; Tate, Jillian R; Merlini, Giampaolo

    2016-06-01

    The serum free light chain (FLC) assay has proven to be an important complementary test in the management of patients with monoclonal gammopathies. The serum FLC assay has value for patients with plasma cell disorders in the context of screening and diagnosis, prognostic stratification, and quantitative monitoring. Nonetheless, serum FLC measurements have analytical limitations which give rise to differences in FLC reporting depending on which FLC assay and analytical platform is used. As the FLC measurements are incorporated in the International Myeloma Working Group guidelines for the evaluation and management of plasma cell dyscrasias, this may directly affect clinical decisions. As new certified methods for serum FLC assays emerge, the need to harmonise patient FLC results becomes increasingly important. In this opinion paper we provide an overview of the current lack of accuracy and harmonisation in serum FLC measurements. The clinical consequence of non-harmonized FLC measurements is that an individual patient may or may not meet certain diagnostic, prognostic, or response criteria, depending on which FLC assay and platform is used. We further discuss whether standardisation of serum FLC measurements is feasible and provide an overview of the steps needed to be taken towards harmonisation of FLC measurements. PMID:26641970

  1. Myosin light chain phosphorylation during the contraction cycle of frog muscle.

    PubMed

    Bárány, K; Bárány, M; Gillis, J M; Kushmerick, M J

    1980-04-01

    Changes in the [32P]phosphate content of proteins during contraction were investigated with sartorius and semitendinosus muscles dissected from live frogs injected with [32P]orthophosphate. During a single tetanus, the only significant change was the increase in the [32P]phosphate content of the 18,000-dalton light chain of myosin. The extent of light chain phosphorylation was a function of stimulus duration and it amounted maximally to 0.35 mol of [32P]phosphate transferred per mol of light chain. The extent of phosphorylation in stimulated and stretched semitendinosus muscles, which were unable to produce active tension, was nearly identical to that in muscles stimulated at standard rest length, when the time of stimulation was over a half-second. Maximal light chain phosphorylation was also observed in muscles treated with caffein. These results provide evidence for the activation of the light chain kinase in the intact muscle through a process involving Ca2+. The phosphorylation of the light chain associated with tetanic stimulation was reversible. After short tetanuses, dephosphorylation of light chain approximately followed relaxation and after longer tetanuses, dephosphorylation lagged behind relaxation. The role of light chain phosphorylation was investigated in caffeine-treated and untreated muscles by measuring the Ca content of actin and the [32P]phosphate content of light chain. Phosphorylation of light chain protected the actin-bound Ca against removal by EDTA stoichiometrically. It is postulated that the physiological role of light chain phosphorylation is to increase the rate of combination of the cross-bridges with the actin filaments in the contracting phase of the mechanical activity. PMID:7364050

  2. neu protooncogene fused to an immunoglobulin heavy chain gene requires immunoglobulin light chain for cell surface expression and oncogenic transformation.

    PubMed Central

    Flanagan, J G; Leder, P

    1988-01-01

    The protein encoded by the neu protooncogene (human gene symbol NGL for neuro/glioblastoma-derived) is a member of the surface receptor/tyrosine kinase family. Though its structure suggests that it can transduce a transmembrane signal, neither its extracellular ligand nor its critical intracellular substrates are known. To explore the functional properties of the protein encoded by neu, we created a fusion gene that joins the cytoplasmic domain of neu to the extracellular portion of an immunoglobulin heavy chain. The localization of the fusion polypeptide can then be controlled by coexpression with immunoglobulin light chain. In the absence of light chain, the heavy chain-neu polypeptide is expressed intracellularly and has no transforming activity. By contrast, in the presence of light chain the fusion polypeptide is expressed at the cell surface and produces tumorigenic foci. Thus, transformation apparently requires expression at the cell surface, where the neu intracellular domain can interact with components that are localized to the plasma membrane. The fusion protein is active in cellular transformation when the transmembrane domain is derived either from neu or from immunoglobulin, indicating that the neu transmembrane domain is not specifically required for transformation, although neu activation in tumors is known to result from a point mutation in this region. The extracellular immunoglobulin heavy and light chain domains of the fusion protein form a functional binding site that allows antigen to modulate its activity, reversing the transforming effect. Images PMID:2903500

  3. Immunoglobulin K light chain deficiency: A rare, but probably underestimated, humoral immune defect.

    PubMed

    Sala, Pierguido; Colatutto, Antonio; Fabbro, Dora; Mariuzzi, Laura; Marzinotto, Stefania; Toffoletto, Barbara; Perosa, Anna R; Damante, Giuseppe

    2016-04-01

    Human immunoglobulin molecules are generated by a pair of identical heavy chains, which identify the immunoglobulin class, and a pair of identical light chains, Kappa or Lambda alternatively, which characterize the immunoglobulin type. In normal conditions, Kappa light chains represent approximately 2/3 of the light chains of total immunoglobulins, both circulating and lymphocyte surface bound. Very few cases of immunoglobulin Kappa or Lambda light chain defects have been reported. Furthermore, the genetic basis of this defect has been extensively explored only in a single case. We report a case of a patient suffering of serious recurrent bacterial infections, which was caused by a very rare form of immunoglobulin disorder, consisting of a pure defect of Kappa light chain. We evaluated major serum immunoglobulin concentrations, as well as total and free Kappa and Lambda light chain concentrations. Lymphocyte phenotyping was also performed and finally we tested the Kappa chain VJ rearrangement as well as the constant Kappa region sequence. Studies performed on VJ rearrangement showed a polyclonal genetic arrangement, whereas the gene sequencing for the constant region of Kappa chain showed a homozygous T to G substitution at the position 1288 (rs200765148). This mutation causes a substitution from Cys to Gly in the protein sequence and, therefore, determines the abnormal folding of the constant region of Kappa chain. We suggest that this defect could lead to an effective reduction of the variability of total antibody repertoire and a consequent defect of an apparently normal immunoglobulin response to common antigens. PMID:26853951

  4. Recurrent Light Chain Proximal Tubulopathy in a Kidney Allograft.

    PubMed

    Angioi, Andrea; Amer, Hatem; Fervenza, Fernando C; Sethi, Sanjeev

    2016-09-01

    We describe a rare case of light chain proximal tubulopathy developing in a kidney transplant 12 months following transplantation. The patient was known to have a monoclonal gammopathy of undetermined significance (MGUS) for more than 15 years. A kidney biopsy done to determine the cause of decline in kidney transplant function showed light chain proximal tubulopathy characterized by numerous eosinophilic and fuchsinophilic granules in proximal tubular epithelial cells, which stained for κ light chains on pronase-based immunofluorescence studies. Electron microscopy confirmed the diagnosis and showed numerous amorphous and geometrically shaped inclusions in proximal tubular epithelial cells. Evaluation of free light chains revealed markedly elevated κ light chains and bone marrow biopsy showed 5% to 10% κ light chain-restricted plasma cells. Retrospective evaluation of the native kidney biopsy performed 15 years earlier also showed numerous fuchsinophilic granules in proximal tubules that stained brightly for κ light chains on pronase-based immunofluorescence studies. The patient was treated with a regimen of bortezomib and dexamethasone with good partial hematologic response and improvement of kidney function. To summarize, we describe a case of recurrent light chain proximal tubulopathy in the transplant, which is an unusual but important cause of decreased kidney function in the setting of a monoclonal gammopathy. PMID:27321964

  5. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  6. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  7. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  8. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  9. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  10. Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain.

    PubMed

    Boettner, Douglas R; Segarra, Verónica A; Moorthy, Balaji T; de León, Nagore; Creagh, John; Collette, John R; Malhotra, Arun; Lemmon, Sandra K

    2016-07-01

    Clathrin facilitates vesicle formation during endocytosis and sorting in the trans-Golgi network (TGN)/endosomal system. Unlike in mammals, yeast clathrin function requires both the clathrin heavy (CHC) and clathrin light (CLC) chain, since Chc1 does not form stable trimers without Clc1. To further delineate clathrin subunit functions, we constructed a chimeric CHC protein (Chc-YR) , which fused the N-terminus of yeast CHC (1-1312) to the rat CHC residues 1318-1675, including the CHC trimerization region. The novel CHC-YR allele encoded a stable protein that fractionated as a trimer. CHC-YR also complemented chc1Δ slow growth and clathrin TGN/endosomal sorting defects. In strains depleted for Clc1 (either clc1Δ or chc1Δ clc1Δ), CHC-YR, but not CHC1, suppressed TGN/endosomal sorting and growth phenotypes. Chc-YR-GFP (green fluorescent protein) localized to the TGN and cortical patches on the plasma membrane, like Chc1 and Clc1. However, Clc1-GFP was primarily cytoplasmic in chc1Δ cells harboring pCHC-YR, indicating that Chc-YR does not bind yeast CLC. Still, some partial phenotypes persisted in cells with Chc-YR, which are likely due either to loss of CLC recruitment or chimeric HC lattice instability. Ultimately, these studies have created a tool to examine non-trimerization roles for the clathrin LC. PMID:27062026

  11. Serum Immunoglobulin Free Light Chain Assessment in IgG4-Related Disease

    PubMed Central

    Grados, Aurélie; Boucraut, José; Vély, Frédéric; Aucouturier, Pierre; Rigolet, Aude; Terrier, Benjamin; Saadoun, David; Ghillani-Dalbin, Pascale; Costedoat-Chalumeau, Nathalie; Harlé, Jean Robert; Schleinitz, Nicolas

    2013-01-01

    Immunoglobulin free light chains are produced in excess during normal antibody synthesis. Their evaluation is commonly used in case of a monoclonal gammopathy. In polyclonal hypergammaglobulinemia related to the Sjögren syndrome or systemic lupus, erythematosus serum free light chain levels are increased and could correlate with disease activity. We show here that the κ (P < 0.0001) and λ (P = 0.0003) free light chains and the κ : λ ratio (P = 0.0049) are increased in sixteen patients with IgG4-related disease when compared to healthy controls. The increase of κ and λ free light chains probably reflects the marked polyclonal B cell activation of the disease. We could not assess in this small cohort of patients a significative correlation of serum free light chain levels and disease activity or extension. PMID:23878543

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

    PubMed

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

    2016-05-27

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

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

    PubMed Central

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

    2014-01-01

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

  14. Involvement of myosin light-chain kinase in endothelial cell retraction

    SciTech Connect

    Wysolmerski, R.B.; Lagunoff, D. )

    1990-01-01

    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 thiophosphorylation 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.

  15. Light and heavy chain deposition disease associated with CH1 deletion

    PubMed Central

    Cohen, Camille; El-Karoui, Khalil; Alyanakian, Marie-Alexandra; Noel, Laure-Hélène; Bridoux, Franck; Knebelmann, Bertrand

    2015-01-01

    Light and heavy chain deposition disease (LHCDD) is a rare complication of monoclonal gammopathy. In all documented cases, LHCDD is the association of deposits of a monoclonal light chain with a normal heavy chain, especially in the kidneys. We describe here a 78-year-old woman whose renal biopsy showed nodular glomerulosclerosis, initially diagnosed as diabetic nephropathy. Detailed kidney biopsy immunofluorescence study corrected the diagnosis to γ1-κ-LHCDD. Advanced immunoblot analysis showed deletion of CH1 in the both blood and kidney heavy chain. We report here, to our knowledge, the first case of γ1 LHCDD associated with a deletion of CH1. PMID:25815184

  16. Light and heavy chain deposition disease associated with CH1 deletion.

    PubMed

    Cohen, Camille; El-Karoui, Khalil; Alyanakian, Marie-Alexandra; Noel, Laure-Hélène; Bridoux, Franck; Knebelmann, Bertrand

    2015-04-01

    Light and heavy chain deposition disease (LHCDD) is a rare complication of monoclonal gammopathy. In all documented cases, LHCDD is the association of deposits of a monoclonal light chain with a normal heavy chain, especially in the kidneys. We describe here a 78-year-old woman whose renal biopsy showed nodular glomerulosclerosis, initially diagnosed as diabetic nephropathy. Detailed kidney biopsy immunofluorescence study corrected the diagnosis to γ1-κ-LHCDD. Advanced immunoblot analysis showed deletion of CH1 in the both blood and kidney heavy chain. We report here, to our knowledge, the first case of γ1 LHCDD associated with a deletion of CH1. PMID:25815184

  17. Deletion of the Synechocystis sp. PCC 6803 kaiAB1C1 gene cluster causes impaired cell growth under light-dark conditions.

    PubMed

    Dörrich, Anja K; Mitschke, Jan; Siadat, Olga; Wilde, Annegret

    2014-11-01

    In contrast to Synechococcus elongatus PCC 7942, few data exist on the timing mechanism of the widely used cyanobacterium Synechocystis sp. PCC 6803. The standard kaiAB1C1 operon present in this organism was shown to encode a functional KaiC protein that interacted with KaiA, similar to the S. elongatus PCC 7942 clock. Inactivation of this operon in Synechocystis sp. PCC 6803 resulted in a mutant with a strong growth defect when grown under light-dark cycles, which was even more pronounced when glucose was added to the growth medium. In addition, mutants showed a bleaching phenotype. No effects were detected in mutant cells grown under constant light. Microarray experiments performed with cells grown for 1 day under a light-dark cycle revealed many differentially regulated genes with known functions in the ΔkaiABC mutant in comparison with the WT. We identified the genes encoding the cyanobacterial phytochrome Cph1 and the light-repressed protein LrtA as well as several hypothetical ORFs with a complete inverse behaviour in the light cycle. These transcripts showed a stronger accumulation in the light but a weaker accumulation in the dark in ΔkaiABC cells in comparison with the WT. In general, we found a considerable overlap with microarray data obtained for hik31 and sigE mutants. These genes are known to be important regulators of cell metabolism in the dark. Strikingly, deletion of the ΔkaiABC operon led to a much stronger phenotype under light-dark cycles in Synechocystis sp. PCC 6803 than in Synechococcus sp. PCC 7942. PMID:25139948

  18. Recurrence of light chain deposit disease after renal allograft transplantation: potential role of rituximab?

    PubMed

    Kuypers, Dirk R J; Lerut, Evelyne; Claes, Kathleen; Evenepoel, Pieter; Vanrenterghem, Yves

    2007-04-01

    Light chain deposit disease (LCDD) is a monoclonal plasma cell disorder characterized by tissue deposition of nonamyloid immunoglobulin light chains, predominantly kappa chains, causing renal insufficiency. LCDD reoccurs almost invariably after renal grafting, leading to early graft loss, usually within a time span of months to years. We describe a female patient with LCDD who lost her first living donor graft after 1 year due to extensive recurrence of kappa chain deposition. Rituximab was administered on the seventh day after her second transplantation with a graft from a deceased donor, in order to prevent early recurrence of LCDD. The 2-year protocol biopsy - similarly to the completely normal 1-year protocol biopsy - revealed persistent absence of light chain deposition on light microscopy but immunohistochemical staining and electron microscopy showed very mild recurrence of light chain deposits. A second 4-week course of rituximab was repeated because of these electron microscopic findings. Subsequently, free kappa light chain concentration decreased from 693 to 74 mg/l and remained low 4 months after completion of therapy. Rituximab could be considered for delaying early LCDD recurrence in patients in whom treatment of the underlying bone marrow disorder failed or is contraindicated, but maintenance therapy is apparently necessary to consolidate this response. PMID:17326779

  19. Reconstitution of heavy chain and light chain 1 in cardiac subfragment-1 from hyperthyroid and euthyroid rabbit hearts.

    PubMed

    Ueda, S; Yamaoki, K; Nagai, R; Yazaki, Y

    1983-01-01

    It is now established that cardiac myosin from hyperthyroid rabbit hearts (TXM) exhibits high Ca2+ ATPase activity. The high Ca2+ ATPase activity of TXM was completely retained in cardiac myosin subfragment-1 (S-1) (1.33 +/- 0.04 mumol Pi/mg per min; euthyroid, 0.51 +/- 0.04). Cardiac S-1 from hyperthyroid and euthyroid rabbits (TXS-1 and NS-1) had the same pattern in SDS-polyacrylamide gel electrophoresis. The possible influence of heavy and light chains of TXM on increasing the ATPase activity was examined by reconstitution in the S-1 preparation. Crosswise reconstitution was performed using cardiac S-1 heavy chain (90,000 daltons) and light chain 1 (LC1) (27,000 daltons) from hyperthyroid and euthyroid hearts. Reconstitution was verified by using radiolabeled LC1. More than 95% of S-1 was recovered with full ATPase activity. When TXS-1 was reconstituted with LC1 from euthyroid hearts, the reconstituted molecule retained high ATPase activity. On the other hand, NS-1 reconstituted with LC1 from hyperthyroid hearts failed to increase the ATPase activity. The ATPase activity of S-1 was determined by the source of the heavy chain. These results suggest that the high Ca2+ ATPase activity of cardiac myosin and S-1 from hyperthyroid animals arises from the molecular alteration of the heavy chain induced by thyroxine administration. PMID:6304826

  20. Biased Immunoglobulin Light Chain Gene Usage in the Shark.

    PubMed

    Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

    2015-10-15

    This study of a large family of κ L chain clusters in nurse shark completes the characterization of its classical Ig gene content (two H chain isotypes, μ and ω, and four L chain isotypes, κ, λ, σ, and σ-2). The shark κ clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over an ~500-bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ~39 κ clusters are prerearranged in the germline (germline 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, nonproductive, and sterile transcripts of the κ clusters compared with the other three L chain isotypes. κ cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and nonproductive rearrangements. These results show that the individual activation of the spatially distant κ clusters is nonrandom. Although both split and germline-joined κ 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

  1. Cloning, functional expression, and characterization of CYP709C1, the first sub-terminal hydroxylase of long chain fatty acid in plants. Induction by chemicals and methyl jasmonate.

    PubMed

    Kandel, Sylvie; Morant, Marc; Benveniste, Irène; Blée, Elizabeth; Werck-Reichhart, Danièle; Pinot, Franck

    2005-10-28

    We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We showed here that CYP709C1 is capable of hydroxylating fatty acids at the omega-1 and omega-2 positions. This work was performed after recoding and heterologous expression of a full-length cDNA isolated from a wheat cDNA library in an engineered yeast strain. Investigation on substrate specificity indicates that CYP709C1 metabolizes different fatty acids varying in their chain length (C12 to C18) and unsaturation. CYP709C1 is the first identified plant cytochrome P450 that can catalyze sub-terminal hydroxylation of C18 fatty acids. cis-9,10-Epoxystearic acid is metabolized with the highest efficiency, i.e. K((m)(app)) of 8 microM and V(max(app)) of 328 nmol/min/nmol P450. This, together with the fact that wheat possesses a microsomal peroxygenase able to synthesize this compound from oleic acid, strongly suggests that it is a physiological substrate. Hydroxylated fatty acids are implicated in plant defense events. We postulated that CYP709C1 could be involved in plant defense by producing such compounds. This receives support from the observation that (i) sub-terminal hydroxylation of 9,10-epoxystearic acid is induced (15-fold after 3 h) in microsomes of wheat seedlings treated with the stress hormone methyl jasmonate and (ii) CYP709C1 is enhanced at the transcriptional level by this treatment. CYP709C1 transcript also accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital. This indicates a possible detoxifying function for CYP709C1 that we discussed. PMID:16120613

  2. IMMUNOGLOBULIN LIGHT CHAIN IMMUNOHISTOCHEMISTRY REVISITED, WITH EMPHASIS ON REACTIVE FOLLICULAR HYPERPLASIA VS. FOLLICULAR LYMPHOMA

    PubMed Central

    Weiss, Lawrence M.; Loera, Sofia; Bacchi, Carlos E.

    2009-01-01

    The identification of monotypic light chains is an important adjunct to the diagnosis of B-cell lymphoma, yet is often difficult to reliably perform on formalin-fixed paraffin sections. We have evaluated a new set of monoclonal antibodies to kappa and lambda light chain that are reactive in paraffin sections. In reactive lymphoid tissues, polytypic staining was noted in greater than 95% of cases, with strong staining of plasma cells, moderate staining of the follicular dendritic cell network, and weak staining of mantle zone cells. Strong staining for the appropriate light chain was seen in each of 7 cases of multiple myeloma. In a series of 58 cases of B-cell lymphoma, correlation between the results of immunohistochemistry and flow cytometry was obtained in 36 cases (62%), including 32 cases (21 kappa and 11 lambda) in which a single light chain was expressed. Monotypic staining also seen in 6 additional cases (10%) in which the flow cytometry had been negative. Thirty of 46 cases (65%) of follicular lymphoma showed monotypic light chain expression, in contrast to 64 of 67 cases (95%) of reactive lymphoid hyperplasia, which showed polytypic light chain expression. These antibodies may provide an effective adjunct to the diagnosis of B-cell lymphoma in routine diagnostic work. PMID:20042853

  3. Plasmonic graded-chains as deep-subwavelength light concentrators.

    PubMed

    Esteves-López, Natalia; Pastawski, Horacio M; Bustos-Marún, Raúl A

    2015-04-01

    We have studied the plasmonic properties of aperiodic arrays of identical nanoparticles (NPs) formed by two opposite and equal graded-chains (a chain where interactions change gradually). We found that these arrays concentrate the external electromagnetic fields even in the long wavelength limit. The phenomenon was understood by identifying the system with an effective cavity where plasmonics excitations are trapped between effective band edges, resulting from the change of passband with the NP's position. Dependence of excitation concentration on several system parameters was also assessed. This includes different gradings as well as NP couplings, damping, and resonant frequencies. In the spirit of the scaling laws in condensed matter physics, we developed a theory that allows us to rationalize all these system parameters into universal curves. The theory is quite general and can also be used in many other situations (different arrays for example). Additionally, we also provided an analytical solution, in the tight-binding limit, for the plasmonic response of homogeneous linear chains of NPs illuminated by a plane wave. Our results can find applications in sensing, near field imaging, plasmon-enhanced photodetectors, as well as to increase solar cell efficiency. PMID:25740978

  4. Plasmonic graded-chains as deep-subwavelength light concentrators

    NASA Astrophysics Data System (ADS)

    Esteves-López, Natalia; Pastawski, Horacio M.; Bustos-Marún, Raúl A.

    2015-04-01

    We have studied the plasmonic properties of aperiodic arrays of identical nanoparticles (NPs) formed by two opposite and equal graded-chains (a chain where interactions change gradually). We found that these arrays concentrate the external electromagnetic fields even in the long wavelength limit. The phenomenon was understood by identifying the system with an effective cavity where plasmonics excitations are trapped between effective band edges, resulting from the change of passband with the NP's position. Dependence of excitation concentration on several system parameters was also assessed. This includes different gradings as well as NP couplings, damping, and resonant frequencies. In the spirit of the scaling laws in condensed matter physics, we developed a theory that allows us to rationalize all these system parameters into universal curves. The theory is quite general and can also be used in many other situations (different arrays for example). Additionally, we also provided an analytical solution, in the tight-binding limit, for the plasmonic response of homogeneous linear chains of NPs illuminated by a plane wave. Our results can find applications in sensing, near field imaging, plasmon-enhanced photodetectors, as well as to increase solar cell efficiency.

  5. Characterization of the subclasses and light chain types of IgG antibodies to rubella.

    PubMed Central

    Skvaril, F; Schilt, U

    1984-01-01

    IgG subclasses of antibodies to rubella were determined in indirect enzyme linked immunoassay (ELISA) with monoclonal antibodies specific for human IgG1, IgG2, IgG3 and IgG4. Eleven sera from women with long past history of rubella, two hyperimmune and five non-hyperimmune immunoglobulin preparations were tested. Light chain types of the antibodies were tested in ELISA with polyclonal specific antibodies to kappa and lambda chains. Antibodies to rubella in the sera as well as in the immunoglobulin preparations were found in the IgG1 subclass only. Both light chain types were present in the antibodies. PMID:6423327

  6. Monoclonal light chain deposits within the stomach manifesting as immunotactoid gastropathy.

    PubMed

    Jen, Kuang-Yu; Fix, Oren K; Foster, Erina N; Laszik, Zoltan G; Ferrell, Linda D

    2015-02-01

    Immunotactoid deposits are defined by their ultrastructural appearance and are characterized by microtubular or cylindrical structures typically measuring greater than 30 nm in diameter. Although a rare entity, immunotactoid deposition most often manifests as immunotactoid glomerulopathy and is associated with underlying lymphoplasmacytic disorders. Corneal immunotactoid deposition known as immunotactoid keratopathy has also been reported in patients with paraproteinemia. Here, we describe the first reported case of immunotactoid deposition in the stomach. The deposits were composed solely of kappa immunoglobulin light chains without significant lambda light chain or immunoglobulin heavy chain components. The patient displayed no renal signs or symptoms, and additional thorough clinical examination failed to detect any evidence of a paraproteinemia or plasma cell dyscrasia. Thus, the gastric immunotactoid deposits in this case appear to be an isolated finding of light chain deposition, of which the significance and etiology are unclear. PMID:25191812

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

    PubMed

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

    1984-05-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains.

    PubMed Central

    Raffen, R.; Dieckman, L. J.; Szpunar, M.; Wunschl, C.; Pokkuluri, P. R.; Dave, P.; Wilkins Stevens, P.; Cai, X.; Schiffer, M.; Stevens, F. J.

    1999-01-01

    The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant kappa4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V(L) from a benign light chain. The amyloidogenic V(L)s were significantly less stable than the benign V(L). Furthermore, only the amyloidogenic V(L)s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V(L) could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V(L) that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V(L) beta-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation. PMID:10091653

  11. Functional Material Features of Bombyx mori Silk Light vs. Heavy Chain Proteins

    PubMed Central

    Zafar, Muhammad S.; Belton, David J.; Hanby, Benjamin; Kaplan, David L.; Perry, Carole C.

    2016-01-01

    Bombyx mori (BM) silk fibroin is composed of two different subunits; heavy chain and light chain fibroin linked by a covalent disulphide bond. Current methods of separating the two silk fractions is complicated and produces inadequate quantities of the isolated components for the study of the individual light and heavy chain silks with respect to new materials. We report a simple method of separating silk fractions using formic acid. The formic acid treatment partially releases predominately the light chain fragment (soluble fraction) and then the soluble fraction and insoluble fractions can be converted into new materials. The regenerated original (total) silk fibroin and the separated fractions (soluble vs. insoluble) had different molecular weights and showed distinctive pH stabilities against aggregation/precipitation based on particle charging. All silk fractions could be electrospun to give fibre mats with viscosity of the regenerated fractions being the controlling factor for successful electrospinning. The silk fractions could be mixed to give blends with different proportions of the two fractions to modify the diameter and uniformity of the electrospun fibres formed. The soluble fraction containing the light chain was able to modify the viscosity by thinning the insoluble fraction containing heavy chain fragments, perhaps analogous to its role in natural fibre formation where the light chain provides increased mobility and the heavy chain producing shear thickening effects. The simplicity of this new separation method should enable access to these different silk protein fractions and accelerate the identification of methods, modifications and potential applications of these materials in biomedical and industrial applications. PMID:25565556

  12. Optimization of Serum Immunoglobulin Free Light Chain Analysis for Subclassification of Cardiac Amyloidosis.

    PubMed

    Halushka, Marc K; Eng, George; Collins, A Bernard; Judge, Daniel P; Semigran, Marc J; Stone, James R

    2015-06-01

    Accurate and rapid classification of cardiac amyloidosis is important for patient management. We have optimized the use of serum free light chain kappa and lambda values to differentiate immunoglobulin light chain amyloid (AL) amyloidosis from transthyretin amyloid and amyloid A using 85 cases of tissue-proven cardiac amyloidosis, in which there was direct classification of amyloidosis by mass spectrometry or immunofluorescence. The serum free light chain kappa/lambda ratios were non-overlapping for the three major groups: AL-lambda (0.01-0.41, n = 30), non-AL (0.52-2.7, n = 43), and AL-kappa (6.7-967, n = 12). A kappa/lambda ratio value between 0.5 and 5.0 had 100 % sensitivity and 100 % specificity for distinguishing AL amyloidosis from non-AL amyloidosis. This optimized range for serum light chain kappa/lambda ratio provides extremely robust classification of cardiac amyloidosis. Cases of cardiac amyloidosis in which the serum kappa/lambda free light chain ratio falls close to these new cutoff values may benefit most from direct amyloid subtyping. PMID:25925232

  13. Orientation of the N- and C-terminal lobes of the myosin regulatory light chain in cardiac muscle.

    PubMed

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2015-01-20

    The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1-N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1-C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface. PMID:25606679

  14. 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

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

    DOE PAGESBeta

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

    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

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

    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.

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

    PubMed

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

    2011-11-01

    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. PMID:21942220

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

    PubMed

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

    2013-11-01

    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

  20. Rapid specific amplification of rat antibody cDNA from nine hybridomas in the presence of myeloma light chains.

    PubMed

    Brady, Jamie L; Corbett, Alexandra J; McKenzie, Brent S; Lew, Andrew M

    2006-08-31

    Most monoclonal antibodies to mouse antigens have been derived from rat spleen-mouse myeloma fusions. Many resultant hybridomas express one of several myeloma kappa chain transcripts, even though the parent myeloma may have been ascribed as not expressing light chain protein. Previous reports have only differentiated against one of these mouse light chains. We have found at least three different myeloma kappa transcripts in the panel of nine hybridomas that were derived from four different myeloma parents. We have designed an amplification strategy that differentiates the rearranged rat kappa chain from all mouse light chains. Moreover, this method is expedient as it requires minimal downstream manipulation. PMID:16901500

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

    PubMed

    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

  2. Equine cutaneous amyloidosis derived from an immunoglobulin lambda-light chain. Immunohistochemical, immunochemical and chemical results.

    PubMed

    Linke, R P; Geisel, O; Mann, K

    1991-09-01

    Amyloid deposits from equine cutaneous nodular amyloidosis associated with extramedullary plasmacytoma were classified immunohistochemically as equine immunoglobulin lambda-light chain-derived and designated eA lambda (HIP). For chemical identification, the amyloid fibril proteins were separated on Sephadex G-100 in 6M guanidine.HCl. Polypeptides of predominantly 24 kDa and 50 kDa were found by polyacrylamide gel electrophoresis. They have preponderance of immunoglobulin lambda-antigenic determinants as detected by immunodiffusion and immunoblotting. Since the N-terminus of the major proteins was blocked, peptides were generated with trypsin and endoproteinase Asp-N and then isolated using reversed-phase high-performance liquid chromatography. Automatic amino-acid sequence determination of seven peptides showed novel sequences. Data bank comparison indicated that these peptides were derived from a monoclonal immunoglobulin lambda-light and a gamma-heavy chain. The light chain was considered to be the leading amyloidogenic polypeptide, since it was the predominant component in a virtually pure amyloid fibril preparation. Thus, immunoglobulin lambda-light chain-derived amyloidosis, so far established only in man and cat, has now also been identified in the horse. PMID:1772596

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

    PubMed Central

    Vanam, Ram P; Schneider, Michael A; Marlow, Michael S

    2015-01-01

    An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown. PMID:26305772

  5. Two Essential Light Chains Regulate the MyoA Lever Arm To Promote Toxoplasma Gliding Motility

    PubMed Central

    Williams, Melanie J.; Alonso, Hernan; Enciso, Marta; Egarter, Saskia; Sheiner, Lilach; Meissner, Markus; Striepen, Boris; Smith, Brian J.

    2015-01-01

    ABSTRACT Key to the virulence of apicomplexan parasites is their ability to move through tissue and to invade and egress from host cells. Apicomplexan motility requires the activity of the glideosome, a multicomponent molecular motor composed of a type XIV myosin, MyoA. Here we identify a novel glideosome component, essential light chain 2 (ELC2), and functionally characterize the two essential light chains (ELC1 and ELC2) of MyoA in Toxoplasma. We show that these proteins are functionally redundant but are important for invasion, egress, and motility. Molecular simulations of the MyoA lever arm identify a role for Ca2+ in promoting intermolecular contacts between the ELCs and the adjacent MLC1 light chain to stabilize this domain. Using point mutations predicted to ablate either the interaction with Ca2+ or the interface between the two light chains, we demonstrate their contribution to the quality, displacement, and speed of gliding Toxoplasma parasites. Our work therefore delineates the importance of the MyoA lever arm and highlights a mechanism by which this domain could be stabilized in order to promote invasion, egress, and gliding motility in apicomplexan parasites. PMID:26374117

  6. A patient with AL amyloidosis with negative free light chain results.

    PubMed

    Milani, Paolo; Valentini, Veronica; Ferraro, Giovanni; Basset, Marco; Russo, Francesca; Foli, Andrea; Palladini, Giovanni; Merlini, Giampaolo

    2016-06-01

    The detection and quantification of amyloidogenic monoclonal light chains are necessary for the diagnosis and evaluation of response to treatment in AL amyloidosis. However, the amyloid clone is often small and difficult to detect. We report the case of a 68-year-old man who was referred to our Center in April 2013 after syncope and the identification of left ventricular hypertrophy at echocardiography, suspected for amyloidosis. A commercial agarose gel electrophoresis immunofixation (IFE) did not reveal monoclonal components in serum and urine. The κ serum free light chain (FLC) concentration was 21.5 mg/L, λ 33 mg/L (κ/λ ratio 0.65), NT-proBNP 9074 ng/L (u.r.l. <332 ng/L) and an echocardiogram confirmed characteristic features of amyloidosis. The abdominal fat aspiration was positive and the amyloid typing by immune-electron microscopy revealed λ light chains deposits. A high-resolution (hr) IFE of serum and urine showed a faint monoclonal λ component in the urine. A bone marrow biopsy showed 8% plasma cells (BMPC) and a kappa/lambda light-chain restriction with λ light chain on immunofluorescence. The diagnosis of AL (λ) amyloidosis with cardiac involvement was made. In May 2013, patient was started on cyclophosphamide, bortezomib and dexamethasone. After six cycles, serum and urine hr-IFE were negative, the bone marrow biopsy showed 3% BMPC without light chain restriction by immunofluorescence, and a decrease of NT-proBNP was observed (5802 ng/L).Thus, treatment was discontinued. In this patient the amyloid clone could be detected only by in house hr-IFE of urine and bone marrow examination. The detection of the small dangerous amyloidogenic clone should be pursued with a combination of high-sensitivity techniques, including assessment of BMPC clonality. Studies of novel tools, such as mass spectrometry on serum and next-generation flow cytometry analysis of the bone marrow, for detecting plasma cell clones in AL amyloidosis and other monoclonal light

  7. Light-induced magnetization in a spin S =1 easy-plane antiferromagnetic chain

    NASA Astrophysics Data System (ADS)

    Herbrych, J.; Zotos, X.

    2016-04-01

    The time evolution of magnetization induced by circularly polarized light in an S =1 Heisenberg chain with large easy-plane anisotropy is studied numerically and analytically. Results at constant light frequency Ω =Ω0 are interpreted in terms of absorption lines of the electronic spin resonance spectrum. The application of time-dependent frequency Ω =Ω (t ) light, so called chirping, is shown to be an efficient procedure in order to obtain within a short time a large, controlled value of the magnetization Mz. Furthermore, comparison with a two-level model provides a qualitative understanding of the induced magnetization process.

  8. A role for destabilizing amino acid replacements in light-chain amyloidosis.

    PubMed Central

    Hurle, M R; Helms, L R; Li, L; Chan, W; Wetzel, R

    1994-01-01

    Light-chain (L-chain) amyloidosis is characterized by deposition of fibrillar aggregates composed of the N-terminal L-chain variable region (VL) domain of an immunoglobulin, generally in individuals overproducing a monoclonal L chain. In addition to proteolytic fragmentation and high protein concentration, particular amino acid substitutions may also contribute to the tendency of an L chain to aggregate in L-chain amyloidosis, although evidence in support of this has been limited and difficult to interpret. In this paper we identify particular amino acid replacements at specific positions in the VL domain that are occupied at frequencies significantly higher in those L chains associated with amyloidosis. Analysis of the structural model for the VL domain of the Bence-Jones protein REI suggests that these positions play important roles in maintaining domain structure and stability. Using an Escherichia coli expression system, we prepared single-point mutants of REI VL incorporating amyloid-associated amino acid replacements that are both rare and located at structurally important positions. These mutants support ordered aggregate formation in an in vitro L-chain fibril formation model in which wild-type REI VL remains soluble. Moreover, the ability of these sequences to aggregate in vitro correlates well with the extent to which domain stability is decreased in denaturant-induced unfolding. The results are consistent with a mechanism for the disease process in which the VL domain, either before or after proteolytic cleavage from the L-chain constant region domain, unfolds by virtue of one or more destabilizing amino acid replacements to generate an aggregation-prone nonnative state. Images PMID:8202506

  9. Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans

    PubMed Central

    Wojtovich, Andrew P.; Wei, Alicia Y.; Sherman, Teresa A.; Foster, Thomas H.; Nehrke, Keith

    2016-01-01

    Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the ‘singlet oxygen generator’ miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology. PMID:27440050

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

  11. Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans.

    PubMed

    Wojtovich, Andrew P; Wei, Alicia Y; Sherman, Teresa A; Foster, Thomas H; Nehrke, Keith

    2016-01-01

    Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the 'singlet oxygen generator' miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology. PMID:27440050

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

    PubMed

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X; Zheng, Lu; Pereira, Natasha A; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  13. Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells

    PubMed Central

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X.; Zheng, Lu; Pereira, Natasha A.; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  14. Masticatory (;superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles.

    PubMed

    Reiser, Peter J; Bicer, Sabahattin; Chen, Qun; Zhu, Ling; Quan, Ning

    2009-08-01

    Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style. PMID:19648394

  15. Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain

    PubMed Central

    Ichikawa, Muneyoshi; Saito, Kei; Yanagisawa, Haru-aki; Yagi, Toshiki; Kamiya, Ritsu; Yamaguchi, Shin; Yajima, Junichiro; Kushida, Yasuharu; Nakano, Kentaro; Numata, Osamu; Toyoshima, Yoko Y.

    2015-01-01

    The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (α, β, and γ HCs) and >10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas γ head domain, but its precise localization within the γ head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the γ head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the γ MTBD region. Together with observations that LC1 decreased the affinity of the γ MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating γ MTBD's affinity for the doublet microtubule. PMID:26399296

  16. Daily postdilutional hemodiafiltration with FX800 polysulfone dialyzers for removing kappa light chains in multiple myeloma-induced kidney injury

    PubMed Central

    Tillmann, F. P.

    2015-01-01

    Multiple myeloma is an increasing cause of renal failure in the elderly. Early diagnosis of myeloma-associated acute renal failure is paramount and rapid initiation of disease-specific treatments with a combination of chemotherapy and dialytic therapies for instant removal of free light chains have been proposed. For immediate light chain removal, high cut-off dialyzers have been reported to yield superior light chain clearance parameters, but these dialyzers are not widely used due to increased treatment costs. In addition, the clinical virtue of hemodiafiltration (HDF) has not yet been definitively determined. We hereby present the case of a 70-year-old female patient with kappa light chain myeloma and acute on chronic renal failure. Daily HDF for 1-week using standard polysulfone high-flux dialyzers was implemented and led to remarkable and effective light chain reduction ratios between 87% and 95%. PMID:26199476

  17. Effect of clathrin light chains on the stiffness of clathrin lattices and membrane budding.

    PubMed

    Dannhauser, Philip N; Platen, Mitja; Böning, Heike; Ungewickell, Huberta; Schaap, Iwan A T; Ungewickell, Ernst J

    2015-05-01

    Clathrin-dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor-coated convex-, planar- and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes. PMID:25652138

  18. Glycosaminoglycans promote fibril formation by amyloidogenic immunoglobulin light chains through a transient interaction

    PubMed Central

    Martin, Douglas J.; Ramirez-Alvarado, Marina

    2011-01-01

    Amyloid formation occurs when a precursor protein misfolds and aggregates, forming a fibril nucleus that serves as a template for fibril growth. Glycosaminoglycans are highly charged polymers known to associate with tissue amyloid deposits that have been shown to accelerate amyloidogenesis in vitro. We studied two immunoglobulin light chain variable domains from light chain amyloidosis patients with 90% sequence identity, analyzing their fibril formation kinetics and binding properties with different glycosaminoglycan molecules. We find that the less amyloidogenic of the proteins shows a weak dependence on glycosaminoglycan size and charge, while the more amyloidogenic protein responds only minimally to changes in the glycosaminoglycan. These glycosaminoglycan effects on fibril formation do not depend on a stable interaction between the two species but still show characteristic traits of an interaction-dependent mechanism. We propose that transient, predominantly electrostatic interactions between glycosaminoglycans and the precursor proteins mediate the acceleration of fibril formation in vitro. PMID:21640469

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

    SciTech Connect

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    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 result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  20. A novel antibody light chain dimer: Implications for T-cell receptor structure

    SciTech Connect

    Schiffer, M.; Chang, Chong-Hwan; Solomon, A.; Stevens, F.J.

    1989-01-01

    The dimeric structures of antibody light chains produced in patients with multiple myeloma (Bence Jones proteins) have for some time been studied chemically and crystallographically as models of the antigen binding fragment (Fab) of an antibody. The conformational concordance of Fabs and a Bence Jones dimer was demonstrated by the initial immunoglobulin crystallographic structures. We have recently described the structure of a second intact light chain, the lambda-type protein Loc. The Loc protein exhibits an unanticipated protruding arrangement of its complementarity-determining residues. Grooves on each side of the protrusion may function as separate binding sites. In this report, we examine the Loc structure and its intracrystalline interactions in more detail and consider aspects of this structure that may possess implications for models of a nonantibody constituent of the immunoglobulin superfamily, the T-cell antigen receptor. 26 refs., 3 figs., 1 tab.

  1. Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase.

    PubMed

    Inaba, Kazuo

    2002-10-01

    Sperm flagellar movements are regulated by cAMP-dependent protein phosphorylation. Tctex2-related light chain of outer arm dynein is a well-defined phosphorylated protein that is phosphorylated at activation of sperm motility. Here, the protein phosphatase that dephosphorylates Tctex2-related dynein light chain (LC2) has been characterized in salmonid fish sperm. Most of the phosphatase activity against LC2 is found in Triton-soluble fraction of flagella but trace extent of the activity is retained in the axoneme. The dephosphorylation of LC2 is inhibited by okadaic acid at more than 1nM, whereas that of dynein alpha heavy chain is inhibited at more than 10nM. The addition of Ca(2+) gives no direct effect on LC2 dephosphorylation, but it accelerates the dephosphorylation of the regulatory subunit of cAMP-dependent protein kinase, resulting in the decrease of LC2 phosphorylation. The activity to dephosphorylate the LC2 is separated by MonoQ ion-exchange column chromatography along with the immunoreactivity to the antibody against the catalytic subunit of type 2A protein phosphatase. These results suggest that LC2 is dephosphorylated by type 2A protein phosphatase and that dynein alpha heavy chain and the regulatory subunit of cAMP-dependent protein kinase are dephosphorylated by other types of protein phosphatases. PMID:12359223

  2. Sequence analysis of the 3' non-coding region of mouse immunoglobulin light chain messenger RNA.

    PubMed Central

    Hamlyn, P H; Gillam, S; Smith, M; Milstein, C

    1977-01-01

    Using an oligonucleotide d(pT10-C-A) as primer, cDNA has been transcribed from the 3' non-coding region of mouse immunoglobulin light chain mRNA and sequenced by a modification1 of the 'plus-minus' gel method2. The sequence obtained has partially corrected and extended a previously obtained sequence3. The new data contains an unusual sequence in which a trinucleotide is repeated seven times. Images PMID:405661

  3. The complexity of expressed kappa light chains in egg-laying mammals.

    PubMed

    Nowak, Melissa A; Parra, Zuly E; Hellman, Lars; Miller, Robert D

    2004-11-01

    Complementary DNAs encoding immunoglobulin light chains were isolated from two monotreme species, Ornithorhynchus anatinus (duckbill platypus) and Tachyglossus aculeatus (echidna). The sequences of both the variable and constant regions of these clones had greater similarity to IGK than to other light chain classes and phylogenetic analyses place them squarely within the mammalian IGK group, establishing them as monotreme IGK homologues. The constant region sequences of all clones were essentially identical within each species and, along with Southern blot results, the data are consistent with a single IGKC in each species. The expressed IGKV repertoires from both platypus and echidna were randomly sampled and there appear to be at least four platypus and at least nine echidna IGKV subgroups. The IGKV subgroups are highly divergent within species, in some cases sharing as little as 57% nucleotide identity. Two of the IGKV subgroups are present in both species, so there is some degree of overlap in the germline repertoires of these two monotremes. Overall the complexity seen in platypus and echidna IGK light chains is comparable with that of other mammals considered to have high levels of germline diversity and is in contrast to what has been found so far for monotreme IGL. PMID:15448942

  4. Detection of free immunoglobulin light chains in cerebrospinal fluids of patients with central nervous system lymphomas.

    PubMed

    Schroers, Roland; Baraniskin, Alexander; Heute, Christoph; Kuhnhenn, Jan; Alekseyev, Andriy; Schmiegel, Wolff; Schlegel, Uwe; Pels, Hendrik-Johannes

    2010-09-01

    Diagnosis of central nervous system (CNS) lymphoma depends on histopathology of brain biopsies, because no reliable disease marker in the cerebrospinal fluid (CSF) has been identified yet. B-cell lymphomas such as CNS lymphomas are clonally restricted and express either kappa or lambda immunoglobulin light chains. The aim of this study was to find out a potential diagnostic value of free immunoglobulin light chains released into the CSF of CNS lymphoma patients. Kappa (kappa) and lambda (lambda) free immunoglobulin light chains (FLC) were measured in CSF and serum samples collected from 21 patients with primary and secondary CNS lymphomas and 14 control patients with different neurologic disorders. FLC concentrations and ratios were compared between patient groups and were further analyzed in correlation with clinical, cytopathological, and radiological findings. FLC concentrations for all patients were lower in CSF when compared to serum. In patients with CNS lymphoma, the FLC ratios in CSF were higher (range 392-0.3) compared to control patients (range 3.0-0.3). Irrespective of cytopathological proven lymphomatous meningitis, in 11/21 lymphoma CSF samples the FLC ratios were markedly above 3.0 indicating a clonally restricted B-cell population. Increased FLC ratios in CSF were found in those patients showing subependymal lymphoma contact as detected in magnetic resonance imaging. In summary, this is the first report demonstrating that a significant proportion of patients with CNS lymphomas display a markedly increased FLC ratio in the CSF. PMID:20528903

  5. What is new in diagnosis and management of light chain amyloidosis?

    PubMed

    Palladini, Giovanni; Merlini, Giampaolo

    2016-07-14

    Light chain (AL) amyloidosis is caused by a usually small plasma cell clone producing a misfolded light chain that deposits in tissues. Survival is mostly determined by the severity of heart involvement. Recent studies are clarifying the mechanisms of cardiac damage, pointing to a toxic effect of amyloidogenic light chains and offering new potential therapeutic targets. The diagnosis requires adequate technology, available at referral centers, for amyloid typing. Late diagnosis results in approximately 30% of patients presenting with advanced, irreversible organ involvement and dying in a few months despite modern treatments. The availability of accurate biomarkers of clonal and organ disease is reshaping the approach to patients with AL amyloidosis. Screening of early organ damage based on biomarkers can help identify patients with monoclonal gammopathy of undetermined significance who are developing AL amyloidosis before they become symptomatic. Staging systems and response assessment based on biomarkers facilitate the design and conduction of clinical trials, guide the therapeutic strategy, and allow the timely identification of refractory patients to be switched to rescue therapy. Treatment should be risk-adapted. Recent studies are linking specific characteristics of the plasma cell clone to response to different types of treatment, moving toward patient-tailored therapy. In addition, novel anti-amyloid treatments are being developed that might be combined with anti-plasma cell chemotherapy. PMID:27053535

  6. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis

    PubMed Central

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G.; González-Reyes, Acaimo; Martín-Bermudo, María D.

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  7. Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

    PubMed Central

    Fry, Madeline; Greene, Madelyne; Chernaya, Olga; Hu, Wen-Yang; Chew, Teng-Leong; Mahmud, Nadim; Kadkol, Shrihari S.; Glover, Sarah; Prins, Gail; Strakova, Zuzana; de Lanerolle, Primal

    2013-01-01

    Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors. PMID:24224004

  8. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

    PubMed

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G; González-Reyes, Acaimo; Martín-Bermudo, María D

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  9. Molecular mechanisms of cardiomyopathy phenotypes associated with myosin light chain mutations.

    PubMed

    Huang, Wenrui; Szczesna-Cordary, Danuta

    2015-12-01

    We discuss here the potential mechanisms of action associated with hypertrophic (HCM) or dilated (DCM) cardiomyopathy causing mutations in the myosin regulatory (RLC) and essential (ELC) light chains. Specifically, we focus on four HCM mutations: RLC-A13T, RLC-K104E, ELC-A57G and ELC-M173V, and one DCM RLC-D94A mutation shown by population studies to cause different cardiomyopathy phenotypes in humans. Our studies indicate that RLC and ELC mutations lead to heart disease through different mechanisms with RLC mutations triggering alterations of the secondary structure of the RLC which further affect the structure and function of the lever arm domain and impose changes in the cross bridge cycling rates and myosin force generation ability. The ELC mutations exert their detrimental effects through changes in the interaction of the N-terminus of ELC with actin altering the cross talk between the thick and thin filaments and ultimately resulting in an altered force-pCa relationship. We also discuss the effect of mutations on myosin light chain phosphorylation. Exogenous myosin light chain phosphorylation and/or pseudo-phosphorylation were explored as potential rescue tools to treat hypertrophy-related cardiac phenotypes. PMID:26385864

  10. 2.3 A crystal structure of tetanus neurotoxin light chain.

    PubMed

    Breidenbach, Mark A; Brunger, Axel T

    2005-05-24

    TeNT is the causative agent of the neuroparalytic disease tetanus. A key component of TeNT is its light chain, a Zn(2+) endopeptidase that targets SNAREs. Recent structural studies of closely related BoNT endopeptidases indicate that substrate-binding exosites remote from a conserved active site are the primary determinants of substrate specificity. Here we report the 2.3 A X-ray crystal structure of TeNT-LC, determined by combined molecular replacement and MAD phasing. As expected, the overall structure of TeNT-LC is similar to the other known CNT light chain structures, including a conserved thermolysin-like core inserted between structurally distinct amino- and carboxy-terminal regions. Differences between TeNT-LC and the other CNT light chains are mainly limited to surface features such as unique electrostatic potential profiles. An analysis of surface residue conservation reveals a pattern of relatively high variability matching the path of substrate binding around BoNT/A, possibly serving to accommodate the variations in different SNARE targets of the CNT group. PMID:15895988

  11. A novel method of preparing the monoform structure of catalytic antibody light chain.

    PubMed

    Hifumi, Emi; Matsumoto, Shingo; Nakashima, Hiroki; Itonaga, Shogo; Arakawa, Mitsue; Katayama, Yoshiki; Kato, Ryuichi; Uda, Taizo

    2016-02-01

    Along with the development of antibody drugs and catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several catalytic antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet-visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure. PMID:26527062

  12. Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats.

    PubMed Central

    Rijken, D C; Emeis, J J

    1986-01-01

    In order to assess which part of the tissue-type plasminogen activator (t-PA) molecule should be (genetically) modified to obtain more-slowly-clearing mutants, two-chain t-PA and its isolated heavy and light chains were radiolabelled and injected into rats. The vast majority of t-PA and the heavy chain disappeared from the blood circulation with half-lives of 2.3 and 1.0 min respectively. The clearance of the light chain was biphasic, owing to complex-formation with plasma proteinase inhibitors. The disappearance of di-isopropylphospho-light chain, which has a blocked active site, was nearly monophasic, with a half-life of 5.7 min. Organ distribution studies showed that hepatic clearance constituted the major pathway in all cases. These results strongly suggest that t-PA is recognized by the liver primarily through the heavy chain. PMID:3099771

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

    PubMed Central

    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. PMID:25838650

  14. Selective induction of light chain synthesis in cultures of blood lymphocytes from patients with IgG myelomatosis.

    PubMed Central

    Walker, L A; Johnson, G D; MacLennan, I C

    1988-01-01

    In the present study evidence is provided that neoplastic B cells from the blood from four of 24 patients with myelomatosis were activated selectively with polyclonal B cell mitogens. In three of these patients the activated cells produced light chains without heavy chains; of these, two patients had IgGK paraproteins and one had free lambda light chain disease. The ratio of kappa-expressing to lambda-expressing B cells in the initial blood B cell preparations was within the range for healthy controls for all four patients where neoplastic B cells were selectively activated. It is concluded that in some patients with myelomatosis the neoplastic clone is a mosaic of: (1) cells capable of synthesizing both light and heavy chains with (2) cells producing light chains only. PMID:2832107

  15. The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

    PubMed Central

    Rowe, T; Kendrick-Jones, J

    1993-01-01

    In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified. Images PMID:8223496

  16. Production and characterization of monoclonal antibodies specific for kappa and. gamma. light chain types of porcine immunoglobulins

    SciTech Connect

    McCauley, I.; Kim, Y.B.

    1986-03-05

    It has been difficult to raise specific antisera to the light chain types of pigs because of the difficulty in isolating sufficient pure material from polyclonal immunoglobulin. The authors have taken an approach based upon the characterization of a number of monoclonal antibodies (MoAb) raised against porcine IgG in order to obtain antisera specific for light chain types. Spleen cells from mice immunized with porcine IgG were fused with myeloma P3x63-Ag 653. Hybridomas were screened by an ELISA technique against pure porcine light chains coated on microtiter plates. Five clones specific for light chains were isolated. MoAb from these clones have been characterized by sequential immunoprecipitation of /sup 125/I labelled light chains. The pattern of reactivities show that the MoAb can be classified into two mutually exclusive groups, each of which precipitate approximately equal amounts of the labelled light chains. The type specificity of these groups has been determined by utilizing the cross-reaction between anti-human kappa and ..gamma.. with porcine light chains and the groups of MoAb in sequential immunoprecipitations. The MoAb were used in an immunofluorescence study of porcine B lymphocytes. The anti-..gamma.. MoAb stained 57% and the anti-kappa, 43% of total B lymphocytes.

  17. Clinical course of light-chain smouldering multiple myeloma (idiopathic Bence Jones proteinuria): a retrospective cohort study

    PubMed Central

    Kyle, Robert A; Larson, Dirk R; Therneau, Terry M; Dispenzieri, Angela; Melton, L Joseph; Benson, Joanne T; Kumar, Shaji; Rajkumar, S Vincent

    2014-01-01

    Summary Background Bence Jones proteinuria is a disorder that is defined by the excretion of monoclonal light-chain protein. About 15–20% of patients with multiple myeloma secrete monoclonal light chains only, without expression of the normal immunoglobulin heavy chain, which constitutes light-chain multiple myeloma. The definition, prevalence, and progression of these premalignant phases of light-chain multiple myeloma have not been fully characterised. We aimed to identify a subset of patients with idiopathic Bence Jones proteinuria who had a high risk of progression to light-chain multiple myeloma analogous to that seen in patients with smouldering multiple myeloma. Methods In this retrospective cohort study, we studied all patients seen at the Mayo Clinic (Rochester, MN, USA) within 30 days of diagnosis of idiopathic Bence Jones proteinuria between Jan 1, 1960, and June 30, 2004. Inclusion criteria were monoclonal light chain in the urine (≥0·2 g/24 h), absence of intact monoclonal immunoglobulin (M protein) in the serum, and no evidence of multiple myeloma, light-chain amyloidosis, or other related plasma-cell proliferative disorders. The primary endpoint was progression to symptomatic multiple myeloma or light-chain amyloidosis. We examined the cumulative probability of progression and the association of potential risk factors on progression rates to identify patients with a high risk of progression to multiple myeloma or light-chain amyloidosis. Findings We identified 101 patients with idiopathic Bence Jones proteinuria. During 901 total person-years of follow-up, 27 (27%) patients developed multiple myeloma and seven (7%) developed light-chain amyloidosis. The major risk factors for progression were amount of urinary excretion of M protein per 24 h, proportion of bone marrow plasma cells, presence of a markedly abnormal free-light-chain ratio (<0·01 or >100), and reduction of all three uninvolved immunoglobulins. Based on the risk of progression

  18. Inhibition by small-molecule ligands of formation of amyloid fibrils of an immunoglobulin light chain variable domain

    PubMed Central

    Brumshtein, Boris; Esswein, Shannon R; Salwinski, Lukasz; Phillips, Martin L; Ly, Alan T; Cascio, Duilio; Sawaya, Michael R; Eisenberg, David S

    2015-01-01

    Overproduction of immunoglobulin light chains leads to systemic amyloidosis, a lethal disease characterized by the formation of amyloid fibrils in patients' tissues. Excess light chains are in equilibrium between dimers and less stable monomers which can undergo irreversible aggregation to the amyloid state. The dimers therefore must disassociate into monomers prior to forming amyloid fibrils. Here we identify ligands that inhibit amyloid formation by stabilizing the Mcg light chain variable domain dimer and shifting the equilibrium away from the amyloid-prone monomer. DOI: http://dx.doi.org/10.7554/eLife.10935.001 PMID:26576950

  19. A Unique Role for Endothelial Cell Kinesin Light Chain 1, Variant 1 in Leukocyte Transendothelial Migration.

    PubMed

    Cyrus, Bita F; Muller, William A

    2016-05-01

    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

  20. Plural light chains in a single plasma cell of a monoclonal gammopathy undetermined significance case: an ultrastructural study.

    PubMed

    Saito, Nagahito; Konishi, Kohei; Ohta, Shuichi; Kondo, Takeshi; Kato, Mototsugu; Hashino, Satoshi; Takeda, Hiroshi; Asaka, Masahiro; Ooi, Hong-Kean

    2007-02-01

    A 44-year-old man was found to have M-proteins of IgG consisting of kappa- and lambda-chains in serum without lymphadenopathy or splenomegaly. The serum concentrations of IgG, IgA and IgM were within normal limits. Bone marrow examination showed normal cellular marrow containing 6.3% of plasma cells with no abnormal features. No chromosomal abnormality was observed at all. The patient was diagnosed as having monoclonal gammopathy of undetermined significance. The bone marrow plasma cells possessed free kappa- and lambda-chains in Golgi apparatus, rough endoplasmic reticula and cytoplasmic matrices. Plural light chains were simultaneously produced with the same heavy chain in a plasma cell by immunoelectron microscopy. This is the first report in the world of a monoclonal gammopathy of undetermined significance producing plural light chains with the same heavy chain. PMID:17506772

  1. Discrete renal deposition of IgM heavy chain and κ light chain in Waldenström macroglobulinemia (IgM-κ).

    PubMed

    Komatsuda, Atsushi; Masai, Rie; Togashi, Masaru; Ohtani, Hiroshi; Sawada, Kenichi; Wakui, Hideki

    2012-10-01

    We report previously undescribed renal lesions associated with monoclonal gammopathy in a 59-year-old man with Waldenström macroglobulinemia (IgM-κ). Light microscopy showed mesangial proliferation and thickening of glomerular basement membranes (GBMs) and tubular basement membranes (TBMs). Neither intraglomerular thrombi nor nodular glomerulosclerosis was observed. Immunofluorescence studies disclosed essentially discrete localization of IgM heavy chain within the mesangial area and κ light chain along GBMs and TBMs. Electron microscopy showed continuous linear deposits of finely granular electron-dense material along the inner aspect of GBMs and TBMs. Repeated rituximab treatment and chemotherapy (melphalan and prednisolone) led to the improvement of proteinuria. PMID:26019823

  2. Discrete renal deposition of IgM heavy chain and κ light chain in Waldenström macroglobulinemia (IgM-κ)

    PubMed Central

    Komatsuda, Atsushi; Masai, Rie; Togashi, Masaru; Ohtani, Hiroshi; Sawada, Kenichi; Wakui, Hideki

    2012-01-01

    We report previously undescribed renal lesions associated with monoclonal gammopathy in a 59-year-old man with Waldenström macroglobulinemia (IgM-κ). Light microscopy showed mesangial proliferation and thickening of glomerular basement membranes (GBMs) and tubular basement membranes (TBMs). Neither intraglomerular thrombi nor nodular glomerulosclerosis was observed. Immunofluorescence studies disclosed essentially discrete localization of IgM heavy chain within the mesangial area and κ light chain along GBMs and TBMs. Electron microscopy showed continuous linear deposits of finely granular electron-dense material along the inner aspect of GBMs and TBMs. Repeated rituximab treatment and chemotherapy (melphalan and prednisolone) led to the improvement of proteinuria. PMID:26019823

  3. Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril.

    PubMed

    Schmidt, Andreas; Annamalai, Karthikeyan; Schmidt, Matthias; Grigorieff, Nikolaus; Fändrich, Marcus

    2016-05-31

    Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles. PMID:27185936

  4. Multiple qualitative and quantitative methods for free light chain analysis are necessary as first line tests for AL amyloidosis.

    PubMed

    Sečník, Peter; Honsová, Eva; Jabor, Antonín; Lavríková, Petra; Franeková, Janka

    2016-06-01

    The objective of this study was to demonstrate the necessity of using different methods for amyloidogenic light chain detection. Serum and urine agarose gel electrophoresis and immunofixation, as well as serum free light chain (FLC) immunoassay measurements, were evaluated in a patient with verified multiple myeloma and consequent AL amyloidosis confirmed by Congo red staining and immunofluorescence techniques. Conventional chemistry tests [serum and urine electrophoresis (SPE and UPE); serum and urine immunofixation (SIFE and UIFE)] were inconclusive. Only quantitative FLC immunoassay (serum free light chain immunoanalysis, SFLC) provided correct diagnostic information. A combination of gel-based SIFE and UIFE with more novel quantitative FLC immunoassays appears necessary when searching for monoclonal immunoglobulin light chain-related diseases. PMID:26760309

  5. Recruitment of Light Chains by Homologous and Heterologous Fibrils Shows Distinctive Kinetic and Conformational Specificity.

    PubMed

    Blancas-Mejía, Luis M; Ramirez-Alvarado, Marina

    2016-05-31

    Light chain amyloidosis is a protein misfolding disease in which immunoglobulin light chains aggregate as insoluble fibrils that accumulate in extracellular deposits. Amyloid fibril formation in vitro has been described as a nucleation-polymerization, autocatalytic reaction in which nascent fibrils catalyze formation of new fibrils, recruiting soluble protein into the fibril. In this context, it is also established that preformed fibrils or "seeds" accelerate fibril formation. In some cases, seeds with a substantially different sequence are able to accelerate the reaction, albeit with a lower efficiency. In this work, we studied the recruitment and addition of monomers in the presence of seeds of five immunoglobulin light chain proteins, covering a broad range of protein stabilities and amyloidogenic properties. Our data reveal that in the presence of homologous or heterologous seeds, the fibril formation reactions become less stochastic than de novo reactions. The kinetics of the most amyloidogenic proteins (AL-T05 and AL-09) do not present significant changes in the presence of seeds. Amyloidogenic protein AL-103 presented fairly consistent acceleration with all seeds. In contrast, the less amyloidogenic proteins (AL-12 and κI) presented dramatic differential effects that are dependent on the kind of seed used. κI had a poor efficiency to elongate preformed fibrils. Together, these results indicate that fibril formation is kinetically determined by the conformation of the amyloidogenic precursor and modulated by the differential ability of each protein to either nucleate or elongate fibrils. We observe morphological and conformational properties of some seeds that do not favor elongation with some proteins, resulting in a delay in the reaction. PMID:27158939

  6. Clathrin light chain B: gene structure and neuron-specific splicing.

    PubMed Central

    Stamm, S; Casper, D; Dinsmore, J; Kaufmann, C A; Brosius, J; Helfman, D M

    1992-01-01

    The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing. Images PMID:1408826

  7. 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

  8. Light chain amyloidosis of the urinary bladder. A site restricted deposition of an externally produced immunoglobulin

    PubMed Central

    Livneh, A; Shtrasburg, S; Martin, B; Baniel, J; Gal, R; Pras, M

    2001-01-01

    Aims—To identify the amyloid protein in a patient with amyloidosis localised to the urinary bladder, and to see whether subtyping of the protein by sequence analysis increases the understanding of the selection of the urinary bladder as the site of amyloid deposition. Methods—A patient with gross haematuria and a congophilic mass in his urinary bladder was evaluated further. Characterisation of the amyloid protein was performed using conventional histological and immunohistochemical methods. Determination of the N-terminal amino acid sequence of the amyloid protein was performed using protein sequencers. Results—The patient's history, physical examination, and laboratory evaluation excluded the involvement of other organs, justifying a diagnosis of amyloidosis localised to the urinary bladder. Histological and immunological studies showed that the amyloid protein deposited in the urinary bladder of the patient was probably of the amyloid light chain type. No plasma cells or lymphocytes were seen in sections of the urinary bladder and lower ureter adjacent to the amyloid deposits. Molecular analysis showed the sequence NFMLTQPHSISGSPG, which assigned the amyloid protein to either the VλI or the VλVI immunoglobulin (Ig) light chain families. Conclusions—The findings suggest that the amyloid protein in this patient originated outside the urinary bladder. The heterogeneity of the Ig proteins in known cases of amyloidosis of the lower urinary tract suggests that the amino acid residues, which determine the Vλ subtyping, have no major role in restricting the deposited protein to the urinary bladder. Key Words: primary amyloidosis • urinary bladder • λ light chain • amino acid sequence PMID:11729210

  9. Light chain-dependent myosin structural dynamics in solution investigated by transient electrical birefringence.

    PubMed Central

    Eden, D; Highsmith, S

    1997-01-01

    The technique of transient electrical birefringence was used to compare some of the electric and structural dynamic properties of myosin subfragment 1 (S1(elc, rlc)), which has both the essential and regulatory light chains bound, to S1(elc), which has only an essential light chain. The rates of rotational Brownian motion indicate that S1(elc, rlc) is larger, as expected. The permanent electric dipole moment of S1(elc, rlc) is also larger, indicating that the regulatory light chain portion of S1(elc, rlc) has a dipole moment and that it is aligned head-to-tail with the dipole moment of the S1(elc) portion. The permanent electric dipoles decrease with increasing ionic strength, apparently because of ion binding to surface charges. Both S1(elc, rlc) and S1(elc) have intrinsic segmental flexibility, as detected by the ability to selectively align segments with a brief weak electric field. However, unlike S1(elc), which can be structurally distorted by the action of a brief strong electric field, S1(elc, rlc) is stiffer and cannot be distorted by fields as high as 7800 V/cm applied to its approximately 8000 D permanent electric dipole moment. The S1 . MgADP . Pi analog S1 . MgADP . Vi is smaller than S1 . MgADP, for both S1(elc, rlc) and S1(elc). Interestingly, the smaller, stiffer S1(elc, rlc) . MgADP . Vi complex retains intrinsic segmental flexibility. These results are discussed within a framework of current hypotheses of force-producing mechanisms that involve S1 segmental motion and/or the loss of cross-bridge flexibility during force production. PMID:9251811

  10. Structural studies on the zinc-endopeptidase light chain of tetanus neurotoxin.

    PubMed

    De Filippis, V; Vangelista, L; Schiavo, G; Tonello, F; Montecucco, C

    1995-04-01

    Tetanus neurotoxin (TeNT) blocks neuroexocytosis via a zinc-endopeptidase activity highly specific for vescicle-associated membrane protein(VAMP)/synaptobrevin. TeNT is the prototype of clostridial neurotoxins, a new family of metalloproteinases. They consist of three domains and the proteolytic activity is displayed by the 50-kDa light chain (L chain). The L chain was isolated here in the native state from bacterial filtrates of Clostridium tetani and its structure was studied via circular dichroism (CD) and fluorescence spectroscopy. The secondary structure content (27% alpha-helix and 43% beta-sheet), estimated by far-ultraviolet CD measurements, was in reasonable agreement with that obtained by standard predictive methods (25% alpha-helix and 49% beta-sheet). Moreover, the hypothetical zinc-binding motif, encompassing residues His-Glu-Leu-Ile-His, was correctly predicted to be in alpha-helical conformation, as also expected on the basis of the geometrical requirements for a correct coordination of the zinc ion. Both near-ultraviolet CD and fluorescence data strongly suggest that the single Trp43 residue is buried and constrained in a hydrophobic environment, likely distant from the zinc ion located in the active-site cleft. The contribution of the bound zinc ion to the overall conformation of TeNT L chain was investigated by different and complementary techniques, including spectroscopic (far- and near-ultraviolet CD, fluorescence, second derivative absorption spectroscopy) as well as proteolytic probes. The results indicate that the zinc ion plays little, if any, role in determining the structural properties of the L chain molecule. Similarly, the metal-free apo-enzyme and the holo-protein share common stability features evaluated in respect to different physico-chemical parameters (pH, temperature and urea concentration). These results parallel those obtained on thermolysin, a zinc-dependent neutral endoprotease from Bacillus thermoproteolyticus, where both

  11. Induction of antiphosphocholine antibodies utilizing lambda light chains in BALB/c mice.

    PubMed

    Hall, T J

    1987-01-01

    BALB/c mice immunized with 1 or 100 micrograms of phosphocholine (PC)-keyhole-limpet hemocyanin absorbed on aluminum hydroxide gel (alum) produced up to 50% lambda 1-light-chain-containing anti-PC antibodies in their serum. Only 10% lambda 1 antibodies were seen when complete Freund's adjuvant or bentonite were used as the adjuvant or if PC-bovine serum albumin was used with alum. Thus, the induction of large lambda-antibody responses to PC (a response usually dominated by kappa-antibodies) was both adjuvant- and carrier-dependent. PMID:3108165

  12. Nephrotic Syndrome Secondary to Proliferative Glomerulonephritis with Monoclonal Immunoglobulin Deposits of Lambda Light Chain

    PubMed Central

    Yun, Seongseok; Braunhut, Beth L.; Walker, Courtney N.; Bhati, Waheed; Sussman, Amy N.; Anwer, Faiz

    2014-01-01

    We describe a rare case of a 46-year-old woman with history of refractory nephrotic syndrome and hypertension who presented with worsening proteinuria and kidney function. Work-up for both autoimmune and infectious diseases and hematologic malignancies including multiple myeloma were negative. Kidney biopsy demonstrated glomerular sclerotic change with lambda light chain deposits in the subendothelial space, which is consistent with proliferative glomerulonephritis with monoclonal immunoglobulin deposit (PGNMID). The patient was treated with bortezomib and dexamethasone without clinical improvement and eventually became hemodialysis dependent. PMID:25136462

  13. Structural Characterization of the Partially Folded Intermediates of An Immunoglobulin Light Chain Leading to Amyloid Fibrillation And Amorphous Aggregation

    SciTech Connect

    Qin, Z.; Hu, D.; Zhu, M.; Fink, A.L.; /UC, Santa Cruz

    2007-07-12

    Immunoglobulin light chain deposition diseases involve various types of extracellular deposition of light chain variable domains, including amyloid fibrils and amorphous deposits. The decreased thermodynamic stability of the light chain is believed to be the major factor leading to fibrillation. However, the differences in the nature of the deposits among the light chain deposition diseases raise the question of whether the mechanisms leading to fibrillar or amorphous aggregation is different. In this study, we generated two partially folded intermediates of the light chain variable domain SMA in the presence of guanidine hydrochloride (GuHCl) and characterized their conformations. The more unfolded intermediate formed fibrils most rapidly, while the more native-like intermediate predominantly led to amorphous deposits. The results also show that the monomeric, rather than the dimeric state, was critical for fibrillation. The data also indicate that fibril elongation involves addition of a partially unfolded intermediate, rather than the native state. We postulate that a more highly unfolded intermediate is more suited to undergo the topological rearrangements necessary to form amyloid fibrils than a more structured one and that this also correlates with increased destabilization. In the case of light chain aggregation, it appears that more native-like intermediate conformations are more prone to form amorphous deposits.

  14. Preferential combination between the light and heavy chain isotypes of fish immunoglobulins.

    PubMed

    Zhang, Nu; Zhang, Xu-Jie; Song, Yu-Long; Lu, Xiao-Bing; Chen, Dan-Dan; Xia, Xiao-Qin; Sunyer, J Oriol; Zhang, Yong-An

    2016-08-01

    Immunoglobulin light chain (IgL) is necessary for the assembly of an Ig molecule, which plays important roles in the immune response. IgL genes were identified in various teleost species, but the basic functions of different IgL isotypes and the preferential combination between IgL and IgH (Ig heavy chain) isotypes remain unclear. In the current study, by EST database searching and cDNA cloning in rainbow trout, 8 IgL sequences were obtained, which could be classified into the IgLκF, IgLκG, IgLσ and IgLλ isotypes, respectively. Trout IgL isotypes were highly expressed in the immune-related tissues, and participated in the immune responses in spleen and gut by stimulation with LPS and poly (I:C). The results of FACS and LC-MS/MS indicated that the IgLκG and IgLσ isotypes preferentially bonded with the heavy chains of IgM and IgT, respectively, in trout B cells and serum. In addition, the genomic organization of trout IgL isotypes and the utilization of recombination signal sequences were studied. PMID:27057962

  15. Achievement and steering of light-induced sub-wavelength longitudinal magnetization chain.

    PubMed

    Nie, Zhongquan; Ding, Weiqiang; Shi, Guang; Li, Dongyu; Zhang, Xueru; Wang, Yuxiao; Song, Yinglin

    2015-08-10

    The light-induced magnetization distributions for a high numerical aperture focusing configuration with an azimuthally polarized Bessel-Gaussian beam modulated by optimized vortex binary filters are investigated based on the inverse Faraday effect. It is found that, by adjusting the radii of different rings of the single/ cascaded vortex binary filters, super-long (12λ) and sub-wavelength (0.416λ) longitudinal magnetization chain with single/dual channels can be achieved in the focal region. Such well-behaved magnetization trait is attributed to the mutual effect between the optical polarization singularities of the azimuthally polarized beam and single/cascaded spiral optical elements. In addition, we find that the displacement distance of the longitudinal magnetization chain is proportional to the phase difference between the inner circle and outer ring of the vortex binary filters, thus giving rise to the steerable magnetization chain. It is expected that the research outcomes can be applied in multiple atoms trapping and transport, multilayer magneto-optical data storage, fabrication of magnetic lattices for spin wave operation and development of ultra-compact optomagnetic devices. PMID:26367978

  16. Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2

    PubMed Central

    Cadete, Virgilio J. J.; Sawicka, Jolanta; Jaswal, Jagdip; Lopaschuk, Gary D.; Schulz, Richard; Szczesna-Cordary, Danuta; Sawicki, Grzegorz

    2012-01-01

    Summary Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia. PMID:22564771

  17. A Caenorhabditis elegans–based assay recognizes immunoglobulin light chains causing heart amyloidosis

    PubMed Central

    Rognoni, Paola; Lavatelli, Francesca; Romeo, Margherita; del Favero, Elena; Cantù, Laura; Ghibaudi, Elena; di Fonzo, Andrea; Corbelli, Alessandro; Fiordaliso, Fabio; Palladini, Giovanni; Valentini, Veronica; Perfetti, Vittorio; Salmona, Mario; Merlini, Giampaolo

    2014-01-01

    Poor prognosis and limited therapeutic options characterize immunoglobulin light-chain (AL) amyloidosis with major heart involvement. Reliable experimental models are needed to study light-chain (LC)/heart interactions and to explore strategies for prevention of cardiac damage. We have exploited the nematode Caenorhabditis elegans as a novel tool, because its pharynx is evolutionarily related to the vertebrate heart. Our data demonstrate that the pharyngeal pumping of C elegans is significantly and selectively reduced by LCs from AL patients suffering from cardiomyopathy, but not by amyloid LCs with different organ tropism or nonamyloidogenic LCs from multiple myeloma. This functional alteration is dependent on the LC concentration and results in persistent pharyngeal dysfunction and in a significant reduction of the worms’ lifespan. These manifestations are paralleled by an increase of mitochondrial reactive oxygen species and can be prevented by treatment with antioxidant agents. In conclusion, these data indicate that this nematode-based assay is a promising surrogate model for investigating the heart-specific toxicity of amyloidogenic LCs and for a rapid screening of new therapeutic strategies. PMID:24665135

  18. Multiple myeloma-associated skin light chain amyloidosis: A case of misdiagnosis

    PubMed Central

    XIAO, LI; LIN, FENGXIA; XIAO, RONG; HU, CHUN; DENG, MINGYANG; LI, DAIQIANG; SHE, XIAOLING; LIU, FUYOU; SUN, LIN

    2016-01-01

    The present study reports the case of a 42-year-old male with multiple myeloma (MM)-associated skin light chain amyloidosis who presented with skin purpura as the initial symptom, which was misdiagnosis as Henoch-Schönlein purpura nephritis prior to admission to the Second Xiangya Hospital (Changsha, Hunan, China). The patient presented with purpura, papules petechiae and spontaneous ecchymosis, which was located scattered around the neck, chest and limbs, accompanied by a small amount of bleeding in the conjunctival and oral mucosa, and a swollen tongue. Upon laboratory examination, the serum immunological change showed increased serum immunoglobulin G and λ light chain levels, and a urine Bence Jones protein level of >1 g/24 h. This was accompanied with an abnormal result for immunofixation electrophoresis, and positive staining with Congo red showing apple-green birefringence in skin biopsy specimens. Thus, the patient was diagnosed with MM-associated skin amyloidosis with the initial symptom of skin purpura. Following treatment with chemotherapy consisting of prednisone and bortezomib, the skin lesions markedly improved. The present study indicates that the presentation of skin purpura in systemic amyloidosis associated with MM may be an important aid in the diagnosis and direct treatment of this disease in the clinic. PMID:27284363

  19. Restrictions among heavy and light chain determinants of granulocyte-specific antinuclear factors

    PubMed Central

    Wiik, A.; Munthe, E.

    1972-01-01

    In fifty-five rheumatoid arthritis sera with positive granulocyte-specific antinuclear factors (GS-ANF) tests were made to further characterize these antibodies. All sera contained GS-ANF of the IgG class and most of the sera also of the IgM and IgA classes, whereas only about 10 per cent of the sera contained GS-ANF of the IgD class. Most of the sera contained GS-ANF carrying both κ and λ light chain determinants, but in five cases only one of the light chain subclasses could be found. The distribution of the GS-ANF among the four subclasses of IgG showed marked variations. From one to three subclasses could be lacking or noticeably depressed. There was no predominance of any one or two subclasses. Complement (C3) fixing properties correlated with GS-ANF of the IgG1 and/or IgG3 subclasses. These properties make the GS-ANF interesting as possible pathogenic factors in rheumatoid arthritis. Some evidence is presented that the GS-ANF may be directed against several different antigens in the polymorphonuclear granulocyte nuclei. PMID:4114648

  20. Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

    PubMed

    Wu, Shuang; Majeed, Sophia R; Evans, Timothy M; Camus, Marine D; Wong, Nicole M L; Schollmeier, Yvette; Park, Minjong; Muppidi, Jagan R; Reboldi, Andrea; Parham, Peter; Cyster, Jason G; Brodsky, Frances M

    2016-08-30

    Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor β receptor 2 (TGFβR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the β2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules. PMID:27540116

  1. Russell body gastritis/duodenitis: a case series and description of immunoglobulin light chain restriction.

    PubMed

    Zhang, Hejun; Jin, Zhu; Cui, Rongli

    2014-10-01

    Russell body esophago-gastro-duodenitis is an unusual form of chronic inflammation, with only 22 cases being reported in PubMed. However, the prevalence and clinical significance remain unknown. This report describes the clinico-pathological characteristics of nine cases of Russell body gastritis (RBG) and one case of Russell body duodentitis (RBD), with nonspecific endoscopic appearance. The Mott cells (plasma cells with Russell bodies) showed κ light chain restriction in eight gastritis cases and λ light chain restriction in the duodentitis case, and there were no histological features that suggested lymphoma. Thus, a diagnosis of monoclonal RBG/RBD was made. Helicobacter pylori infection was found in 55.6% of RBG cases and in the RBD case. And, the clinical follow-up evaluations were uneventful. This report is the first study to describe this benign disease entity with monoclonality on a large-scale basis. In addition, the monoclonality of Mott cells cannot be used as evidence of an existing neoplastic lesion, and taken together, these findings may indicate a reactive process. PMID:25001185

  2. CaMKII in addition to MLCK contributes to phosphorylation of regulatory light chain in cardiomyocytes.

    PubMed

    Eikemo, Hilde; Moltzau, Lise Román; Hussain, Rizwan I; Nguyen, Cam H T; Qvigstad, Eirik; Levy, Finn Olav; Skomedal, Tor; Osnes, Jan-Bjørn

    2016-02-26

    The aim was to identify kinase activities involved in the phosphorylation of regulatory light chain (RLC) in situ in cardiomyocytes. In electrically stimulated rat cardiomyocytes, phosphatase inhibition by calyculin A unmasked kinase activities evoking an increase of phosphorylated RLC (P-RLC) from about 16% to about 80% after 80 min. The phosphorylation rate in cardiomyocytes was reduced by about 40% by the myosin light chain kinase (MLCK) inhibitor, ML-7. In rat ventricular muscle strips, calyculin A induced a positive inotropic effect that correlated with P-RLC levels. The inotropic effect and P-RLC elevation were abolished by ML-7 treatment. The kinase activities phosphorylating RLC in cardiomyocytes were reduced by about 60% by the non-selective kinase inhibitor staurosporine and by about 50% by the calmodulin antagonist W7. W7 eliminated the inhibitory effect of ML-7, suggesting that the cardiac MLCK is Ca(2+)/calmodulin (CaM)-dependent. The CaM-dependent kinase II (CaMKII) inhibitor KN-93 attenuated the calyculin A-induced RLC phosphorylation by about 40%, indicating a contribution from CaMKII. The residual phosphorylation in the presence of W7 indicated that also CaM-independent kinase activities might contribute. RLC phosphorylation was insensitive to protein kinase C inhibition. In conclusion, in addition to MLCK, CaMKII phosphorylates RLC in cardiomyocytes. Involvement of other kinases cannot be excluded. PMID:26809094

  3. Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain

    PubMed Central

    Cooley, Christina B.; Ryno, Lisa M.; Plate, Lars; Morgan, Gareth J.; Hulleman, John D.; Kelly, Jeffery W.; Wiseman, R. Luke

    2014-01-01

    Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation. PMID:25157167

  4. 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. PMID:25585662

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

    PubMed Central

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

    2013-01-01

    Background/purpose 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. Methods 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. Results 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. Conclusions 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. PMID:23385633

  6. Myosin Light-Chain Kinase Is Necessary for Membrane Homeostasis in Cochlear Inner Hair Cells

    PubMed Central

    Chen, Chen; Xu, Lin; Zhang, Wen-Cheng; Fan, Chi; Peng, Ya-Jing; Chen, Jie; He, Wei-Qi; Guo, Shi-Ying; Zuo, Jian; Gao, Xia; Zhu, Min-Sheng

    2012-01-01

    The structural homeostasis of the cochlear hair cell membrane is critical for all aspects of sensory transduction, but the regulation of its maintenance is not well understood. In this report, we analyzed the cochlear hair cells of mice with specific deletion of myosin light chain kinase (MLCK) in inner hair cells. MLCK-deficient mice showed impaired hearing, with a 5- to 14-dB rise in the auditory brainstem response (ABR) thresholds to clicks and tones of different frequencies and a significant decrease in the amplitude of the ABR waves. The mutant inner hair cells produced several ball-like structures around the hair bundles in vivo, indicating impaired membrane stability. Inner hair cells isolated from the knockout mice consistently displayed less resistance to hypoosmotic solution and less membrane F-actin. Myosin light-chain phosphorylation was also reduced in the mutated inner hair cells. Our results suggest that MLCK is necessary for maintaining the membrane stability of inner hair cells. PMID:22485190

  7. Synthesis and secretion of light-chain immunoglobulin in two successive cycles of synchronized plasmacytoma cells

    PubMed Central

    1976-01-01

    Suspension-cultured mouse plasmacytoma cells (MPC-11) were accumulated in the late G1 phase by exposure to isoleucine-deficient medium for 20- 24 h. The arrested culture was fed with complete medium enabling the cells to continue the cell cycle synchronously, undergo mitosis, and enter a second cycle of growth. This method of synchronization left the protein-synthesizing ability intact as judged by the polysome profile and the capacity of the cells to incorporate labeled amino acids into protein after the restoration of isoleucine. After incubation in isoleucine-deficient medium and the addition of isoleucine to the culture, cells entered the S phase after a short lag, as judged by [3H]thymidine incorporation into nucleic acid and by spectrophotometric measurement of nuclear DNA. The cells were in mitosis between 12 and 18 h as judged by the increase in cell count and analysis of cell populations on albumin gradients. Synthesis and secretion of light- chain immunoglobulin were maximal in the late G1/early S phase of the first cycle. During late S phase, G2 phase, and mitosis, both synthesis and secretion were observed to be at a low level; however, immediately after motosis the cells which then entered the G1 phase apparently commenced synthesis of light chain immunoglobulin straight away, although secretion of labeled material remained at a low level. PMID:812877

  8. Inhibition of pathologic immunoglobulin free light chain production by small interfering RNA molecules

    PubMed Central

    Phipps, Jonathan E.; Kestler, Daniel P.; Foster, James S.; Kennel, Stephen J.; Donnell, Robert; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2010-01-01

    Objectives Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal Ig light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved anti-plasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference (RNAi). Materials and Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the CMV promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the ATCC. Both were treated with small interfering RNA (siRNA) directed specifically to the V, J, or C portions of the molecules and then analyzed by ELISA, flow cytometry and real time PCR. Results Transfected cells were found to constitutively express detectable quantities of mRNA and protein Wil and, after exposure to siRNAs, an ~40% reduction in mRNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. Conclusion Our results have shown that RNAi can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma. PMID:20637260

  9. Effect of Lysine Modification on the Stability and Cellular Binding of Human Amyloidogenic Light Chains

    SciTech Connect

    O'Neill, Hugh Michael; Davern, Sandra M.; Murphy, Charles L.; Wall, Jonathan; Deborah, Weiss T.; Solomon, Alan

    2011-01-01

    AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescent microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichorism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

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

    SciTech Connect

    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, 37 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.

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

    PubMed Central

    Jancso, A; Szent-Györgyi, A G

    1994-01-01

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

  12. Proliferative glomerulonephritis with monoclonal immunoglobulin deposition disease: The utility of routine staining with immunoglobulin light chains

    PubMed Central

    Gowda, K. K.; Nada, R.; Ramachandran, R.; Joshi, K.; Tewari, R.; Kohli, H. S.; Jha, V.; Gupta, K. L.

    2015-01-01

    Proliferative glomerulonephritis occurring as a consequence of monoclonal glomerular deposits of IgG is uncommon. It is a form of renal involvement in monoclonal gammopathy that mimics immune complex glomerulonephritis. Here, we report the first series of proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) from the Indian subcontinent highlighting use of light chain immunofluorescence (IF) in routine renal biopsy interpretation. We retrieved 6 patients diagnosed as proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) out of 160 biopsies (3.7%) with membranoproliferative patterns over 5 1/2 years (2009–2014), one of whom had recurrence 6 months post-renal transplant. Four (67%) patients presented with rapidly progressive renal failure and two (33%) with nephrotic syndrome. None of these patients had overt multiple myeloma. The predominant histologic pattern was membranoproliferative with all the biopsies showing IgG3 Kappa deposits on IF. The deposits were primarily subendothelial on electron microscopy. PMID:26664209

  13. Light chain crystalline kidney disease: diagnostic urine microscopy as the "liquid kidney biopsy".

    PubMed

    Luciano, Randy L; Castano, Ekaterina; Fogazzi, Giovanni B; Perazella, Mark A

    2014-12-01

    Multiple myeloma (MM) is a plasma cell disorder, which often causes parenchymal kidney disease. Light chain (LC) cast nephropathy represents the most common renal lesion. In some instances, LC crystals precipitate within renal tubular lumens and deposit within proximal tubular cell cytoplasms. Importantly, urine microscopy in such patients can provide insight into the underlying LC-related lesion. Here we present two patients with MM complicated by acute kidney injury (AKI) where LC crystalline casts were observed on urinary sediment analysis. Kidney biopsy revealed acute tubular injury with LC crystal casts within both tubular lumens and renal tubular epithelial cell cytoplasms. These findings suggest that the urinary sediment may be a non-invasive way to diagnose LC crystalline-induced AKI in patients with MM. PMID:25295579

  14. Proliferative glomerulonephritis with monoclonal immunoglobulin deposition disease: The utility of routine staining with immunoglobulin light chains.

    PubMed

    Gowda, K K; Nada, R; Ramachandran, R; Joshi, K; Tewari, R; Kohli, H S; Jha, V; Gupta, K L

    2015-01-01

    Proliferative glomerulonephritis occurring as a consequence of monoclonal glomerular deposits of IgG is uncommon. It is a form of renal involvement in monoclonal gammopathy that mimics immune complex glomerulonephritis. Here, we report the first series of proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) from the Indian subcontinent highlighting use of light chain immunofluorescence (IF) in routine renal biopsy interpretation. We retrieved 6 patients diagnosed as proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) out of 160 biopsies (3.7%) with membranoproliferative patterns over 5 1/2 years (2009-2014), one of whom had recurrence 6 months post-renal transplant. Four (67%) patients presented with rapidly progressive renal failure and two (33%) with nephrotic syndrome. None of these patients had overt multiple myeloma. The predominant histologic pattern was membranoproliferative with all the biopsies showing IgG3 Kappa deposits on IF. The deposits were primarily subendothelial on electron microscopy. PMID:26664209

  15. Measurement of free light chains with assays based on monoclonal antibodies.

    PubMed

    Te Velthuis, Henk; Drayson, Mark; Campbell, John P

    2016-06-01

    Recently, serum free light chain (FLC) assays incorporating anti-kappa (κ) and anti-lambda (λ) FLC monoclonal antibodies have become available: N Latex FLC assay (Siemens) and Seralite® (Abingdon Health). The purpose of this review is to provide an overview of these two new monoclonal antibody-based methods. In doing so, the review will outline the performance characteristics of each method, including a summary of: assay principles, antibody specificity, analytical performance and assay performance in disease. Additionally, the review will describe the potential user benefits of adopting these new generation FLC assays, which are designed to overcome the established limitations of existing polyclonal antibody based FLC assays. PMID:27010775

  16. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  17. Recurrent Syncope and Cardiac Arrest in a Patient with Systemic Light Chain Amyloidosis Treated with Bortezomib.

    PubMed

    Jaipaul, Navin; Pi, Alexander; Zhang, Zhiwei

    2016-05-10

    About 10-15% of patients with multiple myeloma develop light chain (AL) amyloidosis. AL amyloidosis is a systemic disease that may involve multiple organs, often including the heart. It may present clinically with bradyarrhythmia and syncope. The proteasome inhibitor bortezomib has been used with clinical efficacy in treating patients with AL amyloidosis but also implicated as a possible cause of cardiomyocyte injury. We report a case of a 48-year-old man with AL amyloidosis and increased frequency of syncope and cardiac arrest after starting bortezomib. The biologic and clinical plausibility of a heightened risk for cardiac arrest in patients with cardiac AL amyloidosis and history of syncope being treated with bortezomib is a possibility that is not well documented in the medical literature and warrants further investigation. PMID:27499835

  18. Recurrent Syncope and Cardiac Arrest in a Patient with Systemic Light Chain Amyloidosis Treated with Bortezomib

    PubMed Central

    Jaipaul, Navin; Pi, Alexander; Zhang, Zhiwei

    2016-01-01

    About 10-15% of patients with multiple myeloma develop light chain (AL) amyloidosis. AL amyloidosis is a systemic disease that may involve multiple organs, often including the heart. It may present clinically with bradyarrhythmia and syncope. The proteasome inhibitor bortezomib has been used with clinical efficacy in treating patients with AL amyloidosis but also implicated as a possible cause of cardiomyocyte injury. We report a case of a 48-year-old man with AL amyloidosis and increased frequency of syncope and cardiac arrest after starting bortezomib. The biologic and clinical plausibility of a heightened risk for cardiac arrest in patients with cardiac AL amyloidosis and history of syncope being treated with bortezomib is a possibility that is not well documented in the medical literature and warrants further investigation. PMID:27499835

  19. Kinesin's light chains inhibit the head- and microtubule-binding activity of its tail.

    PubMed

    Wong, Yao Liang; Rice, Sarah E

    2010-06-29

    Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail's regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1's role in microtubule transport/sliding. PMID:20547877

  20. Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

    PubMed Central

    Wong, Yao Liang; Rice, Sarah E.

    2010-01-01

    Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail’s regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1’s role in microtubule transport/sliding. PMID:20547877

  1. Conventional Kinesin Holoenzymes Are Composed of Heavy and Light Chain Homodimers†

    PubMed Central

    DeBoer, Scott R.; You, YiMei; Szodorai, Anita; Kaminska, Agnieszka; Pigino, Gustavo; Nwabuisi, Evelyn; Wang, Bin; Estrada-Hernandez, Tatiana; Kins, Stefan; Brady, Scott T.; Morfini, Gerardo

    2009-01-01

    Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations. PMID:18361505

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

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII. PMID:23244324

  3. The regulation of RhoGEF Lfc by dynein light chain Tctex-1

    NASA Astrophysics Data System (ADS)

    Balan, Marc

    Lfc is a guanine nucleotide exchange factor (GEF) that activates the small GTPase RhoA, and its GEF activity is tightly regulated through protein-protein interactions, phosphorylation, and cellular localization. Lfc is anchored to microtubules through its interaction with the dynein light chain Tctex-1, which results in inhibition of Lfc's GEF activity. Here we present a crystallographic structure of Tctex-1 in complex with Lfc with residues 143-155 of Lfc bound at the Tctex-1 dimer interface. Structural alignment of our structure with Tctex-1 in complex with the dynein intermediate chain (DIC) shows the binding site of the DIC peptide and Lfc substantially overlap. Biochemical evidence, NMR perturbations assays and intrinsic fluorescence provide structural validation and support an extension of the Lfc binding site to the andalpha;-helices that may accommodate additional contact points with Tctex-1. We postulate a potential mechanism for Lfcandrsquo;s recruitment to the microtubules through a tripartite complex with Tctex-1 and DIC.

  4. Cooperative exosite-dependent cleavage of synaptobrevin by tetanus toxin light chain.

    PubMed

    Cornille, F; Martin, L; Lenoir, C; Cussac, D; Roques, B P; Fournie-Zaluski, M C

    1997-02-01

    The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 and COOH terminus of synaptobrevin have shown that the sequence 39-88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S1) and S 82-93 (S2), to the synaptobrevin fragment S 56-81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins. PMID:9013591

  5. Structure and function of outer dynein arm intermediate and light chain complex

    PubMed Central

    Oda, Toshiyuki; Abe, Tatsuki; Yanagisawa, Haruaki; Kikkawa, Masahide

    2016-01-01

    The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella. Chlamydomonas ODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo–electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs. PMID:26864626

  6. Prevalence and progression of monoclonal gammopathy of undetermined significance and light-chain MGUS in Germany.

    PubMed

    Eisele, Lewin; Dürig, Jan; Hüttmann, Andreas; Dührsen, Ulrich; Assert, Roland; Bokhof, Beate; Erbel, Raimund; Mann, Klaus; Jöckel, Karl-Heinz; Moebus, Susanne

    2012-02-01

    We determined the prevalence and progression rate of monoclonal gammopathy of undetermined significance (MGUS) and light-chain MGUS (LCMGUS) in Germany utilizing the biobank of the population-based Heinz Nixdorf Recall Study. The Heinz Nixdorf Recall Study comprises 4,814 men and women aged 45-75 years. To detect monoclonal proteins, standard serum electrophoresis was combined with parallel screening immunofixation using pentavalent antisera. Additionally, free light chains (FLC) were measured in all samples. Definition of MGUS included M-protein concentration, laboratory results, and disease history. LCMGUS was defined as abnormal FLC ratio, increase in FLC causing the abnormal ratio, and lack of intact immunoglobulin. One hundred sixty-five MGUS cases were identified among 4,702 screened samples (prevalence 3.5%, 95% confidence interval (CI) 3.0-4.1; median age 63 years, range 47-75 years; 103 (62%) male; IgG 59%, IgA 17%, IgM 17%, biclonal 4.8%, kappa 56%, and lambda 44%). Five cases progressed (0.6%/year, 95% CI 0.2-1.4). An abnormal FLC ratio was detected in 220 samples. Thirty-nine of these showed intact immunoglobulin. Thirty-four of the remaining met LCMGUS criteria (prevalence 0.7%, 95% CI 0.5-1.0). None of the LCMGUS cases progressed. We demonstrate a MGUS prevalence of 3.5% and a LCMGUS prevalence of 0.7% in the general population aged 45-75 years in Germany using a sensitive screening approach. PMID:21789623

  7. Association between Free Light Chain Levels, and Disease Progression and Mortality in Chronic Kidney Disease

    PubMed Central

    Desjardins, Lucie; Liabeuf, Sophie; Lenglet, Aurélie; Lemke, Horst-Dieter; Vanholder, Raymond; Choukroun, Gabriel; Massy, Ziad A.

    2013-01-01

    Immunoglobulin free light chains (FLCs) form part of the middle molecule group of uremic toxins. Accumulation of FLCs has been observed in patients with chronic kidney disease (CKD). The aim of the present study was to measure FLC levels in patients at different CKD stages and to assess putative associations between FLC levels on one hand and biochemical/clinical parameters and mortality on the other. One hundred and forty patients at CKD stages 2-5D were included in the present study. Routine clinical biochemistry assays and assays for FLC kappa (κ) and lambda (λ) and other uremic toxins were performed. Vascular calcification was evaluated using radiological techniques. The enrolled patients were prospectively monitored for mortality. Free light chain κ and λ levels were found to be elevated in CKD patients (especially in those on hemodialysis). Furthermore, FLC κ and λ levels were positively correlated with inflammation, aortic calcification and the levels of various uremic toxins levels. A multivariate linear regression analysis indicated that FLC κ and λ levels were independently associated with CKD stages and β2 microglobulin levels. Elevated FLC κ and λ levels appeared to be associated with mortality. However, this association disappeared after adjustment for a propensity score including age, CKD stage and aortic calcification. In conclusion, our results indicate that FLC κ and λ levels are elevated in CKD patients and are associated with inflammation, vascular calcification and levels of other uremic toxins. The observed link between elevated FLC levels and mortality appears to depend on other well-known factors. PMID:24217396

  8. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

    PubMed Central

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta

    2011-01-01

    The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-Δ43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing (∼1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I1,1/I1,0, indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-Δ43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-Δ43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.—Muthu, P., Wang, L., Yuan, C.-C., Kazmierczak, K., Huang, W., Hernandez, O. M., Kawai, M., Irving, T. C., Szczesna-Cordary, D. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction. PMID:21885653

  9. Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

    SciTech Connect

    Zhu, Haizhong; Lee, Han Youl; Tong, Yufeng; Hong, Bum-Soo; Kim, Kyung-Phil; Shen, Yang; Lim, Kyung Jik; Mackenzie, Farrell; Tempel, Wolfram; Park, Hee-Won

    2012-10-23

    Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form 'a carboxylate clamp' with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins.

  10. Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice.

    PubMed

    Lai, Shaojuan; Collins, Brittany C; Colson, Brett A; Kararigas, Georgios; Lowe, Dawn A

    2016-05-01

    Impairment of skeletal muscle function has been associated with changes in ovarian hormones, especially estradiol. To elucidate mechanisms of estradiol on skeletal muscle strength, the hormone's effects on phosphorylation of the myosin regulatory light chain (pRLC) and muscle contractility were investigated, hypothesizing an estradiol-specific beneficial impact. In a skeletal muscle cell line, C2C12, pRLC was increased by 17β-estradiol (E2) in a concentration-dependent manner. In skeletal muscles of C57BL/6 mice that were E2 deficient via ovariectomy (OVX), pRLC was lower than that from ovary-intact, sham-operated mice (Sham). The reduced pRLC in OVX muscle was reversed by in vivo E2 treatment. Posttetanic potentiation (PTP) of muscle from OVX mice was low compared with that from Sham mice, and this decrement was reversed by acute E2 treatment, demonstrating physiological consequence. Western blot of those muscles revealed that low PTP corresponded with low pRLC and higher PTP with greater pRLC. We aimed to elucidate signaling pathways affecting E2-mediated pRLC using a kinase inhibitor library and C2C12 cells as well as a specific myosin light chain kinase inhibitor in muscles. PI3K/Akt, MAPK, and CamKII were identified as candidate kinases sensitive to E2 in terms of phosphorylating RLC. Applying siRNA strategy in C2C12 cells, pRLC triggered by E2 was found to be mediated by estrogen receptor-β and the G protein-coupled estrogen receptor. Together, these results provide evidence that E2 modulates myosin pRLC in skeletal muscle and is one mechanism by which this hormone can affect muscle contractility in females. PMID:26956186

  11. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

    PubMed

    Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H Lee; Kamm, Kristine E; Stull, James T

    2016-07-01

    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  12. Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle.

    PubMed

    Kampourakis, Thomas; Irving, Malcolm

    2015-08-01

    The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone. PMID:26057075

  13. Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle

    PubMed Central

    Kampourakis, Thomas; Irving, Malcolm

    2015-01-01

    The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone. PMID:26057075

  14. Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: is resting calcium responsible?

    PubMed

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

    2013-03-01

    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 Ca(2+) concentration ([Ca(2+)](i)) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca(2+)-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca(2+) 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 [Ca(2+)](i). Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca(2+) 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 [Ca(2+)](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

  15. In Vitro Aggregation Behavior of a Non-Amyloidogenic λ Light Chain Dimer Deriving from U266 Multiple Myeloma Cells

    PubMed Central

    Arosio, Paolo; Owczarz, Marta; Müller-Späth, Thomas; Rognoni, Paola; Beeg, Marten; Wu, Hua; Salmona, Mario; Morbidelli, Massimo

    2012-01-01

    Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature Tm at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO4−≫Cl−>H2PO4−, confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding. PMID:22432016

  16. In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells.

    PubMed

    Arosio, Paolo; Owczarz, Marta; Müller-Späth, Thomas; Rognoni, Paola; Beeg, Marten; Wu, Hua; Salmona, Mario; Morbidelli, Massimo

    2012-01-01

    Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T(m) at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4)(-)≫Cl(-)>H(2)PO(4)(-), confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding. PMID:22432016

  17. Biphenotypic plasma cell myeloma: two cases of plasma cell neoplasm with a coexpression of kappa and lambda light chains

    PubMed Central

    Jiwani, Shahanawaz; Bornhost, Joshua; Alapat, Daisy

    2015-01-01

    Plasma cell neoplasm (PCM) is a medullary and extra medullary proliferation of clonal plasma cells that occurs due to accidental translocation of proto-oncogenes into immunoglobulin (Ig) gene loci. While the majority of plasma cell neoplasms are monoclonal, up to 2% of the PCMs [1] considered being biclonal based on electrophoretic analysis, characterized by secretion of paraprotein with two distinct heavy chains or light chains are possible and present unique diagnostic challenges. Methods: Traditionally protein electrophoresis has been used to diagnose, characterize, and monitor progression of plasma cell neoplasm. To characterize neoplastic plasma cells, in our institution, other ancillary studies, including in situ hybridization, flow cytometric analyses of plasma cell surface markers and cytoplasmic immunoglobulins with DNA ploidy, are also utilized routinely. Results: We present two cases of plasma cell myeloma in which the neoplastic plasma cells shows production of cytoplasmic kappa and lambda light chain, with secretion of free lambda light chain only. Co-expression of kappa and lambda light chain by the same neoplastic plasma cells is a rare but reported phenomenon. Conclusions: Our study indicates that serum electrophoresis alone could mischaracterize biphenotypic myeloma as monotypic plasma cell myelomas in the absence of additional testing methods. PMID:26339430

  18. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG

    PubMed Central

    Fischer, Nicolas; Elson, Greg; Magistrelli, Giovanni; Dheilly, Elie; Fouque, Nicolas; Laurendon, Amélie; Gueneau, Franck; Ravn, Ulla; Depoisier, Jean-François; Moine, Valery; Raimondi, Sylvain; Malinge, Pauline; Di Grazia, Laura; Rousseau, François; Poitevin, Yves; Calloud, Sébastien; Cayatte, Pierre-Alexis; Alcoz, Mathias; Pontini, Guillemette; Fagète, Séverine; Broyer, Lucile; Corbier, Marie; Schrag, Delphine; Didelot, Gérard; Bosson, Nicolas; Costes, Nessie; Cons, Laura; Buatois, Vanessa; Johnson, Zoe; Ferlin, Walter; Masternak, Krzysztof; Kosco-Vilbois, Marie

    2015-01-01

    Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies. PMID:25672245

  19. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG.

    PubMed

    Fischer, Nicolas; Elson, Greg; Magistrelli, Giovanni; Dheilly, Elie; Fouque, Nicolas; Laurendon, Amélie; Gueneau, Franck; Ravn, Ulla; Depoisier, Jean-François; Moine, Valery; Raimondi, Sylvain; Malinge, Pauline; Di Grazia, Laura; Rousseau, François; Poitevin, Yves; Calloud, Sébastien; Cayatte, Pierre-Alexis; Alcoz, Mathias; Pontini, Guillemette; Fagète, Séverine; Broyer, Lucile; Corbier, Marie; Schrag, Delphine; Didelot, Gérard; Bosson, Nicolas; Costes, Nessie; Cons, Laura; Buatois, Vanessa; Johnson, Zoe; Ferlin, Walter; Masternak, Krzysztof; Kosco-Vilbois, Marie

    2015-01-01

    Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies. PMID:25672245

  20. Genetically engineering cyanobacteria to convert CO₂, water, and light into the long-chain hydrocarbon farnesene.

    PubMed

    Halfmann, Charles; Gu, Liping; Gibbons, William; Zhou, Ruanbao

    2014-12-01

    Genetically engineered cyanobacteria offer a shortcut to convert CO2 and H2O directly into biofuels and high value chemicals for societal benefits. Farnesene, a long-chained hydrocarbon (C15H24), has many applications in lubricants, cosmetics, fragrances, and biofuels. However, a method for the sustainable, photosynthetic production of farnesene has been lacking. Here, we report the photosynthetic production of farnesene by the filamentous cyanobacterium Anabaena sp. PCC 7120 using only CO2, mineralized water, and light. A codon-optimized farnesene synthase gene was chemically synthesized and then expressed in the cyanobacterium, enabling it to synthesize farnesene through its endogenous non-mevalonate (MEP) pathway. Farnesene excreted from the engineered cyanobacterium volatilized into the flask head space and was recovered by adsorption in a resin column. The maximum photosynthetic productivity of farnesene was 69.1 ± 1.8 μg·L(-1)·O.D.(-1)·d(-1). Compared to the wild type, the farnesene-producing cyanobacterium also exhibited a 60 % higher PSII activity under high light, suggesting increased farnesene productivity in such conditions. We envision genetically engineered cyanobacteria as a bio-solar factory for photosynthetic production of a wide range of biofuels and commodity chemicals. PMID:25301585

  1. The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

    PubMed Central

    Blisnick, Thierry; Buisson, Johanna; Absalon, Sabrina; Marie, Alexandra; Cayet, Nadège; Bastin, Philippe

    2014-01-01

    Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex. PMID:24989795

  2. The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions.

    PubMed

    Blisnick, Thierry; Buisson, Johanna; Absalon, Sabrina; Marie, Alexandra; Cayet, Nadège; Bastin, Philippe

    2014-09-01

    Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140(RNAi) mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex. PMID:24989795

  3. Antibody light chain variable domains and their biophysically improved versions for human immunotherapy.

    PubMed

    Kim, Dae Young; To, Rebecca; Kandalaft, Hiba; Ding, Wen; van Faassen, Henk; Luo, Yan; Schrag, Joseph D; St-Amant, Nadereh; Hefford, Mary; Hirama, Tomoko; Kelly, John F; MacKenzie, Roger; Tanha, Jamshid

    2014-01-01

    We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (T(m)), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their T(m)s, by 5.5-17.5 ° C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach's acidic pH and pepsin. PMID:24423624

  4. MODULAR STRUCTURE OF SMOOTH MUSCLE MYOSIN LIGHT CHAIN KINASE: HYDRODYNAMIC MODELING AND FUNCTIONAL IMPLICATIONS†

    PubMed Central

    Mabuchi, Yasuko; Mabuchi, Katsuhide; Stafford, Walter F.; Grabarek, Zenon

    2010-01-01

    Smooth muscle myosin light chain kinase (smMLCK) is a calcium/calmodulin dependent enzyme that activates contraction of smooth muscle. The polypeptide chain of rabbit uterine smMLCK (Swiss-Prot: P29294) contains the catalytic/regulatory domain, three immunoglobulin related motifs (Ig), one fibronectin related motif (Fn3), a repetitive, proline rich segment (PEVK) and, at the N-terminus, a unique F-actin binding domain. We have evaluated the spatial arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its two catalytically active C-terminal fragments (77 kDa, residues 461-1147 and 61 kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to F-actin show particles at variable distance (11-55 nm) from the filament, suggesting that a well-structured C-terminal segment of smMLCK is connected to the actin-binding domain by a long, flexible tether. We have used structural homology and molecular dynamics methods to construct various all-atom representation models of smMLCK and its two fragments. The theoretical sedimentation coefficients computed with the program HYDROPRO were compared with those determined by sedimentation velocity. We found agreement between the predicted and observed sedimentation coefficients for models in which the independently folded catalytic domain, Fn3 and Ig domains are aligned consecutively on the long axis of the molecule. The PEVK segment is modeled as an extensible linker that enables smMLCK to remain bound to F-actin and simultaneously activate the myosin heads of adjacent myosin filaments at a distance of 40 nm or more. The structural properties of smMLCK may contribute to the elasticity of smooth muscle cells. PMID:20196616

  5. Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

    PubMed Central

    Kim, Dae Young; To, Rebecca; Kandalaft, Hiba; Ding, Wen; van Faassen, Henk; Luo, Yan; Schrag, Joseph D; St-Amant, Nadereh; Hefford, Mary; Hirama, Tomoko; Kelly, John F; MacKenzie, Roger; Tanha, Jamshid

    2014-01-01

    We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (Tm), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their Tms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin. PMID:24423624

  6. A Strategy for Synthesis of Pathogenic Human Immunoglobulin Free Light Chains in E. coli

    PubMed Central

    Rognoni, Paola; Lavatelli, Francesca; Casarini, Simona; Palladini, Giovanni; Verga, Laura; Pedrazzoli, Paolo; Valentini, Giovanna; Merlini, Giampaolo; Perfetti, Vittorio

    2013-01-01

    Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine (“free” LC). Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders. PMID:24086679

  7. Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis.

    PubMed

    Jaskowski, Troy D; Litwin, Christine M; Hill, Harry R

    2006-02-01

    Recently, turbidimetric immunoassays for detecting and quantifying kappa and lambda free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound kappa and lambda light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the kappa-FLC and lambda-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal kappa/lambda FLC ratios using the turbidimetric immunoassays. In conclusion, the kappa and lambda FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the kappa/lambda ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal kappa/lambda FLC ratios. PMID:16467338

  8. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

    PubMed

    Del Coso, Juan; Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  9. Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice.

    PubMed

    Yuan, Chen-Ching; Muthu, Priya; Kazmierczak, Katarzyna; Liang, Jingsheng; Huang, Wenrui; Irving, Thomas C; Kanashiro-Takeuchi, Rosemeire M; Hare, Joshua M; Szczesna-Cordary, Danuta

    2015-07-28

    Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 → Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. We hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In support of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca(2+) sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype. PMID:26124132

  10. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon

    PubMed Central

    Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  11. C1q and systemic lupus erythematosus.

    PubMed

    Walport, M J; Davies, K A; Botto, M

    1998-08-01

    In this chapter we review the association between SLE and C1q. In the first part of the chapter we discuss the clinical associations of C1q deficiency, and tabulate the available information in the literature relating to C1q deficiency and autoimmune disease. Other clinical associations of C1q deficiency are then considered, and we mention briefly the association between other genetically determined complement deficiencies and lupus. In the review we explore the relationship between C1q consumption and lupus and we discuss the occurrence of low molecular weight (7S) C1q in lupus, which raises the possibility that increased C1q turnover in the disease may result in unbalanced chain synthesis of the molecule. Anti-C1q antibodies are also strongly associated with severe SLE affecting the kidney, and with hypocomplementaemic urticarial vasculitis, and these associations are also examined. We address the question of how C1q deficiency may cause SLE, discussing the possibility that this may be due to abnormalities of immune complex processing, which have been well characterised in a umber of different human models. There is clear evidence that immune complex processing is abnormal in patients with hypocomplementaemia, and this is compatible with the hypothesis that ineffective immune complex clearance could cause tissue injury, and this may in turn stimulate an autoantibody response. We have also considered the possibility that C1q-C1q receptor interactions are critical in the regulation of apoptosis, and we explore the hypothesis that dysregulation of apoptosis could explain important features in the development of autoimmune disease associated with C1q deficiency. An abnormally high rate of apoptosis, or defective clearance of apoptotic cells, could promote the accumulation of abnormal cellular products that might drive an autoimmune response. Anti-C1q antibodies have been described in a number of murine models of lupus, and these are also briefly discussed. We focus

  12. pH and light-controlled self-assembly of bistable [c2] daisy chain rotaxanes.

    PubMed

    Wolf, Adrian; Moulin, Emilie; Cid, Juan-José; Goujon, Antoine; Du, Guangyan; Busseron, Eric; Fuks, Gad; Giuseppone, Nicolas

    2015-03-11

    A logic gate based on a bistable [c2] daisy chain rotaxane decorated with lateral triarylamine units is described, giving rise to an INHIBIT logic function using proton concentration and light as inputs, and producing dual color change and supramolecular self-assembly as outputs. PMID:25661046

  13. Hematogones With Lambda Light Chain Restriction in a 4-Year-Old Boy With Burkitt Lymphoma: A Potential Diagnostic Pitfall.

    PubMed

    Guillory, Tesha; Li, Shiyong; Bergsagel, Daniel J; Weinzierl, Elizabeth; Bunting, Silvia T

    2016-05-01

    Hematogones are immature normal B cell precursors with a characteristic immunophenotype profile on flow cytometry that typically do not express surface immunoglobulin light chains. In this report, we describe a case in which the hematogones exhibit light chain restriction. Our patient was a 4-year-old boy with a complicated medical history involving treatment for a presumed bilateral Wilms tumor of the kidney that on later resection was diagnosed as Burkitt lymphoma. Flow cytometry analysis of his bone marrow revealed a small distinct population of cells expressing dim cluster of differentiation (CD)10, CD19, CD22, CD38, dim CD58, human leukocyte antigen-D related (HLA-DR), and dim CD45, which are characteristic of hematogones. These cells, however, demonstrated dim surface immunoglobulin lambda light-chain restriction. Molecular study results for immunoglobulin heavy and kappa light-chain gene rearrangements were negative. We present this case to raise awareness of the potential pitfalls of working up bone marrow for involvement by B cell lymphoproliferative disorder. PMID:27069035

  14. Hematogones With Lambda Light Chain Restriction in a 4-Year-Old Boy With Burkitt Lymphoma: A Potential Diagnostic Pitfall

    PubMed Central

    Guillory, Tesha; Li, Shiyong; Bergsagel, Daniel J.; Weinzierl, Elizabeth; Bunting, Silvia T.

    2016-01-01

    Hematogones are immature normal B cell precursors with a characteristic immunophenotype profile on flow cytometry that typically do not express surface immunoglobulin light chains. In this report, we describe a case in which the hematogones exhibit light chain restriction. Our patient was a 4-year-old boy with a complicated medical history involving treatment for a presumed bilateral Wilms tumor of the kidney that on later resection was diagnosed as Burkitt lymphoma. Flow cytometry analysis of his bone marrow revealed a small distinct population of cells expressing dim cluster of differentiation (CD)10, CD19, CD22, CD38, dim CD58, human leukocyte antigen–D related (HLA-DR), and dim CD45, which are characteristic of hematogones. These cells, however, demonstrated dim surface immunoglobulin lambda light-chain restriction. Molecular study results for immunoglobulin heavy and kappa light-chain gene rearrangements were negative. We present this case to raise awareness of the potential pitfalls of working up bone marrow for involvement by B cell lymphoproliferative disorder. PMID:27069035

  15. Sequence analysis of the myosin regulatory light chain gene of the vestimentiferan Riftia pachyptila.

    PubMed

    Ravaux, J; Hassanin, A; Deutsch, J; Gaill, F; Markmann-Mulisch, U

    2001-01-24

    We have isolated and characterized a cDNA (DNA complementary to RNA) clone (Rf69) from the vestimentiferan Riftia pachyptila. The cDNA insert consists of 1169 base pairs. The aminoacid sequence deduced from the longest reading frame is 193 residues in length, and clearly characterized it as a myosin regulatory light chain (RLC). The RLC primary structure is described in relation to its function in muscle contraction. The comparison with other RLCs suggested that Riftia myosin is probably regulated through its RLC either by phosphorylation like the vertebrate smooth muscle myosins, and/or by Ca2+-binding like the mollusk myosins. Riftia RLC possesses a N-terminal extension lacking in all other species besides the earthworm Lumbricus terrestris. Aminoacid sequence comparisons with a number of RLCs from vertebrates and invertebrates revealed a relatively high identity score (64%) between Riftia RLC and the homologous gene from Lumbricus. The relationships between the members of the myosin RLCs were examined by two phylogenetic methods, i.e. distance matrix and maximum parsimony. The resulting trees depict the grouping of the RLCs according to their role in myosin activity regulation. In all trees, Riftia RLC groups with RLCs that depend on Ca2+-binding for myosin activity regulation. PMID:11223252

  16. Investigating heart-specific toxicity of amyloidogenic immunoglobulin light chains: A lesson from C. elegans

    PubMed Central

    Diomede, Luisa; Rognoni, Paola; Lavatelli, Francesca; Romeo, Margherita; di Fonzo, Andrea; Foray, Claudia; Fiordaliso, Fabio; Palladini, Giovanni; Valentini, Veronica; Perfetti, Vittorio; Salmona, Mario; Merlini, Giampaolo

    2014-01-01

    Abnormalities in protein folding are involved in many localized and systemic diseases, all of which are characterized by insoluble amyloid formation and deposition. In immunoglobulin light chain (LC) amyloidosis, the most frequent systemic form of amyloidosis, the amyloid involvement of the heart dictates the prognosis and the elucidation of the mechanism of heart targeting and toxicity is essential for designing and testing new effective treatments. To this end, the availability of an appropriate animal model is crucial. We recently described the use of C. elegans as an innovative experimental system to investigate in vivo the pathogenic effects of monoclonal LC. This idea stems from the knowledge that the worm's pharynx is an “ancestral heart” with the additional ability to recognize stressor compounds. The feeding of worms with LC purified from patients suffering from cardiomyopathy, selectively and permanently impaired the pharyngeal function. This irreversible damage resulted in time, in a significant reduction in the lifespan of worms. We also reported that the ability of LC to generate reactive oxygen species was associated with their toxic effects and was counteracted by anti-oxidant compounds. This new nematode-based assay represents a promising model for elucidating the heart-specific toxicity of LC and for a rapid screening of new therapeutic strategies. PMID:26430549

  17. Burden of cytogenetically abnormal plasma cells in light chain amyloidosis and their prognostic relevance.

    PubMed

    Kim, Seon Young; Im, Kyongok; Park, Si Nae; Kim, Jung-Ah; Yoon, Sung-Soo; Lee, Dong Soon

    2016-05-01

    We performed cytoplasmic fluorescence in situ hybridization assays of light chain amyloidosis (AL). In total, 234 patients were enrolled: 28 patients with AL, 24 with monoclonal gammopathy of undetermined significance (MGUS), and 182 with multiple myeloma (MM). Chromosomal abnormalities were detected in 13 of 22 (59%) AL patients without MM. All 13 patients demonstrated IGH rearrangement, and t(11;14)/IGH-CCND1 was most frequent (32%). Chromosome gain was not observed in AL patients without MM. These findings were dissimilar to findings in MGUS patients, in whom trisomy 9 was the most frequent abnormality. Of 6 AL patients with MM, 5 (83%) patients had cytogenetic abnormalities: 1q gain (4/6, 67%), gains of chromosome 9 (3/6, 50%), IGH rearrangement and RB1 (13q) deletions (2/6 each, 33%). The percentage of clonal plasma cells among total plasma cells was variable (median, 75%; range, 16-100%) for AL patients without MM, which was lower than the results for MM patients (median 100%). The overall survival of AL patients without MM was not significantly different according to the presence of cytogenetic abnormalities (P=0.510). In summary, among Korean AL patients, IGH rearrangement was the most frequent cytogenetic abnormality and cytogenetic aberration patterns differ compared with MGUS and MM patients. PMID:27015231

  18. Phenotypic, transcriptomic, and genomic features of clonal plasma cells in light-chain amyloidosis.

    PubMed

    Paiva, Bruno; Martinez-Lopez, Joaquin; Corchete, Luis A; Sanchez-Vega, Beatriz; Rapado, Inmaculada; Puig, Noemi; Barrio, Santiago; Sanchez, Maria-Luz; Alignani, Diego; Lasa, Marta; García de Coca, Alfonso; Pardal, Emilia; Oriol, Alberto; Garcia, Maria-Esther Gonzalez; Escalante, Fernando; González-López, Tomás J; Palomera, Luis; Alonso, José; Prosper, Felipe; Orfao, Alberto; Vidriales, Maria-Belen; Mateos, María-Victoria; Lahuerta, Juan-Jose; Gutierrez, Norma C; San Miguel, Jesús F

    2016-06-16

    Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n = 11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs. PMID:27069257

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

    PubMed

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

    2016-07-01

    POEMS syndrome is a rare plasma cell dyscrasia. Serum concentrations of the monoclonal protein in this disorder are typically low, and inapplicable to monitor disease activity in most cases, resulting in limited practical and prognostic values. Novel immunoassays measuring isotype-specific heavy/light chain (HLC) pairs showed its utility in disease monitoring and outcome prediction in several plasma cell dyscrasias. We report results of HLC measurements in 90 patients with POEMS syndrome. Sixty-six patients (73%; 95% confidence interval, 63-82%) had an abnormal HLC ratio at baseline. It could stratify the risk of disease relapse and was strongly associated with worse progression-free survival in a multivariate analysis (P = 0.021; hazard ratio [HR] 6.89, 95% CI 1.34-35.43). After therapy, HLC ratios improved, with 43 patients (48%) remaining abnormal. The post-therapeutic HLC ratio, if abnormal, also remained as an independent prognostic factor associated with worse progression-free survival (P = 0.019; HR 4.30, 95% CI 1.27-14.56). These results suggest the prognostic utility of HLC ratios in clinical management of POEMS patients. PMID:26383741

  20. Essential myosin light chain as a target for caspase-3 in failing myocardium

    PubMed Central

    Moretti, Alessandra; Weig, Hans-Jörg; Ott, Thomas; Seyfarth, Melchior; Holthoff, Hans-Peter; Grewe, Diana; Gillitzer, Angelika; Bott-Flügel, Lorenz; Schömig, Albert; Ungerer, Martin; Laugwitz, Karl-Ludwig

    2002-01-01

    Programmed cell death involves the activation of caspase proteases that can mediate the cleavage of vital cytoskeletal proteins. We have recently reported that, in failing cardiac myocytes, caspase-3 activation is associated with a reduction in contractile performance. In this study we used a modified yeast two-hybrid system to screen for caspase-3 interacting proteins of the cardiac cytoskeleton. We identified ventricular essential myosin light chain (vMLC1) as a target for caspase-3. By sequencing and site-directed mutagenesis, a noncanonical cleavage site for caspase-3 was mapped to the C-terminal DFVE135G motif. We demonstrated that vMLC1 cleavage in failing myocardium in vivo is associated with a morphological disruption of the organized vMLC1 staining of sarcomeres, and with a reduction in myocyte contractile performance. Adenoviral gene transfer of the caspase inhibitor p35 in vivo prevented caspase-3 activation and vMLC1 cleavage, with positive impact on contractility. These data suggest that direct cleavage of vMLC1 by activated caspase-3 may contribute to depression of myocyte function by altering cross-bridge interaction between myosin and actin molecules. Therefore, activation of apoptotic pathways in the heart may lead to contractile dysfunction before cell death. PMID:12186978

  1. Immunoglobulin Heavy-And Light-chain Repertoire in Splenic Marginal Zone Lymphoma

    PubMed Central

    Stamatopoulos, Kostas; Belessi, Chrysoula; Papadaki, Theodora; Kalagiakou, Evangelia; Stavroyianni, Niki; Douka, Vassiliki; Afendaki, Stavroula; Saloum, Riad; Parasi, Aikaterini; Anagnostou, Dimitra; Laoutaris, Nikolaos; Fassas, Athanasios; Anagnostopoulos, Achilles

    2004-01-01

    The considerable heterogeneity in morphology, immunophenotype, genotype, and clinical behavior of splenic marginal zone lymphoma (SMZL) hinders firm conclusions on the origin and differentiation stage of the neoplastic cells. Immunoglobulin (IG) gene usage and somatic mutation patterns were studied in a series of 43 SMZL cases. Clonal IGHV-D-J rearrangements were amplified in 42/43 cases (4 cases carried double rearrangements). Among IGHV-D-J rearrangements, IGHV3 and IGHV4 subgroup genes were used with the highest frequency. Nineteen IGHV genes were unmutated (>98% homology to the closest germline IGHV gene), whereas 27/46 were mutated. Clonal IGKV-J and IGLV-J gene rearrangements were amplified in 36/43 cases, including 31 IGKV-J (8/31 in lambda light-chain expressing cases) and 12 IGLV-J rearrangements; 9/31 IGKV and 6/12 IGLV sequences were mutated. IGKV-J and IGLV-J rearrangements used 14 IGKV and 9 IGLV different germline genes. Significant evidence for positive selection by classical T-dependent antigen was found in only 5/27 IGHV and 6/15 IGKV+IGLV mutated genes. These results provide evidence for the diverse B-cell subpopulations residing in the SMZ, which could represent physiologic equivalents of distinct SMZL subtypes. Furthermore, they indicate that in SMZL, as in other B cell malignancies, a complementarity imprint of antigen selection might be witnessed either by IGHV, IGKV, or IGLV rearranged sequences. PMID:15706403

  2. Plasma membrane localization signals in the light chain of botulinum neurotoxin

    PubMed Central

    Fernández-Salas, Ester; Steward, Lance E.; Ho, Helen; Garay, Patton E.; Sun, Sarah W.; Gilmore, Marcella A.; Ordas, Joseph V.; Wang, Joanne; Francis, Joseph; Aoki, K. Roger

    2004-01-01

    Botulinum neurotoxin (BoNT) is a potent biological substance used to treat neuromuscular and pain disorders. Both BoNT type A and BoNT type E display high-affinity uptake into motor neurons and inhibit exocytosis through cleavage of the synaptosome-associated protein of 25 kDa (SNAP25). The therapeutic effects of BoNT/A last from 3 to 12 months, whereas the effects of BoNT/E last less than 4 weeks. Using confocal microscopy and site-specific mutagenesis, we have determined that the protease domain of BoNT/A light chain (BoNT/A-LC) localizes in a punctate manner to the plasma membrane, colocalizing with the cleaved product, SNAP25197. In contrast, the short-duration BoNT/E serotype is cytoplasmic. Mutations in the BoNT/A-LC have revealed sequences at the N terminus necessary for plasma membrane localization, and an active dileucine motif in the C terminus that is likely involved in trafficking and interaction with adaptor proteins. These data support sequence-specific signals as determinants of intracellular localization and as a basis for the different durations of action in these two BoNT serotypes. PMID:14982988

  3. Olanzapine May Inhibit Colonic Motility Associated with the 5-HT Receptor and Myosin Light Chain Kinase

    PubMed Central

    Zhang, Jiarui; Qiao, Ying; Le, Jingjing

    2016-01-01

    Objective To study whether the effects of olanzapine on gastrointestinal motility is related to the serotonin antagonism and myosin light chain kinase. Methods Male Sprague-Dawley rats were randomly divided into four groups. Olanzapine gavage was performed for each treatment group during the course of 30 continuous days, while the same volume of saline was given to the rats in the control group. Defecation of the rats was observed on days 7 and 30 after olanzapine gavage. The effects of olanzapine on contraction of colonic smooth muscles were observed in ex vivo experiments. A Western blot was used to evaluate expression levels of the serotonin transporter (SERT) and MLCK in colon segments of the rats. Results ResultsaaCompared to the control group, 5-160 µ M of olanzapine could inhibit dose-dependently the contraction of colonic smooth muscle ex vivo experiments. The maximum smooth muscle contraction effects of 5-HT and acetylcholine significantly decreased after treatment with 40-160 µ M of olanzapine. Constipation was found in the olanzapine-treated rats on day 7 and have sustained day 30 after gavage. Expression of MLCK in olanzapine-treated rats was significantly decreased, whereas the expression of SERT significantly increased on the day 7, then significantly decreased on the day 30 after olanzapine gavage. Conclusion SERT and MLCK may involve in the inhibition of colonic contraction induced by olanzapine. PMID:27081386

  4. Biofunctionalized gold nanoparticles for colorimetric sensing of botulinum neurotoxin A light chain.

    PubMed

    Liu, Xiaohu; Wang, Yi; Chen, Peng; Wang, Yusong; Zhang, Jinling; Aili, Daniel; Liedberg, Bo

    2014-03-01

    Botulinum neurotoxin is considered as one of the most toxic food-borne substances and is a potential bioweapon accessible to terrorists. The development of an accurate, convenient, and rapid assay for botulinum neurotoxins is therefore highly desirable for addressing biosafety concerns. Herein, novel biotinylated peptide substrates designed to mimic synaptosomal-associated protein 25 (SNAP-25) are utilized in gold nanoparticle-based assays for colorimetric detection of botulinum neurotoxin serotype A light chain (BoLcA). In these proteolytic assays, biotinylated peptides serve as triggers for the aggregation of gold nanoparticles, while the cleavage of these peptides by BoLcA prevents nanoparticle aggregation. Two different assay strategies are described, demonstrating limits of detection ranging from 5 to 0.1 nM of BoLcA with an overall assay time of 4 h. These hybrid enzyme-responsive nanomaterials provide rapid and sensitive detection for one of the most toxic substances known to man. PMID:24484451

  5. Organization and genomic complexity of sheep immunoglobulin light chain gene loci.

    PubMed

    Qin, Tong; Liu, Zhihong; Zhao, Huijing

    2015-12-01

    Sheep is the representative of the artiodactyla and is an agriculturally important animal, but limited knowledge is available with regard to its immunoglobulin genes and their expression mechanisms in the sheep. Based on the recently released sheep genome, we have characterized the genomic organization of the sheep immunoglobulin light gene loci. The sheep Igλ locus, located on chromosome 17, contains 2Cλ segments each preceded by a Jλ, but the Cλ2 appears to be a pseudogene. A total of 42 Vλ segments (14 potentially functional genes, 1 ORF and 27 pseudogenes) were identified. In contrast, the Igκ locus on chromosome 3 contains only a single Cκ gene, 3 Jk segments and 13 Vκ segments (8 potentially functional genes and 5 pseudogenes). Analysis of junctions of the recombined VJ transcripts revealed a restricted Vλ4-Jλ1-Cλ1 recombination and Vk6-Jk3-Cκ recombination, respectively encode most of λ and κ chain antibody repertoire in the sheep despite more potential germline encoded combinatorial diversity. Therefore, the sheep may use gene conversion in combination with somatic hypermutation for antibody repertoire formation. PMID:26499865

  6. Quantitative measurement of immunoglobulins and free light chains using mass spectrometry.

    PubMed

    VanDuijn, Martijn M; Jacobs, Joannes F M; Wevers, Ron A; Engelke, Udo F; Joosten, Irma; Luider, Theo M

    2015-08-18

    Serum free light chain (sFLC) assays are well established in the diagnosis and monitoring of plasma cell disorders. However, current FLC immunoassays are subject to several analytical issues, which results in a lack of harmonized results. To facilitate sFLC standardization, we investigated the strengths and limitations of mass spectrometry as a novel technological platform for sFLC quantification. Stable isotope labeled reference peptides are added to serum samples for quantitation by selected reaction monitoring (SRM). The use of redundant peptide sets allows for quality control measures during data analysis. Measurements on serum provide information on intact immunoglobulins, but depletion of these intact molecules from the sera during sample processing permits the quantitation of sFLC. sFLC concentrations measured with SRM were comparable to those obtained by nephelometry and showed excellent linearity (r(2) > 0.99). In samples with high levels of sFLC, SRM data was more consistent with serum protein electrophoresis than nephelometric data and SRM is unaffected by antigen excess. The lower limits of quantitation were 3.8 and 2.7 mg/L for κ and λ sFLC. Errors due to polymorphic sequences were prevented by comparison of redundant peptide pairs. The application of stable isotope labeling combined with SRM can overcome many of the current potential analytical issues of sFLC analysis. We describe which hurdles still need to be taken to make SRM a robust and more accurate method for sFLC measurements. PMID:26168337

  7. Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

    PubMed Central

    Lou, Sunny S.; Diz-Muñoz, Alba; Weiner, Orion D.; Fletcher, Daniel A.

    2015-01-01

    Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions. PMID:25918227

  8. Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

    PubMed

    Pulcastro, Hannah C; Awinda, Peter O; Breithaupt, Jason J; Tanner, Bertrand C W

    2016-07-01

    Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles. PMID:26763941

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

    PubMed

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

    2016-08-01

    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

  10. Regulation of Myosin II Dynamics by Phosphorylation and Dephosphorylation of Its Light Chain in Epithelial Cells

    PubMed Central

    Watanabe, Toshiyuki; Hosoya, Hiroshi

    2007-01-01

    Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase. PMID:17151359

  11. Urine Free Light Chains as a Novel Biomarker of Acute Kidney Allograft Injury

    PubMed Central

    Zhang, Rubin; Li, Min; Chouhan, Kanwaljit K.; Simon, Eric E.; Hamm, L. Lee; Batuman, Vecihil

    2015-01-01

    Background We evaluated urine free light chains (FLC) as a potential biomarker for acute kidney allograft injury (AKAI). Methods Urine κ and λ FLC were compared with urine β-2 microglobulin (β2-M), RBP, KIM-1, NGAL and microalbuminuria (MAB) in biopsy-confirmed acute rejection (AR) and ATN. Healthy volunteers (Normal) and transplant recipients with normal allograft function (Control) were used as references. Results Compared to Control or Normal group (N=15), urine FLC, MAB and RBP were higher in ATN (N=29) and AR (N=41) groups (p<0.05). There was no difference in KIM-1, NGAL or β2-M between 4 groups. In AR group, urine κFLC demonstrated the highest predictive value with sensitivity of 95.12% and specificity of 87.5% (p<0.0001). Urine κFLC also performed best with a sensitivity of 96.55% and specificity of 93.33% (p<0.0001) in ATN group. The AUC by ROC analysis is greatest in urine RBP (100%) and FLC (99%), and lowest in KIM-1 (53.5%), then NGAL (71.5%) in AR group. The AUC is also greatest in urine FLC (100%) and RBP (99%), and lowest in urine KIM-1 (55.6%) and NGAL (69.9%) in ATN group. Conclusions Urine FLC appears sensitive for both AR and ATN, and it may be a novel AKAI biomarker. PMID:24304377

  12. Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

    PubMed Central

    Adekar, Sharad P.; Takahashi, Tsuyoshi; Jones, R. Mark; Al-Saleem, Fetweh H.; Ancharski, Denise M.; Root, Michael J.; Kapadnis, B. P.; Simpson, Lance L.; Dessain, Scott K.

    2008-01-01

    Background Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure. PMID:18714390

  13. Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury

    PubMed Central

    Wang, Ting; Mathew, Biji; Wu, Xiaomin; Shimizu, Yuka; Rizzo, Alicia N.; Dudek, Steven M.; Weichselbaum, Ralph R.; Jacobson, Jeffrey R.; Hecker, Louise

    2016-01-01

    Abstract Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK−/− mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK. PMID:27252850

  14. Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation.

    PubMed

    Hoar, P E; Kerrick, W G

    1988-08-01

    Two effects of Mn2+ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence or absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a "latch"-like state, and the other involves direct Mn2+ activation of contractile proteins. Cells activated by Mn2+ in the presence of ATP and the absence of Ca2+, Mg2+ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn2+ contracting solution to prevent protein oxidation by Mn2+, the cells still contracted when exposed to Mn2+, but relaxed rapidly when the Mn2+ was removed. In the presence of DTT both the Mn2+ activation and the relaxation following removal of Mn2+ were more rapid than normal Ca2+-activated contractions and relaxations. The skinned fibers activated by Mn2+ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50 mM) and then contracted again in response to Ca2+ and relaxed normally. The Mn2+ activation was not associated with myosin light chain phosphorylation, in contrast to Ca2+-activated contractions. PMID:3186428

  15. Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

    PubMed

    Wang, Ting; Mathew, Biji; Wu, Xiaomin; Shimizu, Yuka; Rizzo, Alicia N; Dudek, Steven M; Weichselbaum, Ralph R; Jacobson, Jeffrey R; Hecker, Louise; Garcia, Joe G N

    2016-06-01

    Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK(-/-) mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK. PMID:27252850

  16. Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity.

    PubMed

    Xue, Song; Javor, Sacha; Hixon, Mark S; Janda, Kim D

    2014-11-01

    Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo. PMID:25295706

  17. 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

    2015-12-01

    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. PMID:26598961

  18. Studies on human plasma C1 inactivator-enzyme interactions. I. Mechanisms of interaction with C1s, plasmin, and trypsin.

    PubMed Central

    Harpel, P C; Cooper, N R

    1975-01-01

    This study has explored the nature of the molecular events which occur when C1 inactivator, a human plasma inhibitor of the complement, kinin-forming, coagulation, and fibrinolytic enzyme systems, interacts with C1s, plasmin, and trypsin. Purified inhibitor preparations demonstrated two bands, when examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The molecular weights of the major and minor bands were 105,000 and 96,000 daltons, respectively. The minor component appeared to be immunologically and functionally identical to the main C1 inactivator component. Loss of C1s and plasmin functional activity was associated with the formation of a 1:1 molar complex between the inhibitor and each enzyme. These complexes were stable in the presence of SDS and urea. The light chain of both these enzymes provided the binding site for C1 inactivator. Complex formation and enzyme inhibition occurred only with native and not with an inhibitor preparation denatured by acid treatment, thereby demonstrating the importance of conformational factors in the enzyme-inhibitor reaction. Although peptide bond cleavage of the C1 inactivator molecule by C1s was not documented, plasmin was found to degrade the inhibitor with the production of several characteristic derivatives. At least one of these products retained the ability to complex with C1s and plasmin. Trypsin, which failed to form a complex with C1 inactivator, degraded the inhibitor in a limited and sequential manner with the production of nonfunctional derivatives one of which appeared structurally similar to a plasmin-induced product. These studies therefore, provide new information concerning the molecular interactions between C1 inactivator and several of the proteases which it inhibits. Images PMID:123251

  19. 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

  20. Circulating serum free light chains as predictive markers of AIDS-related lymphoma.

    PubMed

    Landgren, Ola; Goedert, James J; Rabkin, Charles S; Wilson, Wyndham H; Dunleavy, Kieron; Kyle, Robert A; Katzmann, Jerry A; Rajkumar, S Vincent; Engels, Eric A

    2010-02-10

    PURPOSE HIV-infected persons have an elevated risk of developing non-Hodgkin's lymphoma (NHL); this risk remains increased in the era of effective HIV therapy. We evaluated serum immunoglobulin (Ig) proteins as predictors of NHL risk among HIV-infected individuals. PATIENTS AND METHODS By using three cohorts of HIV-infected persons (from 1982 to 2005), we identified 66 individuals who developed NHL and 225 matched (by cohort, sex, ethnicity, age, and CD4 count), HIV-infected, lymphoma-free controls who had available stored prediagnostic blood samples. Serum/plasma samples obtained 0 to 2 years and 2 to 5 years before diagnosis/selection were assayed for IgG, IgM, and IgA levels; monoclonal (M) Igs; and kappa and lambda free light chain (FLC) levels. Patients and matched controls were compared by using conditional logistic regression. Results The kappa and lambda FLCs were both significantly higher in patients (eg, in 2- to 5-year window: median kappa, 4.24 v 3.43 mg/dL; median lambda, 4.04 v 3.09 mg/dL) and strongly predicted NHL in a dose-response manner up to 2 to 5 years before diagnosis/selection (eg, NHL risk 3.76-fold higher with kappa concentration at least 2.00 times the upper limit of normal, and 8.13-fold higher with lambda concentration at least 2.00 times the upper limit of normal compared with normal levels). In contrast, IgG, IgM, and IgA levels were similar in patients and controls. M proteins were detected in only two patients with NHL (3%) and in nine controls (4%), and they were not significantly associated with NHL risk. CONCLUSION Elevated FLCs may represent sensitive markers of polyclonal B-cell activation and dysfunction and could be useful for identifying HIV-infected persons at increased NHL risk. PMID:20048176

  1. Localization of dynein light chains 1 and 2 and their pro-apoptotic ligands.

    PubMed Central

    Day, Catherine L; Puthalakath, Hamsa; Skea, Gretchen; Strasser, Andreas; Barsukov, Igor; Lian, Lu-Yun; Huang, David C S; Hinds, Mark G

    2004-01-01

    The dynein and myosin V motor complexes are multi-protein structures that function to transport molecules and organelles within the cell. DLC (dynein light-chain) proteins, found as components of both dynein and myosin V motor complexes, connect the complexes to their cargoes. One of the roles of these motor complexes is to selectively sequester the pro-apoptotic 'BH3-only' (Bcl-2 homology 3-only) proteins, Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor), and so regulate their cell death-inducing function. In vivo DLC2 is found exclusively as a component of the myosin V motor complex and Bmf binds DLC2 selectively. On the other hand, Bim interacts with DLC1 (LC8), an integral component of the dynein motor complex. The two DLCs share 93% sequence identity yet show unambiguous in vivo specificity for their respective BH3-only ligands. To investigate this specificity the three-dimensional solution structure of DLC2 was elucidated using NMR spectroscopy. In vitro structural and mutagenesis studies show that Bmf and Bim have identical binding characteristics to recombinant DLC2 or DLC1. Thus the selectivity shown by Bmf and Bim for binding DLC1 or DLC2, respectively, does not reside in their DLC-binding domains. Remarkably, mutational analysis of DLC1 and DLC2 indicates that a single surface residue (residue 41) determines the specific localization of DLCs with their respective motor complexes. These results suggest a molecular mechanism for the specific compartmentalization of DLCs and their pro-apoptotic cargoes and implicate other protein(s) in defining the specificity between the cargoes and the DLC proteins. PMID:14561217

  2. Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility.

    PubMed

    Rao, P Vasantha; Deng, Peifeng; Sasaki, Yasuharu; Epstein, David L

    2005-02-01

    Cellular contraction and relaxation and integrity of the actin cytoskeleton in trabecular meshwork (TM) tissue have been thought to influence aqueous humour outflow. However, the cellular pathways that regulate these events in TM cells are not well understood. In this study, we investigated physiological agonist-mediated regulation of myosin light chain (MLC) phosphorylation in the TM, and correlated such effects with alterations in aqueous outflow facility, since MLC phosphorylation is a critical biochemical determinant of cellular contraction in TM cells. Treatment of serum starved human TM cells with endothelin-1 (0.1 microM), thromboxane A2 mimetic U-46619 (1.0 microM), or angiotensin II (1 microM), all of which are agonists of G-protein coupled receptors, triggered activation of MLC phosphorylation, as determined by urea/glycerol-based Western blot analysis. Agonist-stimulated increase in MLC phosphorylation was associated with activation of Rho GTPase in TM cells, as determined in pull-down assays. In contrast, treatment of human TM cells with a novel Rho-kinase inhibitor H-1152 (0.1-2 microM), in the presence of serum reduced basal MLC phosphorylation. H-1152 also increased aqueous outflow facility significantly in a dose-dependent fashion, in perfusion studies with cadaver porcine eyes. This effect of H-1152 on outflow facility was associated with decreased MLC phosphorylation in TM tissue of drug-perfused eyes. Collectively, this study identifies potential physiological regulators of MLC phosphorylation in human TM cells and demonstrates the significance of Rho/Rho-kinase pathway-mediated MLC phosphorylation in modulation of aqueous outflow facility through TM. PMID:15670798

  3. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

    SciTech Connect

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta

    2012-04-02

    The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-{Delta}43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing ({approx} 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I{sub 1,1}/I{sub 1,0}, indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-{Delta}43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-{Delta}43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

  4. The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription

    PubMed Central

    Tan, Gene S.; Preuss, Mirjam A. R.; Williams, John C.; Schnell, Matthias J.

    2007-01-01

    Recent studies indicate that the interaction between rabies virus (RV) phosphoprotein and the dynein light chain 8 (LC8) is essential for RV pathogenesis. Through its association with the dynein motor complex, LC8 has been suggested as a molecular factor that links the viral ribonucleoprotein to the host cell transport system. Recent structural investigations, however, dispute this model. To understand the role of LC8 in RV pathogenesis, we generated recombinant RVs with or without the LC8 binding domain (LC8-BD) deleted from the RV phosphoprotein. Peripheral infection of adult mice showed that removal of the LC8-BD did not inhibit entry into the CNS, although it prevented onset of RV-induced CNS disease. However, deletion of the LC8-BD significantly attenuated viral transcription and replication in the CNS. Studies in RAG2 knockout (KO) mice infected with the same recombinant RVs confirmed this finding and indicated that the adaptive immune system is not a factor in the attenuation of viral replication early in the infection. In cell culture, the deletion of the LC8-BD greatly attenuated growth on neuronal cells whereas the growth pattern on nonneuronal cells remained unchanged. However, deletion of the LC8-BD did not affect production of RV virions. We provide evidence that removal of the LC8-BD decreases primary transcription. In this study, we propose that LC8 does not play a role in the retrograde axonal transport of RV and that the deletion of the LC8-BD impairs the infectivity of the virions by reducing early transcription and replication in neurons. PMID:17438267

  5. Early Reduction of Serum-Free Light Chains Associates with Renal Recovery in Myeloma Kidney

    PubMed Central

    Cockwell, Paul; Stringer, Stephanie; Bradwell, Arthur; Cook, Mark; Gertz, Morie A.; Dispenzieri, Angela; Winters, Jeffrey L.; Kumar, Shaji; Rajkumar, S. Vincent; Kyle, Robert A.; Leung, Nelson

    2011-01-01

    Myeloma kidney is the major cause of severe irreversible renal failure in patients with multiple myeloma. This tubulointerstitial injury is a direct consequence of high concentrations of circulating monoclonal free light chains (FLCs) produced by a clonal expansion of plasma cells. Early reduction of serum FLCs associates with renal recovery, but the target threshold of reduction to facilitate renal recovery is unknown. To determine the relationship between the achieved FLC reduction and renal recovery, we identified 39 patients with biopsy-proven myeloma kidney, the majority of whom had severe renal failure at presentation (median estimated GFR 9 ml/min per 1.73 m2). In a multivariable analysis incorporating demographic, hematologic, and renal variables, only the achieved FLC reduction significantly predicted renal recovery (P = 0.003). The relationship between renal recovery and FLC reduction was linear with no absolute threshold for FLC reduction. A 60% reduction in FLCs by day 21 associated with recovery of renal function for 80% of the population. Patient survival strongly associated with renal recovery: the median survival was 42.7 months (range 0 to 80) among those who recovered function compared with 7.8 months (range 0 to 54) among those who did not (P < 0.02). Cox-regression analysis demonstrated that the first presentation of myeloma, the kappa isotype of FLC, and renal recovery were independent predictors of survival. In conclusion, recovery of renal function in myeloma kidney depends on early reduction of serum FLCs, and this recovery associates with a significant survival advantage. PMID:21511832

  6. T1 mapping and survival in systemic light-chain amyloidosis

    PubMed Central

    Banypersad, Sanjay M.; Fontana, Marianna; Maestrini, Viviana; Sado, Daniel M.; Captur, Gabriella; Petrie, Aviva; Piechnik, Stefan K.; Whelan, Carol J.; Herrey, Anna S.; Gillmore, Julian D.; Lachmann, Helen J.; Wechalekar, Ashutosh D.; Hawkins, Philip N.; Moon, James C.

    2015-01-01

    Aims To assess the prognostic value of myocardial pre-contrast T1 and extracellular volume (ECV) in systemic amyloid light-chain (AL) amyloidosis using cardiovascular magnetic resonance (CMR) T1 mapping. Methods and results One hundred patients underwent CMR and T1 mapping pre- and post-contrast. Myocardial ECV was calculated at contrast equilibrium (ECVi) and 15 min post-bolus (ECVb). Fifty-four healthy volunteers served as controls. Patients were followed up for a median duration of 23 months and survival analyses were performed. Mean ECVi was raised in amyloid (0.44 ± 0.12) as was ECVb (mean 0.44 ± 0.12) compared with healthy volunteers (0.25 ± 0.02), P < 0.001. Native pre-contrast T1 was raised in amyloid (mean 1080 ± 87 ms vs. 954 ± 34 ms, P < 0.001). All three correlated with pre-test probability of cardiac involvement, cardiac biomarkers, and systolic and diastolic dysfunction. During follow-up, 25 deaths occurred. An ECVi of >0.45 carried a hazard ratio (HR) for death of 3.84 [95% confidence interval (CI): 1.53–9.61], P = 0.004 and pre-contrast T1 of >1044 ms = HR 5.39 (95% CI: 1.24–23.4), P = 0.02. Extracellular volume after primed infusion and ECVb performed similarly. Isolated post-contrast T1 was non-predictive. In Cox regression models, ECVi was independently predictive of mortality (HR = 4.41, 95% CI: 1.35–14.4) after adjusting for E:E′, ejection fraction, diastolic dysfunction grade, and NT-proBNP. Conclusion Myocardial ECV (bolus or infusion technique) and pre-contrast T1 are biomarkers for cardiac AL amyloid and they predict mortality in systemic amyloidosis. PMID:25411195

  7. Myosin Light Chain Kinase Mediates Intestinal Barrier Disruption following Burn Injury

    PubMed Central

    Chen, Chuanli; Wang, Pei; Su, Qin; Wang, Shiliang; Wang, Fengjun

    2012-01-01

    Background Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction. Methodology/Principal Findings Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression. Conclusions/Significance The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury. PMID:22529961

  8. N-Terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size.

    PubMed

    Wang, Yihua; Ajtai, Katalin; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Burghardt, Thomas P

    2016-01-12

    Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ∼19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method. PMID:26671638

  9. Impaired Lysosomal Function Underlies Monoclonal Light Chain-Associated Renal Fanconi Syndrome.

    PubMed

    Luciani, Alessandro; Sirac, Christophe; Terryn, Sara; Javaugue, Vincent; Prange, Jenny Ann; Bender, Sébastien; Bonaud, Amélie; Cogné, Michel; Aucouturier, Pierre; Ronco, Pierre; Bridoux, Frank; Devuyst, Olivier

    2016-07-01

    Monoclonal gammopathies are frequently complicated by kidney lesions that increase the disease morbidity and mortality. In particular, abnormal Ig free light chains (LCs) may accumulate within epithelial cells, causing proximal tubule (PT) dysfunction and renal Fanconi syndrome (RFS). To investigate the mechanisms linking LC accumulation and PT dysfunction, we used transgenic mice overexpressing human control or RFS-associated κLCs (RFS-κLCs) and primary cultures of mouse PT cells exposed to low doses of corresponding human κLCs (25 μg/ml). Before the onset of renal failure, mice overexpressing RFS-κLCs showed PT dysfunction related to loss of apical transporters and receptors and increased PT cell proliferation rates associated with lysosomal accumulation of κLCs. Exposure of PT cells to RFS-κLCs resulted in κLC accumulation within enlarged and dysfunctional lysosomes, alteration of cellular dynamics, defective proteolysis and hydrolase maturation, and impaired lysosomal acidification. These changes were specific to the RFS-κLC variable (V) sequence, because they did not occur with control LCs or the same RFS-κLC carrying a single substitution (Ala30→Ser) in the V domain. The lysosomal alterations induced by RFS-κLCs were reflected in increased cell proliferation, decreased apical expression of endocytic receptors, and defective endocytosis. These results reveal that specific κLCs accumulate within lysosomes, altering lysosome dynamics and proteolytic function through defective acidification, thereby causing dedifferentiation and loss of reabsorptive capacity of PT cells. The characterization of these early events, which are similar to those encountered in congenital lysosomal disorders, provides a basis for the reported differential LC toxicity and new perspectives on LC-induced RFS. PMID:26614382

  10. Clinical and echocardiographic characteristics for differentiating between transthyretin-related and light-chain cardiac amyloidoses.

    PubMed

    Mori, Minako; An, Yoshimori; Katayama, Oju; Kitagawa, Tomoya; Sasaki, Yuya; Onaka, Takashi; Yonezawa, Akihito; Murata, Kenichiro; Yokota, Tadaaki; Ando, Kenji; Imada, Kazunori

    2015-11-01

    Differential diagnosis between transthyretin (TTR) and immunoglobulin light-chain (AL) cardiac amyloidoses is essential due to significantly different prognoses and therapeutic options. Therefore, clinical characteristics of patients with biopsy-proven cardiac amyloidosis were investigated to differentiate TTR from AL amyloidosis. From September 2006 to May 2014, 46 patients were confirmed to have cardiac amyloidosis (TTR, n = 28; AL, n = 18) in our institute. The median age of patients with TTR amyloidosis was 78 years (range 61-90) with 27 (96 %) males, while that of patients with AL amyloidosis was 66 (range 52-76) with 12 (67 %) males. There were no statistically significant differences in echocardiographic findings regarding left ventricular (LV) systolic function or diastolic dysfunction between the two groups. Interestingly, serum brain natriuretic peptide (BNP) levels in patients with AL amyloidosis were significantly higher than those in TTR amyloidosis patients. In contrast, the LV wall was significantly thicker in patients with TTR amyloidosis than in those with AL amyloidosis. Therefore, the ratio of BNP to LV mass index (LVMI) at presentation in AL amyloidosis patients was significantly higher than that in TTR patients (6.7 vs 2.9, p = 0.0006). A BNP-LVMI ratio of less than 3.5 had a diagnostic sensitivity and specificity for TTR amyloidosis of 71 and 83 %, respectively. One-year overall survival was 88.7 % in the patients with TTR amyloidosis and 23.7 % in the patients with AL amyloidosis. Our analysis indicates that the BNP-LVMI ratio, as well as age and sex, may be useful parameters for distinguishing TTR from AL cardiac amyloidosis. PMID:26251157

  11. [Usefulness of serum cardiac myosin light chain I for the estimation of acute myocardial infarction size].

    PubMed

    Narita, M; Kurihara, T; Murano, K; Usami, M

    1991-09-01

    To evaluate the usefulness of serum level of cardiac myosin light chain I (LC I) for the estimation of the extent of acute myocardial infarction (AMI), peak LC I level was compared with myocardial infarction weight (AMI weight) which was obtained by myocardial emission tomography with Tc-99m pyrophosphate (PYP). In 11 patients with AMI, serum LC I levels were measured once a day in most cases, and plasma CPK levels were measured serially (every 4 hours at least 48 hours after admission). Tc-99m PYP imagings were performed at second or third day of AMI, and AMI weight was calculated from the voxel numbers of myocardial hot spot in which Tc-99m PYP had accumulated. Peak LC I level correlated well with AMI weight (r = 0.72, p less than 0.02). As well as peak LC I level, peak CPK level correlated well with AMI weight (r = 0.68, p less than 0.05). But the estimation of the infarct size from peak LC I level had the following advantages over the estimation from peak CPK level. 1) We could compare peak LC I level with AMI weight in all 11 patients, but peak CPK level was able to compared with AMI weight in only 9 of them. This was because CPK level changed rapidly and reached maximum within 24 hours after the onset of AMI, while LC I level peaked after 3 to 5 days. 2) A good correlation between LC I and AMI weight was obtained by the determination of serum LC I level once a day.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1836269

  12. Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions.

    PubMed

    Duggal, D; Nagwekar, J; Rich, R; Huang, W; Midde, K; Fudala, R; Das, H; Gryczynski, I; Szczesna-Cordary, D; Borejdo, J

    2015-05-15

    Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC. PMID:25770245

  13. Effects of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin regulatory light chain.

    PubMed

    Espinoza-Fonseca, L Michel; Colson, Brett A; Thomas, David D

    2014-10-01

    We have performed 50 independent molecular dynamics (MD) simulations to determine the effect of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin (SMM) regulatory light chain (RLC). We previously showed that the N-terminal phosphorylation domain of RLC simultaneously populates two structural states in equilibrium, closed and open, and that phosphorylation at S19 induces a modest shift toward the open state, which is sufficient to activate smooth muscle. However, it remains unknown why pseudophosphorylation mutants poorly mimic phosphorylation-induced activation of SMM. We performed MD simulations of unphosphorylated, phosphorylated, and three pseudophosphorylated RLC mutants: S19E, T18D/S19D and T18E/S19E. We found that the S19E mutation does not shift the equilibrium toward the open state, indicating that simple charge replacement at position S19 does not mimic the activating effect of phosphorylation, providing a structural explanation for previously published functional data. In contrast, mutants T18D/S19D and T18E/S19E shift the equilibrium toward the open structure and partially activate in vitro motility, further supporting the model that an increase in the mol fraction of the open state is coupled to SMM motility. Structural analyses of the doubly-charged pseudophosphorylation mutants suggest that alterations in an interdomain salt bridge between residues R4 and D100 results in impaired signal transmission from RLC to the catalytic domain of SMM, which explains the low ATPase activity of these mutants. Our results demonstrate that phosphorylation produces a unique structural balance in the RLC. These observations have important implications for our understanding of the structural aspects of activation and force potentiation in smooth and striated muscle. PMID:25091814

  14. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load.

    PubMed

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2015-08-15

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  15. Lenalidomide and dexamethasone for acute light chain-induced renal failure: a phase II study

    PubMed Central

    Ludwig, Heinz; Rauch, Elisabeth; Kuehr, Thomas; Adam, Zdeněk; Weißmann, Adalbert; Kasparu, Hedwig; Autzinger, Eva-Maria; Heintel, Daniel; Greil, Richard; Poenisch, Wolfram; Müldür, Ercan; Zojer, Niklas

    2015-01-01

    We prospectively evaluated the activity and tolerance of lenalidomide-dexamethasone in 35 patients with acute light chain-induced renal failure. The lenalidomide dose was adapted to the estimated glomerular filtration rate and dexamethasone was given at high dose in cycle one and at low dose thereafter. Four patients died within the first two cycles, and five discontinued therapy leaving 26 patients for the per-protocol analysis. Responses were observed in 24/35 (68.6%) patients of the intent-to-treat population. Complete response was noted in seven patients (20%), very good partial response in three patients (8.6%), partial response in 14 patients (40%), and minimal response in one patient (2.9%). Renal response was observed in 16 (45.7%) patients: five (14.2%) achieved complete, four (11.4%) partial and seven (20%) minor renal responses. Five of 13 patients who were dialysis dependent at baseline became dialysis independent. The median time to myeloma and to renal response was 28 days for both parameters, while the median time to best myeloma and best renal response was 92 and 157 days, respectively. The median estimated glomerular filtration rate increased significantly in patients with partial response or better from 17.1 mL/min at baseline to 39.1 mL/min at best response (P=0.001). The median progression-free and overall survival was 5.5 and 21.8 months, respectively, in the intent-to-treat population and 12.1 and 31.4 months, respectively, in the per-protocol group. Infections, cardiotoxicity, anemia and thrombocytopenia were the most frequent toxicities. In conclusion, the lenalidomide-dexamethasone regimen achieved rapid and substantial myeloma and renal responses. The trial was registered under EUDRACT number 2008-006497-15. PMID:25398836

  16. Prognostic Significance of Serum Free Light Chains in Chronic Lymphocytic Leukemia

    PubMed Central

    Sarris, Katerina; Koulieris, Efstathios; Bartzis, Vassiliki; Tzenou, Tatiana; Sachanas, Sotirios; Nikolaou, Eftychia; Efthymiou, Anna; Bitsani, Katerina; Dimou, Maria; Vassilakopoulos, Theodoros P.; Angelopoulou, Maria K.; Kontopidou, Flora; Tsaftaridis, Panagiotis; Kafasi, Nikolitsa; Pangalis, Gerasimos A.; Panayiotidis, Panayiotis P.; Harding, Stephen

    2013-01-01

    Background. Serum free light chains (sFLC), the most commonly detected paraprotein in CLL, were recently proposed as useful tools for the prognostication of CLL patients. Objective. To investigate the prognostic implication of sFLC and the summated FLC-kappa plus FLC-lambda in a CLL patients' series. Patients and Methods. We studied 143 CLL patients of which 18 were symptomatic and needed treatment, while 37 became symptomatic during follow-up. Seventy-two percent, 18%, and 10% were in Binet stage A, B and C, respectively. Median patients' followup was 32 months (range 4–228). Results. Increased involved (restricted) sFLC (iFLC) was found in 42% of patients, while the summated FLC-kappa plus FLC-lambda was above 60 mg/dL in 14%. Increased sFLC values as well as those of summated FLC above 60 were related to shorter time to treatment (P = 0.0005 and P = 0.000003, resp.) and overall survival (P = 0.05 and P = 0.003, resp.). They also correlated with β2-microglobulin (P = 0.009 and P = 0.03, resp.), serum albumin (P = 0.009 for summated sFLC), hemoglobin (P < 0.001), abnormal LDH (P = 0.037 and P = 0.001, resp.), Binet stage (P < 0.05) and with the presence of beta symptoms (P = 0.004 for summated sFLC). Conclusion. We confirmed the prognostic significance of sFLC in CLL regarding both time to treatment and survival and showed their relationship with other parameters. PMID:24288537

  17. Myosin light chain phosphorylation in contraction of gastric antral smooth muscle from neonate and adult rabbits.

    PubMed

    Ierardi, J A; Paul, D A; Ryan, J P

    1996-01-01

    The decreased contractility of gastric antral smooth muscle in the neonate has been attributed to reduced levels of activator calcium. It is generally accepted that calcium-dependent myosin light chain phosphorylation (MLCP) is the key step in the initiation of force development in smooth muscle. In this study, we investigated the relationship between MLCP and force development in gastric antral smooth muscle from neonatal (4-6 d old) and adult rabbits. We tested the hypothesis that the reduced force development of circular smooth muscle from the neonate would be accompanied by decreased levels of MLCP, as compared with data from adult animals. Full thickness muscle strips oriented parallel to the circular muscle layer were examined for their contractile response to acetylcholine (ACh) (10(-8) M to 10(-3) M) or 10(-4) M ACh only. In the latter study, tissues were rapidly frozen in a dry ice-acetone slurry for subsequent MLCP determination. MLCP was determined at times corresponding to 5, 10, 15, 30, and 60 s of stimulation. For each age group, maximal active force developed at an ACh concentration of 10(-4) M and was significantly greater in tissues from adults (1.86 +/- 0.24 N/m2, adult; 0.95 +/- 0.05 N/m2, neonate; p < 0.05). In contrast, no significant differences were observed with respect to basal or agonist-stimulated levels of MLCP. The data suggest that factors other than levels of MLCP contribute to the reduced force-generating capacity of antral smooth muscle from the neonate. PMID:8825402

  18. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load

    PubMed Central

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2016-01-01

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin’s ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31–41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  19. Measurement of free light chains - pros and cons of current methods.

    PubMed

    Graziani, Maria Stella

    2016-06-01

    The measurement of the serum free light chains (FLC) is of paramount importance in the management of patients with plasma cell dyscrasias (PSD). The immunoassays for FLC measurement require adequate precision, accuracy, specificity and reproducibility between batches to prevent under or over estimation of FLC concentration and for an adequate patient monitoring. Considering the peculiarity of the measurand (monoclonal proteins), the optimization of any analytical aspect is difficult to achieve. Three methods are currently available for the assay. The first one has been on the market for over 15 years, and it is based on polyclonal antibodies. The vast majority of the clinical studies demonstrating the utility of the serum FLC measurement have been performed using this assay. A second method based on monoclonal antibodies (mAbs) was marketed in 2011; a third one, also employing mAbs and allowing the simultaneous measurement of κ and λ FLC is in the process of publication. These methods show relevant differences in the type of antibodies used and in the assay design and it is not possible to identify an immunoassay that is superior to the others in any analytical aspect. The comparison studies show that the three methods differ significantly in terms of quantitative values, especially when samples containing monoclonal proteins are compared. Hence the methods cannot be used interchangeably, in particular when the assay is used to monitor the patient response to therapy. In the absence of an international standard for FLC measurement, it is impossible, at this stage to establish, which method shows the best accuracy. PMID:26812876

  20. Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila

    SciTech Connect

    Leicht, B.G.; Muse, S.V.; Hanczyc, M.

    1995-01-01

    Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two Mlc1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium. does seem to be present in the 5{prime} half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule. 48 refs., 5 figs., 3 tabs.

  1. Factor VII Light Chain-Targeted Lidamycin Shows Intensified Therapeutic Efficacy for Liver Cancer

    PubMed Central

    Liu, Xiujun; Xu, Shuangshuang; Li, Caihong; Zhang, Yang; Yang, Jie; Zheng, Junnian

    2012-01-01

    Abstract The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers makes TF an ideal target for cancer therapy. The purpose of this study is to develop a TF-targeting energized fusion protein hlFVII-LDP-AE, which is composed of a human Factor VII light chain (hlFVII) as the targeting domain conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE) as the effector domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was tested in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays and in vivo with a BALB/c nude mouse xenograft model of human liver cancer line HepG2. The inhibitory concentration (IC50) value of hlFVII-LDP-AE varied from 0.15 to 0.64 nM for the various human tumor lines. hlFVII-LDP-AE showed a tumor growth inhibition rate of 90.6% at the dose of 0.6 mg/kg in in vivo animal experiments. The mechanism through which hlFVII-LDP-AE inhibits tumor growth also was determined by Hoechst 33342 staining and Tdt-mediated dUTP nick-end labeling (TUNEL) assay. hlFVII-LDP-AE causes tumor cell death through inducing chromatin condensation and cleavage of genomic DNA. These findings suggest that the hlFVII-LDP-AE protocol is efficacious and tolerated in the mouse model of human liver cancer HepG2 and has clinical applicability for treating cancer patients. PMID:22651685

  2. Significant Influences of Elaborately Modulating Electron Donors on Light Absorption and Multichannel Charge-Transfer Dynamics for 4-(Benzo[c][1,2,5]thiadiazol-4-ylethynyl)benzoic Acid Dyes.

    PubMed

    Wang, Erfeng; Yao, Zhaoyang; Zhang, Yiqiang; Shao, Guosheng; Zhang, Min; Wang, Peng

    2016-07-20

    4-(Benzo[c][1,2,5]thiadiazol-4-ylethynyl)benzoic acid (BTEBA) as a promising electron acceptor has been used in the highly efficient organic dye-sensitized solar cells (DSCs) recently. Because of its strong electron-deficient character, BTEBA could bring forth a remarkable decline in the energy level of the lowest unoccupied molecular orbital (LUMO) and further reduce the energy gap of dye molecules significantly. In this contribution, two metal-free organic dyes WEF1 and WEF2 were synthesized by simply combining BTEBA with two slightly tailored electron-releasing moieties: 4-hexylphenyl substituted indaceno[1,2-b:5,6-b']dithiophene (IDT) and cyclopenta[1,2-b:5,4-b']dithiophene[2',1':4,5]thieno[2,3-d]thiophene (CPDTDT), which were screened rationally from an electron-donor pool via computational simulation. With respect to those of WEF1, WEF2-sensitized solar cells demonstrate a far better short-circuit photocurrent density (JSC) and open-circuit photovoltage (VOC), resulting in a ∼50% improved power conversion efficiency of 10.0% under irradiance of 100 mW cm(-2) AM1.5G sunlight. We resorted to theoretical calculations, electrical measurements, steady-state, and time-resolved spectroscopic methods to shed light on the fatal influences of elaborately modulating electron donors on light absorption, interfacial energetics, and multichannel charge-transfer dynamics. PMID:27331621

  3. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain.

    PubMed

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D

    2015-04-14

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  4. [Secondary monoclonal gammopathy after bone marrow autotransplantation as a cause of worse renal function in light chain immunoglobulin deposition disease].

    PubMed

    Rekhtina, I G; Mendeleeva, L P; Stolyarevich, E S; Gal'tseva, I V; Povilaitite, P E; Biryukova, L S

    2016-01-01

    The paper describes a clinical case of a female woman with nephropathy due to light chain deposition disease caused by secretion of κ Bence-Jones protein. Complete immunochemical remission was achieved after induction therapy using a bortezomib + cyclophosphamide + dexamethasone regimen. Renal function remained unchanged (glomerular filtration rate 16 ml/min), there was a reduction in proteinuria from 5.8 to 2.6 g/day. High-dose melphalan (200 mg/m2) chemotherapy with peripheral blood stem cell autotransplantation was performed as consolidation of remission. A year posttransplantation, there was no secretion of κ light chains; however, monoclonal IgG lambda emerged in a quantity of 3.2 g/l. At the same period, nephrotic syndrome became progressive (daily proteinuria 12 g) and dialysis-dependent renal failure developed. A repeat renal biopsy specimen revealed changes, suggesting that there was a decrease in renal deposits of κ light chains. Simultaneously with this, the obvious negative trend as progressive nephrosclerosis and fixation of IgG and λ light chains in the glomeruli (in the sclerotic areas) cause IgGλ monoclonal protein to be involved in the genesis of further kidney injury. Attention is also paid to different characteristics of capillary wall deposits by density (according to the electron microscopic findings), which may point to their different qualitative composition and possibly different formation duration. Papaprotein Gλ disappeared after a year without therapy, suggesting its reactivity. The findings confirm that worse renal function is caused by the action of paraprotein Gλ due to secondary (after autologous hematopoietic stem cells transplantation) monoclonal gammopathy. PMID:27296267

  5. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

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

    PubMed Central

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

    2015-01-01

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

  7. 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

  8. Biochemical features and antiviral activity of a monomeric catalytic antibody light-chain 23D4 against influenza A virus.

    PubMed

    Hifumi, Emi; Arakawa, Mitsue; Matsumoto, Shingo; Yamamoto, Tatsuhiro; Katayama, Yoshiki; Uda, Taizo

    2015-06-01

    Catalytic antibodies have exhibited interesting functions against some infectious viruses such as HIV, rabies virus, and influenza virus in vitro as well as in vivo. In some cases, a catalytic antibody light chain takes on several structures from the standpoint of molecular size (monomer, dimer, etc.) and/or isoelectronic point. In this study, we prepared a monomeric 23D4 light chain by mutating the C-terminal Cys to Ala of the wild-type. The mutated 23D4 molecule took a simple monomeric form, which could hydrolyze synthetic 4-methyl-coumaryl-7-amide substrates and a plasmid DNA. Because the monomeric 23D4 light chain suppressed the infection of influenza virus A/Hiroshima/37/2001 in an in vitro assay, the corresponding experiments were conducted in vivo, after the virus strain (which was taken from a human patient) was successfully adapted into BALB/cN Sea mice. In the experiments, a mixture of the monomeric 23D4 and the virus was nasally administered 1) with preincubation and 2) without preincubation. As a result, the monomeric 23D4 clearly exhibited the ability to suppress the influenza virus infection in both cases, indicating a potential drug for preventing infection of the influenza A virus. PMID:25713031

  9. CaMKII and at least two unidentified kinases phosphorylate regulatory light chain in non-contracting cardiomyocytes.

    PubMed

    Eikemo, Hilde; Moltzau, Lise Román; Nguyen, Cam H T; Levy, Finn Olav; Skomedal, Tor; Osnes, Jan-Bjørn

    2016-08-12

    In cardiac tissue, regulatory light chain (RLC, myosin light chain 2) phosphorylation (Ser(15)) leads to modulation of muscle contraction through Ca(2+)-sensitization. To elucidate which kinases that are involved in the basal (diastolic phase) RLC phosphorylation, we studied non-contracting adult rat cardiomyocytes. RLC kinase activities in situ were unmasked by maximally inhibiting myosin light chain phosphatase (MLCP) by calyculin A in the absence and presence of various protein kinase inhibitors. Surprisingly MLCK did not contribute to the phosphorylation of RLC in the non-contracting cardiomyocytes. Two kinase activity groups were revealed by different sensitivities to staurosporine. The fraction with the highest sensitivity to staurosporine was inhibited by KN-93, a selective CaMKII inhibitor, producing a 23% ± 7% reduction in RLC phosphorylation. Calmodulin antagonism (W7) and reduction in Ca(2+) (EGTA) combined with low concentration of staurosporine caused a larger decrease in RLC phosphorylation than staurosporine alone. These data strongly suggest that in addition to CaMKII, there is another Ca(2+)/calmodulin-dependent kinase and a Ca(2+)/calmodulin-independent kinase phosphorylating RLC. Thus the RLC phosphorylation seems to be ensured by redundant kinase activities. PMID:27237977

  10. No detectable reiteration of genes coding for mouse MOPC 41 immunoglobulin light-chain mRNA.

    PubMed Central

    Farace, M G; Aellen, M F; Briand, P A; Faust, C H; Vassalli, P; Mach, B

    1976-01-01

    RNA fractions rich in immunoglobulin light (L)-chain mRNA were isolated from mouse myeloma MOPC 41 by procedures previously described, and chemically labeled with 125I. These RNA fractions were hybridized with MOPC 41 DNA under conditions of DNA excess. Hybridization conditions were chosen under which the entire sequence of the L-chain mRNA probe, thus including the variable region, remains available for hybridization throughout the reaction. The hybridization (C0t) curve showed double transition kinetics, with one component corresponding to about 250 gene copies and the other to about two to four copies. In contrast, when MOPC 41 L-chain mRNA was further purified as a single band by gel elecptrophoresis in 99% formamide, the hybridization curve showed only a single transition, corresponding to about two to four genes, with the disappearance of the "reiterated" component. That component resulted therefore from contaminating RNA species. The data indicate that no reiteration can be detected by RNase or by hydroxylapatite for the genes corresponding to the entire sequence of MOPC 41 L-chain mRNA, including the untranslated segments, within the limits of detectability of short reiterated segments. It thus appears that there is only one or very few genes corresponding to the 41 L-chain variable region "subgroup" in MOPC 41 DNA. The possibility that the variable genes of plasmocytes might result frm a combination of several nonreiterated germline genes is discussed. Images PMID:815907

  11. Cerebrospinal fluid neurofilament light chain protein levels in subtypes of frontotemporal dementia

    PubMed Central

    2013-01-01

    Background Frontotemporal dementia (FTD) is recognised as a clinically and morphologically heterogeneous group of interrelated neurodegenerative conditions. One of the subtypes within this disease spectrum is the behavioural variant FTD (bvFTD). This is known to be a varied disorder with a mixture of tau-positive and tau-negative underlying pathologies. The other subtypes include semantic dementia (SD), which generally exhibits tau-negative pathology, and progressive non-fluent aphasia (PNFA), which is usually tau-positive. As the clinical presentation of these subtypes may overlap, a specific diagnosis can be difficult to attain and today no specific biomarker can predict the underlying pathology. Neurofilament light chain protein (NFL), a cytoskeletal constituent of intermediate filaments, is thought to reflect neuronal and axonal death when appearing in the cerebrospinal fluid (CSF). NFL has been shown to be elevated in CSF in patients with FTD compared with AD and controls. Our hypothesis was that the levels of NFL also differ between the subtypes of FTD and may indicate the underlying pathological subtype. Methods We retrospectively analysed data from previous CSF analyses in 34 FTD cases (23 bvFTD, seven SD, four PNFA), 20 AD cases, and 26 healthy controls. A separate group of 10 neuropathologically verified and subtyped FTD cases (seven tau-negative, three tau-positive) were also analysed. Result NFL levels were significantly higher in FTD compared with both AD (p<0.001) and controls (p<0.001). The NFL levels of SD and bvFTD were significantly higher (p<0.001) compared with AD. The biomarker profiles of PNFA and AD were similar. In the neuropathologically verified FTD cases, NFL was higher in the tau-negative than in the tau-positive cases (exact p=0.017). Conclusions The marked NFL elevation in some but not all FTD cases is likely to reflect the different underlying pathologies. The highest NFL values found in the SD group as well as in the

  12. Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation

    PubMed Central

    Taverner, Alistair; Dondi, Ruggero; Almansour, Khaled; Laurent, Floriane; Owens, Siân-Eleri; Eggleston, Ian M.; Fotaki, Nikoletta; Mrsny, Randall J.

    2015-01-01

    The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein–protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4 kDa fluorescent dextran but not 70 kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70 kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the

  13. Mutations of the Drosophila Myosin Regulatory Light Chain Affect Courtship Song and Reduce Reproductive Success

    PubMed Central

    Chakravorty, Samya; Vu, Hien; Foelber, Veronica; Vigoreaux, Jim O.

    2014-01-01

    The Drosophila indirect flight muscles (IFM) rely on an enhanced stretch-activation response to generate high power output for flight. The IFM is neurally activated during the male courtship song, but its role, if any, in generating the small amplitude wing vibrations that produce the song is not known. Here, we examined the courtship song properties and mating behavior of three mutant strains of the myosin regulatory light chain (DMLC2) that are known to affect IFM contractile properties and impair flight: (i) Dmlc2Δ2–46 (Ext), an N-terminal extension truncation; (ii) Dmlc2S66A,S67A (Phos), a disruption of two MLC kinase phosphorylation sites; and (iii) Dmlc2Δ2–46;S66A,S67A (Dual), expressing both mutations. Our results show that the Dmlc2 gene is pleiotropic and that mutations that have a profound effect on flight mechanics (Phos and Dual) have minimal effects on courtship song. None of the mutations affect interpulse interval (IPI), a determinant of species-specific song, and intrapulse frequency (IPF) compared to Control (Dmlc2+ rescued null strain). However, abnormalities in the sine song (increased frequency) and the pulse song (increased cycles per pulse and pulse length) evident in Ext males are not apparent in Dual males suggesting that Ext and Phos interact differently in song and flight mechanics, given their known additive effect on the latter. All three mutant males produce a less vigorous pulse song and exhibit impaired mating behavior compared to Control males. As a result, females are less receptive to Ext, Phos, and Dual males when a Control male is present. These results open the possibility that DMLC2, and perhaps contractile protein genes in general, are partly under sexual selection. That mutations in DMLC2 manifest differently in song and flight suggest that this protein fulfills different roles in song and flight and that stretch activation plays a smaller role in song production than in flight. PMID:24587213

  14. Long term outcomes of cardiac transplant for immunoglobulin light chain amyloidosis: The Mayo Clinic experience

    PubMed Central

    Grogan, Martha; Gertz, Morie; McCurdy, Arleigh; Roeker, Lindsey; Kyle, Robert; Kushwaha, Sudhir; Daly, Richard; Dearani, Joseph; Rodeheffer, Richard; Frantz, Robert; Lacy, Martha; Hayman, Suzanne; McGregor, Christopher; Edwards, Brooks; Dispenzieri, Angela

    2016-01-01

    AIM: To determine the outcome of orthotopic heart transplantation (OHT) in immunoglobulin light chain (AL) amyloidosis. METHODS: The medical records of patients with AL who underwent orthotopic heart transplantation at the Mayo Clinic in Rochester Minnesota from 1992 to 2011 were reviewed. Patients met at least one of the following at: New York Heart Association class IV heart failure, ventricular thickness > 15 mm, ejection fraction < 40%. Selection guidelines for heart transplant included age < 60 years, absence of multiple myeloma and significant extra-cardiac organ involvement. Baseline characteristics including age, gender, organ involvement, and New York Heart Association functional class were recorded. Laboratory data, waiting time until heart transplant, and type of treatment of the underlying plasma cell disorder were recorded. Survival from the time of OHT was calculated using Kaplan-Meier survival curves. Survival of patients undergoing OHT for AL was compared to that of non-amyloid patients undergoing OHT during the same time period. RESULTS: Twenty-three patients (median age 53 years) with AL received OHT. There were no deaths in the immediate perioperative period. Twenty patients have died post OHT. For the entire cohort, the median overall survival was 3.5 years (95%CI: 1.2, 8.2 years). The 1-year survival post OHT was 77%, the 2-year survival 65%, and the 5-year survival 43%. The 5-year survival for non-amyloid patients undergoing OHT during the same era was 85%. Progressive amyloidosis contributed to death in twelve patients. Of those without evidence of progressive amyloidosis, the cause of death included complications of autologous hematopoietic stem cell transplantation for 3 patients, post-transplant lymphoproliferative disorder for 2 patients; and for the remaining one death was related to each of the following causes: acute rejection; cardiac vasculopathy; metastatic melanoma; myelodysplastic syndrome; and unknown. Eight patients had

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. Stimulation of kappa light-chain gene rearrangement by the immunoglobulin mu heavy chain in a pre-B-cell line.

    PubMed Central

    Shapiro, A M; Schlissel, M S; Baltimore, D; DeFranco, A L

    1993-01-01

    B-lymphocyte development exhibits a characteristic order of immunoglobulin gene rearrangements. Previous work has led to the hypothesis that expression of the immunoglobulin mu heavy chain induces rearrangement activity at the kappa light-chain locus. To examine this issue in more detail, we isolated five matched pairs of mu- and endogenously rearranged mu+ cell lines from the Abelson murine leukemia virus-transformed pro-B-cell line K.40. In four of the five mu+ cell lines, substantial expression of mu protein on the cell surface was observed, and this correlated with an enhanced frequency of kappa immunoglobulin gene rearrangement compared with that in the matched mu- cell lines. This increased kappa gene rearrangement frequency was not due to a general increase in the amount of V(D)J recombinase activity in the mu+ cells. Consistently, introduction of a functionally rearranged mu gene into one of the mu- pre-B-cell lines resulted in a fivefold increase in kappa gene rearrangements. In three of the four clonally matched pairs with increased kappa gene rearrangements, the increase in rearrangement frequency was not accompanied by a significant increase in germ line transcripts from the C kappa locus. However, in the fourth pair, K.40D, we observed an increase in germ line transcription of the kappa locus after expression of mu protein encoded by either an endogenously rearranged or a transfected functional heavy-chain allele. In these cells, the amount of the germ line C kappa transcript correlated with the measured frequency of rearranged kappa genes. These results support a regulated model of B-cell development in which mu protein expression in some way targets the V(D)J recombinase to the kappa gene locus. Images PMID:8355709

  18. Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis.

    PubMed

    Feng, Zhonghui; Okada, Satoshi; Cai, Guoping; Zhou, Bing; Bi, Erfei

    2015-04-01

    MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament-dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species. PMID:25631819

  19. Heavy chain (LvH) and light chain (LvL) of lipovitellin (Lv) of zebrafish can both bind to bacteria and enhance phagocytosis.

    PubMed

    Liang, Xue; Hu, Yu; Feng, Shuoqi; Zhang, Shicui; Zhang, Yu; Sun, Chen

    2016-10-01

    Lipovitellin (Lv) is an apoprotein in oviparous animals. Lv consists of a heavy chain (LvH) and a light chain (LvL) which are traditionally regarded as energy reserves for developing embryos. Recently, Lv has been shown to be involved in immune defense of developing embryos in fish. However, it remains unknown if each of LvH and LvL possesses immune activity; and if so, whether or not they function similarly. Here we clearly demonstrated that recombinant LvH (rLvH) and LvL (rLvL) from zebrafish vg1 gene bound to both the Gram-negative bacteria Escherichia coli and Vibrio anguillarum and the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus as well as the pathogen-associated molecular patterns LPS, LTA and PGN. In addition, both rLvH and rLvL were able to enhance the phagocytosis of bacteria E. coli and S. aureus by macrophages. All these data suggest that both LvH and LvL, in addition to being energy reserves, are also maternal immune-relevant factors capable of interacting with invading bacteria in zebrafish embryos/larvae. PMID:27185202

  20. 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

  1. Immunoglobulin kappa light chain variable region gene complex organization and immunoglobulin genes encoding anti-DNA autoantibodies in lupus mice.

    PubMed Central

    Kofler, R; Strohal, R; Balderas, R S; Johnson, M E; Noonan, D J; Duchosal, M A; Dixon, F J; Theofilopoulos, A N

    1988-01-01

    We have investigated the genetic origin of autoantibody production in several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease. Restriction fragment length polymorphism analyses of gene loci encoding kappa light chain variable regions (Igk-V) demonstrated, as shown previously for the Ig heavy chain locus, that autoantibody production and disease occur in different Igk-V haplotypes. Moreover, autoimmune mice with known genetic derivation inherited their Igk-V loci essentially unaltered from their nonautoimmune ancestors. New Zealand black lupus mice, with unknown genetic derivation, had a possibly recombinant Igk-V haplotype, composed of V kappa loci that were primarily indistinguishable from those of nonautoimmune strains from either of the two potential donor haplotypes. The heavy and light chain gene segments (variable, diversity, joining) encoding anti-DNA antibodies were diverse and often closely related, or even identical, to those found in antibodies to foreign antigens in normal mice. Only 1 of 11 sequenced variable region genes could not be assigned to existing variable region gene families; however, corresponding germline genes were present in the genome of normal mice as well. These data argue against abnormalities in the genes and mechanisms generating antibody diversity in lupus mice and suggest a remarkable genetic and structural diversity in the generation of anti-DNA binding sites. Images PMID:3138286

  2. Four things to know about myosin light chains as reporters for non-muscle myosin-2 dynamics in live cells.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2015-02-01

    The interplay between non-muscle myosins-2 and filamentous actin results in cytoplasmic contractility which is essential for eukaryotic life. Concomitantly, there is tremendous interest in elucidating the physiological function and temporal localization of non-muscle myosin-2 in cells. A commonly used method to study the function and localization of non-muscle myosin-2 is to overexpress a fluorescent protein (FP)-tagged version of the regulatory light chain (RLC) which binds to the myosin-2 heavy chain by mass action. Caveats about this approach include findings from recent studies indicating that the RLC does not bind exclusively to the non-muscle myosin-2 heavy chain. Rather, it can also associate with the myosin heavy chains of several other classes as well as other targets than myosin. In addition, the presence of the FP moiety may compromise myosin's enzymatic and mechanical performance. This and other factors to be discussed in this commentary raise questions about the possible complications in using FP-RLC as a marker for the dynamic localization and regulatory aspects of non-muscle myosin-2 motor functions in cell biological experiments. PMID:25712372

  3. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites. PMID:27318258

  4. Thermodynamic and fibril formation studies of full length immunoglobulin light chain AL-09 and its germline protein using scan rate dependent thermal unfolding.

    PubMed

    Blancas-Mejía, Luis M; Horn, Timothy J; Marin-Argany, Marta; Auton, Matthew; Tischer, Alexander; Ramirez-Alvarado, Marina

    2015-12-01

    Light chain (AL) amyloidosis is a fatal disease where monoclonal immunoglobulin light chains deposit as insoluble amyloid fibrils. For many years it has been considered that AL amyloid deposits are formed primarily by the variable domain, while its constant domain has been considered not to be amyloidogenic. However recent studies identify full length (FL) light chains as part of the amyloid deposits. In this report, we compare the stabilities and amyloidogenic properties of two light chains, an amyloid-associated protein AL-09 FL, and its germline protein κ I O18/O8 FL (IGKV 1-33). We demonstrate that the thermal unfolding for both proteins is irreversible and scan rate dependent, with similar stability parameters compared to their VL counterparts. In addition, the constant domain seems to modulate their amyloidogenic properties and affect the morphology of the amyloid fibrils. These results allow us to understand the role of the kappa constant domain in AL amyloidosis. PMID:26263488

  5. Novel familial dilated cardiomyopathy mutation in MYL2 affects the structure and function of myosin regulatory light chain.

    PubMed

    Huang, Wenrui; Liang, Jingsheng; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Zhou, Zhiqun; Morales, Ana; McBride, Kim L; Fitzgerald-Butt, Sara M; Hershberger, Ray E; Szczesna-Cordary, Danuta

    2015-06-01

    Dilated cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. Here we describe a novel DCM mutation in the myosin regulatory light chain (RLC), in which aspartic acid at position 94 is replaced by alanine (D94A). The mutation was identified by exome sequencing of three adult first-degree relatives who met formal criteria for idiopathic DCM. To obtain insight into the functional significance of this pathogenic MYL2 variant, we cloned and purified the human ventricular RLC wild-type (WT) and D94A mutant proteins, and performed in vitro experiments using RLC-mutant or WT-reconstituted porcine cardiac preparations. The mutation induced a reduction in the α-helical content of the RLC, and imposed intra-molecular rearrangements. The phosphorylation of RLC by Ca²⁺/calmodulin-activated myosin light chain kinase was not affected by D94A. The mutation was seen to impair binding of RLC to the myosin heavy chain, and its incorporation into RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared with ATPase of wild-type. No changes in the myofibrillar ATPase-pCa relationship were observed in wild-type- or D94A-reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross-section of muscle, with no change in the calcium sensitivity of force in D94A-reconstituted skinned porcine papillary muscle strips compared with wild-type. Our data indicate that subtle structural rearrangements in the RLC molecule, followed by its impaired interaction with the myosin heavy chain, may trigger functional abnormalities contributing to the DCM phenotype. PMID:25825243

  6. Myosin light chain phosphorylation in sup 32 P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

    SciTech Connect

    Singer, H.A.; Oren, J.W.; Benscoter, H.A. )

    1989-12-15

    The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.

  7. FhCaBP3: a Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains.

    PubMed

    Banford, Samantha; Drysdale, Orla; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J

    2013-04-01

    A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism. PMID:23142130

  8. Surgical Management of Pulmonary Mucosa-Associated Lymphoid Tissue Lymphoma Associated With Light-Chain Deposition Disease.

    PubMed

    Muñoz-Largacha, Juan A; O'Hara, Carl J; Sloan, J Mark; Fernando, Hiran C; Litle, Virginia R

    2016-06-01

    A 52-year-old woman presented with a right middle lobe (RML) lung nodule suspicious for malignancy. Thoracoscopic middle lobectomy was performed. The pathology report revealed a pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma in association with light-chain deposition disease (LCDD). Pulmonary MALT lymphoma and LCDD are unusual disorders presenting in the lung, and the association between these 2 conditions is even more uncommon. The optimal management for these patients is controversial, although surgical resection of localized well-circumscribed lesions may represent an effective therapeutic approach. PMID:27211983

  9. A Ras-like domain in the light intermediate chain bridges the dynein motor to a cargo-binding region

    PubMed Central

    Schroeder, Courtney M; Ostrem, Jonathan ML; Hertz, Nicholas T; Vale, Ronald D

    2014-01-01

    Cytoplasmic dynein, a microtubule-based motor protein, transports many intracellular cargos by means of its light intermediate chain (LIC). In this study, we have determined the crystal structure of the conserved LIC domain, which binds the motor heavy chain, from a thermophilic fungus. We show that the LIC has a Ras-like fold with insertions that distinguish it from Ras and other previously described G proteins. Despite having a G protein fold, the fungal LIC has lost its ability to bind nucleotide, while the human LIC1 binds GDP preferentially over GTP. We show that the LIC G domain binds the dynein heavy chain using a conserved patch of aromatic residues, whereas the less conserved C-terminal domain binds several Rab effectors involved in membrane transport. These studies provide the first structural information and insight into the evolutionary origin of the LIC as well as revealing how this critical subunit connects the dynein motor to cargo. DOI: http://dx.doi.org/10.7554/eLife.03351.001 PMID:25272277

  10. TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

    PubMed

    Xu, Chang; Wu, Xiaoyan; Hack, Bradley K; Bao, Lihua; Cunningham, Patrick N

    2015-12-01

    A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway. PMID:26634902

  11. Quantification of β region IgA paraproteins - should we include immunochemical "heavy/light chain" measurements? Counterpoint.

    PubMed

    Paolini, Lucia

    2016-06-01

    Serum protein electrophoresis (SPE), serum immunofixation (s-IFE), free light chain measurement (FLC) and nephelometric measurements of total immunoglobulin in serum (IgTot) are some of the laboratory tests required for the management of plasma cell proliferative disorders. The monoclonal protein is usually visible on SPE as a spike (M-spike) in the γ region and the derived densitogram is used to quantify it relative to serum total protein concentration. IgA M-protein, however, often migrates in the β region on SPE and its quantification can be masked by other serum proteins that migrate in this region. The immunoassay Hevylite™ (heavy/light chain, HLC) seems to solve this problem: it quantifies the involved/uninvolved isotype, calculating the ratio IgAκ/IgAλ, considered indicative of clonal proliferation. However, this test seems redundant in the case of artifacts on SPE such as obvious hemolysis or lipemia, or if the IgA M-spike is clearly visible in the β region. In conclusion whereas the IgA HLC assay does not represent an alternative to SPE and s-IFE in the diagnostic patient workup, it may prove to be an alternative to SPE, s-IFE and total IgA quantification in risk stratification and evaluation of response to therapy in patients affected by MM and other monoclonal plasma proliferative disorders. PMID:26812795

  12. [The concurrence of light-chain deposition disease, AL-amyloidosis, and cast nephropathy in a patient with multiple myeloma].

    PubMed

    Rekhtina, I G; Zakharova, E V; Stoliarevich, E S; Sinitsina, M N; Denisova, E N

    2015-01-01

    Despite of the fact that their clinical manifestations are similar, AL-amyloidosis (AL-A) and light chain deposition disease (LCDD) are individual nosological entities in view of considerable differences in their pathogenesis and pathomorphology. The paper describes a rare case of the concurrence of LCDD and AL-A in a patient with multiple myeloma. Clinically, there was dialysis-dependent renal failure, flail leg syndrome, myocardiopathy, and rhabdomyolysis. At the disease onset, his nephrobiopsy specimen could diagnose LCDD and myeloma or cast nephropathy. The disease was characterized by an aggressive course. Despite the administration of innovative agents, the patient had a short-term remission and died from disease progression. Autopsy additionally revealed amyloid deposition in the heart and kidney. The development of AL-A in the presence of prior LCDD may reflect the progression of the tumor and the appearance of an additional subclone of plasma cells that produce amyloidogenic light chains. The uncommonness of this case is that renal amyloid was found in the tubular casts and absent in the glomeruli, which may be considered as a special form--tubular AL-amyloidosis. PMID:26281203

  13. The double polymethylmethacrylate filter (DELETE system) in the removal of light chains in chronic dialysis patients with multiple myeloma.

    PubMed

    Santoro, Antonio; Grazia, Michele; Mancini, Elena

    2013-01-01

    Multiple myeloma (MM) is still one of the most common haematological diseases and is associated with a poor prognosis. It is frequently worsened by acute kidney failure that, in turn, aggravates the risk of death. In the past few years, the idea has made headway that the removal of free light chains (FLC) by means of extracorporeal blood purification systems may facilitate the recovery of renal function. Up to now, many different extracorporeal techniques have been put forward in FLC removal, such as plasma exchange, dialysis with super-flux filters, and adsorption by means of cartridge of resins. In this paper, we illustrate the use of polymethylmethacrylate (PMMA) dialysis membranes with a high adsorptive capacity (Toray BK-F; Toray Industries, Inc., Tokyo, Japan). We have evaluated light chain removal by means of an original dialysis procedure using a double-filter circuit made of PMMA working in sequential dialysis (DELETE system). The system provides satisfactory results in terms of FLC removal and, at the same time, ensures an adequate dialysis treatment (Kt/V >1.5) with significant reduction in urea, creatinine, and β2-microglobulin. The dual PMMA filter system combines an acceptable cost/efficiency ratio when compared with other methods and constitutes a concrete prospect in FLC removal. Its preferential setting of use is in patients with MM or with monoclonal gammopathies, who are on chronic dialysis and maintain high circulating levels of FLC. PMID:23676829

  14. European trial of free light chain removal by extended haemodialysis in cast nephropathy (EuLITE): A randomised control trial

    PubMed Central

    Hutchison, Colin A; Cook, Mark; Heyne, Nils; Weisel, Katja; Billingham, Lucinda; Bradwell, Arthur; Cockwell, Paul

    2008-01-01

    Background Renal failure is a frequent complication of multiple myeloma and when severe is associated with a greatly increased morbidity and mortality. The principal cause of severe renal failure is cast nephropathy, a direct consequence of high concentrations of monoclonal free light chains (FLCs) in patients' sera. FLC removal by extended haemodialysis, using a high cut-off dialyser, has recently been described as a novel therapeutic option. Methods The EUropean trial of free LIght chain removal by exTEnded haemodialysis in cast nephropathy (EuLITE) trial is a prospective, randomised, multicentre, open label clinical trial to investigate the clinical benefits of FLC removal haemodialysis in patients with cast nephropathy, dialysis dependent acute renal failure and de novo multiple myeloma. Recruitment commenced in May 2008. In total, 90 patients will be recruited. Participants will be randomised, centrally, upon enrolment, to either trial chemotherapy and FLC removal haemodialysis or trial chemotherapy and standard high flux haemodialysis. Trial chemotherapy consists of bortezomib, doxorubicin and dexamethasone. FLC removal haemodialysis is undertaken with two Gambro HCO 1100 dialysers in series using an intensive treatment schedule. The primary outcome for the study is independence of dialysis at 3 months. Secondary outcomes are: duration of dialysis, reduction in serum FLC concentrations; myeloma response and survival. Hypothesis FLC removal haemodialysis will increase the rate of renal recovery in patients with severe renal failure secondary to cast nephropathy in de novo multiple myeloma. Trial registration ISRCTN45967602 PMID:18822172

  15. IgD multiple myeloma: Clinical, biological features and prognostic value of the serum free light chain assay.

    PubMed

    Djidjik, R; Lounici, Y; Chergeulaïne, K; Berkouk, Y; Mouhoub, S; Chaib, S; Belhani, M; Ghaffor, M

    2015-09-01

    IgD multiple myeloma (MM) is a rare subtype of myeloma, it affects less than 2% of patients with MM. To evaluate the clinical and prognostic attributes of serum free light chains (sFLCs) analysis, we examined 17 cases of IgD MM. From 1998 to 2012, we obtained 1250 monoclonal gammapathies including 590 multiple myeloma and 17 patients had IgD MM. With preponderance of men patients with a mean age at diagnosis of: 59±12years. Patients with IgD MM have a short survival (Median survival=9months). The presenting features included: bone pain (75%), lymphadenopathy (16%), hepatomegaly (25%), splenomegaly (8%), associated AL amyloidosis (6%), renal impairment function (82%), infections (47%), hypercalcemia (37%) and anemia (93%). Serum electrophoresis showed a subtle M-spike (Mean=13.22±10g/L) in all patients associated to a hypogammaglobulinemia. There was an over-representation of Lambda light chain (65%); high serum β2-microglobulin in 91% and Bence Jones proteinuria was identified in 71%. The median rate of sFLCs κ was 19.05mg/L and 296.75mg/L for sFLCs λ. sFLCR was abnormal in 93% of patients and it showed concordance between baseline sFLCR and the survival (P=0.034). The contribution of FLC assay is crucial for the prognosis of patients with IgD MM. PMID:26294067

  16. IQ motif selectivity in human IQGAP1: binding of myosin essential light chain and S100B.

    PubMed

    Pathmanathan, Sevvel; Elliott, Sarah F; McSwiggen, Sara; Greer, Brett; Harriott, Pat; Irvine, G Brent; Timson, David J

    2008-11-01

    IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition. PMID:18587628

  17. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study.

    PubMed

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  18. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study

    PubMed Central

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  19. Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin.

    PubMed

    Bowman, B F; Peterson, J A; Stull, J T

    1992-03-15

    Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process. PMID:1544916

  20. RELATIONSHIP BETWEEN ELEVATED IMMUNOGLOBULIN FREE LIGHT CHAIN AND THE PRESENCE OF IgH TRANSLOCATIONS IN MULTIPLE MYELOMA

    PubMed Central

    Kumar, S.; Zhang, L.; Dispenzieri, A.; Van Wier, S.; Katzmann, J.A.; Snyder, M.; Blood, E.; DeGoey, R.; Henderson, K.; Kyle, R.A.; Bradwell, A.R.; Greipp, P.R.; Rajkumar, S.V.; Fonseca, R

    2013-01-01

    Elevated immunoglobulin free light chain (FLC) level and abnormal FLC ratio is commonly seen in multiple myeloma (MM) and have prognostic implications. We hypothesized that presence of IgH translocations leads to unbalanced production of light chains and more extreme abnormalities of FLC and may explain the prognostic value of FLC. We studied 314 patients with newly diagnosed MM enrolled on a phase-III trial, in whom results of FISH testing and serum FLC were available. Cytogenetic analyses and FLC estimates were performed on stored samples and results correlated with clinical data. The median ratio (FLC-ratio) and the absolute difference (FLC-diff) between the involved and uninvolved FLC was higher among those with IgH translocations, especially t(14;16). In multivariate analysis, the prognostic value of FLC estimates on progression free and overall survival were independent of high-risk IgH translocations t(4;14) and t(14;16). A combination of the risk factors; either abnormal FLC estimate and/or the presence of high-risk IgH translocation, achieved better prognostic stratification. In conclusion, patients with IgH translocations tend to have higher FLC levels and abnormal ratios, but the prognostic effect of FLC is only partially explained by translocation status. A system including both these risk factors allows better prediction of outcome. PMID:20520636

  1. Resonant transmission of light in chains of high-index dielectric particles

    NASA Astrophysics Data System (ADS)

    Savelev, Roman S.; Filonov, Dmitry S.; Petrov, Mihail I.; Krasnok, Alexander E.; Belov, Pavel A.; Kivshar, Yuri S.

    2015-10-01

    We study numerically, analytically, and experimentally the resonant transmission of light in a waveguide formed by a periodic array of high-index dielectric nanoparticles with a side-coupled resonator. We demonstrate that a resonator with high enough Q -factor provides the conditions for the Fano-type interference allowing one to control the resonant transmission of light. We suggest a practical realization of this resonant effect based on the quadrupole resonance of a dielectric particle and demonstrate it experimentally for ceramic disks at microwave frequencies.

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

    SciTech Connect

    Hernández-Santoyo, A.; Del Pozo Yauner, L; Fuentes-Silva, D; Ortiz, E; Rudiño-Piñera, E; Sánchez-López, R; Horjales, E; Becerril, B; Rodríguez-Romero, A

    2010-01-01

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

  3. Local light-induced spin manipulation in two magnetic centre metallic chains

    NASA Astrophysics Data System (ADS)

    Hartenstein, T.; Li, C.; Lefkidis, G.; Hübner, W.

    2008-08-01

    In this paper localized optically induced spin dynamics is presented, based on highly correlational ab initio calculations. Two-magnetic-centre metallic chains are chosen as a material on which the total spin is always found to lie on one of the magnetic centres only. Switching is achieved through a Λ-process driven by a laser pulse whose parameters are optimized with a genetic algorithm. Locally switching the spin on the iron side of a Co-Na-Fe cluster is given as an example of local spin manipulation.

  4. Generation and Control of Chains of Entangled Atom-Ion Pairs with Quantum Light

    SciTech Connect

    Shapiro, Moshe; Brumer, Paul

    2011-04-15

    Coherent control using quantum light incident upon molecules in an optical lattice is shown to give rise to a direct way of writing arbitrary sequences of entangled atom-ion pairs. There is no evident limitation on the length of the word (i.e., the number of qbits) that can be formed.

  5. The Mechanism of Dynein Light Chain LC8-mediated Oligomerization of the Ana2 Centriole Duplication Factor*

    PubMed Central

    Slevin, Lauren K.; Romes, Erin M.; Dandulakis, Mary G.; Slep, Kevin C.

    2014-01-01

    Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156–251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ∼34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis. PMID:24920673

  6. The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor.

    PubMed

    Slevin, Lauren K; Romes, Erin M; Dandulakis, Mary G; Slep, Kevin C

    2014-07-25

    Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ~34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis. PMID:24920673

  7. 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. PMID:27056571

  8. Neuregulin1-β decreases interleukin-1β-induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability.

    PubMed

    Wu, Limin; Ramirez, Servio H; Andrews, Allison M; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. We propose the following events for neuregulin-1-mediated effects on Interleukin-1 β (IL-1β)-induced endothelial hyperpermeability: IL-1β leads to RhoA activation, resulting in an increase in phosphorylation of myosin light chain (MLC). Phosphorylation of MLC is known to result in actin contraction and alterations in the f-actin cytoskeletal structure. These changes are associated with increased endothelial permeability

  9. Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers.

    PubMed Central

    Hambly, B; Franks, K; Cooke, R

    1991-01-01

    Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which

  10. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

    SciTech Connect

    Silvaggi,N.; Wilson, D.; Tzipori, S.; Allen, K.

    2008-01-01

    The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.

  11. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    PubMed Central

    Lima, V.V.; Lobato, N.S.; Filgueira, F.P.; Webb, R.C.; Tostes, R.C.; Giachini, F.R.

    2014-01-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  12. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

    PubMed

    Lima, V V; Lobato, N S; Filgueira, F P; Webb, R C; Tostes, R C; Giachini, F R

    2014-10-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  13. Regulation of calcium channels in smooth muscle: New insights into the role of myosin light chain kinase

    PubMed Central

    Martinsen, A; Dessy, C; Morel, N

    2014-01-01

    Smooth muscle myosin light chain kinase (MLCK) plays a crucial role in artery contraction, which regulates blood pressure and blood flow distribution. In addition to this role, MLCK contributes to Ca2+ flux regulation in vascular smooth muscle (VSM) and in non-muscle cells, where cytoskeleton has been suggested to help Ca2+ channels trafficking. This conclusion is based on the use of pharmacological inhibitors of MLCK and molecular and cellular techniques developed to down-regulate the enzyme. Dissimilarities have been observed between cells and whole tissues, as well as between large conductance and small resistance arteries. A differential expression in MLCK and ion channels (either voltage-dependent Ca2+ channels or non-selective cationic channels) could account for these observations, and is in line with the functional properties of the arteries. A potential involvement of MLCK in the pathways modulating Ca2+ entry in VSM is described in the present review. PMID:25483583

  14. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells

    PubMed Central

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K.; Das, Mahua R.; Sen, Shamik; Sarkar, Debi P.; Jana, Siddhartha S.

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (−) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  15. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

    PubMed

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K; Das, Mahua R; Sen, Shamik; Sarkar, Debi P; Jana, Siddhartha S

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  16. Resonance Assignments and Secondary Structure Analysis of Dynein Light Chain 8 by Magic-angle Spinning NMR Spectroscopy

    SciTech Connect

    Sun, Shangjin; Butterworth, Andrew H.; Paramasivam, Sivakumar; Yan, Si; Lightcap, Christine M.; Williams, John C.; Polenova, Tatyana E.

    2011-08-04

    Dynein light chain LC8 is the smallest subunit of the dynein motor complex and has been shown to play important roles in both dynein-dependent and dynein-independent physiological functions via its interaction with a number of its binding partners. It has also been linked to pathogenesis including roles in viral infections and tumorigenesis. Structural information for LC8-target proteins is critical to understanding the underlying function of LC8 in these complexes. However, some LC8-target interactions are not amenable to structural characterization by conventional structural biology techniques owing to their large size, low solubility, and crystallization difficulties. Here, we report magic-angle spinning (MAS) NMR studies of the homodimeric apo-LC8 protein as a first effort in addressing more complex, multi-partner, LC8-based protein assemblies. We have established site-specific backbone and side-chain resonance assignments for the majority of the residues of LC8, and show TALOS+-predicted torsion angles ø and ψ in close agreement with most residues in the published LC8 crystal structure. Data obtained through these studies will provide the first step toward using MAS NMR to examine the LC8 structure, which will eventually be used to investigate protein–protein interactions in larger systems that cannot be determined by conventional structural studies.

  17. A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

    PubMed Central

    Caglič, Dejan; Bompiani, Kristin M.; Krutein, Michelle C.; Čapek, Petr; Dickerson, Tobin J.

    2013-01-01

    Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds. PMID:24430674

  18. Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils.

    PubMed Central

    Nelson, S R; Lyon, M; Gallagher, J T; Johnson, E A; Pepys, M B

    1991-01-01

    Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1902087

  19. Clathrin Light Chains Regulate Clathrin-Mediated Trafficking, Auxin Signaling, and Development in Arabidopsis[C][W][OA

    PubMed Central

    Wang, Chao; Yan, Xu; Chen, Qian; Jiang, Nan; Fu, Wei; Ma, Bojun; Liu, Jianzhong; Li, Chuanyou; Bednarek, Sebastian Y.; Pan, Jianwei

    2013-01-01

    Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development. PMID:23424247

  20. Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model

    PubMed Central

    Tarigopula, Madhusudhan; Davis, Robert T.; Mungai, Paul T.; Ryba, David M.; Wieczorek, David F.; Cowan, Conrad L.; Violin, Jonathan D.; Wolska, Beata M.; Solaro, R. John

    2015-01-01

    Aims Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca2+-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca2+ transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca2+ response. Methods and results We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure–volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation. Conclusion Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM. PMID:26045475

  1. Immunoglobulin heavy/light chain analysis enhances the detection of residual disease and monitoring of multiple myeloma patients

    PubMed Central

    Batinić, Josip; Perić, Zinaida; Šegulja, Dragana; Last, James; Prijić, Sanja; Dubravčić, Klara; Volarić, Lidija; Sertić, Dubravka; Radman, Ivo; Bašić-Kinda, Sandra; Matišić, Danica; Batinić, Drago; Labar, Boris; Nemet, Damir

    2015-01-01

    Aim To evaluate the clinical utility of incorporating a novel heavy/light chain immunoassay (HLC) into the existing methods for the assessment of multiple myeloma (MM) patients. Methods Convenience sera samples from 90 previously treated IgG and IgA MM patients in different disease stages were analyzed. The study was conducted in Clinical Hospital Center Zagreb between 2011 and 2013. The collected sera were analyzed by standard laboratory techniques (serum protein electrophoresis, quantification of total immunoglobulins, serum immunofixation, serum free light chain [FLC] assay) and HLC assay. Results HLC ratios outside the normal range were found in 58 of 90 patients, including 28 out of 61 patients with total immunoglobulin measurements within the normal range and 5 out of 23 patients in complete response. Both elevated HLC isotype level and abnormal HLC ratio correlated with the parameters of tumor burden, including percentage of plasma cells in the bone marrow (P < 0.001 and P = 0.002, respectively) and an abnormal serum FLC ratio (for both P < 0.001). In addition, abnormal HLC isotype level correlated with serum beta-2-microglobulin level (P = 0.038). In terms of prognosis, abnormal HLC isotype level and abnormal HLC ratio were significantly associated with shorter overall survival (P < 0.001 and P = 0.002, respectively). Interestingly, suppression of the uninvolved (polyclonal) isotype pair, but not other non-myeloma immunoglobulin isotypes, was also associated with a shorter overall survival (P = 0.021). In a multivariate analysis, an abnormal HLC ratio and β2-microglobulin level >3.5mg/L were independent risk factors for survival. Conclusion The new HLC assay has greater sensitivity in detecting monoclonal protein, correlates with tumor burden markers, and affects patients' outcome. PMID:26088851

  2. IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain.

    PubMed

    Atcheson, Erwan; Hamilton, Elaine; Pathmanathan, Sevvel; Greer, Brett; Harriott, Pat; Timson, David J

    2011-10-01

    The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins. PMID:21299499

  3. IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain

    PubMed Central

    Atcheson, Erwan; Hamilton, Elaine; Pathmanathan, Sevvel; Greer, Brett; Harriott, Pat; Timson, David J.

    2011-01-01

    The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins. PMID:21299499

  4. Distinct tissue distributions and subcellular localizations of differently phosphorylated forms of the myosin regulatory light chain in Drosophila.

    PubMed

    Zhang, Liang; Ward, Robert E

    2011-01-01

    Nonmuscle myosin II (myosin hereafter) has well-established roles in generating contractile force on actin filaments during morphogenetic processes in all metazoans. Myosin activation is regulated by phosphorylation of the myosin regulatory light chain (MRLC, encoded by spaghettisquash or sqh in Drosophila) first on Ser21 and subsequently on Thr20. These phosphorylation events are positively controlled by a variety of kinases including myosin light chain kinase, Rho kinase, citron kinase, and AMP kinase and are negatively regulated by myosin phosphatase. The activation of myosin is thus highly regulated and likely developmentally controlled. In order to monitor the activity of myosin during development, we have generated antibodies against the monophosphorylated (Sqh1P) and diphosphorylated (Sqh2P) forms of Sqh. We first show that the antibodies are highly specific. We then used these antibodies to monitor myosin activation in wild type Drosophila tissues. Interestingly, Sqh1P and Sqh2P show distinct patterns of expression in embryos. Sqh1P is expressed nearly ubiquitously and outlines cells consistent with a junctional localization, whereas Sqh2P is strongly expressed on the apical surfaces and in filopodia of tissues undergoing extensive cell shape change or cell movements including the invaginating fore- and hindgut, the invaginating tracheal system, the dorsal pouch and the dorsal most row of epidermal (DME) cells during dorsal closure. In imaginal discs, Sqh1P predominantly localizes in the adherens junction, whereas Sqh2P locates to the apical domain. These antibodies thus have the potential to be very useful in monitoring myosin activation for functional studies of morphogenesis in Drosophila. PMID:20920606

  5. Comparison of crystal structures of two homologous proteins: structural origin of altered domain interactions in immunoglobulin light-chain dimers.

    PubMed

    Huang, D B; Chang, C H; Ainsworth, C; Brünger, A T; Eulitz, M; Solomon, A; Stevens, F J; Schiffer, M

    1994-12-13

    The sequence and structure of a second human kappa 1 immunoglobulin light-chain variable domain, Wat, has been determined. The R-factor is 15.7% for 1.9-A data. One hundred and ninety-five water molecules were identified; 30 water molecules were located in identical positions in each of the monomers. Some of the water molecules are integral parts of the domains. This light chain is encoded by the same variable domain gene that encoded the previously characterized kappa I variable domain, Rei. Due to limited somatic mutation, the two highly homologous proteins differ in only 20 of the 108 residues. Wat crystallized in space group P6(4) while Rei crystallized in space group P6(1); in both crystals, the asymmetric unit was the noncovalent dimer. Although the basic domain structure is the same for both proteins, the relative positions of the domains within the two dimers differ. This difference is most likely accounted for by the replacement of Tyr36 in Rei by Phe in the Wat protein. Residue Tyr36 is part of the hydrogen-bonding network in the interface between the domains in Rei. Losing the hydrogen-bonding capability of residue 36 by replacement of Tyr by Phe alters the network of hydrogen bonds between the domains, resulting in a different domain-domain contact. The details of lattice contacts in the two crystals were compared. One type of contact that extends the beta-sheet of the individual domains was conserved, but because it involved different symmetry elements within the crystal, different crystal packing resulted. In the Wat crystal, one of the contacts shows an example of how a symmetrical binding site can "bind" an asymmetrical object. Further, the examination of the Wat crystal also illustrates how the different crystalline environments of the domains of the dimer results in different distributions of temperature factors for the residues within the domains. PMID:7993911

  6. Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway.

    PubMed

    Sorigué, Damien; Légeret, Bertrand; Cuiné, Stéphan; Morales, Pablo; Mirabella, Boris; Guédeney, Geneviève; Li-Beisson, Yonghua; Jetter, Reinhard; Peltier, Gilles; Beisson, Fred

    2016-08-01

    Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway. PMID:27288359

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

    PubMed Central

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

    2015-01-01

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

  8. Light Chain Deposition Disease in an Older Adult Patient Successfully Treated with Long-term Administration of Bortezomib, Melphalan and Prednisone.

    PubMed

    Hiyamuta, Hiroto; Yamada, Shunsuke; Matsukuma, Yuta; Tsuchimoto, Akihiro; Nakano, Toshiaki; Taniguchi, Masatomo; Masutani, Kosuke; Yoshimoto, Goichi; Muta, Tsuyoshi; Akashi, Koichi; Kitazono, Takanari; Tsuruya, Kazuhiko

    2016-01-01

    A 70-year-old woman was admitted to our hospital because of fatigue and renal dysfunction and was diagnosed with light chain deposition disease (LCDD) with multiple organ involvement (kidney, thyroid gland, heart and eyes). After chemotherapy with bortezomib, cyclophosphamide and dexamethasone, hepatobiliary enzyme levels increased abruptly. A liver biopsy showed light chain deposition in Disse spaces. After two years of treatment with bortezomib, melphalan and prednisone (VMP) administered at shorter intervals relative to regular cycles, the patient showed a hematological and organ response. This case indicates that a relatively low dose intensity VMP regimen is preferable for elderly patients with LCDD with multiple organ involvement. PMID:27181540

  9. Immunoglobulin analysis tool: a novel tool for the analysis of human and mouse heavy and light chain transcripts.

    PubMed

    Rogosch, Tobias; Kerzel, Sebastian; Hoi, Kam Hon; Zhang, Zhixin; Maier, Rolf F; Ippolito, Gregory C; Zemlin, Michael

    2012-01-01

    Sequence analysis of immunoglobulin (Ig) heavy and light chain transcripts can refine categorization of B cell subpopulations and can shed light on the selective forces that act during immune responses or immune dysregulation, such as autoimmunity, allergy, and B cell malignancy. High-throughput sequencing yields Ig transcript collections of unprecedented size. The authoritative web-based IMGT/HighV-QUEST program is capable of analyzing large collections of transcripts and provides annotated output files to describe many key properties of Ig transcripts. However, additional processing of these flat files is required to create figures, or to facilitate analysis of additional features and comparisons between sequence sets. We present an easy-to-use Microsoft(®) Excel(®) based software, named Immunoglobulin Analysis Tool (IgAT), for the summary, interrogation, and further processing of IMGT/HighV-QUEST output files. IgAT generates descriptive statistics and high-quality figures for collections of murine or human Ig heavy or light chain transcripts ranging from 1 to 150,000 sequences. In addition to traditionally studied properties of Ig transcripts - such as the usage of germline gene segments, or the length and composition of the CDR-3 region - IgAT also uses published algorithms to calculate the probability of antigen selection based on somatic mutational patterns, the average hydrophobicity of the antigen-binding sites, and predictable structural properties of the CDR-H3 loop according to Shirai's H3-rules. These refined analyses provide in-depth information about the selective forces acting upon Ig repertoires and allow the statistical and graphical comparison of two or more sequence sets. IgAT is easy to use on any computer running Excel(®) 2003 or higher. Thus, IgAT is a useful tool to gain insights into the selective forces and functional properties of small to extremely large collections of Ig transcripts, thereby assisting a researcher to mine a data set

  10. Metachronous/concomitant B-cell neoplasms with discordant light-chain or heavy-chain isotype restrictions: evidence of distinct B-cell neoplasms rather than clonal evolutions.

    PubMed

    Wei, Qiang; Sebastian, Siby; Papavassiliou, Paulie; Rehder, Catherine; Wang, Endi

    2014-10-01

    Metachronous/concomitant B-cell neoplasms with distinct morphology are usually considered clonally related. We retrospectively analyzed 4 cases of metachronous/concomitant B-cell neoplasms with discordant light-chain/heavy-chain restrictions. The primary diagnoses included chronic lymphocytic leukemia (CLL; n = 2), lymphoplasmacytic lymphoma (n = 1), and pediatric follicular lymphoma (FL; n = 1). The respective secondary diagnoses included diffuse large B-cell lymphoma (DLBCL; n = 2), plasmablastic myeloma, and pediatric FL. The secondary B-cell neoplasm occurred after the primary diagnosis in 3 cases, with the median interval of 120 months (range, 21-216), whereas the remaining 1 case had the 2 neoplasms (CLL/DLBCL) diagnosed concurrently. Histology suggested aggressive transformation in 3 cases and recurrence in 1 case (FL). Nonetheless, 3 cases showed discordant light-chain restrictions between the 2 B-cell neoplasms, whereas in the remaining case (lymphoplasmacytic lymphoma/plasmablastic myeloma), the 2 neoplasms shared κ light-chain restriction but expressed different heavy-chain isotypes (IgM versus IgA). The 2 CLL/DLBCL cases had polymerase chain reaction-based IGH/K gene rearrangement study and amplicon sequence analysis performed, which demonstrated distinct clonal amplicons between the 2 B-cell neoplasms in each case. Concomitant/metachronous B-cell neoplasms may be clonally unrelated, which can be confirmed by immunoglobulin isotype analysis and/or genotypic studies. We advocate analysis of clonal identities in large cell transformation or recurrent disease compared with primary indolent B-cell neoplasm because of a potential difference in prognosis between clonally related and unrelated secondary B-cell neoplasms. PMID:25179408

  11. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    SciTech Connect

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L.

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  12. Localization of the human kinesin light chain gene (KNS2) to chromosome 14q32.3 by fluorescence in situ hybridization

    SciTech Connect

    Goedert, M.; Marsh, S.; Carter, N.

    1996-02-15

    This article reports on the localization of human kinesin light chain gene (KNS2) to human chromosome 14q32.3 using fluorescence in situ hybridization. Further studies will need to be conducted to see whether mutations in the KNS2 gene are associated with hereditary diseases. 10 refs., 1 fig.

  13. Myosin light chain phosphatase activation is involved in the hydrogen sulfide-induced relaxation in mouse gastric fundus.

    PubMed

    Dhaese, Ingeborg; Lefebvre, Romain A

    2009-03-15

    The relaxant effect of hydrogen sulfide (H(2)S) in the vascular tree is well established but its influence and mechanism of action in gastrointestinal smooth muscle was hardly investigated. The influence of H(2)S on contractility in mouse gastric fundus was therefore examined. Sodium hydrogen sulfide (NaHS; H(2)S donor) was administered to prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse gastric fundus, before and after incubation with interfering drugs. NaHS caused a concentration-dependent relaxation of the pre-contracted mouse gastric fundus strips. The K(+) channels blockers glibenclamide, apamin, charybdotoxin, 4-aminopyridin and barium chloride had no influence on the NaHS-induced relaxation. The relaxation by NaHS was also not influenced by L-NAME, ODQ and SQ 22536, inhibitors of the cGMP and cAMP pathway, by nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The myosin light chain phosphatase (MLCP) inhibitor calyculin-A reduced the NaHS-induced relaxation, but the Rho-kinase inhibitor Y-27632 had no influence. We show that NaHS is able to relax PGF(2alpha)-contracted mouse gastric fundus strips. The results suggest that in the mouse gastric fundus, H(2)S causes relaxation at least partially via activation of MLCP. PMID:19374871

  14. Fasciola hepatica calcium-binding protein FhCaBP2: structure of the dynein light chain-like domain.

    PubMed

    Nguyen, Thanh H; Thomas, Charlotte M; Timson, David J; van Raaij, Mark J

    2016-07-01

    The common liver fluke Fasciola hepatica causes an increasing burden on human and animal health, partly because of the spread of drug-resistant isolates. As a consequence, there is considerable interest in developing new drugs to combat liver fluke infections. A group of potential targets is a family of calcium-binding proteins which combine an N-terminal domain with two EF-hand motifs and a C-terminal domain with predicted similarity to dynein light chains (DLC-like domain). The function of these proteins is unknown, although in several species, they have been localised to the tegument, an important structure at the host-parasite interface. Here, we report the X-ray crystal structure of the DLC-like domain of F. hepatica calcium-binding protein 2 (FhCaBP2), solved using single-wavelength anomalous diffraction and refined at 2.3 Å resolution in two different crystal forms. The FhCaBP2 DLC-like domain has a structure similar to other DLC domains, with an anti-parallel β-sheet packed against an α-helical hairpin. Like other DLC domains, it dimerises through its β2-strand, which extends in an arch and forms the fifth strand in an extended β-sheet of the other monomer. The structure provides molecular details of the dimerisation of FhCaBP2, the first example from this family of parasite proteins. PMID:27083189

  15. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation.

    PubMed

    Orr, Nathan; Arnaout, Rima; Gula, Lorne J; Spears, Danna A; Leong-Sit, Peter; Li, Qiuju; Tarhuni, Wadea; Reischauer, Sven; Chauhan, Vijay S; Borkovich, Matthew; Uppal, Shaheen; Adler, Arnon; Coughlin, Shaun R; Stainier, Didier Y R; Gollob, Michael H

    2016-01-01

    Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect. PMID:27066836

  16. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation.

    PubMed

    Hansen, Charlotte Toftmann

    2016-06-01

    Measurements of immunoglobulins and serum free light chains (sFLC) are frequently used in patients with monoclonal plasma cell dyscrasia (PCD). For optimum patient care, well-defined performance standards or goals for the measured concentrations of immunoglobulins and sFLC are required. Generally, data based on biological variation is a good and reliable method for setting desirable performance standards; this also applies for the measurements of paraprotein and sFLC. The benefits of this approach are several. Among others, it is independent of the clinician, and it provides us with information about reference change value and index of individuality. Several studies on biological variation of both immunoglobulins and sFLC have been published, and mostly the studies are well performed. The studies normally show small within-subject biological variation resulting in strict analytical goals, which in most cases are difficult to meet. Nevertheless, we still need further information on biological variation of immunoglobulins and sFLC in patients with PCD and in the elderly, which are the main target populations for the two measurands. Furthermore, to improve data on biological variation of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed. PMID:26824979

  17. Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

    PubMed

    Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan; Jin, Yutong; Wei, Liming; Chen, Albert J; McKiernan, Susan H; Aiken, Judd M; Moss, Richard L; Diffee, Gary M; Ge, Ying

    2016-08-01

    Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia. PMID:27362462

  18. Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in Neurodegenerative Diseases.

    PubMed

    Bacioglu, Mehtap; Maia, Luis F; Preische, Oliver; Schelle, Juliane; Apel, Anja; Kaeser, Stephan A; Schweighauser, Manuel; Eninger, Timo; Lambert, Marius; Pilotto, Andrea; Shimshek, Derya R; Neumann, Ulf; Kahle, Philipp J; Staufenbiel, Matthias; Neumann, Manuela; Maetzler, Walter; Kuhle, Jens; Jucker, Mathias

    2016-07-01

    A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases. PMID:27292537

  19. 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. PMID:25412230

  20. Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

    PubMed Central

    Mahale, Sagar P.; Sharma, Amit; Mylavarapu, Sivaram V. S.

    2016-01-01

    The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC). However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division. PMID:27441562

  1. Variant translocation of the bcl-2 gene to immunoglobulin. lambda. light chain gene in chronic lymphocytic leukemia

    SciTech Connect

    Adachi, M.; Cossman, J.; Longo, D.; Croce, C.M.; Tsujimoto, Y. )

    1989-04-01

    The bcl-2 gene has been identified as a gene directly involved in the consistent chromosome translocation t(14;18), which is found in {approx} 90% of human follicular lymphoma cases, and is a prime candidate for the oncogene playing a crucial role in follicular lymphomagenesis. In this paper, the authors describe a case of chronic lymphocytic leukemia showing the juxtaposition of the bcl-2 gene on chromosome 18 to immunoglobulin {lambda} light chain (Ig{lambda}) gene on chromosome 22 in a head-to-head configuration. Sequencing analysis of the joining site of the bcl-2 gene and Ig{lambda} gene has shown that the breakpoint is within the 5{prime} flanking region of the bcl-2 gene and about 2.2 kilobases 5{prime} to the joining segment of Ig{lambda} locus in a germ-line configuration. The extranucleotide, commonly appearing at the joining site of the t(14;18) translocation involving the IgH locus, is absent from the joining site of bcl-2 and Ig{lambda}. The lack of extranucleotide suggests that the juxtaposition of the bcl-2 and Ig{lambda} genes occurred during physiological rearrangement of the Ig{lambda} gene since it has been shown that the rearrangement of the Ig{lambda} locus is not accompanied by extranucleotides.

  2. The human myosin light chain kinase (MLCK) from hippocampus: Cloning, sequencing, expression, and localization to 3qcen-q21

    SciTech Connect

    Potier, M.C.; Rossier, J.; Turnell, W.G.; Pekarsky, Y.; Gardiner, K.

    1995-10-10

    Myosin light chain kinase (MLCK), a key enzyme in muscle contraction, has been shown by immunohistology to be present in neurons and glia. We describe here the cloning of the cDNA for human MLCK from hippocampus, encoding a protein sequence 95% similar to smooth muscle MLCKs but less than 60% similar to skeletal muscle MLCKs. The cDNA clone detected two RNA transcripts in human frontal and entorhinal cortex, in hippocampus, and in jejunum, one corresponding to MLCK and the other probably to telokin, the carboxy-terminal 154 codons of MLCK expressed as an independent protein in smooth muscle. Levels of expression were lower in brain compared to smooth muscle. We show that within the protein sequence, a motif of 28 or 24 residues is repeated five times, the second repeat ending with the putative methionine start codon. These repeats overlap with a second previously reported module of 12 residues repeated five times in the human sequence. In addition, the acidic C-terminus of all MLCKs from both brain and smooth muscle resembles the C-terminus of tubulins. The chromosomal localization of the gene for human MLCK is shown to be at 3qcen-q21, as determined by PCR and Southern blotting using two somatic cell hybrid panels. 33 refs., 8 figs.

  3. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

    PubMed Central

    Bhargava, Amol; Cotton, James A.; Dixon, Brent R.; Gedamu, Lashitew; Yates, Robin M.; Buret, Andre G.

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections. PMID:26334299

  4. Gene expression patterns in transgenic mouse models of hypertrophic cardiomyopathy caused by mutations in myosin regulatory light chain.

    PubMed

    Huang, Wenrui; Kazmierczak, Katarzyna; Zhou, Zhiqun; Aguiar-Pulido, Vanessa; Narasimhan, Giri; Szczesna-Cordary, Danuta

    2016-07-01

    Using microarray and bioinformatics, we examined the gene expression profiles in transgenic mouse hearts expressing mutations in the myosin regulatory light chain shown to cause hypertrophic cardiomyopathy (HCM). We focused on two malignant RLC-mutations, Arginine 58→Glutamine (R58Q) and Aspartic Acid 166 → Valine (D166V), and one benign, Lysine 104 → Glutamic Acid (K104E)-mutation. Datasets of differentially expressed genes for each of three mutants were compared to those observed in wild-type (WT) hearts. The changes in the mutant vs. WT samples were shown as fold-change (FC), with stringency FC ≥ 2. Based on the gene profiles, we have identified the major signaling pathways that underlie the R58Q-, D166V- and K104E-HCM phenotypes. The correlations between different genotypes were also studied using network-based algorithms. Genes with strong correlations were clustered into one group and the central gene networks were identified for each HCM mutant. The overall gene expression patterns in all mutants were distinct from the WT profiles. Both malignant mutations shared certain classes of genes that were up or downregulated, but most similarities were noted between D166V and K104E mice, with R58Q hearts showing a distinct gene expression pattern. Our data suggest that all three HCM mice lead to cardiomyopathy in a mutation-specific manner and thus develop HCM through diverse mechanisms. PMID:26906074

  5. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    PubMed

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  6. Immunoglobulin-free light chain monomer-dimer patterns help to distinguish malignant from premalignant monoclonal gammopathies: a pilot study.

    PubMed

    Kaplan, Batia; Golderman, Sizilia; Aizenbud, Boris; Esev, Konstantin; Kukuy, Olga; Leiba, Merav; Livneh, Avi; Ben-Zvi, Ilan

    2014-09-01

    Multiple myeloma (MM) and AL amyloidosis (AL) are two malignant forms of monoclonal gammopathies. For the purposes of prognosis and treatment, it is important to distinguish these diseases from the premalignant forms of monoclonal gammopathies, such as monoclonal gammopathy of unknown significance (MGUS) and smoldering myeloma (SMM). Routine serum/urine tests for monoclonal protein are insufficient for differential diagnosis. Thus, invasive procedures, such as tissue aspiration or biopsy, are applied. In this study, we aimed at characterization of serum-free light chain (FLC) monomer-dimer patterns to distinguish the malignant from the premalignant forms of monoclonal gammopathies. A quantitative Western blotting was applied to estimate the FLC monomer and dimer levels in AL, MM, MGUS, and SMM patients, and in control subjects (healthy individuals and patients with AA amyloidosis). AL and MM patients displayed an abnormally increased dimerization of monoclonal FLC, accompanied by higher clonality values of FLC dimers, as compared to that of monomers. These abnormalities of FLC patterns were not observed in patients with MGUS, SMM, AA amyloidosis, and healthy individuals. Analysis of FLC patterns helped to differentiate AL and MM from MGUS and SMM, a goal difficult to achieve using routine serum tests. Also, our technique might serve as a complimentary diagnostic tool in the cases with suspected AL amyloidosis, where the diagnosis of MM is excluded, while the results of amyloid typing by routine immunohistochemical techniques are inconclusive. PMID:24866208

  7. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation

    PubMed Central

    Orr, Nathan; Arnaout, Rima; Gula, Lorne J.; Spears, Danna A.; Leong-Sit, Peter; Li, Qiuju; Tarhuni, Wadea; Reischauer, Sven; Chauhan, Vijay S.; Borkovich, Matthew; Uppal, Shaheen; Adler, Arnon; Coughlin, Shaun R.; Stainier, Didier Y. R.; Gollob, Michael H.

    2016-01-01

    Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect. PMID:27066836

  8. Evaluation of Newcastle disease virus immunoassays for waterfowl using a monoclonal antibody specific for the duck immunoglobulin light chain.

    PubMed

    Kothlow, Sonja; Haüslaigner, Rafaela; Kaspers, Bernd; Grund, Christian

    2008-06-01

    In the present study a monoclonal antibody (mAb 14A3) was tested for its reactivity against serum immunoglobulin Y (IgY) of several waterfowl species, and subsequently for its applicability as anti-species antibody in common immunoassays. Western blot analyses demonstrated its broad cross-reactivity with the serum IgY light chain of different duck species: Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), white-winged wood duck (Asarcornis scutulatus), common pintail (Dafila acuta). Reactivity was also evident with IgY of two swan species--mute swan (Cygnus olor) and black-necked swan (Sthenelides melanocoryphus)--and two goose species--domestic goose (Anser anser var. domestica) and red-breasted goose (Rufibrenta ruficollis). Applying the mAb for Newcastle disease virus (avian paramyxovirus serotype 1 [APMV-1]) test systems, its functionality within indirect immunoassays was evaluated. Using APMV-1-positive sera of domestic geese and Muscovy ducks, mAb 14A3 facilitated specific staining of APMV-1-infected cells in an immunofluorescence test. In addition, it proved to be functional in an indirect enzyme-linked immunosorbent assay (ELISA) and a western blot assay. Thus, the analysed mAb represents an attractive and versatile reagent that offers the opportunity to develop serological tests for waterfowl, allowing a high sample throughput using the ELISA technique or the fine analysis of humoral immune responses using the western blot. PMID:18568660

  9. The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

    PubMed Central

    Kazmierczak, Katarzyna; Xu, Yuanyuan; Jones, Michelle; Guzman, Georgianna; Hernandez, Olga M.; Kerrick, W. Glenn L.; Szczesna-Cordary, Danuta

    2011-01-01

    Summary To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43 amino acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of non-pathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin. PMID:19361417

  10. Ferritin binds to light chain of human H-kininogen and inhibits kallikrein-mediated bradykinin release.

    PubMed Central

    Parthasarathy, Narayanan; Torti, Suzy V; Torti, Frank M

    2002-01-01

    Ferritin is an iron-storage protein that exists in both intracellular and extracellular compartments. We have previously identified H-kininogen (high-molecular-weight kininogen) as a ferritin-binding protein [Torti and Torti (1998) J. Biol. Chem. 273, 13630-13635]. H-Kininogen is a precursor of the potent pro-inflammatory peptide bradykinin, which is released from H-kininogen following cleavage of H-kininogen by the serine protease kallikrein. In this report, we demonstrate that binding of ferritin to H-kininogen occurs via the modified light chain of H-kininogen, and that ferritin binds preferentially to activated H-kininogen. We further demonstrate that binding of ferritin to H-kininogen retards the proteolytic cleavage of H-kininogen by kallikrein and its subsequent release of bradykinin from H-kininogen. Ferritin does not interfere with the ability of kallikrein to digest a synthetic substrate, suggesting that ferritin specifically impedes the ability of kallikrein to digest H-kininogen, perhaps by steric hindrance. Based on these results, we propose a model of sequential H-kininogen cleavage and ferritin binding. These results are consistent with the hypothesis that the binding of ferritin to H-kininogen may serve to modulate bradykinin release. PMID:12071855

  11. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    SciTech Connect

    Silvaggi,N.; Boldt, G.; Hixon, M.; Kennedy, J.; Tzipori, S.; Janda, K.; Allen, K.

    2007-01-01

    The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

  12. Chlamydia trachomatis inclusion membrane protein CT850 interacts with the dynein light chain DYNLT1 (Tctex1).

    PubMed

    Mital, Jeffrey; Lutter, Erika I; Barger, Alexandra C; Dooley, Cheryl A; Hackstadt, Ted

    2015-06-26

    Chlamydia trachomatis actively subverts the minus-end directed microtubule motor, dynein, to traffic along microtubule tracks to the Microtubule Organizing Center (MTOC) where it remains within a membrane bound replicative vacuole for the duration of its intracellular development. Unlike most substrates of the dynein motor, disruption of the dynactin cargo-linking complex by over-expression of the p50 dynamitin subunit does not inhibit C. trachomatis transport. A requirement for chlamydial protein synthesis to initiate this process suggests that a chlamydial product supersedes a requirement for p50 dynamitin. A yeast 2-hybrid system was used to screen the chlamydia inclusion membrane protein CT850 against a HeLa cell cDNA library and identified an interaction with the dynein light chain DYNLT1 (Tctex1). This interaction was at least partially dependent upon an (R/K-R/K-X-X-R/K) motif that is characteristic of DYNLT1 binding domains. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 disrupts the characteristic association of the inclusion membrane with centrosomes. Collectively, the results suggest that CT850 interacts with DYNLT1 to promote appropriate positioning of the inclusion at the MTOC. PMID:25944661

  13. 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

  14. Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    PubMed Central

    Yun, Woo Jin; Kim, Eun-Young; Park, Ji-Eun; Jo, Soo Youn; Bang, Seung Hyun; Chang, Eun-Ju; Chang, Sung Eun

    2016-01-01

    Although autophagy plays a role in melanogenesis by regulating melanosome degradation and biogenesis in melanocytes, a detailed understanding of the regulatory functions of autophagy factors is lacking. Here, we report a mechanistic link between microtubule-associated protein light chain 3 (LC3) activation and melanogenesis. We observed high expression of LC3 in melanosome-associated pigment-rich melanocytic nevi of sun-exposed skin, as indicated by patterns of melanosomal protein MART1 expression. Rapamycin-induced autophagy significantly increased the melanin index, tyrosinase activity and expression of several proteins linked to melanosome biogenesis, including microphthalmia transcription factor (MITF), pre-melanosome protein and tyrosinase, in Melan-a melanocytes. siRNA-mediated knockdown of LC3, but not beclin-1 or ATG5, decreased melanin content and tyrosinase activity. LC3 knockdown also markedly inhibited MITF expression and subsequent rapamycin-induced melanosome formation. More importantly, LC3 knockdown suppressed α-MSH-mediated melanogenesis by attenuating cAMP response element-binding protein (CREB) phosphorylation and MITF expression in Melan-a cells via decreased extracellular signal-regulated kinase (ERK) activity. Overexpression of constitutively active ERK reversed the effect of LC3 knockdown on CREB phosphorylation and MITF expression. These findings demonstrate that LC3 contributes to melanogenesis by increasing ERK-dependent MITF expression, thereby providing a mechanistic insight into the signaling network that links autophagy to melanogenesis. PMID:26814135

  15. Compensatory Aspects of Allele Diversity at Immunoglobulin Loci: Gene Correlations in Rabbit Populations Devoid of Light Chain Diversity (Oryctolagus Cuniculus L.; Kerguelen Islands)

    PubMed Central

    van-der-Loo, W.; Bousses, P.; Arthur, C. P.; Chapuis, J. L.

    1996-01-01

    Is there a selective advantage of increased diversity at one immunoglobulin locus when diversity at another locus is low? A previous paper demonstrated excess heterozygosity at the rabbit light chain b locus when heterozygosity was low at the heavy chain constant region e locus. Here we consider the reverse situation by analyzing allele distributions at heavy chain loci in populations fixed for the light chain b locus. We analyzed the a locus that encodes the predominantly expressed heavy chain variable region, and the d and e loci that control different parts of the Ig gamma class constant region. While there was excess heterozygosity, genetic differentiation between localities was extensive and was most pronounced for females. This was in marked contrast with observations in areas where b-locus diversity was important and confirms a negative correlation between e- and b-locus heterozygosity. Trigenic disequilibria corresponded to a significant negative correlation between e- and a-locus heterozygosity due mainly to strong variation among localities within the context of pronounced (digenic) linkage disequilibria. Although substantial, the average increase in a/e-locus single heterozygosity implemented by higher order disequilibria within localities was not significant. PMID:8913759

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

    SciTech Connect

    Wall, J.S.; Gupta, V.; Wilkerson, M.; Schell, M.; Loris, R.; Adams, P.; Solomon, A.; Stevens, F.; Dealwis, C.

    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 acids 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

  17. New surface-modified zinc oxide nanoparticles with aminotriethylene oxide chains linked by 1,2,3-triazole ring: Preparation, and visible light-emitting and noncytotoxic properties

    NASA Astrophysics Data System (ADS)

    Sato, Moriyuki; Shimatani, Kanako; Iwasaki, Yuko; Morito, Shigekazu; Tanaka, Hidekazu; Fujita, Yasuhisa; Nakamura, Morihiko

    2011-11-01

    Novel surface-modified, visible light-emitting and noncytotoxic ZnO nanoparticles (NPs) (ZPAZ) having aminotriethylene oxide chains linked by 1,4- and/or 1,5-disubstituted 1,2,3-triazole rings were prepared from ZnO NPs (ZPA) with ethynyl groups on the surfaces and an azide derivative of triethylene oxide chain linking terminal amino group (ATA) via 1,3-dipolar azide/alkyne click reaction by heating without Cu(I) catalyst. FTIR spectroscopy, elemental analysis, XRD analysis and TEM observation suggested that the resulting ZPA and ZPAZ NPs have the particle sizes below 10 nm in diameters, triethylene oxide chains linking the terminal amino groups and wurtzite crystal structure. UV-vis absorption spectrum of the ZPAZ NPs in methanol showed maximum absorption band at 346.5 nm, supporting the TEM observation. PL spectra depicted that the ZPA and ZPAZ NPs display broad light green and lightly greenish yellow visible light emitting bands in methanol. Zeta potentials measured in distilled water suggested that the ZPAZ NPs have a low tendency to aggregate and possess better stability than the ZPA NPs. Cytotoxicity assay revealed that the ZPAZ NPs, having water-dispersion properties, are noncytotoxic at low concentrations and almost all RAW264.7 cells are alive after 24 h of treatment.

  18. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

    PubMed Central

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-01-01

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease. PMID:27162358

  19. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    PubMed

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-05-24

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease. PMID:27162358

  20. Reversible suppression of glutamatergic neurotransmission of cerebellar granule cells in vivo by genetically manipulated expression of tetanus neurotoxin light chain.

    PubMed

    Yamamoto, Mutsuya; Wada, Norio; Kitabatake, Yasuji; Watanabe, Dai; Anzai, Masayuki; Yokoyama, Minesuke; Teranishi, Yutaka; Nakanishi, Shigetada

    2003-07-30

    We developed a novel technique that allowed reversible suppression of glutamatergic neurotransmission in the cerebellar network. We generated two lines of transgenic mice termed Tet and TeNT mice and crossed the two transgenic lines to produce the Tet/TeNT double transgenic mice. In the Tet mice, the tetracycline-controlled reverse activator (rtTA) was expressed selectively in cerebellar granule cells by the promoter function of the GABA(A) receptor alpha6 subunit gene. In the TeNT mice, the fusion gene of tetanus neurotoxin light chain (TeNT) and enhanced green fluorescent protein (EGFP) was designed to be induced by the interaction of doxycycline (DOX)-activated rtTA with the tetracycline-responsive promoter. The Tet/TeNT mice grew normally even after DOX treatment and exhibited a restricted DOX-dependent expression of TeNT in cerebellar granule cells. Along with this expression, TeNT proteolytically cleaved the synaptic vesicle protein VAMP2 (also termed synaptobrevin2) and reduced glutamate release from granule cells. Both cleavage of VAMP2/synaptobrevin2 and reduction of glutamate release were reversed by removal of DOX. Among the four genotypes generated by heterozygous crossing of Tet and TeNT mice, only Tet/TeNT mice showed DOX-dependent reversible motor impairments as analyzed with fixed bar and rota-rod tests. Reversible suppression of glutamatergic neurotransmission thus can be manipulated with spatiotemporal accuracy by DOX treatment and removal. These transgenic mice will serve as an animal model to study the cerebellar function in motor coordination and learning. PMID:12890769

  1. Immunoglobulin Free Light Chains and GAGs Mediate Multiple Myeloma Extracellular Vesicles Uptake and Secondary NfκB Nuclear Translocation

    PubMed Central

    Di Noto, Giuseppe; Chiarini, Marco; Paolini, Lucia; Mazzoldi, Elena Laura; Giustini, Viviana; Radeghieri, Annalisa; Caimi, Luigi; Ricotta, Doris

    2014-01-01

    Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. Monoclonal plasma cells often secrete high amounts of immunoglobulin free light chains (FLCs) that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles (EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS) serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs with anti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished after anti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure. The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular re-distribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches. PMID:25386176

  2. Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

    PubMed Central

    Patel, J R; Diffee, G M; Moss, R L

    1996-01-01

    To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers. Images FIGURE 1 FIGURE 5 PMID:9172757

  3. Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

    PubMed Central

    Diffee, G M; Patel, J R; Reinach, F C; Greaser, M L; Moss, R L

    1996-01-01

    We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment. Images FIGURE 2 PMID:8804617

  4. Light chain of botulinum A neurotoxin expressed as an inclusion body from a synthetic gene is catalytically and functionally active.

    PubMed

    Ahmed, S A; Smith, L A

    2000-08-01

    Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. To facilitate the structural and functional characterization of these unique endopeptidases, we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A), overexpressed it in Escherichia coli, and purified the gene product from inclusion bodies. Our procedure can provide 1.1 g of the LC from 1 L of culture. The LC product was stable in solution at 4 degrees C for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency kcat/Km was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn2+ to a Zn2+-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing. For the first time, results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form. PMID:11195972

  5. On the quantum phase fluctuations of coherent light in a chain of two anharmonic oscillators coupled through a linear one

    NASA Astrophysics Data System (ADS)

    Alam, Nasir; Mandal, Swapan

    2016-05-01

    We investigate the quantum phase fluctuations of input coherent light involving two quartic anharmonic oscillators coupled through a linear one. The analytical expressions for various phase fluctuation parameters due to Carruthers and Nieto are expressed as functions of coupling constant, anharmonic constant, initial excitation numbers, and the initial phase of the input coherent field. By using some numerical estimates of the analytical expressions, the effects of anharmonic and coupling constants are clearly indicated. In one of the two anharmonic modes (say mode a1), it is found that the presence of coupling causes the reduction of phase fluctuation parameters U1, and S1, compared to their counterparts at t=0. In sharp contrast to these results, the increase and the decrease (at least in the axis range of kt) of the phase fluctuation parameters U1, S1 and Q1 compared to their initial value counterpart are attributed by the strong field and hence the nonlinearity. We establish that the signature of anharmonicity (β ≠ 0) is realized only for intense field situations. It corroborates the fact that the nonlinearity of the medium is invoked only if the field strength is quite strong. Interestingly, for significantly strong field situation, the reduction of the phase fluctuation parameters compared to their initial values is exhibited for the harmonic mode. These reductions are attributed partly by the strong field and partly by the coupling between the oscillators. In spite of the modes corresponding to two anharmonic oscillators which are in vacuum, we report the generation of excitation for nonzero coupling constant with β=0. It may be attributed by the quantum state transfer through the chain of harmonic oscillators.

  6. A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

    PubMed

    Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Chomwong, Sudarat; Senapin, Saengchan; Tassanakajon, Anchalee; Amparyup, Piti

    2016-01-01

    Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp. PMID:26271600

  7. 18F-Florbetapir Binds Specifically to Myocardial Light Chain and Transthyretin Amyloid Deposits: An Autoradiography Study

    PubMed Central

    Park, Mi-Ae; Padera, Robert F.; Belanger, Anthony; Dubey, Shipra; Hwang, David H.; Veeranna, Vikas; Falk, Rodney H.; Di Carli, Marcelo F.; Dorbala, Sharmila

    2015-01-01

    Background 18F-florbetapir is a promising imaging biomarker for light chain (AL) and transthyretin (ATTR) cardiac amyloidosis. Our aim, using human autopsy myocardial specimens, was to test the hypothesis that 18F-florbetapir binds specifically to myocardial AL and ATTR amyloid deposits. Methods and Results We studied myocardial sections from 30 subjects with autopsy documented AL (N = 10), ATTR (N = 10) and non-amyloid controls (N = 10), using 18F-florbetapir and cold florbetapir compound and digital autoradiography. Total and non-specific binding of 18F-florbetapir was determined using the maximum signal intensity values. Specific binding of 18F-florbetapir was calculated by subtracting non-specific from total binding measurements (in decays per minute/mm2, DPM mm2), and was compared to cardiac structure and function on echocardiography and the histological extent of amyloid deposits. Diffuse or focally increased 18F-florbetapir uptake was noted in all AL and ATTR samples and in none of the control samples. Compared to control samples, mean 18F-florbetapir specific uptake was significantly higher in the amyloid samples (0.94 ± 0.43 vs. 2.00 ± 0.58 DPM/mm2, p < 0.001), and in the AL compared to the ATTR samples (2.48 ± 0.40 vs. 1.52 ± 0.22 DPM/mm2, p < 0.001). The samples from subjects with atypical echocardiographic features of amyloidosis showed quantitatively more intense 18F-florbetapir specific uptake compared to control samples (1.50 ± 0.17 vs. 0.94 ± 0.43 DPM/mm2, p = 0.004), despite smaller amyloid extent than in subjects with typical echocardiograms. Conclusions 18F-florbetapir specifically binds to myocardial AL and ATTR deposits in humans and offers the potential to screen for the two most common types of myocardial amyloid. PMID:26259579

  8. [Estimation of destruction of necrotic myocardium with serial PYP SPECT images and serum myosin light chain I level].

    PubMed

    Tanaka, T; Aizawa, T; Kato, K; Hosoi, H

    1992-02-01

    PYP SPECT images were underwent in 15 patients with acute myocardial infarction 2-5 times in three weeks. PYP SPECT images were reconstructed as to include both vertebral images and myocardial images. Quantitative estimation of PYP images was performed by the ratio of maximal PYP myocardial uptake to maximal PYP vertebral uptake in the central sagittal images (%PYP). Disappearance of PYP images was defined as the day, when %PYP reached 50%. Normalization of serum myosin light chain I (LCI) level was defined as the day, when LCI level reached 2.5 ng/ml. %PYP decreased continuously and maximal PYP point remained at the same area. Shape of PYP images varied and diminished. In case of anterior wall infarction apical PYP uptake persisted longer than basal uptake. In case of inferior wall infarction basal PYP uptake persisted longer than apical uptake. The mean period from onset to the disappearance of PYP images was 9 +/- 3 days. Pattern of serial serum MB level was simple, however corresponding pattern of serial serum LCI level showed various types. The mean period from onset to the peak level was 4.1 +/- 1 day. Normalization of LCI level was 9.3 +/- 2.9 days. It showed that process of destruction of necrotic myocardium vary in each case. Weak relation was noted between disappearance of PYP images (DAY-PYP) and normalization of LCI level (DAY-LCI). DAY-PYP = 4.4 +/- 0.46 DAY-LCI (n = 13, r = 0.4). Quantitative PYP images were useful for detecting ongoing necrotic myocardium and serum LCI level was useful for estimating destruction of necrotic myocardium.2+ level were useful to study the process of destruction of necrotic myocardium. PMID:1532996

  9. Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice.

    PubMed

    Huang, Wenrui; Liang, Jingsheng; Kazmierczak, Katarzyna; Muthu, Priya; Duggal, Divya; Farman, Gerrie P; Sorensen, Lars; Pozios, Iraklis; Abraham, Theodore P; Moore, Jeffrey R; Borejdo, Julian; Szczesna-Cordary, Danuta

    2014-09-01

    We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)-associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results were compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5 to 6month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype. PMID:24992035

  10. Discrete effects of A57G-myosin essential light chain mutation associated with familial hypertrophic cardiomyopathy

    PubMed Central

    Kazmierczak, Katarzyna; Paulino, Ellena C.; Huang, Wenrui; Muthu, Priya; Liang, Jingsheng; Yuan, Chen-Ching; Rojas, Ana I.; Hare, Joshua M.

    2013-01-01

    The functional consequences of the familial hypertrophic cardiomyopathy A57G (alanine-to-glycine) mutation in the myosin ventricular essential light chain (ELC) were assessed in vitro and in vivo using previously generated transgenic (Tg) mice expressing A57G-ELC mutant vs. wild-type (WT) of human cardiac ELC and in recombinant A57G- or WT-protein-exchanged porcine cardiac muscle strips. Compared with the Tg-WT, there was a significant increase in the Ca2+ sensitivity of force (ΔpCa50 ≅ 0.1) and an ∼1.3-fold decrease in maximal force per cross section of muscle observed in the mutant preparations. In addition, a significant increase in passive tension in response to stretch was monitored in Tg-A57G vs. Tg-WT strips indicating a mutation-induced myocardial stiffness. Consistently, the hearts of Tg-A57G mice demonstrated a high level of fibrosis and hypertrophy manifested by increased heart weight-to-body weight ratios and a decreased number of nuclei indicating an increase in the two-dimensional size of Tg-A57G vs. Tg-WT myocytes. Echocardiography examination showed a phenotype of eccentric hypertrophy in Tg-A57G mice, enhanced left ventricular (LV) cavity dimension without changes in LV posterior/anterior wall thickness. Invasive hemodynamics data revealed significantly increased end-systolic elastance, defined by the slope of the pressure-volume relationship, indicating a mutation-induced increase in cardiac contractility. Our results suggest that the A57G allele causes disease by means of a discrete modulation of myofilament function, increased Ca2+ sensitivity, and decreased maximal tension followed by compensatory hypertrophy and enhanced contractility. These and other contributing factors such as increased myocardial stiffness and fibrosis most likely activate cardiomyopathic signaling pathways leading to pathologic cardiac remodeling. PMID:23748425

  11. ALBUMIN CAUSES INCREASED MYOSIN LIGHT CHAIN KINASE EXPRESSION IN ASTROCYTES VIA P38 MITOGEN ACTIVATED PROTEIN KINASE

    PubMed Central

    Rossi, Janet L.; Ralay Ranaivo, Hantamala; Patel, Fatima; Chrzaszcz, MaryAnn; Venkatesan, Charu; Wainwright, Mark S.

    2011-01-01

    Myosin light chain kinase (MLCK) plays an important role in the reorganization of the cytoskeleton leading to disruption of vascular barrier integrity in multiple organs including the blood brain barrier (BBB) after traumatic brain injury (TBI). MLCK has been linked to transforming growth factor (TGF) and rho kinase signaling pathways, but the mechanisms regulating MLCK expression following TBI are not well understood. Albumin leaks into the brain parenchyma following TBI, activates glia and has been linked to TGF-β receptor signaling. We investigated the role of albumin in the increase in MLCK in astrocytes and the signaling pathways involved in this increase. Following midline closed-skull TBI in mice, there was a significant increase in MLCK-immunoreactive (IR) cells and albumin extravasation, which was prevented by treatment with the MLCK inhibitor ML-7. Using immunohistochemical methods, we identified the MCLK-IR cells as astrocytes. In primary astrocytes, exposure to albumin increased both isoforms of MLCK, 130 and 210. Inhibition of the TGF-β receptor partially prevented the albumin-induced increase in both isoforms, which was not prevented by inhibition of smad3. Inhibition of p38 MAPK, but not ERK, JNK or rho kinase also prevented this increase. These results are further evidence of a role of MCLK in the mechanisms of BBB compromise following TBI, and identify astrocytes as a cell type, in addition to endothelium in the BBB which express MLCK. These findings implicate albumin, acting through p38 MAPK, in a novel mechanism by which activation of MLCK following TBI may lead to compromise of the BBB. PMID:21360574

  12. p62 sequestosome 1/light chain 3b complex confers cytoprotection on lung epithelial cells after hyperoxia.

    PubMed

    Liang, Xiaoliang; Wei, Shu-Quan; Lee, Seon-Jin; Fung, James K; Zhang, Meng; Tanaka, Akihiko; Choi, Augustine M K; Jin, Yang

    2013-04-01

    Lung epithelial cell death is a prominent feature of hyperoxic lung injury, and has been considered a very important underlying mechanism of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Here we report on a novel mechanism involved in epithelial cytoprotection and homeostasis after oxidative stress. p62 (sequestosome 1; SQSTM1) is a ubiquitously expressed cellular protein. It interacts with ubiquitinated proteins and autophagic marker light chain 3b (LC3b), thus mediating the degradation of selective targets. In this study, we explored the role of p62 in mitochondria-mediated cell death after hyperoxia. Lung alveolar epithelial cells demonstrate abundant p62 expression, and p62 concentrations are up-regulated by oxidative stress at both the protein and mRNA levels. The p62/LC3b complex interacts with Fas and truncated BID (tBID) physically. These interactions abruptly diminish after hyperoxia. The deletion of p62 robustly increases tBID and cleaved caspase-3, implying an antiapoptotic effect. This antiapoptotic effect of p62 is further confirmed by measuring caspase activities, cleaved poly ADP ribose polymerase, and cell viability. The deletion of the p62 PBI domain or the ubiquitin-associated domain both lead to elevated tBID, cleaved caspase-3, and significantly more cell death after hyperoxia. Moreover, p62 traffics in an opposite direction with LC3b after hyperoxia, leading to the dissociation of the p62/Cav-1/LC3b/BID complex. Subsequently, the LC3b-mediated lysosomal degradation of tBID is eliminated. Taken together, our data suggest that the p62/LC3b complex regulates lung alveolar epithelial cell homeostasis and cytoprotection after hyperoxia. PMID:23333919

  13. Kinesin Light Chain 1 Suppression Impairs Human Embryonic Stem Cell Neural Differentiation and Amyloid Precursor Protein Metabolism

    PubMed Central

    Killian, Rhiannon L.; Flippin, Jessica D.; Herrera, Cheryl M.; Almenar-Queralt, Angels; Goldstein, Lawrence S. B.

    2012-01-01

    The etiology of sporadic Alzheimer disease (AD) is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aβ) and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aβ and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC) expressing reduced levels of the kinesin light chain 1 (KLC1) Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aβ, supporting the previously established connection between KLC1, Tau and Aβ. Intriguingly, KLC1-suppressed neural precursors (NPs), isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s) of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases. PMID:22272245

  14. Plerixafor and abbreviated-course granulocyte colony-stimulating factor for mobilizing hematopoietic progenitor cells in light chain amyloidosis.

    PubMed

    Dhakal, Binod; Strouse, Christopher; D'Souza, Anita; Arce-Lara, Carlos; Esselman, Jeanie; Eastwood, Daniel; Pasquini, Marcelo; Saber, Wael; Drobyski, William; Rizzo, J Douglas; Hari, Parameswaran N; Hamadani, Mehdi

    2014-12-01

    Cytokine-based mobilization in light chain (AL) amyloidosis is frequently complicated by fluid overload, weight gain, cardiac arrhythmias, and peri-mobilization mortality. We analyzed hematopoietic progenitor cells (HPC) mobilization outcomes in 49 consecutive AL amyloidosis patients at our institution between 2004 and 2013 with granulocyte colony-stimulating factor (G) (10 μg/kg/day) (n = 25) versus an institutional protocol to limit G exposure using plerixafor (P) (.24 mg/kg s.c. starting day 3 of G 10 μg/kg) (n = 24). G+P strategy yielded higher total CD34(+) cells/kg (12.8 × 10(6) versus 6.3 × 10(6); P < .001) and CD34(+) cells/kg collected on day 1 (10.8 × 10(6) versus 4.9 × 10(6), P = .004) compared with the G cohort. More G+P patients collected ≥5 × 10(6) CD34(+) HPCs/kg (22 versus 16, P = .02) and ≥ 10 × 10(6) CD34(+) HPCs/kg (13 versus 5, P = .01). Four patients (16%) had mobilization failure with G; none with G+P. Peri-mobilization weight gain was lower with G+P strategy (median weight gain 1 versus 7 pounds, P = .009). Numbers of apheresis sessions (median, 1 versus 1, P = .52), number of hospitalization days (median, 1.1 versus 1.6, P = .52), transfusions, use of intravenous antibiotics, and cardiac arrhythmias were similar. In conclusion, our study demonstrates that upfront use of G+P as a mobilization strategy results in superior HPC collection, no mobilization failures, and less weight gain than G alone. PMID:25111581

  15. Microtubule-Associated Protein 1 Light Chain 3 Interacts with and Contributes to Growth Inhibiting Effect of PML

    PubMed Central

    Hou, Jia-Kai; Fan, Li; Xu, Yi-Wei; Liu, Man-Hua; Yan, Shu-Yang; Chen, Guo-Qiang; Huang, Ying

    2014-01-01

    Previously we reported that the expression of promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARα) fusion gene, which is caused by specific translocation (15;17) in acute promyelocytic leukemia, can enhance constitutive autophagic activity in leukemic and nonleukemic cells, and PML overexpression can sequestrate part of microtubule-associated protein light chain 3 (LC3) protein in PML nuclear bodies, suggesting that LC3 protein also distributes into nuclei although it is currently thought to function primarily in the cytoplasm, the site of autophagosomal formation. However, its potential significance of nucleoplasmic localizations remains greatly elusive. Here we demonstrate that PML interacts with LC3 in a cell type-independent manner as assessed by Co-IP assay and co-localization observation. Overexpressed PML significantly coprecipitates with endogenous and nuclear LC3 protein. Furthermore, a fraction of endogenous PML protein is found to be co-localized with LC3 protein under steady state condition, which is further enhanced by IFNα induction, indicating that PML up-regulation potentiates this interaction. Additionally, DsRed-PML associates with EGFP-LC3 during telophase and G1 phase but not in metaphase and anaphase. Two potential LC3-interacting region (LIR) motifs in PML are required for interaction of PML with LC3 while this association is independent of autophagic activity. Finally, we show that interaction between PML and LC3 contributes to cell growth inhibition function of PML. Considering that PML is an important tumor suppressor, we propose that nuclear portion of LC3 protein may associate with PML to control cell growth for prevention and inhibition of cancer occurrence and development. PMID:25419843

  16. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

    PubMed Central

    Hong, Feng; Brizendine, Richard K.; Carter, Michael S.; Alcala, Diego B.; Brown, Avery E.; Chattin, Amy M.; Haldeman, Brian D.; Walsh, Michael P.; Facemyer, Kevin C.; Baker, Josh E.

    2015-01-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot–labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto–myosin and MLCK–myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting “stuck” on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle. PMID:26415568

  17. Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library.

    PubMed

    Miethe, Sebastian; Rasetti-Escargueil, Christine; Liu, Yvonne; Chahboun, Siham; Pelat, Thibaut; Avril, Arnaud; Frenzel, André; Schirrmann, Thomas; Thullier, Philippe; Sesardic, Dorothea; Hust, Michael

    2014-01-01

    Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease. PMID:24492304

  18. Neonatal asphyxia induces the nitration of cardiac myosin light chain 2 that is associated with cardiac systolic dysfunction.

    PubMed

    Doroszko, Adrian; Polewicz, Dorota; Cadete, Virgilio J J; Sawicka, Jolanta; Jones, Michelle; Szczesna-Cordary, Danuta; Cheung, Po-Yin; Sawicki, Grzegorz

    2010-12-01

    Hypoxia followed by reoxygenation (H-R) observed during perinatal asphyxia is a serious complication with high mortality and morbidity rates that may cause adverse cardiovascular effects in neonates. Our aim was to determine if oxidative stress related to H-R induces peroxynitrite-dependent modifications of the cardiac contractile protein, myosin regulatory light chain 2 (MLC2), and whether this is associated with development of cardiac systolic dysfunction. Twelve newborn piglets were acutely instrumented for hemodynamic monitoring and randomized to a control group ventilated with only atmospheric air or to the H-R study group exposed to alveolar normocapnic hypoxia followed by reoxygenation. Afterward, animals were euthanized, and the hearts were harvested for biochemical analyses. Systolic function as well as cardiac MLC2 levels decreased in H-R animals, whereas nitrates and nitrotyrosine levels increased. Negative correlations between nitrates, nitrotyrosine, and MLC2 levels were observed. Moreover, H-R induced nitration of two tyrosine residues within the MLC2 protein. Similarly, in vitro exposure of MLC2 to peroxynitrite resulted in the nitration of tyrosine, which increased the susceptibility of MLC2 to subsequent degradation by matrix metalloproteinase 2. Substitution of this tyrosine with phenylalanine prevented the matrix metalloproteinase 2-dependent degradation of MLC2. In addition, a large decrease in MLC2 phosphorylation caused by H-R was observed. Oxidative stress related to asphyxia induces nitration of cardiac MLC2 protein and thus increases its degradation. This and a large decrease in MLC2 phosphorylation contribute to the development of systolic dysfunction. Inhibition of MLC2 nitration and/or direct inhibition of its degradation by MMP-2 could be potential therapeutic targets aiming at reduction of myocardial damage during resuscitation of asphyxiated newborns. PMID:20386496

  19. Non–Muscle Myosin Light Chain Kinase Isoform Is a Viable Molecular Target in Acute Inflammatory Lung Injury

    PubMed Central

    Mirzapoiazova, Tamara; Moitra, Jaideep; Moreno-Vinasco, Liliana; Sammani, Saad; Turner, Jerry R.; Chiang, Eddie T.; Evenoski, Carrie; Wang, Ting; Singleton, Patrick A.; Huang, Yong; Lussier, Yves A.; Watterson, D. Martin; Dudek, Steven M.; Garcia, Joe G. N.

    2011-01-01

    Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non–muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (∼50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody–conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6–signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation. PMID:20139351

  20. Lighting.

    SciTech Connect

    United States. Bonneville Power Administration.

    1992-09-01

    Since lighting accounts for about one-third of the energy used in commercial buildings, there is opportunity to conserve. There are two ways to reduce lighting energy use: modify lighting systems so that they used less electricity and/or reduce the number of hours the lights are used. This booklet presents a number of ways to do both. Topics covered include: reassessing lighting levels, reducing lighting levels, increasing bulb & fixture efficiency, using controls to regulate lighting, and taking advantage of daylight.

  1. Pair breaking by chain oxygen disorder in light-ion irradiated YBa2Cu3Ox thin films

    NASA Astrophysics Data System (ADS)

    Arias, D.; Sefrioui, Z.; Loos, G. D.; Agullo-Rueda, F.; Garcia-Barriocanal, J.; Leon, C.; Santamaria, J.

    2003-09-01

    We report on the effect of oxygen disorder in the CuO chains on the superconducting properties of oxygen depleted YBa2Cu3Ox(YBCO). While moderate disorder, induced thermally, depresses the critical temperature as a result of a reduced carrier concentration, strong oxygen disorder and chain fragmentation induced by low energy (80 keV) He+ irradiation suppresses the critical temperature beyond the values expected from the reduction of the carrier density. This provides an experimental evidence of pair breaking by chain disorder and outlines the importance of chain states in the pairing mechanism in the YBCO family.

  2. Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement.

    PubMed

    Illy, C; Thielens, N M; Arlaud, G J

    1993-12-01

    The C1 complex of human complement comprises two loosely interacting subunits, C1q and the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer. With a view to gain information on the nature of the ionic interactions involved in C1 assembly, we have studied the effects of the chemical modifications of charged residues of C1q or the tetramer on their ability to reconstitute the C1 complex. Treatment of C1q with pyridoxal-5'-phosphate, acetic anhydride, and citraconic anhydride, as well as with cyclohexanedione and diethylpyrocarbonate, inhibited its ability to associate with C1s-C1r-C1r-C1s. Treatment of the collagen-like fragments of C1q with the same reagents yielded the same effects. Treatment of C1s-C1r-C1r-C1s with 1-ethyl-3-[-3-(dimethylamino) propyl] carbodiimide also prevented C1 assembly, through modification of acidic amino acids which were shown to be located in C1r. Further studies on the location of the interaction sites within C1q, using ligand-blotting and competition experiments with synthetic peptides, were unsuccessful, suggesting that these sites are contributed to by two or three of the C1q chains. It is concluded that C1 assembly involves interactions between acidic amino acids of C1r and lysine (hydroxylysine) and arginine residues located within the collagen-like region of C1q. Sequence comparison with mannan binding protein, another collagen-like molecule which binds the C1s-C1r-C1r-C1s tetramer, suggests Arg A38, and HyL B32, B65, and C29 of C1q as possible interaction sites. PMID:8136028

  3. An efficient nitration of light alkanes and the alkyl side-chain of aromatic compounds with nitrogen dioxide and nitric acid catalyzed by N-hydroxyphthalimide.

    PubMed

    Nishiwaki, Yoshiki; Sakaguchi, Satoshi; Ishii, Yasutaka

    2002-08-01

    Nitration of light alkanes and the alkyl side-chain of aromatic compounds with NO(2) and HNO(3) was successfully achieved by the use of N-hydroxyphthalimide (NHPI) as a catalyst under relatively mild conditions. For example, the nitration of propane with NO(2) catalyzed by NHPI at 100 degrees C for 14 h gave 2-nitropropane in good yield without formation of 1-nitropropane and cleaved products such as nitroethane and nitromethane. Various aliphatic nitroalkanes, which are difficult to prepare by conventional methods, could be selectively obtained by means of the present methodology by using NHPI as the key catalyst. In addition, the side-chain nitration of alkylbenzenes such as toluene was selectively carried out to lead to alpha-nitrotoluene without the ring nitration. The present reaction provides an efficient selective method for the nitration of light alkanes and alkylbenzenes, which has been very difficult to carry out so far. PMID:12153265

  4. Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry.

    PubMed

    Schaefer, Wolfgang; Völger, Hans R; Lorenz, Stefan; Imhof-Jung, Sabine; Regula, Jörg T; Klein, Christian; Mølhøj, Michael

    2016-01-01

    The quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution. PMID:26496506

  5. Confocal Cornea Microscopy Detects Involvement of Corneal Nerve Fibers in a Patient with Light-Chain Amyloid Neuropathy Caused by Multiple Myeloma: A Case Report

    PubMed Central

    Sturm, Dietrich; Schmidt-Wilcke, Tobias; Greiner, Tineke; Maier, Christoph; Schargus, Marc; Tegenthoff, Martin; Vorgerd, Matthias

    2016-01-01

    Changes in the subbasal corneal plexus detected by confocal cornea microscopy (CCM) have been described for various types of neuropathy. An involvement of these nerves within light-chain (AL) amyloid neuropathy (a rare cause of polyneuropathy) has never been shown. Here, we report on a case of a patient suffering from neuropathy caused by AL amyloidosis and underlying multiple myeloma. Small-fiber damage was detected by CCM. PMID:27482195

  6. The Dictyostelium class I myosin, MyoD, contains a novel light chain that lacks high-affinity calcium-binding sites.

    PubMed Central

    De La Roche, Marc A; Lee, Sheu-Fen; Côté, Graham P

    2003-01-01

    Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains. PMID:12826013

  7. A Rare Case of Waldenström Macroglobulinemia/Lymphoplasmacytic Lymphoma with Light Chain Discrepancy between B Lymphocyte Population and Serum Paraprotein.

    PubMed

    Kim, Hyun Jeong; Hur, Mina; Kim, Hanah; Moon, Hee-Won; Yun, Yeo-Min; Kim, Sung Yong; Lee, Mark Hong

    2015-01-01

    Waldenström macroglobulinemia (WM) is a subset of lymphoplasmacytic lymphoma with bone marrow involvement, monotypic immunoglobulin (Ig) M and a light chain of neoplastic cells. A 68-year-old woman presented with fever, nausea, vomiting, and pancytopenia. Her serum albumin/globulin ratio was reversed, and monoclonal gammopathy of IgM, lambda type (23.20%, 1.58 g/dL) was detected. In her bone marrow, increased small lymphocytes were admixed with plasmacytoid lymphocytes and plasma cells. She was diagnosed as having lymphoplasmacytic variant of WM. Immunohistochemical stains and flow cytometic analysis revealed two distinct populations; monoclonal B cells (kappa+) and abnormal plasma cells (CD19-/CD56+/lambda+). She expired 19 days after admission due to septic shock. This is a rare case of WM exhibiting a light chain discrepancy between monoclonal B lymphocytes and paraprotein-secreting plasma cells. Light chain restriction may occur distinctly between lymphocyte and plasma cell populations in WM. PMID:26586715

  8. Identification and functional analysis of the essential and regulatory light chains of the only type II myosin Myo1p in Saccharomyces cerevisiae

    PubMed Central

    Luo, Jianying; Vallen, Elizabeth A.; Dravis, Christopher; Tcheperegine, Serguei E.; Drees, Becky; Bi, Erfei

    2004-01-01

    Cytokinesis in Saccharomyces cerevisiae involves coordination between actomyosin ring contraction and septum formation and/or targeted membrane deposition. We show that Mlc1p, a light chain for Myo2p (type V myosin) and Iqg1p (IQGAP), is the essential light chain for Myo1p, the only type II myosin in S. cerevisiae. However, disruption or reduction of Mlc1p–Myo1p interaction by deleting the Mlc1p binding site on Myo1p or by a point mutation in MLC1, mlc1-93, did not cause any obvious defect in cytokinesis. In contrast, a different point mutation, mlc1-11, displayed defects in cytokinesis and in interactions with Myo2p and Iqg1p. These data suggest that the major function of the Mlc1p–Myo1p interaction is not to regulate Myo1p activity but that Mlc1p may interact with Myo1p, Iqg1p, and Myo2p to coordinate actin ring formation and targeted membrane deposition during cytokinesis. We also identify Mlc2p as the regulatory light chain for Myo1p and demonstrate its role in Myo1p ring disassembly, a function likely conserved among eukaryotes. PMID:15210731

  9. The Association between Polyclonal Combined Serum Free Light Chain Concentration and Mortality in Individuals with Early Chronic Kidney Disease

    PubMed Central

    Assi, Lakhvir K.; McIntyre, Natasha; Fraser, Simon; Harris, Scott; Hutchison, Colin A.; McIntyre, Chris W.; Cockwell, Paul; Taal, Maarten W.

    2015-01-01

    A major component of increased mortality risk in people with chronic kidney disease (CKD) is associated with non-traditional cardiovascular risk factors including markers of inflammation. We studied whether a novel marker of systemic inflammation, elevated serum combined polyclonal immunoglobulin free light chains (cFLC), was an independent risk factor for increased all-cause mortality in people with CKD stage 3. In a prospective community based cohort study, 1695 participants with stage 3 CKD and no cases of monoclonal gammopathy had cFLC concentrations measured. cFLC levels were determined using the summation of Freelite kappa and lambda assays. All other bioclinical variables were collected at the time of sample collection. Kaplan-Meier plots and Cox proportional hazards analysis was used to assess the relationship between high cFLC levels (>43.3 mg/L) and mortality. There were 167 deaths (10%) after a median of 1375 days. cFLC levels at recruitment were higher in participants who died compared with those who were alive at the end of the study; median: 46.5 mg/L (IQR: 36.1-65.4 mg/L) and 35.4 mg/L (28.1-46.6 mg/L) respectively, P <0.001. Kaplan-Meier survival analysis demonstrated participants with cFLC >43.3 mg/L levels had an increased risk of mortality compared to people with normal cFLC levels (P <0.001). Elevated cFLC levels were independently associated with worse survival (Hazard ratio: 1.50; 95% confidence interval: 1.04-2.16; P=0.03). Other independent risk factors for worse survival were: older age, male gender, previous cardiovascular event, lower eGFR and higher high sensitivity C-reactive protein (hsCRP). To conclude, high cFLC levels predict increased mortality in people with stage 3 CKD, independent of established risk factors and other markers of inflammation. PMID:26132658

  10. Extracorporeal Membrane Oxygenation as Bridge-to-Decision in Acute Heart Failure due to Systemic Light-Chain Amyloidosis

    PubMed Central

    Silva, Jennifer Mancio; Fontes-Carvalho, Ricardo; Valente, Dília; Almeida, Cristiana; Cruz, António José; Tente, David; Coelho, Henrique; Oliveira, Marco; Albuquerque, Aníbal; Ribeiro, Vasco Gama

    2015-01-01

    Patient: Female, 58 Final Diagnosis: Acute hear failure Symptoms: Dispnoea • edema • fatigue Medication: — Clinical Procedure: Bone marrow biopsy • endomyocardial biopsy • abdominal subcutaneous fat biopsy under ECMO support Specialty: Cardiology Objective: Rare disease Background: Cardiac amyloidosis results from the amyloid deposition in heart tissue, either in the context of a systemic disease or as a localized form. Several pro-amyloid proteins can produce amyloid deposits in the heart. Each of these amyloidoses has characteristic clinical (cardiac and extracardiac) features, and a specific diagnosis and treatment. Case Report: A 58-year-old woman who presented with acute heart failure and echocardiographic findings strongly suggestive of infiltrative cardiomyopathy needed percutaneous veno-arterial extracorporeal membrane oxygenation (ECMO) as bridge-to-decision. Amyloid deposition was found on endomyocardial and bone marrow biopsies. Bone marrow plasma cell infiltrate with acute renal lesion and hypercalcemia confirmed the diagnosis of multiple myeloma-associated systemic light-chain amyloidosis (AL). Refractory shock with multi-organic failure syndrome persisted and no improvements in left ventricular function and structure were seen. After extensive discussion by a multidisciplinary team, and with the patients’ family, she was not considered eligible for high-dose chemotherapy and/or autologous stem cell transplantation, heart transplantation, or sequential heart with autologous stem cell transplantation. The patient died a few hours after ECMO withdrawal. During the 14 days of ECMO support no major bleeding or thrombotic complications occurred. Conclusions: The clinician must consider a diagnosis of cardiac amyloidosis in patients with heart failure, a restrictive type of cardiomyopathy with ventricular hypertrophy in the absence of valve abnormalities, or uncontrolled arterial hypertension. Although developments in chemotherapy have greatly

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

    DOE PAGESBeta

    Kumaran, D.; Adler, M.; Levit, M.; Krebs, M.; Sweeney, R.; Swaminathan, S.

    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 Ki 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 Zn2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter« less

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

    SciTech Connect

    Kumaran, D.; Adler, M.; Levit, M.; Krebs, M.; Sweeney, R.; Swaminathan, S.

    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; 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 Ki 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 Zn2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter

  13. 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. PMID:26522088

  14. Fluid-phase interaction of C1 inhibitor (C1 Inh) and the subcomponents C1r and C1s of the first component of complement, C1.

    PubMed

    Chesne, S; Villiers, C L; Arlaud, G J; Lacroix, M B; Colomb, M G

    1982-01-01

    Interactions between proenzymic or activated complement subcomponents of C1 and C1 Inh (C1 inhibitor) were analysed by sucrose-density-gradient ultracentrifugation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The interaction of C1 Inh with dimeric C1r in the presence of EDTA resulted into two bimolecular complexes accounting for a disruption of C1r. The interaction of C1 Inh with the Ca2+-dependent C1r2-C1s2 complex (8.8 S) led to an 8.5 S inhibited C1r-C1s-C1 Inh complex (1:1:2), indicating a disruption of C1r2 and of C1s2 on C1 Inh binding. The 8.5 S inhibited complex was stable in the presence of EDTA; it was also formed from a mixture of C1r, C1s and C1 Inh in the presence of EDTA or from bimolecular complexes of C1r-C1 Inh and C1s-C1 Inh. C1r II, a modified C1r molecule, deprived of a Ca2+-binding site after autoproteolysis, did not lead to an inhibited tetrameric complex on incubation with C1s and C1 Inh. These findings suggest that, when C1 Inh binds to C1r2-C1s2 complex, the intermonomer links inside C1r2 or C1s2 are weakened, whereas the non-covalent Ca2+-independent interaction between C1r2 and C1s2 is strengthened. The nature of the proteinase-C1 Inh link was investigated. Hydroxylamine (1M) was able to dissociate the complexes partially (pH 7.5) or totally (pH 9.0) when the incubation was performed in denaturing conditions. An ester link between a serine residue at the active site of C1r or C1s and C1 Inh is postulated. PMID:6282262

  15. Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

    PubMed Central

    Nabeshima, Y; Fujii-Kuriyama, Y; Muramatsu, M; Ogata, K

    1982-01-01

    We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA. PMID:6128725

  16. 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. PMID:27285777

  17. 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

  18. [Hereditary angioneurotic edema: a molecular disease caused by a defect in the O-glycosylation of C1 esterase inhibitor (C1-INH)].

    PubMed

    Ollier-Hartmann, M P; Strecker, G; Montreuil, J; Hartmann, L

    1984-01-01

    A quantitative and qualitative study of neutral and aminosaccharides in C 1-esterase inhibitor (C 1-INH), protein of the complement system, was performed. We observe a mixed glycosylation of the molecule with an N-glycosylated: O-glycosylated chain ratio of 1: 4. The loss of the inhibitory activity of the molecule in hereditary angioedema (O ANH) is associated with an O-glycosylation deficiency which differs according to the two molecular variants: C 1-INH (1 A) and C 1-INH (II) previously described. PMID:6440668

  19. Aggregates of Isotactic Poly(methacrylic acid) Chains in Aqueous CsCl Solutions: a Static and Dynamic Light Scattering Study.

    PubMed

    Hočevar, Katarina; Sitar, Simona; Kogej, Ksenija

    2015-01-01

    Properties of isotactic polymethacrylic acid, iPMA, chains were studied at 25°C in aqueous solutions at various CsCl concentrations, c(s) (= 0.05-0.20 M), in dependence on degree of neutralization of the polyion's carboxyl groups, α(N), using static, SLS, and dynamic light scattering, DLS, measurements. It was demonstrated that iPMA chains with α(N) somewhat above the solubility limit of iPMA in aqueous solutions (in the present case at α(N) ≈ 0.27) are strongly aggregated. The size of the aggregates increases with increasing c(s), whereas the shape parameter, ρ, is approximately constant (ρ ≈ 0.6), irrespective of c(s). The low ρ value suggests that the aggregates have characteristics of microgel particles with a dense core surrounded by a less dense corona. The diffusion of iPMA chains was investigated also at higher α(N), up to α(N) = 1. The polyion slow mode arising from electrostatic interactions between charged chains was observed for α(N) exceeding the value 0.27 even at the highest c(s) (= 0.20 M). The diffusion coefficients for the show mode were nearly independent of α(N) and cs at the studied polymer concentration. PMID:26454588

  20. A Markov Chain-based quantitative study of angular distribution of photons through turbid slabs via isotropic light scattering

    NASA Astrophysics Data System (ADS)

    Li, Xuesong; Northrop, William F.

    2016-04-01

    This paper describes a quantitative approach to approximate multiple scattering through an isotropic turbid slab based on Markov Chain theorem. There is an increasing need to utilize multiple scattering for optical diagnostic purposes; however, existing methods are either inaccurate or computationally expensive. Here, we develop a novel Markov Chain approximation approach to solve multiple scattering angular distribution (AD) that can accurately calculate AD while significantly reducing computational cost compared to Monte Carlo simulation. We expect this work to stimulate ongoing multiple scattering research and deterministic reconstruction algorithm development with AD measurements.

  1. Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle.

    PubMed

    Sutton, T A; Haeberle, J R

    1990-02-15

    In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase

  2. 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

  3. The novel zinc finger protein dASCIZ regulates mitosis in Drosophila via an essential role in dynein light-chain expression.

    PubMed

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

    2014-02-01

    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

  4. Structure of the Small Dictyostelium discoideum Myosin Light Chain MlcB Provides Insights into MyoB IQ Motif Recognition*

    PubMed Central

    Liburd, Janine; Chitayat, Seth; Crawley, Scott W.; Munro, Kim; Miller, Emily; Denis, Chris M.; Spencer, Holly L.; Côté, Graham P.; Smith, Steven P.

    2014-01-01

    Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca2+. However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca2+ binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin. PMID:24790102

  5. Autoantibodies against complement C1q specifically target C1q bound on early apoptotic cells.

    PubMed

    Bigler, Cornelia; Schaller, Monica; Perahud, Iryna; Osthoff, Michael; Trendelenburg, Marten

    2009-09-01

    Autoantibodies against complement C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE). They strongly correlate with the occurrence of severe lupus nephritis, suggesting a pathogenic role in SLE. Because anti-C1q are known to recognize a neoepitope on bound C1q, but not on fluid-phase C1q, the aim of this study was to clarify the origin of anti-C1q by determining the mechanism that renders C1q antigenic. We investigated anti-C1q from serum and purified total IgG of patients with SLE and hypocomplementemic urticarial vasculitis as well as two monoclonal human anti-C1q Fab from a SLE patient generated by phage display. Binding characteristics, such as their ability to recognize C1q bound on different classes of Igs, on immune complexes, and on cells undergoing apoptosis, were analyzed. Interestingly, anti-C1q did not bind to C1q bound on Igs or immune complexes. Neither did we observe specific binding of anti-C1q to C1q bound on late apoptotic/necrotic cells when compared with binding in the absence of C1q. However, as shown by FACS analysis and confocal microscopy, anti-C1q specifically targeted C1q bound on early apoptotic cells. Anti-C1q were found to specifically target C1q bound on cells undergoing apoptosis. Our observations suggest that early apoptotic cells are a major target of the autoimmune response in SLE and provide a direct link between human SLE, apoptosis, and C1q. PMID:19648280

  6. Microgel-like aggregates of isotactic and atactic poly(methacrylic acid) chains in aqueous alkali chloride solutions as evidenced by light scattering.

    PubMed

    Sitar, Simona; Aseyev, Vladimir; Kogej, Ksenija

    2014-10-21

    A comparative light-scattering study of isotactic and atactic poly(methacrylic acid), iPMA and aPMA, respectively, in aqueous solutions with added alkali chlorides, XCl (X = Li, Na, Cs), at 25 °C and XCl concentration of 0.1 mol L(-1), demonstrates that both PMA isomers are strongly associated at low degrees of neutralization, αN (= 0 for aPMA and 0.25 for iPMA), in the presence of all XCls. The shape parameter ρ and the scattering functions suggest that aggregates have the characteristics of microgel particles, with a dense core surrounded by a less dense shell. The extent of aggregation depends on the stereoregular structure of the polymer and on the type of the added cation. Li(+) and Na(+) ions support aggregation better than Cs(+) ions. Besides, iPMA chains are more strongly aggregated than aPMA chains and form particles with a denser core. A model of the aggregation process is suggested for iPMA. At high αN, a slow diffusive process (so-called extraordinary or anomalous mode in diffusion of polyelectrolytes), arising from electrostatic interactions between charged chains, is observed for both PMAs. Results suggest that under the same experimental conditions iPMA is effectively more charged than aPMA. The role of ions in the slow-mode phenomenon is less pronounced than in aggregation. PMID:25137480

  7. A nationwide study of acquired C1-inhibitor deficiency in France: Characteristics and treatment responses in 92 patients.

    PubMed

    Gobert, Delphine; Paule, Romain; Ponard, Denise; Levy, Pierre; Frémeaux-Bacchi, Véronique; Bouillet, Laurence; Boccon-Gibod, Isabelle; Drouet, Christian; Gayet, Stéphane; Launay, David; Martin, Ludovic; Mekinian, Arsène; Leblond, Véronique; Fain, Olivier

    2016-08-01

    Acquired angioedema (AAE) due to C1-inhibitor (C1INH) deficiency is rare. Treatment options for acute attacks are variable and used off-label. Successful treatment of the associated lymphoma with rituximab seems to prevent acute attacks in subjects with AAE. The aim of this study was to describe AAE manifestations, its associated diseases, and patients' responses to treatments in a representative cohort.A retrospective nationwide study was conducted in France. The inclusion criteria were recurrent angioedema attacks and an acquired decrease in functional C1INH <50% of the reference value.A total of 92 cases were included, with a median age at onset of 62 years. Facial edema and abdominal pain were the most frequent symptoms. Fifteen patients were hospitalized in the intensive care unit because of laryngeal edema, and 1 patient died. Anti-C1INH antibodies were present in 43 patients. The associated diseases were primarily non-Hodgkin lymphoma (n = 44, with 24 splenic marginal zone lymphomas) and monoclonal gammopathy of undetermined significance (n = 24). Three patients had myeloma, 1 had amyloid light-chain (of immunoglobulin) (AL) amyloidosis, 1 patient had a bronchial adenocarcinoma, and 19 patients had no associated disease. Icatibant relieved the symptoms in all treated patients (n = 26), and plasma-derived C1INH concentrate in 19 of 21 treated patients. Six patients experienced thromboembolic events under tranexamic acid prophylaxis. Rituximab prevented angioedema in 27 of 34 patients as a monotherapy or in association with chemotherapy. Splenectomy controlled AAE in 7 patients treated for splenic marginal zone lymphoma. After a median follow-up of 4.2 years, angioedema was on remission in 52 patients.AAE cases are primarily associated with indolent lymphoma-especially splenic marginal zone lymphoma-and monoclonal gammopathy of undetermined significance but not with autoimmune diseases or other conditions. Icatibant and plasma-derived C1INH concentrate control

  8. C1 neurons: the body's EMTs.

    PubMed

    Guyenet, Patrice G; Stornetta, Ruth L; Bochorishvili, Genrieta; Depuy, Seth D; Burke, Peter G R; Abbott, Stephen B G

    2013-08-01

    The C1 neurons reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM, IVLM). They use glutamate as a fast transmitter and synthesize catecholamines plus various neuropeptides. These neurons regulate the hypothalamic pituitary axis via direct projections to the paraventricular nucleus and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. The presympathetic C1 cells, located in the RVLM, are probably organized in a roughly viscerotopic manner and most of them regulate the circulation. C1 cells are variously activated by hypoglycemia, infection or inflammation, hypoxia, nociception, and hypotension and contribute to most glucoprivic responses. C1 cells also stimulate breathing and activate brain stem noradrenergic neurons including the locus coeruleus. Based on the various effects attributed to the C1 cells, their axonal projections and what is currently known of their synaptic inputs, subsets of C1 cells appear to be differentially recruited by pain, hypoxia, infection/inflammation, hemorrhage, and hypoglycemia to produce a repertoire of stereotyped autonomic, metabolic, and neuroendocrine responses that help the organism survive physical injury and its associated cohort of acute infection, hypoxia, hypotension, and blood loss. C1 cells may also contribute to glucose and cardiovascular homeostasis in the absence of such physical stresses, and C1 cell hyperactivity may contribute to the increase in sympathetic nerve activity associated with diseases such as hypertension. PMID:23697799

  9. 42 CFR 52c.1 - Applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Applicability. 52c.1 Section 52c.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS MINORITY BIOMEDICAL RESEARCH... Biomedical Research Support Program) awarded in accordance with section 301(a)(3) of the Public...

  10. 42 CFR 52c.1 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Applicability. 52c.1 Section 52c.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS MINORITY BIOMEDICAL RESEARCH... Biomedical Research Support Program) awarded in accordance with section 301(a)(3) of the Public...

  11. C1 neurons: the body's EMTs

    PubMed Central

    Stornetta, Ruth L.; Bochorishvili, Genrieta; DePuy, Seth D.; Burke, Peter G. R.; Abbott, Stephen B. G.

    2013-01-01

    The C1 neurons reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM, IVLM). They use glutamate as a fast transmitter and synthesize catecholamines plus various neuropeptides. These neurons regulate the hypothalamic pituitary axis via direct projections to the paraventricular nucleus and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. The presympathetic C1 cells, located in the RVLM, are probably organized in a roughly viscerotopic manner and most of them regulate the circulation. C1 cells are variously activated by hypoglycemia, infection or inflammation, hypoxia, nociception, and hypotension and contribute to most glucoprivic responses. C1 cells also stimulate breathing and activate brain stem noradrenergic neurons including the locus coeruleus. Based on the various effects attributed to the C1 cells, their axonal projections and what is currently known of their synaptic inputs, subsets of C1 cells appear to be differentially recruited by pain, hypoxia, infection/inflammation, hemorrhage, and hypoglycemia to produce a repertoire of stereotyped autonomic, metabolic, and neuroendocrine responses that help the organism survive physical injury and its associated cohort of acute infection, hypoxia, hypotension, and blood loss. C1 cells may also contribute to glucose and cardiovascular homeostasis in the absence of such physical stresses, and C1 cell hyperactivity may contribute to the increase in sympathetic nerve activity associated with diseases such as hypertension. PMID:23697799

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

    PubMed Central

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

    2013-01-01

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

  13. Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography

    PubMed Central

    Wollacott, Rachel B; Casaz, Paul L; Morin, Trevor J; Zhu, H Lily; Anderson, Roger S; Babcock, Gregory J; Que, John; Thomas Jr, William D; Ozturk, Sadettin S

    2013-01-01

    Size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mAb) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species, referred to as “shoulder,” indicated that it was a mAb containing an extra light chain and had a molecular weight of approximately 175 kDa. The extra light chain was found to be non-covalently associated with the Fab portion of the protein. The relative amount of shoulder (typically 1−3% of the total mAb present) varied with the Chinese hamster ovary cell line producing the mAb and was not influenced by the growth conditions. Our three-step mAb purification platform using protein A, anion exchange, and cation exchange process steps was successful at removing dimer and higher and lower molecular weight species, but not the shoulder impurity. It was found that hydrophobic interaction chromatography could be used in place of cation exchange to exploit the subtle differences in hydrophobicity between monomer and shoulder. We developed an antibody polishing process using Butyl Sepharose HP resin that is capable of removing the majority of high and low molecular weight impurities yielding 99% pure mAb monomer, virtually devoid of the shoulder species, with a step recovery of about 80%. PMID:23995619

  14. Antibody light chain-restricted recognition of the site of immune pressure in the RV144 HIV-1 vaccine trial is phylogenetically conserved

    PubMed Central

    Wiehe, Kevin; Easterhoff, David; Luo, Kan; Nicely, Nathan I.; Bradley, Todd; Jaeger, Frederick H.; Dennison, S. Moses; Zhang, Ruijun; Lloyd, Krissey E.; Stolarchuk, Christina; Parks, Robert; Sutherland, Laura L.; Scearce, Richard M.; Morris, Lynn; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Sinangil, Faruk; Phogat, Sanjay; Michael, Nelson L.; Kim, Jerome H.; Kelsoe, Garnett; Montefiori, David C.; Tomaras, Georgia D.; Bonsignori, Mattia; Santra, Sampa; Kepler, Thomas B.; Alam, S. Munir; Moody, M. Anthony; Liao, Hua-Xin; Haynes, Barton F.

    2015-01-01

    SUMMARY 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 pre-configured for ED motif-mediated recognition prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. These data indicate 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. PMID:25526306

  15. Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-{kappa}{beta} and myosin light-chain kinase pathways

    SciTech Connect

    Zhang, Ying; Li, Jianguo

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Carbachol reduced the lipopolysaccharide-induced intestinal barrier breakdown. Black-Right-Pointing-Pointer Carbachol ameliorated the lipopolysaccharide-induced ileal tight junction damage. Black-Right-Pointing-Pointer Carbachol prevented the LPS-induced NF-{kappa}{beta} and myosin light-chain kinase activation. Black-Right-Pointing-Pointer Carbachol exerted its beneficial effects in an {alpha}7 nicotinic receptor-dependent manner. -- Abstract: Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10 mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-{kappa}{beta}) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the {alpha}7 nicotinic acetylcholine receptor ({alpha}7nAchR) antagonist {alpha}-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-{kappa}{beta} and MLCK pathways in an {alpha}7nAchR-dependent manner.

  16. Protein kinase C enhances myosin light-chain kinase effects on force development and ATPase activity in rat single skinned cardiac cells.

    PubMed Central

    Clement, O; Puceat, M; Walsh, M P; Vassort, G

    1992-01-01

    Many neurohormones alter the force of cardiac contraction by variations in the intracellular Ca2+ concentration. alpha 1-Adrenergic and muscarinic stimulations, rather, modify the sensitivity of contractile proteins to Ca(2+)-calmodulin-myosin light-chain kinase (MLCK) complex induces a large increase in Ca2+ sensitivity (0.14 pCa unit) of these easily accessible myofilaments. This increase is further enhanced by up to 0.19 pCa unit when protein kinase C (PKC) is added together with MLCK. Similarly, the Ca2+ ATPase activity of skinned cells in suspension is increased in the presence of MLCK and further in the presence of both kinases. 32P-labelling and SDS/PAGE show that these changes are associated with light-chain 2 (LC2) phosphorylation together with phosphorylation of troponin I and troponin T when PKC is added. Although to a smaller extent than in smooth muscle, phosphorylation of cardiac myosin LC2 may be involved in the modulation of heart contractility. Images Fig. 4. Fig. 5. Fig. 6. PMID:1386218

  17. A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

    PubMed Central

    Heaslip, Aoife T.; Leung, Jacqueline M.; Carey, Kimberly L.; Catti, Federica; Warshaw, David M.; Westwood, Nicholas J.; Ballif, Bryan A.; Ward, Gary E.

    2010-01-01

    Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains. PMID:20084115

  18. Cerebral low-grade lymphoma and light chain deposition disease: exceedingly high IgG levels in the cerebrospinal fluid as a diagnostic clue.

    PubMed

    Pantazis, G; Psaras, T; Krope, K; von Coelln, R; Fend, F; Bock, T; Schittenhelm, J; Melms, A; Meyermann, R; Bornemann, A

    2010-01-01

    Herein, we report the case of a 72-year-old male with an exceedingly rare manifestation of a low-grade lymphoma in the brain associated with light chain deposition disease (LCDD). The patient presented with epileptic seizures. Magnetic resonance imaging (MRI) of the brain revealed multiple hyperintense lesions in the right parietal lobe that were suspicious of vasculitis, low-grade glioma, or neurosarcoidosis. In the cerebrospinal fluid (CSF), but not in the serum, highly elevated IgG was found. A stereotactic biopsy of one cerebral lesion was performed. Histopathology revealed a low grade lymphoplasmacytic B-cell lymphoma with light chain deposition disease (LCDD). Bone marrow biopsy and laboratory workup did not show any systemic involvement. LCDD exclusively affecting the brain is an exceedingly rare finding. It can be associated with low-grade B-cell lymphoma. This is the first report of LCDD exclusively affecting the brain in an elderly patient. Compared with the two younger patients previously reported, the course of the disease was of a slow-evolving nature. In constellations of highly elevated IgG in CSF and multiple white matter lesions, LCDD should be considered as underlying pathology. PMID:21073842

  19. Evaluation of serum markers in the LRF CLL4 trial: β2-microglobulin but not serum free light chains, is an independent marker of overall survival.

    PubMed

    Pratt, Guy; Thomas, Peter; Marden, Nicola; Alexander, Denis; Davis, Zadie; Hussey, David; Parry, Helen; Harding, Stephen; Catovsky, Daniel; Begley, Joe; Oscier, David

    2016-10-01

    Chronic lymphocytic leukemia (CLL) is characterized by heterogeneous clinical behavior and there is a need for improved biomarkers. The current study evaluated the prognostic significance of serum free light chains (sFLC, kappa, and lambda) and other serum markers (bar, serum thymidine kinase (sTK), soluble CD23, and LDH) together with established biomarkers in 289 patients enrolled into the LRF CLL4 trial. In a multivariable analysis of serum markers alone, higher big and kappa light chains were statistically significant in predicting disease progression and higher blg, and sTK in predicting mortality. In multivariable analysis for overall survival the following were independently significant: β2M levels, immunoglobulin gene (IGHV) mutational status (>98% homology), age, 17p13 deletions (>10%), and CD38 expression. β2M is the only serum marker that retained clear independent value as a biomarker in the LRF CLL4 trial and remains powerfully prognostic requiring evaluation in any future method of risk stratifying patients. PMID:26732125

  20. Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter-alpha-trypsin inhibitor and saposin B but increased expression of an inter-alpha-trypsin inhibitor heavy chain 4 fragment.

    PubMed

    Jayapalan, Jaime J; Ng, Keng L; Shuib, Adawiyah S; Razack, Azad H A; Hashim, Onn H

    2013-06-01

    The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required. PMID:23417432

  1. Markov chain formalism for polarized light transfer in plane-parallel atmospheres, with numerical comparison to the Monte Carlo method.

    PubMed

    Xu, Feng; Davis, Anthony B; West, Robert A; Esposito, Larry W

    2011-01-17

    Building on the Markov chain formalism for scalar (intensity only) radiative transfer, this paper formulates the solution to polarized diffuse reflection from and transmission through a vertically inhomogeneous atmosphere. For verification, numerical results are compared to those obtained by the Monte Carlo method, showing deviations less than 1% when 90 streams are used to compute the radiation from two types of atmospheres, pure Rayleigh and Rayleigh plus aerosol, when they are divided into sublayers of optical thicknesses of less than 0.03. PMID:21263634

  2. C1q Nephropathy: The Unique Underrecognized Pathological Entity.

    PubMed

    Devasahayam, Joe; Erode-Singaravelu, Gowrishankar; Bhat, Zeenat; Oliver, Tony; Chandran, Arul; Zeng, Xu; Dakshinesh, Paramesh; Pillai, Unni

    2015-01-01

    C1q nephropathy is a rare glomerular disease with characteristic mesangial C1q deposition noted on immunofluorescence microscopy. It is histologically defined and poorly understood. Light microscopic features are heterogeneous and comprise minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), and proliferative glomerulonephritis. Clinical presentation is also diverse, and ranges from asymptomatic hematuria or proteinuria to frank nephritic or nephrotic syndrome in both children and adults. Hypertension and renal insufficiency at the time of diagnosis are common findings. Optimal treatment is not clear and is usually guided by the underlying light microscopic lesion. Corticosteroids are the mainstay of treatment, with immunosuppressive agents reserved for steroid resistant cases. The presence of nephrotic syndrome and FSGS appear to predict adverse outcomes as opposed to favorable outcomes in those with MCD. Further research is needed to establish C1q nephropathy as a universally recognized distinct clinical entity. In this paper, we discuss the current understanding of pathogenesis, histopathology, clinical features, therapeutic options, and outcomes of C1q nephropathy. PMID:26640759

  3. Similarities and Differences Between the Light and Heavy Chain Ig Variable Region Gene Repertoires in Chronic Lymphocytic Leukemia

    PubMed Central

    Ghiotto, Fabio; Fais, Franco; Albesiano, Emilia; Sison, Cristina; Valetto, Angelo; Gaidano, Gianluca; Reinhardt, Janine; Kolitz, Jonathan E; Rai, Kanti; Allen, Steven L; Ferrarini, Manlio; Chiorazzi, Nicholas

    2006-01-01

    Analyses of Ig VHDJH rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the VL and JL gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express VL genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific VL and JL segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the VH repertoire, VL gene use was not significantly different than that of normal B-lymphocytes. In addition, Vκ genes that lie more upstream on the germline locus were less frequently mutated than those at the 3′ end of the locus; this was not the case for Vλ genes and is not for VH genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire. PMID:17380195

  4. Aeolus: A Markov Chain Monte Carlo Code for Mapping Ultracool Atmospheres. An Application on Jupiter and Brown Dwarf HST Light Curves

    NASA Astrophysics Data System (ADS)

    Karalidi, Theodora; Apai, Dániel; Schneider, Glenn; Hanson, Jake R.; Pasachoff, Jay M.

    2015-11-01

    Deducing the cloud cover and its temporal evolution from the observed planetary spectra and phase curves can give us major insight into the atmospheric dynamics. In this paper, we present Aeolus, a Markov chain Monte Carlo code that maps the structure of brown dwarf and other ultracool atmospheres. We validated Aeolus on a set of unique Jupiter Hubble Space Telescope (HST) light curves. Aeolus accurately retrieves the properties of the major features of the Jovian atmosphere, such as the Great Red Spot and a major 5 μm hot spot. Aeolus is the first mapping code validated on actual observations of a giant planet over a full rotational period. For this study, we applied Aeolus to J- and H-band HST light curves of 2MASS J21392676+0220226 and 2MASS J0136565+093347. Aeolus retrieves three spots at the top of the atmosphere (per observational wavelength) of these two brown dwarfs, with a surface coverage of 21% ± 3% and 20.3% ± 1.5%, respectively. The Jupiter HST light curves will be publicly available via ADS/VIZIR.

  5. Posterior osseous bridging of C1.

    PubMed

    Simsek, Serkan; Yigitkanli, Kazim; Comert, Ayhan; Acar, Halil I; Seckin, Hakan; Er, Uygur; Belen, Deniz; Tekdemir, Ibrahim; Elhan, Alaittin

    2008-06-01

    The sulcus of the vertebral artery is located behind the lateral mass of the atlas and in some cases is converted into a foramen by anomalous ossification known as the posterior ponticulus (osseous bridge). This study involved anatomical observations of 158 isolated anatomical specimens of dry C1 vertebrae. The incidence and types of posterior osseous bridging were identified for the 158 dry samples of atlas vertebrae. In nine (5.6%) dry C1 vertebrae, partial osseous bridging was detected (bilaterally in eight vertebrae and unilaterally on the left in one). Complete osseous bridging (arcuate foramen) was observed in six (3.8%) dry C1 vertebrae (bilaterally in one vertebra, unilaterally on the left in three, and on the right in two). Awareness of the types of posterior osseous bridging of C1 in craniocervical junction surgery is essential, and may be helpful in surgical interventions in this region. PMID:18378457

  6. Anti-C1q autoantibodies.

    PubMed

    Kallenberg, Cees G M

    2008-09-01

    Autoantibodies to complement components are associated with various diseases. Anti-C1q antibodies are present in all patients with hypocomplementemic urticarial vasculitis, but also, with varying prevalence, in other conditions. In SLE, these antibodies are neither sensitive nor specific for this condition. They occur, however, more frequently in (proliferative) lupus nephritis, particularly during active disease. Furthermore, levels of anti-C1q rise, in many cases, prior to a relapse of lupus nephritis, suggesting a pathogenic role for the autoantibodies. Indeed, experimental studies strongly support a pathogenic role for anti-C1q in immune complex-mediated renal disease. In addition, anti-C1q may interfere with the clearance of apoptotic cells, so influencing induction and expression of autoimmunity. PMID:18606253

  7. LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

    SciTech Connect

    Rapali, Peter; Garcia-Mayoral, Maria Flor; Martinez-Moreno, Monica; Tarnok, Krisztian; Schlett, Katalin; Albar, Juan Pablo; Bruix, Marta; Nyitray, Laszlo; Rodriguez-Crespo, Ignacio

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We have screened a human library with dynein light chain DYNLL1 (DLC8) as bait. Black-Right-Pointing-Pointer Dynein light chain DYNLL1 binds to ATM-kinase interacting protein (ATMIN). Black-Right-Pointing-Pointer ATMIN has 17 SQ/TQ motifs, a motif frequently found in DYNLL1-binding partners. Black-Right-Pointing-Pointer The two proteins interact in vitro, with ATMIN displaying at least five binding sites. Black-Right-Pointing-Pointer The interaction of ATMIN and DYNNL1 in transfected cells can also be observed. -- Abstract: LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1

  8. Frequent N addition and clonal relatedness among immunoglobulin lambda light chains expressed in rheumatoid arthritis synovia and PBL, and the influence of V lambda gene segment utilization on CDR3 length.

    PubMed Central

    Bridges, S. L.

    1998-01-01

    BACKGROUND: In rheumatoid arthritis (RA), B-lineage cells in the synovial membrane secrete large amounts of immunoglobulin that contribute to tissue destruction. The CDR3 of an immunoglobulin light chain is formed by rearrangements of VL and JL gene segments. Addition of non-germline-encoded (N) nucleotides at V(D)J joins by the enzyme terminal deoxynucleotidyl transferase (TdT) enhances antibody diversity. TdT was previously thought to be active in B cells only during heavy chain rearrangement, but we and others reported unexpectedly high levels of N addition in kappa light chains. We also found clonally related kappa chains bearing unusually long CDR3 intervals in RA synovium, suggesting oligoclonal expansion of a set of atypical B lymphocytes. In this study, we analyzed lambda light chain expression to determine if N addition occurs throughout immunoglobulin gene rearrangement and to compare CDR3 lengths of lambda and kappa light chains in RA patients and normal individuals. MATERIALS AND METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) amplification of V lambda III transcripts was performed on RA synovia and peripheral blood lymphocytes (PBL) and normal PBL for which kappa repertoires were previously analyzed. Representative lambda + PCR products were cloned and sequenced. RESULTS: Analysis of 161 cDNA clones revealed that N addition occurs in lambda light chains of RA patients and normal controls. The lambda light chain repertoires in RA were enriched for long CDR3 intervals. In both RA and controls, CDR3 lengths were strongly influenced by which V lambda gene segment was present in the rearrangement. Five sets of clonally related sequences were found in RA synovia and PBL; one set was found in normal PBL. CONCLUSIONS: In humans, unlike mice, N addition enhances antibody diversity at all stages of immunoglobulin assembly, and the structural diversity of lambda CDR3 intervals is greater than that of kappa light chains. Clonally related V lambda

  9. Differences in the nuclease sensitivity between the two alleles of the immunoglobulin kappa light chain genes in mouse liver and myeloma nuclei.

    PubMed Central

    Weischet, W O; Glotov, B O; Schnell, H; Zachau, H G

    1982-01-01

    In mouse myeloma T the productive kappa light chain gene differs from its aberrantly rearranged allele in the patterns of DNAase I hypersensitive sites. In the region of the alleles where they are identical in sequence they have one site in common which lies 0.8 kb downstream of the coding region; but two sites upstream of and within the C gene segment (2) are found only on the non-productive allele. Within the region of different sequences both alleles have analogously located DNAase I hypersensitive sites; they lie 0.15 kb upstream of the respective leader segments and cover putative promoter sequences. Only one of the six DNAase I hypersensitive sites is also very sensitive towards micrococcal nuclease due to its particular DNA sequence. The non-rearranged gene studied in liver nuclei has no DNAase I hypersensitive sites but is preferentially cleaved in A/T rich regions. Images PMID:6287416

  10. Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus.

    PubMed

    Kothapalli, Nagarama; Norton, Darrell D; Fugmann, Sebastian D

    2008-02-15

    Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3' regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus. PMID:18250404

  11. Crystallization and Preliminary X-ray Analysis of the Human Long Myosin Light-Chain Kinase 1-Specific Domain IgCAM3

    SciTech Connect

    W Vallen Graham; A Magis; K Bailey; J Turner; D Ostrov

    2011-12-31

    Myosin light-chain kinase-dependent tight junction regulation is a critical event in inflammatory cytokine-induced increases in epithelial paracellular permeability. MLCK is expressed in human intestinal epithelium as two isoforms, long MLCK1 and long MLCK2, and MLCK1 is specifically localized to the tight junction, where it regulates paracellular permeability. The sole difference between these long MLCK splice variants is the presence of an immunoglobulin-like cell-adhesion molecule domain, IgCAM3, in MLCK1. To gain insight into the structure of the IgCAM3 domain, the IgCAM3 domain of MLCK1 has been expressed, purified and crystallized. Preliminary X-ray diffraction data were collected to 2.0 {angstrom} resolution and were consistent with the primitive trigonal space group P2{sub 1}2{sub 1}2{sub 1}.

  12. Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity.

    PubMed

    Watson, C T; Steinberg, K M; Graves, T A; Warren, R L; Malig, M; Schein, J; Wilson, R K; Holt, R A; Eichler, E E; Breden, F

    2015-01-01

    Germline variation at immunoglobulin (IG) loci is critical for pathogen-mediated immunity, but establishing complete haplotype sequences in these regions has been problematic because of complex sequence architecture and diploid source DNA. We sequenced BAC clones from the effectively haploid human hydatidiform mole cell line, CHM1htert, across the light chain IG loci, kappa (IGK) and lambda (IGL), creating single haplotype representations of these regions. The IGL haplotype generated here is 1.25 Mb of contiguous sequence, including four novel IGLV alleles, one novel IGLC allele, and an 11.9-kb insertion. The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap. Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters. Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH. PMID:25338678

  13. Synthesis of kappa light chains by cell lines containing an 8;22 chromosomal translocation derived from a male homosexual with Burkitt's lymphoma.

    PubMed

    Magrath, I; Erikson, J; Whang-Peng, J; Sieverts, H; Armstrong, G; Benjamin, D; Triche, T; Alabaster, O; Croce, C M

    1983-12-01

    Three cell lines were derived from a homosexual patient with probable acquired immunodeficiency syndrome and Burkitt's lymphoma. The cell lines produce an unusual strain of Epstein-Barr virus which will both transform cord blood lymphocytes and induce early antigens in Raji cells. Translocations between chromosomes 8 and 22 have occurred in all three lines, but the cells synthesize immunoglobulin M with light chains of the kappa type, in contrast to the usual concordance between a translocation involving chromosome 22 and lambda chain synthesis. Both kappa genes and one lambda gene are rearranged. These findings indicate either that translocation may occur as a separate event from immunoglobulin gene rearrangement or that the proposed hierarchical sequence of immunoglobulin gene rearrangements is not always adhered to. The data also imply that in cells containing a translocation between the long arm of chromosome 8 and a chromosome bearing an immunoglobulin gene, alteration of cellular myc expression may occur regardless of the immunoglobulin gene that is expressed. PMID:6316501

  14. Somatic mutation and CDR3 lengths of immunoglobulin kappa light chains expressed in patients with rheumatoid arthritis and in normal individuals.

    PubMed Central

    Bridges, S L; Lee, S K; Johnson, M L; Lavelle, J C; Fowler, P G; Koopman, W J; Schroeder, H W

    1995-01-01

    Immunoglobulin secretion by plasma cells infiltrating synovial membranes is a prominent feature of RA. Previous analyses of a cDNA library generated from synovium of RA patient BC revealed immunoglobulin kappa light chain transcripts with extensive somatic mutation, frequent N region addition, and unexpected variation in the lengths of CDR3 regions which form the center of the antigen binding site. To determine if these characteristics are present in other individuals, we performed reverse transcription-polymerase chain reaction amplification and sequenced > or = 10 V kappa-containing amplicons from nine tissue samples: synovia of three individuals with long-standing RA (including patient BC), PBLs of two of these individuals, and PBLs or splenocytes of four normal individuals. Increased levels of somatic mutation in PBLs appeared to correlate with increased age, which may reflect accumulation of circulating memory cells and/or decreased bone marrow production of naive B lymphocytes. Two of three RA synovial samples and both RA PBL samples exhibited increased proportions of clones with unusual CDR3 lengths. Enrichment for these antibody binding sites could be due to abnormal regulation of the emerging repertoire or to selection for B lymphocytes bearing antibodies of unusual specificity, and may play a role in the pathogenesis of RA. Images PMID:7635977

  15. [Efficacy of serum immunoglobulin free light chain and 18F-FDG PET/CT for therapeutic evaluation in a case with multiple plasmacytoma of bone].

    PubMed

    Inoue, Takashi; Nagai, Koichi; Momoi, Akihito; Isahai, Noriatsu; Sakai, Takeshi; Sekiya, Masao

    2015-04-01

    Solitary/multiple plasmacytoma of bone, a rare disease as compared to multiple myeloma, is characterized by monoclonal proliferation of plasma cells in local legion (s) of bone, with no bone marrow abnormalities. Monoclonal gammagloblinemia is often absent in these conditions, and useful examinations allowing evaluation of responses to treatment are as yet lacking. Recently, 18F-FDG PET/CT (PET/CT) was reported to be useful for detecting bone lesions. PET/CT is also valuable for predicting the outcomes of patients with solitary plasmacytoma, when applied in combination with the serum free light chain (sFLC) κ/λ ratio. We present a 62-year-old male with multiple plasmacytoma of bone (MPB), in whom PET/CT and the sFLC κ/λ ratio were useful for evaluating the response to treatment. The patient was diagnosed with MPB, with PET/CT showing multiple abnormal tracer uptakes in the scapula, spine, and ribs. The sFLC ratio was markedly elevated, with the κ chain level being especially high. We administered bortezomib and dexamethasone, after which the abnormal uptakes on PET/CT disappeared. The sFLC ratio and the FLC value also normalized, and have remained stable for more than one year, to date, since treatment. In our case, PET/CT and the sFLC κ/λ ratio were found to be extremely useful for monitoring treatment responses. PMID:25971273

  16. Heterogeneity of myofibrillar proteins in lobster fast and slow muscles: variants of troponin, paramyosin, and myosin light chains comprise four distinct protein assemblages

    SciTech Connect

    Mykles, D.L.

    1985-01-01

    Fast and slow muscles from the claws and abdomen of the American lobster Homarus americanus were examined for adenosine triphosphatase (ATPase) activity and for differences in myofibrillar proteins. Both myosin and actomyosin ATPase were correlated with fiber composition and contractile speed. Four distinct patterns of myofibrilla proteins observed in sodium dodecyl sulfate-polyacrylamide gels were distinguished by different assemblages of regulatory and contractile protein variants. A total of three species of troponin-T, five species of troponin-I, and three species of troponin-C were observed. Lobster myosins contained two groups of light chains (LC), termed alpha and beta. There were three ..cap alpha..-LC variants and two ..beta..-LC variants. There were no apparent differences in myosin heavy chain, actin, and tropomyosin. Only paramyosin showed a pattern completely consistent with muscle fiber type: slow fibers contained a species (105 kD) slightly smaller than the principle variant (110 kD) in fast fibers. It is proposed that the type of paramyosin present could provide a biochemical marker to identify the fiber composition of muscles that have not been fully characterized. The diversity of troponin and myosin LC variants suggests that subtle differences in physiological performance exist within the broader categories of fast- and slow-twitch muscles. 31 references, 6 figures, 2 tables.

  17. N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points

    PubMed Central

    Islam, Md Ariful; Sharif, Syeda Ridita; Lee, HyunSook; Seog, Dae-Hyun; Moon, Il Soo

    2015-01-01

    N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74–96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching. PMID:26272270

  18. Melatonin alleviates myosin light chain kinase expression and activity via the mitogen-activated protein kinase pathway during atherosclerosis in rabbits.

    PubMed

    Cheng, Xiaowen; Wan, Yufeng; Xu, Yuanhong; Zhou, Qing; Wang, Yuan; Zhu, Huaqing

    2015-01-01

    Melatonin (MLT) is an endogenous indole compound with numerous biological activities that has been associated with atherosclerosis (AS). In the present study, rabbits were used as an AS model in order to investigate whether MLT affects endothelial cell permeability, myosin light chain kinase (MLCK) activity and MLCK expression via the mitogen-activated protein kinase (MAPK) pathway. Expression and activity of MLCK were measured using western blot analysis, quantitative polymerase chain reaction, immunohistochemistry and γ-32P-adenosine triphosphate incorporation. Endothelial permeability was detected using rhodamine phalloidin fluorescence staining. The phosphorylation of extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 in endothelial cells were also analyzed using western blot analysis. Atheromatous plaques were formed in rabbits with a high cholesterol diet; however, following treatment with MLT, the number and areas of atheromatous plaques were significantly reduced. In addition, MLT treatment reversed the increase of MLCK activity and expression that occurred in rabbits with high cholesterol intake. Furthermore, levels of phosphorylated ERK, JNK and p38 decreased following MLT treatment. In conclusion, the results of the present study indicated that AS may be associated with increased MLCK expression and activity, which was reduced following treatment with MLT. The mechanism of action of MLT was thought to proceed via modulating MAPK pathway signal transduction; however, further studies are required in order to fully elucidate the exact regulatory mechanisms involved. PMID:25339116

  19. Dissect Kif5b in nuclear positioning during myogenesis: The light chain binding domain and the autoinhibitory peptide are both indispensable

    SciTech Connect

    Wang, Zai; Xue, Wenqian; Li, Xiuling; Lin, Raozhou; Cui, Ju; Huang, Jian-Dong

    2013-03-08

    Highlights: ► Kif5b localizes at myonuclear membrane and is responsible for nuclear dispersion. ► Kif5b stalk/tail domain contains signal for nuclear membrane targeting. ► Kif5b stalk/tail domain directly binds to a nesprin 4 in vitro. ► KLC binding domain and autoinhibitory peptide are both functionally indispensable. -- Abstract: The microtubule motor kinesin-1 is responsible for the nuclear positioning during myogenesis. Here we show that the coiled-coil stalk/tail domain containing the kinesin light chain (KLC) binding sites targets to the perinuclear region like endogenous Kif5b, while the globular tail domain cannot. To investigate which fragments of kinesin heavy chain (Kif5b) is responsible for the myonuclear positioning, we transfect Kif5b expression constructs into Kif5b deficient myoblasts and test their ability to rescue the myonuclear phenotype. We find that the KLC binding domain and the autoinhibitory peptide in the globular tail region are both indispensable for the nuclear membrane localization of Kif5b and the kinesin-1-mediated myonuclear positioning. These results suggest that while the KLC binding domain may directly targets Kif5b to the myonuclear membrane, the autoinhibitory peptide may play an indirect role in regulating the kinesin-1-mediated myonuclear positioning.

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

    PubMed Central

    Ponomarenko, Natalia; Chatziefthimiou, Spyros D.; Kurkova, Inna; Mokrushina, Yuliana; Mokrushina, Yuliana; Stepanova, Anastasiya; Smirnov, Ivan; Avakyan, Marat; Bobik, Tatyana; Mamedov, Azad; Mitkevich, Vladimir; Belogurov, Alexey; Fedorova, Olga S.; Dubina, Michael; Golovin, Andrey; Lamzin, Victor; Friboulet, Alain; Makarov, Alexander A.; Wilmanns, Matthias; Gabibov, Alexander

    2014-01-01

    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 found 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 VL and VH domains. These VL/VH 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. PMID:24598740

  1. A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species

    PubMed Central

    Lu, Yan; Zhang, Chenglin; Wu, Xiaobing; Han, Haitang; Zhao, Yaofeng; Ren, Liming

    2016-01-01

    Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates. PMID:26901135

  2. A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species.

    PubMed

    Wang, Xifeng; Cheng, Gang; Lu, Yan; Zhang, Chenglin; Wu, Xiaobing; Han, Haitang; Zhao, Yaofeng; Ren, Liming

    2016-01-01

    Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates. PMID:26901135

  3. New patterns of relapse in multiple myeloma: a case of "light chain escape" in which FLC predicted relapse earlier than urine and serum immunofixation.

    PubMed

    Caldini, Anna; Nozzoli, Chiara; Terreni, Alessandro; Staderini, Michela; Berardi, Margherita; Biagioli, Tiziana; Brogi, Marco; Bosi, Alberto

    2016-06-01

    Multiple myeloma (MM) is characterized, in about 80% of cases, by the production of monoclonal intact immunoglobulin and more than 95% of them have elevated concentrations of involved (i.e. of the same class of intact immunoglobulin) free light chain (FLC). The introduction of novel therapeutic strategies has changed the natural history of the disease, leading to new manifestations of relapse. Light chain escape (LCE) is a pattern of relapse in which the FLC increase is not accompanied by a concomitant raise of the original monoclonal component (MC). Here we present a case of a 55-year-old man with an IgG kappa MM stage III diagnosed in September 2007. At presentation an IgG kappa MC and urine Bence Jones protein (BJP) kappa were present. Bone marrow biopsy (BMB) showed the presence of 80% monotypic kappa plasma cells (PCs). The patient received bortezomib, thalidomide, dexamethasone before undergoing a double autologous stem cell transplantation (ASCT) in October 2008 and April 2009. In May 2011 he relapsed showing the same pattern of presentation and treatment with lenalidomide and dexamethasone was started. ln May 2013 serum and urine immunofixation and FLC became negative. In September 2014, an increase of kappa FLC was observed, while serum and urine immunofixations remained negative until January 2015, when urine immunofixation became positive. Eventually, in February 2015, serum immunofixation revealed the presence of a free kappa MC. After a new BMB showing 80% of monotypic kappa PCs, a LCE relapse was diagnosed and the patient started the treatment with bendamustine, bortezomib and dexamethasone. In the present case, the increase of kappa FLC has indicated relapse 4 and 5 months earlier than urine and serum IFE, respectively. Our observation confirms that it is advisable to routinely perform FLC or BJP during follow up of MM patients undergoing ASCT and/or treatment with biological drugs to ensure that LCE is not missed. PMID:26581069

  4. 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 Central

    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

  5. X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

    PubMed

    Yamaguchi, Maki; Kimura, Masako; Li, Zhao-Bo; Ohno, Tetsuo; Takemori, Shigeru; Hoh, Joseph F Y; Yagi, Naoto

    2016-04-15

    The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments. PMID:26911280

  6. Neuregulin1–β decreases interleukin–1β–induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability

    PubMed Central

    Wu, Limin; Ramirez, Servio H.; Andrews, Allison M.; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H.; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. PMID:26438054

  7. Coexistent Multiple Myeloma or Increased Bone Marrow Plasma Cells Define Equally High-Risk Populations in Patients With Immunoglobulin Light Chain Amyloidosis

    PubMed Central

    Kourelis, Taxiarchis V.; Kumar, Shaji K.; Gertz, Morie A.; Lacy, Martha Q.; Buadi, Francis K.; Hayman, Suzanne R.; Zeldenrust, Steven; Leung, Nelson; Kyle, Robert A.; Russell, Stephen; Dingli, David; Lust, John A.; Lin, Yi; Kapoor, Prashant; Rajkumar, S. Vincent; McCurdy, Arleigh; Dispenzieri, Angela

    2013-01-01

    Purpose There is consensus that patients with light chain (AL) amyloidosis with hypercalcemia, renal failure, anemia, and lytic bone lesions attributable to clonal expansion of plasma cells (CRAB criteria) also have multiple myeloma (MM). The aim of this study was to examine the spectrum of immunoglobulin AL amyloidosis with and without MM, with a goal of defining the optimal bone marrow plasma cell (BMPC) number to qualify as AL amyloidosis with MM. Patients and Methods We identified 1,255 patients with AL amyloidosis seen within 90 days of diagnosis between January 1, 2000, and December 31, 2010. We defined a population of patients with coexisting MM on the basis of the existence of CRAB criteria (AL-CRAB). Receiver operating characteristic analysis determined the optimal BMPC cut point to predict for 1-year mortality in patients with AL amyloidosis without CRAB to produce two additional groups: AL only (≤ 10% BMPCs) and AL plasma cell MM (AL-PCMM; > 10% BMPCs). Results Among the 1,255 patients, 100 (8%) had AL-CRAB, 476 (38%) had AL-PCMM, and 679 (54%) had AL only. Their respective median overall survival rates were 10.6, 16.2, and 46 months (P < .001). Because the outcomes of AL-CRAB and AL-PCMM were similar, they were pooled for univariate and multivariate analyses. On multivariate analysis, pooled AL-CRAB and AL-PCMM retained negative prognostic value independent of age, Mayo Clinic AL amyloidosis stage, prior autologous stem-cell transplantation, and difference between the involved and uninvolved free light chain. Conclusion Patients with AL amyloidosis who have more than 10% BMPCs have a poor prognosis, similar to that of patients with AL-CRAB, and should therefore be considered together as AL amyloidosis with MM. PMID:24145344

  8. Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway1[OPEN

    PubMed Central

    Légeret, Bertrand; Mirabella, Boris; Guédeney, Geneviève; Jetter, Reinhard; Peltier, Gilles

    2016-01-01

    Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway. PMID:27288359

  9. Identification of N-terminal methionine in the precursor of immunoglobulin light chain. Initiation of translation of messenger ribonucleic acid in plants and animals.

    PubMed Central

    Schechter, I; Burstein, Y

    1976-01-01

    The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-321 mouse myeloma L (light) chain were labelled with [35S]methionine, [4,5-3H]leucine or [3-3H]serine, and were subjected to amino acid-sequence analyses. Over 95% of the total cell-free product was sequenced as one homogeneous protein, which corresponds to the precursor of the L-chain protein. In the precursor, 20 amino acid residues precede the N-terminus of the mature protein. This extra piece contains one methionine residue at the N-terminus, one serine residue at position 18, and six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13. The identification of methionine at the N-terminus of the precursor is in agreement with the evidence showing that unblocked methionine is the initiator residue for protein synthesis in eukaryotes. The absence of methionine at position 20, which precedes the N-terminal residue of the mature protein, suggests that myeloma cells synthesize the precursor. However, within the cell the precursor should be rapidly processed to the mature L chain, since precursor molecules have not yet been found in the intact animal. The abundance (30%) of leucine residues indicates that the extra-piece moiety is quite hydrophobic. The extra piece of the MOPC-321 L-chain precursor synthesized with the aid of the Krebs II ascites cell-free system is of identical size and it has the same leucine sequence [Schechter et al. (1975) Science 188, 160-162]. This indicates that cell-free systems derived from the plant and animal kingdom initiate mRNA translation from the same point. It is shown that the amino acid sequence of minute amounts of a highly labelled protein (0.1 pmol) can be faithfully determined in the presence of a large excess (over 2000 000-fold) of unrelated non-radioactive proteins. Images PLATE 2 PLATE 1 PMID:821467

  10. Hereditary angioedema with normal C1 inhibitor.

    PubMed

    Bork, Konrad

    2013-11-01

    Until recently it was assumed that hereditary angioedema was a disease that results exclusively from a genetic deficiency of the C1 inhibitor. In 2000, families with hereditary angioedema, normal C1 inhibitor activity, and protein in plasma were described. Since then, numerous patients and families with that condition have been reported. Most of the patients were women. In many of the affected women, oral contraceptives, hormone replacement therapy containing estrogens, and pregnancies triggered the clinical symptoms. In some families mutations in the coagulation factor XII (Hageman factor) gene were detected. PMID:24176211

  11. Preliminary crystallographic studies of a Schistosoma mansoni antigen (Sm21.7) dynein light-chain (DLC) domain

    PubMed Central

    Costa, M. A. F.; Rodrigues, F. T. G.; Chagas, B. C. A.; Rezende, C. M. F.; Goes, A. M.; Nagem, R. A. P.

    2014-01-01

    Schistosomiasis is an inflammatory chronic disease that represents a major health problem in tropical and subtropical countries. The drug of choice for treatment, praziquantel, is effective in killing adult worms but fails to kill immature forms and prevent reinfection. One prominent antigen candidate for an anti-schistosomiasis vaccine is the protein Sm21.7 (184 amino-acid residues) from Schistosoma mansoni, a tegumental protein capable of reducing the worm burden in a murine immunization model. In the present work, the Sm21.7 gene was cloned and expressed in Escherichia coli and the full-length protein was purified to homogeneity. Crystals of recombinant Sm21.7 suitable for X-ray diffraction were obtained using PEG monomethyl ether 2000 as a precipitant. X-ray diffraction images of a native crystal (at 2.05 Å resolution) and a quick-cryosoaked NaI derivative (at 1.95 Å resolution) were collected on the W01B-MX2 beamline at the Laboratório Nacional de Luz Síncrotron (LNLS, Brazilian Synchrotron Light Laboratory/MCT). Both crystals belonged to the hexagonal space group P6122, with similar unit-cell parameters a = b = 108.5, c = 55.8 Å. SIRAS-derived phases were used to generate the first electron-density map, from which a partial three-dimensional model of Sm21.7 (from Gln89 to Asn184) was automatically constructed. Anaysis of dissolved crystals by SDS–PAGE confirmed that the protein was cleaved in the crystallization drop and only the Sm21.7 C-terminal domain was crystallized. The structure of the Sm21.7 C-terminal domain will help in the localization of the epitopes responsible for its protective immune responses, constituting important progress in the development of an anti-schistosomiasis vaccine. PMID:24915098

  12. Effects of anti-heat diet and inverse lighting on growth performance, immune organ, microorganism and short chain fatty acids of broiler chickens under heat stress.

    PubMed

    Ryu, Seung-Tae; Park, Byung-Sung; Bang, Han-Tae; Kang, Hwan-Ku; Hwangbo, Jong

    2016-03-01

    The present study investigated the effects of night restricted feeding of extreme heat diet (EHD) containing heat stress resistance nutrients, with inverse lighting program, on their growth performance in broiler chickens exposed to extreme heat stress (33 ± 2°C). EHD 1 contained soy oil, molasses, methionine and lysine. EHD 2 contained all nutrients of EHD 1 and vitamin C additionally. Three hundred broiler chickens (Ross 308) were randomized into four dietary treatment groups according to a randomized block design on the day they were hatched. The treatment groups were: T1 (EHD 1, 10:00-19:00 dark, 19:00-10:00 light), T2 (EHD 2, 10:00-19:00 dark, 19:00-10:00 light), T3 (EHD 1, 09:00-18:00 dark, 18:00-09:00 light) and T4 (EHD 2, 09:00-18:00 dark, 18:00-09:00 light). The body weight gain of the broilers increased most significantly in T2, followed by T1, T4 and T3 (p < 0.05). Weights of the immune system, thymus and bursa of Fabricius recorded higher in T1 and T2 than in T3 and T4. The spleen was higher in T1, T2 and T3 than in T4 (p < 0.05). Blood triglyceride, total cholesterol and blood sugar were higher in T1 and T2 than in T3 and T4 (p < 0.05). LDL-C recorded high in the order of T4, T3, T2 and T1, but HDL-C showed the inverse order (p < 0.05). IgG, IgA and IgM were higher in T1 and T2 than in T3 and T4, however, the corticosterone concentration showed the inverse order (p<0.05). Lactobacillus in feces was higher in T1 and T2 than in T3 and T4, but total aerobic bacteria, E. coli, Coliform bacteria was higher in T4 and T3 than in T2 and T1 (p < 0.05). Contents of acetic acid, propionic acid and total Short chain fatty acid were significantly higher in the order of T2, T1, T3 and T4. Butyric acid, isobutyric acid, valeric acid and isovaleric acid were higher in T4 and T3 than in T1 and T2 (p < 0.05). PMID:27097436

  13. Lighting

    SciTech Connect

    Audin, L.

    1994-12-31

    EPAct covers a vast territory beyond lighting and, like all legislation, also contains numerous {open_quotes}favors,{close_quotes} compromises, and even some sleight-of-hand. Tucked away under Title XIX, for example, is an increase from 20% to 28% tax on gambling winnings, effective January 1, 1993 - apparently as a way to help pay for new spending listed elsewhere in the bill. Overall, it is a landmark piece of legislation, about a decade overdue. It remains to be seen how the Federal Government will enforce upgrading of state (or even their own) energy codes. There is no mention of funding for {open_quotes}energy police{close_quotes} in EPAct. Merely creating such a national standard, however, provides a target for those who sincerely wish to create an energy-efficient future.

  14. Chain formation and chain dynamics in a dilute magnetorheological fluid.

    PubMed

    Hagenbüchle, M; Liu, J

    1997-10-20

    Magnetorheological fluids are suspensions of magnetizable particles that reversibly change from liquid to solid when subjected to a magnetic field. A field-induced structure of dipolar chains is responsible for these changes. Our work aimed at understanding chain dynamics and the kinetics of chain formation by using dynamic light scattering. Chain length is determined by measurement of the diffusion coefficient. Chain-length growth shows a Smoluchowski behavior. PMID:18264283

  15. Association of a Rare Haplotype in Kinesin Light Chain 1 Gene with Age-Related Cataract in a Han Chinese Population

    PubMed Central

    Qu, Xin; Liu, Dong-Rui; Liu, Ping; Zhao, Xin-Zhi

    2013-01-01

    Purpose The causal genes for congenital cataract are good candidates for the genetic susceptibility for age-related cataract (ARC). The aim of this study was to investigate association between the polymorphisms in the causal genes for congenital cataract and ARC in a Chinese population. Meanwhile, we performed the replication study for previous identified risk genes for ARC. Methods We recruited 212 sporadic Han Chinese patients with age-related cataracts (ARC) and 172 normal controls in this study. We analyzed 31 SNPs from 13 genes which mostly possible contributes the progress of ARC in a Chinese population, comprising 212 cataract patients and 172 controls. Polymorphism-spanning fragments were amplified by using the multiplex polymerase chain reaction (PCR) and genotyped using primer extension method in MassARRAY platform. Allelic and haplotypic difference in the frequencies were estimated using the SHEsis software platform. P-value was adjusted by the Bonferroni correction. Results There was no difference in the frequencies of the genotype and allele of the all SNPs between the patients with ARC and the controls. In the haplotypic analysis, the haplotypes consisting of rs7154572, rs7150141 and rs12432994 in Kinesin Light Chain 1 Gene (KLC1) showed significant association with ARC (p = 0.000878). A rare haplotype CGT was more frequent in patients (p = 0.000106, and p = 0.00795 after corrected for 75 tests). Conclusions Our study provides evidence that the combined effect of three variants within the KLC1 gene may predispose to ARC, but the precise mechanism needs further investigating. PMID:23776437

  16. 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

  17. Robust Reference Intervals for Serum Kappa and Lambda Free Light Chains from a Multi Centre Study Population from Hyderabad, India: Myeloma Diagnostic Implications.

    PubMed

    Mohammed, Noorjahan; Chandran, Priscilla Abraham; Kandregula, Madhavi; Mattaparthi, Ratna Deepika; Gundeti, Sadasivudu; Volturi, Jyotsna; Darapuneni, Radhika; Raju, Sree Bhushan; Dattatreya, Palanki Satya

    2016-01-01

    The International Myeloma Working Group considers the serum free light chain (SFLC) assay to be an adjunct to traditional tests. Apart from the FLC ratio, the absolute values of individual free light chains also are gaining importance as they appear to be more relevant in certain clinical settings. Automated assays are available for their determination. As laboratories put new test systems into use catering to different disease populations, they are required by accreditation and certification bodies to verify or establish performance specifications, including reference intervals (RIs) representative of their population. Our aim was to establish local RIs for SFLC in a multicentre representative healthy population using a robust method. There was no significant relationship between SFLC levels and age, gender and creatinine levels. The 95% RI for κSFLC was 4.81 to 33.86mg/L, for ? SFLC was 5.19 to 23.67mg/L and for κ/?SFLC was 0.36 to 2.33, significantly higher than the values given by the manufacturer. The κ/? SFLC ratio at 2.23, covering 100% of the data, showed 72% sensitivity (95% CI=39.0 - 94.0), 100% specificity (95% CI=71.5 - 100.0), 100% PPV (95% CI=21.5 - 100.0), 95% NPV (95% CI=75.4 - 99.9), and 79% accuracy (95% CI=56.0 - 93.0). In the patient group, kit RI for κ /? SFLC ratio classified 45.5% (n=5) as positive vs 9.1% (n=1) positive by the study RI, while the kit RI for kappa FLC classified 90.9% (n=10) as positive vs 54.5% (n=6) , indicating increased probability of false positive test results with the kit RI when applied to our patient population. Appropriate and specific reference intervals and criteria values result in fewer false-positive and false-negative results which means fewer wrong or missed diagnoses. PMID:27268638

  18. New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphoryl