WorldWideScience

Sample records for dynein light chain

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

    Directory of Open Access Journals (Sweden)

    Sean M O'Rourke

    2007-08-01

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

  2. Analysis of dynein intermediate chains, light intermediate chains and light chains in a cohort of hereditary peripheral neuropathies.

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    Tey, Shelisa; Ahmad-Annuar, Azlina; Drew, Alexander P; Shahrizaila, Nortina; Nicholson, Garth A; Kennerson, Marina L

    2014-10-01

    The cytoplasmic dynein heavy chain (DYNC1H1) gene has been increasingly associated with neurodegenerative disorders including axonal Charcot-Marie-Tooth disease (CMT2), intellectual disability and malformations of cortical development. In addition, evidence from mouse models (Loa, catabolite repressor-activator (Cra) and Sprawling (Swl)) has shown that mutations in Dync1h1 cause a range of neurodegenerative phenotypes with motor and sensory neuron involvement. In this current study, we examined the possible contribution of other cytoplasmic dynein subunits that bind to DYNC1H1 as a cause of inherited peripheral neuropathy. We focused on screening the cytoplasmic dynein intermediate, light intermediate and light chain genes in a cohort of families with inherited peripheral neuropathies. Nine genes were screened and ten variants were detected, but none was identified as pathogenic, indicating that cytoplasmic dynein intermediate, light intermediate and light chains are not a cause of neuropathy in our cohort.

  3. Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

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    DiBella, Linda M.; Gorbatyuk, Oksana; Sakato, Miho; Wakabayashi, Ken-ichi; Patel-King, Ramila S.; Pazour, Gregory J.; Witman, George B.; King, Stephen M.

    2005-01-01

    Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum. PMID:16195342

  4. Identification of the t Complex–encoded Cytoplasmic Dynein Light Chain Tctex1 in Inner Arm I1 Supports the Involvement of Flagellar Dyneins in Meiotic Drive

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    Harrison, Alistair; Olds-Clarke, Patricia; King, Stephen M.

    1998-01-01

    The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell–specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme. PMID:9490726

  5. Dynein light intermediate chains maintain spindle bipolarity by functioning in centriole cohesion.

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    Jones, Laura A; Villemant, Cécile; Starborg, Toby; Salter, Anna; Goddard, Georgina; Ruane, Peter; Woodman, Philip G; Papalopulu, Nancy; Woolner, Sarah; Allan, Victoria J

    2014-11-24

    Cytoplasmic dynein 1 (dynein) is a minus end-directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs' role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis.

  6. An outer arm dynein light chain acts in a conformational switch for flagellar motility

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    Patel-King, Ramila S.

    2009-01-01

    A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity. PMID:19620633

  7. The LC7 Light Chains of Chlamydomonas Flagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation

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    DiBella, Linda M.; Sakato, Miho; Patel-King, Ramila S.; Pazour, Gregory J.; King, Stephen M.

    2004-01-01

    Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced ∼20% in axonemes isolated from strains lacking inner arm I1 and are ∼80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles ∼30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm γ heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins. PMID:15304520

  8. Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint.

    Directory of Open Access Journals (Sweden)

    Sagar P Mahale

    Full Text Available 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.

  9. Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia.

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    Rashid, Sajid; Grzmil, Pawel; Drenckhahn, Joerg-Detlef; Meinhardt, Andreas; Adham, Ibrahim; Engel, Wolfgang; Neesen, Juergen

    2010-01-01

    To elucidate the role of the mouse gene Tcte3 (Tctex2), which encodes a putative light chain of the outer dynein arm of cilia and sperm flagella, we have inactivated this gene in mice using targeted disruption. Breeding of heterozygous males and females resulted in normal litter size; however, we were not able to detect homozygous Tcte3-deficent mice using standard genotype techniques. In fact, our results indicate the presence of at least three highly similar copies of the Tcte3 gene (Tcte3-1, Tcte3-2, and Tcte3-3) in the murine genome. Therefore, quantitative real-time PCR was established to differentiate between mice having one or two targeted Tcte3-3 alleles. By this approach, Tcte3-3(-/-) animals were identified, which were viable and revealed no obvious malformation. Interestingly, some homozygous Tcte3-3-deficient male mice bred with wild-type female produced no offspring while other Tcte3-3-deficient males revealed decreased sperm motility but were fertile. In infertile Tcte3-3(-/-) males, spermatogenesis was affected and sperm motility was reduced, too, resulting in decreased ability of Tcte3-3-deficient spermatozoa to move from the uterus into the oviduct. Impaired flagellar motility is not correlated with any gross defects in the axonemal structure, since outer dynein arms are detectable in sperm of Tcte3-3(-/-) males. However, in infertile males, deficient Tcte3-3 function is correlated with increased apoptosis during male germ cell development, resulting in a reduction of sperm number. Moreover, multiple malformations in developing haploid germ cells are present. Our results support a role of Tcte3-3 in generation of sperm motility as well as in male germ cell differentiation.

  10. Dynein light chain binding to a 3′-untranslated sequence mediates parathyroid hormone mRNA association with microtubules

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    Epstein, Eyal; Sela-Brown, Alin; Ringel, Israel; Kilav, Rachel; King, Stephen M.; Benashski, Sharon E.; Yisraeli, Joel K.; Silver, Justin; Naveh-Many, Tally

    2000-01-01

    The 3′-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3′-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3′-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3′-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell. PMID:10683380

  11. Structure–function–folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights

    Indian Academy of Sciences (India)

    P M Krishna Mohan; Ramakrishna V Hosur

    2009-09-01

    The detailed characterization of the structure, dynamics and folding process of a protein is crucial for understanding the biological functions it performs. Modern biophysical and nuclear magnetic resonance (NMR) techniques have provided a way to obtain accurate structural and thermodynamic information on various species populated on the energy landscape of a given protein. In this context, we review here the structure–function–folding relationship of an important protein, namely, dynein light chain protein (DLC8). DLC8, the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions and dissociates into a pure monomer below pH 4. Cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface are anticipated to be playing crucial roles in the variety of functions the protein performs. NMR investigations have provided great insights into these aspects of DLC8 in recent years.

  12. A Chlamydomonas Homologue of the Putative Murine t Complex Distorter Tctex-2 Is an Outer Arm Dynein Light Chain

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    Patel-King, Ramila S.; Benashski, Sharon E.; Harrison, Alistair; King, Stephen M.

    1997-01-01

    Molecular analysis of a 19,000-Mr protein from the Chlamydomonas flagellum reveals that it is homologous to the t complex–encoded protein Tctex-2, which is a candidate for one of the distorter products that cause the extreme transmission ratio distortion (meiotic drive) of the murine t complex. The 19,000-Mr protein is extracted from the axoneme with 0.6 M NaCl and comigrates with the outer dynein arm in sucrose density gradients. This protein also is specifically missing in axonemes prepared from a mutant that does not assemble the outer arm. These data raise the possibility that Tctex-2 is a sperm flagellar dynein component. Combined with the recent identification of Tctex-1 (another distorter candidate) as a light chain of cytoplasmic dynein, these results lead to a biochemical model for how differential defects in spermiogenesis that result in the phenomenon of meiotic drive might be generated in wild-type vs t-bearing sperm. PMID:9166408

  13. Dynein light chain DLC-1 promotes localization and function of the PUF protein FBF-2 in germline progenitor cells.

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    Wang, Xiaobo; Olson, Jenessa R; Rasoloson, Dominique; Ellenbecker, Mary; Bailey, Jessica; Voronina, Ekaterina

    2016-12-15

    PUF family translational repressors are conserved developmental regulators, but the molecular function provided by the regions flanking the PUF RNA-binding domain is unknown. In C. elegans, the PUF proteins FBF-1 and FBF-2 support germline progenitor maintenance by repressing production of meiotic proteins and use distinct mechanisms to repress their target mRNAs. We identify dynein light chain DLC-1 as an important regulator of FBF-2 function. DLC-1 directly binds to FBF-2 outside of the RNA-binding domain and promotes FBF-2 localization and function. By contrast, DLC-1 does not interact with FBF-1 and does not contribute to FBF-1 activity. Surprisingly, we find that the contribution of DLC-1 to FBF-2 activity is independent of the dynein motor. Our findings suggest that PUF protein localization and activity are mediated by sequences flanking the RNA-binding domain that bind specific molecular partners. Furthermore, these results identify a new role for DLC-1 in post-transcriptional regulation of gene expression.

  14. Tissue Specific Roles of Dynein Light Chain 1 in Regulating Germ Cell Apoptosis in Ceanorhabditis elegans

    DEFF Research Database (Denmark)

    Morthorst, Tine Hørning

    2015-01-01

    (dlc-1) in apoptosis are described. DLC-1 is a part of the motor complex dynein, which moves along microtubules inside the cell. DLC-1 has been demonstrated to have both dynein dependent and independent functions in mammalian cells, which is also apparent from the studies presented here. Specifically......, DLC-1 was found to play a cell-nonautonomous role in somatic tissue to negatively regulate the apoptotic response to ironizing radiation-induced apoptosis upstream of the KRIT1/CCM1 homolog KRI-1. Depletion of dlc-1 results in ectopic apoptosis in the germline, which is dependent on the BH3-only...... proteins EGL-1 and CED-13. These proteins are normally regulated by the p53 homolog CEP-1, however, DLC-1 regulates apoptosis independently of the function of CEP-1. Furthermore, the function of DLC-1 is independent of its association with dynein. The other apoptotic mechanism of DLC-1 regulates...

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

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

    2012-01-01

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

  16. Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

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    Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

    2017-09-01

    The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier.

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    Ashokkumar, Balasubramaniem; Nabokina, Svetlana M; Ma, Thomas Y; Said, Hamid M

    2009-09-01

    The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC.

  18. Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A.

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    Kirkham, Justin K; Park, Sung Hee; Nguyen, Tu N; Lee, Ju Huck; Günzl, Arthur

    2016-01-01

    Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

  19. LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Rapali, Peter [Dept. Biochemistry, Eoetvoes Lorand University, Budapest (Hungary); Garcia-Mayoral, Maria Flor [Dept. Biological Physical Chemistry, IQFR, CSIC, Madrid (Spain); Martinez-Moreno, Monica [Dept. Biochemistry and Molecular Biology I, Universidad Complutense, Madrid (Spain); Tarnok, Krisztian; Schlett, Katalin [Dept. Physiology and Neurobiology, Eoetvoes Lorand University, Budapest (Hungary); Albar, Juan Pablo [Proteomics Facility, CNB, CSIC, Madrid (Spain); Bruix, Marta [Dept. Biological Physical Chemistry, IQFR, CSIC, Madrid (Spain); Nyitray, Laszlo, E-mail: nyitray@elte.hu [Dept. Biochemistry, Eoetvoes Lorand University, Budapest (Hungary); Rodriguez-Crespo, Ignacio, E-mail: nacho@bbm1.ucm.es [Dept. Biochemistry and Molecular Biology I, Universidad Complutense, Madrid (Spain)

    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

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

    Science.gov (United States)

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

    2013-01-01

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

  1. Overexpression of the dynein light chain km23-1 in human ovarian carcinoma cells inhibits tumor formation in vivo and causes mitotic delay at prometaphase/metaphase.

    Science.gov (United States)

    Pulipati, Nageswara R; Jin, Qunyan; Liu, Xin; Sun, Baodong; Pandey, Manoj K; Huber, Jonathan P; Ding, Wei; Mulder, Kathleen M

    2011-08-01

    km23-1 is a dynein light chain that was identified as a TGFβ receptor-interacting protein. To investigate whether km23-1 controls human ovarian carcinoma cell (HOCC) growth, we established a tet-off inducible expression system in SKOV-3 cells in which the expression of km23-1 is induced upon doxycycline removal. We found that forced expression of km23-1 inhibited both anchorage-dependent and anchorage-independent growth of SKOV-3 cells. More importantly, induction of km23-1 expression substantially reduced the tumorigenicity of SKOV-3 cells in a xenograft model in vivo. Fluorescence-activated cell sorting analysis of SKOV-3 and IGROV-1 HOCCs demonstrated that the cells were accumulating at G2/M. Phospho-MEK, phospho-ERK and cyclin B1 were elevated, as was the mitotic index, suggesting that km23-1 suppresses HOCCs growth by inducing a mitotic delay. Immunofluorescence analyses demonstrated that the cells were accumulating at prometaphase/metaphase with increases in multipolar and multinucleated cells. Further, although the mitotic spindle assembly checkpoint protein BubR1 was present at the prometaphase kinetochore in Dox+/- cells, it was inappropriately retained at the metaphase kinetochore in Dox- cells. Thus, the mechanism by which high levels of km23-1 suppress ovarian carcinoma growth in vitro and inhibit ovary tumor formation in vivo appears to involve a BubR1-related mitotic delay.

  2. FhCaBP2: a Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains.

    Science.gov (United States)

    Thomas, Charlotte M; Timson, David J

    2015-09-01

    FhCaBP2 is a Fasciola hepatica protein which belongs to a family of helminth calcium-binding proteins which combine an N-terminal domain containing two EF-hand motifs and a C-terminal dynein light chain-like (DLC-like) domain. Its predicted structure showed two globular domains joined by a flexible linker. Recombinant FhCaBP2 interacted reversibly with calcium and manganese ions, but not with magnesium, barium, strontium, copper (II), colbalt (II), iron (II), nickel, lead or potassium ions. Cadmium (II) ions appeared to bind non-site-specifically and destabilize the protein. Interaction with either calcium or magnesium ions results in a conformational change in which the protein's surface becomes more hydrophobic. The EF-hand domain alone was able to interact with calcium and manganese ions; the DLC-like domain was not. Alteration of a residue (Asp-58 to Ala) in the second EF-hand motif in this domain abolished ion-binding activity. This suggests that the second EF-hand is the one responsible for ion-binding. FhCaBP2 homodimerizes and the extent of dimerization was not affected by calcium ions or by the aspartate to alanine substitution in the second EF-hand. The isolated EF-hand and DLC-like domains are both capable of homodimerization. FhCaBP2 interacted with the calmodulin antagonists trifluoperazine, chlorpromazine, thiamylal and W7. Interestingly, while chlorpromazine and thiamylal interacted with the EF-hand domain (as expected), trifluoperazine and W7 bound to the DLC-like domain. Overall, FhCaBP2 has distinct biochemical properties compared with other members of this protein family from Fasciola hepatica, a fact which supports the hypothesis that these proteins have different physiological roles.

  3. Subunit organization in cytoplasmic dynein subcomplexes

    Science.gov (United States)

    King, Stephen J.; Bonilla, Myriam; Rodgers, Michael E.; Schroer, Trina A.

    2002-01-01

    Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo. PMID:11967380

  4. WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia.

    Science.gov (United States)

    Patel-King, Ramila S; Gilberti, Renée M; Hom, Erik F Y; King, Stephen M

    2013-09-01

    Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle-like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

  5. WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia

    Science.gov (United States)

    Patel-King, Ramila S.; Gilberti, Renée M.; Hom, Erik F. Y.; King, Stephen M.

    2013-01-01

    Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle–like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly. PMID:23864713

  6. Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

    Science.gov (United States)

    Martinez, J M; Elfarissi, H; De Velasco, B; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

    2000-01-01

    Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

  7. Dyneins across eukaryotes: a comparative genomic analysis.

    Science.gov (United States)

    Wickstead, Bill; Gull, Keith

    2007-12-01

    Dyneins are large minus-end-directed microtubule motors. Each dynein contains at least one dynein heavy chain (DHC) and a variable number of intermediate chains (IC), light intermediate chains (LIC) and light chains (LC). Here, we used genome sequence data from 24 diverse eukaryotes to assess the distribution of DHCs, ICs, LICs and LCs across Eukaryota. Phylogenetic inference identified nine DHC families (two cytoplasmic and seven axonemal) and six IC families (one cytoplasmic). We confirm that dyneins have been lost from higher plants and show that this is most likely because of a single loss of cytoplasmic dynein 1 from the ancestor of Rhodophyta and Viridiplantae, followed by lineage-specific losses of other families. Independent losses in Entamoeba mean that at least three extant eukaryotic lineages are entirely devoid of dyneins. Cytoplasmic dynein 2 is associated with intraflagellar transport (IFT), but in two chromalveolate organisms, we find an IFT footprint without the retrograde motor. The distribution of one family of outer-arm dyneins accounts for 2-headed or 3-headed outer-arm ultrastructures observed in different organisms. One diatom species builds motile axonemes without any inner-arm dyneins (IAD), and the unexpected conservation of IAD I1 in non-flagellate algae and LC8 (DYNLL1/2) in all lineages reveals a surprising fluidity to dynein function.

  8. Drosophila Dynein intermediate chain gene, Dic61B, is required for spermatogenesis.

    Directory of Open Access Journals (Sweden)

    Roshan Fatima

    Full Text Available This study reports the identification and characterization of a novel gene, Dic61B, required for male fertility in Drosophila. Complementation mapping of a novel male sterile mutation, ms21, isolated in our lab revealed it to be allelic to CG7051 at 61B1 cytogenetic region, since two piggyBac insertion alleles, CG7051(c05439 and CG7051(f07138 failed to complement. CG7051 putatively encodes a Dynein intermediate chain. All three mutants, ms21, CG7051(c05439 and CG7051(f07138, exhibited absolute recessive male sterility with abnormally coiled sperm axonemes causing faulty sperm individualization as revealed by Phalloidin staining in Don Juan-GFP background. Sequencing of PCR amplicons uncovered two point mutations in ms21 allele and confirmed the piggyBac insertions in CG7051(c05439 and CG7051(f07138 alleles to be in 5'UTR and 4(th exon of CG7051 respectively, excision of which reverted the male sterility. In situ hybridization to polytene chromosomes demonstrated CG7051 to be a single copy gene. RT-PCR of testis RNA revealed defective splicing of the CG7051 transcripts in mutants. Interestingly, expression of cytoplasmic dynein intermediate chain, α, β, γ tubulins and α-spectrin was normal in mutants while ultra structural studies revealed defects in the assembly of sperm axonemes. Bioinformatics further highlighted the homology of CG7051 to axonemal dynein intermediate chain of various organisms, including DNAI1 of humans, mutations in which lead to male sterility due to immotile sperms. Based on these observations we conclude that CG7051 encodes a novel axonemal dynein intermediate chain essential for male fertility in Drosophila and rename it as Dic61B. This is the first axonemal Dic gene of Drosophila to be characterized at molecular level and shown to be required for spermatogenesis.

  9. Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport.

    Science.gov (United States)

    Niclas, J; Allan, V J; Vale, R D

    1996-05-01

    Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.

  10. Serum Free Light Chains

    Science.gov (United States)

    ... or of one of its component parts – a kappa or lambda light chain, or rarely, a heavy chain. Traditionally, plasma cell ... protein (M-protein) production and to calculate a kappa/lambda free light chain ratio. If the protein electrophoresis test is abnormal, ...

  11. Epidermal Growth Factor Stimulates Extracellular-Signal Regulated Kinase Phosphorylation of a Novel Site on Cytoplasmic Dynein Intermediate Chain 2

    Directory of Open Access Journals (Sweden)

    Andrew D. Catling

    2013-02-01

    Full Text Available Extracellular-signal regulated kinase (ERK signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2 as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts. IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin.

  12. Protein-Protein Interactions between Intermediate Chains and the Docking Complex of Chlamydomonas Flagellar Outer Arm Dynein

    Science.gov (United States)

    Ide, Takahiro; Owa, Mikito; King, Stephen M.; Kamiya, Ritsu; Wakabayashi, Ken-ichi

    2013-01-01

    Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association. PMID:23747306

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Melanie Brunsch

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

  15. Light chain nephropathy

    Directory of Open Access Journals (Sweden)

    Sihem Darouich

    2015-01-01

    Full Text Available 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.

  16. Neurofilament light chain

    OpenAIRE

    Lu, CH; Macdonald-Wallis, C.; Gray, E; Pearce, N; Petzold, A; Norgren, N.; Giovannoni, G; Fratta, P.; Sidle, K.; Fish, M.; Orrell, R; Howard, R; Talbot, K.; Greensmith, L.; Kuhle, J

    2015-01-01

    OBJECTIVE: To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). METHODS: Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals...

  17. Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome).

    Science.gov (United States)

    Guichard, C; Harricane, M C; Lafitte, J J; Godard, P; Zaegel, M; Tack, V; Lalau, G; Bouvagnet, P

    2001-04-01

    Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

  18. Neurofilament light chain

    Science.gov (United States)

    Lu, Ching-Hua; Macdonald-Wallis, Corrie; Gray, Elizabeth; Pearce, Neil; Petzold, Axel; Norgren, Niklas; Giovannoni, Gavin; Fratta, Pietro; Sidle, Katie; Fish, Mark; Orrell, Richard; Howard, Robin; Talbot, Kevin; Greensmith, Linda; Kuhle, Jens

    2015-01-01

    Objective: To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). Methods: Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals for up to 3 years. Change in ALS Functional Rating Scale–Revised score was used to assess disease progression. Survival was evaluated using Cox regression and Kaplan–Meier analysis. Results: CSF, serum, and plasma NfL discriminated patients with ALS from healthy controls with high sensitivity (97%, 89%, 90%, respectively) and specificity (95%, 75%, 71%, respectively). CSF NfL was highly correlated with serum levels (r = 0.78, p NfL levels were approximately 4 times as high in patients with ALS compared with controls in both cohorts, and maintained a relatively constant expression during follow-up. Blood NfL levels at recruitment were strong, independent predictors of survival. The highest tertile of blood NfL at baseline had a mortality hazard ratio of 3.91 (95% confidence interval 1.98–7.94, p NfL level is an easily accessible biomarker with prognostic value in ALS. The individually relatively stable levels longitudinally offer potential for NfL as a pharmacodynamic biomarker in future therapeutic trials. Classification of evidence: This report provides Class III evidence that the NfL electrochemiluminescence immunoassay accurately distinguishes patients with sporadic ALS from healthy controls. PMID:25934855

  19. Functional Architecture of the Outer Arm Dynein Conformational Switch*

    Science.gov (United States)

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

    2012-01-01

    Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn → Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia. PMID:22157010

  20. N-Acetyl-D-Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division.

    Science.gov (United States)

    Sharif, Syeda Ridita; Islam, Ariful; Moon, Il Soo

    2016-09-01

    N-acetyl-D-glucosamine kinase (GlcNAc kinase or NAGK) primarily catalyzes phosphoryl transfer to GlcNAc during amino sugar metabolism. Recently, it was shown NAGK interacts with dynein light chain roadblock type 1 (DYNLRB1) and upregulates axo-dendritic growth, which is an enzyme activity-independent, non-canonical structural role. The authors examined the distributions of NAGK and NAGK-dynein complexes during the cell cycle in HEK293T cells. NAGK was expressed throughout different stages of cell division and immunocytochemistry (ICC) showed NAGK was localized at nuclear envelope, spindle microtubules (MTs), and kinetochores (KTs). A proximity ligation assay (PLA) for NAGK and DYNLRB1 revealed NAGK-dynein complex on nuclear envelopes in prophase cells and on chromosomes in metaphase cells. NAGK-DYNLRB1 PLA followed by Lis1/NudE1 immunostaining showed NAGK-dynein complexes were colocalized with Lis1 and NudE1 signals, and PLA for NAGK-Lis1 showed similar signal patterns, suggesting a functional link between NAGK and dynein-Lis1 complex. Subsequently, NAGK-dynein complexes were found in KTs and on nuclear membranes where KTs were marked with CENP-B ICC and nuclear membrane with lamin ICC. Furthermore, knockdown of NAGK by small hairpin (sh) RNA was found to delay cell division. These results indicate that the NAGK-dynein interaction with the involvements of Lis1 and NudE1 plays an important role in prophase nuclear envelope breakdown (NEB) and metaphase MT-KT attachment during eukaryotic cell division.

  1. Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function

    Science.gov (United States)

    Tanner, Christopher A.; Rompolas, Panteleimon; Patel-King, Ramila S.; Gorbatyuk, Oksana; Wakabayashi, Ken-ichi; Pazour, Gregory J.

    2008-01-01

    The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located ∼2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3′ end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors. PMID:18579685

  2. The ciliary inner dynein arm, I1 dynein, is assembled in the cytoplasm and transported by IFT before axonemal docking.

    Science.gov (United States)

    Viswanadha, Rasagnya; Hunter, Emily L; Yamamoto, Ryosuke; Wirschell, Maureen; Alford, Lea M; Dutcher, Susan K; Sale, Winfield S

    2014-10-01

    To determine mechanisms of assembly of ciliary dyneins, we focused on the Chlamydomonas inner dynein arm, I1 dynein, also known as dynein f. I1 dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein preassembly in the cytoplasm. Unlike I1 dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1-deficient axonemes in vitro. To test the hypothesis that I1 dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild-type and I1 dynein mutant cells. Rescue of I1 dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1 revealed that transport of I1 dynein is dependent on kinesin-2 activity. Thus, I1 dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme.

  3. Light chain immunofluorescence in various nephropathies

    Directory of Open Access Journals (Sweden)

    Megha S Uppin

    2011-01-01

    Full Text Available Context: Light chain immunofluoresence (IF in renal biopsy is routinely used in the diagnosis of light chain deposition disease (LCDD, amyloidosis and cast nephropathy. Light chain predominance has also been reported in certain glomerulopathies like IgA nephropathy. However, pathogenesis of this pattern of deposition in various glomerulopathies is uncertain. Aim: To discuss the pathogenesis and utility of light chain IF in nephropathies. Setting and Design: Retrospective study. Materials and Methods: The pattern of light chain IF and light microscopic diagnosis in 306 cases of various nephropathies was reviewed. Direct IF was done in all these cases with commercial fluorescence (Fluoresciene Isothiocynate conjugated polyclonal rabbit anti-human antisera against IgM, IgG, IgA, C3, C1q, kappa and lambda light chains. Results: Light chain deposits were seen in 240 (78.43% cases. In IgA nephropathy, lupus nephritis and post-infectious glomerulonephritis (PIGN, lambda positivity was more as compared to kappa. Light chain deposits in LCDD and membranous nephropathy were more kappa type. The IF pattern in amyloidosis was not consistent. Conclusion: The pathogenesis of light chain predominance in glomerulopathies is not clear and it depends on isoelectric point and size of the immune complex. Light chain IF should be performed routinely in all the renal biopsies.

  4. Lymphoplasmacytic lymphoma causing light chain cast nephropathy.

    Science.gov (United States)

    Pérez, Nuria S; Garcia-Herrera, Adriana; Rosiñol, Laura; Palos, Lily; Santiago, Evelyn; Espinosa, Gerard; Solé, Manel; Campistol, Josep M; Quintana, Luis F

    2012-01-01

    Plasma cell dyscrasias are frequently associated with kidney disease through the production of monoclonal immunoglobulin but with a diverse set of pathologic renal patterns. While almost all patients with a renal biopsy showing a cast nephropathy have myeloma, kidney involvement associated with pathological immunoglobulin light chains and lymphoma is rare. To our knowledge, this is the first report of a cast nephropathy associated with lymphoplasmacytic lymphoma. We emphasize the relation between light chain deposition and renal dysfunction in this disease with production of light chains. A therapeutic approach that decreases light chain production appears to be warranted in these patients.

  5. Regulation of Cytoplasmic Dynein ATPase by Lis1

    Science.gov (United States)

    Mesngon, Mariano T.; Tarricone, Cataldo; Hebbar, Sachin; Guillotte, Aimee M.; Schmitt, E. William; Lanier, Lorene; Musacchio, Andrea; King, Stephen J.; Smith, Deanna S.

    2015-01-01

    Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dynein in vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulation in vitro is not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport. PMID:16481446

  6. Cytoplasmic dynein nomenclature

    Science.gov (United States)

    Pfister, K. Kevin; Fisher, Elizabeth M.C.; Gibbons, Ian R.; Hays, Thomas S.; Holzbaur, Erika L.F.; McIntosh, J. Richard; Porter, Mary E.; Schroer, Trina A.; Vaughan, Kevin T.; Witman, George B.; King, Stephen M.; Vallee, Richard B.

    2005-01-01

    A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms. PMID:16260502

  7. Localized Lymph Node Light Chain Amyloidosis

    Directory of Open Access Journals (Sweden)

    Binod Dhakal

    2015-01-01

    Full Text Available Immunoglobulin-derived light chain amyloidosis can occasionally be associated with localized disease. We present a patient with localized lymph node light chain amyloidosis without an underlying monoclonal protein or lymphoproliferative disorder and review the literature of lymph node amyloidosis discussing work-up and risk factors for systemic progression.

  8. Cyclin B3 and dynein heavy chain cooperate to increase fitness in the absence of mdf-1/MAD1 in Caenorhabditis elegans.

    Science.gov (United States)

    Tarailo-Graovac, Maja; Wong, Tammy; Qin, Zhaozhao; Flibotte, Stephane; Taylor, Jon; Moerman, Donald G; Rose, Ann M; Chen, Nansheng

    2014-01-01

    Spindle assembly checkpoint (SAC) ensures genome stability by delaying anaphase onset until all the chromosomes have achieved proper spindle attachment. Once correct attachment has been achieved, SAC must be silenced. In the absence of mdf-1/MAD1, an essential SAC component, Caenorhabditis elegans cannot propagate beyond 3 generations. Previously, in a dog-1(gk10)/FANCJ mutator background, we isolated a suppressor of mdf-1(gk2) sterility (such-4) which allowed indefinite propagation in the absence of MDF-1. We showed that such-4 is a Cyclin B3 (cyb-3) duplication. Here we analyze mdf-1 such-4; dog-1, which we propagated for 470 generations, with freezing of samples for long time storage at F170 and F270. Phenotypic analysis of this strain revealed additional suppression of sterility in the absence of MDF-1, beyond the effects of such-4. We applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) and whole genome sequencing (WGS) and identified a further amplification of cyb-3 (triplication) and a new missense mutation in dynein heavy chain (dhc-1). We show that dhc-1(dot168) suppresses the mdf-1(gk2), and is the second cloned suppressor, next to cyb-3 duplication, that does not cause a delay in anaphase onset. We also show that amplification of cyb-3 and dhc-1(dot168) cooperate to increase fitness in the absence of MDF-1.

  9. Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

    Science.gov (United States)

    King, Stephen J.; Dutcher, Susan K.

    1997-01-01

    To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. PMID:9008712

  10. Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo

    Science.gov (United States)

    King, Stephen J.; Brown, Christa L.; Maier, Kerstin C.; Quintyne, Nicholas J.; Schroer, Trina A.

    2003-01-01

    Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms. PMID:14565986

  11. Polyclonal light chains in cerebrovascular disease

    Directory of Open Access Journals (Sweden)

    Patrizia Fiori

    2010-08-01

    Full Text Available Patrizia Fiori1, Maria Giannetti Luigi2, Linda Iurato1, Carminantonio Tammaro3, Gigliola Esposito3, Antonio Monaco11Central Operative Unit of Neurology (Dir. A Monaco, 2Infantile Neuropsychiatry and Social Service (Dir. LM Giannetti, 3Laboratory (Dir. CA Tammaro, ASL AV, Civil Hospital of Ariano Irpino, University of Naples, ItalyAbstract: Altered membrane permeability is a hallmark of inflammation and ischemia with systemic spreading. Renal dysfunction is a risk factor for cardiovascular, cerebrovascular, and metabolic diseases. The aim of the present study was to assess proteinuria and urinary polyclonal light chains in acute stroke and chronic cerebrovascular disease compared with other neurologic diseases. Our results showed significantly increased levels of urinary polyclonal light chains in cerebrovascular disease compared with other neurologic diseases. The highest values of urinary polyclonal κ chains were found in acute stroke compared with chronic cerebrovascular disease and other neurologic diseases, while the level of λ chains was mainly increased in chronic cerebrovascular diseases. The shift to chronic renal failure seems to be signaled by a decreased polyclonal light chain/creatinemia ratio. The absence of a significant correlation with blood pressure and other seric parameters suggests that polyclonal light chains are an early marker of reversible vascular impairment with renal dysfunction before progression to irreversible renal failure and need for dialysis and/or intensive care.Keywords: polyclonal light chains, cerebrovascular disease, renal failure

  12. Mutant glycyl-tRNA synthetase (Gars ameliorates SOD1(G93A motor neuron degeneration phenotype but has little affect on Loa dynein heavy chain mutant mice.

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    Gareth T Banks

    Full Text Available BACKGROUND: In humans, mutations in the enzyme glycyl-tRNA synthetase (GARS cause motor and sensory axon loss in the peripheral nervous system, and clinical phenotypes ranging from Charcot-Marie-Tooth neuropathy to a severe infantile form of spinal muscular atrophy. GARS is ubiquitously expressed and may have functions in addition to its canonical role in protein synthesis through catalyzing the addition of glycine to cognate tRNAs. METHODOLOGY/PRINCIPAL FINDINGS: We have recently described a new mouse model with a point mutation in the Gars gene resulting in a cysteine to arginine change at residue 201. Heterozygous Gars(C201R/+ mice have locomotor and sensory deficits. In an investigation of genetic mutations that lead to death of motor and sensory neurons, we have crossed the Gars(C201R/+ mice to two other mutants: the TgSOD1(G93A model of human amyotrophic lateral sclerosis and the Legs at odd angles mouse (Dync1h1(Loa which has a defect in the heavy chain of the dynein complex. We found the Dync1h1(Loa/+;Gars(C201R/+ double heterozygous mice are more impaired than either parent, and this is may be an additive effect of both mutations. Surprisingly, the Gars(C201R mutation significantly delayed disease onset in the SOD1(G93A;Gars(C201R/+ double heterozygous mutant mice and increased lifespan by 29% on the genetic background investigated. CONCLUSIONS/SIGNIFICANCE: These findings raise intriguing possibilities for the study of pathogenetic mechanisms in all three mouse mutant strains.

  13. Cytoplasmic Dynein Promotes HIV-1 Uncoating

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    Paulina Pawlica

    2014-11-01

    Full Text Available Retroviral capsid (CA cores undergo uncoating during their retrograde transport (toward the nucleus, and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.

  14. Structural repertoire of immunoglobulin λ light chains

    KAUST Repository

    Chailyan, Anna

    2011-03-01

    The immunoglobulin λ isotype is present in nearly all vertebrates and plays an important role in the human immune system. Despite its importance, few systematic studies have been performed to analyze the structural conformation of its variable regions, contrary to what is the case for κ and heavy chains. We show here that an analysis of the structures of λ chains allows the definition of a discrete set of recurring conformations (canonical structures) of their hypervariable loops and, most importantly, the identification of sequence constraints that can be used to predict their structure. We also show that the structural repertoire of λ chains is different and more varied than that of the κ chains, consistently with the current view of the involvement of the two major light-chain families in complementary strategies of the immune system to ensure a fine tuning between diversity and stability in antigen recognition. © 2011 Wiley-Liss, Inc.

  15. Quantitative Analysis of Pac1/LIS1-mediated Dynein Targeting: Implications for Regulation of Dynein Activity in Budding Yeast

    OpenAIRE

    Markus, Steven M.; Plevock, Karen M.; St. Germain, Bryan J.; Punch, Jesse J.; Meaden, Christopher W.; Lee, Wei-Lih

    2011-01-01

    LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either. Dyn1, Pac1 and Bik1 interact in vivo at the plus ends, where an excess amount of Bik...

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

    Science.gov (United States)

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

    2012-01-01

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

  17. Systemic Light Chain Amyloidosis Mimicking Rheumatic Disorders

    Science.gov (United States)

    2016-01-01

    Secondary amyloidosis can complicate chronic inflammatory autoimmune diseases. However, the clinical findings of primary amyloidosis may mimic those of primary rheumatologic disorders. We present the case of a 53-year-old woman who presented with dystrophic nail changes, dry eyes, bilateral carpal tunnel syndrome, Raynaud's phenomenon, and high titer positive nucleolar pattern antinuclear antibody. She was initially misdiagnosed as having Undifferentiated Connective Tissue Disease (UCTD). On further workup, she was eventually diagnosed with lambda light chain systemic amyloidosis by abdominal fat pad biopsy. Her symptoms completely resolved after autologous stem cell transplantation. With this case, we would like to highlight the similarities in the clinical features between light chain amyloidosis and rheumatological disorders. We would also like to emphasize the importance of the prompt recognition of the clinical features of amyloidosis which are crucial to triggering appropriate diagnostic procedures, since early diagnosis is a key to improving outcomes in this disease with an otherwise poor prognosis. PMID:28042297

  18. Systemic Light Chain Amyloidosis Mimicking Rheumatic Disorders

    Directory of Open Access Journals (Sweden)

    Rohit R. Rao

    2016-01-01

    Full Text Available Secondary amyloidosis can complicate chronic inflammatory autoimmune diseases. However, the clinical findings of primary amyloidosis may mimic those of primary rheumatologic disorders. We present the case of a 53-year-old woman who presented with dystrophic nail changes, dry eyes, bilateral carpal tunnel syndrome, Raynaud’s phenomenon, and high titer positive nucleolar pattern antinuclear antibody. She was initially misdiagnosed as having Undifferentiated Connective Tissue Disease (UCTD. On further workup, she was eventually diagnosed with lambda light chain systemic amyloidosis by abdominal fat pad biopsy. Her symptoms completely resolved after autologous stem cell transplantation. With this case, we would like to highlight the similarities in the clinical features between light chain amyloidosis and rheumatological disorders. We would also like to emphasize the importance of the prompt recognition of the clinical features of amyloidosis which are crucial to triggering appropriate diagnostic procedures, since early diagnosis is a key to improving outcomes in this disease with an otherwise poor prognosis.

  19. Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

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    Takuya Kobayashi

    Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

  20. Myeloma light chains are ligands for cubilin (gp280).

    Science.gov (United States)

    Batuman, V; Verroust, P J; Navar, G L; Kaysen, J H; Goda, F O; Campbell, W C; Simon, E; Pontillon, F; Lyles, M; Bruno, J; Hammond, T G

    1998-08-01

    Although myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, the molecular identity of the receptor has not been characterized. We examined the interaction between cubilin (gp280) and four species of light chains isolated from the urine of patients with multiple myeloma. Four lines of evidence identify cubilin, a giant glycoprotein receptor, which is restricted in distribution to endocytic scavenger pathways and which has potent effects on endosomal trafficking, as a potentially physiologically relevant binding site for light chains: 1) light chains coeluted during immunoaffinity purification of cubilin; 2) polyclonal antisera to cubilin but not control sera, displaced human light chain binding from rat renal brush-border membranes; 3) cubilin bound to multiple species of light chains during surface plasmon resonance; 4) anti-cubilin antiserum interfered with light chain endocytosis by visceral yolk sac epithelial cells. However, both binding of light chains to brush-border membranes and endocytosis of light chains by yolk sac epithelial cells were only partially inhibited by anticubilin antibodies, suggesting presence of additional or alternate binding sites for light chains. Excess light chain had a potent inhibitory effect on endosomal fusion in vitro. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. These data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.

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

    Science.gov (United States)

    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

  2. Nucleoporin translocated promoter region (Tpr) associates with dynein complex, preventing chromosome lagging formation during mitosis.

    Science.gov (United States)

    Nakano, Hiroshi; Funasaka, Tatsuyoshi; Hashizume, Chieko; Wong, Richard W

    2010-04-01

    Gain or loss of whole chromosomes is often observed in cancer cells and is thought to be due to aberrant chromosome segregation during mitosis. Proper chromosome segregation depends on a faithful interaction between spindle microtubules and kinetochores. Several components of the nuclear pore complex/nucleoporins play critical roles in orchestrating the rapid remodeling events that occur during mitosis. Our recent studies revealed that the nucleoporin, Rae1, plays critical roles in maintaining spindle bipolarity. Here, we show association of another nucleoporin, termed Tpr (translocated promoter region), with the molecular motors dynein and dynactin, which both orchestrate with the spindle checkpoints Mad1 and Mad2 during cell division. Overexpression of Tpr enhanced multinucleated cell formation. RNA interference-mediated knockdown of Tpr caused a severe lagging chromosome phenotype and disrupted spindle checkpoint proteins expression and localization. Next, we performed a series of rescue and dominant negative experiments to confirm that Tpr orchestrates proper chromosome segregation through interaction with dynein light chain. Our data indicate that Tpr functions as a spatial and temporal regulator of spindle checkpoints, ensuring the efficient recruitment of checkpoint proteins to the molecular motor dynein to promote proper anaphase formation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: burghardt@mayo.edu [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)

    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

  4. Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Luthra, Priya; Jordan, David S.; Leung, Daisy W.; Amarasinghe, Gaya K.; Basler, Christopher F.; Lyles, D. S.

    2015-03-04

    Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

  5. 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.

    Science.gov (United States)

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

    2016-03-01

    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. (Clinical Trials Register.eu identifier: 2007-005204-40).

  6. Increased Serum Free Light Chains Precede the Presentation of Immunoglobulin Light Chain Amyloidosis

    Science.gov (United States)

    Weiss, Brendan M.; Hebreo, Joseph; Cordaro, Daniel V.; Roschewski, Mark J.; Baker, Thomas P.; Abbott, Kevin C.; Olson, Stephen W.

    2014-01-01

    Purpose Patients with immunoglobulin light chain amyloidosis (AL amyloidosis) generally present with advanced organ dysfunction and have a high risk of early death. We sought to characterize monoclonal immunoglobulin (M-Ig) light chains before clinical presentation of AL amyloidosis. Patients and Methods We obtained prediagnostic sera from 20 cases with AL amyloidosis and 20 healthy controls matched for age, sex, race, and age of serum sample from the Department of Defense Serum Repository. Serum protein electrophoresis with immunofixation and serum free light chain (FLC) analysis were performed on all samples. Results An M-Ig was detected in 100% of cases and 0% of controls (P diff) was higher in cases compared with controls at all time periods, less than 4 years (174.8 v 0.3 mg/L; P diff was greater than 23 mg/L in 85% of cases and 0% of controls (P diff level increased more than 10% per year in 84% of cases compared with 16% of controls (P < .001). Conclusion Increase of FLCs, including within the accepted normal range, precedes the development of AL amyloidosis for many years. PMID:25024082

  7. Complete nucleotide sequence of primitive vertebrate immunoglobulin light chain genes.

    Science.gov (United States)

    Shamblott, M J; Litman, G W

    1989-06-01

    Antibody to Heterodontus francisci (horned shark) immunoglobulin light chain was used to screen a spleen cDNA expression library, and recombinant clones encoding light chain genes were isolated. The complete sequences of the mature coding regions of two light chain genes in this phylogenetically distant vertebrate have been determined and are reported here. Comparisons of the sequences are consistent with the presence of mammalian-like framework and complementarity-determining regions. The predicted amino acid sequences of the genes are more related to mammalian lambda than to kappa light chains. The nucleotide sequences of the genes are most related to mammalian T-cell antigen receptor beta chain. Heterodontus light chain genes may reflect characteristics of the common ancestor of immunoglobulin and T-cell antigen receptors before its evolutionary diversification.

  8. Recurrence of light-chain deposition disease after renal transplantation

    DEFF Research Database (Denmark)

    Larsen, Thomas; Hammer, Anne; Jørgensen, Kaj Anker

    2008-01-01

    A 51-year-old male with a history of chronic renal disease received a renal allograft, in which disease recurred. Light-chain deposition disease was confirmed through biopsies of the native kidney and graft, and detection of free kappa light chains in serum. Udgivelsesdato: 2007-Sep-6...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-01-12

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

  10. Increased serum free light chains precede the presentation of immunoglobulin light chain amyloidosis.

    Science.gov (United States)

    Weiss, Brendan M; Hebreo, Joseph; Cordaro, Daniel V; Roschewski, Mark J; Baker, Thomas P; Abbott, Kevin C; Olson, Stephen W

    2014-09-01

    Patients with immunoglobulin light chain amyloidosis (AL amyloidosis) generally present with advanced organ dysfunction and have a high risk of early death. We sought to characterize monoclonal immunoglobulin (M-Ig) light chains before clinical presentation of AL amyloidosis. We obtained prediagnostic sera from 20 cases with AL amyloidosis and 20 healthy controls matched for age, sex, race, and age of serum sample from the Department of Defense Serum Repository. Serum protein electrophoresis with immunofixation and serum free light chain (FLC) analysis were performed on all samples. An M-Ig was detected in 100% of cases and 0% of controls (P < .001). The M-Ig was present in 100%, 80%, and 42% of cases at less than 4 years, 4 to 11 years, and more than 11 years before diagnosis, respectively. The median FLC differential (FLC-diff) was higher in cases compared with controls at all time periods, less than 4 years (174.8 v 0.3 mg/L; P < .001), 4 to 11 years (65.1 v 2.2 mg/L; P < .001), and more than 11 years (4.5 v 0.4 mg/L; P = .03) before diagnosis. The FLC-diff was greater than 23 mg/L in 85% of cases and 0% of controls (P < .001). The FLC-diff level increased more than 10% per year in 84% of cases compared with 16% of controls (P < .001). Increase of FLCs, including within the accepted normal range, precedes the development of AL amyloidosis for many years. © 2014 by American Society of Clinical Oncology.

  11. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister

    2006-01-01

    Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  12. Flow cytometric detection of immunoglobulin light chain in hematolymphoid immunophenotyping

    Institute of Scientific and Technical Information of China (English)

    Xu Dongsheng

    2011-01-01

    During B cell development and maturation, the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH)and light chain genes,rearrange to associate one of a number of variable, diverse, and joining gene segments. A single mature Bcell expresses an IgH chain and either a kappa or lambda light chain, which is known as allelic or isotypic exclusion. In normal or reactive conditions, lymphoid cells comprise the mixtures of lymphocytes with either kappa or lambda expression. The norreal proportion of kappa to lambda (κ/λ ratio) is within the range of 0. 5 - 3. 0 in peripheral blood or bone marrow and 1.2 2.7 in lymph nodes[1].The most useful feature for diagnosing mature B cell neoplasm is light chain restriction or monotypic staining with κ or λ light chain. Currently, it is a common assumption that demonstration of light chain restriction in a B lymphocyte population is generally considered proof of monoclonality and indicates malignancy although monotypic B cell populations have been infrequently demonstrated in patients with no definitive evidence ofB cell malignancy[2-4]. The most common flow cytometric analysis for determining B cell monotype is the percent κ and λimmunoglobulin light chains.Because of the importance of light chain restriction in the diagnosis of B cell neoplasm, anti immunoglobulin antibodies (e. g. anti-κ and anti-λ) are vital tools in the detection of monotypic B cell populations. Accurate determination of surface light chain expression depends on many factors, such as proper washing procedure, lysing solution, type of antibody used, specimen type or lymphoma type[5]. This article will discuss some common problems encountered in flow cytometric(FCM)deterruination of surface immunoglobulin light chain expression inhematolymphoid immunophenotyping.%@@ During B-cell development and maturation,the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH) and light-chain genes,rearrange to associate one of a number of

  13. Tetany and osteolysis in light chain myeloma with amyloidosis.

    Science.gov (United States)

    Bhakri, H. L.; Cundy, T. F.; Pettingale, K. W.

    1983-01-01

    A patient is described with light chain myeloma and amyloidosis in whom 2 unusual complications occurred, diffuse osteolytic lesions and tetany. These observations extend the previously recognized clinical spectrum of this disorder. Images Fig. 1 PMID:6413962

  14. Preliminary research on myosin light chain kinase in rabbit liver

    Institute of Scientific and Technical Information of China (English)

    Bin Ren; Hua-Qing Zhu; Zhao-Feng Luo; Qing Zhou; Yuan Wang; Yu-Zhen Wang

    2001-01-01

    AIM: To study preliminarily the properties of myosin light chain kinase (MLCK) in rabbit liver. METHODS: The expression of MLCK was detected by reverse transcription-polymerase chain reaction (RT-PCR);the MLCK was obtained from rabbit liver, and its activity was analyzed by γ-32P incorporation technique to detect the phosphorylation of myosin light chain. RESULTS: MLCK was expressed in rabbit liver, and the activity of the enzyme was similar to rabbit smooth muscle MLCK, and calmodulin-dependent. When the concentration was 0.65 mg-L-1, the activity was at the highest level. CONCLUSION: MLCK expressed in rabbit liver may catalyze the phosphorylation of myosin light chain, which may play important roles in the regulation of hepatic cell functions.

  15. Myosin light chain genes in the turkey (Meleagris gallopavo).

    Science.gov (United States)

    Chaves, L D; Ostroski, B J; Reed, K M

    2003-01-01

    Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.

  16. Role of kinesin-1 and cytoplasmic dynein in endoplasmic reticulum movement in VERO cells

    Science.gov (United States)

    Woźniak, Marcin J.; Bola, Becky; Brownhill, Kim; Yang, Yen-Ching; Levakova, Vesselina; Allan, Victoria J.

    2009-01-01

    Summary Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that ∼50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought. PMID:19454478

  17. Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate

    Energy Technology Data Exchange (ETDEWEB)

    Caly, Leon [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia); Kassouf, Vicki T. [Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145 (Australia); Moseley, Gregory W. [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia); Diefenbach, Russell J.; Cunningham, Anthony L. [Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145 (Australia); Jans, David A., E-mail: david.jans@monash.edu [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia)

    2016-02-12

    Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications. - Highlights: • HIV-1 Vpr utilizes the microtubule network to traffic towards the nucleus. • Mechanism relies on interaction between Vpr and dynein light chain protein DYNLT1. • Long-term non-progressor derived mutation (F72L) impairs this interaction. • Key residues in the vicinity of F72 contribute to interaction with DYNLT1.

  18. Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

    Directory of Open Access Journals (Sweden)

    Amanda E Siglin

    Full Text Available Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC and the dynactin p150(Glued; however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued. Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued. Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

  19. Light scattering of thin azobenzene side-chain polyester layers

    DEFF Research Database (Denmark)

    Kerekes, Á.; Lörincz, E.; Ramanujam, P.S.

    2002-01-01

    characteristics than the liquid crystalline polyester. The amorphous samples have negligible polarization part orthogonal to the incident beam. the liquid crystalline samples have relative high orthogonal polarization part in light scattering, The light scattering results can be used to give a lower limit...... for the domain size in thin liquid crystalline polyester layers being responsible for the dominant light scattering. The characteristic domain Sizes obtained from the Fourier transformation of polarization microscopic Pictures confirm these values.......Light scattering properties of liquid crystalline and amorphous azobenzene side-chain polyester layers used for optical data storage were examined by means of transmissive scatterometry. Comparative experiments show that the amorphous polyester has significantly lower light scattering...

  20. The immunoglobulin light chain locus of the turkey, Meleagris gallopavo.

    Science.gov (United States)

    Bao, Yonghua; Wu, Sun; Zang, Yunlong; Wang, Hui; Song, Xiangfeng; Xu, Chunyang; Xie, Bohong; Guo, Yongchen

    2012-06-15

    To date, most jawed vertebrate species encode more than one immunoglobulin light (IgL) chain isotypes. It has been shown that several bird species (chickens, white Pekin or domestic duck, and zebra finches) exclusively express lambda isotype. We analyze here the genomic organization of another bird species turkey IgL genes based on the recently released genome data. The turkey IgL locus located on chromosome 17 spans approximately 75.2kb and contains a single functional V(λ) gene, twenty V(λ) pseudogenes, and a single functional J(λ)-C(λ) block. These data suggest that the genomic organization of bird IgL chain genes seems to be conserved. Ten cDNA clones from turkey Igλ chain containing almost full-length V(λ), J(λ) and C(λ) segments were acquired. The comparison of V(λ) cDNA sequences to all the germline V(λ) segments suggests that turkey species may be generating IgL chain diversity by gene conversion and somatic hypermutation like the chicken. This study provides insights into the immunoglobulin light chain genes in another bird species.

  1. LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

    Science.gov (United States)

    Pazour, Gregory J.; Koutoulis, Anthony; Benashski, Sharon E.; Dickert, Bethany L.; Sheng, Hong; Patel-King, Ramila S.; King, Stephen M.; Witman, George B.

    1999-01-01

    Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility. PMID:10512883

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

    Science.gov (United States)

    Kraj, Maria

    2014-01-01

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

  3. Integrated Control of Axonemal Dynein AAA+ Motors

    Science.gov (United States)

    King, Stephen M.

    2012-01-01

    Axonemal dyneins are AAA+ enzymes that convert ATP hydrolysis to mechanical work. This leads to the sliding of doublet microtubules with respect to each other and ultimately the generation of ciliary/flagellar beating. However, in order for useful work to be generated, the action of individual dynein motors must be precisely controlled. In addition, cells modulate the motility of these organelles through a variety of second messenger systems and these signals too must be integrated by the dynein motors to yield an appropriate output. This review describes the current status of efforts to understand dynein control mechanisms and their connectivity focusing mainly on studies of the outer dynein arm from axonemes of the unicellular biflagellate green alga Chlamydomonas. PMID:22406539

  4. DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes.

    Science.gov (United States)

    Hoang, Ha Thi; Schlager, Max A; Carter, Andrew P; Bullock, Simon L

    2017-02-28

    Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein's core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

  5. Neurofilament light chain: a biomarker for genetic frontotemporal dementia

    OpenAIRE

    Meeter, Lieke H.; Dopper, Elise G.; Jiskoot, Lize C.; Sanchez-Valle, Raquel; Graff, Caroline; Benussi, Luisa; Ghidoni, Roberta; Pijnenburg, Yolande A; Borroni, Barbara; Galimberti, Daniela; Laforce, Robert Jr; Masellis, Mario; Vandenberghe, Rik; Le Ber, Isabelle; Otto, Markus

    2016-01-01

    Abstract Objective To evaluate cerebrospinal fluid (CSF) and serum neurofilament light chain (NfL) levels in genetic frontotemporal dementia (FTD) as a potential biomarker in the presymptomatic stage and during the conversion into the symptomatic stage. Additionally, to correlate NfL levels to clinical and neuroimaging parameters. Methods In this multicenter case?control study, we investigated CSF NfL in 174 subjects (48 controls, 40 presymptomatic carriers and 86 patients with microtubule?as...

  6. CSF neurofilament light chain reflects corticospinal tract degeneration in ALS

    OpenAIRE

    Menke, Ricarda A.L.; Gray, Elizabeth; Lu, Ching-Hua; Kuhle, Jens; Talbot, Kevin; Malaspina, Andrea; Turner, Martin R.

    2015-01-01

    Objective Diffusion tensor imaging (DTI) is sensitive to white matter tract pathology. A core signature involving the corticospinal tracts (CSTs) has been identified in amyotrophic lateral sclerosis (ALS). Raised neurofilament light chain protein (NfL) in cerebrospinal fluid (CSF) is thought to reflect axonal damage in a range of neurological disorders. The relationship between these two measures was explored. Methods CSF and serum NfL concentrations and DTI acquired at 3?Tesla on the same da...

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  8. Light chain (AL amyloidosis: update on diagnosis and management

    Directory of Open Access Journals (Sweden)

    Rosenzweig Michael

    2011-11-01

    Full Text Available Abstract Light chain (AL amyloidosis is a plasma cell dyscrasia characterized by the pathologic production of fibrillar proteins comprised of monoclonal light chains which deposit in tissues and cause organ dysfunction. The diagnosis can be challenging, requiring a biopsy and often specialized testing to confirm the subtype of systemic disease. The goal of treatment is eradication of the monoclonal plasma cell population and suppression of the pathologic light chains which can result in organ improvement and extend patient survival. Standard treatment approaches include high dose melphalan (HDM followed by autologous hematopoietic stem cell transplantation (SCT or oral melphalan with dexamethasone (MDex. The use of novel agents (thalidomide, lenalidomide and bortezomib alone and in combination with steroids and alkylating agents has shown efficacy and continues to be explored. A risk adapted approach to SCT followed by novel agents as consolidation reduces treatment related mortality with promising outcomes. Immunotherapeutic approaches targeting pathologic plasma cells and amyloid precursor proteins or fibrils are being developed. Referral of patients to specialized centers focusing on AL amyloidosis and conducting clinical trials is essential to improving patient outcomes.

  9. Role of myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability in vitro and in vivo.

    Science.gov (United States)

    Wu, Fan; Guo, Xiaohua; Xu, Jing; Wang, Weiju; Li, Bingling; Huang, Qiaobing; Su, Lei; Xu, Qiulin

    2016-03-01

    We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen-activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase-targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product-induced myosin light chain phosphorylation. Advanced glycation end product-induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product-induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability.

  10. Mutations in specific structural regions of immunoglobulin light chains are associated with free light chain levels in patients with AL amyloidosis.

    Directory of Open Access Journals (Sweden)

    Tanya L Poshusta

    Full Text Available BACKGROUND: The amyloidoses are protein misfolding diseases characterized by the deposition of amyloid that leads to cell death and tissue degeneration. In immunoglobulin light chain amyloidosis (AL, each patient has a unique monoclonal immunoglobulin light chain (LC that forms amyloid deposits. Somatic mutations in AL LCs make these proteins less thermodynamically stable than their non-amyloidogenic counterparts, leading to misfolding and ultimately the formation of amyloid fibrils. We hypothesize that location rather than number of non-conservative mutations determines the amyloidogenicity of light chains. METHODOLOGY/PRINCIPAL FINDINGS: We performed sequence alignments on the variable domain of 50 kappa and 91 lambda AL light chains and calculated the number of non-conservative mutations over total number of patients for each secondary structure element in order to identify regions that accumulate non-conservative mutations. Among patients with AL, the levels of circulating immunoglobulin free light chain varies greatly, but even patients with very low levels can have very advanced amyloid deposition. CONCLUSIONS: Our results show that in specific secondary structure elements, there are significant differences in the number of non-conservative mutations between normal and AL sequences. AL sequences from patients with different levels of secreted light chain have distinct differences in the location of non-conservative mutations, suggesting that for patients with very low levels of light chains and advanced amyloid deposition, the location of non-conservative mutations rather than the amount of free light chain in circulation may determine the amyloidogenic propensity of light chains.

  11. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... the reagents used to measure by immunochemical techniques both kappa and lambda types of light chain... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin (light chain specific... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  12. Lis1 regulates dynein by sterically blocking its mechanochemical cycle.

    Science.gov (United States)

    Toropova, Katerina; Zou, Sirui; Roberts, Anthony J; Redwine, William B; Goodman, Brian S; Reck-Peterson, Samara L; Leschziner, Andres E

    2014-11-07

    Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is known to keep dynein bound to microtubules; however, how this is accomplished mechanistically remains unknown. We have used three-dimensional electron microscopy, single-molecule imaging, biochemistry, and in vivo assays to help establish this mechanism. The three-dimensional structure of the dynein-Lis1 complex shows that binding of Lis1 to dynein's AAA+ ring sterically prevents dynein's main mechanical element, the 'linker', from completing its normal conformational cycle. Single-molecule experiments show that eliminating this block by shortening the linker to a point where it can physically bypass Lis1 renders single dynein motors insensitive to regulation by Lis1. Our data reveal that Lis1 keeps dynein in a persistent microtubule-bound state by directly blocking the progression of its mechanochemical cycle.

  13. Dynein mutations associated with hereditary motor neuropathies impair mitochondrial morphology and function with age.

    Science.gov (United States)

    Eschbach, Judith; Sinniger, Jérôme; Bouitbir, Jamal; Fergani, Anissa; Schlagowski, Anna-Isabel; Zoll, Joffrey; Geny, Bernard; René, Frédérique; Larmet, Yves; Marion, Vincent; Baloh, Robert H; Harms, Matthew B; Shy, Michael E; Messadeq, Nadia; Weydt, Patrick; Loeffler, Jean-Philippe; Ludolph, Albert C; Dupuis, Luc

    2013-10-01

    Mutations in the DYNC1H1 gene encoding for dynein heavy chain cause two closely related human motor neuropathies, dominant spinal muscular atrophy with lower extremity predominance (SMA-LED) and axonal Charcot-Marie-Tooth (CMT) disease, and lead to sensory neuropathy and striatal atrophy in mutant mice. Dynein is the molecular motor carrying mitochondria retrogradely on microtubules, yet the consequences of dynein mutations on mitochondrial physiology have not been explored. Here, we show that mouse fibroblasts bearing heterozygous or homozygous point mutation in Dync1h1, similar to human mutations, show profoundly abnormal mitochondrial morphology associated with the loss of mitofusin 1. Furthermore, heterozygous Dync1h1 mutant mice display progressive mitochondrial dysfunction in muscle and mitochondria progressively increase in size and invade sarcomeres. As a likely consequence of systemic mitochondrial dysfunction, Dync1h1 mutant mice develop hyperinsulinemia and hyperglycemia and progress to glucose intolerance with age. Similar defects in mitochondrial morphology and mitofusin levels are observed in fibroblasts from patients with SMA-LED. Last, we show that Dync1h1 mutant fibroblasts show impaired perinuclear clustering of mitochondria in response to mitochondrial uncoupling. Our results show that dynein function is required for the maintenance of mitochondrial morphology and function with aging and suggest that mitochondrial dysfunction contributes to dynein-dependent neurological diseases, such as SMA-LED.

  14. Schwann cell myelination requires Dynein function

    Directory of Open Access Journals (Sweden)

    Langworthy Melissa M

    2012-11-01

    Full Text Available Abstract Background Interaction of Schwann cells with axons triggers signal transduction that drives expression of Pou3f1 and Egr2 transcription factors, which in turn promote myelination. Signal transduction appears to be mediated, at least in part, by cyclic adenosine monophosphate (cAMP because elevation of cAMP levels can stimulate myelination in the absence of axon contact. The mechanisms by which the myelinating signal is conveyed remain unclear. Results By analyzing mutations that disrupt myelination in zebrafish, we learned that Dynein cytoplasmic 1 heavy chain 1 (Dync1h1, which functions as a motor for intracellular molecular trafficking, is required for peripheral myelination. In dync1h1 mutants, Schwann cell progenitors migrated to peripheral nerves but then failed to express Pou3f1 and Egr2 or make myelin membrane. Genetic mosaic experiments revealed that robust Myelin Basic Protein expression required Dync1h1 function within both Schwann cells and axons. Finally, treatment of dync1h1 mutants with a drug to elevate cAMP levels stimulated myelin gene expression. Conclusion Dync1h1 is required for retrograde transport in axons and mutations of Dync1h1 have been implicated in axon disease. Our data now provide evidence that Dync1h1 is also required for efficient myelination of peripheral axons by Schwann cells, perhaps by facilitating signal transduction necessary for myelination.

  15. Model for bidirectional movement of cytoplasmic dynein

    CERN Document Server

    Sumathy, S

    2014-01-01

    Cytoplasmic dynein exhibits a directional processive movement on microtubule filaments and is known to move in steps of varying length based on the number of ATP molecules bound to it and the load that it carries. It is experimentally observed that dynein takes occasional backward steps and the frequency of such backward steps increases as the load approaches the stall force. Using a stochastic process model, we investigate the bidirectional movement of single head of a dynein motor. The probability for backward step is implemented based on Crook's fluctuation theorem of non-equilibrium statistical mechanics. We find that the movement of dynein motor is characterized with negative velocity implying backward motion beyond stall force. We observe that the motor moves backward for super stall forces by hydrolyzing the ATP exactly the same way as it does while moving forward for sub stall forces.

  16. Characterization and localization of dynein and myosins V and VI in the ovaries of queen bees.

    Science.gov (United States)

    Patricio, Karina; Calábria, Luciana Karen; Peixoto, Pablo Marco; Espindola, Foued Salmen; Da Cruz-Landim, Carminda

    2010-10-01

    The presence of myosin and dynein in the ovaries of both Apis mellifera and Scaptotrigona postica was investigated in extracts and in histological sections. In the ovary extracts, motor proteins, myosins V, VI and dynein were detected by Western blot. In histological sections, they were detected by immunocytochemistry, using a mouse monoclonal antibody against the intermediary chain of dynein and a rabbit polyclonal antibody against the myosin V head domain. The myosin VI tail domain was recognized by a pig polyclonal antibody. The results show that these molecular motors are expressed in the ovaries of both bee species with few differences in location and intensity, in regions where movement of substances is expected during oogenesis. The fact that antibodies against vertebrate proteins recognize proteins of bee species indicates that the specific epitopes are evolutionarily well preserved.

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

    Science.gov (United States)

    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.

  18. Dynein at kinetochores: Making the connection.

    Science.gov (United States)

    McHugh, Toni; Welburn, Julie P I

    2017-04-03

    Dynein removes the checkpoint proteins from kinetochores once chromosomes are bioriented. In this issue, Gama et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201610108) and Mosalaganti et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201611060) reveal the molecular basis for how dynein and its adaptor protein Spindly are recruited to the ROD-Zw10-Zwilch complex in the fibrous corona of unattached kinetochores.

  19. Super-light pearl-chain arch vaults

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl; Halding, Philip Skov

    2014-01-01

    in the vault. This means that the structure can sustain 4 hours standard fire. That it has a very high level of sound insulation, efficient sound damping properties, optimized production, a low production of CO2 that has already given rise to several awards, and easy transport of plane elements instead of what......Arch vaults are known as optimal and impressive structures, but due to their curved shapes they are extremely costly to produce in countries, where the cost of labor is high. By means of super-light pearl-chain technology arch vaults can be constructed from equal plane prefabricated elements, which...... are assembled on the erection site by means of post-tensioning and lifted in place in buildings and bridges. The super-light SL-deck element is developed by the company Abeo Ltd and The Technical University of Denmark for plane floor structures in buildings. However, this deck element is designed in such a way...

  20. Immunoglobulin Free Light Chain Dimers in Human Diseases

    Directory of Open Access Journals (Sweden)

    Batia Kaplan

    2011-01-01

    Full Text Available Immunoglobulin free light chain (FLC kappa (κ and lambda (λ isotypes exist mainly in monomeric and dimeric forms. Under pathological conditions, the level of FLCs as well as the structure of monomeric and dimeric FLCs and their dimerization properties might be significantly altered. The abnormally high fractions of dimeric FLCs were demonstrated in the serum of patients with multiple myeloma (MM and primary systemic amyloidosis (AL, as well as in the serum of anephric patients. The presence of tetra- and trimolecular complexes formed due to dimer-dimer and dimer-monomer interactions was detected in the myeloma serum. Analysis of the amyloidogenic light chains demonstrated mutations within the dimer interface, thus raising the possibility that these mutations are responsible for amyloidogenicity. Increased κ monomer and dimer levels, as well as a high κ/λ monomer ratio, were typically found in the cerebrospinal fluid from patients with multiple sclerosis (MS. In many MS cases, the elevation of κ FLCs was accompanied by an abnormally high proportion of λ dimers. This review focuses on the disease-related changes of the structure and level of dimeric FLCs, and raises the questions regarding their formation, function, and role in the pathogenesis and diagnosis of human diseases.

  1. Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport

    Science.gov (United States)

    Chanduri, Manasa; Rai, Ashim; Malla, Aushaq Bashir; Wu, Mingxuan; Fiedler, Dorothea; Mallik, Roop; Bhandari, Rashna

    2016-01-01

    Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP7), are conserved eukaryotic signaling molecules that possess pyrophosphate and monophosphate moieties. Generated predominantly by inositol hexakisphosphate kinases (IP6Ks), inositol pyrophosphates can modulate protein function by posttranslational serine pyrophosphorylation. Here, we report inositol pyrophosphates as novel regulators of cytoplasmic dynein-driven vesicle transport. Mammalian cells lacking IP6K1 display defects in dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance. Expression of catalytically active but not inactive IP6K1 reverses these defects, suggesting a role for inositol pyrophosphates in these processes. Endosomes derived from slime mold lacking inositol pyrophosphates also display reduced dynein-directed microtubule transport. We demonstrate that Ser51 in the dynein intermediate chain (IC) is a target for pyrophosphorylation by IP7, and this modification promotes the interaction of the IC N-terminus with the p150Glued subunit of dynactin. IC–p150Glued interaction is decreased, and IC recruitment to membranes is reduced in cells lacking IP6K1. Our study provides the first evidence for the involvement of IP6Ks in dynein function and proposes that inositol pyrophosphate-mediated pyrophosphorylation may act as a regulatory signal to enhance dynein-driven transport. PMID:27474409

  2. Super-light concrete with pearl-chains

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl

    2009-01-01

    half that of similar structures in concrete and steel. They are heat-insulating, fire-resistant and they open up the possibility for large spans and the creation of advanced shapes. Fields of application include roofs, shells, beams, columns, walls, façades, offshore structures, tunnels and structures......-scale applications, such as façade elements, crash barriers and secondary structures. One special small-scale application of the principle is in frame building, where super-light frames with pearl-chain-reinforced components have some of the same advantages as timber-frame structures with regard to stability, heat...... insulation, economy and simple erection processes, but without the disadvantages of fire and rot....

  3. Intrathecal synthesis of free immunoglobulin light chains in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, M; Schaldemose Nielsen, H; Jensen, J

    1998-01-01

    OBJECTIVE: The detection of oligoclonal immunoglobulin free light chains (FLC) in the diagnosis of multiple sclerosis (MS) was compared to IgG isoelectric focusing. MATERIAL AND METHODS: Cerebrospinal fluid and serum samples from 69 patients with possible first attacks of MS, 50 patients...... with clinically definite MS (CDMS), and 118 patients with other neurological diseases (OND) were analyzed. IgG and FLC oligoclonal bands were detected by isoelectric focusing and immunoperoxidase staining. RESULTS: Intrathecal synthesis of IgG, kappa FLC, and lambda FLC oligoclonal bands, respectively, was seen...... in 92%, 92%, and 86% of MS patients; in 61%, 62%, and 64% of patients with possible first attacks of MS; and in 3%, 3%, and 8% of the patients with OND. In control patients without IgG synthesis intrathecal lambda FLC synthesis was more common than kappa FLC synthesis (P=0.03). CONCLUSION: Kappa FLC...

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

    Energy Technology Data Exchange (ETDEWEB)

    Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Wall, Jonathan S. [Departments of Radiology and Medicine, The University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN (United States); González Andrade, Martín [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Sánchez-López, Rosana [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa Cuernavaca, Morelos C.P. 62210 (Mexico); Rodríguez-Ambriz, Sandra L. [Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Calle CEPROBI No. 8, Col. San Isidro, Yautepec, Morelos C.P. 62731 (Mexico); Pérez Carreón, Julio I. [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); 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.

  5. A Unified Taxonomy for Ciliary Dyneins

    Science.gov (United States)

    Hom, Erik F.Y.; Witman, George B.; Harris, Elizabeth H.; Dutcher, Susan K.; Kamiya, Ritsu; Mitchell, David R.; Pazour, Gregory J.; Porter, Mary E.; Sale, Winfield S.; Wirschell, Maureen; Yagi, Toshiki; King, Stephen M.

    2011-01-01

    The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, fifty-four proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologues of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles and orthologues has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans. PMID:21953912

  6. Enhancing lean supply chain through traffic light quality management system

    Directory of Open Access Journals (Sweden)

    Md. Mazharul Islam

    2013-03-01

    Full Text Available Lean is a continuous journey to grow and excel the company. Any company want to develop and cope with the world pace must adopt lean. However, in most of the organizations the management culture or people’s mentality is not so good to embrace change. They have predestined mind set where no change is normally allowed. Lean is a cooperative way of working that involves all departments and all personnel to work together in a team for the betterment of the entire company. Without providing fixed solution of any problem it suggests the best way that people willingly accept to do. Lean normally deals with highest quality, shorter lead time and lowest cost. In Bangladesh, most of the garment manufacturing companies are experiencing a massive quality problem. We describe a case where traffic light, a tool of lean quality system was adopted to a garment manufacturing company in Bangladesh. We also provide the charts to contrast the before and after scenario in detail, in order to illustrate the company benefits. After the traffic light system being implemented, the quality status was improved, production capacity was increased; significant days were saved that enhanced the lead time and thus strengthen the supply chain.

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

    Science.gov (United States)

    De Ioannes, A E; Aguila, H L

    1989-01-01

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

  8. Verification of serum reference intervals for free light chains in a local South African population.

    Science.gov (United States)

    Zemlin, Annalise E; Rensburg, Megan A; Ipp, Hayley; Germishuys, Jurie J; Erasmus, Rajiv T

    2013-11-01

    Monoclonal serum free light chain measurements are used to follow up and manage patients with monoclonal gammopathies, and abnormal serum free light chain ratios are associated with risk of progression in certain diseases. We aimed to validate the reference intervals in our population. Reference intervals for κ and λ free light chains were established on 120 healthy adults. Creatinine levels were measured to exclude renal dysfunction and serum protein electrophoresis was performed. All creatinine values were within normal limits. After exclusion of subjects with abnormal serum protein electrophoreses, 113 subjects were available for analysis. The 95% reference interval was 6.3-20.6 mg/L for κ free light chains, 8.7-25.9 mg/L for λ free light chains and 0.46-1.23 for free light chain ratio. Most of the values fell within the manufacturer's recommended limits and therefore could be used for our population.

  9. Judging disease activity in rheumatoid arthritis by serum free kappa and lambda light chain levels.

    Science.gov (United States)

    Ye, Yun; Li, Su-Liang; Xie, Ming; Jiang, Ping; Liu, Kai-Ge; Li, Ya-Jun

    2013-10-01

    The study aimed to evaluate the levels of serum free kappa (κ) and lambda (λ) light chains in patients with rheumatoid arthritis (RA) as well as exploring the association between serum free κ and λ light chains and activity of RA. For this purpose, healthy individuals and patients with active RA and RA in remission were enrolled, and their serum levels of free κ and λ light chains were measured using rate nephelometry. The diagnostic accuracy of serum free κ and λ light chains was evaluated by receiver operating characteristic curves and 95% confidence intervals for areas under the curve (AUC). The results obtained indicated that the levels of serum free κ and λ light chains in patients with active RA were significantly higher than those of patients in remission and of healthy controls (p light chain and 0.781 for free λ light chain. When the optimal cut-off point for serum κ light chain was 8.02 g/L, the maximum sensitivity and specificity were 82.5% and 82.5%, respectively, and when the optimal cut-off point for serum λ light chain was 3.57 g/L, the maximum sensitivity and specificity were 80% and 82.5%, respectively. It was thus found that serum levels of free κ and λ light chains were positively correlated with disease activity in RA, the Disease Activity Score 28 (DAS28), and values for C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), platelet count (PLT), rheumatoid factor (RF), and anticitrullinated protein antibody (ACPA) (p light chains in patients with active RA are closely correlated with disease activity parameters including DAS28, CRP, ESR, PLT, RF, and ACPA. Thus, the above-mentioned levels of serum free κ and λ light chains may be used as important indicators of activity of RA.

  10. B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

    OpenAIRE

    J. Chen(Florida State University, Tallahassee, U.S.A.); Trounstine, M; Kurahara, C.; Young, F.; Kuo, C C; Y. Xu; Loring, J.F.; Alt, F W; Huszar, D

    1993-01-01

    We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, ...

  11. Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny.

    Science.gov (United States)

    Rast, J P; Anderson, M K; Ota, T; Litman, R T; Margittai, M; Shamblott, M J; Litman, G W

    1994-01-01

    The prototypic chondrichthyan immunoglobulin (Ig) light chain type (type I) isolated from Heterodontus francisci (horned shark) has a clustered organization in which variable (V), joining (J), and constant (C) elements are in relatively close linkage (V-J-C). Using a polymerase chain reaction-based approach on a light chain peptide sequence from the holocephalan, Hydrolagus colliei (spotted ratfish), it was possible to isolate members of a second light chain gene family. A probe to this light chain (type II) detects homologs in two orders of elasmobranchs, Heterodontus, a galeomorph and Raja erinacea (little skate), a batoid, suggesting that this light chain type may be present throughout the cartilaginous fishes. In all cases, V, J, and C regions of the type II gene are arranged in closely linked clusters typical of all known Ig genes in cartilaginous fishes. All representatives of this type II gene family are joined in the germline. A third (kappa-like) light chain type from Heterodontus is described. These findings establish that a degree of light chain class complexity comparable to that of the mammals is present in the most phylogenetically distant extant jawed vertebrates and that the phenomenon of germline-joined (pre-rearranged) genes, described originally in the heavy chain genes of cartilaginous fishes, extends to light chain genes.

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

    Science.gov (United States)

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

    2009-08-01

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

  13. Ciliobrevins as Tools for Studying Dynein Motor Function

    Directory of Open Access Journals (Sweden)

    Douglas eRoossien

    2015-07-01

    Full Text Available Dyneins are a small class of molecular motors that bind to microtubules and walk towards their minus ends. They are essential for the transport and distribution of organelles, signaling complexes and cytoskeletal elements. In addition dyneins generate forces on microtubule arrays that power the beating of cilia and flagella, cell division, migration and growth cone motility. Classical approaches to the study of dynein function in axons involve the depletion of dynein, expression of mutant/truncated forms of the motor, or interference with accessory subunits. By necessity, these approaches require prolonged time period for the expression or manipulation of cellular dynein levels. With the discovery of the ciliobrevins, a class of cell permeable small molecule inhibitors of dynein, it is now possible to acutely disrupt dynein both globally and locally. In this review, we briefly summarize recent work using ciliobrevins to inhibit dynein and discuss the insights ciliobrevins have provided about dynein function in various cell types with a focus on neurons. We temper this with a discussion of the need for studies that will elucidate the mechanism of action of ciliobrevin and as well as the need for experiments to further analyze the specificity of ciliobreviens for dynein. Although much remains to be learned about ciliobrevins, these small molecules are proving themselves to be valuable novel tools to assess the cellular functions of dynein.

  14. Neurofilament light chain: a biomarker for genetic frontotemporal dementia.

    Science.gov (United States)

    Meeter, Lieke H; Dopper, Elise G; Jiskoot, Lize C; Sanchez-Valle, Raquel; Graff, Caroline; Benussi, Luisa; Ghidoni, Roberta; Pijnenburg, Yolande A; Borroni, Barbara; Galimberti, Daniela; Laforce, Robert Jr; Masellis, Mario; Vandenberghe, Rik; Ber, Isabelle Le; Otto, Markus; van Minkelen, Rick; Papma, Janne M; Rombouts, Serge A; Balasa, Mircea; Öijerstedt, Linn; Jelic, Vesna; Dick, Katrina M; Cash, David M; Harding, Sophie R; Jorge Cardoso, M; Ourselin, Sebastien; Rossor, Martin N; Padovani, Alessandro; Scarpini, Elio; Fenoglio, Chiara; Tartaglia, Maria C; Lamari, Foudil; Barro, Christian; Kuhle, Jens; Rohrer, Jonathan D; Teunissen, Charlotte E; van Swieten, John C

    2016-08-01

    To evaluate cerebrospinal fluid (CSF) and serum neurofilament light chain (NfL) levels in genetic frontotemporal dementia (FTD) as a potential biomarker in the presymptomatic stage and during the conversion into the symptomatic stage. Additionally, to correlate NfL levels to clinical and neuroimaging parameters. In this multicenter case-control study, we investigated CSF NfL in 174 subjects (48 controls, 40 presymptomatic carriers and 86 patients with microtubule-associated protein tau (MAPT), progranulin (GRN), and chromosome 9 open reading frame 72 (C9orf72) mutations), and serum NfL in 118 subjects (39 controls, 44 presymptomatic carriers, 35 patients). In 55 subjects both CSF and serum was determined. In two subjects CSF was available before and after symptom onset (converters). Additionally, NfL levels were correlated with clinical parameters, survival, and regional brain atrophy. CSF NfL levels in patients (median 6762 pg/mL, interquartile range 3186-9309 pg/mL) were strongly elevated compared with presymptomatic carriers (804 pg/mL, 627-1173 pg/mL, P NfL correlated highly with CSF NfL (r s = 0.87, P NfL after disease onset. Additionally, NfL levels in patients correlated with disease severity, brain atrophy, annualized brain atrophy rate and survival. NfL in both serum and CSF has the potential to serve as a biomarker for clinical disease onset and has a prognostic value in genetic FTD.

  15. CSF neurofilament light chain reflects corticospinal tract degeneration in ALS

    Science.gov (United States)

    Menke, Ricarda A L; Gray, Elizabeth; Lu, Ching-Hua; Kuhle, Jens; Talbot, Kevin; Malaspina, Andrea; Turner, Martin R

    2015-01-01

    Objective Diffusion tensor imaging (DTI) is sensitive to white matter tract pathology. A core signature involving the corticospinal tracts (CSTs) has been identified in amyotrophic lateral sclerosis (ALS). Raised neurofilament light chain protein (NfL) in cerebrospinal fluid (CSF) is thought to reflect axonal damage in a range of neurological disorders. The relationship between these two measures was explored. Methods CSF and serum NfL concentrations and DTI acquired at 3 Tesla on the same day were obtained from ALS patients (n = 25 CSF, 40 serum) and healthy, age-similar controls (n = 17 CSF, 25 serum). Within-group correlations between NfL and DTI measures of microstructural integrity in major white matter tracts (CSTs, superior longitudinal fasciculi [SLF], and corpus callosum) were performed using tract-based spatial statistics. Results NfL levels were higher in patients compared to controls. CSF levels correlated with clinical upper motor neuron burden and rate of disease progression. Higher NfL levels were significantly associated with lower DTI fractional anisotropy and increased radial diffusivity in the CSTs of ALS patients, but not in controls. Interpretation Elevated CSF and serum NfL is, in part, a result of CST degeneration in ALS. This highlights the wider potential for combining neurochemical and neuroimaging-based biomarkers in neurological disease. PMID:26273687

  16. Prognostic Value of Serum Free Light Chain in Multiple Myeloma.

    Science.gov (United States)

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

    2015-01-01

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

  17. Surface-mediated light transmission in metal nanoparticle chains

    NARCIS (Netherlands)

    Compaijen, P. Jasper; Malyshev, Victor A.; Knoester, Jasper

    2013-01-01

    We study theoretically the efficiency of the transmission of optical signals through a linear chain consisting of identical and equidistantly spaced silver metal nanoparticles. Two situations are compared: the transmission efficiency through an isolated chain and through a chain in close proximity o

  18. Identification of Tctex2 beta, a novel dynein light chain family member that interacts with different transforming growth factor-beta receptors

    NARCIS (Netherlands)

    Meng, QingJun; Lux, Andreas; Holloschi, Andreas; Li, Jian; Hughes, John M. X.; Foerg, Tassilo; McCarthy, John E. G.; Heagerty, Anthony M.; Kioschis, Petra; Hafner, Mathias; Garland, John M.

    2006-01-01

    Endoglin is a membrane-inserted protein that is preferentially synthesized in angiogenic vascular endothelial and smooth muscle cells. Endoglin associates with members of the transforming growth factor-beta(TGF-beta) receptor family and has been identified as the gene involved in hereditary hemorrha

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Aurélie Grados

    2013-01-01

    Full Text Available 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 ( and λ ( free light chains and the κ : λ ratio ( 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.

  1. Neurofilament light chain: A prognostic biomarker in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Lu, Ching-Hua; Macdonald-Wallis, Corrie; Gray, Elizabeth; Pearce, Neil; Petzold, Axel; Norgren, Niklas; Giovannoni, Gavin; Fratta, Pietro; Sidle, Katie; Fish, Mark; Orrell, Richard; Howard, Robin; Talbot, Kevin; Greensmith, Linda; Kuhle, Jens; Turner, Martin R; Malaspina, Andrea

    2015-06-02

    To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals for up to 3 years. Change in ALS Functional Rating Scale-Revised score was used to assess disease progression. Survival was evaluated using Cox regression and Kaplan-Meier analysis. CSF, serum, and plasma NfL discriminated patients with ALS from healthy controls with high sensitivity (97%, 89%, 90%, respectively) and specificity (95%, 75%, 71%, respectively). CSF NfL was highly correlated with serum levels (r = 0.78, p NfL levels were approximately 4 times as high in patients with ALS compared with controls in both cohorts, and maintained a relatively constant expression during follow-up. Blood NfL levels at recruitment were strong, independent predictors of survival. The highest tertile of blood NfL at baseline had a mortality hazard ratio of 3.91 (95% confidence interval 1.98-7.94, p NfL level is an easily accessible biomarker with prognostic value in ALS. The individually relatively stable levels longitudinally offer potential for NfL as a pharmacodynamic biomarker in future therapeutic trials. This report provides Class III evidence that the NfL electrochemiluminescence immunoassay accurately distinguishes patients with sporadic ALS from healthy controls. © 2015 American Academy of Neurology.

  2. Plasma neurofilament light chain predicts progression in progressive supranuclear palsy.

    Science.gov (United States)

    Rojas, Julio C; Karydas, Anna; Bang, Jee; Tsai, Richard M; Blennow, Kaj; Liman, Victor; Kramer, Joel H; Rosen, Howard; Miller, Bruce L; Zetterberg, Henrik; Boxer, Adam L

    2016-03-01

    Blood-based biomarkers for neurodegenerative conditions could improve diagnosis and treatment development. Neurofilament light chain (NfL), a marker of axonal injury, is elevated in cerebrospinal fluid (CSF) of patients with progressive supranuclear palsy (PSP). The goal of this study was to determine the diagnostic and prognostic value of plasma NfL in patients with PSP. Plasma NfL was measured with ultrasensitive digital immunoassay-based technology at baseline and 1-year follow-up in a pilot cohort of 15 PSP patients and 12 healthy controls, and a validation cohort of 147 PSP patients. Mixed linear models tested the ability of plasma NfL to predict neurological, cognitive and functional decline, and brain atrophy. Baseline mean plasma NfL levels were elevated in PSP patients (31 ± 4 pg/mL, vs. control, 17.5 ± 1 pg/mL, P NfL levels had more severe neurological (PSPRS, -36.9% vs. -28.9%, P = 0.04), functional (SEADL, -38.2% vs. -20%, P = 0.03), and neuropsychological (RBANS, -23.9% vs. -12.3%, P = 001) deterioration over 1 year. Higher baseline NfL predicted greater whole-brain and superior cerebellar peduncle volume loss. Plasma and CSF NfL were significantly correlated (r = 0.74, P = 0.002). Plasma NfL is elevated in PSP and could be of value as a biomarker both to assist clinical diagnosis and to monitor pharmacodynamic effects on the neurodegenerative process in clinical trials.

  3. Sensing the Mechanical State of the Axoneme and Integration of Ca2+ Signaling by Outer Arm Dynein

    Science.gov (United States)

    King, Stephen M.

    2010-01-01

    Axonemal dyneins have been demonstrated to monitor the mechanical state of the axoneme and must also alter activity in response to various signaling pathways. The central pair/radial spoke systems are clearly involved in controlling inner dynein arm function; however, the mechanisms by which the outer dynein arm transduces regulatory signals appear quite distinct at the molecular level. In Chlamydomonas, these regulatory components include thioredoxins involved in response to redox changes, molecules that tether the γ heavy chain motor unit to the A-tubule of the outer doublet and a Ca2+-binding protein that controls the structure of the γ heavy chain N-terminal domain. Together, these studies now suggest that the γ heavy chain acts as a key regulatory node for controlling outer arm function in response to alterations in curvature and ligand binding. Furthermore, they allow us to propose a testable molecular mechanism by which altered Ca2+ levels might lead to a change in ciliary waveform by controlling whether one heavy chain of outer arm dynein acts as a microtubule translocase or as an ATP-dependent brake that limits the amount of inter-doublet sliding. PMID:20186692

  4. Light-chain nephropathy in patient with renal carcinoma.

    OpenAIRE

    Enia, G.; Maringhini, S; L'Abbate, A; Zoccali, C.; Maggiore, Q

    1981-01-01

    Paraneoplastic syndromes are often associated with renal parenchymal tumours. This report describes a case of renal-cell carcinoma with kappa-chain nephropathy. The patient, a 60-year-old man, had renal tubular dysfunction, shown by low serum concentrations of urate and phosphate. Kappa-chains were found in both serum and urine, but no lambda-chains were found. Investigations showed a clear-cell carcinoma, and the patient underwent a radical nephrectomy. Two years after operation serum phosph...

  5. Exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus.

    Science.gov (United States)

    Suikkanen, Sanna; Aaltonen, Tuula; Nevalainen, Marjukka; Välilehto, Outi; Lindholm, Laura; Vuento, Matti; Vihinen-Ranta, Maija

    2003-10-01

    Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptor-mediated endocytosis, escapes from endosomal vesicles to the cytosol, and then replicates in the nucleus. We examined the role of the microtubule (MT)-mediated cytoplasmic trafficking of viral particles toward the nucleus. Immunofluorescence and immunoelectron microscopy showed that capsids were transported through the cytoplasm into the nucleus after cytoplasmic microinjection but that in the presence of MT-depolymerizing agents, viral capsids were unable to reach the nucleus. The nuclear accumulation of capsids was also reduced by microinjection of an anti-dynein antibody. Moreover, electron microscopy and light microscopy experiments demonstrated that viral capsids associate with tubulin and dynein in vitro. Coprecipitation studies indicated that viral capsids interact with dynein. When the cytoplasmic transport process was studied in living cells by microinjecting fluorescently labeled capsids into the cytoplasm of cells containing fluorescent tubulin, capsids were found in close contact with MTs. These results suggest that intact MTs and the motor protein dynein are required for the cytoplasmic transport of CPV capsids and contribute to the accumulation of the capsid in the nucleus.

  6. Partially Functional Outer-Arm Dynein in a Novel Chlamydomonas Mutant Expressing a Truncated γ Heavy Chain▿

    Science.gov (United States)

    Liu, Zhongmei; Takazaki, Hiroko; Nakazawa, Yuki; Sakato, Miho; Yagi, Toshiki; Yasunaga, Takuo; King, Stephen M.; Kamiya, Ritsu

    2008-01-01

    The outer dynein arm of Chlamydomonas flagella contains three heavy chains (α, β, and γ), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose γ heavy chain is truncated at about 30% of the sequence. While the previously isolated γ chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain α and β heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the γ heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the α heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the β heavy chain). Thus, the outer-arm dynein lacking the γ heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein. PMID:18487347

  7. Frequent lambda light chain gene rearrangement and expression in a Ly-1 B lymphoma with a productive kappa chain allele.

    OpenAIRE

    Hardy, R R; Dangl, J L; Hayakawa, K.; Jager, G.; Herzenberg, L.A.

    1986-01-01

    We describe here a murine Ly-1-bearing pre-B-cell tumor that, when induced for kappa light chain expression with bacterial lipopolysaccharide, also gives rise spontaneously to a few percent of cells expressing surface lambda light chains. These lambda-positive cells have undergone DNA rearrangements involving either V lambda 1 or V lambda 2 genes. Nearly all clones of lambda-bearing cells express mu and lambda on their surface (but not kappa). However, all these lambda-positive clones continu...

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

    Science.gov (United States)

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

    2016-05-27

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

  9. Increased neurofilament light chain blood levels in neurodegenerative neurological diseases.

    Directory of Open Access Journals (Sweden)

    Johanna Gaiottino

    Full Text Available Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf are cytoskeletal proteins of neurons and their release into cerebrospinal fluid has shown encouraging results as a biomarker for neurodegeneration. This study aimed to validate the quantification of the Nf light chain (NfL in blood samples, as a biofluid source easily accessible for longitudinal studies.We developed and applied a highly sensitive electrochemiluminescence (ECL based immunoassay for quantification of NfL in blood and CSF.Patients with Alzheimer's disease (AD (30.8 pg/ml, n=20, Guillain-Barré-syndrome (GBS (79.4 pg/ml, n=19 or amyotrophic lateral sclerosis (ALS (95.4 pg/ml, n=46 had higher serum NfL values than a control group of neurological patients without evidence of structural CNS damage (control patients, CP (4.4 pg/ml, n=68, p<0.0001 for each comparison, p=0.002 for AD patients and healthy controls (HC (3.3 pg/ml, n=67, p<0.0001. Similar differences were seen in corresponding CSF samples. CSF and serum levels correlated in AD (r=0.48, p=0.033, GBS (r=0.79, p<0.0001 and ALS (r=0.70, p<0.0001, but not in CP (r=0.11, p=0.3739. The sensitivity and specificity of serum NfL for separating ALS from healthy controls was 91.3% and 91.0%.We developed and validated a novel ECL based sandwich immunoassay for the NfL protein in serum (NfL(Umea47:3; levels in ALS were more than 20-fold higher than in controls. Our data supports further longitudinal studies of serum NfL in neurodegenerative diseases as a potential biomarker of on-going disease progression, and as a potential surrogate to quantify effects of neuroprotective drugs in clinical trials.

  10. Spectroscopic properties of light-chain derivatives of murine MOPC-315 immunoglobulin A

    DEFF Research Database (Denmark)

    Zidovetzki, R; Farver, O; Pecht, I

    1981-01-01

    Three light-chain derivatives of the homogeneous IgA, secreted by the mouse myeloma MOPC-315, were studied employing circular dichroism and thermal-perturbation spectroscopy: (a) the light-chain dimer with intact native inter-chain disulfide bond, L2,cov; (b) the light-chain dimer with this bond...... reduced and alkylated, L2,ncov; and (c) the dimer of only the variable regions of the light chains, (VL)2. Comparison of the well resolved circular dichroism spectra of these derivatives allowed the assignments of the bands above 290 nm to the following chromophores: Trp-35L and Trp-91L in the variable...... are found between these light-chain derivatives ant the light chain--heavy chain associates, namely the intact protein M-315 and FV fragment. The comparison between the CD spectra of the free and the hapten-bound L2,cov, L2,ncov and (VL)2 directly demonstrates the existence of the conformational transitions...

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

    Science.gov (United States)

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

    2014-02-10

    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.

  12. Fragments of the constant region of immunoglobulin light chains are constituents of AL-amyloid proteins

    DEFF Research Database (Denmark)

    Olsen, K E; Sletten, K; Westermark, Per

    1998-01-01

    Immunoglobulin light chains are the precursor proteins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin...... light chain are a constituent of the AL-amyloid proteins of kappa type. A specific antiserum has identified these fragments in gel filtration fractions where the absorbance approached the base line after the main retarded peak. The fragments are small and have been overlooked previously...

  13. Super-light Structures with Pearl-chain Reinforcement

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl; Bagger, Anne

    2010-01-01

    of prefabricated segments of a strong material, assembled by one or more prestressing wires. By means of pearl-chain reinforcement, it becomes affordable to provide structures with a wide variety of shapes. This allows a more resource-economical design, because it removes practical hindrances for application...... of compression zones with optimal curved shapes. Some benefits of SLS with pearl-chains are: considerable savings on materials, energy, and CO2, reduction of costs, no scaffolding, cheaper moulds, faster assembly on site, improved durability, good thermal insulation, good indoor-climate, user friendly operation...

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

    Science.gov (United States)

    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.

  15. Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.

    Science.gov (United States)

    Hohman, V S; Schluter, S F; Marchalonis, J J

    1992-01-01

    A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.

  16. DIAGNOSTIC AND PROGNOSTIC SIGNIFICANCE OF INTRATHECAL SYNTHESIS OF IMMUNOGLOBULIN FREE LIGHT CHAINS IN MULTIPLE SCLEROSIS

    Directory of Open Access Journals (Sweden)

    V. D. Nazarov

    2015-01-01

    Full Text Available Increased intrathecal synthesis and oligoclonal banding of immunoglobulins (Ig in cerebrospinal fluid (CSF are major immunological findings in multiple sclerosis. Free light chains are fragments of Ig molecules that are produced in parallel to oligoclonal Ig, and their production reflects total Ig synthesis within central nervous system. Assessment of intrathecal kappaand lambda free light chain production within CNS compartment will help to improve diagnosis and prediction of outcomes in multiple sclerosis. Concentrations of kappa and lambda free light chains and clonality of immunoglobulin pattern synthesis were studied in paired CSF and serum samples of 151 patients, including 92 patients with multiple sclerosis, and 33 patients with clinically isolated syndrome which later was transformed into the definite multiple sclerosis. A control group consisted of 26 patients with other inflammatory diseases of central nervous system. Several Ig parameters have been determined in the patients, i.e., Ig clonality in CSF; concentrations of free light chains (both kappaand lambda- in CSF; as well as their indexes and ratios. It was established that synthesis of kappa free light chains were significantly elevated in patients with multiple sclerosis. Moreover, the amounts of free light Ig chains in patients with positive oligoclonality test were significantly higher than in cases without intrathecal oligoclonal synthesis. With respect to diagnostic significance, the kappa quotient proved the best available option for diagnosis of multiple sclerosis. Its combination with Ig oligoclonality assays caused a decrease in false-negative diagnostics by 42%. Diagnostic benefit of the kappa quotient could be also confirmed by the data of ROC analysis. Also concentration of lambda free light chains in cerebrospinal fluid showed a negative correlation with conversion terms of clinically isolated syndrome to evident multiple sclerosis. Therefore, the indexes of free Ig

  17. Immunoglobulin light chain immunohistochemistry revisited, with emphasis on reactive follicular hyperplasia versus follicular lymphoma.

    Science.gov (United States)

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

    2010-05-01

    The identification of monotypic light chains is an important adjunct to the diagnosis of B-cell lymphoma, yet to reliably perform it on formalin-fixed paraffin sections is often difficult. We have evaluated a new set of monoclonal antibodies to kappa and lambda light chains 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 of the appropriate light chain was seen in each of the 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 was also seen in 6 additional cases (10%) in which flow cytometry was 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.

  18. Structure of the microtubule-binding domain of flagellar dynein.

    Science.gov (United States)

    Kato, Yusuke S; Yagi, Toshiki; Harris, Sarah A; Ohki, Shin-ya; Yura, Kei; Shimizu, Youské; Honda, Shinya; Kamiya, Ritsu; Burgess, Stan A; Tanokura, Masaru

    2014-11-04

    Flagellar dyneins are essential microtubule motors in eukaryotes, as they drive the beating motions of cilia and flagella. Unlike myosin and kinesin motors, the track binding mechanism of dyneins and the regulation between the strong and weak binding states remain obscure. Here we report the solution structure of the microtubule-binding domain of flagellar dynein-c/DHC9 (dynein-c MTBD). The structure reveals a similar overall helix-rich fold to that of the MTBD of cytoplasmic dynein (cytoplasmic MTBD), but dynein-c MTBD has an additional flap, consisting of an antiparallel b sheet. The flap is positively charged and highly flexible. Despite the structural similarity to cytoplasmic MTBD, dynein-c MTBD shows only a small change in the microtubule- binding affinity depending on the registry change of coiled coil-sliding, whereby lacks the apparent strong binding state. The surface charge distribution of dynein-c MTBD also differs from that of cytoplasmic MTBD, which suggests a difference in the microtubule-binding mechanism.

  19. Exploring single chain amphiphile self-assembly and their possible roles in light transduction

    DEFF Research Database (Denmark)

    Monnard, Pierre-Alain

    2011-01-01

    Self-assembled structures of single-chain amphiphiles have been used as hosts for biochemical, and chemical reactions. Their use as models for protocells (i.e., precursors to the first biological cells) has been extensively researched by various groups because the availability of single chain...... pigments, polycyclic aromatic hydrocarbons (PAHs) which can function as light harvesting systems1. Thus, mixed membranes formed by single chain amphiphile and PAHs could represent simple primitive energy uptake systems which transduce light into chemical energy that can power an internalized catalytic...

  20. The Oligomeric Outer Dynein Arm Assembly Factor CCDC103 Is Tightly Integrated within the Ciliary Axoneme and Exhibits Periodic Binding to Microtubules*

    Science.gov (United States)

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

    2015-01-01

    CCDC103 is an ∼29-kDa protein consisting of a central RPAP3_C domain flanked by N- and C-terminal coiled coils. Defects in CCDC103 lead to primary ciliary dyskinesia caused by the loss of outer dynein arms. This protein is present along the entire length of the ciliary axoneme and does not require other dynein or docking complex components for its integration. Unlike other known dynein assembly factors within the axoneme, CCDC103 is not solubilized by 0.6 m NaCl and requires more chaotropic conditions, such as 0.5 m KI. Alternatively, it can be extracted using 0.3% sarkosyl. CCDC103 forms stable dimers and other oligomers in solution through interactions involving the central domain. The smallest particle observed by dynamic light scattering has a hydrodynamic diameter of ∼25 nm. Furthermore, CCDC103 binds microtubules directly, forming ∼9-nm diameter particles that exhibit a 12-nm spacing on the microtubule lattice, suggesting that there may be two CCDC103 units per outer arm dynein repeat. Although the outer dynein arm docking complex is necessary to form arrays of dyneins along microtubules, it is not sufficient to set up a single array in a precise location on each axonemal doublet. We propose that CCDC103 helps generate a high-affinity site on the doublets for outer arm assembly, either through direct interactions or indirectly, perhaps by modifying the underlying microtubule lattice. PMID:25572396

  1. Light-switching-light optical transistor based on metallic nanoparticle cross-chains geometry incorporating Kerr nonlinearity

    Energy Technology Data Exchange (ETDEWEB)

    AbdelMalek, Fathi; Aroua, Walid [National Institute of Applied Science and Technology, University of Carthage, Tunis (Tunisia); Haxha, Shyqyri [Computer Science and Technology Department, Bedfordshire University, Luton (United Kingdom); Flint, Ian [Selex ES Ltd, Luton, Bedfordshire (United Kingdom)

    2016-08-15

    In this research work, we propose all-optical transistor based on metallic nanoparticle cross-chains geometry. The geometry of the proposed device consists of two silver nanoparticle chains arranged along the x- and z-axis. The x-chain contains a Kerr nonlinearity, the source beam is set at the left side of the later, while the control beam is located at the top side of the z-chain. The control beam can turn ON and OFF the light transmission of an incoming light. We report a theoretical model of a very small all-optical transistor proof-of-concept made of optical 'light switching light' concept. We show that the transmission efficiency strongly depends on the control beam and polarization of the incoming light. We investigate the influence of a perfect reflector and reflecting substrate on the transmission of the optical signal when the control beam is turned ON and OFF. These new findings make our unique design a potential candidate for future highly-integrated optical information processing chips. (copyright 2016 by WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

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

    KAUST Repository

    Chailyan, Anna

    2011-06-28

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

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

    Directory of Open Access Journals (Sweden)

    Maria Kraj

    2015-01-01

    Full Text Available 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.

  4. Super-light concrete with pearl-chains

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl

    2009-01-01

    half that of similar structures in concrete and steel. They are heat-insulating, fire-resistant and they open up the possibility for large spans and the creation of advanced shapes. Fields of application include roofs, shells, beams, columns, walls, façades, offshore structures, tunnels and structures...... the applicability and to reveal any potential hidden problems. Continuing research will aim to reduce the weight and thereby the resource consumption further. In addition, the paper introduces pearl-chain reinforcement, which is a new principle for creating compression and tension zones. A small number of simple...

  5. BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

    NARCIS (Netherlands)

    D. Splinter (Daniël); D.S. Razafsky (David); M.A. Schlager (Max); A. Serra-Marques (Andrea); I. Grigoriev (Ilya); J.A.A. Demmers (Jeroen); N. Keijzer (Nanda); K. Jiang (Kai); S. Poser; A. Hyman (Anthony); C.C. Hoogenraad (Casper); S.J. King (Stephen); A.S. Akhmanova (Anna)

    2012-01-01

    textabstractCytoplasmic dynein is the major microtubule minus-end-directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein-dynactin interaction are poorly understood. In this study, we focus on dynein-dynactin recruitment to cargo by the

  6. BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

    NARCIS (Netherlands)

    D. Splinter (Daniël); D.S. Razafsky (David); M.A. Schlager (Max); A. Serra-Marques (Andrea); I. Grigoriev (Ilya); J.A.A. Demmers (Jeroen); N. Keijzer (Nanda); K. Jiang (Kai); S. Poser; A. Hyman (Anthony); C.C. Hoogenraad (Casper); S.J. King (Stephen); A.S. Akhmanova (Anna)

    2012-01-01

    textabstractCytoplasmic dynein is the major microtubule minus-end-directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein-dynactin interaction are poorly understood. In this study, we focus on dynein-dynactin recruitment to cargo by the

  7. Prognostic value of serum free light chain ratio at diagnosis in multiple myeloma.

    Science.gov (United States)

    Kyrtsonis, Marie-Christine; Vassilakopoulos, Theodoros P; Kafasi, Nicoletta; Sachanas, Sotirios; Tzenou, Tatiana; Papadogiannis, Argiroula; Galanis, Zacharoula; Kalpadakis, Christina; Dimou, Maria; Kyriakou, Elias; Angelopoulou, Maria K; Dimopoulou, Maria N; Siakantaris, Marina P; Dimitriadou, Evangelia M; Kokoris, Styliani I; Panayiotidis, Panayiotis; Pangalis, Gerassimos A

    2007-05-01

    The prognostic value of baseline serum free light chain ratio (sFLCR) was investigated in 94 multiple myeloma (MM) patients. sFLCR was calculated as kappa/lambda or lambda/kappa, depending on the patients' dominating monoclonal light chain. Median baseline sFLCR was 3.57 in kappa-MM patients, 45.09 in lambda-MM. 'High' sFLCR (> or = the observed median value for kappa- and lambda-MM respectively) correlated with elevated serum creatinine and lactate dehydrogenase, extensive marrow infiltration and light chain type MM. The 5-year disease-specific survival was 82% and 30% in patients with sFLCR lower than and equal or greater than the median, respectively (P = 0.0001). sFLCR was an independent prognostic factor.

  8. Cast nephropathy and light-chain deposition disease in Waldenström macroglobulinemia.

    Science.gov (United States)

    Gnemmi, Viviane; Leleu, Xavier; Provot, François; Moulonguet, Florence; Buob, David

    2012-09-01

    Waldenström macroglobulinemia is a rare low-grade hematologic malignancy due to clonal proliferation of B lymphocytes responsible for immunoglobulin M (IgM) monoclonal gammopathy secreted in serum. This disease is characterized by lymphoplasmacytic tumoral infiltration of bone marrow and various organs, especially the liver and spleen. Kidney involvement in Waldenström macroglobulinemia has been described previously with reports of various forms of glomerular injury: large intracapillary IgM pseudothrombi, cryoglobulinemia-associated membranoproliferative glomerulonephritis, or amyloidosis. Interstitial infiltration by tumoral B lymphocytes is another classic pattern. Conversely, tubular involvement in the form of myeloma-like casts or basement membrane deposition of monoclonal light chain (light-chain deposition disease) is unusual. We report the occurrence of cast nephropathy associated with light-chain deposition disease in 2 patients with Waldenström macroglobulinemia, which resulted in severe and prolonged kidney failure.

  9. Lambda Light Chain Myeloma with Oliguric Cast Nephropathy and Remission with Bortezomib, Doxorubicin and Dexamethasone

    Directory of Open Access Journals (Sweden)

    R Hada

    2012-12-01

    We report the clinical course and outcome of a patient presenting with acute kidney injury and oliguria, preceded by acute gastroenteritis and intake of Chinese medications and dialysis dependent state for eight weeks. Kidney biopsy revealed cast nephropathy with lambda light chain restriction and severe tubular injury. Serum protein electrophoresis was normal with no “M spike” but serum free light chain ratio was altered with very high lambda and normal kappa light chain levels. Bone marrow biopsy showed >85% atypical plasma cells. Haemodialysis was continued and chemotherapy with bortezomib, doxorubicin and dexamethasone was started. Kidney function gradually improved with discontinuation of dialysis after 1 month and complete remission of acute kidney injury and myeloma in 4 months of chemotherapy. Keywords: acute kidney injury; bortezomib; cast nephropathy; multiple myeloma.

  10. Higher Serum Levels of Free ĸ plus λ Immunoglobulin Light Chains Ameliorate Survival of Hemodialysis Patients

    DEFF Research Database (Denmark)

    Thilo, Florian; Caspari, Christina; Scholze, Alexandra;

    2011-01-01

    Background/Aims: Impaired immune function is common in patients with chronic renal failure. Now, we determined whether serum levels of free immunoglobulin light chains predict mortality in patients with chronic kidney disease stage 5 on hemodialysis. Methods: We performed a prospective cohort study...... of 160 hemodialysis patients with a median follow-up of 15 months (interquartile range, 3-44 months). Serum levels of free κ and λ immunoglobulin light chains were measured at the start of the study. The primary end point was mortality from any cause. Results: In survivors, median serum levels of free κ....../l (χ(2) = 5.91; p = 0.015 by log-rank, Mantel-Cox, test). We performed univariate and multivariate regression analysis showing that older age and lower serum levels of free κ plus λ immunoglobulin light chains predicted mortality in hemodialysis patients. Conclusion: Higher serum levels of free κ plus...

  11. Dynein at the kinetochore: Timing, Interactions and Functions.

    Science.gov (United States)

    Bader, Jason R; Vaughan, Kevin T

    2010-05-01

    Kinetochores have been proposed to play multiple roles in mitotic chromosome alignment, including initial microtubule (MT) capture, monitoring MT attachments, prometaphase and anaphase chromosome movement and tension generation at metaphase. In addition, kinetochores are essential components of the spindle assembly checkpoint (SAC), and couple chromosome alignment with SAC silencing at metaphase. Although the molecular details of these activities remain under investigation, cytoplasmic dynein has been implicated in several aspects of MT and SAC regulation. Recent work clarifies the contribution of dynein to MT interactions and to events that drive anaphase onset. This review summarizes these studies and provides new models for dynein function. Copyright 2010 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2017-06-01

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

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

    Science.gov (United States)

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

    2014-05-01

    Cardiac amyloidosis represents an archetypal form of restrictive heart disease, characterized by profound diastolic dysfunction. As ejection fraction is preserved until the late stage of the disease, the majority of patients do fulfill the definition of diastolic heart failure, that is, heart failure with preserved ejection fraction (HFpEF). In another clinical model of HFpEF, that is, pressure-overload hypertrophy, depressed midwall fractional shortening (mFS) has been shown to be a powerful prognostic factor. To assess the potential prognostic role of mFS in cardiac light-chain amyloidosis with preserved ejection fraction, we enrolled 221 consecutive untreated patients, in whom a first diagnosis of cardiac light-chain amyloidosis was concluded between 2008 and 2010. HFpEF was present in 181 patients. Patients in whom cardiac involvement was excluded served as controls (n = 121). Prognosis was assessed after a median follow-up of 561 days. When compared with light-chain amyloidosis patients without myocardial involvement, cardiac light-chain amyloidosis was characterized by increased wall thickness (P chain amyloidosis with normal ejection fraction, depressed circumferential mFS, a marker of myocardial contractile dysfunction, is a powerful predictor of survival.

  14. A prospective study of nutritional status in immunoglobulin light chain amyloidosis

    DEFF Research Database (Denmark)

    Sattianayagam, Prayman T; Lane, Thirusha; Fox, Zoe;

    2013-01-01

    Weight loss is common in systemic immunoglobulin light chain amyloidosis but there are limited data on the impact of nutritional status on outcome. Using the Patient-Generated Subjective Global Assessment (PG-SGA) score, we prospectively examined nutritional status in 110 consecutive newly......-diagnosed, treatment-naïve patients with immunoglobulin light chain amyloidosis attending the UK National Amyloidosis Centre. At study entry, 72 of 110 (66%) patients had a PG-SGA score of 4 or over, indicating malnutrition requiring specialist nutritional intervention. Number of amyloidotic organs, elevated alkaline...... phosphatase, presence of autonomic neuropathy and advanced Mayo disease stage were independently associated with poor nutritional status (P...

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

    Science.gov (United States)

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

    2008-11-25

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

  16. Human lambda light-chain constant region gene CMor lambda: the primary structure of lambda VI Bence Jones protein Mor.

    OpenAIRE

    Frangione, B.; Moloshok, T; Prelli, F; Solomon, A

    1985-01-01

    Serologic, structural, and genetic analyses have shown that the constant (C) region of human kappa light chains is encoded by a single gene, whereas that of lambda chains is encoded by multiple genes. We have determined the complete C region amino acid sequence of two monoclonal lambda VI light chains, Bence Jones proteins Sut and Mor. The C region of lambda chains Sut and Mor consists of 105 residues, as is characteristic for human lambda light chains, of which 102 are identical in sequence....

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

    Science.gov (United States)

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

    2016-01-01

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

  18. Self-organization of dynein motors generates meiotic nuclear oscillations.

    Directory of Open Access Journals (Sweden)

    Sven K Vogel

    2009-04-01

    Full Text Available Meiotic nuclear oscillations in the fission yeast Schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. We report a mechanism of these oscillations on the basis of collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. By combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. The redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. Our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, owing to the intrinsic motor properties, generate regular large-scale movements in the cell.

  19. Catch bond mechanism in Dynein motor driven collective transport

    CERN Document Server

    Nair, Anil; Mitra, Mithun K; Muhuri, Sudipto; Chaudhuri, Abhishek

    2016-01-01

    Recent experiments have demonstrated that dynein motor exhibits catch bonding behaviour, in which the unbinding rate of a single dynein decreases with increasing force, for a certain range of force. Motivated by these experiments, we propose a model for catch bonding in dynein using a threshold force bond deformation (TFBD) model wherein catch bonding sets in beyond a critical applied load force. We study the effect of catch bonding on unidirectional transport properties of cellular cargo carried by multiple dynein motors within the framework of this model. We find catch bonding can result in dramatic changes in the transport properties, which are in sharp contrast to kinesin driven unidirectional transport, where catch bonding is absent. We predict that, under certain conditions, the average velocity of the cellular cargo can actually increase as applied load is increased. We characterize the transport properties in terms of a velocity profile phase plot in the parameter space of the catch bond strength and ...

  20. Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells.

    Science.gov (United States)

    Li, Min; Balamuthusamy, Saravanan; Simon, Eric E; Batuman, Vecihi

    2008-07-01

    Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to kappa-light chain for up to 24 h, light chain endocytosis was reduced in either megalin- or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring kappa-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled kappa-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.

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

    Science.gov (United States)

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

    2017-03-16

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

  2. Intramanchette transport during primate spermiogenesis:expression of dynein, myosin Va, motor recruiter myosin Va,Ⅶa-Rab27a/b interacting protein, and Rab27b in the manchette during human and monkey spermiogenesis

    Institute of Scientific and Technical Information of China (English)

    Shinichi Hayasaka; Yukihiro Terada; Kichiya Suzuki; Haruo Murakawa; Ikuo Tachibana; Tadashi Sankai; Takashi Murakami; Nobuo Yaegashi; Kunihiro Okamura

    2008-01-01

    Aim: To show whether molecular motor dynein on a microtubule track, molecular motor myosin Va, motor recruiter myosin Va, Ⅶa-Rab27a/b interacting protein (MyRIP), and vesicle receptor Rab27b on an F-actin track were present during human and monkey spermiogenesis involving intramanchette transport (IMT). Methods: Spermiogenic cells were obtained from three men with obstructive azoospermia and normal adult cynomolgus monkey (Macaca fascieularis). Immunocytochemical detection and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the pro- teins were carried out. Samples were analyzed by light microscope. Results: Using RT-PCR, we found that dynein, myosin Va, MyRIP and Rab27b were expressed in monkey testis. These proteins were localized to the manchette, as shown by immunofluorescence, particularly during human and monkey spermiogenesis. Conclusion: We speculate that during primate spermiogenesis, those proteins that compose microtubule-based and actin-based vesicle transport systems are actually present in the manchette and might possibly be involved in intramanchette transport. (Asian J Androl 2008 Jul; 10: 561-568)

  3. Immunoglobulin free light chains: new insights in mast cell activation and immunology

    NARCIS (Netherlands)

    Thio, M.

    2011-01-01

    In this thesis, several studies are described that elaborate on the biological properties of immunoglobulin free light chains (Ig-fLC) related to the activation of mast cells and effects on other cells. Mast cell degranulation through Ig-fLC requires two events. At first, mast cell-bound Ig-fLCs sho

  4. Bone marrow lambda-type light chain crystalline structures associated with multiple myeloma.

    Science.gov (United States)

    Schvartz, H; Bonhomme, P; Caulet, S; Beorchia, A; Patey, M; Caulet, T

    1985-01-01

    A 58-year-old man showed bone marrow crystalline structures associated with a lambda light chain producing multiple myeloma. Analysis and processing of electron images clearly displayed the periodic structure of the crystals. Immunochemistry suggested that they contained the whole or a fragmented constant portion of immunoglobulin.

  5. Constant region of a kappa III immunoglobulin light chain as a major AL-amyloid protein

    DEFF Research Database (Denmark)

    Engvig, J P; Olsen, K E; Gislefoss, R E

    1998-01-01

    and the corresponding AL protein as a kappa III immunoglobulin light chain from material of a patient with systemic AL-amyloidosis presenting as a local inguinal tumour. The two proteins showed some unique features. The major part of the AL amyloid fibril protein consisted of C-terminal fragments of the Bence...

  6. Light-induced circular birefringence in cyanoazobenzene side-chain liquid-crystalline polyester films

    DEFF Research Database (Denmark)

    Naydenova, I; Nikolova, L; Ramanujam, P.S.;

    1999-01-01

    We report the inducement of large circular birefringence (optical activity) in films of a cyanoazobenzene side-chain liquid-crystalline polyester on illumination with circularly polarized light. The polyester has no chiral groups and is initially isotropic. The induced optical rotation is up to 5...

  7. Diagnostic performance of measuring free light chains in fat tissue of patients with AL amyloidosis

    NARCIS (Netherlands)

    Bijzet, J.; Van Gameren, I.I.; Limburg, P.C.; Bos, R.; Vellenga, E.; Hazenberg, B.P.C.

    2010-01-01

    Background: The diagnostic performance of measuring immunoglobulin free light chains in abdominal subcutaneous fat tissue was studied for detection and characterisation of AL amyloidosis. Methods: Fat tissue specimens of 107 consecutive patients with AL amyloidosis (23 k and 84 l) and 95 controls (3

  8. Quantitative Measurement of Immunoglobulins and Free Light Chains Using Mass Spectrometry

    NARCIS (Netherlands)

    VanDuijn, M.M.; Jacobs, J.F.M.; Wevers, R.A.; Engelke, U.F.H.; Joosten, I.; Luider, T.M.

    2015-01-01

    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 stren

  9. A novel approach for the chromatographic purification and peptide mass fingerprinting of urinary free light chains.

    Science.gov (United States)

    Mali, Bhupesh C; Badgujar, Shamkant B; Shukla, Kunal K; Bhanushali, Paresh B

    2017-02-01

    We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

  10. Systemic immunoglobulin light-chain amyloidosis presenting hematochezia as the initial symptom.

    Science.gov (United States)

    Kon, Tetsuo; Nakagawa, Naoki; Yoshikawa, Fumitsugu; Haba, Kazunao; Kitagawa, Nagako; Izumi, Michihiro; Kumazaki, Setsuo; Ishida, Satoshi; Aikawa, Ryuichi

    2016-08-01

    Immunoglobulin light-chain (AL) amyloidosis is characterized by the deposition of insoluble fibrils composed of immunoglobulin light chains secreted by monoclonal plasma cells. Given the recent advances in the therapy of AL amyloidosis, it is important to diagnose this disease as early as possible. Herein, we describe the case of a 62-year-old man with hepatitis C virus (HCV)-related cirrhosis presenting with hematochezia. Colonoscopy showed multiple submucosal hematomas within the region ranging from the transverse colon to the sigmoid colon. Kappa immunoglobulin light-chain amyloid deposition was also detected. Bone marrow examination revealed a monoclonal abnormal plasma cell population. Thus, the patient was diagnosed with systemic immunoglobulin light-chain amyloidosis. The hematochezia was conservatively managed. However, because of liver failure caused by liver cirrhosis, the patient developed massive pleural effusion and died of respiratory failure. Postmortem examination revealed amyloid deposition in the esophagus, stomach, duodenum, ileum, descending colon, pancreas, heart, and lung. In these organs, amyloid deposition was limited to the vascular wall. We concluded that AL amyloidosis can present hematochezia arising from submucosal hematoma in the large colon before other systemic symptoms appear.

  11. Spectroscopic properties of light-chain derivatives of murine MOPC-315 immunoglobulin A

    DEFF Research Database (Denmark)

    Zidovetzki, R; Farver, O; Pecht, I

    1981-01-01

    domains, and Trp-148L, Trp-185L and the disulfide of Cys-214L in the constant domains. The differences in the spectral characteristics of L2,cov as compared to those of L2,ncov and (VL)2 illustrate the significant influence of the disulfide bridge on the conformation of the L2,cov. Pronounced differences...... are found between these light-chain derivatives ant the light chain--heavy chain associates, namely the intact protein M-315 and FV fragment. The comparison between the CD spectra of the free and the hapten-bound L2,cov, L2,ncov and (VL)2 directly demonstrates the existence of the conformational transitions...

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

    Science.gov (United States)

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

    2013-09-01

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

  13. Dlic1 deficiency impairs ciliogenesis of photoreceptors by destabilizing dynein

    Institute of Scientific and Technical Information of China (English)

    Shanshan Kong; Xinrong Du; Chao Peng; Yiming Wu; Huirong Li; Xi Jin; Ling Hou

    2013-01-01

    Cytoplasmic dynein 1 is fundamentally important for transporting a variety of essential cargoes along microtubules within eukaryotic cells.However,in mammals,few mutants are available for studying the effects of defects in dynein-controlled processes in the context of the whole organism.Here,we deleted mouse Dlic1 gene encoding DLIC1,a subunit of the dynein complex.Dlic1-/-mice are viable,but display severe photoreceptor degeneration.Ablation of Dlic1 results in ectopic accumulation of outer segment (OS) proteins,and impairs OS growth and ciliogenesis of photoreceptors by interfering with Rabll-vesicle trafficking and blocking efficient OS protein transport from Golgi to the basal body.Our studies show that Dlic1 deficiency partially blocks vesicle export from endoplasmic reticulum (ER),but seems not to affect vesicle transport from the ER to Golgi.Further mechanistic study reveals that lack of Dlic1 destabilizes dynein subunits and alters the normal subcellular distribution of dynein in photoreceptors,probably due to the impaired transport function of dynein.Our results demonstrate that Dlic1 plays important roles in ciliogenesis and protein transport to the OS,and is required for photoreceptor development and survival.The Dlic1-/-mice also provide a new mouse model to study human retinal degeneration.

  14. Value of serum free light chain testing for the diagnosis and monitoring of monoclonal gammopathies in hematology.

    Science.gov (United States)

    Jagannath, Sundar

    2007-09-01

    The automated quantification of serum free kappa and lambda light chain concentrations provides a highly sensitive tool for the diagnosis and monitoring of monoclonal gammopathies. An abnormal kappa:lambda ratio supports the presence of clonal plasma cell expansion and requires further investigation. More than 94% of myeloma, light chain myeloma, and AL amyloidosis and, likewise, a majority of patients with light chain deposition disease are detectable with this technology. Importantly, these assays identify M-proteins in most patients with oligosecretory disease and permit their recruitment into clinical trials from which they have been previously excluded. Combining serum free light chain testing with traditional electrophoresis provides > 99% accuracy in the first-line diagnosis of monoclonal gammopathies and eliminates the need for urine testing in most instances. One third of patients with monoclonal gammopathy of undetermined significance have an abnormal free light chain ratio, and these patients harbor a greater risk of progression to plasma cell dyscrasia. For monitoring response to therapy, the international uniform response criteria define a normal free light chain ratio as an essential element of the "stringent complete response" category. Because the half-life of free light chains is chain measurements at short sampling intervals allow real-time measurement of treatment-induced tumor kill, and provide prompt indications of chemosensitivity, dose adequacy, need for alternative approaches, and even prognosis, as demonstrated in AL amyloidosis if the involved free light chain concentration normalizes. Clinical applications of these assays will likely increase as their utility is more widely explored.

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

    Science.gov (United States)

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

    2014-11-07

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

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

    Science.gov (United States)

    Toshchevikov, V; Saphiannikova, M; Heinrich, G

    2009-04-16

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

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

    Science.gov (United States)

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

    2014-09-10

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

  18. NCBI nr-aa BLAST: CBRC-TSYR-01-0241 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TSYR-01-0241 ref|XP_001915362.1| PREDICTED: similar to Dynein heavy chain 17, axonemal (Ciliary dynei...n heavy chain 17) (Axonemal beta dynein heavy chain 17) (Ciliary dynein heavy chain-l...ike protein 1) (Axonemal dynein heavy chain-like protein 1) (Dynein light chain 2, axonemal) [Equus caballus] XP_001915362.1 2e-59 92% ...

  19. NCBI nr-aa BLAST: CBRC-PHAM-01-1210 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-1210 ref|XP_001915362.1| PREDICTED: similar to Dynein heavy chain 17, axonemal (Ciliary dynei...n heavy chain 17) (Axonemal beta dynein heavy chain 17) (Ciliary dynein heavy chain-l...ike protein 1) (Axonemal dynein heavy chain-like protein 1) (Dynein light chain 2, axonemal) [Equus caballus] XP_001915362.1 7e-39 97% ...

  20. NCBI nr-aa BLAST: CBRC-PHAM-01-1072 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-1072 ref|XP_001915362.1| PREDICTED: similar to Dynein heavy chain 17, axonemal (Ciliary dynei...n heavy chain 17) (Axonemal beta dynein heavy chain 17) (Ciliary dynein heavy chain-l...ike protein 1) (Axonemal dynein heavy chain-like protein 1) (Dynein light chain 2, axonemal) [Equus caballus] XP_001915362.1 3e-69 76% ...

  1. NCBI nr-aa BLAST: CBRC-MMUR-01-0834 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MMUR-01-0834 ref|XP_001915362.1| PREDICTED: similar to Dynein heavy chain 17, axonemal (Ciliary dynei...n heavy chain 17) (Axonemal beta dynein heavy chain 17) (Ciliary dynein heavy chain-l...ike protein 1) (Axonemal dynein heavy chain-like protein 1) (Dynein light chain 2, axonemal) [Equus caballus] XP_001915362.1 1e-36 97% ...

  2. NCBI nr-aa BLAST: CBRC-PHAM-01-0917 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-0917 ref|XP_001915362.1| PREDICTED: similar to Dynein heavy chain 17, axonemal (Ciliary dynei...n heavy chain 17) (Axonemal beta dynein heavy chain 17) (Ciliary dynein heavy chain-l...ike protein 1) (Axonemal dynein heavy chain-like protein 1) (Dynein light chain 2, axonemal) [Equus caballus] XP_001915362.1 3e-69 76% ...

  3. NCBI nr-aa BLAST: CBRC-PHAM-01-0234 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-0234 ref|XP_001915362.1| PREDICTED: similar to Dynein heavy chain 17, axonemal (Ciliary dynei...n heavy chain 17) (Axonemal beta dynein heavy chain 17) (Ciliary dynein heavy chain-l...ike protein 1) (Axonemal dynein heavy chain-like protein 1) (Dynein light chain 2, axonemal) [Equus caballus] XP_001915362.1 4e-57 84% ...

  4. NCBI nr-aa BLAST: CBRC-MDOM-02-0177 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-02-0177 ref|XP_001915362.1| PREDICTED: similar to Dynein heavy chain 17, axonemal (Ciliary dynei...n heavy chain 17) (Axonemal beta dynein heavy chain 17) (Ciliary dynein heavy chain-l...ike protein 1) (Axonemal dynein heavy chain-like protein 1) (Dynein light chain 2, axonemal) [Equus caballus] XP_001915362.1 0.0 86% ...

  5. NCBI nr-aa BLAST: CBRC-OPRI-01-0158 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-0158 ref|XP_001915362.1| PREDICTED: similar to Dynein heavy chain 17, axonemal (Ciliary dynei...n heavy chain 17) (Axonemal beta dynein heavy chain 17) (Ciliary dynein heavy chain-l...ike protein 1) (Axonemal dynein heavy chain-like protein 1) (Dynein light chain 2, axonemal) [Equus caballus] XP_001915362.1 2e-47 75% ...

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

    Science.gov (United States)

    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

  7. Distribution of kappa and lambda light chain isotypes among human blood immunoglobulin-secreting cells after vaccination with pneumococcal polysaccharides

    DEFF Research Database (Denmark)

    Heilmann, C; Barington, T

    1989-01-01

    The light chain isotype of immunoglobulin-secreting blood cells was investigated by means of monolayer plaque-forming cell assays allowing direct immunofluorescence staining for cytoplasmic kappa and lambda light chains in centre cells. The study revealed that cultured, polyclonally activated pok...

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

    Science.gov (United States)

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

    2005-10-01

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

  9. Deposition of kappa and lambda light chains in amyloid filaments of dialysis-related amyloidosis.

    Science.gov (United States)

    Brancaccio, D; Ghiggeri, G M; Braidotti, P; Garberi, A; Gallieni, M; Bellotti, V; Zoni, U; Gusmano, R; Coggi, G

    1995-10-01

    beta 2-Microglobulin (beta 2m) is considered to be the amyloidogenic precursor in dialysis-related amyloidosis, although the implication of other relevant cofactors in the pathogenesis of this disease has also been hypothesized. It is conceivable that substances found in amyloid deposits might represent something more than simple codeposition, possibly playing a pathogenic role in amyloidogenesis. Along these lines, a detailed analysis of the protein composition of amyloid fibrils purified from synovial material surgically obtained from nine patients on long-term dialysis was carried out. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several other protein components, in addition to beta 2m, were found. These were characterized by NH2 amino-terminal sequencing and immunoblotting. In fibrils obtained by water extraction, which fulfill the electron microscopy criteria of highly pure amyloid material, polyclonal kappa and lambda light chains were detected with a concentration of 15 micrograms/mL in the water extraction material; the beta 2m concentration was 200 micrograms/mL. Light microscopy immunohistochemistry was performed on samples from five patients. Amyloid deposits reacted with anti-beta 2m, and anti-light (kappa, lambda), chain antibodies. The immunoreaction of amyloid filaments to anti-beta 2m, anti-lambda, and anti-kappa light chain antibodies was also tested by electron microscopy by use of the immunogold staining procedure. Amyloid filaments were labeled by the three antibodies and showed a different intensity of immunostaining apparently related to their different aggregation pattern. These observations demonstrate that polyclonal immunoglobulin light chains (kappa and lambda) are not contaminants but, together with beta 2m, represent a major constituent of amyloid deposits in dialysis-related osteoarticular amyloidosis, thus indicating their possible role in amyloidogenesis.

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

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

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

  11. IMMUNOMETRIC ASSAY TO DETERMINE FREE LIGHT CHAIN CONCENTRATIONS OF HUMAN IMMUNOGLOBULINS

    Directory of Open Access Journals (Sweden)

    M. P. Samoylovich

    2016-01-01

    Full Text Available Detection of total free light chains (FLC of immunoglobulins and their ratio (kappa/lambda quotient are used in diagnostics and monitoring of multiple myeloma and other gammapathies, primary amyloidosis and multiple sclerosis. Previously described immunoassays with monoclonal antibodies (Mabs against cryptic and constantly exposed epitopes of FLC failed to recognize rare variants of lambda Bence-Jones proteins and a significant proportion of lambda chains excreted with urine. Aiming to improve this approach, a novel murine Mab (IgG2b coded as 1C8 was employed, which specifically binds free lambda chains but doesn’t interact with native IgA, IgG, and IgM. The novel Mab recognized an epitope exposed at free lambda chains in peripheral blood of healthy donors and patients with multiple myeloma. It is not destroyed or masked upon renal filtration.The aim of this study was to determine basic features of improved assay system, and to estimate its potential in diagnostics of monoclonal gammapathies. The mixtures of three Bence-Jones proteins of either kappa- or lambda- types purified from the urine of multiple myeloma patients were used as calibrator samples.Improved immunometric assay is able to detect free kappa and lambda chains in serum and urine at a scale of 1 to 100 ng/ml, thus being three orders more sensitive than, e.g., detection levels of Freelite method based on polyclonal antibodies.A novel assay allows to detect free kappa and lambda chains at comparable levels in serum or urine, and to deduce kappa/lambda ratio. The proposed assay is able to detect FLC in 10,000-fold excess of whole IgG molecules. The calibrating plots for both antigens are linear on log-log scales, with very similar slopes. Detection thresholds for kappa or lambda chains proved to be 5 and 3 ng/ml, respectively. Mean concentrations of free kappa chains in sera of healthy donors were 6.7±2.1, in urine, 4.2±3.8 mcg/ml. Mean concentrations of free lambda chains were 4

  12. Power-Law Behavior in Signal Scattering Process in Vertical Granular Chain with Light Impurities

    Institute of Scientific and Technical Information of China (English)

    XU Ai-Guo

    2001-01-01

    We investigate the scattering process of impulse in vertical granular chain with light impurities. When the perturbation is weak, the quantities describing the reflection rate exhibit power-law behavior with the impurity depth. The exponent is nearly independent of vi. When the perturbation is very strong, the vertical chain shows similar behavior to that of the horizontal chain, so the exponent is nearly zero. Our numerical investigation begins from the weak perturbation region and extends to the nonlinear region and found a peak of the exponent. The difficulty in extending the numerical investigation to a stronger perturbation region is analyzed.PACS numbers: 45.70.-n, 43.25.+y, 46.40.Cd

  13. Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

    DEFF Research Database (Denmark)

    Hougs, L; Barington, T; Madsen, HO

    1993-01-01

    of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the Hib......CP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain...... reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody...

  14. Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

    DEFF Research Database (Denmark)

    Hougs, L; Barington, T; Madsen, HO

    1993-01-01

    reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody......CP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain...... of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the Hib...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Light-chain deposition disease of the kidney: a case report.

    Science.gov (United States)

    Darouich, Sihem; Goucha, Rym; Jaafoura, Mohamed Habib; Zekri, Semy; Kheder, Adel; Maiz, Hedi Ben

    2012-04-01

    A 41-year-old man was admitted for evaluation of nephrotic syndrome associated with microhematuria, hypertension, and moderate renal failure. In serum and urine samples, monoclonal IgG-lambda was detected. Bone marrow examination showed normal representation of all cell lines with normal range of plasma cells. Renal biopsy demonstrated diabetes-like nodular glomerulosclerosis. Immunofluorescence failed to demonstrate the presence of kappa or lambda light chains in the kidney. Electron microcopy showed granular electron-dense deposits along the glomerular basement membranes and in the mesangial nodules. The patient was diagnosed as having light-chain deposition disease (LCDD) without evidence of plasma cell dyscrasia. This report was designed to stress the significant challenges that remain in the diagnosis of LCDD-related glomerulopathy. The salient morphological features that help in making an accurate diagnosis are discussed.

  17. Unique morphology of intratubular light chain casts in multiple myeloma: The amyloid cast nephropathy

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2014-01-01

    Full Text Available Cast nephropathy is the most frequent pattern of renal involvement in multiple myeloma characterized by presence of tubular casts with characteristic morphology that are composed of monotypic (either kappa or lambda light chains as seen by immunofluorescence microscopy. Rarely these casts may show evidence of amyloidogenesis and assume a unique morphology, which needs to be appreciated for arriving at accurate diagnosis. We present the case of an elderly male presenting with features of acute kidney injury and detected with extensive inspissation of intratubular casts with lambda light chain restriction and a unique morphology with spiculated congophilic periphery. Further investigations confirmed the presence of systemic myeloma. Presence of intratubular amyloid casts is a rare occurrence which needs to be recognized by the pathologist and forms a vital element in timely diagnosis of the systemic disease which often presents with renal involvement.

  18. Dynein associates with oskar mRNPs and is required for their efficient net plus-end localization in Drosophila oocytes.

    Directory of Open Access Journals (Sweden)

    Paulomi Sanghavi

    Full Text Available In order for eukaryotic cells to function properly, they must establish polarity. The Drosophila oocyte uses mRNA localization to establish polarity and hence provides a genetically tractable model in which to study this process. The spatial restriction of oskar mRNA and its subsequent protein product is necessary for embryonic patterning. The localization of oskar mRNA requires microtubules and microtubule-based motor proteins. Null mutants in Kinesin heavy chain (Khc, the motor subunit of the plus end-directed Kinesin-1, result in oskar mRNA delocalization. Although the majority of oskar particles are non-motile in khc nulls, a small fraction of particles display active motility. Thus, a motor other than Kinesin-1 could conceivably also participate in oskar mRNA localization. Here we show that Dynein heavy chain (Dhc, the motor subunit of the minus end-directed Dynein complex, extensively co-localizes with Khc and oskar mRNA. In addition, immunoprecipitation of the Dynein complex specifically co-precipitated oskar mRNA and Khc. Lastly, germline-specific depletion of Dhc resulted in oskar mRNA and Khc delocalization. Our results therefore suggest that efficient posterior localization of oskar mRNA requires the concerted activities of both Dynein and Kinesin-1.

  19. Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers

    OpenAIRE

    Das, Sabyasachi; Nikolaidis, Nikolas; Klein, Jan; Nei, Masatoshi

    2008-01-01

    The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups (κ, λ, and σ isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess thr...

  20. Therapeutic Approaches for Botulinum Intoxication Targeting Degradation of the Light Chain

    Science.gov (United States)

    2013-04-01

    Synaptic Research will develop dichain hybrids consisting of Clostridium botulinum toxin light chains (LCs) from serotypes A (long-lived) and E...provided this plasmid to the McNutt laboratory that will use a modified version of this virus to produce transgenic mice expressing the designer...interactions via FRET using 24 transgenic lines that co-express fluorescently tagged LCs and target proteins. After optimization of pull-down

  1. 6th International Symposium on Clinical Applications of Serum Free Light Chain Analysis (plus Hevylite

    Directory of Open Access Journals (Sweden)

    Guest Editor: A.R. Bradwell

    2011-08-01

    Full Text Available The 6th International Symposium on Clinical Applications of Serum Free Light Chain Analysis (plus Hevylite was held in Bath, UK on 23rd and 24th September 2010. This Symposium included measurements in monoclonal gammopathies, myeloma kidney and inflammatory diseases; data from the Multi-centre, Phase III, Randomised control trial on extended haemodialysis with the Gambro protein-leaking HCO 1100 dialyser; and presentations on Hevylite (intact immunoglobulin kappa/lambda ratios in a variety of clinical situations.

  2. 6th International Symposium on Clinical Applications of Serum Free Light Chain Analysis (plus Hevylite)

    OpenAIRE

    Guest Editor: A.R. Bradwell

    2011-01-01

    The 6th International Symposium on Clinical Applications of Serum Free Light Chain Analysis (plus Hevylite) was held in Bath, UK on 23rd and 24th September 2010. This Symposium included measurements in monoclonal gammopathies, myeloma kidney and inflammatory diseases; data from the Multi-centre, Phase III, Randomised control trial on extended haemodialysis with the Gambro protein-leaking HCO 1100 dialyser; and presentations on Hevylite (intact immunoglobulin kappa/lambda ratios) in a variety ...

  3. Spinon and bound-state excitation light cones in Heisenberg XXZ chains

    Science.gov (United States)

    de Paula, A. L.; Bragança, H.; Pereira, R. G.; Drumond, R. C.; Aguiar, M. C. O.

    2017-01-01

    We investigate the out-of-equilibrium dynamics after a local quench that connects two spin-1/2 XXZ chains prepared in the ground state of the Hamiltonian in different phases, one in the ferromagnetic phase and the other in the critical phase. We analyze the time evolution of the on-site magnetization and bipartite entanglement entropy via adaptive time-dependent density matrix renormalization group. In systems with short-range interactions, such as the one we consider, the velocity of information transfer is expected to be bounded, giving rise to a light-cone effect. Interestingly, our results show that, when the anisotropy parameter of the critical chain is sufficiently close to that of the isotropic ferromagnet, the light cone is determined by the velocity of spin-wave bound states that propagate faster than single-particle ("spinon") excitations. Furthermore, we investigate how the system approaches equilibrium in the inhomogeneous ground state of the connected system, in which the ferromagnetic chain induces a nonzero magnetization in the critical chain in the vicinity of the interface.

  4. Dynein prevents erroneous kinetochore-microtubule attachments in mitosis.

    Science.gov (United States)

    Barisic, Marin; Maiato, Helder

    2015-01-01

    Equal distribution of the genetic material during cell division relies on efficient congression of chromosomes to the metaphase plate. Prior to their alignment, the Dynein motor recruited to kinetochores transports a fraction of laterally-attached chromosomes along microtubules toward the spindle poles. By doing that, Dynein not only contributes to chromosome movements, but also prevents premature stabilization of end-on kinetochore-microtubule attachments. This is achieved by 2 parallel mechanisms: 1) Dynein-mediated poleward movement of chromosomes counteracts opposite polar-ejection forces (PEFs) on chromosome arms by the microtubule plus-end-directed motors chromokinesins. Otherwise, they could stabilize erroneous syntelic kinetochore-microtubule attachments and lead to the random ejection of chromosomes away from the spindle poles; and 2) By transporting chromosomes to the spindle poles, Dynein brings the former to the zone of highest Aurora A kinase activity, further destabilizing kinetochore-microtubule attachments. Thus, Dynein plays an important role in keeping chromosome segregation error-free by preventing premature stabilization of kinetochore-microtubule attachments near the spindle poles.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2017-08-08

    Objective: To investigate the value of serum free light chain (FLC) in differential diagnosis of monoclonal gammopathy of renal significance (MGRS). Methods: Forty-nine hospitalized patients who underwent renal biopsy in Peking Union Medical College Hospital between January 2013 and December 2015 were included. Monoclonal gammopathy was detected by serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE), urine IFE and serum FLC. All patients were classified as MGRS (n=32) and monoclonal gammopathy of undetermined significance (MGUS) (n=17). Results: Renal lesions in MGRS subgroup included light chain amyloidosis (n=24, 75.0%), light chain deposition disease (n=7, 21.9%), and fibrillary glomerulopathy (n=1, 3.1%). Renal diseases in MGUS subgroup included membranous nephropathy (n=10), focal segmental glomerulosclerosi (FSGS) (n=3), diabetic glomerulopathy (n=1), Henoch-Schonlein purpura nephritis (n=1), anti-GBM disease concurrent with membranous nephropathy (n=1) and glomerulomegaly (n=1). Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGRS subgroup were 12, 16, 23 and 30, respectively. Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGUS subgroup were 11, 17, 6 and 3, respectively. MGRS and MGUS subgroups differed significantly in positive rate of serum IFE (Pvalue for MGRS, which was helpful for differential diagnosis of patients who had contraindication to renal biopsy.

  7. Computational design of a specific heavy chainlight chain interface for expressing fully IgG bispecific antibodies.

    Science.gov (United States)

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

    2017-07-20

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

  8. Genomic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest coevolution of immunoglobulin gene organization.

    Science.gov (United States)

    Shamblott, M J; Litman, G W

    1989-01-01

    The genomic organization and sequence of immunoglobulin light chain genes in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, have been characterized. Light chain variable (VL) and joining (JI) segments are separated by 380 nucleotides and together with the single constant region exon (CI), occupy less than 2.7 kb, the closest linkage described thus far for a rearranging gene system. The VL segment is flanked by a characteristic recombination signal sequence possessing a 12 nucleotide spacer; the recombination signal sequence flanking the JL segment is 23 nucleotides. The VL genes, unlike heavy chain genes, possess a typical upstream regulatory octamer as well as conserved enhancer core sequences in the intervening sequence separating JL and CL. Restriction mapping and genomic Southern blotting are consistent with the presence of multiple light chain gene clusters. There appear to be considerably fewer light than heavy chain genes. Heavy and light chain clusters show no evidence of genomic linkage using field inversion gel electrophoresis. The findings of major differences in the organization and functional rearrangement properties of immunoglobulin genes in species representing different levels of vertebrate evolution, but consistent similarity in the organization of heavy and light chain genes within a species, suggests that these systems may be coevolving. Images PMID:2511000

  9. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

    DEFF Research Database (Denmark)

    Merveille, Anne-Christine; Davis, Erica E; Becker-Heck, Anita

    2011-01-01

    cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre...... with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex....

  10. Identifikation und Charakterisierung von Medium Chain Acyl-CoA Dehydrogenase und Myosin Light Chain 4 als neuartige Interaktionspartner des Östrogenrezeptor alpha

    OpenAIRE

    Schanz, Miriam A.

    2012-01-01

    Estrogens (E2) are key regulators of growth, differentiation and physiological processes in various target tissues, including the human heart. E2 exerts its effects mainly through its cognate receptors, e.g.estrogen receptor (ER) α. ERα acts in concert with other interaction partners to mediate estrogenic effects. So far, only few interaction partners of ERα are known in the human myocardium. This project identified Medium Chain Acyl-CoA Dehydrogenase (MCAD) and atrial Myosin Light Chain ...

  11. Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements.

    Science.gov (United States)

    Toropova, Katerina; Mladenov, Miroslav; Roberts, Anthony J

    2017-05-01

    Cilia are multifunctional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multimotor assemblies with kinesin-II. Combining these data with insights from single-particle EM, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2's mechanical 'linker' and 'stalk' domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2's recycling activity during IFT.

  12. Building complexity: an in vitro study of cytoplasmic dynein with in vivo implications.

    Science.gov (United States)

    Mallik, Roop; Petrov, Dmitri; Lex, S A; King, S J; Gross, S P

    2005-12-06

    Cytoplasmic dynein is the molecular motor responsible for most retrograde microtubule-based vesicular transport. In vitro single-molecule experiments suggest that dynein function is not as robust as that of kinesin-1 or myosin-V because dynein moves only a limited distance (approximately 800 nm) before detaching and can exert a modest (approximately 1 pN) force. However, dynein-driven cargos in vivo move robustly over many microns and exert forces of multiple pN. To determine how to go from limited single-molecule function to robust in vivo transport, we began to build complexity in a controlled manner by using in vitro experiments. We show that a single cytoplasmic dynein motor frequently transitions into an off-pathway unproductive state that impairs net transport. Addition of a second (and/or third) dynein motor, so that cargos are moved by two (or three) motors rather than one, is sufficient to recover several properties of in vivo motion; such properties include long cargo travels, robust motion, and increased forces. Part of this improvement appears to arise from selective suppression of the unproductive state of dynein rather than from a fundamental change in dynein's mechanochemical cycle. Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.

  13. Clinical assessment of serum myosin light chain I in patients with dilated cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Tsuda, Takashi; Izumi, Tohru; Shibata, Akira (Niigata Univ. (Japan). School of Medicine)

    1992-08-01

    Serum cardiac myosin light chain I (LCI) levels were quantitated using a radioimmunoassay kit in patients suspected of dilated cardiomyopathy (DCM). In this study, 55 patients were evaluated between 1986 and 1991. They were composed of 40 males and 15 females, and their age was 27-75 years (51[+-]11 years). The patients with renal dysfunction were excluded due to their serum creatinine levels (>2.0 mg/dl). After cardiac catheterization, endomyocardial biopsy and echocardiography, 44 patients were diagnosed as DCM, 2 as ischemic heart disease, 2 as chronic myocarditis, 1 as restrictive cardiomyopathy, 1 as dilated hypertrophic cardiomyopathy, 1 as cardiac amyloidosis, 2 as myopathy, 1 as polymyositis and 1 as hypothyroidism. Only two patients with DCM had elevated LCI. Besides, two patients with myopathy or hypothyroidism had elevated LCI. In the follow-up, one patient died suddenly 6 months later and another showed normal value of LCI four years later. LCI elevation in DCM was not related to either the severity of heart failure or cardiac function and it showed no finding of [sup 201]Tl myocardial defect or elevated CPK. The mechanism for elevated LCI in myopathy is related to a crossreaction with myosin light chain in the skeletal muscle. In hypothyroidism, it may be related to decreased clearance of normal LCI concentration or increased myosin light chain from damaged skeletal muscle. In conclusion, it is evident that the measurement of LCI is not helpful in clinical assessment of patients with DCM, but may be useful in detection of secondary cardiomyopathy. (author).

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

    OpenAIRE

    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 ...

  15. Super-light and pearl-chain technology for support of ancient structures

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl; Schmidt, Jacob Wittrup; Goltermann, Per

    2014-01-01

    optimal and often curved paths of strong concrete assembled from smaller segments by prestressing wires, so that expensive curved moulds and supports can be avoided. Pearl-chains can provide a resistance to impact and earthquake of ancient structures. High-strength concrete and prestressed carbon fibre...... reinforcement may be applied, because the new technology solves the main problems for that, since the light aggregate concrete provides a fire protection needed for both materials and provides a stabilization of the slender cores in compression. The paper explains more about the new technology...

  16. Effect of specimen type on free immunoglobulin light chains analysis on the Roche Diagnostics cobas 8000 analyzer.

    Science.gov (United States)

    Nelson, Louis S; Steussy, Bryan; Morris, Cory S; Krasowski, Matthew D

    2015-01-01

    The measurement of free immunoglobulin light chains is typically performed on serum; however, the use of alternative specimen types has potential benefits. Using the Freelite™ kappa and lambda free light chains assay on a Roche Diagnostics cobas 8000 c502 analyzer, we compared three specimen types (serum, EDTA-plasma and lithium heparin plasma separator gel-plasma) on 100 patients. Using Deming regression and eliminating outliers (limiting data to light chain concentrations below 400 mg/L), the three specimen types showed comparable results for kappa light chain concentration, lambda light chain concentration, and kappa/lambda ratio with slopes close to 1.0 and y-intercepts close to zero. EDTA-plasma showed slightly more positive bias relative to serum than lithium heparin. Analysis using EDTA-plasma and lithium heparin plasma showed comparable linearity, precision, and temperature stability. A single sample showing hook effect (not in the comparison set) gave comparable results using either plasma specimen type. For the Freelite™ kappa and lambda free light chains assay, both EDTA-plasma or lithium heparin-plasma can serve as acceptable substitutes for serum, at least for the Roche cobas 8000 analyzer.

  17. Reversible Addition-Fragmentation Chain Transfer Polymerization of Acrylonitrile under Irradiation of Blue LED Light

    Directory of Open Access Journals (Sweden)

    Zhicheng Huang

    2016-12-01

    Full Text Available Compared to unhealthy UV or γ-ray and high-energy-consumption thermal external stimuli, the promising light emitting diode (LED external stimulus has some outstanding technological merits such as narrow wavelength distribution, low heat generation and energy consumption, and safety for human beings. In this work, a novel reversible addition-fragmentation transfer (RAFT polymerization system for acrylonitrile (AN was developed under the irradiation of blue LED light at room temperature, using 1,2,3,5-tetrakis(carbazol-9-yl-4,6-dicyanobenzene (4CzIPN as a novel radical initiator and 2-cyanoprop-2-yl-1-dithionaphthalate (CPDN as the typical chain transfer agent. Well-defined polyacrylonitrile (PAN with a controlled molecular weight and narrow molecular weight distribution was successfully synthesized. This strategy may provide another effective method for scientific researchers or the industrial community to synthesize a PAN-based precursor of carbon fibers.

  18. Bos taurus genome sequence reveals the assortment of immunoglobulin and surrogate light chain genes in domestic cattle

    Directory of Open Access Journals (Sweden)

    Liljavirta Jenni

    2009-04-01

    Full Text Available Abstract Background The assortment of cattle immunoglobulin and surrogate light chain genes has been extracted from the version 3.1 of Bos taurus genome sequence as a part of an international effort to sequence and annotate the bovine genome. Results 63 variable lambda chain and 22 variable kappa chain genes were identified and phylogenetically assigned to 8 and 4 subgroups, respectively. The specified phylogenetic relationships are compatible with the established ruminant light chain variable gene families or subgroups. Because of gaps and uncertainties in the assembled genome sequence, the number of genes might change in the future versions of the genome sequence. In addition, three bovine surrogate light chain genes were identified. The corresponding cDNAs were cloned and the expression of the surrogate light chain genes was demonstrated from fetal material. Conclusion The bovine kappa gene locus is compact and simple which may reflect the preferential use of the lambda chain in cattle. The relative orientation of variable and joining genes in both loci are consistent with a deletion mechanism in VJ joining. The orientation of some variable genes cannot be determined from the data available. The number of functional variable genes is moderate when compared to man or mouse. Thus, post-recombinatorial mechanisms might contribute to the generation of the bovine pre-immune antibody repertoire. The heavy chains probably contribute more to recombinational immunoglobulin repertoire diversity than the light chains but the heavy chain locus could not be annotated from the version 3.1 of Bos taurus genome.

  19. Immunohistochemical and morphometric analysis of immunoglobulin light-chain immunoreactive amyloid in psammoma bodies of the human choroid plexus.

    Science.gov (United States)

    Jovanović, Ivan; Ugrenović, Sladjana; Vasović, Ljiljana; Stojanović, Ivan

    2014-03-01

    The aim of this research was to establish the presence of amyloid and to quantify immunohistochemical reactions of kappa and lambda light chains of psammoma bodies of the choroid plexus. Choroid plexus tissue obtained from 14 right lateral ventricles postmortem was processed histologically and stained with Congo red, thioflavin T, and monoclonal antibodies for kappa and lambda light chains. Morphological analysis was performed with a light microscope at lens magnifications of 4×, 10×, 20×, 25×, and 40×. The morphometric characteristics of psammoma bodies that were kappa and lambda positive and negative were analyzed with ImageJ. Histological analysis showed that the psammoma bodies, stromal blood vessel walls, and some epithelial cells reacted positively with Congo red and thioflavin T. Psammoma bodies were predominantly positive for lambda light chains. Lambda positivity was detected inside some stromal blood vessels, which pointed to a probable systemic origin for these light chains. Morphometric analysis showed that the mean optical densities of lambda- and kappa-positive psammoma bodies were significantly higher than those that gave a negative reaction. The percentage of lambda-positive psammoma bodies was significantly higher than the percentage of lambda-negative psammoma bodies in 80% of the cases, while the reaction with kappa light chains was negative in the majority of the cases. Linear regression analysis showed a significant increase in the percentage of lambda-positive psammoma bodies and their mean optical density with age. Finally, it can be concluded that the positive reaction of psammoma bodies in the choroid plexus with respect to amyloid and lambda light chains may point to the presence of light-chain amyloid in their structures.

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

    Science.gov (United States)

    Glenney, J R

    1990-01-01

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

  1. Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age.

    Science.gov (United States)

    Belik, J; Kerc, Ewa; Pato, Mary D

    2006-03-01

    We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cdelta) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cdelta contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.

  2. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    Science.gov (United States)

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

  3. Regulatory light chain phosphorylation increases eccentric contraction-induced injury in skinned fast-twitch fibers.

    Science.gov (United States)

    Childers, Martin K; McDonald, Kerry S

    2004-02-01

    During contraction, activation of Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) results in phosphorylation of myosin's regulatory light chain (RLC), which potentiates force and increases speed of force development over a wide range of [Ca(2+)]. We tested the hypothesis that RLC phosphorylation by MLCK mediates the extent of eccentric contraction-induced injury as measured by force deficit in skinned fast-twitch skeletal muscle fibers. Results indicated that RLC phosphorylation in single skinned rat psoas fibers significantly increased Ca(2+) sensitivity of isometric force; isometric force from 50 +/- 16 to 59 +/- 18 kN/m(2) during maximal Ca(2+) activation; peak absolute power output from 38 +/- 15 to 48 +/- 14 nW during maximal Ca(2+) activation; and the magnitude of contraction-induced force deficit during maximal (pCa 4.5) activation from 26 +/- 9.8 to 35 +/- 9.6%. We conclude that RLC phosphorylation increases force deficits following eccentric contractions, perhaps by increasing the number of force-generating cross-bridges.

  4. A Novel Myosin Essential Light Chain Mutation Causes Hypertrophic Cardiomyopathy with Late Onset and Low Expressivity

    Directory of Open Access Journals (Sweden)

    Paal Skytt Andersen

    2012-01-01

    Full Text Available Hypertrophic cardiomyopathy (HCM is caused by mutations in genes encoding sarcomere proteins. Mutations in MYL3, encoding the essential light chain of myosin, are rare and have been associated with sudden death. Both recessive and dominant patterns of inheritance have been suggested. We studied a large family with a 38-year-old asymptomatic HCM-affected male referred because of a murmur. The patient had HCM with left ventricular hypertrophy (max WT 21 mm, a resting left ventricular outflow gradient of 36 mm Hg, and left atrial dilation (54 mm. Genotyping revealed heterozygosity for a novel missense mutation, p.V79I, in MYL3. The mutation was not found in 300 controls, and the patient had no mutations in 10 sarcomere genes. Cascade screening revealed a further nine heterozygote mutation carriers, three of whom had ECG and/or echocardiographic abnormalities but did not fulfil diagnostic criteria for HCM. The penetrance, if we consider this borderline HCM the phenotype of the p.V79I mutation, was 40%, but the mean age of the nonpenetrant mutation carriers is 15, while the mean age of the penetrant mutation carriers is 47. The mutation affects a conserved valine replacing it with a larger isoleucine residue in the region of contact between the light chain and the myosin lever arm. In conclusion, MYL3 mutations can present with low expressivity and late onset.

  5. Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

    Science.gov (United States)

    Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B. Cicero; Zarzoso, Manuel; Ramirez, Rafael J.; Sener, Michelle F.; Mundada, Lakshmi V.; Klos, Matthew; Devaney, Eric J.; Vikstrom, Karen L.; Herron, Todd J.; Jalife, José

    2014-01-01

    Applications of human induced pluripotent stemcell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricularmyocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. PMID:24095945

  6. [Ontogenetic and phylogenetic analysis of myosin light chain proteins from skeletal muscles of loach Misgurnus fossilis].

    Science.gov (United States)

    Miuge, N S; Tikhonov, A V; Ozerniuk, N D

    2005-01-01

    mRNAs of all three types of myosin light chain proteins are expressed in skeletal muscles of both larval and adult stages of loach Misgurnus fossilis (Cobitidae) and these proteins are encoded by different genes (mlc1, mlc2, and mlc3). No difference was revealed between transcripts from larval stage and adult fish for all three mlc proteins. Our approach (RT-PCR with fish-specific mlc1, mlc2, and mlc3 primers) failed to reveal the larval form of myosin light chain protein found previously by protein electrophoresis of loach fry muscle extract. Comparative analysis of the protein structure shows high homology of MLC1 and MLC3 proteins sharing a large EF-hand calcium-binding domain. Phylogenetic analysis of MLC1 from skeletal muscles of fish and other vertebrate species is concordant with the traditional phylogeny of the group. Within the Teleostei, loach MLC1 had the highest homology with other Cyprinidae, and least with Salmonidae fishes.

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

    Science.gov (United States)

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

    2004-04-20

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

  8. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis

    NARCIS (Netherlands)

    Eisel, Ulrich; Reynolds, Kay; Riddick, Michelle; Zimmer, Anne; Niemann, Heiner; Zimmer, Andreas; Gruss, P.

    Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light

  9. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis

    NARCIS (Netherlands)

    Eisel, Ulrich; Reynolds, Kay; Riddick, Michelle; Zimmer, Anne; Niemann, Heiner; Zimmer, Andreas; Gruss, P.

    1993-01-01

    Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light c

  10. Inhibition of microtubules and dynein rescues human immunodeficiency virus type 1 from owl monkey TRIMCyp-mediated restriction in a cellular context-specific fashion.

    Science.gov (United States)

    Pawlica, Paulina; Dufour, Caroline; Berthoux, Lionel

    2015-04-01

    IFN-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. TRIM5α (tripartite motif protein 5, isoform α) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5α maps to the N-terminal domain of the retroviral capsid proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA)-coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits human immunodeficiency virus type 1 (HIV-1). Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5α. Here, we showed that efficient restriction of HIV-1 by omkTRIMCyp was similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by small interfering RNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggested the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp. © 2015 The Authors.

  11. Differences in potential for amino acid change after mutation reveals distinct strategies for kappa and lambda light-chain variation.

    Science.gov (United States)

    Hershberg, Uri; Shlomchik, Mark J

    2006-10-24

    B cells generate varied yet functional clones under high rates of mutation of their V genes. It has been proposed that as a result of the opposing demands of diversification and preservation of integrity, the V genes of heavy and light chains have evolved to overexpress codons prone to amino acid change in their complementarity determining regions (CDR) compared with the framework (FW) regions. We have analyzed the germ-line V genes of heavy and light chains (both kappa and lambda), comparing codons of CDR and FW of the germ-line V regions both to each other and to control regions. We found that in both germ-line heavy chains and lambda chains, CDR codons are prone to replacement mutations, whereas in the FW, the opposite is true. Furthermore, the difference between CDR and FW in heavy chains and lambda chains is based on codons that are prone to nonconservative changes of amino acid. In contrast, in germ-line kappa chains, the codons in both CDR and FW are more prone to replacement mutations. We also demonstrated that negative selection during immune responses is more sensitive to nonconservative amino acid substitutions than overall amino acid change, demonstrating the applicability of our analysis to real-time process of selection in the immune system. The differences in germ-line kappa and lambda light chains' potential reaction to mutation suggests that via these two differently evolved light-chain types, the B cell repertoire encompasses two different strategies to balance diversity and stability in an immune response.

  12. Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons.

    Science.gov (United States)

    del Castillo, Urko; Winding, Michael; Lu, Wen; Gelfand, Vladimir I

    2015-12-28

    In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides 'minus-end-out' microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein.

  13. Four primordial immunoglobulin light chain isotypes, including lambda and kappa, identified in the most primitive living jawed vertebrates.

    Science.gov (United States)

    Criscitiello, Michael F; Flajnik, Martin F

    2007-10-01

    The discovery of a fourth immunoglobulin (Ig) light (L) chain isotype in sharks has revealed the origins and natural history of all vertebrate L chains. Phylogenetic comparisons have established orthology between this new shark L chain and the unique Xenopus L chain isotype sigma. More importantly, inclusion of this new L chain family in phylogenetic analyses showed that all vertebrate L chains can be categorized into four ancestral clans originating prior to the emergence of cartilaginous fish: one restricted to elasmobranchs (sigma-cart/type I), one found in all cold-blooded vertebrates (sigma/teleost type 2/elasmobranch type IV), one in all groups except bony fish (lambda/elasmobranch type II), and one in all groups except birds (kappa/elasmobranch type III/teleost type 1 and 3). All four of these primordial L chain isotypes (sigma, sigma-cart, lambda and kappa) have maintained separate V region identities since their emergence at least 450 million years ago, suggestive of an ancient physiological distinction of the L chains. We suggest that, based upon unique, discrete sizes of complementarity determining regions 1 and 2 and other features of the V region sequences, the different L chain isotypes arose to provide different functional conformations in the Ig binding site when they pair with heavy chains.

  14. Characteristics and Long-Term Outcome of Patients with Systemic Immunoglobulin Light-Chain Amyloidosis

    DEFF Research Database (Denmark)

    Nelson, Lærke Marie; Gustafsson, Finn; Gimsing, Peter

    2014-01-01

    and predominantly originate from highly selected centers. We aimed to evaluate patient characteristics and outcomes in a cohort treated at a single center. Methods: This is a single-center retrospective study of 63 consecutive patients diagnosed with AL amyloidosis between January 2000 and December 2012. Patients......Background/Aims: Immunoglobulin light-chain (AL) amyloidosis is a systemic disorder that causes progressive organ dysfunction. The optimal treatment strategy requires accurate patient stratification with an emphasis on the extent of cardiac involvement. Reports on its prognosis are sparse...... were evaluated by treatment strategy and cardiac involvement. Results: The mean age at diagnosis was 61.4 years (±8.9), and 39 patients (62%) were male. Thirty-two (51%) patients presented with cardiac amyloid involvement (CA) and the remaining 31 (49%) had noncardiac amyloidosis (NCA). The median...

  15. Measurement of diversification in the immunoglobulin light chain gene of DT40 cells.

    Science.gov (United States)

    Sale, Julian E

    2012-01-01

    The immunoglobulin loci of the genetically tractable chicken B cell line DT40 provide a unique opportunity to study the cellular response to endogenously generated DNA damage in a chromosomal context. Abasic sites generated by the concerted action of Activation-Induced Deaminase (AID) and Uracil DNA Glycosylase result in both homologous recombination-dependent gene conversion and translesion synthesis-dependent point mutations. The system has provided important insights into both the early stages of AID-dependent immunoglobulin gene diversification and into the relationship between pathways of DNA damage bypass. Here we describe the assays that can be employed to monitor the rate and pattern of immunoglobulin gene diversification at the light chain locus of DT40.

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

    Science.gov (United States)

    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.

  17. Induction therapy pre-autologous stem cell transplantation in immunoglobulin light chain amyloidosis: a retrospective evaluation.

    Science.gov (United States)

    Hwa, Yi L; Kumar, Shaji K; Gertz, Morie A; Lacy, Martha Q; Buadi, Francis K; Kourelis, Taxiarchis V; Gonsalves, Wilson I; Rajkumar, S Vincent; Go, Ronald S; Leung, Nelson; Kapoor, Prashant; Dingli, David; Kyle, Robert A; Russell, Stephen; Lust, John A; Hayman, Suzanne R; Lin, Yi; Zeldenrust, Steven; Dispenzieri, Angela

    2016-10-01

    There is no consensus on whether patients with immunoglobulin light chain amyloidosis (AL) should receive induction therapy prior to an autologous stem cell transplant (ASCT). This study investigated the relationships between baseline bone marrow plasmacytosis (BMPC), cardiac staging, and pre-transplant induction in AL patients. All patients who received ASCT for AL within 12 months of diagnosis were included. Patient characteristics and outcomes were abstracted. Univariate and multivariate modeling was performed. Among 415 AL patients, 35% had induction prior to ASCT. Post-ASCT hematologic CR plus VGPR rates were significantly higher in those with baseline BMPC ≤ 10% compared to BMPC >10% (58% versus 40%, P = 0.0013). Significant risk factors for lack of attainment of CR included attenuated dose melphalan conditioning, baseline BMPC > 10%, no induction, and male gender. The 5-year OS for the entire group was 65%. On multivariate analysis, risk factors for inferior OS included no induction therapy, advanced AL amyloid staging, BMPC > 10%, attenuated conditioning melphalan dose, and male gender. Patients with Mayo 2012 stage I-II patients with BMPC ≤ 10%, who comprised 56% of the ASCT population fared exceedingly well regardless of whether or not they received induction therapy with a 5-year OS of 81 to 83%. Induction therapy pre-ASCT may improve outcomes among AL patients due to a rapid reduction of toxic light chains or alternatively by elimination of less fit patients by "testing" their ability to tolerate chemotherapy. Prospective studies will be required to sort out these and other questions. Am. J. Hematol. 91:984-988, 2016. © 2016 Wiley Periodicals, Inc.

  18. Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish

    Science.gov (United States)

    Mishra, Shikha; Guan, Jian; Plovie, Eva; Seldin, David C.; Connors, Lawreen H.; Merlini, Giampaolo; Falk, Rodney H.; MacRae, Calum A.

    2013-01-01

    Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies. PMID:23624626

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

    Directory of Open Access Journals (Sweden)

    European Uremic Toxin (EUTox Work Group

    2013-11-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  1. 5DFRXXL region of long myosin light chain kinase causes F-actin bundle formation

    Institute of Scientific and Technical Information of China (English)

    YANG Chunxiang; WEI Dongmei; CHEN Chen; YU Weiping; ZHU Minsheng

    2005-01-01

    Long myosin light chain kinase (L-MLCK) contains five DFRXXL motifs with ability to bind F-actin. Binding stoichiometry data indicated that each DFRXXL motif might bind each G-actin, but its biological significance remained unknown. We hypothesized that L-MLCK might act as an F-actin bundle peptides by its multiple binding sites of 5DFRXXL motifs to actin. In order to characterize F-actin-bundle formation properties of 5DFRXXL region of long myosin light chain kinase, we expressed and purified 5DFRXXL peptides tagged with HA in vitro. The properties of 5DFRXXL peptides binding to myofilaments or F-actin were analyzed by binding stoichiometries assays. The results indicated that 5DFRXXL peptides bound to myofilaments or F-actin with high affinity. KD values of 5DFRXXL binding to myofilaments and F-actin were 0.45 and 0.41 μmol/L, re- spectively. Cross-linking assay demonstrated that 5DFRXXL peptides could bundle F-actin efficiently. Typical F-actin bundles were observed morphologically through determina- tion of confocal and electron microscopy after adding 5DFRXXL peptides. After transfection of pEGFP-5DFRXXL plasmid into eukaryocyte, spike structure was observed around cell membrane edge. We guess that such structure formation may be attributable to F-actin over-bundle forma- tion caused by 5DFRXXL peptides. Therefore, we suppose that L-MLCK may be a new bundling protein and somehow play a certain role in organization of cell skeleton besides mediating cell contraction by it kinase activity.

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

    Science.gov (United States)

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

    2012-12-01

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

  3. Elevated neurofilament light chain (NFL) mRNA levels in prediabetic peripheral neuropathy.

    Science.gov (United States)

    Celikbilek, Asuman; Tanik, Nermin; Sabah, Seda; Borekci, Elif; Akyol, Lutfi; Ak, Hakan; Adam, Mehmet; Suher, Murat; Yilmaz, Neziha

    2014-06-01

    Evidence suggests that peripheral nerve injury occurs during the early stages of disease with mild glycemic dysregulation. Two proteins, neuron-specific enolase (NSE) and neurofilament light chain (NFL), have been examined previously as possible markers of neuronal damage in the pathophysiology of neuropathies. Herein, we aimed to determine the potential value of circulatory NSE and NFL mRNA levels in prediabetic patients and in those with peripheral neuropathy. This prospective clinical study included 45 prediabetic patients and 30 age- and sex-matched controls. All prediabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy and nephropathy. mRNA levels of NSE and NFL were determined in the blood by real-time polymerase chain reaction. NSE mRNA levels were similar between prediabetic and control groups (p > 0.05), whereas NFL mRNA levels were significantly higher in prediabetics than in controls (p 0.05), while NFL mRNA levels were significantly higher in prediabetics with peripheral neuropathy than in those without (p = 0.038). According to correlation analysis, NFL mRNA levels were positively correlated with the Douleur Neuropathique 4 questionnaire score in prediabetic patients (r = 0.302, p = 0.044). This is the first study to suggest blood NFL mRNA as a surrogate marker for early prediction of prediabetic peripheral neuropathy, while NSE mRNA levels may be of no diagnostic value in prediabetic patients.

  4. BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

    Science.gov (United States)

    Splinter, Daniël; Razafsky, David S.; Schlager, Max A.; Serra-Marques, Andrea; Grigoriev, Ilya; Demmers, Jeroen; Keijzer, Nanda; Jiang, Kai; Poser, Ina; Hyman, Anthony A.; Hoogenraad, Casper C.; King, Stephen J.; Akhmanova, Anna

    2012-01-01

    Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors. PMID:22956769

  5. Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

    Science.gov (United States)

    Karak, Somdatta; Jacobs, Julie S; Kittelmann, Maike; Spalthoff, Christian; Katana, Radoslaw; Sivan-Loukianova, Elena; Schon, Michael A; Kernan, Maurice J; Eberl, Daniel F; Göpfert, Martin C

    2015-11-26

    Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

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

    Science.gov (United States)

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

    2014-03-01

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

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

    Science.gov (United States)

    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.

  8. Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains.

    Science.gov (United States)

    del Pozo Yauner, Luis; Ortiz, Ernesto; Sánchez, Rosalba; Sánchez-López, Rosana; Güereca, Leopoldo; Murphy, Charles L; Allen, Amy; Wall, Jonathan S; Fernández-Velasco, D Alejandro; Solomon, Alan; Becerril, Baltazar

    2008-08-01

    Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.

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

    Directory of Open Access Journals (Sweden)

    Paolo Arosio

    Full Text Available 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(2PO(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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  12. Chlamydomonas axonemal dynein assembly locus ODA8 encodes a conserved flagellar protein needed for cytoplasmic maturation of outer dynein arm complexes.

    Science.gov (United States)

    Desai, Paurav B; Freshour, Judy R; Mitchell, David R

    2015-01-01

    The Chlamydomonas reinhardtii oda8 mutation blocks assembly of flagellar outer dynein arms (ODAs), and interacts genetically with ODA5 and ODA10, which encode axonemal proteins thought to aid dynein binding onto axonemal docking sites. We positionally cloned ODA8 and identified the gene product as the algal homolog of vertebrate LRRC56. Its flagellar localization depends on ODA5 and ODA10, consistent with genetic interaction studies, but phylogenomics suggests that LRRC56 homologs play a role in intraflagellar transport (IFT)-dependent assembly of outer row dynein arms, not axonemal docking. ODA8 distribution between cytoplasm and flagella is similar to that of IFT proteins and about half of flagellar ODA8 is in the soluble matrix fraction. Dynein extracted in vitro from wild type axonemes will rebind efficiently to oda8 mutant axonemes, without re-binding of ODA8, further supporting a role in dynein assembly or transport, not axonemal binding. Assays comparing preassembled ODA complexes from the cytoplasm of wild type and mutant strains show that dynein in oda8 mutant cytoplasm has not properly preassembled and cannot bind normally onto oda axonemes. We conclude that ODA8 plays an important role in formation and transport of mature dynein complexes during flagellar assembly.

  13. Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself

    National Research Council Canada - National Science Library

    McCrudden, Cian M; O'Rourke, Martin G; Cherry, Kim E; Yuen, Hiu-Fung; O'Rourke, Declan; Babur, Muhammad; Telfer, Brian A; Thomas, Huw D; Keane, Patrick; Nambirajan, Thiagarajan; Hagan, Chris; O'Sullivan, Joe M; Shaw, Chris; Williams, Kaye J; Curtin, Nicola J; Hirst, David G; Robson, Tracy

    2015-01-01

    ...), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK...

  14. Vasoactivity of Rucaparib, a PARP-1 Inhibitor, is a Complex Process that Involves Myosin Light Chain Kinase, P2 Receptors, and PARP Itself: e0118187

    National Research Council Canada - National Science Library

    Cian M McCrudden; Martin G O'Rourke; Kim E Cherry; Hiu-Fung Yuen; Declan O'Rourke; Muhammad Babur; Brian A Telfer; Huw D Thomas; Patrick Keane; Thiagarajan Nambirajan; Chris Hagan; Joe M O'Sullivan; Chris Shaw; Kaye J Williams; Nicola J Curtin; David G Hirst; Tracy Robson

    2015-01-01

    ...), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK...

  15. A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  17. A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli.

    Directory of Open Access Journals (Sweden)

    Paola Rognoni

    Full Text Available 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.

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

    Science.gov (United States)

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

    2016-10-01

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

  19. Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

    Science.gov (United States)

    Kokovic, Ira; Jezersek Novakovic, Barbara; Novakovic, Srdjan

    2015-03-01

    Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2011-09-30

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

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

    Science.gov (United States)

    Zhao, Lei; Tian, Zhuang; Fang, Quan

    2016-03-01

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

  3. CCDC151 Mutations Cause Primary Ciliary Dyskinesia by Disruption of the Outer Dynein Arm Docking Complex Formation

    NARCIS (Netherlands)

    Hjeij, R.; Onoufriadis, A.; Watson, C.M.; Slagle, C.E.; Klena, N.T.; Dougherty, G.W.; Kurkowiak, M.; Loges, N.T.; Diggle, C.P.; Morante, N.F.; Gabriel, G.C.; Lemke, K.L.; Li, Y.; Pennekamp, P.; Menchen, T.; Konert, F.; Marthin, J.K.; Mans, D.A.; Letteboer, S.J.F.; Werner, C.; Burgoyne, T.; Westermann, C.; Rutman, A.; Carr, I.M.; O'Callaghan, C.; Moya, E.; Chung, E.M.; Consortium, U.K.; Sheridan, E.; Nielsen, K.G.; Roepman, R.; Bartscherer, K.; Burdine, R.D.; Lo, C.W.; Omran, H.; Mitchison, H.M.

    2014-01-01

    A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, p

  4. An Association between Neutrophils and Immunoglobulin Free Light Chains in the Pathogenesis of Chronic Obstructive Pulmonary Disease

    NARCIS (Netherlands)

    Braber, Saskia; Thio, Marco; Blokhttis, Bart R.; Henricks, Paul A. J.; Koelink, Pim J.; Kormelink, Tom Groot; Bezemer, Gillina F. G.; Kerstjens, Huib A. M.; Postma, Dirkje S.; Garssen, Johan; Kraneveld, Aletta D.; Redegeld, Frank A.; Folkerts, Gert

    2012-01-01

    Rationale: Neutrophils are key players in chronic obstructive pulmonary disease (COPD), and increased numbers of neutrophils are present in sputum and lung tissue of patients with COPD. Interestingly, immunoglobulin free light chains (IgLC) are able to prolong the life of neutrophils; therefore, IgL

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

    Science.gov (United States)

    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.

  6. Characterization and ontogenetic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi.

    Science.gov (United States)

    Chu, W Y; Chen, J; Zhou, R X; Zhao, F L; Meng, T; Chen, D X; Nong, X X; Liu, Z; Lu, S Q; Zhang, J S

    2011-04-01

    Three full-length complementary DNA (cDNA) clones were isolated encoding the skeletal myosin light chain 1 (MLC1; 1237 bp), myosin light chain 2 (MLC2; 1206 bp) and myosin light chain 3 (MLC3; 1079 bp) from the fast white muscle cDNA library of mandarin fish Siniperca chuatsi. The sequence analysis indicated that MLC1 and MLC3 were not produced from differentially spliced messenger RNAs (mRNA) as reported in birds and rodents but were encoded by different genes. The MLC2 encodes 170 amino acids, which include four EF-hand (helix-loop-helix) structures. The primary structures of the Ca(2+)-binding domain were well conserved among the MLC2s of seven other fish species. The ontogenetic expression analysis by real-time PCR showed that the three light-chain mRNAs were first detected in the gastrula stage, and their expression increased from the tail bud stage to the larval stage. All three MLC mRNAs showed longitudinal expression variation in the fast white muscle of S. chuatsi, especially MLC1 which was highly expressed at the posterior area. Taken together, the study provides a better understanding about the MLC gene structure and their expression pattern in muscle development of S. chuatsi.

  7. Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins

    Science.gov (United States)

    Namdeo, S.; Onck, P. R.

    2016-10-01

    Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dyneins. The dyneins deliver the required mechanical work through the hydrolysis of ATP. Although the dynein-ATP cycle, the axoneme microstructure, and the flagellar-beating kinematics are well studied, their integration into a coherent picture of ATP-powered flagellar beating is still lacking. Here we show that a time-delayed negative-work-based switching mechanism is able to convert the individual sliding action of hundreds of dyneins into a regular overall beating pattern leading to propulsion. We developed a computational model based on a minimal representation of the axoneme consisting of two representative doublet microtubules connected by nexin links. The relative sliding of the microtubules is incorporated by modeling two groups of ATP-powered dyneins, each responsible for sliding in opposite directions. A time-delayed switching mechanism is postulated, which is key in converting the local individual sliding action of multiple dyneins into global beating. Our results demonstrate that an overall nonreciprocal beating pattern can emerge with time due to the spatial and temporal coordination of the individual dyneins. These findings provide insights in the fundamental working mechanism of axonemal dyneins and could possibly open new research directions in the field of flagellar motility.

  8. Removal of free light chains in hemodialysis patients without multiple myeloma: a crossover comparison of three different dialyzers.

    Science.gov (United States)

    Donati, Gabriele; Moretti, Maria Ilaria; Baraldi, Olga; Spazzoli, Alessandra; Capelli, Irene; Comai, Giorgia; Marchetti, Antonio; Sarma, Maria; Mancini, Rita; La Manna, Gaetano

    2016-11-25

    Immunoglobulin light chains are classified as middle molecule uremic toxins able to interact with B lymphocyte membranes leading to the activation of transmembrane signaling. The ensuing impairment of neutrophil function can contribute to the chronic inflammation state of uremic patients, and the increased risk of bacterial infections or vascular calcifications. The aim of this crossover observational study was to assess the difference in free light chain removal by three different hemodialysis filters in patients not affected by multiple myeloma. Free light chain removal was compared in the polymethylmethacrylate (PMMA) membrane Filtryzer BK-F, the polyphenylene HFR17 filter and the conventional polysulfone filter F7HPS. Twenty chronic hemodialysis patients were enrolled: mean age was 67.7 ± 17.0 years, M/F = 14/6, dialysis vintage (months) 25.5 ± 32.0. The patients were randomized into two groups of treatment lasting 6 weeks each. The dialysis sessions checked were the midweek sessions and the blood was drawn at times 0, 120' and 240'. Kappa (k) and lambda (λ) light chain levels, β2microglobulin (β2M), C reactive protein (CRP) and albumin were checked. K light chain levels were 345.0 ± 100.0 mg/L, λ light chains were 121.4 ± 27.0 mg/L. The values of k light chains at times 120' and 240' were significantly lower with PMMA and HFR17 than those obtained with F7. The reduction ratio per session (RRs) for k light chains was 44.1 ± 4.3% with HFR17, 55.3 ± 3.4% with PMMA, 25.7 ± 8.3% with F7 (p = 0.018). The RRs for λ light chains was 30.3 ± 2.9% with HFR17, 37.8 ± 17.3% with PMMA, 14.0 ± 3.9% with F7 (p = 0.032). As to β2M, RRs was 42.4 ± 3.2% with HFR17 vs. 33.9 ± 2.8% with PMMA vs. 6.3 ± 1.9% with F7 (p = 0.022). The three filters tested showed no differences in CRP or albumin levels. In terms of light chain and β2M removal, the PMMA and on-line HFR filters are similar and

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

    Science.gov (United States)

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

    2001-08-01

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

  10. Prolonged Cerebrospinal Fluid Neurofilament Light Chain Increase in Patients with Post-Traumatic Disorders of Consciousness.

    Science.gov (United States)

    Bagnato, Sergio; Grimaldi, Luigi M E; Di Raimondo, Giorgio; Sant'Angelo, Antonino; Boccagni, Cristina; Virgilio, Vittorio; Andriolo, Maria

    2017-08-15

    The mechanisms involved in secondary brain injury after the acute phase of severe traumatic brain injury (TBI) are largely unknown. Ongoing axonal degeneration, consequent to the initial trauma, may lead to secondary brain injury. To test this hypothesis, we evaluated the cerebrospinal fluid (CSF) level of neurofilament light chain (NF-L), a proposed marker of axonal degeneration, in 10 patients who developed a severe disorder of consciousness after a TBI, including 7 in a minimally conscious state and 3 with unresponsive wakefulness syndrome (time since brain injury, 309 ± 169 days). CSF NF-L level was measured with a commercially available NF-L enzyme-linked immunosorbent assay. CSF NF-L level was very high in all 10 patients, ranging from 2.4- to 60.5-fold the upper normal limit (median value, 4458 pg/mL; range, 695-23,000). Moreover, NF-L level was significantly higher after a severe TBI than in a reference group of 9 patients with probable Alzheimer's disease, a population with elevated levels of CSF NF-L attributed to neuronal degeneration (median value, 1173 pg/mL; range, 670-3643; p < 0.01). CSF NF-L level was correlated with time post-TBI (p = 0.04). These results demonstrate prolonged secondary brain injury, suggesting that patients exhibit ongoing axonal degeneration up to 19 months after a severe TBI.

  11. [Effect of free light chain ratio normalization after treatment on prognosis of patients with multiple myeloma].

    Science.gov (United States)

    Meng, Qing-Qi; Wang, Jian-Ning; Song, Min; Bao, Hong-Yu; Hou, Yan-Qiu; Zhang, Liu-Bo; Jiang, Su-Yu

    2014-12-01

    This study was aimed to investigate the normalization of serum free light chain ratio (sPLCR) after treatment of multiple myeloma (MM) and its influence on the prognosis of MM patients. The clinical data of 42 patients with MM were analyzed retrospectively from January 2009 to November 2013 in out department. According to sPLCR consecutive normalization for more 4 weeks or not after treatment, the patients were classified in patients with mormalized sPLCR and patients with abnormalized sPLCR, then the influence of traditional prognostic factors of MM on sPLCR and effect of sPLCR on overall survival (OS) time of MM patients were analyzed. The results showed that the influence of age, ISS stage displayed statistical difference between sFLCR normalization group and abnormalization group, the age ≥ 65 years and ISS stage III negatively impacted on sFLCR normalization (P normalized sFLCR were as follows: CR - 60%, VGPR - 38.89%; PR - 28.57%; 17 patients (40.48%) with sFLCR normalization showed superior OS, as compared with patients with sPLCR abnormalization (P normalization is the independent prognostic factor for MM, suggesting that the MM patients with sPLCR normalization after treatment have superior prognosis.

  12. Cerebrospinal fluid neurofilament light chain levels predict visual outcome after optic neuritis

    DEFF Research Database (Denmark)

    Modvig, Signe; Degn, M; Sander, B

    2016-01-01

    BACKGROUND: Optic neuritis is a good model for multiple sclerosis relapse, but currently no tests can accurately predict visual outcome. OBJECTIVE: The purpose of this study was to examine whether cerebrospinal fluid (CSF) biomarkers of tissue damage and remodelling (neurofilament light chain (NF-L...... cell layer+inner plexiform layer (GC-IPL) thicknesses. RESULTS: CSF NF-L levels at onset predicted inter-ocular differences in follow-up LCVA (β=13.8, p=0.0008), RNFL (β=5.6, p=0.0004) and GC-IPL (β=4.0, p=0.0008). The acute-phase GC-IPL thickness also predicted follow-up LCVA (β=12.9, p=0.0021 for NF-L......, β=-1.1, p=0.0150 for GC-IPL). Complete/incomplete remission was determined based on LCVA from 30 healthy controls. NF-L had a positive predictive value of 91% and an area under the curve (AUC) of 0.79 for incomplete remission. CONCLUSION: CSF NF-L is a promising biomarker of visual outcome after...

  13. Serum neurofilament light chain levels are increased in patients with a clinically isolated syndrome

    DEFF Research Database (Denmark)

    Disanto, Giulio; Adiutori, Rocco; Dobson, Ruth

    2016-01-01

    BACKGROUND: Neurofilament light chain (NfL) represents a promising biomarker for axonal injury. We present the first exploratory study on serum NfL in patients with a clinically isolated syndrome (CIS) and healthy controls. METHODS: We investigated serum NfL levels in 100 patients with CIS...... with a short conversion interval to clinically definite multiple sclerosis (MS) (fast converters (FC), median (IQR) conversion time: 110 days (79-139)); 98 patients with non-converting CIS (non-converters (NC), follow-up: 6.5 years (5.3-7.9)); and 92 healthy controls. RESULTS: NfL levels were higher in FC (24.......1 pg/mL (13.5-51.8)) and NC (19.3 pg/mL (13.6-35.2)) than in healthy controls (7.9 pg/mL (5.6-17.2)) (OR=5.85; 95% CI 2.63 to 13.02; p=1.5×10(-5) and OR=7.03; 95% CI 2.85 to 17.34; p=2.3×10(-5), respectively). When grouping FC and NC, increased serum NfL concentration was also associated...

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

    Science.gov (United States)

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

    2014-11-04

    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.

  15. Immunoparesis status in immunoglobulin light chain amyloidosis at diagnosis affects response and survival by regimen type

    Science.gov (United States)

    Muchtar, Eli; Dispenzieri, Angela; Kumar, Shaji K.; Dingli, David; Lacy, Martha Q.; Buadi, Francis K.; Hayman, Suzanne R.; Kapoor, Prashant; Leung, Nelson; Chakraborty, Rajshekhar; Russell, Stephen; Lust, John A.; Lin, Yi; Go, Ronald S.; Zeldenrust, Steven; Kyle, Robert A.; Rajkumar, S. Vincent; Gertz, Morie A.

    2016-01-01

    Clinical tools to guide in the appropriate treatment selection in immunoglobulin light chain (AL) amyloidosis are not well developed. We evaluated the response and outcome for various regimens at first-line treatment (n=681) and first progression (n=240) stratified by the immunoparesis status at diagnosis. Immunoparesis was assessed by the average relative difference of the uninvolved immunoglobulins, classifying patients into a negative average relative difference (i.e. significant immunoparesis) or a positive average relative difference (no/modest immunoparesis). Treatment was categorized as autologous stem cell transplant and four non-transplant regimens (melphalan-based; bortezomib-based, immunomodulatory drug-based and dexamethasone alone). Patients with significant immunoparesis who underwent stem cell transplant had a significantly lower rate of very good partial response or better response (58%), progression-free survival (median 30 months) and overall survival (108 months), compared to those without significant immunoparesis (80%, 127 months, median not reached, respectively; Pcomparisons). Among the non-transplant regimens, melphalan resulted in an unfavorable progression-free survival (11 vs. 27 months; Pcompared to those without significant immunoparesis. In contrast, no significant difference in outcomes between the immunoparesis groups was seen for those treated with bortezomib or immunomodulatory drugs. At first progression, immunoparesis status did not impact response or survival of any regimen. Melphalan at first-line provided poorer outcomes for patients with significant immunoparesis, while bortezomib or immunomodulatory drugs were more likely to overcome the adverse prognosis associated with significant immunoparesis. PMID:27479823

  16. Clinical and prognostic differences among patients with light chain deposition disease, myeloma cast nephropathy and both.

    Science.gov (United States)

    Zand, Ladan; Nasr, Samih H; Gertz, Morie A; Dispenzieri, Angela; Lacy, Martha Q; Buadi, Francis K; Kumar, Shaji; Kyle, Robert A; Fervenza, Fernando C; Sethi, Sanjeev; Dingli, David; Rajkumar, S Vincent; Kapoor, Prashant; McCurdy, Arleigh; Leung, Nelson

    2015-01-01

    In some patients with light chain deposition disease (LCDD) there is also evidence of myeloma cast nephropathy (MCN) on renal biopsy. The purpose of this study was to evaluate the renal and survival outcome of patients with concomitant diagnosis of MCN and LCDD to LCDD and MCN alone. Eighty seven patients were identified and divided into LCDD (n=45), MCN (n=29), and LCDD+ MCN (n=13). Patients with LCDD+ MCN had a worse overall survival (OS) compared to patients with LCDD (p=0.03), but similar to patients with MCN (p=0.4). Death-censored renal survival was no different amongst the groups. Presenting with acute renal failure at time of renal biopsy (HR 7.2, p=0.0002) was an independent poor renal prognostic factor while older age (HR 1.06, p=0.0002), presence of osteolytic lesions (HR 4.4, p<0.0001), and requirement for dialysis or creatinine≥5 mg/dL (HR 3.2, p=0.0006) at time of renal biopsy were independent poor prognostic factors for OS.

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

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

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

  18. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    Science.gov (United States)

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  19. Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers.

    Science.gov (United States)

    Das, Sabyasachi; Nikolaidis, Nikolas; Klein, Jan; Nei, Masatoshi

    2008-10-28

    The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups (kappa, lambda, and sigma isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess three IGL isotypes: kappa, lambda, and sigma. Of these, two (kappa and lambda) are also found in reptiles and some mammals. The lambda isotype is found in all tetrapods tested to date, whereas the kappa isotype seems to have been lost at least in some birds and in the microbat. Conservation of the cladistic molecular markers suggests that they are associated with functional specialization of the three IGL isotypes. The genomic maps of IGL loci reveal multiple gene rearrangements that occurred in the evolution of tetrapod species. These rearrangements have resulted in interspecific variation of the genomic lengths of the IGL loci and the number and order of IGL constituent genes, but the overall organization of the IGL loci has not changed.

  20. The clinical utility and prognostic value of multiparameter flow cytometry immunophenotyping in light-chain amyloidosis.

    Science.gov (United States)

    Paiva, Bruno; Vídriales, María-Belén; Pérez, José J; López-Berges, María-Consuelo; García-Sanz, Ramón; Ocio, Enrique M; de Las Heras, Natalia; Cuello, Rebeca; García de Coca, Alfonso; Pardal, Emilia; Alonso, José; Sierra, Magdalena; Bárez, Abelardo; Hernández, José; Suárez, Lissbett; Galende, Josefina; Mateos, María-Victoria; San Miguel, Jesús F

    2011-03-31

    The clinical value of multiparameter flow cytometry (MFC) immunophenotyping in primary or light chain amyloidosis (AL) remains unknown. We studied 44 consecutive bone marrow samples from newly diagnosed patients with amyloidosis; 35 patients with AL and 9 with other forms of amyloidosis. Monoclonal plasma cells (PCs) were identifiable by MFC immunophenotyping in 34 of 35 (97%) patients with AL, whereas it was absent from all but 1 of the 9 (11%) patients with other forms of amyloidosis. Quantification of bone marrow plasma cells (BMPCs) by MFC immunophenotyping was a significant prognostic factor for overall survival (OS) (≤ 1% vs > 1% BMPC cutoff; 2-year OS rates of 90% vs 44%, P = .02). Moreover, detecting persistent normal PCs at diagnosis identifies a subgroup of patients with AL with prolonged OS (> 5% vs ≤ 5% normal PC within all BMPC cutoff, 2-year rates of 88% vs 37%, P = .01). MFC immunophenotyping could be clinically useful for the demonstration of PC clonality in AL and for the prognostication of patients with AL.

  1. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.

    Science.gov (United States)

    Thaiparambil, Jose T; Eggers, Carrie M; Marcus, Adam I

    2012-08-01

    The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.

  2. Clinical Comparisons of Two Free Light Chain Assays to Immunofixation Electrophoresis for Detecting Monoclonal Gammopathy

    Directory of Open Access Journals (Sweden)

    Han-Sung Kim

    2014-01-01

    Full Text Available Free light chains (FLCs are useful biomarkers for the diagnosis and monitoring of various plasma cell dyscrasias. One hundred fifty-seven samples from 120 patients for screening or monitoring of monoclonal gammopathy (MG were included. The new N Latex FLC assays (Siemens Healthcare Diagnostics GmbH, Germany were compared with the Freelite FLC assays (The Binding Site Ltd., UK and the results were analyzed with those of immunofixation electrophoresis (IFE. The Freelite FLC assay showed significantly wider assay ranges than the N Latex FLC assay. The correlation coefficients of the two FLC kappa (κ assays, lambda (λ assays, and the κ/λ ratio were 0.9792, 0.8264, and 0.9064, respectively. The concordance rate was 84.7% for the FLC κ assays, 79.6% for FLC λ, and 89.2% for the κ/λ ratio. The clinical sensitivity and specificity of the κ/λ ratios were 72.2% and 93.6% for the Freelite assay and 64.6% and 100% for the N Latex FLC assay. Two FLC assays showed good correlations and concordance. However, the clinical sensitivity of the κ/λ ratio was higher in the Freelite FLC assays; clinical specificity was higher in the N Latex FLC assay. Both FLC assays seem to have limited clinical utility in detecting MG in certain clinical settings.

  3. Circulating antibody free light chains and risk of posttransplant lymphoproliferative disorder.

    Science.gov (United States)

    Engels, E A; Preiksaitis, J; Zingone, A; Landgren, O

    2012-05-01

    Posttransplant lymphoproliferative disorder (PTLD) is a major complication of solid-organ transplantation. With human immunodeficiency virus infection (an analogous immunosuppressive state), elevated kappa and lambda immunoglobulin free light chains (FLCs) in peripheral blood are associated with increased risk of lymphoma. To assess the role of B-cell dysfunction in PTLD, we measured circulating FLCs among Canadian transplant recipients, including 29 individuals with PTLD and 57 matched transplant recipients who were PTLD-free. Compared with controls, PTLD cases had higher kappa FLCs (median 1.53 vs. 1.07 times upper limit of normal) and lambda FLCs (1.03 vs. 0.68). Using samples obtained on average 3.5 months before PTLD diagnosis, cases were more likely to have polyclonal FLC elevations (i.e. elevated kappa and/or lambda with normal kappa/lambda ratio: odds ratio [OR] 4.2, 95%CI 1.1-15) or monoclonal elevations (elevated kappa and/or lambda with abnormal ratio: OR 3.0, 95%CI 0.5-18). Strong FLC-PTLD associations were also observed at diagnosis/selection. Among recipients with Epstein-Barr virus (EBV) DNA measured in blood, EBV DNAemia was associated with FLC abnormalities (ORs 6.2 and 3.2 for monoclonal and polyclonal elevations). FLC elevations are common in transplant recipients and associated with heightened PTLD risk. FLCs likely reflect B-cell dysfunction, perhaps related to EBV-driven lymphoproliferation.

  4. Helicobacter pylori CagA disrupts epithelial patterning by activating myosin light chain.

    Directory of Open Access Journals (Sweden)

    Jonathan B Muyskens

    Full Text Available Helicobacter pylori infection is a leading cause of ulcers and gastric cancer. We show that expression of the H. pylori virulence factor CagA in a model Drosophila melanogaster epithelium induces morphological disruptions including ectopic furrowing. We find that CagA alters the distribution and increases the levels of activated myosin regulatory light chain (MLC, a key regulator of epithelial integrity. Reducing MLC activity suppresses CagA-induced disruptions. A CagA mutant lacking EPIYA motifs (CagA(EPISA induces less epithelial disruption and is not targeted to apical foci like wild-type CagA. In a cell culture model in which CagA(EPISA and CagA have equivalent subcellular localization, CagA(EPISA is equally potent in activating MLC. Therefore, in our transgenic system, CagA is targeted by EPIYA motifs to a specific apical region of the epithelium where it efficiently activates MLC to disrupt epithelial integrity.

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

    Science.gov (United States)

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

    2016-07-01

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

  6. The prognostic value of diagnosing concurrent multiple myeloma in immunoglobulin light chain amyloidosis.

    Science.gov (United States)

    Dinner, Shira; Witteles, Wesley; Witteles, Ronald; Lam, Anthony; Arai, Sally; Lafayette, Richard; George, Tracy I; Schrier, Stanley L; Liedtke, Michaela

    2013-05-01

    The prevalence and prognostic value of a concomitant diagnosis of symptomatic or asymptomatic multiple myeloma (MM), as defined by the current International Myeloma Working Group (IMWG) criteria, in patients with immunoglobulin light chain amyloidosis (AL), are unknown. We studied 46 consecutive patients with AL who underwent quantification of serum M-protein and clonal bone marrow plasma cells, as well as a comprehensive evaluation for end organ damage by MM. Using standard morphology and CD138 immunohistochemical staining, 57% and 80% of patients were found to have concomitant MM, respectively. Nine patients exhibited end organ damage consistent with a diagnosis of symptomatic MM. While overall survival was similar between AL patients with or without concurrent myeloma (1-year overall survival 68% vs. 87%; P = 0.27), a diagnosis of symptomatic myeloma was associated with inferior outcome (1-year overall survival 39% vs. 81%; P = 0.005). Quantification of bone marrow plasma cells by both standard morphology and CD138 immunohistochemistry identified a much higher prevalence of concurrent MM in patients with AL than previously reported. Evaluation of bone marrow plasma cell infiltration and presence of myeloma associated end organ damage could be clinically useful for prognostication of patients with AL. © 2013 Blackwell Publishing Ltd.

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

    Science.gov (United States)

    Ščudla, Vlastimil; Pika, Tomáš; Minařík, Jiří

    2015-01-01

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

  8. Immunophenotypic analysis of abnormal plasma cell clones in bone marrow of primary systemic light chain amyloidosis patients

    Institute of Scientific and Technical Information of China (English)

    Hu Yang; Wang Mangju; Chen Yan; Chen Xue; Fang Fang; Liu Shiqin; Zhang Ying

    2014-01-01

    Background Primary systemic light chain amyloidosis (AL) is a rare plasma cell disease,our purpose was to analyze the immunophenotypic characteristics of the plasma cells in bone marrow in AL patients,and explore whether the detection of abnormal plasma cell clones in bone marrow by flow cytometry (FCM) could be used as an important indicator of AL diagnosis.Methods Fresh bone marrow samples were collected from 51 AL,21 multiple myeloma (MM),and 5 Waldenstr(o)m's macroglobulinemia (WM) patients.The immunophenotype of bone marrow cells were analyzed and compared by FCM using a panel of antibodies including CD45,CD38,CD138,CD117,CD56,and CD19.Results In AL,light chain restriction could be identified in 31 cases (60.9%),in which the λ light chain restriction was found in 24 cases (77.4%).In MM,κ light chain restriction was found in 13 cases (61.9%),and λ light chain restriction in eight cases.CD45 on abnormal plasma cells was negative to weakly positive in both AL and MM,but was positive to strongly positive in WM.In the bone marrow plasma cells of the 51 AL,78.4% were CD56+,68.6% were CD117+,and 88.2% were CD19-.While in the 21 MM cases,66.7% were CD56+,38.1% were CD117+,and 90.4% were CD19-.The plasmacytoid lymphocytes in the five WM patients were CD19+ and CD56-,CD117-.Conclusion Detection of abnormal plasma cell clones in bone marrow by FCM is valuable for the diagnosis of AL.

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

    Science.gov (United States)

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

    2015-12-04

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

  10. Bi-directional transport of the nucleus by dynein and kinesin-1.

    Science.gov (United States)

    Tanenbaum, Marvin E; Akhmanova, Anna; Medema, René H

    2011-01-01

    Proper transport and positioning of cell organelles often depends on the antagonistic activities of dynein and kinesin-1, two microtubule motors with opposite directionality.1 One of the largest known transport cargoes is the cell nucleus. Both dynein and kinesin-1 participate in positioning of the nucleus through binding to the nuclear envelope (NE).2-9 Surprisingly, both dynein and kinesin-1 can be recruited to the NE through multiple pathways, one involving SUN-KASH domain containing proteins and the other involving nuclear pore complexes (NPCs). Here, we discuss the molecular mechanisms of dynein and kinesin recruitment to the NE through NPCs, as well as the functional implications of dynein and kinesin-1 activity at the NE in mammalian cells. Finally, we discuss how motor activities at the NE might be controlled during the cell cycle.

  11. Value of the free light chain analysis in the clinical evaluation of response in multiple myeloma patients receiving anti-myeloma therapy

    DEFF Research Database (Denmark)

    Toftmann Hansen, Charlotte; Pedersen, Per T.; Jensen, Bo Amdi;

    Value of the free light chain analysis in the clinical evaluation of response in multiple myeloma patients receiving anti-myeloma therapy.......Value of the free light chain analysis in the clinical evaluation of response in multiple myeloma patients receiving anti-myeloma therapy....

  12. Immunoglobulin diversity in the phylogenetically primitive shark, Heterodontus francisci. Suggested lack of structural variation between light chains isolated from different animals.

    Science.gov (United States)

    Litman, G W; Scheffel, C; Gerber-Jenson, B

    1980-06-01

    A two-step procedure employing gel filtration and anion exchange chromatography has been utilized to isolate LMW immunoglobulin from the horned shark, Heterodontus francisci. Light chains obtained by complete reduction and alkylation of the parent protein have been compared by several analytical techniques. Amino acid composition data implies a limited degree of variation in the light chains isolated from individual animals. Polyacrylamide gel electrophoresis of the CNBr digests of the light chains reveal indistinguishable banding profiles of the major peptides. Isoelectric focusing indicates limited heterogeneity in the light chain spectrotype and identity in the pI of the majority of bands detectable by staining. The suggested degree of structural similarity in the light chains of this phylogenetically primitive shark is discussed in terms of the evolutionary position of the species and current theories concerning the origins of structural diversity in immunoglobulins.

  13. CSF Neurofilament Light Chain but not FLT3 Ligand Discriminates Parkinsonian Disorders

    Science.gov (United States)

    Herbert, Megan K.; Aerts, Marjolein B.; Beenes, Marijke; Norgren, Niklas; Esselink, Rianne A. J.; Bloem, Bastiaan R.; Kuiperij, H. Bea; Verbeek, Marcel M.

    2015-01-01

    The differentiation between multiple system atrophy (MSA) and Parkinson’s disease (PD) is difficult, particularly in early disease stages. Therefore, we aimed to evaluate the diagnostic value of neurofilament light chain (NFL), fms-like tyrosine kinase ligand (FLT3L), and total tau protein (t-tau) in cerebrospinal fluid (CSF) as biomarkers to discriminate MSA from PD. Using commercially available enzyme-linked immunosorbent assays, we measured CSF levels of NFL, FLT3L, and t-tau in a discovery cohort of 36 PD patients, 27 MSA patients, and 57 non-neurological controls and in a validation cohort of 32 PD patients, 25 MSA patients, 15 PSP patients, 5 CBS patients, and 56 non-neurological controls. Cut-offs obtained from individual assays and binary logistic regression models developed from combinations of biomarkers were assessed. CSF levels of NFL were substantially increased in MSA and discriminated between MSA and PD with a sensitivity of 74% and specificity of 92% (AUC = 0.85) in the discovery cohort and with 80% sensitivity and 97% specificity (AUC = 0.94) in the validation cohort. FLT3L levels in CSF were significantly lower in both PD and MSA compared to controls in the discovery cohort, but not in the validation cohort. t-tau levels were significantly higher in MSA than PD and controls. Addition of either FLT3L or t-tau to NFL did not improve discrimination of PD from MSA above NFL alone. Our findings show that increased levels of NFL in CSF offer clinically relevant, high accuracy discrimination between PD and MSA. PMID:25999911

  14. Serum neurofilament light chain levels are increased in patients with a clinically isolated syndrome.

    Science.gov (United States)

    Disanto, Giulio; Adiutori, Rocco; Dobson, Ruth; Martinelli, Vittorio; Dalla Costa, Gloria; Runia, Tessel; Evdoshenko, Evgeniy; Thouvenot, Eric; Trojano, Maria; Norgren, Niklas; Teunissen, Charlotte; Kappos, Ludwig; Giovannoni, Gavin; Kuhle, Jens

    2016-02-01

    Neurofilament light chain (NfL) represents a promising biomarker for axonal injury. We present the first exploratory study on serum NfL in patients with a clinically isolated syndrome (CIS) and healthy controls. We investigated serum NfL levels in 100 patients with CIS with a short conversion interval to clinically definite multiple sclerosis (MS) (fast converters (FC), median (IQR) conversion time: 110 days (79-139)); 98 patients with non-converting CIS (non-converters (NC), follow-up: 6.5 years (5.3-7.9)); and 92 healthy controls. NfL levels were higher in FC (24.1 pg/mL (13.5-51.8)) and NC (19.3 pg/mL (13.6-35.2)) than in healthy controls (7.9 pg/mL (5.6-17.2)) (OR=5.85; 95% CI 2.63 to 13.02; p = 1.5 × 10(-5) and OR = 7.03; 95% CI 2.85 to 17.34; p = 2.3 × 10(-5), respectively). When grouping FC and NC, increased serum NfL concentration was also associated with increasing numbers of T2 hyperintense MRI lesions (OR = 2.36; 95% CI 1.21 to 4.59; p = 0.011), gadolinium-enhancing lesions (OR = 2.69; 95% CI 1.13 to 6.41; p=0.026) and higher disability scores (OR = 2.54; 95% CI 1.21 to 5.31; p = 0.013) at CIS diagnosis. If replicated in future studies, serum NfL may represent a reliable and easily accessible biomarker of early axonal damage in CIS and MS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

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

    Science.gov (United States)

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

    2016-09-13

    To determine the prognostic value of selected biomarkers in clinically isolated syndromes (CIS) for conversion to multiple sclerosis (MS) and disability accrual. Data were acquired from 2 CIS cohorts. The screening phase evaluated patients developing clinically definite MS (CIS-CDMS) and patients who remained as CIS during a 2-year minimum follow-up (CIS-CIS). We determined levels of neurofascin, semaphorin 3A, fetuin A, glial fibrillary acidic protein, and neurofilament light (NfL) and heavy chains in CSF (estimated mean [95% confidence interval; CI]). We evaluated associations between biomarker levels, conversion, disability, and magnetic resonance parameters. In the replication phase, we determined NfL levels (n = 155) using a 900 ng/L cutoff. Primary endpoints in uni- and multivariate analyses were CDMS and 2010 McDonald MS. The only biomarker showing significant differences in the screening was NfL (CIS-CDMS 1,553.1 [1,208.7-1,897.5] ng/L and CIS-CIS 499.0 [168.8-829.2] ng/L, p NfL did not correlate with disability. In the replication phase, more NfL-positive patients, according to the cutoff, evolved to MS. Every 100-ng/L increase in NfL predicted CDMS (hazard ratio [HR] = 1.009, 95% CI 1.005-1.014) and McDonald MS (HR = 1.009, 95% CI 1.005-1.013), remaining significant for CDMS in the multivariate analysis (adjusted HR = 1.005, 95% CI 1.000-1.011). This risk was lower than the presence of oligoclonal bands or T2 lesions. NfL is a weak independent risk factor for MS. Its role as an axonal damage biomarker may be more relevant as suggested by its association with medium-term brain volume changes. © 2016 American Academy of Neurology.

  16. Myosin light chain kinase mediates intestinal barrier disruption following burn injury.

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    Chuanli Chen

    Full Text Available 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.

  17. Cardiac Light Chain Amyloidosis: The Role of Metal Ions in Oxidative Stress and Mitochondrial Damage.

    Science.gov (United States)

    Diomede, Luisa; Romeo, Margherita; Rognoni, Paola; Beeg, Marten; Foray, Claudia; Ghibaudi, Elena; Palladini, Giovanni; Cherny, Robert A; Verga, Laura; Capello, Gian Luca; Perfetti, Vittorio; Fiordaliso, Fabio; Merlini, Giampaolo; Salmona, Mario

    2017-09-20

    The knowledge of the mechanism underlying the cardiac damage in immunoglobulin light chain (LC) amyloidosis (AL) is essential to develop novel therapies and improve patients' outcome. Although an active role of reactive oxygen species (ROS) in LC-induced cardiotoxicity has already been envisaged, the actual mechanisms behind their generation remain elusive. This study was aimed at further dissecting the action of ROS generated by cardiotoxic LC in vivo and investigating whether transition metal ions are involved in this process. In the absence of reliable vertebrate model of AL, we used the nematode Caenorhabditis elegans, whose pharynx is an "ancestral heart." LC purified from patients with severe cardiac involvement intrinsically generated high levels of ROS and when administered to C. elegans induced ROS production, activation of the DAF-16/forkhead transcription factor (FOXO) pathway, and expression of proteins involved in stress resistance and survival. Profound functional and structural ROS-mediated mitochondrial damage, similar to that observed in amyloid-affected hearts from AL patients, was observed. All these effects were entirely dependent on the presence of metal ions since addition of metal chelator or metal-binding 8-hydroxyquinoline compounds (chelex, PBT2, and clioquinol) permanently blocked the ROS production and prevented the cardiotoxic effects of amyloid LC. Innovation and Conclusion: Our findings identify the key role of metal ions in driving the ROS-mediated toxic effects of LC. This is a novel conceptual advance that paves the way for new pharmacological strategies aimed at not only counteracting but also totally inhibiting the vicious cycle of redox damage. Antioxid. Redox Signal. 27, 567-582.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  19. Expression of Calmodulin and Myosin Light Chain Kinase during Larval Settlement of the Barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan

    2012-02-13

    Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus (= Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca 2+/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite. © 2012 Chen et al.

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

    Science.gov (United States)

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

    2014-01-01

    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 pseudophosphorylatedRLC 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

  1. Remodeling of the heart in hypertrophy in animal models with myosin essential light chain mutations

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    Katarzyna eKazmierczak

    2014-09-01

    Full Text Available Cardiac hypertrophy represents one of the most important cardiovascular problems yet the mechanisms responsible for hypertrophic remodeling of the heart are poorly understood. In this report we aimed to explore the molecular pathways leading to two different phenotypes of cardiac hypertrophy in transgenic mice carrying mutations in the human ventricular myosin essential light chain (ELC. Mutation-induced alterations in the heart structure and function were studied in two transgenic (Tg mouse models carrying the A57G (alanine to glycine substitution or lacking the N-terminal 43 amino acid residues (Δ43 from the ELC sequence. The first model represents an HCM disease as the A57G mutation was shown to cause malignant HCM outcomes in humans. The second mouse model is lacking the region of the ELC that was shown to be important for a direct interaction between the ELC and actin during muscle contraction. Our earlier studies demonstrated that >7 month old Tg-Δ43 mice developed substantial cardiac hypertrophy with no signs of histopathology or fibrosis. Tg mice did not show abnormal cardiac function compared to Tg-WT expressing the full length human ventricular ELC. Previously reported pathological morphology in Tg-A57G mice included extensive disorganization of myocytes and interstitial fibrosis with no abnormal increase in heart mass observed in >6 month-old animals. In this report we show that strenuous exercise can trigger hypertrophy and pathologic cardiac remodeling in Tg-A57G mice as early as 3 months of age. In contrast, no exercise-induced changes were noted for Tg-Δ43 hearts and the mice maintained a non-pathological cardiac phenotype. Based on our results, we suggest that exercise-elicited heart remodeling in Tg-A57G mice follows the pathological pathway leading to HCM, while it induces no abnormal response in Tg-Δ43 mice.

  2. Light chain amyloidosis: Experience in a tertiary hospital: 2005-2013.

    Science.gov (United States)

    Krsnik, I; Cabero, M; Morillo, D; Segovia, J; García-Pavía, P; Gómez-Bueno, M; Salas, C

    2015-01-01

    AL amyloidosis is a rare condition whose management is undergoing changes due to recent advances in diagnosis and treatment. We describe a contemporary series of patients with AL amyloidosis to analyze the features that enable early diagnosis and optimal management. We recruited for analysis 32 patients (19 women; mean age, 63 years) treated consecutively at our center. Eighty-four percent of the patients presented with asthenia, dyspnea or edema, with a previous duration of symptoms of 8 months (median). Cardiac (21/32) and renal impairment were the most common type (11/32). All of the patients, except one, had a monoclonal component in serum/urine or abnormal values for free light chains (78%, λ). The bone marrow (BM) showed clonal plasmacytosis in 29 cases. All of the cardiac biopsies and 50% of the BM biopsies showed amyloid deposits. The results of the echocardiogram and/or cardiac resonance were abnormal in 27/30 cases. The median NT-proBNP value at diagnosis was 5200 ng/ml. Thirteen patients died due to heart failure, 2 due to rejection after heart transplantation, 2 due to pneumonia and 1 after a stroke. Ten patients did not undergo treatment, 12 were treated with bortezomib and 5 were treated with alkylating agents. Five patients underwent heart transplantation and 4 underwent autologous bone marrow transplantation. Fourteen patients achieved a complete hematologic response and 10 achieved organ response. The median survival was 17 months. Cardiac involvement is the major determinant of prognosis. Yield of involved organ biopsy is high (100% heart biopsies). Antineoplastic treatment with bortezomib and/or autologous bone marrow transplantation achieves hematological responses with improvements in organ impairment. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

  3. Immunoglobulin free light chains are increased in hypersensitivity pneumonitis and idiopathic pulmonary fibrosis.

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    Tom Groot Kormelink

    Full Text Available BACKGROUND: Idiopathic pulmonary fibrosis (IPF, a devastating lung disorder of unknown aetiology, and chronic hypersensitivity pneumonitis (HP, a disease provoked by an immunopathologic reaction to inhaled antigens, are two common interstitial lung diseases with uncertain pathogenic mechanisms. Previously, we have shown in other upper and lower airway diseases that immunoglobulin free light chains (FLCs are increased and may be involved in initiating a local inflammation. In this study we explored if such a mechanism may also apply to HP and IPF. METHODS: In this study we examined the presence of FLC in serum and BAL fluid from 21 IPF and 22 HP patients and controls. IgG, IgE and tryptase concentrations were measured in BAL fluid only. The presence of FLCs, plasma cells, B cells and mast cells in lung tissue of 3 HP and 3 IPF patients and 1 control was analyzed using immunohistochemistry. RESULTS: FLC concentrations in serum and BAL fluid were increased in IPF and HP patients as compared to control subjects. IgG concentrations were only increased in HP patients, whereas IgE concentrations were comparable to controls in both patient groups. FLC-positive cells, B cells, plasma cells, and large numbers of activated mast cells were all detected in the lungs of HP and IPF patients, not in control lung. CONCLUSION: These results show that FLC concentrations are increased in serum and BAL fluid of IPF and HP patients and that FLCs are present within affected lung tissue. This suggests that FLCs may be involved in mediating pathology in both diseases.

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

    Science.gov (United States)

    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.

  5. Prognostic significance of serum free light chains in chronic lymphocytic leukemia.

    Science.gov (United States)

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

    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 β 2-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.

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

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    Katerina Sarris

    2013-01-01

    Full Text Available 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.

  7. Expression of calmodulin and myosin light chain kinase during larval settlement of the Barnacle Balanus amphitrite.

    Directory of Open Access Journals (Sweden)

    Zhang-Fan Chen

    Full Text Available Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM, encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca(2+/CaM-dependent kinase II (CaMKII inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite.

  8. CSF neurofilament light chain but not FLT3 ligand discriminates Parkinsonian disorders

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    Megan Kristy Herbert

    2015-05-01

    Full Text Available The differentiation between multiple system atrophy (MSA and Parkinson’s disease (PD is difficult, particularly in early disease stages. Therefore, we aimed to evaluate the diagnostic value of neurofilament light chain (NFL, fms-like tyrosine kinase ligand (FLT3L and total tau protein (t-tau in cerebrospinal fluid (CSF as biomarkers to discriminate MSA from PD. Using commercially available enzyme-linked immunoassays (ELISAs, we measured CSF levels of NFL, FLT3L and t-tau in a discovery cohort of 36 PD patients, 27 MSA patients and 57 non-neurological controls and in a validation cohort of 32 PD patients, 25 MSA patients, 15 PSP patients, 5 CBS patients, and 56 non-neurological controls. Cut-offs obtained from individual assays and binary logistic regression models developed from combinations of biomarkers were assessed. CSF levels of NFL were substantially increased in MSA and discriminated between MSA and PD with a sensitivity of 74% and specificity of 92% (AUC = 0.85 in the discovery cohort and with 80% sensitivity and 97% specificity (AUC = 0.94 in the validation cohort. FLT3L levels in CSF were significantly lower in both PD and MSA compared to controls in the discovery cohort, but not in the validation cohort. T-tau levels were significantly higher in MSA than PD and controls. Addition of either FLT3L or t-tau to NFL did not improve discrimination of PD from MSA above NFL alone. Our findings show that increased levels of NFL in CSF offer clinically relevant, high accuracy discrimination between PD and MSA.

  9. Another way to view the chain conformation broadening of the line-width distribution measured in dynamic light scattering

    Institute of Scientific and Technical Information of China (English)

    吴奇; 牛爱珍

    1999-01-01

    In dynamic laser light scattering (LLS), for a given polydisperse sample, a line-width distribution G(Γ) or the translational diffusion coefficient distribution G(D) can be obtained from the measured time correlation function. For rigid colloid particles, G(Γ) can be directly related to the hydrodynamic size distribution. However, for flexible polymer chains, G(Γ) depends not only on the chain length distribution, but also on the relaxation of the chain conformation; that is, even for a monodisperse polymer sample there still exists a chain conformation distribution. If the time scale of the chain conformation relaxation is comparable to that of the translational diffusion, such as in the case of a very long polymer chain, the conformation relaxation might lead to an additional broadening in G (Γ). This "conformation broadening" has been directly observed for the first time by comparing two G(Γ) s obtained from a poly(N-isopropyl-acrylamide) solution at~25℃ and~32℃ at which the solution is ther

  10. Cargo transport by cytoplasmic Dynein can center embryonic centrosomes.

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    Rafael A Longoria

    Full Text Available To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell's center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.

  11. Cytoplasmic dynein binding, run length, and velocity are guided by long-range electrostatic interactions.

    Science.gov (United States)

    Li, Lin; Alper, Joshua; Alexov, Emil

    2016-08-17

    Dyneins are important molecular motors involved in many essential biological processes, including cargo transport along microtubules, mitosis, and in cilia. Dynein motility involves the coupling of microtubule binding and unbinding to a change in the configuration of the linker domain induced by ATP hydrolysis, which occur some 25 nm apart. This leaves the accuracy of dynein stepping relatively inaccurate and susceptible to thermal noise. Using multi-scale modeling with a computational focusing technique, we demonstrate that the microtubule forms an electrostatic funnel that guides the dynein's microtubule binding domain (MTBD) as it finally docks to the precise, keyed binding location on the microtubule. Furthermore, we demonstrate that electrostatic component of the MTBD's binding free energy is linearly correlated with the velocity and run length of dynein, and we use this linearity to predict the effect of mutating each glutamic and aspartic acid located in MTBD domain to alanine. Lastly, we show that the binding of dynein to the microtubule is associated with conformational changes involving several helices, and we localize flexible hinge points within the stalk helices. Taken all together, we demonstrate that long range electrostatic interactions bring a level of precision to an otherwise noisy dynein stepping process.

  12. Branched-Chain in situ Hybridization for κ and λ Light Chains: A Powerful Ancillary Technique for Determining B-Cell Clonality in Cytology Samples

    Science.gov (United States)

    Arora, Kshitij; Chebib, Ivan; Zukerberg, Lawrence; Gandhi, Manoj; Rivera, Miguel; Ting, David; Deshpande, Vikram

    2017-01-01

    BACKGROUND Current immunohistochemical and in situ hybridization (ISH) assays are generally inconclusive for clonality unless plasmacytic differentiation is present. This study examined a series of cytology specimens and explored the ability of a branched-chain RNA (bRNA) ISH assay for immunoglobulin κ constant (IGKC) and immunoglobulin λ constant (IGLC) to detect a clonal population of B lymphocytes. METHODS Pathology databases were used to identify fine-needle aspiration biopsies (n = 28) and exfoliative cytology samples (n = 20). Demographic, flow cytometry, and excision biopsy results were recorded. bRNA ISH was performed on the Leica Bond platform with the following probes: IGKC, IGLC, immunoglobulin λ-like polypeptide 5 (IGLL5), and a housekeeping gene (HKG). RESULTS The bRNA ISH assay was validated with 30 surgical biopsies. On bRNA ISH, a clonal B-cell population (light-chain ratio > 10:1) was detected in 22 of 28 cases with a final diagnosis of lymphoma. In 2 cases, a κ predominance was present, although the ratio was <10:1. Eleven of the 17 κ-clonal lymphomas also expressed IGLL5, the latter recognized by the presence of an intranuclear signal. Two B-cell lymphomas lacked IGKC and IGLC, whereas 2 cases were negative for the HKG. In 12 of the 20 cases with reactive lymphoid tissue, bRNA ISH identified a polyclonal lymphoid population. No light-chain messenger RNA was detected in 6 cases (typically those associated with very few B cells). CONCLUSIONS The automated bRNA ISH platform is a robust technique for detecting a clonal B-cell population in cytology material. PMID:26524200

  13. Phylogeny, genomic organization and expression of lambda and kappa immunoglobulin light chain genes in a reptile, Anolis carolinensis.

    Science.gov (United States)

    Wu, Qian; Wei, Zhiguo; Yang, Zhi; Wang, Tao; Ren, Liming; Hu, Xiaoxiang; Meng, Qingyong; Guo, Ying; Zhu, Qinghong; Robert, Jacques; Hammarström, Lennart; Li, Ning; Zhao, Yaofeng

    2010-05-01

    The reptiles are the last major taxon of jawed vertebrates in which immunoglobulin light chain isotypes have not been well characterized. Using the recently released genome sequencing data, we show in this study that the reptile Anolis carolinensis expresses both lambda and kappa light chain genes. The genomic organization of both gene loci is structurally similar to their respective counterparts in mammals. The identified lambda locus contains three constant region genes each preceded by a joining gene segment, and a total of 37 variable gene segments. In contrast, the kappa locus contains only a single constant region gene, and two joining gene segments with a single family of 14 variable gene segments located upstream. Analysis of junctions of the recombined VJ transcripts reveals a paucity of N and P nucleotides in both expressed lambda and kappa sequences. These results help us to understand the generation of the immunoglobulin repertoire in reptiles and immunoglobulin evolution in vertebrates.

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

    Science.gov (United States)

    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

  15. Light conditions alter accumulation of long chain polyprenols in leaves of trees and shrubs throughout the vegetation season.

    Science.gov (United States)

    Bajda, Agnieszka; Chojnacki, Tadeusz; Hertel, Józefina; Swiezewska, Ewa; Wójcik, Jacek; Kaczkowska, Alicja; Marczewski, Andrzej; Bojarczuk, Tomasz; Karolewski, Piotr; Oleksyn, Jacek

    2005-01-01

    In many plants belonging to angiosperms and gymnosperms the accumulation in leaves of long chain polyprenols and polyprenyl esters during growth in natural habitats depends on the light intensity. The amount of polyprenols in leaves is also positively correlated with the thickness of the leaf blade (SLA, specific leaf area). The polyprenol content of leaves shows seasonal changes with a maximum in autumn and a minimum in early summer with the difference between poorly and well illuminated plants persisting throughout the vegetation season.

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

    Science.gov (United States)

    Rekhtina, I G; Mendeleeva, L P; Stolyarevich, E S; Galtseva, 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.

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

    Directory of Open Access Journals (Sweden)

    James N. Cobley

    2016-04-01

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

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

    Science.gov (United States)

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

    2009-07-01

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

  19. Mutations of the Drosophila myosin regulatory light chain affect courtship song and reduce reproductive success.

    Science.gov (United States)

    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) Dmlc2(Δ2-46) (Ext), an N-terminal extension truncation; (ii) Dmlc2(S66A,S67A) (Phos), a disruption of two MLC kinase phosphorylation sites; and (iii) Dmlc2(Δ2-46;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.

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Heart failure induced by perinatal ablation of cardiac myosin light chain kinase

    Directory of Open Access Journals (Sweden)

    Yasmin F. K. Islam

    2016-10-01

    Full Text Available Background: Germline knockout mice are invaluable in understanding the function of the targeted genes. Sometimes, however, unexpected phenotypes are encountered, due in part to the activation of compensatory mechanisms. Germline ablation of cardiac myosin light chain kinase (cMLCK causes mild cardiac dysfunction with cardiomyocyte hypertrophy, whereas ablation in adult hearts results in acute heart failure with cardiomyocyte atrophy. We hypothesized that compensation after ablation of cMLCK is dependent on developmental staging and perinatal-onset of cMLCK ablation will result in more evident heart failure than germline ablation, but less profound when compared to adult-onset ablation.Methods and Results: The floxed-Mylk3 gene was ablated at the beginning of the perinatal stage using a single intra-peritoneal tamoxifen injection of 50 mg/kg into pregnant mice on the 19th day of gestation, this being the final day of gestation. The level of cMLCK protein level could no longer be detected 3 days after the injection, with these mice hereafter denoted as the perinatal Mylk3-KO. At postnatal day 19, shortly before weaning age, these mice showed reduced cardiac contractility with a fractional shortening 22.8 ± 1.0% (n = 7 as opposed to 31.4 ± 1.0% (n = 11 in controls. The ratio of the heart weight relative to body weight was significantly increased at 6.68 ± 0.28 mg/g (n = 12 relative to the two control groups, 5.90 ± 0.16 (flox/flox, n = 11 and 5.81 ± 0.33 (wild/wild/Cre, n = 5, accompanied by reduced body weight. Furthermore, their cardiomyocytes were elongated without thickening, with a long-axis of 101.8 ± 2.4 μm (n = 320 as opposed to 87.1 ± 1.6 μm (n = 360 in the controls. Conclusion: Perinatal ablation of cMLCK produces an increase of heart weight/body weight ratio, a reduction of contractility, and an increase in the expression of fetal genes. The perinatal Mylk3-KO cardiomyocytes were elongated in the absence of thickening, differing

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Batata, A; Shen, B

    1993-08-01

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

  4. Exploring single chain amphiphile self-assembly and their possible roles in light transduction

    DEFF Research Database (Denmark)

    Monnard, Pierre-Alain

    2011-01-01

    Self-assembled structures of single-chain amphiphiles have been used as hosts for biochemical, and chemical reactions. Their use as models for protocells (i.e., precursors to the first biological cells) has been extensively researched by various groups because the availability of single chain amp...... network. References 1 Cape, J., et al. (2011) Chem. Sci., 2 (4), 661-667. 2 Maurer, S. E., et al. (2009) Astrobiology, 9, 979-987. 3 Caschera, F., et al. Langmuir, In press....

  5. Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements

    OpenAIRE

    Toropova, K.; Mladenov, M; Roberts, Anthony

    2017-01-01

    Cilia are multi-functional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multi-motor assemblies with kinesin-II. Combining these data with insigh...

  6. Evidence indicating independent assortment of framework and complementarity-determining segments of the variable regions of rabbit light chains. Delineation of a possible J minigene.

    Science.gov (United States)

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

    1980-07-01

    Amino acid sequences of rabbit light chains show considerable evidence of independent assortment of framework (FR) and complementarity-determining (CDR) segments. This suggests that they are coded for by independent genetic units (minigaenes) and that individual light chains are assembled somatically by recombining these units. Identical FR sets with multiple members generally comprise chains with different specificities, whereas identical CDR sets tend to have chains of a single specificity. A J segment, which, by analogy with mouse light chains, is made up of the last two residues of CDR3 plus all of FR4, contained 18 different sets and could contribute to diversity generated by CDR3. The longest segment, FR3, had a very large number of sets. Evidence is presented showing that the number of sets could be substantially reduced by permitting FR3 to be formed by two independently assorting segments comprising residues 57-68 and 69-88.

  7. Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly.

    Science.gov (United States)

    Tanenbaum, Marvin E; Macůrek, Libor; Galjart, Niels; Medema, René H

    2008-12-17

    Bipolar spindle assembly critically depends on the microtubule plus-end-directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus-end-directed motor dynein, or the dynein-binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5-dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus-end-tracking protein CLIP-170. This function of CLIP-170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP-115, a highly homologous protein that lacks the dynein-dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP-170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.

  8. Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains.

    Science.gov (United States)

    Tanenbaum, Marvin E; Vale, Ronald D; McKenney, Richard J

    2013-09-03

    Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins. DOI:http://dx.doi.org/10.7554/eLife.00943.001.

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

    Science.gov (United States)

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

    2012-07-10

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

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

    Science.gov (United States)

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

    2015-07-01

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

  11. [A case of lambda-expressing pulmonary MALT lymphoma with dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene].

    Science.gov (United States)

    Oh, Hye Ryong; Lee, Mi Ja; Park, Geon; Moon, Dae Soo; Park, Young Jin; Jang, Sook Jin

    2009-06-01

    A 70-yr-old woman was hospitalized with a history of dry cough. Bronchial endoscopy and transbronchial lung biopsy were performed. However, the findings of histopathology and immunohistochemistry were not sufficient to decide whether the lesion was benign or malignant, because of the presence of crush artifacts in the biopsy specimens. We performed B-cell clonality studies using BIOMED-2 multiplex PCR (InVivoScribe Technologies, USA) to detect clonal rearrangements in the immunoglobulin gene. The results of multiplex PCR showed clonal rearrangements of both kappa and lambda immunoglobulin light chain genes. The findings of immunochemistry revealed that the lesion expressed lambda light chain, but not kappa light chain. Based on the clinical, pathologic, and molecular findings, this case was diagnosed as pulmonary MALT lymphoma. We report the first case in Korea of lambda-expressing MALT lymphoma that is shown to have dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene by multiplex PCR.

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

    Science.gov (United States)

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

    2015-08-01

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

  13. Evaluation of the serum free light chain (sFLC) analysis in prediction of response in symptomatic multiple myeloma patients

    DEFF Research Database (Denmark)

    Toftmann Hansen, Charlotte; Pedersen, Per T; Nielsen, Lars C;

    2014-01-01

    BACKGROUND: Observational data from clinical studies indicate that the goal of first-line therapy in newly diagnosed patients with symptomatic multiple myeloma (MM) should be very good partial response (VGPR) or better, preferably before high-dose treatment. We evaluated the value of early...... measurements of involved free light chains (iFLC) in prediction of high-quality responses. Measuring iFLC has a potential advantage due to a short half-life compared to the half-life of the M-protein. METHODS: In 36 multiple myeloma (MM) patients, we measured serial changes in iFLC and M-protein after start...

  14. Renal Light Chain Deposition Associated with the Formation of Intracellular Crystalline Inclusion Bodies in Podocytes: A Rare Case Report.

    Science.gov (United States)

    Wang, Yuan-da; Dong, Zhe-yi; Zhang, Xue-guang; Zhang, Wei; Yin, Zhong; Qiu, Qiang; Chen, Xiang-mei

    2016-01-01

    We herein report the case of an elderly woman with bone pain and proteinuria as the main clinical manifestations. The patient was diagnosed with the IgG κ type of multiple myeloma. Her renal pathology consisted of widespread κ light chain protein deposition associated with the formation of large quantities of rod-like crystals in podocytes. This phenomenon is very rare. We explored the significance of this crystal formation via a detailed and descriptive analysis and also performed a literature review, thus providing data to increase the available information about this type of disease.

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

    Science.gov (United States)

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

    2015-10-01

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

  16. Adenovirus Recruits Dynein by an Evolutionary Novel Mechanism Involving Direct Binding to pH-Primed Hexon

    Directory of Open Access Journals (Sweden)

    Julian Scherer

    2011-08-01

    Full Text Available Following receptor-mediated uptake into endocytic vesicles and escape from the endosome, adenovirus is transported by cytoplasmic dynein along microtubules to the perinuclear region of the cell. How motor proteins are recruited to viruses for their own use has begun to be investigated only recently. We review here the evidence for a role for dynein and other motor proteins in adenovirus infectivity. We also discuss the implications of recent studies on the mechanism of dynein recruitment to adenovirus for understanding the relationship between pathogenic and physiological cargo recruitment and for the evolutionary origins of dynein-mediated adenovirus transport.

  17. Multiple myeloma and multiple plasmacytomas associated with free gamma heavy chain, free kappa light chain and IgGk paraproteins: an unusual triple gammopathy.

    Science.gov (United States)

    Deighan, William I; O'Kane, Maurice J; McNicholl, Feargal P; Keren, David F

    2016-11-01

    Multiple myeloma is a malignant plasma cell dyscrasia that is becoming more prevalent in an increasingly ageing population. It is a complex disease with clinical phases ranging from the premalignant monoclonal gammopathy of undetermined significance to asymptomatic (smouldering) myeloma and then symptomatic myeloma; the latter occasionally terminating in the clonal proliferation of plasma cells outside the bone marrow. We present a patient whose clonally evolved disease from monoclonal gammopathy of undetermined significance to multiple myeloma demonstrated the presence of an unusual combination of monoclonal immunoproteins. Capillary electrophoresis demonstrated the presence of three paraproteins in the gamma region (γ-region), two of which were additional to the IgGk paraprotein which migrated in the slow γ-region at initial diagnosis. Subsequent isotypic identification of the new paraproteins was not possible by immunotyping and initial immunofixation studies failed to definitively characterize the monoclonal proteins. After reduction with beta-mercaptoethanol, two paraproteins were detected by both capillary and gel electrophoresis. However, only immunofixation was able to resolve three distinct monoclonal bands, confirming the presence of free monoclonal kappa light chains in the mid-gamma region and free monoclonal heavy chains in the fast gamma region. Triple gammopathies in themselves are uncommon; this case presents a very unusual combination of paraproteins which required various electrophoretical and immunochemical techniques to identify and characterize them. The change of electrophoretic signature from the monoclonal gammopathy of undetermined significance phase to the diagnosis of multiple myeloma suggested that a number of genetically distinct subclones were present in the pretreatment clonal evolution of the disease.

  18. Myosin light chain phosphorylation is required for peak power output of mouse fast skeletal muscle in vitro.

    Science.gov (United States)

    Bowslaugh, Joshua; Gittings, William; Vandenboom, Rene

    2016-11-01

    The skeletal myosin light chain kinase (skMLCK) catalyzed phosphorylation of the myosin regulatory light chain (RLC) is associated with potentiation of force, work, and power in rodent fast twitch muscle. The purpose of this study was to compare concentric responses of EDL from wild-type (WT) and skMLCK devoid (skMLCK(-/-)) muscles at a range of shortening speeds (0.05 to 0.70 V max) around that expected to produce maximal power (in vitro, 25 °C) both before (unpotentiated) and after (potentiated) a potentiating stimulus (PS). When collapsed across all speeds tested, neither unpotentiated force, work, or power differed between genotypes (all data n = 10, P muscles. For example, when collapsed across the six fastest speeds we tested, both concentric force and power were increased 30-34 % in WT but only 15-17 % in skMLCK(-/-) muscles. In contrast, at the two slowest speeds, these parameters were increased in WT but decreased in skMLCK(-/-) muscles (8-10 and 7-9 %, respectively). Intriguingly, potentiation of concentric force and power was optimal near speeds producing maximal power in both genotypes. Because the PS elevated RLC phosphorylation above resting levels in WT but not in skMLCK(-/-) muscles, our data suggest that skMLCK-catalyzed phosphorylation of the RLC is required for maximal concentric power output of mouse EDL muscle stimulated at high frequency in vitro.

  19. Assessment of free light chains in the cerebrospinal fluid of patients with lymphomatous meningitis – a pilot study

    Directory of Open Access Journals (Sweden)

    Scholz C

    2007-10-01

    Full Text Available Abstract Background Lymphomatous meningitis (LM represents a severe complication of malignant lymphomas. While clinical suspicion is raised by symptoms ranging from mild disturbances of sensation to severe pain or impaired consciousness, the definite diagnosis of LM is often difficult to obtain. Since B-cell lymphomas are clonally restricted to express either kappa or lambda immunoglobulin light chain, we hypothesised that analysis of free light chain (FLC ratios might facilitate the diagnosis of LM. Methods Kappa and lambda FLC were measured using a novel nephelometric assay in cerebrospinal fluid (CSF and serum from 17 patients. 5/17 suffered from LM as demonstrated by cytology, immunocytology, and/or imaging procedures. Results Measurement of FLC concentrations in CSF was achieved for all 17 patients. FLC levels in CSF were lower than serum FLC levels in samples for the same patient obtained at the same time (p Conclusion This is the first report demonstrating that a significant proportion of LM patients display an abnormal kappa/lambda FLC ratio in the CSF.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

    Paolini, Lucia

    2016-06-01

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

  2. Effective market influence. An effect chain analysis of NUTEK`s high-frequency lighting campaign

    Energy Technology Data Exchange (ETDEWEB)

    Goeransson, C.; Faugert, S. [SIPU Utvaerdering, Stockholm (Sweden); Baeckman, B.; Arndt, J. [B2B AB, Stockholm (Sweden)

    1994-12-31

    This report is an evaluation of NUTEK`s `Better lighting and more energy-efficient high-frequency fluorescent tube luminaries` campaign. It is concerned with the ways in which NUTEK can influence the spread and use of high-frequency (HF) lighting devices. It also shows that NUTEK actually has affected (contributed to) development in the area. The report analyses and quantifies (as far as possible) how much NUTEK has influenced the various parties concerned in the sector, their attitudes and their actions. 14 figs, 5 tabs

  3. Molecular characterization and expression analysis of four isotypes of immunoglobulin light chain genes in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Wu, Ming-Shan; Cheng, Chao-An; Lin, Chih-Hung; Lee, Chiou-Yueh; Tseng, Shih-Jou; Tzeng, Chyng-Shyan; Chang, Chi-Yao

    2013-03-01

    To date, many immunoglobulin (Ig) genes have been identified in diverse teleost species, but the contributions of different types of light chain (IgL) to the immune response remain unclear. Screening of a stimulated kidney cDNA library from orange-spotted grouper (Osg, Epinephelus coioides) resulted in the identification of 26 full Ig light chain (OsgIgL) coding sequences. These 26 OsgIgLs encoded peptides from 235 to 248 amino acid residues and could be grouped into five variable (V(L)) and four constant (C(L)) isotypes. The C(L) regions contained three conserved cysteine residues that may participate in intra- or inter-chain disulfide bond formation. The four C(L) isotypes could be sub-grouped into two serological types: κ (C(L)-I, C(L)-II and C(L)-III) and σ (C(L)-IV), by phylogenetic analysis. The OsgIgL genes were found to be expressed in various tissues, with greatest levels of expression observed in the head-kidney and spleen. The major expression type was C(L)-I, which comprised 92% and 91% of total OsgIgL gene expression in the head-kidney and spleen, respectively. Transcription of all four C(L) isotypes was differentially affected in response to various immunostimulators, including lipopolysaccharide (LPS), poly I:C and grouper iridovirus (GIV). Induction of OsgIgL genes in response to immunostimulators was particularly dramatic in the spleen, suggesting this organ holds particular importance for the regulation of OsgIgL expression. Furthermore, vaccination of grouper with formalin-inactivated GIV also induced differential patterns of expression in all four OsgIgL isotypes. In summary, the significant and diverse patterns of transcriptional induction observed for OsgIgL isotypes in the spleen and head-kidney imply that each isotype may have unique roles in the immune response.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

    Science.gov (United States)

    Merveille, Anne-Christine; Davis, Erica E; Becker-Heck, Anita; Legendre, Marie; Amirav, Israel; Bataille, Géraldine; Belmont, John; Beydon, Nicole; Billen, Frédéric; Clément, Annick; Clercx, Cécile; Coste, André; Crosbie, Rachelle; de Blic, Jacques; Deleuze, Stephane; Duquesnoy, Philippe; Escalier, Denise; Escudier, Estelle; Fliegauf, Manfred; Horvath, Judith; Hill, Kent; Jorissen, Mark; Just, Jocelyne; Kispert, Andreas; Lathrop, Mark; Loges, Niki Tomas; Marthin, June K; Momozawa, Yukihide; Montantin, Guy; Nielsen, Kim G; Olbrich, Heike; Papon, Jean-François; Rayet, Isabelle; Roger, Gilles; Schmidts, Miriam; Tenreiro, Henrique; Towbin, Jeffrey A; Zelenika, Diana; Zentgraf, Hanswalter; Georges, Michel; Lequarré, Anne-Sophie; Katsanis, Nicholas; Omran, Heymut; Amselem, Serge

    2012-01-01

    Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry1. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex. PMID:21131972

  6. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs.

    Science.gov (United States)

    Merveille, Anne-Christine; Davis, Erica E; Becker-Heck, Anita; Legendre, Marie; Amirav, Israel; Bataille, Géraldine; Belmont, John; Beydon, Nicole; Billen, Frédéric; Clément, Annick; Clercx, Cécile; Coste, André; Crosbie, Rachelle; de Blic, Jacques; Deleuze, Stephane; Duquesnoy, Philippe; Escalier, Denise; Escudier, Estelle; Fliegauf, Manfred; Horvath, Judith; Hill, Kent; Jorissen, Mark; Just, Jocelyne; Kispert, Andreas; Lathrop, Mark; Loges, Niki Tomas; Marthin, June K; Momozawa, Yukihide; Montantin, Guy; Nielsen, Kim G; Olbrich, Heike; Papon, Jean-François; Rayet, Isabelle; Roger, Gilles; Schmidts, Miriam; Tenreiro, Henrique; Towbin, Jeffrey A; Zelenika, Diana; Zentgraf, Hanswalter; Georges, Michel; Lequarré, Anne-Sophie; Katsanis, Nicholas; Omran, Heymut; Amselem, Serge

    2011-01-01

    Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.

  7. Serum immunoglobulin free light chain measurements and heavy chain isotype usage provide insight into disease biology in patients with POEMS syndrome.

    Science.gov (United States)

    Stankowski-Drengler, Trista; Gertz, Morie A; Katzmann, Jerry A; Lacy, Martha Q; Kumar, Shaji; Leung, Nelson; Hayman, Suzanne R; Buadi, Francis; Kyle, Robert A; Rajkumar, S Vincent; Dispenzieri, Angela

    2010-06-01

    POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) syndrome is a rare paraneoplastic syndrome in which nearly all patients have a monoclonal lambda restricted plasma cell disorder. We investigated whether patients with POEMS have abnormal serum immunoglobulin free light chain (FLC) ratios. Fifty patients with newly diagnosed POEMS syndrome were assessed. Cystatin C levels were measured to discern whether subclinical renal insufficiency could account for FLC elevations in the presence of a normal FLC ratio. Forty-five patients (90%) had elevated lambda FLC; however, only nine (18%) had abnormal FLC ratios. The rise in serum FLC of POEMS patients appeared to be multifactorial-both a function of subclinical renal insufficiency and polyclonal activation of medullary and extramedullary plasma cells. Those patients expressing a clonal IgA were more likely to have clonal plasmacytosis observed on iliac crest biopsy than those with IgG. In summary, serum immunoglobulin profiles are unique in POEMS syndrome as compared with other plasma cell disorders.

  8. Kinetochore dynein generates a poleward pulling force to facilitate congression and full chromosome alignment

    Institute of Scientific and Technical Information of China (English)

    Yan Li; Wei Yu; Yun Liang; Xueliang Zhu

    2007-01-01

    For proper chromosome segregation, all kinetochores must achieve bipolar microtubule (MT) attachment and subsequently align at the spindle equator before anaphase onset. The MT minus end-directed motor dynein/dynactin binds kinetochores in prometaphase and has long been implicated in chromosome congression. Unfortunately, inactivation of dynein usually disturbs spindle organization, thus hampering evaluation of its kinetochore roles. Here we specifically eliminated kinetochore dynein/dynactin by RNAi-mediated depletion of ZW10, a protein essential for kinetochore localization of the motor. Time-lapse microscopy indicated markedly-reduced congression efficiency, though congressing chromosomes displayed similar velocities as in control cells. Moreover, cells frequently failed to achieve full chromosome alignment, despite their normal spindles. Confocal microcopy revealed that the misaligned kinetochores were monoori-ented or unattached and mostly lying outside the spindle, suggesting a difficulty to capture MTs from the opposite pole. Kinetochores on monoastral spindles were dispersed farther away from the pole and exhibited only mild oscillation. Furthermore, inactivating dynein by other means generated similar phenotypes. Therefore, kinetochore dynein produces on monooriented kinetochores a poleward pulling force, which may contribute to efficient bipolar attachment by facilitating their proper microtubule captures to promote congression as well as full chromosome alignment.

  9. DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

    Science.gov (United States)

    Awata, Junya; Song, Kangkang; Lin, Jianfeng; King, Stephen M.; Sanderson, Michael J.; Nicastro, Daniela; Witman, George B.

    2015-01-01

    The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo–electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC–dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform. PMID:26063732

  10. Tuning microtubule-based transport via filamentous MAPs: the problem of dynein

    Science.gov (United States)

    Vershinin, Michael; Xu, Jing; Razafsky, David S.; King, Stephen J.; Gross, Steven P.

    2010-01-01

    We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo. Here, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau’s regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels—where kinesin cannot bind microtubules—dynein can still contact microtubules. The difference between tau’s effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to microtubules. We show that this isoform-specific effect is not due to steric interference of tau’s projection domains, but rather due to tau’s interactions with the motor at the microtubule surface. Nonetheless, we do observe a modest steric interference effect of tau away from the microtubule and discuss the potential implications of this for molecular motor structure. PMID:18373727

  11. Misfolded Gβ is recruited to cytoplasmic dynein by Nudel for efficient clearance

    Institute of Scientific and Technical Information of China (English)

    Yihan Wan; Zhenye Yang; Jing Guo; Qiangge Zhang; Liyong Zeng; Wei Song; Yue Xiao; Xueliang Zhu

    2012-01-01

    The Gβγ heterodimer is an important signal transducer.Gβ,however,is prone to misfolding due to its requirement for Gγ and chaperones for proper folding.How cells dispose of misfolded Gβ (mfGβ) is not clear.Here,we showed that mfGβ was able to be polyubiquitinated and subsequently degraded by the proteasome.It was sequestered in aggresomes after the inhibition of the proteasome activity with MG132.Sustained activation of Gβγ signaling further elevated cellular levels of the ubiquitinated Gβ.Moreover,Nudel,a regulator of cytoplasmic dynein,the microtubule minus end-directed motor,directly interacted with both the unubiquitinated and ubiquitinated mfGβ.Increasing the levels of both mfGβ and Nudel promoted the association of Gβ with both Nudel and dynein,resulting in robust aggresome formation in a dynein-dependent manner.Depletion of Nudel by RNAi reduced the dynein-associated mfGβ,impaired the MG132-induced aggresome formation,and markedly prolonged the half-life of nascent Gβ.Therefore,cytosolic mfGβ is recruited to dynein by Nudel and transported to the centrosome for rapid sequestration and degradation.Such a process not only eliminates mfGβ efficiently for the control of protein quality,but may also help to terminate the Gβγ signaling.

  12. DRC3 connects the N-DRC to dynein g to regulate flagellar waveform.

    Science.gov (United States)

    Awata, Junya; Song, Kangkang; Lin, Jianfeng; King, Stephen M; Sanderson, Michael J; Nicastro, Daniela; Witman, George B

    2015-08-01

    The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo-electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC-dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform.

  13. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    Directory of Open Access Journals (Sweden)

    Dick eJaarsma

    2015-10-01

    Full Text Available The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly polarized morphology. The motor protein complex cytoplasmic dynein has an important role in Golgi apparatus positioning and function. Together with dynactin and other regulatory factors it drives microtubule minus-end directed motility of Golgi membranes. Inhibition of dynein results in fragmentation and dispersion of the Golgi ribbon in the neuronal cell body, resembling the Golgi abnormalities observed in some neurodegenerative disorders, in particular motor neuron diseases. Mutations in dynein and its regulatory factors, including the dynactin subunit p150Glued, BICD2 and Lis-1, are associated with several human nervous system disorders, including cortical malformation and motor neuropathy. Here we review the role of dynein and its regulatory factors in Golgi function and positioning, and the potential role of dynein malfunction in causing Golgi apparatus abnormalities in nervous system disorders.

  14. A new era for homolytic aromatic substitution: replacing Bu3SnH with efficient light-induced chain reactions.

    Science.gov (United States)

    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.

  15. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    Energy Technology Data Exchange (ETDEWEB)

    Lima, V.V. [Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT (Brazil); Lobato, N.S.; Filgueira, F.P. [Curso de Medicina, Setor de Fisiologia Humana, Universidade Federal de Goiás, Jataí, GO (Brazil); Webb, R.C. [Department of Physiology, Georgia Regents University, Augusta, GA (United States); Tostes, R.C. [Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Giachini, F.R. [Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT (Brazil)

    2014-08-15

    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{sup 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.

  17. Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

    NARCIS (Netherlands)

    Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

    2011-01-01

    The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

  18. Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

    NARCIS (Netherlands)

    Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

    2011-01-01

    The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

  19. Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration

    OpenAIRE

    Balastik, M.; Ferraguti, F.; A. Pires da Silva; Lee, T; Alvarez-Bolado, G.; Lu, K.; Gruss, P

    2008-01-01

    TRIM RING finger proteins have been shown to play an important role in cancerogenesis, in the pathogenesis of some human hereditary disorders, and in the defense against viral infection, but the function of the majority of TRIM proteins remains unknown. Here, we show that TRIM RING finger protein TRIM2, highly expressed in the nervous system, is an UbcH5a-dependent ubiquitin ligase. We further demonstrate that TRIM2 binds to neurofilament light subunit (NF-L) and regulates NF-L ubiquitination...

  20. An analytical solution describing the propagation of positive injury signals in an axon: effect of dynein velocity distribution.

    Science.gov (United States)

    Kuznetsov, A V

    2013-01-01

    A model describing the propagation of positive injury signals from the lesion site in an axon towards the neuron soma is described. It is assumed that these signals are driven by dynein molecular motors. An analytical solution that accounts for the probability density function (pdf) of a dynein velocity distribution is obtained. Two examples of pdf of dynein velocity distributions that follow from the results published in Ross et al. (2006, Processive bidirectional motion of dynein-dynactin complexes in vitro. Nat Cell Biol. 8:562-570) and Deinhardt et al. (2006, Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway. Neuron 52:293-305) are considered. The effect of dynein velocity distribution on the rate of spreading of the signal wave is discussed. It is demonstrated that the obtained solution can be applied to the problem of how neurons measure the distance between the lesion site and the neuron soma.

  1. Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus)

    DEFF Research Database (Denmark)

    Kim, Young Kyu; Lee, Jung Seok; Jung, Jae Wook

    2017-01-01

    abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder.......Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used...... successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11...

  2. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation.

    Science.gov (United States)

    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

    2016-04-12

    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.

  3. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation

    DEFF Research Database (Denmark)

    Hansen, Charlotte Toftmann

    2016-01-01

    , 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......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...... of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed....

  4. Characterization and recombinant protein expression of ferritin light chain homologue in the silkworm, Bombyx mori.

    Science.gov (United States)

    Hong, Sun Mee; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2014-04-01

    The silkworm genome encodes three iron storage proteins or ferritins, Fer1HCH, Fer2LCH, and Fer3HCH. Probing our EST library constructed from 1-day-old silkworm eggs revealed only Fer2LCH mRNA, which encoded for a protein with a predicted putative N-glycosylation site. Developmental and tissue expression analyses during embryogenesis revealed that Fer2LCH mRNA was abundant from 6 h to 6 days after oviposition. Transcriptional expression of Fer2LCH during the postembryonic stage is also high in the larval fat body and mid-gut, and then is upregulated in all pupal tissues tested. We found that Fer2LCH mRNA contains an iron-responsive element, suggesting this ferritin subunit is subject to translational control. Although ferritin expression has been shown to increase following immune challenge in other insects, the levels of Fer2LCH mRNA were not significantly induced following viral or bacterial infection of Bombyx mori. Using a baculovirus expression system we expressed recombinant BmFer2LCH protein, which was detectable in the cytoplasmic fraction, likely in a compartment of the secretory pathway, and was shown to undergo posttranslational modifications including N-glycosylation. In particular, rBmFer2LCH carbohydrate chains were composed of mannose and GlcNAc. We suggest that Fer2LCH is important for iron homeostasis and maintaining normal organ function in silkworms.

  5. Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes

    Science.gov (United States)

    Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

    2016-01-01

    Summary Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. PMID:26853472

  6. A Rare Case of Waldenström Macroglobulinemia/Lymphoplasmacytic Lymphoma with Light Chain Discrepancy between B Lymphocyte Population and Serum Paraprotein.

    Science.gov (United States)

    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.

  7. Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

    OpenAIRE

    1993-01-01

    Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow...

  8. Detailed structural and biochemical characterization of the nexin-dynein regulatory complex

    OpenAIRE

    Oda, Toshiyuki; Yanagisawa, Haruaki; Kikkawa, Masahide

    2015-01-01

    The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo–electron tomograph...

  9. Plasma vitamin D-binding protein (GC) factors, immunoglobulin G heavy chain (GM) allotypes and immunoglobulin kappa light chain (KM1) allotype in patients with sarcoidosis and in healthy control subjects

    DEFF Research Database (Denmark)

    Milman, Nils; Thymann, Mariann; Graudal, Niels

    2002-01-01

    and immunoglobulin kappa light chain (KM) allotype differed significantly from the distribution in healthy subjects. METHODS: GC 1S, 1F, 2 allotypes, GM 1, 2, 5 allotypes, and KM1 allotype were assessed in 44 patients with sarcoidosis and in healthy control subjects. RESULTS: There were no significant differences...

  10. The normally expressed kappa immunoglobulin light chain gene repertoire and somatic mutations studied by single-sided specific polymerase chain reaction (PCR); frequent occurrence of features often assigned to autoimmunity

    DEFF Research Database (Denmark)

    Juul, L; Hougs, L; Andersen, V

    1997-01-01

    The expressed human kappa light chain gene repertoire utilized by healthy individuals was studied by two different single-sided specific PCR techniques to avoid bias for certain V genes. A total of 103 rearranged kappa sequences from peripheral blood mononuclear cells from healthy individuals were...

  11. Abnormal kappa:lambda light chain ratio in circulating immune complexes as a marker for B cell activity in juvenile idiopathic arthritis.

    Science.gov (United States)

    Low, J M; Chauhan, A K; Moore, T L

    2007-01-01

    Patients with juvenile idiopathic arthritis (JIA) have been shown to have elevated levels of circulating immune complexes (CICs) which correlated with disease activity. Our aim was to assess B cell activity by measuring the amount of and the kappa:lambda chain immunoglobulin light (L) chain ratio in CICs from JIA patients and to determine potential evidence for either an antigen-driven response or B-cell receptor editing. We used an enzyme-linked immunosorbent assay to measure kappa and lambda chains present in the CICs from the sera of patients with JIA. Statistical analysis was performed using Pearson's correlation, one-way ANOVA and Bonferroni post hoc analysis. Sera from 44 JIA patients were examined for the concentration of L chains in CICs. Healthy controls had a kappa:lambda chain ratio of 1.2:1, whereas this ratio was reversed among JIA subgroups with RF-positive polyarthritis (1:1.2), RF-negative polyarthritis (1:1.3), oligoarthritis (1:2.3) and systemic-onset arthritis (1:2.5). In addition, overall lambda chain selection was not significantly associated with a particular immunoglobulin heavy (H) chain and occurred with all immunoglobulin isotypes. We showed preferential selection of lambda chains contributing to the formation of potentially pathogenic CICs from JIA patients, of all onset types compared to healthy controls, in an H chain-independent manner. The reversal of kappa:lambda chain ratio within the JIA CICs and association with all immunoglobulin isotypes demonstrated the potential for L chain editing. Furthermore, we conclude that a reversal of the normal kappa:lambda chain ratio in JIA CICs may be used as a marker for increased B-cell activity.

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

    Directory of Open Access Journals (Sweden)

    Marie Pancera

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

  13. Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms.

    Directory of Open Access Journals (Sweden)

    Ryosuke Yamamoto

    2017-09-01

    Full Text Available Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA as well as a fraction of the outer dynein arms (ODA. A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.

  14. Clinical usefulness of serum free light chains measurement in patients with multiple myeloma: comparative analysis of two different tests

    Directory of Open Access Journals (Sweden)

    Tadeusz Kubicki

    2017-01-01

    Full Text Available Introduction: There are two commercially available tests for measurement of serum free light chains (sFLC in multiple myeloma (MM patients – Freelite and N Latex FLC. The aim of this study was to perform an assessment and direct comparison of the usefulness of the methods in routine clinical practice.Methods: 40 refractory/relapsed MM patients underwent routine disease activity assessment studies, along with sFLC analysis using both assays. Correlation and concordance between the tests and sensitivity of studied methods of sFLC assessment were established. Special attention was focused on sFLC results in patients finally evaluated after completing the treatment. Results: A weak correlation for the measurement of both κ [Passing–Bablok slope (PB = 0.7681] and λ chains [(PB = 1.542] was found. Using Bland–Altman plots, a bias of 0.0467 (κ and -0.2133 (λ between the measurements was documented. The concordance coefficient equaled 0.87 for κ, 0.62 for λ and 0.52 for κ/λ ratio. Ten patients had an abnormal Freelite assay κ/λ ratio and normal N Latex FLC κ/λ ratio. Three of these patients had negative serum protein electrophoresis results and fulfilled diagnostic criteria of stringent complete remission (sCR according to N Latex FLC (but not according to Freelite. When the κ/λ ratio obtained by both methods was compared to patients’ serum/urine protein electrophoresis and immunofixation results, sensitivity of Freelite and N Latex FLC was established to be 62.5% and 41%, respectively. Conclusions: There was no strong correlation or concordance between the two assays, and the sensitivity in terms of sFLC detection was different. This may cause problems when diagnosis of sCR is considered.

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

    Science.gov (United States)

    Andruchov, Oleg; Galler, Stefan

    2008-03-01

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

  16. Trends in Survival and Renal Recovery in Patients with Multiple Myeloma or Light-Chain Amyloidosis on Chronic Dialysis.

    Science.gov (United States)

    Decourt, Alexandre; Gondouin, Bertrand; Delaroziere, Jean Christophe; Brunet, Philippe; Sallée, Marion; Burtey, Stephane; Dussol, Bertrand; Ivanov, Vadim; Costello, Regis; Couchoud, Cecile; Jourde-Chiche, Noemie

    2016-03-07

    Monoclonal gammopathies (MGs) with renal involvement can lead to ESRD caused by myeloma cast nephropathy (MCN), immunoglobulin light chain amyloidosis (ALA), or light-chain deposition disease (LCDD). Few studies have focused on the prognosis of patients with MG on chronic dialysis. We evaluated the outcomes of patients with MG incident on chronic dialysis in France. All incident patients registered in the Renal Epidemiology and Information Network Registry between 2002 and 2011 with ESRD caused by ALA, LCDD, or MCN were included. Patient's survival, censored for renal transplantation, renal recovery, and loss to follow-up, as well as renal outcomes were analyzed and compared with a control group. Risk factors and causes of death were analyzed. We included 1459 patients, comprising 265 (18%) patients with ALA, 334 (23%) patients with LCDD, and 861 (59%) patients with MCN. Median age was 72 years, and 56% were men. Median follow-up was 13.1 months. Renal recovery was observed in 9.1% of patients and more frequent after 2006. Kidney transplantation was rare in this population (2.3%). Among 1272 patients who remained on dialysis, 67% died. Median survival on dialysis was 18.3 months. Main causes of death were malignancies (34.4%), cardiovascular diseases (18%), infections (13.3%), and cachexia (5.2%). Independent risk factors of death were age (hazard ratio [HR], 1.03 per year increase; 95% confidence interval [95% CI], 1.02 to 1.03), frailty (HR, 1.93; 95% CI, 1.58 to 2.36), congestive heart failure (HR, 1.54; 95% CI, 1.23 to 1.93), and dialysis initiation on a central catheter (HR, 1.40; 95% CI, 1.11 to 1.75). Factors associated with a lower risk of death were year of dialysis initiation (HR, 0.95 per year increase; 95% CI, 0.91 to 0.99) and high BP (HR, 0.80; 95% CI, 0.67 to 0.97). Survival of patients with ALA, LCDD, or MCN on chronic dialysis is poor but has improved over time. Progressive malignancy is the main cause of death in this population. Renal recovery

  17. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    Energy Technology Data Exchange (ETDEWEB)

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L. (IIT); (UW-MED)

    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.

  18. Regulation of myosin light chain kinase during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

    Directory of Open Access Journals (Sweden)

    Shelly Woody

    Full Text Available Myosin II (MyoII is required for insulin-responsive glucose transporter 4 (GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC of MyoIIA via myosin light chain kinase (MLCK. The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy ethane-N,N,N',N'-tetra acetic acid, (BAPTA (in the presence of insulin impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

  19. Steady dynein forces induce flutter instability and propagating waves in mathematical models of flagella.

    Science.gov (United States)

    Bayly, P V; Dutcher, S K

    2016-10-01

    Cilia and flagella are highly conserved organelles that beat rhythmically with propulsive, oscillatory waveforms. The mechanism that produces these autonomous oscillations remains a mystery. It is widely believed that dynein activity must be dynamically regulated (switched on and off, or modulated) on opposite sides of the axoneme to produce oscillations. A variety of regulation mechanisms have been proposed based on feedback from mechanical deformation to dynein force. In this paper, we show that a much simpler interaction between dynein and the passive components of the axoneme can produce coordinated, propulsive oscillations. Steady, distributed axial forces, acting in opposite directions on coupled beams in viscous fluid, lead to dynamic structural instability and oscillatory, wave-like motion. This 'flutter' instability is a dynamic analogue to the well-known static instability, buckling. Flutter also occurs in slender beams subjected to tangential axial loads, in aircraft wings exposed to steady air flow and in flexible pipes conveying fluid. By analysis of the flagellar equations of motion and simulation of structural models of flagella, we demonstrate that dynein does not need to switch direction or inactivate to produce autonomous, propulsive oscillations, but must simply pull steadily above a critical threshold force.

  20. Mitochondria-driven assembly of a cortical anchor for mitochondria and dynein.

    Science.gov (United States)

    Kraft, Lauren M; Lackner, Laura L

    2017-10-02

    Interorganelle contacts facilitate communication between organelles and impact fundamental cellular functions. In this study, we examine the assembly of the MECA (mitochondria-endoplasmic reticulum [ER]-cortex anchor), which tethers mitochondria to the ER and plasma membrane. We find that the assembly of Num1, the core component of MECA, requires mitochondria. Once assembled, Num1 clusters persistently anchor mitochondria to the cell cortex. Num1 clusters also function to anchor dynein to the plasma membrane, where dynein captures and walks along astral microtubules to help orient the mitotic spindle. We find that dynein is anchored by Num1 clusters that have been assembled by mitochondria. When mitochondrial inheritance is inhibited, Num1 clusters are not assembled in the bud, and defects in dynein-mediated spindle positioning are observed. The mitochondria-dependent assembly of a dual-function cortical anchor provides a mechanism to integrate the positioning and inheritance of the two essential organelles and expands the function of organelle contact sites. © 2017 Kraft and Lackner.

  1. Understanding the Roles of Nudel/Lis1/Dynein Pathway in Cell Motility

    Institute of Scientific and Technical Information of China (English)

    Xie Lele; Zhu Xueliang

    2007-01-01

    @@ Under the support of multiple grants by NSFC, including General Program, Key Program, National Science Fund for Distinguished Young Scholars, and Fund for Creative Research Groups, the research group explored how the Nudel/Lis1/dynein pathway functions in cell motility.

  2. Stress-Induced CDK5 Activation Disrupts Axonal Transport via Lis1/Ndel1/Dynein

    Directory of Open Access Journals (Sweden)

    Eva Klinman

    2015-07-01

    Full Text Available Axonal transport is essential for neuronal function, and defects in transport are associated with multiple neurodegenerative diseases. Aberrant cyclin-dependent kinase 5 (CDK5 activity, driven by the stress-induced activator p25, also is observed in these diseases. Here we show that elevated CDK5 activity increases the frequency of nonprocessive events for a range of organelles, including lysosomes, autophagosomes, mitochondria, and signaling endosomes. Transport disruption induced by aberrant CDK5 activation depends on the Lis1/Ndel1 complex, which directly regulates dynein activity. CDK5 phosphorylation of Ndel1 favors a high affinity Lis1/Ndel/dynein complex that blocks the ATP-dependent release of dynein from microtubules, inhibiting processive motility of dynein-driven cargo. Similar transport defects observed in neurons from a mouse model of amyotrophic lateral sclerosis are rescued by CDK5 inhibition. Together, these studies identify CDK5 as a Lis1/Ndel1-dependent regulator of transport in stressed neurons, and suggest that dysregulated CDK5 activity contributes to the transport deficits observed during neurodegeneration.

  3. Bicaudal d family adaptor proteins control the velocity of Dynein-based movements

    NARCIS (Netherlands)

    Schlager, M.A.; Serra-Marquez, A.; Grigoriev, Ilya; Gumy, Laura; Estevez da Silva, M.; Wulf, Phebe; Akhmanova, Anna; Hoogenraad, Casper

    2014-01-01

    Cargo transport along microtubules is driven by the collective function of microtubule plus- and minus-end-directed motors (kinesins and dyneins). How the velocity of cargo transport is driven by opposing teams of motors is still poorly understood. Here, we combined inducible recruitment of motors a

  4. CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms

    Science.gov (United States)

    Panizzi, Jennifer R.; Becker-Heck, Anita; Castleman, Victoria H.; Al-Mutairi, Dalal; Liu, Yan; Loges, Niki T.; Pathak, Narendra; Austin-Tse, Christina; Sheridan, Eamonn; Schmidts, Miriam; Olbrich, Heike; Werner, Claudius; Häffner, Karsten; Hellman, Nathan; Chodhari, Rahul; Gupta, Amar; Kramer-Zucker, Albrecht; Olale, Felix; Burdine, Rebecca D.; Schier, Alexander F.; O’Callaghan, Christopher; Chung, Eddie MK; Reinhardt, Richard; Mitchison, Hannah M.; King, Stephen M.; Omran, Heymut; Drummond, Iain A.

    2012-01-01

    Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation, and to establish laterality1. Cilia motility defects cause Primary Ciliary Dyskinesia (PCD, MIM 242650), a disorder affecting 1:15-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive cilia bending2. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD linked loci3. Here we show that the zebrafish cilia paralysis mutant schmalhanstn222 (smh) mutant encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a regulated gene. Screening 146 unrelated PCD families identified patients in six families with reduced outer dynein arms, carrying mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103 functions as a tightly bound, axoneme-associated protein. The results identify Ccdc103 as a novel dynein arm attachment factor that when mutated causes Primary Ciliary Dyskinesia. PMID:22581229

  5. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders

    NARCIS (Netherlands)

    Jaarsma, Dick; Hoogenraad, Casper C

    2015-01-01

    The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmic dynein has

  6. Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins

    NARCIS (Netherlands)

    Namdeo, S.; Onck, P. R.

    2016-01-01

    Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dynein

  7. LRRC6 mutation causes primary ciliary dyskinesia with dynein arm defects.

    Directory of Open Access Journals (Sweden)

    Amjad Horani

    Full Text Available Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6 that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His. LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects.

  8. An Outer Arm Dynein Conformational Switch Is Required for Metachronal Synchrony of Motile Cilia in Planaria

    Science.gov (United States)

    Rompolas, Panteleimon; Patel-King, Ramila S.

    2010-01-01

    Motile cilia mediate the flow of mucus and other fluids across the surface of specialized epithelia in metazoans. Efficient clearance of peri-ciliary fluids depends on the precise coordination of ciliary beating to produce metachronal waves. The role of individual dynein motors and the mechanical feedback mechanisms required for this process are not well understood. Here we used the ciliated epithelium of the planarian Schmidtea mediterranea to dissect the role of outer arm dynein motors in the metachronal synchrony of motile cilia. We demonstrate that animals that completely lack outer dynein arms display a significant decline in beat frequency and an inability of cilia to coordinate their oscillations and form metachronal waves. Furthermore, lack of a key mechanosensitive regulatory component (LC1) yields a similar phenotype even though outer arms still assemble in the axoneme. The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination. In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia. We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony. PMID:20844081

  9. Expression, purification, and partial in vitro characterization of biologically active human coagulation factor VIII light chain (A3-C1-C2) in Pichia pastoris.

    Science.gov (United States)

    A R, Sudheer Reddy; Satheeshkumar, Padikara Kutty; Vijayalakshmi, Mookambeswaran A

    2013-09-01

    Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1-A2) and light chains (A3-C1-C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.

  10. Successful management of refractory pleural effusion due to systemic immunoglobulin light chain amyloidosis by vincristine adriamycin dexamethasone chemotherapy: a case report

    Directory of Open Access Journals (Sweden)

    Mima Akira

    2010-10-01

    Full Text Available Abstract Introduction Refractory pleural effusion in systemic immunoglobulin light chain amyloidosis without cardiac decompensation is rarely reported and has a poor prognosis in general (a median survival of 1.6 months. Moreover, the optimum treatment for this condition is still undecided. This is the first report on the successful use of vincristine, adriamycin and dexamethasone chemotherapy for refractory pleural effusion due to systemic immunoglobulin light chain amyloidosis without cardiac decompensation. Case presentation We report the case of a 68-year old Japanese male with systemic immunoglobulin light chain amyloidosis presenting with bilateral pleural effusion (more severe on the right side in the absence of cardiac decompensation that was refractory to diuretic therapy. The patient was admitted for fatigue, exertional dyspnea, and bilateral lower extremity edema. He had been receiving intermittent melphalan and prednisone chemotherapy for seven years. One month before admission, his dyspnea had got worse, and his chest radiograph showed bilateral pleural effusion; the pleural effusion was ascertained to be a transudate. The conventionally used therapeutic measures, including diuretics and thoracocentesis, failed to control pleural effusion. Administration of vincristine, adriamycin, and dexamethasone chemotherapy led to successful resolution of the effusion. Conclusion Treatment with vincristine, adriamycin, and dexamethasone chemotherapy was effective for the refractory pleural effusion in systemic immunoglobulin light chain amyloidosis without cardiac decompensation and appears to be associated with improvement in our patient's prognosis.

  11. ASSESSMENT OF THE RESIDUAL TUMOR IN PATIENTS WITH MULTIPLE MYELOMA BASED ON THE ANALYSIS OF THE FREE LIGHT CHAINS OF IMMUNOGLOBULINS IN BLOOD SERUM

    Directory of Open Access Journals (Sweden)

    T. A. Мitina

    2013-01-01

    Full Text Available Efficiency of the multiple myeloma treatment with chemotherapy including bortezomib was assessed based on determination of the level of immunoglobulin free light chains in blood serum. The method enables estimation of changes in kinetic parameters of the residual tumor, detection of the disease course prognosis, and the choice of the optimal approach to the disease therapy.

  12. Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway

    NARCIS (Netherlands)

    Wang, Siyuan; Chen, Cheng; Su, Ke; Zha, Dongqing; Liang, Wei; Hillebrands, J L; van Goor, Harry; Ding, Guohua

    2016-01-01

    Aims In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. Methods Eighteen

  13. Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway

    NARCIS (Netherlands)

    Wang, Siyuan; Chen, Cheng; Su, Ke; Zha, Dongqing; Liang, Wei; Hillebrands, J L; van Goor, Harry; Ding, Guohua

    2016-01-01

    Aims In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. Methods Eighteen m

  14. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.

    Science.gov (United States)

    Poetter, K; Jiang, H; Hassanzadeh, S; Master, S R; Chang, A; Dalakas, M C; Rayment, I; Sellers, J R; Fananapazir, L; Epstein, N D

    1996-05-01

    The muscle myosins and hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle. We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three-dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

  15. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

    Science.gov (United States)

    Bookwalter, Carol S; Kelsen, Anne; Leung, Jacqueline M; Ward, Gary E; Trybus, Kathleen M

    2014-10-31

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

  16. A Toxoplasma gondii Class XIV Myosin, Expressed in Sf9 Cells with a Parasite Co-chaperone, Requires Two Light Chains for Fast Motility*

    Science.gov (United States)

    Bookwalter, Carol S.; Kelsen, Anne; Leung, Jacqueline M.; Ward, Gary E.; Trybus, Kathleen M.

    2014-01-01

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors. PMID:25231988

  17. Cherenkov light production from the α-emitting decay chains of (223)Ra, (212)Pb, and (149)Tb for Cherenkov Luminescence Imaging.

    Science.gov (United States)

    Wood, V; Ackerman, N L

    2016-12-01

    Cherenkov Luminescence Imaging (CLI) is a new method to image radioactive therapeutic and diagnostic agents, primarily in preclinical studies. This study used Geant4 and Python to generate the predicted Cherenkov light production as a function of time for a set of isotopic chains of interest for targeted alpha therapy: (223)Ra, (212)Pb, and (149)Tb. All are shown to produce substantial Cherenkov light, though time delays between initial decays and the production of Cherenkov light requires caution in interpreting CLI. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA, electrochemiluminescence immunoassay and Simoa.

    Science.gov (United States)

    Kuhle, Jens; Barro, Christian; Andreasson, Ulf; Derfuss, Tobias; Lindberg, Raija; Sandelius, Åsa; Liman, Victor; Norgren, Niklas; Blennow, Kaj; Zetterberg, Henrik

    2016-10-01

    Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are specific cytoskeletal proteins of neurons and their quantification has shown encouraging results as a biomarker for axonal injury. We aimed at comparing a widely used conventional ELISA for Nf light chain (NfL) with an electrochemiluminescence-based method (ECL assay) and a newly developed single-molecule array (Simoa) method in clinically relevant cerebrospinal fluid (CSF) and serum samples. Analytical sensitivity was 0.62 pg/mL for Simoa, 15.6 pg/mL for the ECL assay, and 78.0 pg/mL for the ELISA. Correlations between paired CSF and serum samples were strongest for Simoa (r=0.88, pNfL measurements between the platforms were highly correlated (r=1.0, pNfL levels were highly related between ECL assay and Simoa (r=0.86, pNfL levels than controls when measured with Simoa (p=0.001) but not with the other platforms. We found Simoa to be more sensitive than ELISA or the ECL assay. Our results support the feasibility of quantifying NfL in serum; the results correlate with the more-established CSF NfL test. The highly sensitive Simoa technology deserves further studies in larger patient cohorts to clarify whether serum NfL could be used in the future to measure disease severity and determine prognosis or response to treatment interventions in neurological diseases.

  19. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in Neurodegenerative Diseases.

    Science.gov (United States)

    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.

  1. Molecular and Functional Analyses of the Fast Skeletal Myosin Light Chain2 Gene of the Korean Oily Bitterling, Acheilognathus koreensis

    Directory of Open Access Journals (Sweden)

    Hyun Kook Cho

    2013-08-01

    Full Text Available We identified and characterized the primary structure of the Korean oily bitterling Acheilognathus koreensis fast skeletal myosin light chain 2 (Akmlc2f, gene. Encoded by seven exons spanning 3955 bp, the deduced 168-amino acid AkMLC2f polypeptide contained an EF-hand calcium-binding motif and showed strong homology (80%–98% with the MLC2 proteins of Ictalurus punctatus and other species, including mammals. Akmlc2f mRNA was highly enriched in skeletal muscles, and was detectable in other tissues. The upstream regions of Akmlc2f included a TATA box, one copy of a putative MEF-2 binding site and several putative C/EBPβ binding sites. The functional activity of the promoter region of Akmlc2f was examined using luciferase and red fluorescent protein reporters. The Akmlc2f promoter-driven reporter expressions were detected and increased by the C/EBPβ transcription factor in HEK293T cells. The activity of the promoter of Akmlc2f was also confirmed in the developing zebrafish embryo. Although the detailed mechanism underlying the expression of Akmlc2f remains unknown, these results suggest the muscle-specific expression of Akmlc2f transcript and the functional activation of Akmlc2f promoter by C/EBPβ.

  2. Identification and characterization of the Bombyx mori myosin II essential light chain and its effect in BmNPV infection

    Directory of Open Access Journals (Sweden)

    L Hao

    2015-02-01

    Full Text Available Myosin, as a type of molecular motor, is mainly involved in muscle contraction. Recently, myosin research has made considerable progress. However, the function of Bombyx mori myosin remains unclear. In this study, we cloned the BmMyosin II essential light chain (BmMyosin II ELC gene from a cDNA library of silkworm, which had an open reading frame (ORF of 444 bp encoding 147 amino acids (about 16 kDa. After analyzing their sequences, BmMyosin II ELC was similar to the ELCs of 27 other Myosin II types, which contained EFh domain that bound Ca2+. In addition, 28 sequences had five motifs, motifs 1 and 3 were relatively conserved. We constructed two vectors with BmMyosin to transfect MGC803 or BmN, monolayer wound healing of cells indicated they can promote cell migration successfully. For three fifth instar silkworms, Bm306, BmNB, BmBC8, we mainly analyzed the change of BmMyosin II ELC from transcription and translation after infecting with nucleopolyhedrovirus (BmNPV. We found that gene expression of resistant strains were higher than susceptible strains at 12 h, while the result of the translation level was opposite that of the transcription level. Through in vitro protein interactions, we found BmMyosin II ELC can interact with BmNPV ubiquitin.

  3. Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

    LENUS (Irish Health Repository)

    Manser, C

    2012-05-31

    A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

  4. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation.

    Science.gov (United States)

    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.

  5. Rebeccamycin Attenuates TNF-α-Induced Intestinal Epithelial Barrier Dysfunction by Inhibiting Myosin Light Chain Kinase Production

    Directory of Open Access Journals (Sweden)

    Akihiro Watari

    2017-04-01

    Full Text Available Background/Aims: Although proinflammatory cytokine–induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α. Methods: To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER and paracellular permeability were measured. Production levels and localization of tight junction (TJ proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC and MLC kinase (MLCK mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively. Results: Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition. Conclusion: Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation.

  6. Rebeccamycin Attenuates TNF-α-Induced Intestinal Epithelial Barrier Dysfunction by Inhibiting Myosin Light Chain Kinase Production.

    Science.gov (United States)

    Watari, Akihiro; Sakamoto, Yuta; Hisaie, Kota; Iwamoto, Kazuki; Fueta, Miho; Yagi, Kiyohito; Kondoh, Masuo

    2017-01-01

    Although proinflammatory cytokine-induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α. To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER) and paracellular permeability were measured. Production levels and localization of tight junction (TJ) proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC) and MLC kinase (MLCK) mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively. Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition. Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation. © 2017 The Author(s)Published by S. Karger AG, Basel.

  7. Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum.

    Science.gov (United States)

    Okada, Chisa; Nakamura, Akio; Tomioka, Shigeo; Kohama, Kazuhiro; Kaneko, Takako S

    2010-08-01

    A phosphatase was purified through a combination of ion-exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340-fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The Km of the enzyme for PMLC was 10 microM, and the V(max) was 1.17 nkat/mg of protein. Ca(2+) (10 microM) inhibited the activity of the enzyme, and Mg(2+) (8.5 microM) activated the dephosphorylation of PMLC. Mn(2+) (1.6 microM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.

  8. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Directory of Open Access Journals (Sweden)

    Amol Bhargava

    Full Text Available 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.

  9. [Effect of vitamin E on myosin light chain kinase activity and endothelial permeability of the artery in atherosclerotic rabbit].

    Science.gov (United States)

    Zhu, Hua-qing; Ren, Bin; Xiong, Jiang-xia; Hu, Ruo-lei; Liu, Xiao-ying; Wang, Xue; Zhang, Su-mei; Liu, Hui-lan; Jiang, Zhi-kui; Zhou, Qing; Gui, Shu-yu; Wang, Yuan

    2003-08-01

    To study the effect of vitamin E (Vit E) on the myosin light chain kinase(MLCK) activity and the endothelial permeability of the artery in atherosclerotic rabbits. The MLCK activity of rabbit artery was measured by incorporation of gamma-(32)P. The endothelial permeability was accessed by immunofluorescence. The model of atherosclerosis was established after rabbits were fed with cholesterol for four weeks. The activity of MLCK increased markedly, and there was significantly statistical difference compared with the normal control (Prabbits were fed with cholesterol for twelve weeks or with cholesterol and Vit E for twelve weeks, the activity of MLCK did not change markedly, and there was no statistical difference compared with the normal control, respectively (P>0.05). The permeability of arterial wall was increased after the rabbits were fed with cholesterol for four weeks, and the permeability increased even more obviously after the rabbits were fed with cholesterol for twelve weeks. The permeability appeared to be decreased when Vit E was added into the cholesterol feeding. The change in integrity of arterial wall may be associated with the increase of the activity of MLCK. Vit E may decrease the MLCK activity. Vit E may decrease the endothelial permeability of atherosclerotic rabbits.

  10. Auto-SCT improves survival in systemic light chain amyloidosis: a retrospective analysis with 14-year follow-up.

    Science.gov (United States)

    Parmar, S; Kongtim, P; Champlin, R; Dinh, Y; Elgharably, Y; Wang, M; Bashir, Q; Shah, J J; Shah, N; Popat, U; Giralt, S A; Orlowski, R Z; Qazilbash, M H

    2014-08-01

    Optimal treatment approach continues to remain a challenge for systemic light chain amyloidosis (AL). So far, Auto-SCT is the only modality associated with long-term survival. However, failure to show survival benefit in randomized study raises questions regarding its efficacy. We present a comparative outcome analysis of Auto-SCT to conventional therapies (CTR) in AL patients treated over a 14-year period at our institution. Out of the 145 AL amyloidosis patients, Auto-SCT was performed in 80 patients with 1-year non-relapse mortality rate of 12.5%. Novel agents were used as part of induction therapy in 56% of transplant recipients vs 46% of CTR patients. Hematological and organ responses were seen in 74.6% and 39% in the Auto-SCT arm vs 53% and 12% in the CTR arm, respectively. The projected 5-year survival for Auto-SCT vs CTR was 63% vs 38%, respectively. Landmark analysis of patients alive at 1-year after diagnosis showed improved 5-year OS of 72% with Auto-SCT vs 65% in the CTR arm. In the multivariate analysis, age Auto-SCT were associated with improved survival. In conclusion, Auto-SCT is associated with long-term survival for patients with AL amyloidosis.

  11. Melphalan, lenalidomide and dexamethasone for the treatment of immunoglobulin light chain amyloidosis: results of a phase II trial.

    Science.gov (United States)

    Sanchorawala, Vaishali; Patel, Jaymin M; Sloan, J Mark; Shelton, Anthony C; Zeldis, Jerome B; Seldin, David C

    2013-05-01

    We report results of a phase II trial of combination of melphalan, lenalidomide, and dexamethasone for the treatment of immunoglobulin light chain (AL) amyloidosis. The primary objectives were tolerability and hematologic response rate; secondary objectives were organ responses and survival. Treatment protocol consisted of melphalan 5 mg/m(2)/day for four days, lenalidomide 10 mg/day for 21 days and dexamethasone 20-40 mg once a week every 28 days for a total of 12 cycles. Sixteen subjects were enrolled of whom 14 completed at least 3 cycles and were evaluable for response. Grade 3/4 toxicities were experienced by 88% (n=14), the most common being myelosuppression (n=7). Dose reductions occurred in 85% (n=12 of 14) of subjects. Hematologic partial and complete responses were achieved by 43% (n=6 of 14) and 7% (n=1 of 14), respectively. The median overall survival has not been reached and median progression-free survival is 24 months. In conclusion, this combination is associated with significant myelosuppression leading to dose modifications and producing minor hematologic responses in AL amyloidosis. http://clinicaltrials.gov/ct2/show/NCT00679367.

  12. Yeast myosin light chain, Mlc1p, interacts with both IQGAP and class II myosin to effect cytokinesis.

    Science.gov (United States)

    Boyne, J R; Yosuf, H M; Bieganowski, P; Brenner, C; Price, C

    2000-12-01

    MLC1 (myosin light chain) acts as a dosage suppressor of a temperature sensitive mutation in the gene encoding the S. cerevisiae IQGAP protein. Both proteins localize to the bud neck in mitosis although Mlc1p localisation precedes Iqg1p. Mlc1p is also found at the incipient bud site in G(1) and the growing bud tip during S and G(2) phases of the cell cycle. A dominant negative GST-Mlc1p fusion protein specifically blocks cytokinesis and prevents Iqg1p localisation to the bud neck, as does depletion of Mlc1p. These data support a direct interaction between the two proteins and immunoprecipitation experiments confirm this prediction. Mlc1p is also shown to interact with the class II conventional myosin (Myo1p). All three proteins form a complex, however, the interaction between Mlc1p and Iqg1p can be separated from the Mlc1p/Myo1p interaction. Mlc1p localisation and maintenance at the bud neck is independent of actin, Myo1p and Iqg1p. It is proposed that Mlc1p therefore functions to recruit Iqg1p and in turn actin to the actomyosin ring and that it is also required for Myo1p function during ring contraction.

  13. Free Light Chains and Intrathecal B Cells Activity in Multiple Sclerosis: A Prospective Study and Meta-Analysis

    Science.gov (United States)

    Passerini, Gabriella; Sangalli, Francesca; Moiola, Lucia; Colombo, Bruno; Locatelli, Massimo; Comi, Giancarlo

    2016-01-01

    Background. The presence of CSF oligoclonal bands (OBs) is an independent prognostic factor for multiple sclerosis (MS), but the difficulties in the standardization of the test and the interlaboratory variation in reporting have contributed to its limited use in the diagnosis of the disease. Standard nephelometric assays to measure free light chains (FLC) levels have been recently developed and the test may improve the detection of intrathecal B cells activity. Methods. The presence of OBs, kappa and lambda FLC levels, and standard indices of intrathecal inflammation were assessed in 100 consecutive patients, including patients with MS, clinically isolated syndromes (CIS), other inflammatory diseases of the CNS, and other noninflammatory diseases. Results. Both KFLC and LFLC correlated strongly with the presence of OCBs and with all common tests for intrathecal inflammation (p < 0.001 for all comparisons). KFLC and LFLC were significantly different in patients with MS and CIS compared to the other groups (p < 0.001 and p < 0.001, resp.) and had a better diagnostic accuracy than all the other tests (area under the curve 82.3 % for KFLC index and 79.3 % for LFLC index). Conclusion. Nephelometric assays for KFLC in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate MS patients. PMID:28116160

  14. Free Light Chains and Intrathecal B Cells Activity in Multiple Sclerosis: A Prospective Study and Meta-Analysis

    Directory of Open Access Journals (Sweden)

    Gabriella Passerini

    2016-01-01

    Full Text Available Background. The presence of CSF oligoclonal bands (OBs is an independent prognostic factor for multiple sclerosis (MS, but the difficulties in the standardization of the test and the interlaboratory variation in reporting have contributed to its limited use in the diagnosis of the disease. Standard nephelometric assays to measure free light chains (FLC levels have been recently developed and the test may improve the detection of intrathecal B cells activity. Methods. The presence of OBs, kappa and lambda FLC levels, and standard indices of intrathecal inflammation were assessed in 100 consecutive patients, including patients with MS, clinically isolated syndromes (CIS, other inflammatory diseases of the CNS, and other noninflammatory diseases. Results. Both KFLC and LFLC correlated strongly with the presence of OCBs and with all common tests for intrathecal inflammation (p<0.001 for all comparisons. KFLC and LFLC were significantly different in patients with MS and CIS compared to the other groups (p<0.001 and p<0.001, resp. and had a better diagnostic accuracy than all the other tests (area under the curve 82.3 % for KFLC index and 79.3 % for LFLC index. Conclusion. Nephelometric assays for KFLC in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate MS patients.

  15. Slow motility in hair cells of the frog amphibian papilla: myosin light chain-mediated shape change.

    Science.gov (United States)

    Farahbakhsh, Nasser A; Narins, Peter M

    2008-07-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

  18. Long-term reversibility of renal dysfunction associated to light chain deposition disease with bortezomib and dexamethasone and high dose therapy and autologous stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Tomás J. González-López

    2011-11-01

    Full Text Available A 63-year-old woman presented with progressive renal insufficiency, until a glomerular filtration rate (GFR of 12 mL/min. A renal biopsy demonstrated glomerular deposition of immunoglobulin k light chain. The presence of a small population of monoclonal plasmacytes producing an only light k monoclonal component was demonstrated and Bortezomib and Dexamethasone (BD was provided as initial therapy. After seven courses of therapy, renal function improved without dialysis requirements up to a GFR 31 mL/min. Under hematological complete response (HCR the patient underwent high dose of melphalan (HDM and autologous peripheral blood stem cell transplant. Fifty-four months later the patient remains in HCR and the GFR has progressively improved up to 48 mL/min. This report describes a notably renal function improvement in a patient with Light Chain Deposition Disease after therapy with BD followed by HDM, which can support this treatment as a future option for these patients.

  19. Lighting

    Data.gov (United States)

    Federal Laboratory Consortium — Lighting Systems Test Facilities aid research that improves the energy efficiency of lighting systems. • Gonio-Photometer: Measures illuminance from each portion of...

  20. Dynein and mast/orbit/CLASP have antagonistic roles in regulating kinetochore-microtubule plus-end dynamics.

    Science.gov (United States)

    Reis, Rita; Feijão, Tália; Gouveia, Susana; Pereira, António J; Matos, Irina; Sampaio, Paula; Maiato, Helder; Sunkel, Claudio E

    2009-07-15

    Establishment and maintenance of the mitotic spindle requires the balanced activity of microtubule-associated proteins and motors. In this study we have addressed how the microtubule plus-end tracking protein mast/orbit/CLASP and cytoplasmic dynein regulate this process in Drosophila melanogaster embryos and S2 cells. We show that mast accumulates at kinetochores early in mitosis, which is followed by a poleward streaming upon microtubule attachment. This leads to a reduction of mast levels at kinetochores during metaphase and anaphase that depends largely on the microtubule minus end-directed motor cytoplasmic dynein. Surprisingly, we also found that co-depletion of dynein rescues spindle bipolarity in mast-depleted cells, while restoring normal microtubule poleward flux. Our results suggest that mast and dynein have antagonistic roles in the local regulation of microtubule plus-end dynamics at kinetochores, which are important for the maintenance of spindle bipolarity and normal spindle length.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  2. Light

    DEFF Research Database (Denmark)

    Prescott, N.B.; Kristensen, Helle Halkjær; Wathes, C.M.

    2004-01-01

    This chapter presents the effect of artificial light environments (light levels, colour, photoperiod and flicker) on the welfare of broilers in terms of vision, behaviour, lameness and mortality......This chapter presents the effect of artificial light environments (light levels, colour, photoperiod and flicker) on the welfare of broilers in terms of vision, behaviour, lameness and mortality...

  3. Optical anisotropy of polyimide and polymethacrylate containing photocrosslinkable chalcone group in the side chain under irradiation of a linearly polarized UV light

    CERN Document Server

    Choi, D H

    2002-01-01

    Photocrosslinkable soluble polyimide and polymethacrylate compound were synthesized for studying the optically induced anisotropy of the thin films. Chalcone group was introduced into the side chain unit of two polymers. We observed a photodimerization behavior between the double bonds in the chalcone group and an optical anisotropy of these materials by irradiation of a linearly polarized UV light (LPL). Optical anisotropy of the thin film was also investigated by using polarized UV absorption spectroscopy.The dynamic property of optical anisotropy in photoreactive polyimide was compared to that in polymethacrylate containing chalcone group in the side chain.

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

    Science.gov (United States)

    Singh, Gurmukh

    2017-01-01

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

  5. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

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    Drescher Uwe

    2010-06-01

    Full Text Available Abstract Background In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A. It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. Results In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK and myosin light chain kinase (MLCK, which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. Conclusions Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

  8. Acute heart failure with cardiomyocyte atrophy induced in adult mice by ablation of cardiac myosin light chain kinase.

    Science.gov (United States)

    Massengill, Michael T; Ashraf, Hassan M; Chowdhury, Rajib R; Chrzanowski, Stephen M; Kar, Jeena; Warren, Sonisha A; Walter, Glenn A; Zeng, Huadong; Kang, Byung-Ho; Anderson, Robert H; Moss, Richard L; Kasahara, Hideko

    2016-07-01

    Under pressure overload, initial adaptive hypertrophy of the heart is followed by cardiomyocyte elongation, reduced contractile force, and failure. The mechanisms governing the transition to failure are not fully understood. Pressure overload reduced cardiac myosin light chain kinase (cMLCK) by ∼80% within 1 week and persists. Knockdown of cMLCK in cardiomyocytes resulted in reduced cardiac contractility and sarcomere disorganization. Thus, we hypothesized that acute reduction of cMLCK may be causative for reduced contractility and cardiomyocyte remodelling during the transition from compensated to decompensated cardiac hypertrophy. To mimic acute cMLCK reduction in adult hearts, the floxed-Mylk3 gene that encodes cMLCK was inducibly ablated in Mylk3(flox/flox)/merCremer mice (Mylk3-KO), and compared with two control mice (Mylk3(flox/flox) and Mylk3(+/+)/merCremer) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). In Mylk3-KO mice, reduction of cMLCK protein was evident by 4 days, with a decline to below the level of detection by 6 days. By 7 days, these mice exhibited heart failure, with reduction of fractional shortening compared with those in two control groups (19.8 vs. 28.0% and 27.7%). Severely convoluted cardiomyocytes with sarcomeric disorganization, wavy fibres, and cell death were demonstrated in Mylk3-KO mice. The cardiomyocytes were also unable to thicken adaptively to pressure overload. Our results, using a new mouse model mimicking an acute reduction of cMLCK, suggest that cMLCK plays a pivotal role in the transition from compensated to decompensated hypertrophy via sarcomeric disorganization. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  9. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm

    Science.gov (United States)

    Li, Liliang; Li, Yuhua; Lin, Junyi; Jiang, Jieqing; He, Meng; Sun, Daming; Zhao, Ziqin; Shen, Yiwen; Xue, Aimin

    2016-01-01

    Background It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. Material/Methods To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. Results The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. Conclusions p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2. PMID:27643564

  10. Kinesin light chain 1 suppression impairs human embryonic stem cell neural differentiation and amyloid precursor protein metabolism.

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    Rhiannon L Killian

    Full Text Available 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.

  11. Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

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    Hong, Feng; Facemyer, Kevin C; Carter, Michael S; Jackson, Del R; Haldeman, Brian D; Ruana, Nick; Sutherland, Cindy; Walsh, Michael P; Cremo, Christine R; Baker, Josh E

    2013-11-26

    During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

  12. Inhibition of Myosin light-chain kinase attenuates cerebral edema after traumatic brain injury in postnatal mice.

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    Rossi, Janet L; Todd, Tracey; Bazan, Nicolas G; Belayev, Ludmila

    2013-10-01

    Traumatic brain injury (TBI) in children less than 8 years of age leads to decline in intelligence and executive functioning. Neurological outcomes after TBI correlate to development of cerebral edema, which affect survival rates after TBI. It has been shown that myosin light-chain kinase (MLCK) increases cerebral edema and that pretreatment with an MLCK inhibitor (ML-7) reduces cerebral edema. The aim of this study was to determine whether inhibition of MLCK after TBI in postnatal day 24 (PND-24) mice would prevent breakdown of the blood-brain barrier (BBB) and development of cerebral edema and improve neurological outcome. We used a closed head injury model of TBI. ML-7 or saline treatment was administered at 4 h and every 24 h until sacrifice or 5 days after TBI. Mice were sacrificed at 24 h, 48 h, and 72 h and 7 days after impact. Mice treated with ML-7 after TBI had decreased levels of MLCK-expressing cells (20.7±4.8 vs. 149.3±40.6), less albumin extravasation (28.3±11.2 vs. 116.2±60.7 mm(2)) into surrounding parenchymal tissue, less Evans Blue extravasation (339±314 vs. 4017±560 ng/g), and showed a significant difference in wet/dry weight ratio (1.9±0.07 vs. 2.2±0.05 g), compared to saline-treated groups. Treatment with ML-7 also resulted in preserved neurological function measured by the wire hang test (57 vs. 21 sec) and two-object novel recognition test (old vs. new, 10.5 touches). We concluded that inhibition of MLCK reduces cerebral edema and preserves neurological function in PND-24 mice.

  13. Structural Dynamics of Actin during Active Interaction with Myosin Depends on the Isoform of the Essential Light Chain

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

    2013-01-01

    We have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of essential light chain (ELC) isoforms (A1 and A2) on the interaction of skeletal muscle myosin with actin, in order to relate structural dynamics to previously reported functional effects. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes S1A1 and S1A2 by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin’s final anisotropy cooperatively and to a similar extent, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (S) state of actomyosin. In contrast, in the presence of saturating ATP, S1A1 increased actin anisotropy much more than S1A2 and with greater cooperativity, indicating that S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin. We conclude that during the active interaction of actin and ATP with myosin, S1A1 is more effective at stabilizing the S state (probably the force-generating state) of actomyosin, while S1A2 tends to stabilize the weak-binding (non-force-generating) W state. When a mixture of isoenzymes is present, S1A1 is dominant in its effects on actin dynamics. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, with significant implications for the contractile function of actomyosin. PMID:23339370

  14. Urinary free light chains may help to identify infection in patients with elevated systemic inflammation due to rheumatic disease.

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

    2017-04-01

    The risk of infection in patients with rheumatic diseases is elevated, but a clear marker to differentiate the cause of the systemic inflammation is missing. We assessed the ability urinary immunoglobulin free light chains (FLCs) to indicate the presence of infection in patients with rheumatic disease. We performed a retrospective analysis of patients with rheumatic disease attending the Georg-August University Hospital in Goettingen, Germany, from January 2011 to December 2013. Subjects were included if they had urine levels of κ and λ FLCs available. A reference group of patients without autoimmune disease, but with documented infection, was constructed. A total of 1500 patients had their urinary FLCs quantified during the study period. Of the 382 patients with rheumatic disease, 172 (45%) displayed no systemic inflammation, 162 (42%) had inflammation due to the underlying disease activity, and 48 (13%) had inflammation due to a confirmed infection. Urinary FLC concentrations were much higher in patients with rheumatic diseases and infection (κ 68.8 ± 81.8 mg/L, λ 31.4 ± 53.5 mg/L) compared to those with inflammation due to rheumatic disease activity (κ 22.7 ± 26.3 mg/L, λ 8.1 ± 9.1 mg/L, κ p inflammation due to rheumatic disease activity from that due to the additional presence of infection. The ability to quantify these proteins in urine provides a simple alternative to the use of blood.

  15. Receptor-associated protein blocks internalization and cytotoxicity of myeloma light chain in cultured human proximal tubular cells.

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    Sule Sengul

    Full Text Available BACKGROUND: Free light chains (LCs are among the many ligands that bind to cubilin/megalin for endocytosis via the clathrin-dependent endosomal/lysosomal pathway. Receptor associated protein (RAP, is a 39 kDA high-affinity, chaperone-like ligand for megalin that assists in the proper folding and functioning of megalin/cubilin. Although RAP is known to inhibit ligand binding to megalin/cubilin, its effect on LC endocytosis has not been shown directly. METHODS AND PRINCIPAL FINDINGS: We investigated whether RAP can block the endocytosis of LC in cultured human proximal tubule cells and whether this can prevent LC cytotoxicity. Immunofluorescence microscopy and flow cytometry showed that fluorescently labeled LC endocytosis was markedly inhibited in HK-2 cells pretreated with human RAP. The effect of RAP was dose-dependent, and was predominantly on endocytosis as it had no effect on the small acid-washable fraction of LC bound to cell membrane. RAP significantly inhibited LC induced cytokine production and phosphorylation of ERK1/2 and p38 MAPK. Prolonged exposure to LC for 48 h resulted in epithelial-to-mesenchymal transformation in HK-2 cells as evidenced by marked reduction in the expression of the epithelial cell marker E-cadherin, and increased the expression of the mesenchymal marker α-SMA, which was also prevented by RAP in the endocytosis medium. CONCLUSIONS: RAP inhibited LC endocytosis by ∼88% and ameliorated LC-induced cytokine responses and EMT in human PTCs. The results not only provide additional evidence that LCs endocytosis occurs via the megalin/cubilin endocytic receptor system, but also show that blocking LC endocytosis by RAP can protect proximal tubule cells from LC cytotoxicity.

  16. Molecular cloning, characterisation and mRNA expression analysis of the sheep myosin light chain 1 gene.

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    Zhang, Chunlan; Wang, Guizhi; Ji, Zhibin; Liu, Zhaohua; Hou, Lei; Liu, Guanqing; Wang, Jianmin

    2015-09-10

    The complete cDNA sequence of the sheep MYL1 (Myosin light chain 1) gene was cloned using RT-PCR, 5' RACE and 3' RACE. We obtained two alternatively spliced isoforms of the MYL1 gene, MYL1a and MYL1b, which are 849 and 1046bp in length and encode proteins composed of 150 and 192 amino acid residues, respectively. And the GenBank accession numbers of MYL1a and MYL1b full-length cDNA sequences that we cloned are KJ700419 and KJ710701, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation and phosphorylation sites. More than half of the secondary structure of these proteins was predicted to be α-helical. The human MYL2 protein (1m8q.1.C) is the most similar in tertiary structure. Sequence alignment showed that the sheep MYL1a protein shares more than 92% amino acid sequence similar with Mus musculus, Homo sapiens, Rattus norvegicus, Sus scrofa and Gallus gallus and that the MYL1b protein shares more than 93% amino acid sequence similar with M. musculus, H. sapiens, R. norvegicus, Bos taurus and Oryctolagus cuniculus. Transcription profile analyses of various tissues indicated that the sheep MYL1a and MYL1b mRNAs were highly but differentially expressed in the longissimus dorsi. Moreover, the expression levels of these genes in the longissimus dorsi differed between Dorper and Small-tailed Han sheep. These results serve as a foundation for further investigations of the function of the sheep MYL1 gene.

  17. The Association between Polyclonal Combined Serum Free Light Chain Concentration and Mortality in Individuals with Early Chronic Kidney Disease.

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    Lakhvir K Assi

    Full Text Available 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 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.

  18. Prognostic value of left atrial function in systemic light-chain amyloidosis: a cardiac magnetic resonance study.

    Science.gov (United States)

    Mohty, Dania; Boulogne, Cyrille; Magne, Julien; Varroud-Vial, Nicolas; Martin, Sylvain; Ettaif, Hind; Fadel, Bahaa M; Bridoux, Frank; Aboyans, Victor; Damy, Thibaud; Jaccard, Arnaud

    2016-09-01

    Cardiac involvement in systemic light-chain amyloidosis (AL) imparts an adverse impact on outcome. The left atrium (LA), by virtue of its anatomical location and muscular wall, is commonly affected by the amyloid process. Although LA infiltration by amyloid fibrils leads to a reduction in its pump function, the infiltration of the left ventricular (LV) myocardium results in diastolic dysfunction with subsequent increase in filling pressures and LA enlargement. Even though left atrial volume (LAV) is an independent prognostic marker in many cardiomyopathies, its value in amyloid heart disease remains to be determined. In addition, few data are available as to the prognostic value of LA function in systemic AL. Using cardiac magnetic resonance (CMR), the current study aims to assess the prognostic significance of the maximal LAV and total LA emptying fraction (LAEF) in patients with AL. Fifty-four consecutive patients (age 66 ± 10 years, 59% males) with confirmed systemic AL and mean LV ejection fraction of 60 ± 12% underwent CMR. As compared with patients with no or minimal cardiac involvement (Mayo Clinic [MC] stage I), those at moderate and high risk (MC stages II and III) had significantly larger indexed maximal LAV (36 ± 15 vs. 46 ± 13 vs. 52 ± 19 mL/m(2), P = 0.03) and indexed minimal LAV (20 ± 6 vs. 34 ± 11 vs. 44 ± 17 mL/m(2), P 16% (37 ± 11 vs. 94 ± 4%, P = 0.001). In multivariate analysis, lower LAEF remained independently associated with a higher risk of 2-year mortality (HR = 1.08 per 1% decrease, 95% CI: 1.02-1.15, P = 0.003). In patients with systemic AL, LAEF as assessed by CMR is associated with NYHA functional class, MC stage, myocardial LGE and 2-year mortality. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  19. The value of serum immunoglobulin free light chain assessment in patients with monoclonal gammopathies and acute renal failure.

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    Cirit, Mustafa; Uzüm, Atilla; Ozen, Pınar; Sentürk, Banu A; Bozkaya, Giray; Payzin, Bahriye; Ural, Orçun

    2012-12-01

    Immunoglobulin free light chain (FLC) abnormalities are common in patients with monoclonal gammopathies and the kidneys are the most affected organs. Immunoassays that provide quantitative measurement of FLC in serum indicate monoclonal FLC production based on the presence of an abnormal FLC kappa:lambda (κ:λ) ratio. The aim of this study was to assess the utility of serum FLC measurement as a diagnostic tool for detecting plasma cell dyscrasias in comparison to standard assays, and to ascertain its sensitivity and specificity in patients with acute renal failure (ARF). Sera from 82 patients with ARF were assessed using serum protein electrophoresis (SPE), serum immunofixation electrophoresis (SIFE), and FLC measurement. The sensitivity and specificity of the FLC ratio in identifying which ARF patients had multiple myeloma (MM) was compared to those of SPE and SIFE. Among the 82 patients with ARF, 7 were diagnosed as MM using SPE, SIFE, and bone marrow biopsy techniques. In total, 8 patients did not have a FLC κ:λ ratio that was within the published reference range (0:26-1:65); the FLC κ:λ ratio based on FLC measurement had a specificity of 96% and sensitivity of 71%, and positive and negative predictive values of 62.9% and 97.3%, respectively, for the diagnosis of MM. The sensitivity and specificity of the FLC κ:λ ratio for diagnosing MM in patients that presented with ARF were lower than those of SPE and SIFE. To further delineate the utility of the FLC κ:λ ratio additional prospective, well-designed large-scale studies are needed. None declared.

  20. A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

    Science.gov (United States)

    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.

  1. Transport and arrangement of the outer-dynein-arm docking complex in the flagella of Chlamydomonas mutants that lack outer dynein arms.

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    Wakabayashi, K; Takada, S; Witman, G B; Kamiya, R

    2001-04-01

    The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC.

  2. The nondigestible disaccharide epilactose increases paracellular Ca absorption via rho-associated kinase- and myosin light chain kinase-dependent mechanisms in rat small intestines.

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    Suzuki, Takuya; Nishimukai, Megumi; Takechi, Maki; Taguchi, Hidenori; Hamada, Shigeki; Yokota, Atsushi; Ito, Susumu; Hara, Hiroshi; Matsui, Hirokazu

    2010-02-10

    We previously showed that epilactose, a nondigestible disaccharide, increased calcium (Ca) absorption in the small intestines of rats. Here, we explored the mechanism(s) underlying the epilactose-mediated promotion of Ca absorption in a ligated intestinal segment of anesthetized rats. The addition of epilactose to the luminal solution increased Ca absorption and chromium (Cr)-EDTA permeability, a paracellular indicator, with a strong correlation (R = 0.93) between these changes. Epilactose induced the phosphorylation of myosin regulatory light chains (MLCs), which is known to activate the paracellular route, without any change in the association of tight junction proteins with the actin cytoskeleton. The epilactose-mediated promotion of the Ca absorption was suppressed by specific inhibitors of myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). These results indicate that epilactose increases paracellular Ca absorption in the small intestine of rats through the induction of MLC phosphorylation via MLCK- and ROCK-dependent mechanisms.

  3. Cytoplasmic Dynein Is Required for the Spatial Organization of Protein Aggregates in Filamentous Fungi

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    Martin J. Egan

    2015-04-01

    Full Text Available Eukaryotes have evolved multiple strategies for maintaining cellular protein homeostasis. One such mechanism involves neutralization of deleterious protein aggregates via their defined spatial segregation. Here, using the molecular disaggregase Hsp104 as a marker for protein aggregation, we describe the spatial and temporal dynamics of protein aggregates in the filamentous fungus Aspergillus nidulans. Filamentous fungi, such as A. nidulans, are a diverse group of species of major health and economic importance and also serve as model systems for studying highly polarized eukaryotic cells. We find that microtubules promote the formation of Hsp104-positive aggregates, which coalesce into discrete subcellular structures in a process dependent on the microtubule-based motor cytoplasmic dynein. Finally, we find that impaired clearance of these inclusions negatively impacts retrograde trafficking of endosomes, a conventional dynein cargo, indicating that microtubule-based transport can be overwhelmed by chronic cellular stress.

  4. Dynamin-1-like protein (Dnm1L) interaction with kinesin light chain 1 (KLC1) through the tetratricopeptide repeat (TPR) domains.

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    Jang, Won Hee; Jeong, Young Joo; Choi, Sun Hee; Kim, Sang-Jin; Urm, Sang-Hwa; Seog, Dae-Hyun

    2014-01-01

    Kinesin light chain 1 (KLC1) mediates binding of KIF5 motor to specific cargo. Using the yeast two-hybrid screening, we found that mitochondrial fission protein dynamin-1-like protein (Dnm1L) interacted with KLC1, but not KIF5. Dnm1L and KLC1 were co-localized in cultured cells. These results suggest that KLC1 may play a potential role in post-fission mitochondrial transport.

  5. A First Report of Endoscopic Ultrasound for the Diagnosis of Pancreatic Amyloid Deposition in Immunoglobulin Light Chain (AL Amyloidosis (Primary Amyloidosis

    Directory of Open Access Journals (Sweden)

    Somashekar G Krishna

    2013-05-01

    Full Text Available Context Pancreatic involvement in systemic light chain (AL-amyloidosis is exceedingly rare. Prior reports of endoscopicultrasound (EUS for the diagnosis of amyloidosis are also limited. Case report We report the first description of EUS-guided fine needle aspiration (FNA for the diagnosis of primary AL-amyloidosis involving the pancreas. Conclusion EUS-FNA can be effectively utilized for the characterization and cytologic diagnosis of pancreatic amyloidosis and potentially other accessible extraluminal amyloid deposits.

  6. The p25 Subunit of the Dynactin Complex is Required for Dynein-Early Endosome Interaction

    Science.gov (United States)

    2011-01-01

    dynein–early endosome interaction in the filamen- tous fungus Aspergillus nidulans. In filamentous fungi , dynein and its regulators are important for...backbone of the dynactin complex, and its loss leads to a disruption of the whole complex. In Drosoph- ila and in filamentous fungi such as N. crassa...how the motor is targeted to these cargoes is still a topic under investigation. In filamentous fungi and higher eukaryotic cells such as neurons

  7. Apparent role of dynein in glucose-6-phosphate dehydrogenase trafficking in neutrophils from pregnant women.

    Science.gov (United States)

    Huang, Ji-Biao; Espinoza, Jimmy; Romero, Roberto; Petty, Howard R

    2006-03-01

    To better understand the mechanisms of metabolic microcompartmentalization associated with neutrophil hexose monophosphate shunt activity during pregnancy, we have studied the intracellular trafficking of glucose-6-phosphate dehydrogenase (G6PDase). Microtubule motor proteins colocalize with G6PDase. Dynein inhibitors block G6PDase accumulation at the microtubule-organizing center in pregnancy cells. On this basis, we conclude that microtubule motor proteins participate in hexose monophosphate shunt enzyme transport within leukocytes.

  8. De novo DNA Methyltransferases Dnmt3a and Dnmt3b regulate the onset of Igκ light chain rearrangement during early B-cell development.

    Science.gov (United States)

    Manoharan, Anand; Du Roure, Camille; Rolink, Antonius G; Matthias, Patrick

    2015-08-01

    Immunoglobulin genes V(D)J rearrangement during early lymphopoiesis is a critical process involving sequential recombination of the heavy and light chain loci. A number of transcription factors act together with temporally activated recombinases and chromatin accessibility changes to regulate this complex process. Here, we deleted the de novo DNA methyltransferases Dnmt3a and Dnmt3b in early B cells of conditionally targeted mice, and monitored the process of V(D)J recombination. Dnmt3a and Dnmt3b deletion resulted in precocious recombination of the immunoglobulin κ light chain without impairing the differentiation of mature B cells or overall B-cell development. Ex vivo culture of IL-7 restricted early B-cell progenitors lacking Dnmt3a and Dnmt3b showed precocious Vκ-Jκ rearrangements that are limited to the proximal Vκ genes. Furthermore, B-cell progenitors deficient in Dnmt3a and Dnmt3b showed elevated levels of germline transcripts at the proximal Vκ genes, alterations in methylation patterns at Igκ enhancer sites and increased expression of the transcription factor E2A. Our data suggest that Dnmt3a and Dnmt3b are critical to regulate the onset of Igκ light chain rearrangement during early B-cell development.

  9. Detailed structural and biochemical characterization of the nexin-dynein regulatory complex.

    Science.gov (United States)

    Oda, Toshiyuki; Yanagisawa, Haruaki; Kikkawa, Masahide

    2015-01-15

    The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo-electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella.

  10. Dynein regulates epithelial polarity and the apical localization of stardust A mRNA.

    Science.gov (United States)

    Horne-Badovinac, Sally; Bilder, David

    2008-01-01

    Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico-basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt) to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico-basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.

  11. Dynein regulates epithelial polarity and the apical localization of stardust A mRNA.

    Directory of Open Access Journals (Sweden)

    Sally Horne-Badovinac

    2008-01-01

    Full Text Available Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico-basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico-basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.

  12. Effect of catch bonding on transport of cellular cargo by dynein motors

    Science.gov (United States)

    Nair, Anil; Chandel, Sameep; Mitra, Mithun K.; Muhuri, Sudipto; Chaudhuri, Abhishek

    2016-09-01

    Recent experiments have demonstrated that dynein motors exhibit catch bonding behavior, in which the unbinding rate of a single dynein decreases with increasing force, for a certain range of force. Motivated by these experiments, we study the effect of catch bonding on unidirectional transport properties of cellular cargo carried by multiple dynein motors. We introduce a threshold force bond deformation (TFBD) model, consistent with the experiments, wherein catch bonding sets in beyond a critical applied load force. We find catch bonding can result in dramatic changes in the transport properties, which are in sharp contrast to kinesin-driven unidirectional transport, where catch bonding is absent. We predict that under certain conditions, the average velocity of the cellular cargo can actually increase as applied load is increased. We characterize the transport properties in terms of a velocity profile plot in the parameter space of the catch bond strength and the stall force of the motor. This plot yields predictions that may be experimentally accessed by suitable modifications of motor transport and binding properties.

  13. Regulation of dynein-mediated autophagosomes trafficking by ASM in CASMCs.

    Science.gov (United States)

    Xu, Ming; Zhang, Qiufang; Li, Pin-Lan; Nguyen, Thaison; Li, Xiang; Zhang, Yang

    2016-01-01

    Acid sphingomyelinase (ASM; gene symbol Smpd1) has been shown to play a crucial role in autophagy maturation by controlling lysosomal fusion with autophagosomes in coronary arterial smooth muscle cells (CASMCs). However, the underlying molecular mechanism by which ASM controls autophagolysosomal fusion remains unknown. In primary cultured CASMCs, lysosomal Ca2+ induced by 7-ketocholesterol (7-Ket, an atherogenic stimulus and autophagy inducer) was markedly attenuated by ASM deficiency or TRPML1 gene silencing suggesting that ASM signaling is required for TRPML1 channel activity and subsequent lysosomal Ca(2+) release. In these CASMCs, ASM deficiency or TRPML1 gene silencing markedly inhibited 7-Ket-induced dynein activation. In addition, 7-Ket-induced autophagosome trafficking, an event associated with lysosomal Ca(2+) release and dynein activity, was significantly inhibited in ASM-deficient (Smpd1(-/-)) CASMCs compared to that in Smpd1(+/+) CASMCs. Finally, overexpression of TRPML1 proteins restored 7-Ket-induced lysosomal Ca(2+) release and autophagosome trafficking in Smpd1-/- CASMCs. Collectively, these results suggest that ASM plays a critical role in regulating lysosomal TRPML1-Ca(2+) signaling and subsequent dynein-mediated autophagosome trafficking, which leads its role in controlling autophagy maturation in CASMCs under atherogenic stimulation.

  14. Degradation of cardiac myosin light chain kinase by matrix metalloproteinase-2 contributes to myocardial contractile dysfunction during ischemia/reperfusion.

    Science.gov (United States)

    Gao, Ling; Zheng, Yan-Jun; Gu, Shan-Shan; Tan, Ji-Liang; Paul, Christian; Wang, Yi-Gang; Yang, Huang-Tian

    2014-12-01

    Although ischemia/reperfusion (I/R)-induced myocardial contractile dysfunction is associated with a prominent decrease in myofilament Ca(2+) sensitivity, the underlying mechanisms have not yet been fully clarified. Phosphorylation of ventricular myosin light chain 2 (MLC-2v) facilitates actin-myosin interactions and enhances contractility, however, its level and regulation by cardiac MLC kinase (cMLCK) and cMLC phosphatase (cMLCP) in I/R hearts are debatable. In this study, the levels and/or effects of MLC-2v phosphorylation, cMLCK, cMLCP, and proteases during I/R were determined. Global myocardial I/R-suppressed cardiac performance in isolated rat hearts was concomitant with decreases of MLC-2v phosphorylation, myofibrillar Ca(2+)-stimulated ATPase activity, and cMLCK content, but not cMLCP proteins. Consistently, simulated I/R in isolated cardiomyocytes inhibited cell shortening, Ca(2+) transients, MLC-2v phosphorylation, and myofilament sensitivity to Ca(2+). These observations were reversed by cMLCK overexpression, while the specific cMLCK knockdown by short hairpin RNA (shRNA) had the opposite effect. Moreover, the inhibition of matrix metalloproteinase-2 (MMP-2, a zinc-dependent endopeptidase) reversed IR-decreased cMLCK, MLC-2v phosphorylation, myofibrillar Ca(2+)-stimulated ATPase activity, myocardial contractile function, and myofilament sensitivity to Ca(2+), while the inhibition or knockdown of cMLCK by ML-9 or specific shRNA abolished MMP-2 inhibition-induced cardioprotection. Finally, the co-localization in cardiomyocytes and interaction in vivo of MMP-2 and cMLCK were observed. Purified recombinant rat cMLCK was concentration- and time-dependently degraded by rat MMP-2 in vitro, and this was prevented by the inhibition of MMP-2. These findings reveal that the I/R-activated MMP-2 leads to the degradation of cMLCK, resulting in a reduction of MLC-2v phosphorylation, and myofibrillar Ca(2+)-stimulated ATPase activity, which subsequently suppresses

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

    Science.gov (United States)

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

    2015-04-01

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

  16. The normally expressed kappa immunoglobulin light chain gene repertoire and somatic mutations studied by single-sided specific polymerase chain reaction (PCR); frequent occurrence of features often assigned to autoimmunity

    DEFF Research Database (Denmark)

    Juul, L; Hougs, L; Andersen, V

    1997-01-01

    The expressed human kappa light chain gene repertoire utilized by healthy individuals was studied by two different single-sided specific PCR techniques to avoid bias for certain V genes. A total of 103 rearranged kappa sequences from peripheral blood mononuclear cells from healthy individuals were....... V genes from the Jkappa-proximal duplication unit of the kappa locus were almost exclusively used. A total of 65% of the sequences could be assigned to four or five genes: A27 (humkv325), L6 (Vg), L2 (humkv328), and A3 and/or A19. N additions and P nucleotides were quite common and found in 32...

  17. Molecular identification of the light brown apple moth (Lepidoptera: Tortricidae) in California using a polymerase chain reaction assay of the internal transcribed spacer 2 locus.

    Science.gov (United States)

    Barr, N B; Ledezma, L A; Vasquez, J D; Epstein, M; Kerr, P H; Kinnee, S; Sage, O; Gilligan, T M

    2009-12-01

    A molecular protocol using a hemi-nested polymerase chain reaction (PCR) of the internal transcribed spacer region 2 (ITS2) is reported for the diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in California. This protocol distinguishes the light brown apple moth from other moths in California based on size differences of PCR amplicons that are visualized on agarose gels. The molecular diagnostic tool generated no false negatives based on analysis of 337 light brown apple moths collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 424 moths representing other tortricid species generated correct identification for >95% of the samples and only two false positives. Of the 761 moths tested only fourteen produced no PCR amplicons and five generated inconclusive data.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2011-01-01

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

  20. Light

    CERN Document Server

    Robertson, William C

    2003-01-01

    Why is left right and right left in the mirror? Baffled by the basics of reflection and refraction? Wondering just how the eye works? If you have trouble teaching concepts about light that you don t fully grasp yourself, get help from a book that s both scientifically accurate and entertaining with Light. By combining clear explanations, clever drawings, and activities that use easy-to-find materials, this book covers what science teachers and parents need to know to teach about light with confidence. It uses ray, wave, and particle models of light to explain the basics of reflection and refraction, optical instruments, polarization of light, and interference and diffraction. There s also an entire chapter on how the eye works. Each chapter ends with a Summary and Applications section that reinforces concepts with everyday examples. Whether you need a deeper understanding of how light bends or a good explanation of why the sky is blue, you ll find Light more illuminating and accessible than a college textbook...

  1. Light-Induced ESR Studies of Quadrimolecular Recombination Kinetics of Photogenerated Charge Carriers in Regioregular Poly(3-alkylthiophene)/C60 Composites: Alkyl Chain Dependence

    Science.gov (United States)

    Tanaka, Hisaaki; Hasegawa, Naoki; Sakamoto, Tomotaka; Marumoto, Kazuhiro; Kuroda, Shin-ichi

    2007-08-01

    Light-induced ESR (LESR) measurements have been performed on the composites of regioregular poly(3-alkylthiophene) (RR-P3AT) and C60 by using polymers having different alkyl chains (CmH2m+1 with m=6, 8, 10, 12). The quadrimolecular recombination (QR) kinetics of photogenerated charge carriers, previously reported, have been confirmed for all the composites from the excitation power (Iex) dependence of the LESR intensity showing an ˜Iex0.25 dependence. The time decay of LESR intensity is also consistent with the QR model. Considering that only bimolecular recombination is observed in regiorandom polymer composites, the occurrence of QR strongly suggests the formation of doubly charged states, either bipolarons or polaron pairs on the regioregular polymer chains. On the other hand, the QR rate constant γ has been found to exhibit weak alkyl chain dependence, contrary to the case of the field-effect mobility of pure regioregular polymers with systematic alkyl chain dependence. This implies the significant contribution of the polymer and fullerene interface in determining γ.

  2. Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.

    Science.gov (United States)

    Reddy, P; Sparvoli, A; Fagioli, C; Fassina, G; Sitia, R

    1996-01-01

    Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi. Images PMID:8641273

  3. A Markov Chain-based quantitative study of angular distribution of photons through turbid slabs via isotropic light scattering

    Science.gov (United States)

    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.

  4. On the role of long-chain aldehydes in the light reaction in Photobacterium phosphoreum enzyme preparations

    NARCIS (Netherlands)

    Terpstra, Willemke

    1960-01-01

    1. (1) Active luciferase-DPNH-oxidase preparations from Photobacterium phosphoreum generally contain some aldehyde-attacking enzyme, probably ADH. Under the experimental conditions applied this enzyme appears to attack decanal, but not palmital. 2. (2) The presence of long-chain aldehydes in the en

  5. Vernier zone residue of 4 mouse subgroup II kappa light chains is a critical determinant for antigen recognition

    NARCIS (Netherlands)

    Haard, de H.; Kazemier, B.; Bent, van der A.; Oudshoorn, P.; Boender, P.; Arends, J.W.; Gemen, van B.

    1999-01-01

    Background: During the conversion of murine monoclonal antibodies directed against the human chorionic gonadotropin (hCG) into bacterially expressed single chain fragments (scFv), we found a major reduction of binding activity upon introduction of a primer encoded mutation. Objectives: In this study

  6. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

    Science.gov (United States)

    Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

    2015-10-01

    Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

  7. Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries.

    Science.gov (United States)

    Injeti, Elisha R; Sandoval, Renan J; Williams, James M; Smolensky, Alexander V; Ford, Lincoln E; Pearce, William J

    2008-12-01

    Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56+/-0.29%) than in fetal preparations (7.39+/-0.53%). In situ MLC20 concentrations (microM) in adult (198+/-28) and fetal arteries (236+/-44) did not differ significantly. In situ MLCK concentrations (microM), however, were significantly greater in adult (8.21+/-0.59) than in fetal arteries (1.83+/-0.13). In situ MLCK activities (ng MLC20 phosphorylated.s(-1).ng MLCK(-1)) were significantly less in adult (0.26+/-0.01) than in fetal arteries (1.52+/-0.11). In contrast, MLCK activities in adult (15.8+/-1.5) and fetal artery homogenates (17.3+/-1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72+/-0.17%) were significantly less than fetal values (9.08+/-0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.

  8. Light

    CERN Document Server

    Ditchburn, R W

    2011-01-01

    This classic study, available for the first time in paperback, clearly demonstrates how quantum theory is a natural development of wave theory, and how these two theories, once thought to be irreconcilable, together comprise a single valid theory of light. Aimed at students with an intermediate-level knowledge of physics, the book first offers a historical introduction to the subject, then covers topics such as wave theory, interference, diffraction, Huygens' Principle, Fermat's Principle, and the accuracy of optical measurements. Additional topics include the velocity of light, relativistic o

  9. DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

    OpenAIRE

    Awata, Junya; Song, Kangkang; Lin, Jianfeng; King, Stephen M.; Sanderson, Michael J.; Nicastro, Daniela; Witman, George B.

    2015-01-01

    The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to o...

  10. Monte Carlo Simulation on Coordinated Movement of Kinesin and Dynein Motors

    Institute of Scientific and Technical Information of China (English)

    WANG Hong; DOU Shuo-Xing; WANG Peng-Ye

    2008-01-01

    Kinesin and dynein are two important classes of molecular motors which are responsible for active organelle trafficking and cell division.They call work together to carry a cargo,moving along the microtubule in a coordinated way.We use Monte Carlo method to simulate the dynamics of this coordinated movement.Based on four essential assumptions,our simulations reproduce some features of the recent in vivo experiments.The fast moving speed of the cargo js simulated and the speed distribution is presented.

  11. Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex.

    Science.gov (United States)

    Gomez Toledo, Alejandro; Nilsson, Jonas; Noborn, Fredrik; Sihlbom, Carina; Larson, Göran

    2015-12-01

    The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)(n), to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally

  12. Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1 Are Essential for the Binding of Clathrin Light Chain

    Directory of Open Access Journals (Sweden)

    Joel A. Ybe

    2009-01-01

    Full Text Available The interaction between HIP family proteins (HIP1 and HIP12/1R and clathrin is fundamental to endocytosis. We used circular dichroism (CD to study the stability of an HIP1 subfragment (aa468-530 that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83C∘. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494. The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae, HIP1 from D. melanogaster, and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.

  13. Flow cytometric analysis of kappa and lambda light chain expression in endoscopic biopsy specimens before the diagnosis of B-cell lymphoma.

    Science.gov (United States)

    Oka, Satoko; Muroi, Kazuo; Sato, Kazuya; Fujiwara, Shin-ichiro; Oh, Iekuni; Matsuyama, Tomohiro; Ohmine, Ken; Suzuki, Takahiro; Ozaki, Katsutoshi; Mori, Masaki; Nagai, Tadashi; Fukushima, Noriyoshi; Fukushima, Noriyoshi; Tanaka, Akira; Ozawa, Keiya

    2012-01-01

    Forty-eight patients with gastrointestinal (GI) tract B-cell lymphoma (BCL) were analyzed retrospectively. The diagnosis was based on the histological examination of specimens obtained by endoscopic biopsy. Before the diagnosis was made, single-color flow cytometry was performed to analyze the expression of light chains and B-cell antigens including CD10 in the specimens. Restricted light chain (RLC) expression, a marker of B-cell clonality, was defined as κ and λ ratios of either more than 3.0 or less than 0.5. The specimens from 30 patients (62.5%) showed RLC expression. No RLC expression or RLC expression not examined was divided into two groups : those showing CD10 positivity in more than 20% of cells (4 patients, 8.3%) and those showing no positivity (14 patients, 29.2%). The cell number analyzed in the latter group was significantly smaller than that in the other two groups. Abnormal karyotypes were found in the specimens from 8 patients (16.7%). These results indicate that the flow cytometric analysis of endoscopic biopsy specimens is useful when BCL is suspected if an adequate number of cells are obtained.

  14. Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-{kappa}{beta} and myosin light-chain kinase pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ying [Department of Anesthesia, Critical Care Medicine and Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, People' s Republic of China (China); Li, Jianguo, E-mail: 2010lijianguo@sina.cn [Department of Anesthesia, Critical Care Medicine and Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, People' s Republic of China (China)

    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.

  15. Cerebral low-grade lymphoma and light chain deposition disease: exceedingly high IgG levels in the cerebrospinal fluid as a diagnostic clue.

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ponomarenko, Natalia [Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117871 (Russian Federation); Chatziefthimiou, Spyros D. [European Molecular Biology Laboratory, Hamburg Unit, c/o DESY, Notkestrasse 85, 22603 Hamburg (Germany); Kurkova, Inna; Mokrushina, Yuliana; Mokrushina, Yuliana; Stepanova, Anastasiya; Smirnov, Ivan; Avakyan, Marat; Bobik, Tatyana; Mamedov, Azad [Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117871 (Russian Federation); Mitkevich, Vladimir [Russian Academy of Sciences, Moscow 119991 (Russian Federation); Belogurov, Alexey Jr [Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117871 (Russian Federation); Institute of Gene Biology, Moscow 117334 (Russian Federation); Fedorova, Olga S. [Russian Academy of Sciences, Novosibirsk 630090 (Russian Federation); Dubina, Michael [St Petersburg Academic University, St Petersburg 194021 (Russian Federation); Golovin, Andrey [Lomonosov Moscow State University, Moscow 119991 (Russian Federation); Lamzin, Victor [European Molecular Biology Laboratory, Hamburg Unit, c/o DESY, Notkestrasse 85, 22603 Hamburg (Germany); Friboulet, Alain [Centre National de la Recherche Scientifique, 60205 Compiègne (France); Makarov, Alexander A. [Russian Academy of Sciences, Moscow 119991 (Russian Federation); Wilmanns, Matthias [European Molecular Biology Laboratory, Hamburg Unit, c/o DESY, Notkestrasse 85, 22603 Hamburg (Germany); Gabibov, Alexander, E-mail: gabibov@mx.ibch.ru [Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117871 (Russian Federation); Institute of Gene Biology, Moscow 117334 (Russian Federation); Lomonosov Moscow State University, Moscow 119991 (Russian Federation)

    2014-03-01

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

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

    Science.gov (United States)

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

    2012-09-18

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

  18. Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter-alpha-trypsin inhibitor and saposin B but increased expression of an inter-alpha-trypsin inhibitor heavy chain 4 fragment.

    Science.gov (United States)

    Jayapalan, Jaime J; Ng, Keng L; Shuib, Adawiyah S; Razack, Azad H A; Hashim, Onn H

    2013-06-01

    The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces

    Science.gov (United States)

    Mitra, Aniruddha; Ruhnow, Felix; Nitzsche, Bert; Diez, Stefan

    2015-01-01