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Sample records for heavy chain vmhc

  1. Clathrin heavy chain, light chain interactions.

    PubMed Central

    Winkler, F K; Stanley, K K

    1983-01-01

    Purified pig brain clathrin can be reversibly dissociated and separated into heavy chain trimers and light chains in the presence of non-denaturing concentrations of the chaotrope thiocyanate. The isolated heavy chain trimers reassemble into regular polygonal cage structures in the absence of light chains. The light chain fraction can be further resolved into its two components L alpha and L beta which give different one-dimensional peptide maps. Radiolabelled light chains bind with high affinity (KD < 10(-10) M) to heavy chain trimers, to heavy chain cages and to a 110,000 mol. wt. tryptic fragment of the heavy chain. Both light chains compete with each other and with light chains from other sources for the same binding sites on heavy chains and c.d. spectroscopy shows that the two pig brain light chains possess very similar structures. We conclude that light chains from different sources, despite some heterogeneity, have a highly conserved, high affinity binding site on the heavy chain but are not essential for the formation of regular cage structures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 8. PMID:10872336

  2. Atwood's Heavy Chain

    NASA Astrophysics Data System (ADS)

    Beeken, Paul

    2011-11-01

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

  3. Atwood's Heavy Chain

    ERIC Educational Resources Information Center

    Beeken, Paul

    2011-01-01

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

  4. Atwood's Heavy Chain

    ERIC Educational Resources Information Center

    Beeken, Paul

    2011-01-01

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

  5. Two Cases of Heavy Chain MGUS

    PubMed Central

    Meijers, Björn; Delforge, Michel; Verhoef, Gregor; Poesen, Koen

    2016-01-01

    Heavy chain diseases are rare variants of B-cell lymphomas that produce one of three classes of immunoglobulin heavy chains, without corresponding light chains. We describe two patients with asymptomatic heavy chain monoclonal gammopathy. The first patient is a 51-year-old woman with alpha paraprotein on serum immunofixation. The second case is a 46-year-old woman with gamma paraprotein on urine immunofixation. Neither patient had corresponding monoclonal light chains. Workup for multiple myeloma and lymphoma was negative in both patients. These two cases illustrate that heavy chain monoclonal gammopathy can exist in the absence of clinically apparent malignancy. Only a few reports of “heavy chain MGUS” have been described before. In the absence of specialized guidelines, we suggest a similar follow-up as for MGUS, while taking into account the higher probability of progression to lymphoma than to myeloma. PMID:27213064

  6. Transchromosomally derived Ig heavy chains

    SciTech Connect

    Knight, K.L.; Kingzette, M.; Crane, M.A.

    1995-07-15

    During an immune response, activated B cells undergo isotype switching and begin to express isotypes other than IgM and IgD. Isotype switching occurs when downstream C{gamma}, C{alpha}, or C{epsilon} genes are rearranged into the S{mu} chromosomal region, resulting in the deletion of the region in between. These rearrangements usually occur in cis, i.e., intrachromosomally. In previous studies, we analyzed allotypic specificities of rabbit secretory IgA and identified a substantial number of IgA heavy chains with V{sub h} and C{alpha} allotypes that were encoded by V{sub h} and C{alpha} genes in trans. In those studies, however, we could not determine whether the trans association of V{sub H} and C{alpha} occurred during VDJ gene rearrangement or during isotype switching. Here, we cloned rabbit cDNA which encodes these trans IgA heavy chains and determined the chromosomal origin of the V{sub H}, J{sub H}, and C{alpha} regions. To determine whether the trans association occurred during VDJ gene rearrangement, we analyzed the nucleotide polymorphism of the J{sub H} region and the V{sub H} allotype encoded by the cDNA. We found that the V{sub H} and J{sub H} genes used in the VDJ gene rearrangements were from the same chromosome, indicating that the V{sub H}, D, and J{sub H} gene rearrangements occurred in cis. Furthermore, we analyzed the DNA polymorphisms of J{sub H} and C{alpha} and showed that the VDJ and C{alpha} genes encoding the trans IgA molecules were derived from different parental chromosomes. We suggest that the trans association occurred during isotype switching. This study shows that V{sub H} and C{sub H} can associate transchromosomally as part of a normal immune response. 34 refs., 5 figs.

  7. Irreversible heavy chain transfer to chondroitin.

    PubMed

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

    2014-10-17

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

  8. Immunoglobulin heavy chains in medaka (Oryzias latipes)

    PubMed Central

    2011-01-01

    Background Bony fish present an immunological system, which evolved independently from those of animals that migrated to land 400 million years ago. The publication of whole genome sequences and the availability of several cDNA libraries for medaka (Oryzias latipes) permitted us to perform a thorough analysis of immunoglobulin heavy chains present in this teleost. Results We identified IgM and IgD coding ESTs, mainly in spleen, kidney and gills using published cDNA libraries but we did not find any sequence that coded for IgT or other heavy chain isotypes described in fish. The IgM - ESTs corresponded with the secreted and membrane forms and surprisingly, the latter form only presented two constant heavy chain domains. This is the first time that this short form of membrane IgM is described in a teleost. It is different from that identified in Notothenioid teleost because it does not present the typical splicing pattern of membrane IgM. The identified IgD-ESTs only present membrane transcripts, with Cμ1 and five Cδ exons. Furthermore, there are ESTs with sequences that do not have any VH which disrupt open reading frames. A scan of the medaka genome using transcripts and genomic short reads resulted in five zones within a region on chromosome 8 with Cμ and Cδ exons. Some of these exons do not form part of antibodies and were at times interspersed, suggesting a recombination process between zones. An analysis of the ESTs confirmed that no antibodies are expressed from zone 3. Conclusions Our results suggest that the IGH locus duplication is very common among teleosts, wherein the existence of a recombination process explains the sequence homology between them. PMID:21676244

  9. The heavy chain has its day

    PubMed Central

    Dulyaninova, Natalya G; Bresnick, Anne R

    2013-01-01

    Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain. PMID:24002531

  10. The immunoglobulin heavy chain locus in the reptile Anolis carolinensis.

    PubMed

    Gambón Deza, Francisco; Sánchez Espinel, Christian; Magadán Mompó, Susana

    2009-05-01

    We describe the entire immunoglobulin heavy chain (IgH) locus from the reptile Anolis carolinensis. The heavy chain constant (C(H)) region includes C mu, C delta and C upsilon genes. This is the first description of a C upsilon gene in the reptilian class. Variable (V(H)), diversity (D(H)) and joining (J(H)) genes are located 5' from the constant (C(H)) chain complex locus. The C mu and C upsilon genes encode antibodies with four immunoglobulin domains. The C delta gene encoded an 11 domain delta heavy chain as in Eublepharis macularius. Seventy V(H) genes, belonging to 28 families, were identified, and they can be sorted into five broader groups. The similarity of the organization of the reptilian genes with those of amphibians and mammals suggests the existence of a process of heavy chain genomic reorganization before the radiation of tetrapod vertebrates.

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

    PubMed Central

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

    1989-01-01

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

  12. In vitro motility from recombinant dynein heavy chain.

    PubMed Central

    Mazumdar, M; Mikami, A; Gee, M A; Vallee, R B

    1996-01-01

    The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692854

  13. Monoclonal free light chains can be found in heavy chain diseases.

    PubMed

    López-Anglada, L; Puig, N; Díez-Campelo, M; Alonso-Ralero, L; Barrena, S; Aparicio, M A; Gutiérrez, N C; García-Sanz, R

    2010-11-01

    Heavy chain diseases (HCDs) are rare B-cell lymphoproliferative neoplasias characterized by the production of a monoclonal component consisting of a truncated monoclonal Ig heavy chain without the associated light chain. Among them, patients with gamma-HCD are so rare that no more than 150 cases can be found in the literature. In this paper, we report one additional case: an 83-year-old man with a gamma-HCD, in whom a kappa light chain component was detected in the serum by using the serum free light-chain assessment and in addition monoclonal kappa cytoplasmic expression was detected in bone marrow plasma cells by flow cytometric analysis. In the work-up of the patient, the underlying anatomopathological lymphoproliferative disease corresponded to a lymphoplasmacytic lymphoma, as it is stated in the current World Health Organization classification (2008), with both lymphadenopathic and bone marrow infiltration. As in other cases, several autoimmune manifestations (antiphospholipidic syndrome and immune thrombocytopenia) were present during the course of the disease in this patient. This case report illustrates a new case of gamma-HCD, in which serum free light-chain analysis and flow cytometry represented a valuable tool for diagnosis, a finding that could be very important for the future management of these patients.

  14. Ig heavy chain class switch recombination: mechanism and regulation

    PubMed Central

    Stavnezer, Janet; Schrader, Carol E.

    2014-01-01

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

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

    PubMed

    De Ioannes, A E; Aguila, H L

    1989-01-01

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

  16. An unconventional myosin heavy chain gene from Drosophila melanogaster.

    PubMed

    Kellerman, K A; Miller, K G

    1992-11-01

    As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, we have undertaken the molecular analysis of a 140-kD ATP-sensitive actin-binding protein (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). Analysis of cDNA clones encoding this protein revealed that it represents a new class of unconventional myosin heavy chains. The amino-terminal two thirds of the protein comprises a head domain that is 29-33% identical (60-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain a approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, we have named the new gene Drosophila 95F myosin heavy chain (95F MHC). The expression profile of the 95F MHC gene is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein. We speculate that the 95F MHC protein isoforms are involved in multiple dynamic processes during Drosophila development.

  17. The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin.

    PubMed

    Patrinou-Georgoulas, M; John, H A

    1977-10-01

    A size class of polysomes was isolated from chick embryonic leg skeletal muscle which synthesized almost exclusively a polypeptide chain with a molecular weight identical to the myosin heavy chain. The mRNA purified from these polysomes was shown to synthesize the 200,000 dalton polypeptide in the wheat germ cell-free translation system. At least 90% of the polypeptide had properties similar to the myosin heavy chain. Isoelectric focusing indicated that the myosin heavy chain synthesized in vitro contained two chains in equal amounts, as did purified embryonic leg skeletal muscle myosin. The kinetics of hybridization of the complementary DNA with an excess of the myosin heavy chain mRNA (MHC mRNA) indicated the presence of two different mRNA sequences. Reassociation of the cDNA to an excess of the DNA of the genome suggest that there is little, if any, reiteration of the myosin heavy chain genes.

  18. Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.

    PubMed

    Ihara, Yoshito; Yasuoka, Chie; Kageyama, Kan; Wada, Yoshinao; Kondo, Takahito

    2002-09-20

    In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking.

  19. Cytoplasmic dynein heavy chain: the servant of many masters

    PubMed Central

    Schiavo, Giampietro; Greensmith, Linda; Hafezparast, Majid; Fisher, Elizabeth M.C.

    2013-01-01

    Cytoplasmic dynein is the main retrograde motor in all eukaryotic cells. This complex comprises different subunits assembled on a cytoplasmic dynein heavy chain 1 (DYNC1H1) dimer. Cytoplasmic dynein is particularly important for neurons because it carries essential signals and organelles from distal sites to the cell body. In the past decade, several mouse models have helped to dissect the numerous functions of DYNC1H1. Additionally, several DYNC1H1 mutations have recently been found in human patients that give rise to a broad spectrum of developmental and midlife-onset disorders. Here, we discuss the effects of mutations of mouse and human DYNC1H1 and how these studies are giving us new insight into the many critical roles DYNC1H1 plays in the nervous system. PMID:24035135

  20. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains.

    PubMed

    Muyldermans, S; Atarhouch, T; Saldanha, J; Barbosa, J A; Hamers, R

    1994-09-01

    We cloned 17 different PCR fragments encoding VH genes of camel (Camelus dromedarius). These clones were derived from the camel heavy chain immunoglobulins lacking the light chain counterpart of normal immunoglobulins. Insight into the camel VH sequences and structure may help the development of single domain antibodies. The most remarkable difference in the camel VH, consistent with the absence of the VL interaction, is the substitution of the conserved Leu45 by an Arg or Cys. Another noteworthy substitution is the Leu11 to Ser. This amino acid normally interacts with the CH1 domain, a domain missing in the camel heavy chain immunoglobulins. The nature of these substitutions agrees with the increased solubility behavior of an isolated camel VH domain. The VH domains of the camels are also characterized by a long CDR3, possibly compensating for the absence of the VL contacts with the antigen. The CDR3 lacks the salt bridge between Arg94 and Asp101. However, the frequent occurrence of additional Cys residues in both the CDR1 and CDR3 might lead to the formation of a second internal disulfide bridge, thereby stabilizing the CDR structure as in the DAW antibody. Within CDRs of the camel VH domains we observe a broad size distribution and a different amino acid pattern compared with the mouse or human VH. Therefore the camel hypervariable regions might adopt structures which differ substantially from the known canonical structures, thereby increasing the repertoire of the camel antigen binding sites within a VH.

  1. The amino acid sequences of the Fd fragments of two human γ heavy chains

    PubMed Central

    Press, E. M.; Hogg, N. M.

    1970-01-01

    The amino acid sequences of the Fd fragments of two human pathological immunoglobulins of the immunoglobulin G1 class are reported. Comparison of the two sequences shows that the heavy-chain variable regions are similar in length to those of the light chains. The existence of heavy chain variable region subgroups is also deduced, from a comparison of these two sequences with those of another γ 1 chain, Eu, a μ chain, Ou, and the partial sequence of a fourth γ 1 chain, Ste. Carbohydrate has been found to be linked to an aspartic acid residue in the variable region of one of the γ 1 chains, Cor. PMID:5449120

  2. Evolutionary conservation of heavy chain protein transfer between glycosaminoglycans.

    PubMed

    Sanggaard, Kristian W; Hansen, Lone; Scavenius, Carsten; Wisniewski, Hans-Georg; Kristensen, Torsten; Thøgersen, Ida B; Enghild, Jan J

    2010-04-01

    The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HC*TSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2*bikunin. In addition, Gg pre-alpha-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein-glycosaminoglycan-protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2*bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-alpha-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of

  3. Production and Characterization of Recombinant Light Chain and Carboxyterminal Heavy Chain Fragments of Tetanus Toxin

    PubMed Central

    Yousefi, Mehdi; Khosravi-Eghbal, Roya; Hemmati, Azam; Shokri, Fazel

    2013-01-01

    Background Light chain (LC) and heavy chain carboxyterminal subdomain (HCC) fragments are the most important parts of tetanus neurotoxin (TeNT) which play key roles in toxicity and binding of TeNT, respectively. In the present study, these two fragments were cloned and expressed in a prokaryotic system and their identity was confirmed using anti-TeNT specific polyclonal and monoclonal antibodies. Methods LC and HCC gene segments were amplified from Clostridium tetani genomic DNA by PCR, cloned into pET28b(+) cloning vector and transformed in Escherichia coli (E. coli) BL21(DE3) expression host. Recombinant proteins were then purified through His-tag using Nickel-based chromatography and characterized by SDS-PAGE, Western blotting and ELISA techniques. Results Recombinant light chain and HCC fragments were successfully cloned and expressed in (E. coli) BL21 (DE3). Optimization of the induction protocol resulted in production of high levels of HCC (~35% of total bacterial protein) and to lesser extends of LC (~5%). Reactivity of the His-tag purified proteins with specific polyclonal and monoclonal antibodies confirmed their renatured structure and identity. Conclusion Our results indicate successful cloning and production of recombinant LC and HCC fragments of TeNT. These two recombinant proteins are potentially useful tools for screening and monitoring of anti-TeNT antibody response and vaccine production. PMID:24285996

  4. Myosin heavy chain expression in respiratory muscles of the rat.

    PubMed

    LaFramboise, W A; Watchko, J F; Brozanski, B S; Daood, M J; Guthrie, R D

    1992-03-01

    Myosin heavy chain (MHC) isoforms of hind limb adult rat muscles and muscles with a range of respiratory activities were analyzed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis technique that allowed electrophoretic separation of the three fast and one slow MHC isoform found in typical rat muscle. Costal and crural diaphragm muscle samples expressed a mixture of MHC beta/slow, MHC2A, and MHC2X but little MHC2B. In contrast, MHC2B was the dominant MHC isoform in the genioglossus, intercostal, and three abdominal muscles, all of which exhibited minimal expression of MHC beta/slow. The amount of MHC2X (relative to total MHC composition) was similar in the diaphragm, genioglossus, and transversus abdominis muscles, while considerably less was detected in the rectus abdominis and external oblique muscles. These results indicate that MHC2X is broadly and variably distributed among respiratory muscles. Furthermore, these data suggest that a large portion of 2X fibers (containing MHC2X), which cannot be detected by standard histochemical analysis, may be present in the genioglossus and transversus abdominis muscles as has been demonstrated for the diaphragm muscle. We speculate that an association exists between the level of MHC2X expression and frequency of respiratory recruitment.

  5. Primary structure of chicken cardiac myosin S-1 heavy chain.

    PubMed

    Nakayama, S; Tanaka, H; Yajima, E; Maita, T

    1994-05-01

    The sequence of the NH2-terminal 830 amino acid residues of chicken cardiac ventricular muscle myosin subfragment-1 (S-1) was determined. S-1 was obtained by limited chymotryptic digestion, and cleaved into three characteristics fragments (23, 41, and 22 kDa fragments) by limited tryptic digestion. These fragments were isolated by gel filtration on a Sephadex G-100 column, followed by cation-exchange chromatography on a CM-52 column and reverse-phase HPLC. The isolated fragments were sequenced completely. Peptides overlapping the 23 and 41 kDa fragments and also overlapping the 41 and 22 kDa fragments were obtained by cleaving S-1 with cyanogen bromide, and sequenced completely. We also obtained a minor fragment, the 20 kDa fragment, in addition to the three characteristic fragments. Amino acid compositions of the cyanogen bromide peptides of the 20 kDa fragment indicated that a portion of S-1 heavy chains had lost their COOH-terminal 21 residues during limited tryptic digestion. Methylated amino acid residues were found at four positions: epsilon-N-monomethyllysine at position 32, epsilon-N-trimethyllysine residues at 127 and 549, and 3-N-methylhistidine at 754.

  6. Adaptations in myosin heavy chain profile in chronically unloaded muscles

    NASA Technical Reports Server (NTRS)

    Talmadge, R. J.; Roy, R. R.; Bodine-Fowler, S. C.; Pierotti, D. J.; Edgerton, V. R.

    1995-01-01

    In this review, myosin heavy chain (MHC) adaptations in response to several models of decreased neuromuscular activity (i.e. electrical activation and loading of a muscle) are evaluated. In each of these "reduced-activity" models it is important to: a) quantify the changes in electrical activation of the muscle as a result of the intervention; b) quantify the forces generated by the muscle; and c) determine whether the neuromuscular junction remains normal. Most of the models, including spaceflight, hindlimb suspension, spinal cord isolation, spinal cord transection, denervation, and limb immobilization in a shortened position, result in increases in the percentage of fast MHCs (or fast MHC mRNA) in normally slow rat muscles. It also can be inferred from histochemical data that increases in fast MHCs occur with TTX application and bed rest. The only "reduced-activity" model to consistently increase slow muscle myosin mRNA, and slow fibers is limb immobilization in a stretched position; however, this model results in at least a temporary increase in tension. It appears that the most common feature of these models that might induce MHC adaptations is the modification in loading rather than a change in the neuromuscular activity.

  7. Adaptations in myosin heavy chain profile in chronically unloaded muscles

    NASA Technical Reports Server (NTRS)

    Talmadge, R. J.; Roy, R. R.; Bodine-Fowler, S. C.; Pierotti, D. J.; Edgerton, V. R.

    1995-01-01

    In this review, myosin heavy chain (MHC) adaptations in response to several models of decreased neuromuscular activity (i.e. electrical activation and loading of a muscle) are evaluated. In each of these "reduced-activity" models it is important to: a) quantify the changes in electrical activation of the muscle as a result of the intervention; b) quantify the forces generated by the muscle; and c) determine whether the neuromuscular junction remains normal. Most of the models, including spaceflight, hindlimb suspension, spinal cord isolation, spinal cord transection, denervation, and limb immobilization in a shortened position, result in increases in the percentage of fast MHCs (or fast MHC mRNA) in normally slow rat muscles. It also can be inferred from histochemical data that increases in fast MHCs occur with TTX application and bed rest. The only "reduced-activity" model to consistently increase slow muscle myosin mRNA, and slow fibers is limb immobilization in a stretched position; however, this model results in at least a temporary increase in tension. It appears that the most common feature of these models that might induce MHC adaptations is the modification in loading rather than a change in the neuromuscular activity.

  8. Mutation of the 4F2 heavy-chain carboxy terminus causes y+ LAT2 light-chain dysfunction.

    PubMed

    Chubb, Sarah; Kingsland, Alice L; Bröer, Angelika; Bröer, Stefan

    2006-01-01

    Heteromeric amino acid transporters are composed of two subunits--a multipass membrane protein called the 'light chain'--and a single pass glycoprotein called the 'heavy chain'. The light chain contains the transport pore, while the heavy chain appears to be necessary for trafficking the light chain to the plasma membrane. In this study, the role of the 4F2hc heavy chain in the function of the y+ LAT2 light chain was investigated. Carboxy terminal truncations and site specific mutants of 4F2hc were co-expressed in Xenopus laevis oocytes with the y+ LAT2 light chain, and the oocytes were analysed for transport activity and surface expression. Truncations of the 4F2hc carboxy terminus ranging between 15 and 404 residues caused a complete loss of light chain function, although all heterodimers were expressed at the cell surface. This indicated that the 15 carboxy-terminal residues of 4F2hc are required for the transport function of the heterodimer. Mutation of the conserved residue leucine 523 to glutamine in the carboxy terminus reduced the Vmax of arginine and leucine uptake. The affinity of the transporter for both arginine and leucine remained unaltered, but the Km-value of Na+, being cotransported with leucine, increased about three-fold. The change of the Na+ Km caused a specific defect of leucine efflux, whereas uptake of leucine at high extracellular NaCl concentration was unaffected.

  9. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    PubMed Central

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  10. SUBGROUPS OF AMINO ACID SEQUENCES IN THE VARIABLE REGIONS OF IMMUNOGLOBULIN HEAVY CHAINS*

    PubMed Central

    Cunningham, Bruce A.; Pflumm, Mollie N.; User, Urs Rutisha; Edelman, Gerald M.

    1969-01-01

    The amino acid sequence of the first 133 residues of the heavy (γ) chain from a human γG immunoglobulin (He) has been determined. This γ-chain is identical in Gm type to that of protein Eu, the complete sequence of which has been reported. Comparison of the two sequences substantiates the previous suggestion that there are subgroups of variable regions of heavy chains. The variable region of Eu has been assigned to subgroup I and that of He to subgroup II; on the other hand, the constant regions of the two proteins appear to be identical. Comparison of the sequence of the heavy chain of He with the heavy chain sequences determined in other laboratories suggests that the variable region of subgroup II is at least 118 residues long. The nature and distribution of amino acid variations in this heavy chain subgroup resemble those observed in light chain subgroups. These studies provide evidence that the translocation hypothesis applies to heavy as well as to light chains, viz., genes for variable regions (V) are somatically translocated to genes for constant regions (C) to form complete VC structural genes. Images PMID:5264153

  11. Allelic inclusion in a pre-B-cell line that generates immunoglobulin heavy chain genes in vitro.

    PubMed Central

    Beck-Engeser, G; Jäck, H M; Wabl, M

    1987-01-01

    In a pre-B-cell line that rearranges its heavy chain gene segments in vitro, we found that the rate of productive rearrangement on one allele was not influenced by the presence of heavy chain protein encoded by the other allele. This shows that allelic exclusion of heavy chain genes is not regulated at the genetic level. Images PMID:3103122

  12. The immunoglobulin heavy chain locus in the platypus (Ornithorhynchus anatinus).

    PubMed

    Gambón-Deza, F; Sánchez-Espinel, C; Magadán-Mompó, S

    2009-08-01

    Immunoglobulins loci in mammals are well known to be organized within a translocon, however their origin remains unresolved. Four of the five classes of immunoglobulins described in humans and rodents (immunoglobulins M, G, E and A-IgM, IgG, IgE and IgA) were found in marsupials and monotremes (immunoglobulin D-IgD was not found) thus showing that the genomic structure of antibodies in mammals has remained constant since its origin. We have recently described the genomic organization of the immunoglobulin heavy chain locus in reptiles (IGHM, IGHD and IGHY). These data and the characterization of the IGH locus in platypus (Ornithorhynchus anatinus), allow us to elucidate the changes that took place in this genomic region during evolution from reptile to mammal. Thus, by using available genome data, we were able to detect that platypus IGH locus contains reptilian and mammalian genes. Besides having an IGHD that is very similar to the one in reptiles and an IGHY, they also present the mammal specific antibody genes IGHG and IGHE, in addition to IGHA. We also detected a pseudogene that originated by recombination between the IGHD and the IGHM (similar to the IGHD2 found in Eublepharis macularius). The analysis of the IGH locus in platypus shows that IGHY was duplicated, firstly by evolving into IGHE and then into IGHG. The IGHA of the platypus has a complex origin, and probably arose by a process of recombination between the IGHM and the IGHY. We detected about 44 VH genes (25 were already described), most of which comprise a single group. When we compared these VH genes with those described in Anolis carolinensis, we find that there is an evolutionary relationship between the VH genes of platypus and the reptilian Group III genes. These results suggest that a fast VH turnover took place in platypus and this gave rise to a family with a high VH gene number and the disappearance of the earlier VH families.

  13. The primary structure of skeletal muscle myosin heavy chain: IV. Sequence of the rod, and the complete 1,938-residue sequence of the heavy chain.

    PubMed

    Maita, T; Yajima, E; Nagata, S; Miyanishi, T; Nakayama, S; Matsuda, G

    1991-07-01

    In the preceding paper [Maita, T., Miyanishi, T., Matsuzono, K., Tanioka, Y., & Matsuda, G. (1991) J. Biochem. 110, 68-74], we reported the amino-terminal 837-residue sequence of the heavy chain of adult chicken pectoralis muscle myosin. This paper describes the carboxyl terminal 1,097-residue sequence and the linkage of the two sequences. Rod obtained by digesting myosin filaments with alpha-chymotrypsin was redigested with the protease at high KCl concentration, and two fragments, subfragment-2 and light meromyosin, were isolated and sequenced by conventional methods. The linkage of the two fragments was deduced from the sequence of an overlapping peptide obtained by cleaving the rod with cyanogen bromide. The rod contained 1,039 amino acid residues, but lacked the carboxyl-terminal 58 residues of the heavy chain. A carboxyl-terminal 63-residue peptide obtained by cleaving the whole heavy chain with cyanogen bromide was sequenced. Thus, the carboxyl terminal 1,097-residue sequence of the heavy chain was completed. The linkage of subfragment-1 and the rod was deduced from the sequence of an overlapping peptide between the two which was obtained by cleaving heavy meromyosin with cyanogen bromide. Comparing the sequence of the adult myosin thus determined with that of chicken embryonic myosin reported by Molina et al. [Molina, M.I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488], we found that the sequence homology is 94%.

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

    PubMed

    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.

  15. Discovery of a gamma heavy chain disease in a patient followed-up for a lymphoplasma cell proliferative disorder.

    PubMed

    Humeau, Camille; Monjanel, Hélène; Schellenberg, François

    2016-06-01

    Gamma-heavy chains disease is a rare disease, with very few cases described in the literature. It is characterized by the presence of a monoclonal gamma-heavy chain without associated light chain. Its prevalence and prognosis are unknown. We report here the accidental discovery of a case of gamma-heavy chain disease during a pancytopenia exploration, performed in the hospital, in a patient known since 2002 for a lymphoplasmacytic type lymphoma first localized in bone marrow.

  16. Autolysis of bovine enteropeptidase heavy chain: evidence of fragment 118-465 involvement in trypsinogen activation.

    PubMed

    Mikhailova, A G; Rumsh, L D

    1999-01-15

    Variations in bovine enteropeptidase (EP) activity were shown to result from autolysis caused by the loss of calcium ions; the cleavage sites were determined. The native enzyme preferred its natural substrate, trypsinogen (KM=2.4 microM), to the peptide and fusion protein substrates (KM=200 and 125 microM, respectively). On the other hand, the truncated enzyme composed of the C-terminal fragment 466-800 of EP heavy chain and intact light chain did not distinguish these substrates. The results suggest that the N-terminal fragment 118-465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.

  17. Complete primary structure of a scallop striated muscle myosin heavy chain. Sequence comparison with other heavy chains reveals regions that might be critical for regulation.

    PubMed

    Nyitray, L; Goodwin, E B; Szent-Györgyi, A G

    1991-10-05

    We have determined the primary structure of the myosin heavy chain (MHC) of the striated adductor muscle of the scallop Aequipecten irradians by cloning and sequencing its cDNA. It is the first heavy chain sequence obtained in a directly Ca(2+)-regulated myosin. The 1938-amino acid sequence has an overall structure similar to other MHCs. The subfragment-1 region of the scallop MHC has a 59-62% sequence identity with sarcomeric and a 52-53% identity with nonsarcomeric (smooth and metazoan nonmuscle) MHCs. The heavy chain component of the regulatory domain (Kwon, H., Goodwin, E. B., Nyitray, L., Berliner, E., O'Neall-Hennessey, E., Melandri, F. D., and Szent-Györgyi, A. G. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4771-4775) starts at either Leu-755 or Val-760. Ca(2+)-sensitive Trp residues (Wells, C., Warriner, K. E., and Bagshaw, C. R. (1985) Biochem. J. 231, 31-38) are located near the C-terminal end of this segment (residues 818-827). More detailed sequence comparison with other MHCs reveals that the 50-kDa domain and the N-terminal two-thirds of the 20-kDa domain differ substantially between sarcomeric and nonsarcomeric myosins. In contrast, in the light chain binding region of the regulatory domain (residues 784-844) the scallop sequence shows greater homology with regulated myosins (smooth muscle, nonmuscle, and invertebrate striated muscles) than with unregulated ones (vertebrate skeletal and heart muscles). The N-terminal 25-kDa domain also contains several residues which are preserved only in regulated myosins. These results indicate that certain heavy chain sites might be critical for regulation. The rod has features typical of sarcomeric myosins. It is 52-60% and 30-33% homologous with sarcomeric and nonsarcomeric MHCs, respectively. A Ser-rich tailpiece (residues 1918-1938) is apparently nonhelical.

  18. Random Mutagenesis by Error-Prone Polymerase Chain Reaction Using a Heavy Water Solvent.

    PubMed

    Minamoto, Toshifumi

    2017-01-01

    Heavy water is a form of water that contains a heavier isotope of hydrogen ((2)H, also known as deuterium, D) or oxygen ((17)O or (18)O). When using heavy water as a solvent, error-prone polymerase chain reaction (epPCR) can induce random mutations independent of the polymerase used or the composition of the PCR reaction mixture. This relatively new method can easily be combined with the existing epPCR methods to increase the rate of mutations.

  19. Immunogenicity and antigenicity of immunoglobulins. XII. Intact light chain and heavy chain isotype-restricted Vk-associated epitopes.

    PubMed

    Walker, M; Hardie, D; Lowe, J; Ling, N R; De Lange, G; Jefferis, R

    1985-06-01

    Immunization with intact IgG has allowed the isolation of four hybridomas producing antibodies recognizing epitopes expressed within subpopulations of human kappa light chains unrelated to known polymorphisms (Km) and previously defined V-region subgroups. The V-region-associated epitopes recognized are conformation-dependent, being expressed on intact light chain but not on isolated VK or CK fragments. The frequency of expression within paraprotein panels of different heavy chain isotypes varied between individual antibodies. An epitope recognized by B2A6, expressed by greater than 85% IgGK paraproteins, was not represented in 16 IgM paraproteins tested, suggesting that association of VK with mu chains does not result in display of the epitope recognized, or alternatively, that selective association between VK and CH gene products occurs. These data contrast with the reactivity of other McAb for CK epitopes which were reactive with isolated CK fragments, and for all kappa-bearing paraproteins, regardless of heavy chain isotypes.

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

    NASA Astrophysics Data System (ADS)

    Bandman, Everett

    1985-02-01

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

  1. Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain.

    PubMed

    Lu, D; Yuan, X; Zheng, X; Sadler, J E

    1997-12-12

    Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean

  2. BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly.

    PubMed

    Lee, Y K; Brewer, J W; Hellman, R; Hendershot, L M

    1999-07-01

    The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP-heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

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

    PubMed Central

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

    2016-01-01

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

  4. Characterization of axolotl heavy and light immunoglobulin chains by monoclonal antibodies.

    PubMed

    Chardin, H; Vilain, C; Charlemagne, J

    1987-12-01

    Axolotl specific antibodies to 2,4-dinitrophenyl (DNP) were purified by affinity chromatography from the sera of animals immunized with 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC). The purified anti-TNP/DNP antibodies, when analyzed by SDS-PAGE, were constituted of high molecular weight molecules, which in reducing conditions, were separated into heavy 72-88 kD and light 27-30 kD polypeptides. The axolotl heavy antibody chains strongly bound Concanavalin-A and migrate faster in SDS-PAGE after endoglycosidase-F (Endo-F) treatment. Using the same techniques, no carbohydrate components were detected onto light chains. Monoclonal antibodies (MAbs) were obtained against these purified axolotl immunoglobulins (Ig) and their specificities were studied by immunoblotting. MAbs 33.45.1 and 33.101.2 respectively recognized heavy and light chains determinants of the Ig molecule. These determinants were resistant to Endo-F digestion, suggesting that the two MAbs were not directed to polypeptide-associated N-linked high mannose or complex oligosaccharides. MAbs 33.45.1 and 33.101.2 were compared to 11.5.2, an anti-axolotl thymocytes MAb which was reactive for both axolotl leucocytes and soluble Ig. MAb 11.5.2 reacted in immunoblotting against several high molecular weight axolotl serum proteins, including heavy Ig chains. Light chains were not recognized. However, 11.5.2 did not further recognize Endo-F treated Ig, suggesting its specificity for a carbohydrate determinant of the heavy chain, and link to a large diversity of soluble or membrane glycoproteins.

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

    PubMed Central

    1988-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed Central

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

    2015-01-01

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

  8. Non-muscle myosin II heavy chain has a cryptic cell-adhesion domain.

    PubMed Central

    Grinnell, F; Ho, C H

    1995-01-01

    We have discovered a cryptic cell-adhesion domain in non-muscle myosin II heavy chain. A 205 kDa cell-adhesion-promoting polypeptide (p205) was extracted from BHK cells by Nonidet P-40 or Dounce homogenization. Adhesion to p205 was specifically inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro, indicating a role for the Arg-Gly-Asp cell-adhesion motif. Purified p205 was identified as non-muscle myosin II heavy chain, based on sequence analysis and on the cross-reactivity of p205 with anti-(bovine trachea myosin) antibodies. Further experiments showed that the heavy chain of purified myosin II has cell-adhesion-promoting activity in a cell-blotting assay, and cross-reacted with anti-p205 antibodies. Finally, the adhesion domain was located in the tail portion of myosin II heavy chain, where an Arg-Gly-Asp-containing sequence can be found. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626021

  9. The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.

    PubMed Central

    Zhang, Z; Tanaka, Y; Nonaka, S; Aizawa, H; Kawasaki, H; Nakata, T; Hirokawa, N

    1993-01-01

    Overlapping cDNA clones encoding the heavy chain of rat brain cytoplasmic dynein have been isolated. The isolated cDNA clones contain an open reading frame of 13,932 bp encoding 4644 aa (M(r), 532,213). The deduced protein sequence of the heavy chain of rat brain dynein shows significant similarity to sea urchin flagellar beta-dynein (27.0% identical) and to Dictyostelium cytoplasmic dynein (53.5% identical) throughout the entire sequence. The heavy chain of rat brain (cytoplasmic) dynein contains four putative nucleotide-binding consensus sequences [GX4GK(T/S)] in the central one-third region that are highly similar to those of sea urchin and Dictyostelium dyneins. The N-terminal one-third of the heavy chain of rat brain (cytoplasmic) dynein shows high similarity (43.8% identical) to that of Dictyostelium cytoplasmic dynein but poor similarity (19.4% identical) to that of sea urchin flagellar dynein. These results suggested that the C-terminal two-thirds of the dynein molecule is conserved and plays an essential role in microtubule-dependent motility activity, whereas the N-terminal regions are different between cytoplasmic and flagellar dyneins. Images Fig. 1 PMID:7690137

  10. Peptide influences the folding and intracellular transport of free major histocompatibility complex class I heavy chains

    PubMed Central

    1995-01-01

    Class I major histocompatibility complex molecules require both beta 2- microglobulin (beta 2m) and peptide for efficient intracellular transport. With the exception of H-2Db and Ld, class I heavy chains have not been detectable at the surface of cells lacking beta 2m. We show that properly conformed class I heavy chains can be detected in a terminally glycosylated form indicative of cell surface expression in H- 2b, H-2d, and H-2s beta 2m-/- concanavalin A (Con A)-stimulated splenocytes incubated at reduced temperature. Furthermore, we demonstrate the presence of Kb molecules at the surface of beta 2m-/- cells cultured at 37 degrees C. The mode of assembly of class I molecules encompasses two major pathways: binding of peptide to preformed "empty" heterodimers, and binding of peptide to free heavy chains, followed by recruitment of beta 2m. In support of the existence of the latter pathway, we provide evidence for a role of peptide in intracellular transport of free class I heavy chains, through analysis of Con A-stimulated splenocytes from transporter associated with antigen processing 1 (TAP1)-/-, beta 2m-/-, and double-mutant TAP1/beta 2m-/- mice. PMID:7869032

  11. Immunoglobulin heavy chain variable gene usage and (super)-antigen drive in chronic lymphocytic leukemia.

    PubMed

    Bühler, Andreas; Zenz, Thorsten; Stilgenbauer, Stephan

    2010-01-15

    Increasing evidence supports the prognostic relevance of specific immunoglobulin heavy chain variable (IGHV) genes or stereotyped B-cell receptors (BCR) in chronic lymphocytic leukemia (CLL). The clonotypic BCRs differ in their specificity and affinity toward classical antigens and/or superantigens. The BCR-triggered mechanisms are distinct but could explain in part the different clinical behavior among CLL subgroups.

  12. Cynomolgus macaque (Macaca fascicularis) immunoglobulin heavy chain locus description.

    PubMed

    Yu, Guo-Yun; Mate, Suzanne; Garcia, Karla; Ward, Michael D; Brueggemann, Ernst; Hall, Matthew; Kenny, Tara; Sanchez-Lockhart, Mariano; Lefranc, Marie-Paule; Palacios, Gustavo

    2016-07-01

    Cynomolgus macaques (Macaca fascicularis) have become an important animal model for biomedical research. In particular, it is the animal model of choice for the development of vaccine candidates associated with emerging dangerous pathogens. Despite their increasing importance as animal models, the cynomolgus macaque genome is not fully characterized, hindering molecular studies for this model. More importantly, the lack of knowledge about the immunoglobulin (IG) locus organization directly impacts the analysis of the humoral response in cynomolgus macaques. Recent advances in next generation sequencing (NGS) technologies to analyze IG repertoires open the opportunity to deeply characterize the humoral immune response. However, the IG locus organization for the animal is required to completely dissect IG repertoires. Here, we describe the localization and organization of the rearranging IG heavy (IGH) genes on chromosome 7 of the cynomolgus macaque draft genome. Our annotation comprises 108 functional genes which include 63 variable (IGHV), 38 diversity (IGHD), and 7 joining (IGHJ) genes. For validation, we provide RNA transcript data for most of the IGHV genes and all of the annotated IGHJ genes, as well as proteomic data to validate IGH constant genes. The description and annotation of the rearranging IGH genes for the cynomolgus macaques will significantly facilitate scientific research. This is particularly relevant to dissect the immune response during vaccination or infection with dangerous pathogens such as Ebola, Marburg and other emerging pathogens where non-human primate models play a significant role for countermeasure development.

  13. Heavy chain transfer by tumor necrosis factor-stimulated gene 6 to the bikunin proteoglycan.

    PubMed

    Lamkin, Elliott; Cheng, Georgiana; Calabro, Anthony; Hascall, Vincent C; Joo, Eun Ji; Li, Lingyun; Linhardt, Robert J; Lauer, Mark E

    2015-02-20

    We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Heavy Chain Transfer by Tumor Necrosis Factor-stimulated Gene 6 to the Bikunin Proteoglycan*

    PubMed Central

    Lamkin, Elliott; Cheng, Georgiana; Calabro, Anthony; Hascall, Vincent C.; Joo, Eun Ji; Li, Lingyun; Linhardt, Robert J.; Lauer, Mark E.

    2015-01-01

    We present data that hyaluronan (HA) polysaccharides, about 14–86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors. PMID:25561734

  15. Purification and quantification of heavy-chain antibodies from the milk of bactrian camels.

    PubMed

    Yao, Hongqiang; Zhang, Min; Li, Yi; Yao, Jirimutu; Meng, He; Yu, Siriguleng

    2017-02-08

    Camel milk has a unique composition with naturally occurring heavy-chain antibodies (HCAbs), which exert rehabilitating potencies in infection and immunity. To characterize HCAb in camel milk, immunoglobulin G (IgG) was isolated from the milk of Camelus bactrianus by a combination of affinity chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis to purify and size-fractionate protein A and protein G, which were further identified by Western blotting, and were quantified by bicinchoninic acid (BCA) and ELISA. The results indicated that IgG1 fraction contains molecules of 50 kDa heavy chains and 36 kDa light chains. The HCAbs (IgG2 and IgG3 fractions) devoid of light chains, contain heavy chains of 45 kDa and 43 kDa, respectively, the amounts of which were significantly higher than that of the IgG1 in the milk of bactrian camels. Above all, we revealed the considerable amounts of HCAbs in the milk of bactrian camels, and developed a novel method for their purification and quantification. These findings provide the basis for developing potential effects of camel milk and its interface with the dairy industry, as well as future investigations of HCAb and its roles in human health and diseases.

  16. Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish, Raja erinacea.

    PubMed

    Harding, F A; Amemiya, C T; Litman, R T; Cohen, N; Litman, G W

    1990-11-11

    Immunoglobulin heavy chain genes in Raja erinacea (little skate) are organized in clusters consisting of VH, DH, JH segments and CH exons (1). An immunoglobulin heavy chain mu-like isotype that exhibits 61-91% nucleotide sequence identity in coding segments to the Heterodontus francisci (horned shark) mu-type immunoglobulin is described. The overall length of the mu-type clusters is approximately 16 kb; transmembrane exons (TM1 and TM2) are located 3 to CH exon 4 (CH4). In three of four TM-containing genomic clones, a significant deletion is present in TM1. A second isotype of Raja immunoglobulin heavy chain genes has been detected by screening a spleen cDNA library with homologous Raja VH- and CH1-specific probes complementing the respective regions of the mu-like isotype. Weak hybridization with VH-specific probes and no discernable hybridization with C mu-specific probes were considered presumptive evidence for a second immunoglobulin isotype that nominally is designated as X-type. The Vx region of the X-type cDNA is approximately 60% identical at the nucleotide (nt) level to other Raja VH segments and thus represents a second VH family. Putative Dx and Jx sequences also have been identified. The constant region of the X-type immunoglobulin heavy chain gene consists of two characteristic immunoglobulin domains and a cysteine-rich carboxy terminal segment that are only partially homologous with the mu-like isotype. Genomic Southern blotting indicates that the V and C segments of both immunoglobulin heavy chain isotypes are encoded by complex multigene families. Vx- and different Cx-specific probes hybridize to different length transcripts in northern blot analyses of Raja spleen RNA suggesting that the regulation of expression of the X-type genes may involve differential RNA processing.

  17. Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish, Raja erinacea.

    PubMed Central

    Harding, F A; Amemiya, C T; Litman, R T; Cohen, N; Litman, G W

    1990-01-01

    Immunoglobulin heavy chain genes in Raja erinacea (little skate) are organized in clusters consisting of VH, DH, JH segments and CH exons (1). An immunoglobulin heavy chain mu-like isotype that exhibits 61-91% nucleotide sequence identity in coding segments to the Heterodontus francisci (horned shark) mu-type immunoglobulin is described. The overall length of the mu-type clusters is approximately 16 kb; transmembrane exons (TM1 and TM2) are located 3 to CH exon 4 (CH4). In three of four TM-containing genomic clones, a significant deletion is present in TM1. A second isotype of Raja immunoglobulin heavy chain genes has been detected by screening a spleen cDNA library with homologous Raja VH- and CH1-specific probes complementing the respective regions of the mu-like isotype. Weak hybridization with VH-specific probes and no discernable hybridization with C mu-specific probes were considered presumptive evidence for a second immunoglobulin isotype that nominally is designated as X-type. The Vx region of the X-type cDNA is approximately 60% identical at the nucleotide (nt) level to other Raja VH segments and thus represents a second VH family. Putative Dx and Jx sequences also have been identified. The constant region of the X-type immunoglobulin heavy chain gene consists of two characteristic immunoglobulin domains and a cysteine-rich carboxy terminal segment that are only partially homologous with the mu-like isotype. Genomic Southern blotting indicates that the V and C segments of both immunoglobulin heavy chain isotypes are encoded by complex multigene families. Vx- and different Cx-specific probes hybridize to different length transcripts in northern blot analyses of Raja spleen RNA suggesting that the regulation of expression of the X-type genes may involve differential RNA processing. Images PMID:2123029

  18. Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum

    PubMed Central

    1994-01-01

    The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain- deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space. PMID:8034739

  19. Looping out and deletion mechanism for the immunoglobulin heavy-chain class switch.

    PubMed Central

    Jäck, H M; McDowell, M; Steinberg, C M; Wabl, M

    1988-01-01

    In the mouse pre-B-cell line 18-81, cells can switch production in vitro from immunoglobulin mu chain to gamma 2b chain. The gene encoding the gamma 2b chain is created by a rearrangement of the mu gene. This rearrangement always takes place within a homolog. In cells with a gamma 2b gene, most of the time the gene segment encoding the constant region of the mu chain is deleted, but often the rearrangement leads to cells that produce no immunoglobulin, and all DNA sequences are retained. The latter result is due to an inversion. Inversions exclude the unequal sister chromatid exchange model of the heavy-chain class switch. Looping out is an intermediate step in the process of generating an inversion. Our findings demonstrate that the switch rearrangement occurs by looping out and deletion. Images PMID:2830623

  20. IgG light chain-independent secretion of heavy chain dimers: consequence for therapeutic antibody production and design.

    PubMed

    Stoyle, Chloe L; Stephens, Paul E; Humphreys, David P; Heywood, Sam; Cain, Katharine; Bulleid, Neil J

    2017-09-07

    Rodent monoclonal antibodies with specificity towards important biological targets are developed for therapeutic use by a process of humanisation. This process involves the creation of molecules, which retain the specificity of the rodent antibody but contain predominantly human coding sequence. Here, we show that some humanised heavy chains (HCs) can fold, form dimers and be secreted even in the absence of a light chain (LC). Quality control of recombinant antibody assembly in vivo is thought to rely upon folding of the HC CH1 domain. This domain acts as a switch for secretion, only folding upon interaction with the LC CL domain. We show that the secreted heavy-chain dimers contain folded CH1 domains and contribute to the heterogeneity of antibody species secreted during the expression of therapeutic antibodies. This subversion of the normal quality control process is dependent on the HC variable domain, is prevalent with engineered antibodies and can occur when only the Fab fragments are expressed. This discovery will have an impact on the efficient production of both humanised antibodies and the design of novel antibody formats. © 2017 The Author(s).

  1. Cloning and characterization of a novel human clathrin heavy chain gene (CLTCL)

    SciTech Connect

    Long, K.R.; Trofatter, J.A.; McCormick, M.K.

    1996-08-01

    An exon representing a novel clathrin heavy chain gene (CLTCL) was isolated during gene identification studies and transcription mapping of human chromosome 22. Isolation and sequencing of cDNA clones corresponding to this exon revealed extensive similarity of the predicted amino acid sequence of this gene product to those of clathrin heavy chain genes of other species. Northern blot analysis has revealed an apparent developmental expression pattern of an approximately 6-kb mRNA. The gene appears to be expressed ubiquitously in the limited number of fetal tissues that were tested, but is selectively expressed in certain adult tissues, particularly in skeletal muscle. In addition, alternative splicing of an exon was observed near the carboxyl terminus of the predicted gene product. Its location overlaps the domain putatively involved in clathrin light chain binding and is adjacent to the heavy chain self-assembly (or trimerization) region, suggesting that alternative splicing may be involved in regulating one or both of these interactions. The expression pattern of this gene, in addition to its potential role in receptor-mediated endocytosis and signal transduction, suggests that is may be important in some developmental processes. The location of CLTCL on human chromosome 22 near the region commonly deleted in DiGeorge and other apparent haploinsufficiency syndromes warrants further investigation into its relationship with these developmental disorders. 34 refs., 3 figs., 1 tab.

  2. Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

    PubMed

    Newell, Jennifer L; Fay, Philip J

    2009-04-24

    The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

  3. Structural differences in the motor domain of temperature-associated myosin heavy chain isoforms from grass carp fast skeletal muscle.

    PubMed

    Tao, Yan; Wang, Sun-Yong; Liang, Chun-Shi; Fukushima, Hideto; Watabe, Shugo

    2009-10-01

    We determined coding sequences for three types of grass carp myosin subfragment-1 (S1) heavy chain by extending 5'-regions of the three known genes encoding light meromyosin isoforms (10 degrees C, intermediate and 30 degrees C types). The primary structures of these three S1 heavy chain isoforms showed 81.4%, 81.2%, and 97.8% identities between the 10 degrees C and intermediate types, between the 10 degrees C and 30 degrees C types, and between the intermediate and 30 degrees C types, respectively. Isoform-specific differences were clearly observed between the 10 degrees C type and the other two types in 97 amino acid residues. Furthermore, among these amino acid mutations, 51 mutations occurred at the conserved residue sites of S1 heavy chain from fish and homoiotherm. Additionally, the 10 degrees C type showed striking differences compared with the other two types in the two surface loops, loop 1 located near the ATP-binding pocket and loop 2, which is one of the actin-binding sites, suggesting that such structural differences possibly affect their motor functions. Interestingly, this 10 degrees C-type myosin heavy chain isolated from adult grass carp skeletal muscle was surprisingly similar to the embryonic fast-type myosin heavy chain from juvenile silver carp in the structure of S1 heavy chain, indicating that it may also function as embryonic fast-type myosin heavy chain in juvenile stage.

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

    PubMed Central

    Wu, Ke; Hoy, Marjorie A.

    2014-01-01

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

  5. Mouse Cmu heavy chain immunoglobulin gene segment contains three intervening sequences separating domains.

    PubMed

    Calame, K; Rogers, J; Early, P; Davis, M; Livant, D; Wall, R; Hood, L

    1980-04-03

    The IgM molecule is composed of subunits made up of two light chain and two heavy chain (mu) polypeptides. The mu chain is encoded by several gene segments--variable (V), joining (J) and constant (Cmu). The Cmu gene segment is of particular interest for several reasons. First, the mu chain must exist in two very different environments--as an integral membrane protein in receptor IgM molecules (micrometer) and as soluble serum protein in IgM molecules into the blood (mus). Second, the Cmu region in mus is composed of four homology units or domains (Cmu1, Cmu2, Cmu3 and Cmu4) of approximately 110 amino acid residues plus a C-terminal tail of 19 residues. We asked two questions concerning the organisation of the Cmu gene segment. (1) Are the homology units separated by intervening DNA sequences as has been reported for alpha (ref. 5), gamma 1 (ref. 6) and gamma 2b (ref. 7) heavy chain genes? (2) Is the C-terminal tail separated from the Cmu4 domain by an intervening DNA sequence? If so, DNA rearrangements or RNA splicing could generate hydrophilic and hydrophobic C-terminal tails for the mus and micrometer polypeptides, respectively. We demonstrate here that intervening DNA sequences separate each of the four coding regions for Cmu domains, and that the coding regions for the Cmu4 domains and the C-terminal tail are directly contiguous.

  6. Gangliosides, Ab1 and Ab2 antibodies II. Light versus heavy chain: An idiotype-anti-idiotype case study.

    PubMed

    López-Requena, Alejandro; Rodríguez, Mabel; de Acosta, Cristina Mateo; Moreno, Ernesto; Puchades, Yaquelin; González, Majela; Talavera, Ariel; Valle, Aisel; Hernández, Tays; Vázquez, Ana María; Pérez, Rolando

    2007-02-01

    The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti-N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands.

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

    PubMed

    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.

  8. Immunoglobulin heavy-chain allotypes play a role in the clinical history of celiac disease.

    PubMed

    Caruso, C; Candore, G; Lio, D; Modica, M A; Cataldo, F; Maltese, I; Marino, V; Albeggiani, A

    1991-01-01

    We studied the role of the immunoglobulin heavy-chain allotypes in the evolution of celiac disease. Particularly the Gm(fb) phenotype was investigated since this phenotype is able to determine the breadth of immune response against alpha-gliadin antigen. The results show that weight percentiles and intestinal absorption as assessed by xylose test are influenced by Gm(fb) phenotype suggesting that Gm(fb) genes contribute to the determination of clinical aspects and evolution of celiac disease.

  9. Vaccination with recombinant whole heavy chain fragments of Clostridium botulinum Type C and D neurotoxins.

    PubMed

    Arimitsu, Hideyuki; Lee, Jae-Chul; Sakaguchi, Yoshihiko; Hayakawa, Yuji; Hayashi, Michiko; Nakaura, Miki; Takai, Hikaru; Lin, Song-Nan; Mukamoto, Masafumi; Murphy, Tom; Oguma, Keiji

    2004-05-01

    Mice and ducks were subcutaneously immunized with recombinant whole heavy (H) chains of Clostridium botulinum type C and D neurotoxins, which were expressed as glutathione S-transferase fusion proteins. In the case of mice, it was confirmed that two immunizations with type C- and D-H chains, 10 microg each time, significantly increased the specific antibodies against 100-kDa H chains of type C and D neurotoxins in an immunoblot analysis and an enzyme-linked immunosorbent assay, respectively. The mice immunized with type C- and D-H chains showed no symptoms of botulism when they were challenged with C- and D-16 S toxins at doses, given intraperitoneally, of up to 10(5) and 10(6) minmum lethal doses (MLD), respectively, per mouse. Ducks were immunized with a total of 100 microg of type C-H chain. The ducks also developed specific antibodies to the type C-H chain and showed significant protection against a challenge with 10(3) duck MLD of C-16 S toxin given intravenously. These results indicate that recombinant whole H chains can be used as an effective and safe vaccine for type C and D botulism in domestic animals.

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

    PubMed

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

    2006-07-01

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

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

    PubMed Central

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

    2008-01-01

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

  12. High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.

    PubMed

    DeKosky, Brandon J; Ippolito, Gregory C; Deschner, Ryan P; Lavinder, Jason J; Wine, Yariv; Rawlings, Brandon M; Varadarajan, Navin; Giesecke, Claudia; Dörner, Thomas; Andrews, Sarah F; Wilson, Patrick C; Hunicke-Smith, Scott P; Willson, C Grant; Ellington, Andrew D; Georgiou, George

    2013-02-01

    Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

  13. Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea.

    PubMed Central

    Harding, F A; Cohen, N; Litman, G W

    1990-01-01

    Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse. Images PMID:2107524

  14. Complete structure and organization of immunoglobulin heavy chain constant region genes in a phylogenetically primitive vertebrate.

    PubMed Central

    Kokubu, F; Hinds, K; Litman, R; Shamblott, M J; Litman, G W

    1988-01-01

    Immunoglobulin (Ig) gene organization in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, is unique. Homologous Ig heavy chain variable (VH) and constant region (CH) specific probes were used to screen a spleen cDNA library constructed in lambda gt11. Both secretory (SEC) and transmembrane (TM) cDNA clones were recovered; the latter were identified by a negative selection strategy. The complete sequence of the CH portion of a Heterodontus genomic DNA-lambda clone also was determined. The sequences of the individual CH genes differ from each other in all exons. When compared to mammalian prototypes, similarities in exon and intron organization as well as conservation of sequences involved with differential splicing of SEC and TM mRNA indicate that Heterodontus heavy chain genes are of the mu type, although intron lengths are uniformly longer in Heterodontus. Heterodontus genes are not associated, however, with the family of DNA sequences that have been implicated in heavy chain class switching in mammals. Spleen cDNA library screening and RNA blot analyses indicate that mRNAs encoding TM Ig are exceedingly rare. The relationship between this quantitative difference and the distribution of polyadenylation signal sequences suggests that regulation of Ig gene expression in Heterodontus may be highly dependent on position effects. Images PMID:3138109

  15. Complete structure and organization of immunoglobulin heavy chain constant region genes in a phylogenetically primitive vertebrate.

    PubMed

    Kokubu, F; Hinds, K; Litman, R; Shamblott, M J; Litman, G W

    1988-07-01

    Immunoglobulin (Ig) gene organization in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, is unique. Homologous Ig heavy chain variable (VH) and constant region (CH) specific probes were used to screen a spleen cDNA library constructed in lambda gt11. Both secretory (SEC) and transmembrane (TM) cDNA clones were recovered; the latter were identified by a negative selection strategy. The complete sequence of the CH portion of a Heterodontus genomic DNA-lambda clone also was determined. The sequences of the individual CH genes differ from each other in all exons. When compared to mammalian prototypes, similarities in exon and intron organization as well as conservation of sequences involved with differential splicing of SEC and TM mRNA indicate that Heterodontus heavy chain genes are of the mu type, although intron lengths are uniformly longer in Heterodontus. Heterodontus genes are not associated, however, with the family of DNA sequences that have been implicated in heavy chain class switching in mammals. Spleen cDNA library screening and RNA blot analyses indicate that mRNAs encoding TM Ig are exceedingly rare. The relationship between this quantitative difference and the distribution of polyadenylation signal sequences suggests that regulation of Ig gene expression in Heterodontus may be highly dependent on position effects.

  16. Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea.

    PubMed

    Harding, F A; Cohen, N; Litman, G W

    1990-02-25

    Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.

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

    PubMed Central

    Rijken, D C; Emeis, J J

    1986-01-01

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

  18. Nested polymerase chain reaction amplification and sequencing analysis of the light-chain and heavy-chain variable regions in the influenza A H1N1 virus hemagglutinin monoclonal antibody gene.

    PubMed

    Li, H J; Guo, C Y; Sun, J Y; Sun, L J; Zhao, P H; Hu, L; Li, Y; Hu, J

    2014-06-11

    The nested polymerase chain reaction (PCR) method was used for the amplification of the influenza A H1N1 virus hemagglutinin monoclonal antibody light-chain and heavy-chain genes. Sequence analysis of the obtained genes was then used to identify common cloning methods of the mouse immunoglobulin-kappa (Igκ) light-chain and heavy-chain variable gene regions. Twenty-two pairs of amplification primers for the mouse Igκ light-chain and heavy-chain variable gene regions were designed, and 6 mouse anti-human H1N1 influenza virus hemagglutinin monoclonal antibody light-chain and heavy-chain variable gene regions were cloned and sequenced. Comparative analysis was conducted between our results and the mouse Ig sequences published in the National Center of Biotechnology Information (NCBI). The nested PCR method effectively avoided cloning the pseudogenes of the monoclonal antibody, and the amino acid sequence obtained was consistent with the characteristics of the mouse Ig variable region. A general method of cloning the mouse Ig light-chain and heavy-chain variable gene regions was established, which provides a basis for further cloning of mouse monoclonal antibody variable gene regions. This study also provides data for further studies of H1N1 influenza virus hemagglutinin antibody binding sites.

  19. Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

    PubMed

    Wang, Yujun; Nakagaki, Masao

    2014-07-18

    The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

  20. Expression of heavy chain‐only antibodies can support B‐cell development in light chain knockout chickens

    PubMed Central

    Schusser, Benjamin; Collarini, Ellen J.; Pedersen, Darlene; Yi, Henry; Ching, Kathryn; Izquierdo, Shelley; Thoma, Theresa; Lettmann, Sarah; Kaspers, Bernd; Etches, Robert J.; van de Lavoir, Marie‐Cecile; Harriman, William

    2016-01-01

    Since the discovery of antibody‐producing B cells in chickens six decades ago, chickens have been a model for B‐cell development in gut‐associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJCL knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL−/−) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy‐chain‐only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4‐week‐old IgL−/− chickens, and antigen‐specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy‐chain‐only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B‐cell development in a gut‐associated lymphoid tissue species. PMID:27392810

  1. Dynein light intermediate chain in Aspergillus nidulans is essential for the interaction between heavy and intermediate chains.

    PubMed

    Zhang, Jun; Li, Shihe; Musa, Shamsideen; Zhou, Henry; Xiang, Xin

    2009-12-11

    Cytoplasmic dynein is a complex containing heavy chains (HCs), intermediate chains (ICs), light intermediate chains (LICs), and light chains (LCs). The HCs are responsible for motor activity. The ICs at the tail region of the motor interact with dynactin, which is essential for dynein function. However, functions of other subunits and how they contribute to the assembly of the core complex are not clearly defined. Here, we analyzed in the filamentous fungus Aspergillus nidulans functions of the only LIC and two LCs, RobA (Roadblock/LC7) and TctexA (Tctex1) in dynein-mediated nuclear distribution (nud). Whereas the deletion mutant of tctexA did not exhibit an apparent nud mutant phenotype, the deletion mutant of robA exhibited a nud phenotype at an elevated temperature, which is similar to the previously characterized nudG (LC8) deletion mutant. Remarkably, in contrast to the single mutants, the robA and nudG double deletion mutant exhibits a severe nud phenotype at various temperatures. Thus, functions of these two LC classes overlap to some extent, but the presence of both becomes important under specific conditions. The single LIC, however, is essential for dynein function in nuclear distribution. This is evidenced by the identification of the nudN gene as the LIC coding gene, and by the nud phenotype exhibited by the LIC down-regulating mutant, alcA-LIC. Without a functional LIC, the HC-IC association is significantly weakened, and the HCs could no longer accumulate at the microtubule plus end. Thus, the LIC is essential for the assembly of the core complex of dynein in Aspergillus.

  2. Dynein Light Intermediate Chain in Aspergillus nidulans Is Essential for the Interaction between Heavy and Intermediate Chains*

    PubMed Central

    Zhang, Jun; Li, Shihe; Musa, Shamsideen; Zhou, Henry; Xiang, Xin

    2009-01-01

    Cytoplasmic dynein is a complex containing heavy chains (HCs), intermediate chains (ICs), light intermediate chains (LICs), and light chains (LCs). The HCs are responsible for motor activity. The ICs at the tail region of the motor interact with dynactin, which is essential for dynein function. However, functions of other subunits and how they contribute to the assembly of the core complex are not clearly defined. Here, we analyzed in the filamentous fungus Aspergillus nidulans functions of the only LIC and two LCs, RobA (Roadblock/LC7) and TctexA (Tctex1) in dynein-mediated nuclear distribution (nud). Whereas the deletion mutant of tctexA did not exhibit an apparent nud mutant phenotype, the deletion mutant of robA exhibited a nud phenotype at an elevated temperature, which is similar to the previously characterized nudG (LC8) deletion mutant. Remarkably, in contrast to the single mutants, the robA and nudG double deletion mutant exhibits a severe nud phenotype at various temperatures. Thus, functions of these two LC classes overlap to some extent, but the presence of both becomes important under specific conditions. The single LIC, however, is essential for dynein function in nuclear distribution. This is evidenced by the identification of the nudN gene as the LIC coding gene, and by the nud phenotype exhibited by the LIC down-regulating mutant, alcA-LIC. Without a functional LIC, the HC-IC association is significantly weakened, and the HCs could no longer accumulate at the microtubule plus end. Thus, the LIC is essential for the assembly of the core complex of dynein in Aspergillus. PMID:19837669

  3. RNA-protein interactions of the 3' untranslated regions of myosin heavy chain transcripts.

    PubMed

    Kiri, Arpna; Goldspink, Geoffrey

    2002-01-01

    The RNA-protein interactions of the myosin heavy chain (MyHC) 3' untranslated regions (3'UTRs) were investigated using gel mobility shift assays. Marine skeletal myosin heavy chain mRNAs were amplified using reverse transcription coupled with the polymerase chain reaction (RT-PCR). Four cloned MyHC sequences were identified as slow type 1, fast 2a, fast 2b and fast 2x. The 3'UTRs of the four MyHC mRNAs were shown to interact with muscle protein in a tissue-specific manner as illustrated by gel retardation assays with protein extracts from various tissues. Competition assays indicate that this interaction is specific to the MyHC 3'UTR sequence. UV cross-linking suggests that several small proteins bind to the 3'UTR's. Peptide sequencing identified aldolase A and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as MyHC 3'UTR RNA-binding proteins. The implications of these interactions and post-transcriptional regulation of the MyHC genes are discussed.

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

    PubMed

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

    2009-08-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. The Enhancing Effects of the Light Chain on Heavy Chain Secretion in Split Delivery of Factor VIII Gene

    PubMed Central

    Chen, Lingxia; Zhu, Fuxiang; Li, Juan; Lu, Hui; Jiang, Haiyan; Sarkar, Rita; Arruda, Valder R; Wang, Jinhui; Zhao, Jennifer; Pierce, Glenn F; Ding, Qiulan; Wang, Xuefeng; Wang, Hongli; Pipe, Steven W; Liu, Xiang-Qin; Xiao, Xiao; Camire, Rodney M; Xiao, Weidong

    2008-01-01

    Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10–100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery. PMID:17653101

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

    PubMed

    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.

  8. Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform

    PubMed Central

    1990-01-01

    Motile activities such as chemotaxis and phagocytosis, which occur in Dictyostelium cells lacking myosin II, may be dependent upon myosin I. To begin to explore this possibility, we have engineered a disruption of the Dictyostelium myosin I heavy chain (DMIHC) gene described recently (Jung, G., C. L. Saxe III, A. R. Kimmel, and J. A. Hammer III. 1989. Proc. Natl. Acad. Sci. USA. 86:6186-6190). The double-crossover, gene disruption event that occurred resulted in replacement of the middle approximate one-third of the gene with the neomycin resistance marker. The resulting cells are devoid of both the 3.6-kb DMIHC gene transcript and the 124-kD DMIHC polypeptide. DMIHC- cells are capable of chemotactic streaming and aggregation, but these processes are delayed. Furthermore, the rate of phagocytosis by DMIHC- cells is reduced, as assessed by growth rate on lawns of heat-killed bacteria and on the initial rate of uptake of FITC-labeled bacteria. Therefore, this Dictyostelium myosin I isoform appears to play a role in supporting chemotaxis and phagocytosis, but it is clearly not required for these processes to occur. Using a portion of the DMIHC gene as a probe, we have cloned three additional Dictyostelium small myosin heavy chain genes. Comparison of these four genes with three genes described recently by Titus et al. (Titus, M. A., H. M. Warrick, and J. A. Spudich. 1989. Cell Reg. 1:55-63) indicates that there are at least five small myosin heavy chain genes in Dictyostelium. The probability that there is considerable overlap of function between these small myosin isoforms indicates that multiple gene disruptions within a single cell may be necessary to generate a more striking myosin I- phenotype. PMID:2141028

  9. cDNA cloning of the immunoglobulin heavy chain binding protein.

    PubMed Central

    Haas, I G; Meo, T

    1988-01-01

    A cDNA library was constructed from size-fractionated poly(A)+ RNA prepared from a murine pre-B-cell hybridoma expressing high levels of immunoglobulin heavy chain binding protein (BiP) and mu heavy chains. Transformed bacterial colonies were screened for recombinant plasmids containing cDNA coding for BiP by hybrid-selected mRNA translation. A clone, pMBiP, containing a 736-base-pair insert was shown to encode the protein. Translation in vitro of hybridoma mRNA selected by hybridization to the pMBiP cDNA yielded a single polypeptide of BiP-like size. The authenticity of this mRNA was verified by comparing the peptides obtained by the limited proteolysis of its in vitro translation product with those obtained from the in vivo produced BiP. Likewise, the authenticity of the cDNA insert was verified by an RNase A protection assay of heteroduplex molecules obtained by annealing a uniformly labeled single-strand copy of the cDNA clone with the same mRNA selected by hybridization and tested by translation. The nucleotide sequence of this clone enabled us to deduce the carboxyl-terminal 142 amino acids of BiP and to establish its kinship with the 70-kDa heat shock protein family. The finding of a single copy of the BiP gene in DNA blots of mouse and rat implies that the BiP-related RNA transcripts constitutively expressed in various murine tissues and cell lines are indeed products of the same gene. These findings imply that BiP plays a more general role than previously anticipated on the basis of the discovery of its association with immunoglobulin heavy chains. Images PMID:2895472

  10. Functional equivalence of the clathrin heavy chains CHC17 and CHC22 in endocytosis and mitosis.

    PubMed

    Hood, Fiona E; Royle, Stephen J

    2009-07-01

    Clathrin is crucial for endocytosis and plays a recently described role in mitosis. Two clathrin heavy chains (CHCs) are found in humans: the ubiquitous CHC17, and CHC22, a CHC that is enriched in skeletal muscle. Functional differences have been proposed for these clathrins despite high sequence similarity. Here, we compared each paralogue in functional assays of endocytosis and mitosis. We find that CHC17 and CHC22 are functionally equivalent. We also describe how previous work on CHC22 has involved a splice variant that is not usually expressed in cells.

  11. Mapping of Heavy Chain Genes for Mouse Immunoglobulins M and D

    NASA Astrophysics Data System (ADS)

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

    1980-09-01

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

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

    PubMed

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

    2014-04-01

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

  13. Kinesin-1 heavy chain mediates microtubule sliding to drive changes in cell shape.

    PubMed

    Jolly, Amber L; Kim, Hwajin; Srinivasan, Divya; Lakonishok, Margot; Larson, Adam G; Gelfand, Vladimir I

    2010-07-06

    Microtubules are typically observed to buckle and loop during interphase in cultured cells by an unknown mechanism. We show that lateral microtubule movement and looping is a result of microtubules sliding against one another in interphase Drosophila S2 cells. RNAi of the kinesin-1 heavy chain (KHC), but not dynein or the kinesin-1 light chain, eliminates these movements. KHC-dependent microtubule sliding powers the formation of cellular processes filled with parallel microtubule bundles. The growth of these cellular processes is independent of the actin cytoskeleton. We further observe cytoplasmic microtubule sliding in Xenopus and Ptk2 cells, and show that antibody inhibition of KHC in mammalian cells prevents sliding. We therefore propose that, in addition to its well established role in organelle transport, an important universal function of kinesin-1 is to mediate cytoplasmic microtubule-microtubule sliding. This provides the cell with a dedicated mechanism to transport long and short microtubule filaments and drive changes in cell shape.

  14. The carboxyl-terminal isoforms of smooth muscle myosin heavy chain determine thick filament assembly properties

    PubMed Central

    Rovner, Arthur S.; Fagnant, Patricia M.; Lowey, Susan; Trybus, Kathleen M.

    2002-01-01

    The alternatively spliced SM1 and SM2 smooth muscle myosin heavy chains differ at their respective carboxyl termini by 43 versus 9 unique amino acids. To determine whether these tailpieces affect filament assembly, SM1 and SM2 myosins, the rod region of these myosin isoforms, and a rod with no tailpiece (tailless), were expressed in Sf 9 cells. Paracrystals formed from SM1 and SM2 rod fragments showed different modes of molecular packing, indicating that the tailpieces can influence filament structure. The SM2 rod was less able to assemble into stable filaments than either SM1 or the tailless rods. Expressed full-length SM1 and SM2 myosins showed solubility differences comparable to the rods, establishing the validity of the latter as a model for filament assembly. Formation of homodimers of SM1 and SM2 rods was favored over the heterodimer in cells coinfected with both viruses, compared with mixtures of the two heavy chains renatured in vitro. These results demonstrate for the first time that the smooth muscle myosin tailpieces differentially affect filament assembly, and suggest that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells. PMID:11781338

  15. The intrinsic hypermutability of antibody heavy and light chain genes decays exponentially

    PubMed Central

    Rada, Cristina; Milstein, César

    2001-01-01

    Somatic hypermutation, essential for the affinity maturation of antibodies, is restricted to a small segment of DNA. The upstream boundary is sharp and is probably related to transcription initiation. However, for reasons unknown, the hypermutation domain does not encompass the whole transcription unit, notably the C-region exon. Since analysis of the downstream decay of hypermutation is obscured by sequence-dependent hot and cold spots, we describe a strategy to minimize these fluctuations by computing mutations of different sequences located at similar distances from the promoter. We pool large databases of mutated heavy and light chains and analyse the decay of mutation frequencies. We define an intrinsic decay of probability of mutation that is remarkably similar for heavy and light chains, faster than anticipated and consistent with an exponential fit. Indeed, quite apart from hot spots, the intrinsic probability of mutation at CDR1 can be almost twice that of CDR3. The analysis has mechanistic implications for current and future models of hypermutation. PMID:11500383

  16. Expression Cloning and Production of Human Heavy-Chain-Only Antibodies from Murine Transgenic Plasma Cells

    PubMed Central

    Drabek, Dubravka; Janssens, Rick; de Boer, Ernie; Rademaker, Rik; Kloess, Johannes; Skehel, John; Grosveld, Frank

    2016-01-01

    Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variable light (VL) domains in combinatorial display libraries or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single-cell sorting, single-cell RT-PCR, and bulk cloning of isolated natural VH–VL pairs. Heavy-chain-only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique, soluble, high-affinity influenza A strain X-31 hemagglutinin-specific HCAbs. PMID:28066429

  17. Susceptibility to multiple sclerosis is associated with the proximal immunoglobulin heavy chain variable region.

    PubMed Central

    Walter, M A; Gibson, W T; Ebers, G C; Cox, D W

    1991-01-01

    15 immunoglobulin heavy chain constant (CH) and variable region (VH) polymorphisms were selected to span the entire length of the heavy chain cluster. These polymorphisms were examined in 34 sib pairs concordant for multiple sclerosis (MS) and in 23 sporadic MS patients. Allele frequencies were calculated for the 2 MS patient groups and compared with those found in a control population from the same geographical location and of similar ethnic background. No significant association was found between MS and the 7 CH region polymorphisms examined. However, a significant correlation between the MS phenotype and a VH2 family polymorphism was observed in both MS patient populations (familial MS patients chi 2 = 8.16, P less than 0.005; sporadic MS patients chi 2 = 8.90, P less than 0.005). One allele of the VH2-5 gene segment was found to be over-represented in both MS groups. VH2-5 has recently been physically mapped close to the CH region, between 180 and 360 kb away. These results indicate that a locus near or within the CH-proximal VH region is associated with increased susceptibility to MS. Images PMID:1672695

  18. Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies.

    PubMed

    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.

  19. Comparative Analysis of Immune Repertoires between Bactrian Camel's Conventional and Heavy-Chain Antibodies

    PubMed Central

    Yang, Kai; Zhang, Wei; Zhang, Changjiang; Fu, Longfei; Ren, Zhe; Wang, Changxi; Wu, Jinghua; Lu, Ruxue; Ye, Yanrui; He, Mengying; Nie, Chao; Yang, Naibo; Wang, Jian; Yang, Huanming; Liu, Xiao

    2016-01-01

    Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P < 0.05). In addition to substitutions at amino acid (AA) residue positions NO.49/50/52 between VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data. PMID:27588755

  20. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    SciTech Connect

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  1. Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

    PubMed Central

    Rutland, Catrin Sian; Polo-Parada, Luis; Ehler, Elisabeth; Alibhai, Aziza; Thorpe, Aaran; Suren, Suganthi; Emes, Richard D.; Patel, Bhakti; Loughna, Siobhan

    2011-01-01

    The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy. PMID:21862559

  2. Evidence that the stalk of Drosophila kinesin heavy chain is an alpha- helical coiled coil

    PubMed Central

    1992-01-01

    Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40- 55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross- linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha- helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha- helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha- helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin. PMID:1734025

  3. High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

    PubMed

    DiMaio, D; Corbin, V; Sibley, E; Maniatis, T

    1984-02-01

    A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

  4. Fiber-type distribution and expression of myosin heavy chain isoforms in newborn heterozygous myostatin-knockout pigs.

    PubMed

    Xing, Xiao-Xu; Xuan, Mei-Fu; Jin, Long; Guo, Qing; Luo, Zhao-Bo; Wang, Jun-Xia; Luo, Qi-Rong; Zhang, Guang-Lei; Cui, Cheng-Du; Cui, Zheng-Yun; Kang, Jin-Dan; Yin, Xi-Jun

    2017-08-31

    To explore the effects of heterozygous myostatin-knockout (MSNT(+/-)) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs. The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN(+/-) pigs at birth than in wild-type (WT) pigs. MSTN(+/-) pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was ~ threefold higher in MSTN(+/-) pigs compared with WT pigs. MSTN(+/-) pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.

  5. [Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.].

    PubMed

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2009-12-25

    Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system

  6. Bioaccumulative and conchological assessment of heavy metal transfer in a soil-plant-snail food chain

    PubMed Central

    2012-01-01

    Background Copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb) can pose serious threats to environmental health because they tend to bioaccumulate in terrestrial ecosystems. We investigated under field conditions the transfer of these heavy metals in a soil-plant-snail food chain in Banat area, Romania. The main goal of this paper was to assess the Roman snail (Helix pomatia) usefulness in environmental monitoring as bioindicator of heavy metal accumulation. Eight sampling sites, selected by different history of heavy metal (HM) exposure, were chosen to be sampled for soil, nettle leaves, and newly matured snails. This study also aimed to identify the putative effects of HM accumulation in the environment on phenotypic variability in selected shell features, which included shell height (SH), relative shell height (RSH), and whorl number (WN). Results Significantly higher amounts of HMs were accumulated in snail hepatopancreas and not in foot. Cu, Zn, and Cd have biomagnified in the snail body, particularly in the hepatopancreas. In contrast, Pb decreased when going up into the food chain. Zn, Cd, and Pb correlated highly with each other at all levels of the investigated food chain. Zn and Pb exhibited an effective soil–plant transfer, whereas in the snail body only foot Cu concentration was correlated with that in soil. There were significant differences among sampling sites for WN, SH, and RSH when compared with reference snails. WN was strongly correlated with Cd and Pb concentrations in nettle leaves but not with Cu and Zn. SH was independent of HM concentrations in soil, snail hepatopancreas, and foot. However, SH correlated negatively with nettle leaves concentrations for each HM except Cu. In contrast, RSH correlated significantly only with Pb concentration in hepatopancreas. Conclusions The snail hepatopancreas accumulates high amounts of HMs, and therefore, this organ can function as a reliable biomarker for tracking HM bioavailability in soil. Long

  7. Dilated Cardiomyopathy Mutation (R134W) in Mouse Cardiac Troponin T Induces Greater Contractile Deficits against α-Myosin Heavy Chain than against β-Myosin Heavy Chain

    PubMed Central

    Gollapudi, Sampath K.; Chandra, Murali

    2016-01-01

    Many studies have demonstrated that depressed myofilament Ca2+ sensitivity is common to dilated cardiomyopathy (DCM) in humans. However, it remains unclear whether a single determinant—such as myofilament Ca2+ sensitivity—is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT), which is known to modulate crossbridge (XB) recruitment dynamics in a myosin heavy chain (MHC)-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W) of the human DCM mutation (R131W), we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC) and transgenic mice (β-MHC). TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca2+ sensitivity to a greater extent in α-MHC (0.14 pCa units) than in β-MHC fiber bundles (0.08 pCa units). Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background. PMID:27757084

  8. Dilated Cardiomyopathy Mutation (R134W) in Mouse Cardiac Troponin T Induces Greater Contractile Deficits against α-Myosin Heavy Chain than against β-Myosin Heavy Chain.

    PubMed

    Gollapudi, Sampath K; Chandra, Murali

    2016-01-01

    Many studies have demonstrated that depressed myofilament Ca(2+) sensitivity is common to dilated cardiomyopathy (DCM) in humans. However, it remains unclear whether a single determinant-such as myofilament Ca(2+) sensitivity-is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT), which is known to modulate crossbridge (XB) recruitment dynamics in a myosin heavy chain (MHC)-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W) of the human DCM mutation (R131W), we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC) and transgenic mice (β-MHC). TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca(2+) sensitivity to a greater extent in α-MHC (0.14 pCa units) than in β-MHC fiber bundles (0.08 pCa units). Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background.

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

    PubMed

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

    2014-09-01

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

  10. Prediction of the secondary structure of myosin light chains from comparison of homologous sequences. Implications for the interaction between myosin heavy and light chains.

    PubMed

    Béchet, J J; Houadjeto, M

    1989-07-06

    The primary sequences of seventeen essential and seventeen regulatory myosin light chains were analyzed and compared, using algorithms based on the different structural properties of their amino acid residues. This process allowed estimation of the structural homology between the proteins studied, and improved the prediction of their mean secondary structure and functionally important segments or residues. On the basis of the crystal structure of troponin C, a model of the myosin essential light chain with a fairly compact form is proposed. The possible sites of interaction between myosin light and heavy chains from rabbit skeletal muscle were also investigated by a complementarity method adapted to helix-rich proteins. Segments 139-149 and 65-75 in the essential light chain and segments 27-37, 67-77 and 97-107 in the regulatory light chain are suggested to constitute some of these sites, as most of them were found to have the features of surface-seeking helices.

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

    PubMed

    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.

  12. Developmental transitions in the myosin heavy chain phenotype of human respiratory muscle.

    PubMed

    Lloyd, J S; Brozanski, B S; Daood, M; Watchko, J F

    1996-01-01

    We studied the expression of myosin heavy chain (MHC) isoforms in the costal diaphragm (DIA) and the genioglossus (GG) muscles from 16 to 42 weeks gestation in the human using Western blotting techniques. Embryonic/neonatal MHC (MHCemb/neo) was the predominant isoform expressed in the DIA and GG at 16-24 weeks gestation. Subsequently, MHCemb/neo expression declined and the expression of MHCslow and MHC2A increased. At term, the DIA MHC phenotype was a composite of MHCemb/neo (15% of the total MHC complement), MHCslow (32%), MHC2A (47%), and MHC2B (6%); whereas, the GG was largely comprised of MHC2A (74%). We conclude that human DIA and GG demonstrate temporally dependent changes in MHC expression during gestation- and muscle-specific MHC phenotypes as they approach term.

  13. Mass spectrometry analyses of rat 2b myosin heavy chain isoform.

    PubMed

    Zurmanová, J; Malácová, D; Půta, F; Novák, P; Rícný, J; Soukup, T

    2007-01-01

    We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDS-PAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDI-TOF MS) and sequenced by liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified.

  14. Myosin heavy chain composition in the rat diaphragm - Effect of age and exercise training

    NASA Technical Reports Server (NTRS)

    Gosselin, Luc E.; Betlach, Michael; Vailas, Arthur C.; Greaser, Marion L.; Thomas, D. P.

    1992-01-01

    The effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Treadmill running at 75 percent maximal oxygen consumption resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23mo) trained animals (P less than 0.05). It was found that the ratio of fast to slow MHC was significantly higher (P less than 0.005) in the crural compared with costal diaphragm region in both age groups. A significant age-related increase in persentage of slow MHC was observed in both diaphragm regions. The relative proportion of slow MHC in either costal or crural region was not changed by exercise training.

  15. Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting

    PubMed Central

    Ho, Mengfei; Chang, Li-Hsin; Pires-Alves, Melissa; Thyagarajan, Baskaran; Bloom, Jordan E.; Gu, Zhengrong; Aberle, Karla K.; Teymorian, Sasha A.; Bannai, Yuka; Johnson, Steven C.; McArdle, Joseph J.; Wilson, Brenda A.

    2011-01-01

    The long half-life of botulinum neurotoxin serotype A (BoNT/A) in cells poses a challenge in developing post-exposure therapeutics complementary to existing antitoxin strategies. Delivery vehicles consisting of the toxin heavy chain (HC), including the receptor-binding domain and translocation domain, connected to an inhibitory cargo offer a possible solution for rescuing intoxicated neurons in victims paralyzed from botulism. Here, we report the expression and purification of soluble recombinant prototype green fluorescent protein (GFP) cargo proteins fused to the entire BoNT/A-HC (residues 544–1295) in Escherichia coli with up to a 40 amino acid linker inserted between the cargo and BoNT/A-HC vehicle. We show that these GFP-HC fusion proteins are functionally active and readily taken up by cultured neuronal cells as well as by neuronal cells in mouse motor nerve endings. PMID:21051321

  16. Multiple DNA sequence elements are necessary for the function of an immunoglobulin heavy chain promoter.

    PubMed Central

    Eaton, S; Calame, K

    1987-01-01

    Sequences required for the function of the mouse V1 immunoglobulin heavy chain variable-region (VH) promoter were identified by transient transfection of the normal and mutated promoters into plasmacytoma cells. Our results identify four regions required for normal promoter function: (i) the octamer ATGCAAAT, previously identified by others; (ii) a heptamer, CTAATGA; (iii) a pyrimidine-rich region; and (iv) a region between positions -125 and -251 relative to the transcription start site. Sequence analysis of 19 mouse and human VH 5' flanking regions shows that the heptamer and pyrimidine stretch are strongly conserved. We have also demonstrated that the octamer functions in an orientation independent manner in the VH promoter. Images PMID:3118372

  17. Evolution of the Iga Heavy Chain Gene in the Genus Mus

    PubMed Central

    Osborne, B. A.; Golde, T. E.; Schwartz, R. L.; Rudikoff, S.

    1988-01-01

    To examine questions of immunoglobulin gene evolution, the IgA α heavy chain gene from Mus pahari, an evolutionarily distant relative to Mus musculus domesticus, was cloned and sequenced. The sequence, when compared to the IgA gene of BALB/c or human, demonstrated that the IgA gene is evolving in a mosaic fashion with the hinge region accumulating mutations most rapidly and the third domain at a considerably lower frequency. In spite of this pronounced accumulation of mutations, the hinge region appears to maintain the conformation of a random coil. A marked propensity to accumulate replacement over silent site changes in the coding regions was noted, as was a definite codon bias. The possibility that these two phenomena are interrelated is discussed. PMID:2842228

  18. Multiple DNA sequence elements are necessary for the function of an immunoglobulin heavy chain promoter

    SciTech Connect

    Eaton, S.; Calame, K.

    1987-11-01

    Sequences required for the function of the mouse V1 immunoglobulin heavy chain variable-region (V/sub H/) promoter were identified by transient transfection of the normal and mutated promoters into plasmacytoma cells. The results identify four regions required for normal promoter function: (i) the octamer ATGCAAAT, previously identified by others; (ii) a heptamer, CTAATGA; (iii) a pyrimidine-rich region; and (iv) a region between positions -125 and -251 relative to the transcription start site. Sequence analysis of 19 mouse and human V/sub H/ 5' flanking regions shows that the heptamer and pyrimidine stretch are strongly conserved. The authors have also demonstrated that the octamer functions in an orientation independent manner in the V/sub H/ promoter.

  19. Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization.

    PubMed

    Vassilopoulos, Stéphane; Gentil, Christel; Lainé, Jeanne; Buclez, Pierre-Olivier; Franck, Agathe; Ferry, Arnaud; Précigout, Guillaume; Roth, Robyn; Heuser, John E; Brodsky, Frances M; Garcia, Luis; Bonne, Gisèle; Voit, Thomas; Piétri-Rouxel, France; Bitoun, Marc

    2014-05-12

    The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM-sarcomere attachment sites also known as costameres. We show that clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.

  20. Passive transfer of maternal immunity in the dromedary (Camelus dromedarius), involvement of heavy chain antibodies.

    PubMed

    Salhi, Imed; Bessalah, Salma; Mbarek, Sonia Ben; Chniter, Mohamed; Seddik, Mabrouk-Mouldi; Khorchani, Touhami; Hammadi, Mohamed

    2015-03-01

    In many mammalian species, newborns are agammaglobulinemic; thus, colostrum and milk are the main sources of early protective antibodies. These antibodies are produced in the mother's serum and transferred to mammalian glands a few days before parturition. Here, we have studied the transfer of immunity from a she-camel immunized with human serum albumin (HSA) to her calf via colostrum and milk. Our results show that HSA-specific antibodies are produced in the mother's serum and are subsequently transferred to her colostrum. These specific antibodies are then transferred by suckling to the calf. The calf serum did not contain HSA-reactive antibodies at parturition and before the first feed, after suckling, a rise in reactivity was observed peaking at 24 h postpartum. The involvement of heavy chain antibodies (HCAbs) in the process of immunity transfer was also examined, and it was found that they were also transferred from the colostrum to the calf serum like conventional antibodies.

  1. Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake

    PubMed Central

    Kasprowicz, Jaroslaw; Kuenen, Sabine; Miskiewicz, Katarzyna; Habets, Ron L.P.; Smitz, Liesbet; Verstreken, Patrik

    2008-01-01

    Synaptic vesicle reformation depends on clathrin, an abundant protein that polymerizes around newly forming vesicles. However, how clathrin is involved in synaptic recycling in vivo remains unresolved. We test clathrin function during synaptic endocytosis using clathrin heavy chain (chc) mutants combined with chc photoinactivation to circumvent early embryonic lethality associated with chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads to substantial membrane internalization visualized by live dye uptake and electron microscopy. However, chc-inactivated membrane cannot recycle and participate in vesicle release, resulting in a dramatic defect in neurotransmission maintenance during intense synaptic activity. Furthermore, inactivation of chc in the context of other endocytic mutations results in membrane uptake. Our data not only indicate that chc is critical for synaptic vesicle recycling but they also show that in the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization. PMID:18762582

  2. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  3. Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition.

    PubMed

    Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Suita, Kenji; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

    2015-01-01

    Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition. We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB. Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.

  4. cDNA cloning of the immunoglobulin heavy chain genes in banded houndshark Triakis scyllium.

    PubMed

    Honda, Yuka; Kondo, Hidehiro; Caipang, Christopher Marlowe A; Hirono, Ikuo; Aoki, Takashi

    2010-11-01

    In this study, cDNAs encoding the secreted forms of the immunoglobulin (Ig) heavy chains of IgM, IgNAR, and IgW were cloned from the banded houndshark Triakis scyllium. Two clones for the IgM heavy chains encoded 569 and 570 amino acids, whose conserved (C) region showed 47-70% amino acid identities to those reported in other cartilaginous fish. Four clones for the IgNAR encoded 673-670 amino acids with conserved Ig-superfamily domains. The IgNAR C region showed 56-69% amino acid identities to those so far reported. High-throughput sequencing revealed that in most of the IgNAR sequences, the two variable regions (CDR1 and CDR3) each possess a cysteine residue. Three types of IgW were identified; one contained Ig-superfamily domains that are in other cartilaginous fish, one lacks the 3rd domain in the constant region, and one lacks the 3rd to 5th domains. Despite these differences, the IgW isoforms clustered with IgWs of other cartilaginous fishes and the C regions showed 47-89% amino acid identities. mRNAs for IgM and IgNAR were detected in various tissues, while IgW mRNA was mainly detected in pancreas. The banded hounded shark also has IgM, IgW and IgNAR as well as the other cartilaginous fish with unique IgW isoform.

  5. Non-Invasive Assessment of Skeletal Muscle Myosin Heavy Chain Expression in Trained and Untrained Men.

    PubMed

    Fry, Andrew C; Housh, Terry J; Cramer, Joel B; Weir, Joseph P; Beck, Travis W; Schilling, Brian K; Miller, Jonathan D; Nicoll, Justin X

    2016-09-20

    Numerous conditions and types of physical activity (e.g., exercise, aging, muscle-related diseases) can influence muscle fiber types and the proteins expressed. To date, muscle fibers can only be characterized by actually obtaining a tissue sample using the invasive muscle biopsy procedure. Mechanomyography (MMG) is the assessment of the vibration properties of contracting skeletal muscle, and has been proposed as a possible non-invasive method for muscle fiber analysis. Therefore, the purpose of this project was to examine the feasibility of using MMG and muscle performance measures to non-invasively assess muscle fiber characteristics. Fifteen men (5 endurance-trained [End], 5 weight-trained [WT], and 5 sedentary [Sed]) provided muscle samples from their vastus lateralis muscle. These samples were analyzed for relative myosin heavy chain protein expression, which is highly correlated with % muscle fiber type areas. Additionally, each subject performed several muscle performance tests, and MMG of the quadriceps was assessed during a knee extension exercise. Multiple regression was used to develop prediction equations for determining relative muscle content of myosin heavy chain (MHC) types I, IIa, and IIx. A combination of MMG and knee extension performance variables estimated types I, IIa, and IIx MHC with approximately 80% accuracy. Although preliminary, these data suggest that muscle performance tests in addition to MMG assessments during a simple muscle performance task (knee extension) can be used to estimate muscle fiber type composition in a healthy male population. Such methods could ultimately be used to non-invasively monitor muscle health and fitness.

  6. Noninvasive Assessment of Skeletal Muscle Myosin Heavy Chain Expression in Trained and Untrained Men.

    PubMed

    Fry, Andrew C; Housh, Terry J; Cramer, Joel B; Weir, Joseph P; Beck, Travis W; Schilling, Brian K; Miller, Jonathan D; Nicoll, Justin X

    2017-09-01

    Fry, AC, Housh, TJ, Cramer, JB, Weir, JP, Beck, TW, Schilling, BK, Miller, JD, and Nicoll, JX. Noninvasive assessment of skeletal muscle myosin heavy chain expression in trained and untrained men. J Strength Cond Res 31(9): 2355-2362, 2017-Numerous conditions and types of physical activity (e.g., exercise, aging, and muscle-related diseases) can influence muscle fiber types and the proteins expressed. To date, muscle fibers can only be characterized by actually obtaining a tissue sample using the invasive muscle biopsy procedure. Mechanomyography (MMG) is the assessment of the vibration properties of contracting skeletal muscle and has been proposed as a possible noninvasive method for muscle fiber analysis. Therefore, the purpose of this project was to examine the feasibility of using MMG and muscle performance measures to noninvasively assess muscle fiber characteristics. Fifteen men (5 endurance-trained, 5 weight-trained, and 5 sedentary) provided muscle samples from their vastus lateralis muscle. These samples were analyzed for relative myosin heavy chain (MHC) protein expression, which is highly correlated with % muscle fiber type areas. Additionally, each subject performed several muscle performance tests, and MMG of the quadriceps was assessed during a knee extension exercise. Multiple regression was used to develop prediction equations for determining relative muscle content of MHC types I, IIa, and IIx. A combination of MMG and knee extension performance variables estimated types I, IIa, and IIx MHCs with approximately 80% accuracy. Although preliminary, these data suggest that muscle performance tests in addition to MMG assessments during a simple muscle performance task (knee extension) can be used to estimate muscle fiber type composition in a healthy male population. Such methods could ultimately be used to noninvasively monitor muscle health and fitness.

  7. Silencing of a putative inner arm dynein heavy chain results in flagellar immotility in Trypanosoma brucei

    PubMed Central

    Springer, Amy L.; Bruhn, David F.; Kinzel, Kathryn W.; Rosenthal, Noël F.; Zukas, Randi; Klingbeil, Michele M.

    2010-01-01

    The Trypanosoma brucei flagellum controls motility and is crucial for cell polarity and division. Unique features of trypanosome motility suggest that flagellar beat regulation in this organism is unusual and worthy of study. The flagellar axoneme, required for motility, has a structure that is highly conserved among eukaryotes. Of the several dyneins in the axonemal inner arm complex, dynein f is thought to control flagellar waveform shape. A T. brucei gene predicted to encode the dynein f alpha heavy chain, TbDNAH10, was silenced using RNA interference in procyclic T. brucei cells. This resulted in immotile flagella, showing no movement except for occasional slight twitches at the tips. Cell growth slowed dramatically and cells were found in large clusters. Microscopic analysis of silenced cultures showed many cells with detached flagella, sometimes entangled between multiple cells. DAPI staining showed an increased frequency of mis-positioned kinetoplasts and multinucleate cells, suggesting that these cells experience disruption at an early cell cycle stage, probably secondary to the motility defect. TEM images showed apparently normal axonemes and no discernable defects in inner arm structure. This study demonstrates use of RNAi as an effective method to study very large genes such as dynein heavy chains (HCs), and the immotility phenotype of these dynein knockdowns suggests that an intact inner arm is necessary for flagellar beating in T. brucei. Since analogous mutants in Chlamydomonas reinhardtii retain motility, this phenotype likely reflects differences in requirements for motility and/or dynein assembly between the two organisms and these comparative studies will help elucidate the mechanisms of flagellar beat regulation. PMID:20888370

  8. Atherosclerosis Susceptibility in Mice Is Independent of the V1 Immunoglobulin Heavy Chain Gene

    PubMed Central

    Centa, Monica; Gruber, Sabrina; Nilsson, Daniel; Polyzos, Konstantinos A.; Johansson, Daniel K.; Hansson, Göran K.; Ketelhuth, Daniel F.J.; Binder, Christoph J.

    2016-01-01

    Objective— The V1 (VHS107.1.42) immunoglobulin heavy chain gene is thought to be critical in producing IgM natural antibodies of the T15-idiotype that protect against both atherosclerosis and infection from Streptococcus pneumoniae. Our aim was to determine whether genetic loss of the V1 gene increased atherosclerotic plaque burden in vivo because of a reduction in the T15-idiotype or other atheroprotective antibodies. Approach and Results— We crossed VHS107.1.42-deficient mice with the atherosclerosis-prone Apoe−/− and Ldlr−/− strains. Although these double knockout strains manifested no defects in B-cell development, we did observe a substantial reduction in early immune responses against phosphocholine after immunization. However, the titers of plasma antibodies reacting against defined atherosclerotic antigens such as oxidized low-density lipoprotein, as well as the T15-idiotype, were unaffected by loss of the VHS107.1.42 gene in hypercholesterolemic mice. Furthermore, we observed no increase in atherosclerotic lesion formation, either within the aortic arch or aortic root. Robust deposition of IgM within atherosclerotic plaques could also be readily observed in both control and experimental mice. Conclusions— Our data indicate that IgM-dependent protection against atherosclerosis is unlikely to be dependent on antibodies that use the VHS107.1.42 gene, in contrast to the acute immune response conferred by this heavy chain in the response to phosphocholine and in providing resistance against lethal S pneumoniae infection. PMID:26564818

  9. Analysis of sense and naturally occurring antisense transcripts of myosin heavy chain in the human myocardium.

    PubMed

    Luther, H P; Podlowski, S; Hetzer, R; Baumann, G

    2001-01-01

    Naturally occurring antisense RNA has the potential to form a duplex with its complementary sense mRNA, thereby regulating protein expression. Previously, we demonstrated considerable amounts of endogenous antisense RNA for both alpha- and beta-myosin heavy chain (MHC) in rat heart suggesting a role in posttranscriptional MHC-regulation (Luther et al. [1997] J Mol Cell Cardiol 29(1):27-35). To evaluate whether antisense RNA is also involved in MHC regulation in human heart we analyzed ventricular myocardium transcripts in nonfailing hearts (n=3) and hearts from patients undergoing heart transplantation (n=5). Investigation of RNA by reverse transcription polymerase chain reaction (RT-PCR) detected an antisense RNA transcript for beta-MHC but none for alpha-MHC. Northern blot analysis of normal and failing hearts detected sense mRNA for beta-MHC, but not alpha-MHC suggesting no functionally relevant levels of alpha-MHC mRNA exist in the human ventricle. The results describe-for the first time-the existence of endogenous polyadenylated MHC antisense transcripts in the human heart. The potential effect of attenuating translation was shown in an in vitro translation assay using a synthetic antisense-oligonucleotide derived from the sequence of the naturally occurring antisense RNA. Copyright 2001 Wiley-Liss, Inc.

  10. Myosin heavy chain mRNA isoforms in masseter muscle before and after orthognathic surgery.

    PubMed

    Harzer, Winfried; Worm, Margret; Gedrange, Tomasz; Schneider, Matthias; Wolf, Peter

    2007-10-01

    Orthognathic surgery leads to changed jaw position and force vector of mastication to which the muscles must adapt. The aim of the present study was to determine the relative expression of myosin heavy chain (MyHC) messenger RNA (mRNA) isoforms in different types of human masseter muscle fiber under consideration of change in the number of occlusal contacts before and 6 months after surgery. Muscle biopsies were taken from the anterior and posterior parts of both sides in 30 patients with prognathic and retrognathic mandibles. Specific mRNA MyHC analysis was made by real-time polymerase chain reaction to quantify the isoforms I, IIa, and IId/x. There was a shift in the relative content from type I (46% before, 37% after) to type IIa (29% before, 42% after). This shift correlates with number of teeth in occlusion. Correlation between isoform shift and number of teeth in occlusion indicates higher mastication force which stabilizes the treatment result.

  11. Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs)

    PubMed Central

    Maass, David R.; Sepulveda, Jorge; Pernthaner, Anton; Shoemaker, Charles B.

    2007-01-01

    Recombinant single domain antibody fragments (VHHs) that derive from the unusual camelid heavy chain only IgG class (HCAbs) have many favourable properties compared with single-chain antibodies prepared from conventional IgG. As a result, VHHs have become widely used as binding reagents and are beginning to show potential as therapeutic agents. To date, the source of VHH genetic material has been camels and llamas despite their large size and limited availability. Here we demonstrate that the smaller, more tractable and widely available alpaca is an excellent source of VHH coding DNA. Alpaca sera IgG consists of about 50% HCAbs, mostly of the short-hinge variety. Sequencing of DNA encoding more than 50 random VHH and hinge domains permitted the design of PCR primers that will amplify virtually all alpaca VHH coding DNAs for phage display library construction. Alpacas were immunized with ovine tumour necrosis factor α (TNFα) and a VHH phage display library was prepared from a lymph node that drains the sites of immunizations and successfully employed in the isolation of VHHs that bind and neutralize ovine TNFα. PMID:17568607

  12. Primary structure of myosin heavy chain from fast skeletal muscle of Chum salmon Oncorhynchus keta.

    PubMed

    Iwami, Yuki; Ojima, Takao; Inoue, Akira; Nishita, Kiyoyoshi

    2002-10-01

    The nucleotide sequence of the cDNA encoding myosin heavy chain of chum salmon Oncorhynchus keta fast skeletal muscle was determined. The sequence consists of 5,994 bp, including 5,814 bp of translated region deducing an amino acid sequence of 1,937 residues. The deduced sequence showed 79% homology to that of rabbit fast skeletal myosin and 84-87% homology to those of fast skeletal myosins from walleye pollack, white croaker and carp. The putative binding-sites for ATP, actin and regulatory light-chains in the subfragment-1 region of the salmon myosin showed high homology with the fish myosins (78-100% homology). However, the Loop-1 and Loop-2 showed considerably low homology (31-60%). On the other hand, the deduced sequences of subfragment-2 (533 residues) and light meromyosin (564 residues) showed 88-93% homology to the corresponding regions of the fish myosins. It becomes obvious that several specific residues of the rabbit LMM are substituted to Gly in the salmon LMM as well as the other fish LMMs. This may be involved in the structural instability of the fish myosin tail region.

  13. Lampreys have a single gene cluster for the fast skeletal myosin heavy chain gene family.

    PubMed

    Ikeda, Daisuke; Ono, Yosuke; Hirano, Shigeki; Kan-no, Nobuhiro; Watabe, Shugo

    2013-01-01

    Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5'-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny.

  14. Lampreys Have a Single Gene Cluster for the Fast Skeletal Myosin Heavy Chain Gene Family

    PubMed Central

    Ikeda, Daisuke; Ono, Yosuke; Hirano, Shigeki; Kan-no, Nobuhiro; Watabe, Shugo

    2013-01-01

    Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5′-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny. PMID:24376886

  15. IgD heavy-chain deposition disease: detection by laser microdissection and mass spectrometry.

    PubMed

    Royal, Virginie; Quint, Patrick; Leblanc, Martine; LeBlanc, Richard; Duncanson, Garrett F; Perrizo, Robert L; Fervenza, Fernando C; Kurtin, Paul; Sethi, Sanjeev

    2015-04-01

    Monoclonal Ig deposition disease (MIDD) is a rare complication of monoclonal gammopathy characterized by deposition of monoclonal Ig light chains and/or heavy chains along the glomerular and tubular basement membranes. Here, we describe a unique case of IgD deposition disease. IgD deposition is difficult to diagnose, because routine immunofluorescence does not detect IgD. A 77-year-old man presented with proteinuria and renal failure, and kidney biopsy analysis showed a nodular sclerosing GN with extensive focal global glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Immunofluorescence was negative for Ig deposits, although electron microscopy showed deposits in the glomeruli and along tubular basement membranes. Laser microdissection of glomeruli and mass spectrometry of extracted peptides showed a large spectra number for IgD, and immunohistochemistry showed intense glomerular and tubular staining for IgD. Together, these findings are consistent with IgD deposition disease. Bone marrow biopsy analysis showed 5% plasma cells, which stained for IgD. The patient was treated with bortezomib and dexamethasone, which resulted in improvement of hematologic parameters but no improvement of renal function. The diagnosis of IgD deposition disease underscores the value of laser microdissection and mass spectrometry in further evaluating renal biopsies when routine assessment fails to reach an accurate diagnosis.

  16. Chronic sleep deprivation alters the myosin heavy chain isoforms in the masseter muscle in rats.

    PubMed

    Cao, Ruihua; Huang, Fei; Wang, Peihuan; Chen, Chen; Zhu, Guoxiong; Chen, Lei; Wu, Gaoyi

    2015-05-01

    To investigate the changes in myosin heavy chain (MyHC) isoforms of rat masseter muscle fibres caused by chronic sleep deprivation and a possible link with the pathogenesis of disorders of the temporomandibular joint (TMJ). A total of 180 male rats were randomly divided into three groups (n=60 in each): cage controls, large platform controls, and chronic sleep deprivation group. Each group was further divided into three subgroups with different observation periods (7, 14, and 21 days). We investigated he expression of MyHC isoforms in masseter muscle fibres by real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunohistochemical staining. In rats with chronic sleep deprivation there was increased MyHC-I expression in layers of both shallow and deep muscles at 7 and 21 days compared with the control groups, whereas sleep deprivation was associated with significantly decreased MyHC-II expression. At 21 days, there were no differences in MyHC-I or MyHC-II expression between the groups and there were no differences between the two control groups at any time point. These findings suggest that chronic sleep deprivation alters the expression of MyHC isoforms, which may contribute to the pathogenesis of disorders of the TMJ.

  17. Clathrin light and heavy chain interface: alpha-helix binding superhelix loops via critical tryptophans.

    PubMed

    Chen, Chih-Ying; Reese, Michael L; Hwang, Peter K; Ota, Nobuyuki; Agard, David; Brodsky, Frances M

    2002-11-15

    Clathrin light chain subunits (LCa and LCb) contribute to regulation of coated vesicle formation to sort proteins during receptor-mediated endocytosis and organelle biogenesis. LC binding to clathrin heavy chain (HC) was characterized by genetic and structural approaches. The core interactions were mapped to HC residues 1267-1522 (out of 1675) and LCb residues 90-157 (out of 228), using yeast two-hybrid assays. The C-termini of both subunits also displayed interactions extending beyond the core domains. Mutations to helix breakers within the LCb core disrupted HC association. Further suppressor mutagenesis uncovered compensatory mutations in HC (K1415E or K1326E) capable of rescuing the binding defects of LCb mutations W127R or W105R plus W138R, thereby pinpointing contacts between HC and LCb. Mutant HC K1415E also rescued loss of binding by LCa W130R, indicating that both LCs interact similarly with HC. Based on circular dichroism data, mapping and mutagenesis, LCa and LCb were represented as alpha-helices, aligned along the HC and, using molecular dynamics, a structural model of their interaction was generated with novel implications for LC control of clathrin assembly.

  18. Expression of Myosin Heavy Chain Isoforms in Rat Soleus Muscle Spindles After 19 Days of Hypergravity

    PubMed Central

    Picquet, Florence; De-Doncker, Laurent; Falempin, Maurice

    2003-01-01

    The aim of this study was to determine whether a period of 19 days in hypergravity was long enough to induce changes in the expression of myosin heavy chain (MyHC) isoforms in the muscle spindles. The soleus muscle of 10 male Wistar rats (control: CONT, n=5; hypergravity: HG, n=5) was frozen, cut into serial sections, and labeled with antibodies against MyHCs: I, IIA, IIA + IIX + IIB, slow-tonic, and α-cardiac. Forty CONT and 45 HG spindles were analyzed. The results from HG spindles compared to CONT showed that there was no change in the cross-sectional area of intrafusal fibers. However, along the entire length of B1 fibers, the expression of both MyHC I and α-cardiac was increased significantly, whereas the labeling against MyHC IIA and MyHC slow-tonic was decreased. In B2 fibers, the labeling against MyHC IIA (region A), slow-tonic (region A), and fast myosins (regions A-C) was statistically decreased. In chain fibers, the labeling against both MyHC IIA and fast MyHC was reduced significantly. We conclude that hypergravity has a real impact on the MyHC content in the muscle spindles and induces some inverse changes of those observed in hypogravity for MyHCs I, α-cardiac, and slow-tonic. PMID:14566020

  19. Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility

    PubMed Central

    Sun, Faneng; Zhu, Chuanmei; Dixit, Ram; Cavalli, Valeria

    2011-01-01

    Neuronal development, function and repair critically depend on axonal transport of vesicles and protein complexes, which is mediated in part by the molecular motor kinesin-1. Adaptor proteins recruit kinesin-1 to vesicles via direct association with kinesin heavy chain (KHC), the force-generating component, or via the accessory light chain (KLC). Binding of adaptors to the motor is believed to engage the motor for microtubule-based transport. We report that the adaptor protein Sunday Driver (syd, also known as JIP3 or JSAP1) interacts directly with KHC, in addition to and independently of its known interaction with KLC. Using an in vitro motility assay, we show that syd activates KHC for transport and enhances its motility, increasing both KHC velocity and run length. syd binding to KHC is functional in neurons, as syd mutants that bind KHC but not KLC are transported to axons and dendrites similarly to wild-type syd. This transport does not rely on syd oligomerization with itself or other JIP family members. These results establish syd as a positive regulator of kinesin activity and motility. PMID:21750526

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

    PubMed Central

    2015-01-01

    Tetrabromobisphenol A (TBBPA) is a ubiquitous flame retardant. A high-throughput immunoassay would allow for monitoring of human and environmental exposures as a part of risk assessment. Naturally occurring antibodies in camelids that are devoid of light chain, show great promise as an efficient tool in monitoring environmental contaminants, but they have been rarely used for small molecules. An alpaca was immunized with a TBBPA hapten coupled to thyroglobulin and a variable domain of heavy chain antibody (VHH) T3–15 highly selective for TBBPA was isolated from a phage displayed VHH library using heterologous coating antigens. Compared to the VHHs isolated using homologous antigens, VHH T3–15 had about a 10-fold improvement in sensitivity in an immunoassay. This assay, under the optimized conditions of 10% methanol in the assay buffer (pH 7.4), had an IC50 for TBBPA of 0.40 ng mL–1 and negligible cross reactivity (<0.1%) with other tested analogues. After heating the VHH at 90 °C for 90 min about 20% of the affinity for coating antigen T3-BSA remained. The recoveries of TBBPA from spiked soil and fetal bovine serum samples ranged from 90.3% to 110.7% by ELISA and agreed well with a liquid chromatography–tandem mass spectrometry method. We conclude the many advantages of VHH make them attractive for the development of immunoassays to small molecules. PMID:25068372

  1. Kinesin-1 heavy chain mediates microtubule sliding to drive changes in cell shape

    PubMed Central

    Jolly, Amber L.; Kim, Hwajin; Srinivasan, Divya; Lakonishok, Margot; Larson, Adam G.; Gelfand, Vladimir I.

    2010-01-01

    Microtubules are typically observed to buckle and loop during interphase in cultured cells by an unknown mechanism. We show that lateral microtubule movement and looping is a result of microtubules sliding against one another in interphase Drosophila S2 cells. RNAi of the kinesin-1 heavy chain (KHC), but not dynein or the kinesin-1 light chain, eliminates these movements. KHC-dependent microtubule sliding powers the formation of cellular processes filled with parallel microtubule bundles. The growth of these cellular processes is independent of the actin cytoskeleton. We further observe cytoplasmic microtubule sliding in Xenopus and Ptk2 cells, and show that antibody inhibition of KHC in mammalian cells prevents sliding. We therefore propose that, in addition to its well established role in organelle transport, an important universal function of kinesin-1 is to mediate cytoplasmic microtubule–microtubule sliding. This provides the cell with a dedicated mechanism to transport long and short microtubule filaments and drive changes in cell shape. PMID:20566873

  2. Characterization of isoform diversity among smooth muscle and nonmuscle myosin heavy chains.

    PubMed

    Kelley, C A

    1997-05-01

    In the last decade, as a result of molecular cloning and the reverse-transcriptase polymerase chain reaction, numerous isoforms of the contractile protein myosin have been discovered. What lags behind their discovery is knowledge of their functions. This review focuses on some of my recent work on the structure, function and regulation of isoforms of the heavy chain of vertebrate smooth muscle and nonmuscle myosin II. Reference to related work in the field is included where appropriate. The particular isoforms discussed are those that are generated by alternative splicing near the 5' end of the pre-mRNA, resulting in either an insertion or a deletion of a cassette of amino acids near the amino-terminus of the myosin heavy chain (MHC) protein. In both the smooth muscle and nonmuscle MHCs, this splicing occurs in the exact same region, which begins at amino acid 212 in the primary sequence. In the three-dimensional structure of the molecule, these inserts are located near the ATP-binding pocket in a region of the MHC that was not resolved in the crystal structure and therefore is believed to represent a flexible loop. In the smooth muscle MHC, the insertion of seven amino acids in this loop confers a higher enzymatic activity on the myosin. The potential mechanism by which this occurs and the significance to smooth muscle contractile diversity is discussed. In the nonmuscle MHC, the insert in this region is a different size and sequence of amino acids than that in the smooth muscle MHC. A serine residue (Ser-214) in the nonmuscle loop is phosphorylated by p34cdc2 kinase in Xenopus during meiotic maturation of oocytes to eggs and is dephosphorylated in interphase egg extracts that are equivalent to the interphase after fertilization of the egg. Thus, MHC-B phosphorylation by cdc2 kinase correlates with the cortical reorganization that occurs during meiosis, and dephosphorylation correlates with the cortical contraction that occurs at fertilization, which aids in

  3. Biosynthesis and characterization of a non-repetitive polypeptide derived from silk fibroin heavy chain.

    PubMed

    Yang, Gaoqiang; Wu, Mingyang; Yi, Honggen; Wang, Jiannan

    2016-02-01

    Silk fibroin heavy chain is the major protein component of Bombyx mori silk fibroin and is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. In order to determine the biomedical function of the non-repetitive domain or potentially use it to modify hydrophobic biomaterials, high-purity isolation is necessary. Previously, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain. Here, this motif and its multimers are inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif f(1) and multimers f(4) and f(8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography, and single bands of purified fusion proteins GST-F(1), GST-F(4), and GST-F(8), were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Target polypeptides F(1), F(4), and F(8), were cleaved clearly from the GST-fusion tag following thrombin digestion. Mass spectrometry results indicate that the molecular weights associated with fusion proteins GST-F(1), GST-F(4), and GST-F(8) are 31.5, 43.8, and 59.0kDa, respectively, and with the cleaved polypeptides F(1), F(4), and F(8) are 4.8, 16.8, and 32.8kDa, respectively. The F(1), F(4), and F(8) polypeptide chains are negatively charged with isoelectric points (pI) of 3.3, 3.2, and 3.0, respectively. The molecular weight and pI values of the polypeptide chains are consistent with the predicted values and the amino acid compositions similar to predicted sequences. FTIR and CD results show the molecular conformation of F(1) was mainly random coil, and more stable α-helix structure formed in longer molecular chain.

  4. Enhanced factor VIII heavy chain for gene therapy of hemophilia A.

    PubMed

    Chen, Lingxia; Lu, Hui; Wang, Jinhui; Sarkar, Rita; Yang, Xiao; Wang, Hongli; High, Katherine A; Xiao, Weidong

    2009-03-01

    Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a "chain imbalance" problem in which LC secretion is approximately 1-2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC(745)). The optimized HC mutein, HC(HL), including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HC(HL)+AAV8-LC or AAV8-HC(745)+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HC(HL)+AAV8-LC achieved three- to fourfold higher HC expression than AAV8-HC(745)+AAV8-LC, consistent with the FVIII functional assays. HC(HL) should be substituted for HC(745) in a dual AAV vector strategy due to its enhanced expression.

  5. Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation*

    PubMed Central

    Bowers, Peter M.; Neben, Tamlyn Y.; Tomlinson, Geoffery L.; Dalton, Jennifer L.; Altobell, Larry; Zhang, Xue; Macomber, John L.; Wu, Betty F.; Toobian, Rachelle M.; McConnell, Audrey D.; Verdino, Petra; Chau, Betty; Horlick, Robert A.; King, David J.

    2013-01-01

    A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods. PMID:23355464

  6. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  7. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  8. Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema

    PubMed Central

    Kim, Dong Kwan; Zhu, Jianliang; Kozyak, Benjamin W; Burkman, James M; Rubinstein, Neal A; Lankford, Edward B; Stedman, Hansell H; Nguyen, Taitan; Levine, Sanford; Shrager, Joseph B

    2003-01-01

    Background Several physiological adaptations occur in the respiratory muscles in rodent models of elastase-induced emphysema. Although the contractile properties of the diaphragm are altered in a way that suggests expression of slower isoforms of myosin heavy chain (MHC), it has been difficult to demonstrate a shift in MHCs in an animal model that corresponds to the shift toward slower MHCs seen in human emphysema. Methods We sought to identify MHC and corresponding physiological changes in the diaphragms of rats with elastase-induced emphysema. Nine rats with emphysema and 11 control rats were studied 10 months after instillation with elastase. MHC isoform composition was determined by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry by using specific probes able to identify all known adult isoforms. Physiological adaptation was studied on diaphragm strips stimulated in vitro. Results In addition to confirming that emphysematous diaphragm has a decreased fatigability, we identified a significantly longer time-to-peak-tension (63.9 ± 2.7 ms versus 53.9 ± 2.4 ms). At both the RNA (RT-PCR) and protein (immunocytochemistry) levels, we found a significant decrease in the fastest, MHC isoform (IIb) in emphysema. Conclusion This is the first demonstration of MHC shifts and corresponding physiological changes in the diaphragm in an animal model of emphysema. It is established that rodent emphysema, like human emphysema, does result in a physiologically significant shift toward slower diaphragmatic MHC isoforms. In the rat, this occurs at the faster end of the MHC spectrum than in humans. PMID:12617755

  9. Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

    PubMed

    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Morgenstern, Sven; Pavlovic, Dragan; Kunert-Keil, Christiane

    2011-12-01

    The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P < 0.01; mean ± standard error of the mean (SEM), Student's unpaired t-test], as well as in the tongue muscle (65.7 versus 73.8 per cent; P < 0.05). Similarly, the content of MyHC-2x isoforms in mdx tongue muscle was lower than in the controls (25.9 versus 30.8 per cent; P < 0.05). The observed down-regulation of the MyHC-2x and MyHC-2b mRNA in the masticatory muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

  10. Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements.

    PubMed

    Singh, B R; DasGupta, B R

    1989-01-23

    The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (Mr 145,000), and its two subunits, the heavy (H) and light (L) chains (Mr 97,000 and 53,000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of alpha helix, beta sheet and beta turns). Also, the alpha helix, beta sheet, beta turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of alpha helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.

  11. Further study of α-decay in heavy isotopic chains considering the isospin effect

    NASA Astrophysics Data System (ADS)

    Qian, Yibin; Ren, Zhongzhou

    2016-06-01

    We have enhanced the deformed density-dependent cluster model to improve the quantitative description of α-decay in heavy even-even nuclei with 84≤slant Z≤slant 92. To preliminarily introduce the isospin effect into α-decay, the neutron excess term is added in the establishment of the crucial α-core potential. The proton and neutron density distributions are respectively considered in different parameterized formulas by combining them with available experimental data of both the charge radius and the neutron skin thickness. The calculated α-decay half-lives are found to be in somewhat better agreement with the experimental data as compared with our previous results. Strikingly, it is noted that the relatively large deviation between theory and experiment, along the tail of the isotopic chain, is obviously reduced and smoother. This may indicate the necessity of considering the isospin effect in α-decay, especially for extremely neutron-rich nuclei, which appears to be essential for the extended study of heaviest nuclei as well.

  12. Possible deletion of a developmentally regulated heavy-chain variable region gene in autoimmune diseases

    SciTech Connect

    Yang, Pei-Ming; Olee, Tsaiwei; Kozin, F.; Carson, D.A.; Chen, P.P. ); Olsen, N.J. ); Siminovitch, K.A. )

    1990-10-01

    Several autoantibody-associated variable region (V) genes are preferentially expressed during early ontogenic development, suggesting strongly that they are of developmental and physiological importance. As such, it is possible that polymorphisms in one or more of these genes may alter susceptibility to autoimmune disease. The authors have searched extensively for a probe related to a developmentally regulated V gene that has the power to differentiate among highly homologous V genes in human populations. Using such a probe (i.e., Humhv3005/P1) related to both anti-DNA and anti-IgG autoantibodies, they studied restriction fragment length polymorphisms in patients with rheumatoid arthritis and systemic lupus erythematosus and found an apparent heavy-chain V (V{sub H}) gene deletion that was nearly restricted to the autoimmune patients. These data suggest that deletions of physiologically important V{sub H} genes may increase the risk of autoimmunity through indirect effects on the development and homeostasis of the B-cell repertoire.

  13. Myosin heavy chain composition of tiger (Panthera tigris) and cheetah (Acinonyx jubatus) hindlimb muscles.

    PubMed

    Hyatt, Jon-Philippe K; Roy, Roland R; Rugg, Stuart; Talmadge, Robert J

    2010-01-01

    Felids have a wide range of locomotor activity patterns and maximal running speeds, including the very fast cheetah (Acinonyx jubatas), the roaming tiger (Panthera tigris), and the relatively sedentary domestic cat (Felis catus). As previous studies have suggested a relationship between the amount and type of activity and the myosin heavy chain (MHC) isoform composition of a muscle, we assessed the MHC isoform composition of selected hindlimb muscles from these three felid species with differing activity regimens. Using gel electrophoresis, western blotting, histochemistry, and immunohistochemistry with MHC isoform-specific antibodies, we compared the MHC composition in the tibialis anterior, medial gastrocnemius (MG), plantaris (Plt), and soleus muscles of the tiger, cheetah, and domestic cat. The soleus muscle was absent in the cheetah. At least one slow (type I) and three fast (types IIa, IIx, and IIb) MHC isoforms were present in the muscles of each felid. The tiger had a high combined percentage of the characteristically slower isoforms (MHCs I and IIa) in the MG (62%) and the Plt (86%), whereas these percentages were relatively low in the MG (44%) and Plt (55%) of the cheetah. In general, the MHC isoform characteristics of the hindlimb muscles matched the daily activity patterns of these felids: the tiger has daily demands for covering long distances, whereas the cheetah has requirements for speed and power. (c) 2009 Wiley-Liss, Inc.

  14. Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state

    NASA Technical Reports Server (NTRS)

    Adams, G. R.; McCue, S. A.; Zeng, M.; Baldwin, K. M.

    1999-01-01

    During the postnatal period, rat limb muscles adapt to weight bearing via the replacement of embryonic (Emb) and neonatal (Neo) myosin heavy chains (MHCs) by the adult isoforms. Our aim was to characterize this transition in terms of the six MHC isoforms expressed in skeletal muscle and to determine the importance of innervation and thyroid hormone status on the attainment of the adult MHC phenotype. Neonatal rats were made hypothyroid via propylthiouracil (PTU) injection. In normal and PTU subgroups, leg muscles were unilaterally denervated at 15 days of age. The MHC profiles of plantaris (PLN) and soleus (Sol) muscles were determined at 7, 14, 23, and 30 days postpartum. At day 7, the Sol MHC profile was 55% type I, 30% Emb, and 10% Neo; in the PLN, the pattern was 60% Neo and 25% Emb. By day 30 the Sol and PLN had essentially attained an adult MHC profile in the controls. PTU augmented slow MHC expression in the Sol, whereas in the PLN it markedly repressed IIb MHC by retaining neonatal MHC expression. Denervation blunted the upregulation of IIb in the PLN and of Type I in the Sol and shifted the pattern to greater expression of IIa and IIx MHCs in both muscles. In contrast to previous observations, these findings collectively suggest that both an intact thyroid and innervation state are obligatory for the attainment of the adult MHC phenotype, particularly in fast-twitch muscles.

  15. Effects of inactivity on myosin heavy chain composition and size of rat soleus fibers

    NASA Technical Reports Server (NTRS)

    Grossman, E. J.; Roy, R. R.; Talmadge, R. J.; Zhong, H.; Edgerton, V. R.

    1998-01-01

    Myosin heavy chain (MHC) and fiber size properties of the adult rat soleus were determined after 4-60 days of complete inactivity, i.e., lumbar spinal cord isolation. Soleus atrophy was rapid and progressive, i.e., 25% and 64% decrease in weight and 33% and 75% decrease in fiber size after 4 and 60 days of inactivity, respectively. Changes in MHC occurred at a slower rate than the atrophic response. After 15 days there was de novo expression of type IIx MHC (approximately 10%). By 60 days, type IIx MHC accounted for 33% of the total MHC content, and 7% of the fibers contained only type IIx MHC. The relative amount of type I MHC was reduced from 93% in control to 49% after 60 days of inactivity. Therefore, the effects of 60 days of inactivity suggest that during this time period at least 75% of fiber size and approximately 40% of type I MHC composition of the adult rat soleus can be attributed to activation-related events.

  16. Identification of myosin heavy chain isoforms in skeletal muscle of four Southern African wild ruminants.

    PubMed

    Kohn, Tertius A; Hoffman, Louw C; Myburgh, Kathryn H

    2007-10-01

    The aim was to separate and characterize the myosin heavy chain (MHC) isoforms of four southern African wild ruminants, namely Blesbuck (Damaliscus dorcas phillipsi), Kudu (Tragelaphus strepsiceros), Black Wildebeest (Connochaetes gnou) and Blue Wildebeest (Connochaetes taurinus). Longissimus dorsi muscle samples were subjected to SDS-PAGE and Western blot analyses using antibodies raised against MHC isoforms. The specificity of these antibodies was assessed using immunohistochemistry combined with ATPase histochemistry, Three MHC isoforms were separated and the bands were identified from fastest to slowest migrating as MHC I, MHC IIx and MHC IIa. The mobility of the MHC isoforms was similar for all four species, including that of bovine, but differed from human muscle. Kudu muscle exhibited the lowest proportion of MHC I and the highest proportion of MHC IIx, whereas Blesbuck muscle had the least MHC IIx. The two Wildebeest species were intermediate in isoform content. In conclusion, when new species are studied, existing electrophoretic protocols may need to be modified to achieve quantifiable separation and isoform migration pattern must be verified in order to reach correct interpretations. Furthermore, antibody specificity may differ between techniques as well as species and needs confirmation.

  17. Dynein axonemal heavy chain 8 promotes androgen receptor activity and associates with prostate cancer progression

    PubMed Central

    Wang, Yu; Ledet, Russell J.; Imberg-Kazdan, Keren; Logan, Susan K.; Garabedian, Michael J.

    2016-01-01

    To gain insight into cellular factors regulating AR action that could promote castration resistant prostate cancer (CRPC), we performed a genome-wide RNAi screen for factors that promote ligand-independent AR transcriptional activity and integrated clinical databases for candidate genes that are positively associated with prostate cancer metastasis and recurrence. From this analysis, we identified Dynein Axonemal Heavy Chain 8 (DNAH8) as an AR regulator that displayed higher mRNA expression in metastatic than in primary tumors, and showed high expression in patients with poor prognosis. Axonemal dyneins function in cellular motility, but the function of DNAH8 in prostate cancer or other cell types has not been reported. DNAH8 is on chromosome 6q21.2, a cancer-associated amplicon, and is primarily expressed in prostate and testis. Its expression is higher in primary tumors compared to normal prostate, and is further increased in metastatic prostate cancers. Patients expressing high levels of DNAH8 have a greater risk of relapse and a poor prognosis after prostatectomy. Depletion of DNAH8 in prostate cancer cells suppressed AR transcriptional activity and proliferation. Androgen treatment increased DNAH8 mRNA expression, and AR bound the DNAH8 promoter sequence indicating DNAH8 is an AR target gene. Thus, DNAH8 is a new regulator of AR associated with metastatic tumors and poor prognosis. PMID:27363033

  18. Bicaudal-D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

    PubMed Central

    Li, Xuan; Kuromi, Hiroshi; Briggs, Laura; Green, David B; Rocha, João J; Sweeney, Sean T; Bullock, Simon L

    2010-01-01

    Cargo transport by microtubule-based motors is essential for cell organisation and function. The Bicaudal-D (BicD) protein participates in the transport of a subset of cargoes by the minus-end-directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co-precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin-mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high-frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin-associated trafficking processes and show a novel requirement for microtubule-based motor transport in the synaptic vesicle cycle. PMID:20111007

  19. Heavy Chain Single Domain Antibodies to Detect Native Human Soluble Epoxide Hydrolase

    PubMed Central

    Cui, Yongliang; Li, Dongyang; Morisseau, Christophe; Yang, Jun; Wan, Debin; Rossotti, Martín A.; Gee, Shirley J.; González-Sapienza, Gualberto G.; Hammock, Bruce D.

    2015-01-01

    The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer and other diseases. However, there is not a simple, inexpensive and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal-variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the ELISA and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney and lung). The VHH based ELISA appears to be a new reliable method for monitoring the sEH, and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research. PMID:26229025

  20. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  1. Developmental Modulation of a beta myosin heavy chain promoter-driven transgene.

    PubMed

    Knotts, S; Sánchez, A; Rindt, H; Robbins, J

    1996-06-01

    The molecular mechanisms underlying heart and skeletal muscle-specific gene expression during development and in response to physioloic stimuli are largely unknown. Using a novel immunohistochemical procedure to detect chloramphenicol acetyltransferase (CAT), we have investigated, in vivo at high resolution, the ability of cis-acting DNA sequences within the 5' flanking region of the mouse beta myosin heavy chain (MyHC) gene (beta-MyHC) to direct appropriate gene expression throughout development. A 5.6-kb fragment 5' to the beta-MyHC's transcriptional start site was linked to the reporter gene encoding CAT (cat) and used to generate transgenic mice. The anti-CAT in situ assay described in this report allowed us to define the ability of the promoter fragment to direct appropriate temporal, tissue- and muscle fiber type-specific gene expression throughout early development. In skeletal muscles, the transgene expression profile mimics the endogenous beta-myHC's at all developmental stages and is appropriately restricted to slow (type I) skeletal fibers in the adult. Surprisingly, transgene expression was detected in both the atria and ventricles during embryonic and fetal development, indicating that ventricular specification involves elements outside the 5.6-kb fragment. In contrast, in the adult, hypothyroid conditions led to transgene induction specifically in the ventricles, suggesting that distinct regulatory mechanisms control fetal versus adult beta-MyHC expression in the cardiac compartment.

  2. In vivo regulation of the mouse beta myosin heavy chain gene.

    PubMed

    Knotts, S; Rindt, H; Neumann, J; Robbins, J

    1994-12-09

    The interactions of trans-acting factors with their respective cis-acting elements in the 5' upstream region of the beta myosin heavy chain gene (MyHC) regulate its tissue- and developmental stage-specific expression. The role of three conserved elements, an MCAT or TEF-1 binding site, a C-rich region, and a beta e3 region, in muscle-specific gene expression was analyzed in vivo. Each cis-acting site was ablated in the context of the beta MyHC promoter, fused to the chloramphenicol acetyltransferase reporter gene, and used to generate transgenic mice. In contrast to results obtained in vitro, the data demonstrate that mutating any one of these cis-acting elements does not affect the level or tissue specificity of transgene expression. Sequences upstream of -600 can functionally substitute for any one of these regulatory cassettes and are important both for high levels of expression as well as for controlled muscle specificity. Mutation of any two of the cis-acting elements also does not affect transgene expression. However, simultaneous mutation of the three sites significantly reduces expression, indicating that these conserved sequences do play an important role and that combinatorial interactions underlie the beta MyHC's regulation.

  3. In vivo analysis of the murine beta-myosin heavy chain gene promoter.

    PubMed

    Rindt, H; Gulick, J; Knotts, S; Neumann, J; Robbins, J

    1993-03-05

    The 5' upstream region of the murine beta-myosin heavy chain (MHC) gene has been isolated and tested for its ability to drive gene expression in transgenic mice. Three classes of transgenic mice were generated. The constructs contained approximately 5000, 2500, and 600 base pairs of beta-MHC upstream sequence fused to the chloramphenicol acetyltransferase gene and were termed beta 5, beta 2.5, and beta .6, respectively. Muscle-specific expression was observed with all three constructs. However, only the beta 5 lines directed high levels of muscle-specific transgene expression in both pre- and postbirth mice. Expression driven by the two shorter constructs was two to three orders of magnitude lower when the chloramphenicol acetyltransferase specific activities were compared. These data suggest that a distal-positive element directs high levels of gene expression in the ventricle and in slow skeletal muscles. Analyses of transgene expression during heart maturation revealed that some of the beta 5 lines were not able to respond in an appropriate manner to developmental transcriptional cues. Unlike the endogenous beta-MHC gene, which is down regulated in the ventricles around the time of birth, reporter gene expression in the majority of the lines generated was not shut off in the ventricles of the adult animals. These data indicate that high levels of muscle-specific beta-MHC gene expression are dependent upon the combinatorial interactions of a number of sequence elements that are distributed over a large region of the gene's upstream sequence.

  4. Transcriptional regulation of the Drosophila melanogaster muscle myosin heavy-chain gene

    PubMed Central

    Hess, Norbert K.; Singer, Phillip A.; Trinh, Kien; Nikkhoy, Massoud; Bernstein, Sanford I.

    2007-01-01

    We show that a 2.6 kb fragment of the muscle myosin heavy-chain gene (Mhc) of Drosophila melanogaster (containing 458 base pairs of upstream sequence, the first exon, the first intron and the beginning of the second exon) drives expression in all muscles. Comparison of the minimal promoter to Mhc genes of ten Drosophila species identified putative regulatory elements in the upstream region and in the first intron. The first intron is required for expression in four small cells of the tergal depressor of the trochanter (jump) muscle and in the indirect flight muscle. The 3′ end of this intron is important for Mhc transcription in embryonic body wall muscle and contains AT–rich elements that are protected from DNase I digestion by nuclear proteins of Drosophila embryos. Sequences responsible for expression in embryonic, adult body wall and adult head muscles are present both within and outside the intron. Elements important for expression in leg muscles and in the large cells of the jump muscle flank the intron. We conclude that multiple transcriptional regulatory elements are responsible for Mhc expression in specific sets of Drosophila muscles. PMID:17194628

  5. Analysis and comparison of the mouse and human immunoglobulin heavy chain JH-Cmu-Cdelta locus.

    PubMed

    Koop, B F; Richards, J E; Durfee, T D; Bansberg, J; Wells, J; Gilliam, A C; Chen, H L; Clausell, A; Tucker, P W; Blattner, F R

    1996-02-01

    We report here 23,686 bases of contiguous DNA sequences from the mouse germline immunoglobulin heavy chain (H) constant (C) mu delta region. The sequence spans the joining (JH) regions, the mu constant region (C mu), the delta constant region (C delta) coding regions, a domain relic, the mu switch region (S mu), seven blocks of simple sequence repeats, a large unique sequence inverted repeat, a large unique sequence forward repeat, and all of the intervening material. A comparison of this 23.7-kb region with the corresponding human C mu/C delta region reveals clear homology in the coding and introns of C mu but not in the 5' flanking J gene segments nor in the intergenic and C delta regions. This mixed pattern of similarity between the human and the mouse sequences contrasts with high levels of similarity found in the T-cell receptor C alpha/C delta region and alpha and beta myosin genes and the very low levels found in the gamma-crystallin, XRCC1, and beta-globin gene clusters. The human and mouse comparison further suggests the incorporation of novel sequences into expressed genes of IgD.

  6. Myosin heavy chain and fibre diameter of extrinsic tongue muscles in rhesus monkey.

    PubMed

    Smith, J Chadwick; Goldberg, Stephen J; Shall, Mary S

    2006-06-01

    The purpose of this investigation was to identify the myosin heavy chain (MHC) phenotype and fibre diameters of hypoglossal innervated extrinsic tongue muscles in rhesus monkey. Genioglossus, styloglossus and hyoglossus muscle samples obtained from three female rhesus monkeys were analysed for MHC isoforms via gel electrophoresis and stained with MHC antibodies to measure least mean diameters. MHC phenotypes were consistent for all three muscles. Each muscle was predominantly composed of MHC type IIa and I. All three isoforms were significantly different from each other in fibre diameter for styloglossus and genioglossus (IIb>IIa and IIx>I; P<0.001). For hyoglossus, the MHC type II isoforms had larger diameters than the MHC type I isoform (P<0.001). While the extrinsic tongue muscle MHC and/or muscle fibre type composition may be different between mammalian species, there are consistent similarities between the intrinsic and extrinsic tongue muscles. We suggest this is necessary for the highly coordinated activities performed by the tongue such as mastication, respiration and swallowing. The differences in fibre diameters among MHC isoforms suggest a large force gradation, which would be consistent with the coordination of these activities. The similarities among primates in MHC and/or muscle fibre composition as well as similar cortical inputs to the hypoglossal nucleus, suggest that we could expect to see similar MHC phenotype for extrinsic tongue muscles in human.

  7. Independent origin of identical [beta] cardiac myosin heavy-chain mutations in hypertrophic cardiomyopathy

    SciTech Connect

    Watkins, H. Harvard Medical School, Boston, MA St. George's Hospital Medical School, London ); Thierfelder, L.; Anan, R.; Jarcho, J.; Seidman, C.E. Harvard Medical School, Boston, MA ); Matsumori, Akira ); McKenna, W. ); Seidman, J.G. Howard Hughes Medical Inst., Boston, MA )

    1993-12-01

    The origins of the [beta] cardiac myosin heavy-chain (MHC) gene missense mutations that cause familial hypertrophic cardiomyopathy (FHC) in 14 families have been evaluated. Of eight different mutations, four were present in single families, while four occurred in two or more families. To investigate the origins of the four shared mutations, the authors defined the [beta] cardiac MHC haplotypes of each of the mutation-bearing chromosomes by determining the alleles present at three intragenic polymorphic loci. Two of the mutations (Arg453Cys and Val606Met) have arisen independently in each of three families, being found on different chromosomal backgrounds. A third mutation (Gly584Arg) is associated with identical haplotypes in two families with Portuguese ancestors, suggesting a founder effect. Haplotype analysis was uninformative for the fourth mutation (Arg403Gln). Thus, FHC-causing mutations have arisen independently in at least 12 of the 14 families studied, suggesting that the majority have arisen relatively recently as new mutations. This finding predicts the prevalence of disease-causing [beta] cardiac MHC mutations to be comparable in all population groups. 21 refs., 4 figs., 1 tab.

  8. A novel disorder reveals clathrin heavy chain-22 is essential for human pain and touch development.

    PubMed

    Nahorski, Michael S; Al-Gazali, Lihadh; Hertecant, Jozef; Owen, David J; Borner, Georg H H; Chen, Ya-Chun; Benn, Caroline L; Carvalho, Ofélia P; Shaikh, Samiha S; Phelan, Anne; Robinson, Margaret S; Royle, Stephen J; Woods, C Geoffrey

    2015-08-01

    Congenital inability to feel pain is very rare but the identification of causative genes has yielded significant insights into pain pathways and also novel targets for pain treatment. We report a novel recessive disorder characterized by congenital insensitivity to pain, inability to feel touch, and cognitive delay. Affected individuals harboured a homozygous missense mutation in CLTCL1 encoding the CHC22 clathrin heavy chain, p.E330K, which we demonstrate to have a functional effect on the protein. We found that CLTCL1 is significantly upregulated in the developing human brain, displaying an expression pattern suggestive of an early neurodevelopmental role. Guided by the disease phenotype, we investigated the role of CHC22 in two human neural crest differentiation systems; human induced pluripotent stem cell-derived nociceptors and TRKB-dependant SH-SY5Y cells. In both there was a significant downregulation of CHC22 upon the onset of neural differentiation. Furthermore, knockdown of CHC22 induced neurite outgrowth in neural precursor cells, which was rescued by stable overexpression of small interfering RNA-resistant CHC22, but not by mutant CHC22. Similarly, overexpression of wild-type, but not mutant, CHC22 blocked neurite outgrowth in cells treated with retinoic acid. These results reveal an essential and non-redundant role for CHC22 in neural crest development and in the genesis of pain and touch sensing neurons.

  9. Association between a common immunoglobulin heavy chain allele and rheumatic heart disease risk in Oceania.

    PubMed

    Parks, Tom; Mirabel, Mariana M; Kado, Joseph; Auckland, Kathryn; Nowak, Jaroslaw; Rautanen, Anna; Mentzer, Alexander J; Marijon, Eloi; Jouven, Xavier; Perman, Mai Ling; Cua, Tuliana; Kauwe, John K; Allen, John B; Taylor, Henry; Robson, Kathryn J; Deane, Charlotte M; Steer, Andrew C; Hill, Adrian V S

    2017-05-11

    The indigenous populations of the South Pacific experience a high burden of rheumatic heart disease (RHD). Here we report a genome-wide association study (GWAS) of RHD susceptibility in 2,852 individuals recruited in eight Oceanian countries. Stratifying by ancestry, we analysed genotyped and imputed variants in Melanesians (607 cases and 1,229 controls) before follow-up of suggestive loci in three further ancestral groups: Polynesians, South Asians and Mixed or other populations (totalling 399 cases and 617 controls). We identify a novel susceptibility signal in the immunoglobulin heavy chain (IGH) locus centring on a haplotype of nonsynonymous variants in the IGHV4-61 gene segment corresponding to the IGHV4-61*02 allele. We show each copy of IGHV4-61*02 is associated with a 1.4-fold increase in the risk of RHD (odds ratio 1.43, 95% confidence intervals 1.27-1.61, P=4.1 × 10(-9)). These findings provide new insight into the role of germline variation in the IGH locus in disease susceptibility.

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

    PubMed

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

    2016-07-01

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

  11. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  12. Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

    NASA Technical Reports Server (NTRS)

    di Maso, N. A.; Caiozzo, V. J.; Baldwin, K. M.

    2000-01-01

    The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

  13. Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions

    PubMed Central

    Perlot, Thomas; Li, Gang; Alt, Frederick W.

    2008-01-01

    Activation-induced cytosine deaminase (AID) is essential for both somatic hypermutation (SHM) and class switch recombination (CSR), two processes involved in antibody diversification. Previously, various groups showed both in vitro and in vivo that AID initiates SHM and CSR by deaminating cytosines in DNA in a transcription-dependent manner. Although in vivo both DNA strands are equally targeted by AID, many in vitro and bacterial experiments found that AID almost exclusively targets the nontemplate strand of a transcribed substrate. Here, we report the detection of antisense transcripts in assembled Ig heavy chain (IgH) variable region exons and their immediate downstream region, as well as in switch regions, sequences that, respectively, are targets for SHM and CSR in vivo. In contrast, we did not detect antisense transcripts from the Cμ constant region exons, which lie between the IgH variable region exons and downstream S regions and which are not normally an AID target. Expression of the antisense variable region/flanking region and the S-region transcripts were found in all lymphocytes that transcribe these sequences in the sense direction. Steady-state levels of antisense transcripts appeared very low, and start sites potentially appeared heterogeneous. We discuss the potential implications of antisense IgH locus transcription for AID targeting or other processes. PMID:18292225

  14. Missense mutation of the {beta}-cardiac myosin heavy-chain gene in hypertrophic cardiomyopathy

    SciTech Connect

    Arai, Shoichi; Matsuoka, Rumiko; Hirayama, Kenji; Sakurai, Hisanao

    1995-09-11

    Hypertrophic cardiomyopathy occurs as an autosomal dominant familial disorder or as a sporadic disease without familial involvement. We describe a missense mutation of the {beta}-cardiac myosin heavy chain (MHC) gene, a G to T transversion (741 Gly{r_arrow}Trp) identified by direct sequencing of exon 20 in four individuals affected with familial hypertrophic cardiomyopathy. Three individuals with sporadic hypertrophic cardiomyopathy, whose parents are clinically and genetically unaffected, had sequence variations of exon 34 of the {alpha}-cardiac MHC gene (a C to T transversion, 1658 Asp{r_arrow}Asp, resulting in FokI site polymorphism), of intron 33 of the {alpha}-cardiac MHC gene (a G to A and an A to T transversion), and also of intron 14 of the {beta}-cardiac MHC gene (a C to T transversion in a patient with Noonan syndrome). Including our case, 30 missense mutations of the {beta}-cardiac MHC gene in 49 families have been reported thus far worldwide. Almost all are located in the region of the gene coding for the globular head of the molecule, and only one mutation was found in both Caucasian and Japanese families. Missense mutations of the {Beta}-cardiac MHC gene in hypertrophic cardiomyopathy may therefore differ according to race. 29 refs., 6 figs., 3 tabs.

  15. Age dependence of myosin heavy chain transitions induced by creatine depletion in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Adams, Gregory R.; Baldwin, Kenneth M.

    1995-01-01

    This study was designed to test the hypothesis that myosin heavy chain (MHC) plasticity resulting from creatine depletion is an age-dependent process. At weaning (age 28 days), rat pups were placed on either standard rat chow (normal diet juvenile group) or the same chow supplemented with 1% wt/wt of the creatine analogue beta-guanidinopropionic acid (creatine depletion juvenile (CDJ) group). Two groups of adult rats (age approximately 8 wk) were placed on the same diet regimens (normal diet adult and creatine depletion adult (CDA) groups). After 40 days (CDJ and normal diet juvenile groups) and 60 days (CDA and normal diet adult groups), animals were killed and several skeletal muscles were removed for analysis of creatine content or MHC ditribution. In the CDJ group, creatine depletion (78%) was accompanied by significant shifts toward expression of slower MHC isoforms in two slow and three fast skeletal muscles. In contrast, creatine depletion in adult animals did not result in similar shifts toward slow MHC isoform expression in either muscle type. The results of this study indicate that there is a differential effect of creatine depletion on MHC tranitions that appears to be age dependent. These results strongly suggest that investigators contemplating experimental designs involving the use of the creatine analogue beta-guanidinopropionic acid should consider the age of the animals to be used.

  16. Relationships between maximal strength, muscle size, and myosin heavy chain isoform composition and postactivation potentiation.

    PubMed

    Seitz, Laurent B; Trajano, Gabriel S; Haff, G Gregory; Dumke, Charles C L S; Tufano, James J; Blazevich, Anthony J

    2016-05-01

    The purpose of this study was to examine the relationships between maximal voluntary postactivation potentiation (PAP) and maximal knee extensor torque, quadriceps cross-sectional area (CSA) and volume, and type II myosin heavy chain (MHC) isoform percentage in human skeletal muscle. Thirteen resistance-trained men completed a test protocol consisting of 2 isokinetic knee extensions at 180°·s(-)(1) performed before and 1, 4, 7, and 10 min after the completion of 4 maximal knee extensions at 60°·s(-)(1) (i.e., a conditioning activity (CA)). Magnetic resonance imaging and muscle microbiopsy procedures were completed on separate days to assess quadriceps CSA and volume and MHC isoform content. Maximal voluntary PAP response was assessed as the ratio of the highest knee extensor torques measured before and after the CA. There were large to very large correlations between maximal voluntary PAP response and maximal knee extensor torque (r = 0.62) and quadriceps CSA (r = 0.68) and volume (r = 0.63). Nonetheless, these correlations were not statistically significant after adjusting for the influence of type II MHC percentage using partial correlation analysis. By contrast, the strongest correlation was observed for type II MHC percentage (r = 0.77), and this correlation remained significant after adjusting for the other variables. Maximal voluntary PAP response is strongly correlated with maximal knee extensor torque and quadriceps CSA and volume, but is mostly clearly associated with the type II myosin isoform percentage in human skeletal muscle.

  17. Mapping of human immunoglobulin heavy chain variable gene segments outside the major IGH locus

    SciTech Connect

    Wintle, R.F.; Cox, D.W. |

    1994-09-01

    Physical mapping of the human immunoglobulin heavy chain gene cluster (IGH) on chromosome 14 has previously shown that the locus includes at least 63 variable region (VH) gene segments. Fifteen VH gene segments are located on six NotI DNA restriction fragments that are not within the mapped region of IGH. We have used human/rodent somatic cell hybrid lines to map these genes segments, as it was previously not proven that they are located in the chromosome 14 IGH locus. Four gene segments map to human chromosome 16 and two to chromosome 15. Apparently, four of the six NotI fragments, representing 11 VH gene segments, are not located within the chromosome 14 IGH locus. In addition, we have demonstrated that a YAC containing a functional human telomere, and mapping to 14qter, is located at the telomeric end of the IGH gene cluster physical map and contains at least four VH gene segments. This YAC is collinear with the existing physical map of genomic DNA. We conclude that our original physical map of IGH represents almost the entire locus on chromosome 14 and that the 11 gene segments newly mapped are not part of the functional IGH locus. 33 refs., 3 figs., 1 tab.

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

    PubMed Central

    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

  19. Conserved protein domains in a myosin heavy chain gene from Dictyostelium discoideum.

    PubMed Central

    Warrick, H M; De Lozanne, A; Leinwand, L A; Spudich, J A

    1986-01-01

    The 2116-amino acid myosin heavy chain sequence from Dictyostelium discoideum was determined from DNA sequence analysis of the cloned gene. The gene product can be divided into two distinct regions, a globular head region and a long alpha-helical, rod-like tail. In comparisons with nematode and mammalian muscle myosins, specific areas of the head region are highly conserved. These areas presumably reflect conserved functional and structural domains. Certain features that are present in the head region of nematode and mammalian muscle myosins, and that have been assumed to be important for myosin function, are missing in the Dictyostelium myosin sequence. The protein sequence of the Dictyostelium tail region is very poorly conserved with respect to the other myosins but displays the periodicities similar to those of muscle myosins. These periodicities are believed to play a role in filament formation. The 196-residue repeating unit that determines the 14.3-nm repeat seen in muscle thick filaments, the 28-residue charge repeating unit, and the 1,4 hydrophobic repeat previously described for the nematode myosin are all present in the Dictyostelium myosin rod sequence, suggesting that the filament structures of muscle and Dictyostelium myosins must be similar. PMID:3540939

  20. Three Routes to Suppression of the Neurodegenerative Phenotypes Caused by Kinesin Heavy Chain Mutations

    PubMed Central

    Djagaeva, Inna; Rose, Debra J.; Lim, Angeline; Venter, Chris E.; Brendza, Katherine M.; Moua, Pangkong; Saxton, William M.

    2012-01-01

    Kinesin-1 is a motor protein that moves stepwise along microtubules by employing dimerized kinesin heavy chain (Khc) subunits that alternate cycles of microtubule binding, conformational change, and ATP hydrolysis. Mutations in the Drosophila Khc gene are known to cause distal paralysis and lethality preceded by the occurrence of dystrophic axon terminals, reduced axonal transport, organelle-filled axonal swellings, and impaired action potential propagation. Mutations in the equivalent human gene, Kif5A, result in similar problems that cause hereditary spastic paraplegia (HSP) and Charcot–Marie–Tooth type 2 (CMT2) distal neuropathies. By comparing the phenotypes and the complementation behaviors of a large set of Khc missense alleles, including one that is identical to a human Kif5A HSP allele, we identified three routes to suppression of Khc phenotypes: nutrient restriction, genetic background manipulation, and a remarkable intramolecular complementation between mutations known or likely to cause reciprocal changes in the rate of microtubule-stimulated ADP release by kinesin-1. Our results reveal the value of large-scale complementation analysis for gaining insight into protein structure–function relationships in vivo and point to possible paths for suppressing symptoms of HSP and related distal neuropathies. PMID:22714410

  1. Cytoskeleton-associated protein 5 and clathrin heavy chain binding regulates spindle assembly in mouse oocytes

    PubMed Central

    Wang, Dong-Hui; Han, Zhe; Kong, Xiang-Wei; Ma, Yu-Zhen; Yun, Zhi-Zhong; Liang, Cheng-Guang

    2017-01-01

    Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to clathrin heavy chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation. PMID:28177917

  2. Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

    NASA Technical Reports Server (NTRS)

    di Maso, N. A.; Caiozzo, V. J.; Baldwin, K. M.

    2000-01-01

    The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

  3. Segmental distribution of myosin heavy chain isoforms within single muscle fibers.

    PubMed

    Zhang, Ming; Gould, Maree

    2017-02-18

    Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the 3 EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I&cenveo_unknown_entity_wingdings_F0F3;IIA&cenveo_unknown_entity_wingdings_F0F3;IIX&cenveo_unknown_entity_wingdings_F0F3;IIB, however in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be due to the myonuclear domain. This article is protected by copyright. All rights reserved.

  4. Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.

    PubMed

    Morin, Nicole A; Oakes, Patrick W; Hyun, Young-Min; Lee, Dooyoung; Chin, Y Eugene; Chin, Eugene Y; King, Michael R; Springer, Timothy A; Shimaoka, Motomu; Tang, Jay X; Reichner, Jonathan S; Kim, Minsoo

    2008-01-21

    Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

  5. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    PubMed

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property.

  6. Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state

    NASA Technical Reports Server (NTRS)

    Adams, G. R.; McCue, S. A.; Zeng, M.; Baldwin, K. M.

    1999-01-01

    During the postnatal period, rat limb muscles adapt to weight bearing via the replacement of embryonic (Emb) and neonatal (Neo) myosin heavy chains (MHCs) by the adult isoforms. Our aim was to characterize this transition in terms of the six MHC isoforms expressed in skeletal muscle and to determine the importance of innervation and thyroid hormone status on the attainment of the adult MHC phenotype. Neonatal rats were made hypothyroid via propylthiouracil (PTU) injection. In normal and PTU subgroups, leg muscles were unilaterally denervated at 15 days of age. The MHC profiles of plantaris (PLN) and soleus (Sol) muscles were determined at 7, 14, 23, and 30 days postpartum. At day 7, the Sol MHC profile was 55% type I, 30% Emb, and 10% Neo; in the PLN, the pattern was 60% Neo and 25% Emb. By day 30 the Sol and PLN had essentially attained an adult MHC profile in the controls. PTU augmented slow MHC expression in the Sol, whereas in the PLN it markedly repressed IIb MHC by retaining neonatal MHC expression. Denervation blunted the upregulation of IIb in the PLN and of Type I in the Sol and shifted the pattern to greater expression of IIa and IIx MHCs in both muscles. In contrast to previous observations, these findings collectively suggest that both an intact thyroid and innervation state are obligatory for the attainment of the adult MHC phenotype, particularly in fast-twitch muscles.

  7. Effects of inactivity on myosin heavy chain composition and size of rat soleus fibers

    NASA Technical Reports Server (NTRS)

    Grossman, E. J.; Roy, R. R.; Talmadge, R. J.; Zhong, H.; Edgerton, V. R.

    1998-01-01

    Myosin heavy chain (MHC) and fiber size properties of the adult rat soleus were determined after 4-60 days of complete inactivity, i.e., lumbar spinal cord isolation. Soleus atrophy was rapid and progressive, i.e., 25% and 64% decrease in weight and 33% and 75% decrease in fiber size after 4 and 60 days of inactivity, respectively. Changes in MHC occurred at a slower rate than the atrophic response. After 15 days there was de novo expression of type IIx MHC (approximately 10%). By 60 days, type IIx MHC accounted for 33% of the total MHC content, and 7% of the fibers contained only type IIx MHC. The relative amount of type I MHC was reduced from 93% in control to 49% after 60 days of inactivity. Therefore, the effects of 60 days of inactivity suggest that during this time period at least 75% of fiber size and approximately 40% of type I MHC composition of the adult rat soleus can be attributed to activation-related events.

  8. Clathrin Heavy Chain Interacts With Estrogen Receptor α and Modulates 17β-Estradiol Signaling

    PubMed Central

    Totta, Pierangela; Pesiri, Valeria; Enari, Masato; Marino, Maria

    2015-01-01

    17β-estradiol (E2)-induced signaling and control of estrogen receptor (ER)α degradation both play a major role in breast cancer cell proliferation. We recently reported the involvement of lysosomal function in both E2-dependent ERα breakdown and E2-induced cell proliferation and thus hypothesized a role for endocytic proteins in ERα signaling. An small interfering RNA screen identified proteins that regulate intracellular endocytic traffic and whose silencing alters E2-induced ERα degradation. One such protein was the clathrin heavy chain (CHC), whose role in E2:ERα signaling to cell proliferation is unknown. Here, we show that CHC physically interacts with ERα in the cytoplasm of breast cancer cells and regulates E2-induced cell proliferation. Surprisingly, the CHC:ERα interaction is required to sustain E2 signaling but is dispensable for ERα degradation. Our data also demonstrate that many membrane trafficking proteins contribute to the regulation of ERα degradation, thus unraveling the contribution of endocytic proteins in E2:ERα signaling. PMID:25860340

  9. Iron deposition and ferritin heavy chain (Fth) localization in rodent teeth.

    PubMed

    Wen, Xin; Paine, Michael L

    2013-01-02

    An iron rich layer on the labial surface is characteristic of the enamel of rodent incisors. In order to address a role for iron content in continuously growing incisors during odontogenesis, we studied iron deposition patterns in enamel and dentine using Perls' blue staining and ferritin heavy chain (Fth) immunolocalization. Fth expression is regulated by iron level; therefore its localization can be used as a sensitive indicator for iron deposition. Sagittal sections of 4-week old rat incisors showed a gradual increase in iron level in the enamel organ from secretory to maturation stages. In addition, iron was detected in ameloblasts of erupting third molars of 4-week old rats, suggesting iron plays a role in both incisor and molar development. In odontoblasts, the presence of iron was demonstrated, and this is consistent with iron's role in collagen synthesis. Using postnatal 3-, 6-, 9-day old mice, the spatial and temporal expression of Fth in tooth development again indicated the presence of iron in mature ameloblasts and odontoblasts. While these data do not explain what functional role iron has in tooth formation, it does highlight a significant molecular activity associated with the formation of the rodent dentition.

  10. Myosin heavy chain is stabilized by BCL-2 interacting cell death suppressor (BIS) in skeletal muscle

    PubMed Central

    Hong, Jin; Park, Jun-Sub; Lee, Hyun; Jeong, Jaemin; Hyeon Yun, Hye; Yun Kim, Hye; Ko, Young-Gyu; Lee, Jeong-Hwa

    2016-01-01

    BCL-2 interacting cell death suppressor (BIS), which is ubiquitously expressed, has important roles in various cellular processes, such as apoptosis, the cellular stress response, migration and invasion and protein quality control. In particular, BIS is highly expressed in skeletal and cardiac muscles, and BIS gene mutations result in human myopathy. In this study, we show that mRNA and protein levels of BIS were markedly increased during skeletal myogenesis in C2C12 cells and mouse satellite cells. BIS knockdown did not prevent the early stage of skeletal myogenesis, but did induce muscle atrophy and a decrease in the diameter of myotubes. BIS knockdown significantly suppressed the expression level of myosin heavy chain (MyHC) without changing the expression levels of myogenic marker proteins, such as Mgn, Cav-3 and MG53. In addition, BIS endogenously interacted with MyHC, and BIS knockdown induced MyHC ubiquitination and degradation. From these data, we conclude that molecular association of MyHC and BIS is necessary for MyHC stabilization in skeletal muscle. PMID:27034027

  11. Plasma neurofilament heavy chain is not a useful biomarker in Charcot-Marie-Tooth disease.

    PubMed

    Rossor, Alexander M; Liu, Ching-Hua; Petzold, Axel; Malaspina, Andreas; Laura, Matilde; Greensmith, Linda; Reilly, Mary M

    2016-06-01

    The negative results in trials of vitamin C in Charcot-Marie-Tooth disease (CMT) type 1A have highlighted the lack of sensitive outcome measures. Neurofilaments are abundant neuronal cytoskeletal proteins, and their concentration in blood is likely to reflect axonal breakdown. We therefore examined plasma neurofilament heavy-chain (NfH) concentration as a potential biomarker in CMT. Blood samples were collected from healthy controls and patients with CMT over a 2-year period. Disease severity was measured using the CMT Examination Score. An in-house enzyme-linked immunoabsorbent assay was used to measure plasma NfH levels. There was no significant difference in plasma NfH concentrations between CMT patients and controls (P = 0.449). There was also no significant difference in plasma NfH levels in the CMT group over 1 year (mean difference = -0.02, SEM = 4.44, P = 0.98). Plasma NfH levels are not altered in patients with CMT and are not a suitable biomarker of disease activity. Muscle Nerve 53: 972-975, 2016. © 2016 Wiley Periodicals, Inc.

  12. Neuromuscular junction defects in mice with mutation of dynein heavy chain 1.

    PubMed

    Courchesne, Stephanie L; Pazyra-Murphy, Maria F; Lee, Daniel J; Segal, Rosalind A

    2011-02-04

    Disruptions in axonal transport have been implicated in a wide range of neurodegenerative diseases. Cramping 1 (Cra1/+) and Legs at odd angles (Loa/+) mice, with hypomorphic mutations in the dynein heavy chain 1 gene, which encodes the ATPase of the retrograde motor protein dynein, were originally reported to exhibit late onset motor neuron disease. Subsequent, conflicting reports suggested that sensory neuron disease without motor neuron loss underlies the phenotypes of Cra1/+ and Loa/+ mice. Here, we present behavioral and anatomical analyses of Cra1/+ mice. We demonstrate that Cra1/+ mice exhibit early onset, stable behavioral deficits, including abnormal hindlimb posturing and decreased grip strength. These deficits do not progress through 24 months of age. No significant loss of primary motor neurons or dorsal root ganglia sensory neurons was observed at ages where the mice exhibited clear symptomatology. Instead, there is a decrease in complexity of neuromuscular junctions. These results indicate that disruption of dynein function in Cra1/+ mice results in abnormal morphology of neuromuscular junctions. The time course of behavioral deficits, as well as the nature of the morphological defects in neuromuscular junctions, suggests that disruption of dynein function in Cra1/+ mice causes a developmental defect in synapse assembly or stabilization.

  13. Neuromuscular Junction Defects in Mice with Mutation of dynein heavy chain 1

    PubMed Central

    Lee, Daniel J.; Segal, Rosalind A.

    2011-01-01

    Disruptions in axonal transport have been implicated in a wide range of neurodegenerative diseases. Cramping 1 (Cra1/+) and Legs at odd angles (Loa/+) mice, with hypomorphic mutations in the dynein heavy chain 1 gene, which encodes the ATPase of the retrograde motor protein dynein, were originally reported to exhibit late onset motor neuron disease. Subsequent, conflicting reports suggested that sensory neuron disease without motor neuron loss underlies the phenotypes of Cra1/+ and Loa/+ mice. Here, we present behavioral and anatomical analyses of Cra1/+ mice. We demonstrate that Cra1/+ mice exhibit early onset, stable behavioral deficits, including abnormal hindlimb posturing and decreased grip strength. These deficits do not progress through 24 months of age. No significant loss of primary motor neurons or dorsal root ganglia sensory neurons was observed at ages where the mice exhibited clear symptomatology. Instead, there is a decrease in complexity of neuromuscular junctions. These results indicate that disruption of dynein function in Cra1/+ mice results in abnormal morphology of neuromuscular junctions. The time course of behavioral deficits, as well as the nature of the morphological defects in neuromuscular junctions, suggests that disruption of dynein function in Cra1/+ mice causes a developmental defect in synapse assembly or stabilization. PMID:21346813

  14. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies

    PubMed Central

    De Genst, Erwin; Silence, Karen; Decanniere, Klaas; Conrath, Katja; Loris, Remy; Kinne, Jörg; Muyldermans, Serge; Wyns, Lode

    2006-01-01

    Clefts on protein surfaces are avoided by antigen-combining sites of conventional antibodies, in contrast to heavy-chain antibodies (HCAbs) of camelids that seem to be attracted by enzymes’ substrate pockets. The explanation for this pronounced preference of HCAbs was investigated. Eight single domain antigen-binding fragments of HCAbs (VHH) with nanomolar affinities for lysozyme were isolated from three immunized dromedaries. Six of eight VHHs compete with small lysozyme inhibitors. This ratio of active site binders is also found within the VHH pool derived from polyclonal HCAbs purified from the serum of the immunized dromedary. The crystal structures of six VHHs in complex with lysozyme and their interaction surfaces were compared to those of conventional antibodies with the same antigen. The interface sizes of VHH and conventional antibodies to lysozyme are very similar as well as the number and chemical nature of the contacts. The main difference comes from the compact prolate shape of VHH that presents a large convex paratope, predominantly formed by the H3 loop and interacting, although with different structures, into the concave lysozyme substrate-binding pocket. Therefore, a single domain antigen-combining site has a clear structural advantage over a conventional dimeric format for targeting clefts on antigenic surfaces. PMID:16537393

  15. Differential susceptibility on myosin heavy chain isoform following eccentric-induced muscle damage.

    PubMed

    Choi, Seung Jun

    2014-12-01

    Based on myosin heavy chain (MHC) isoform, human skeletal muscle fibers can be categorized into three fiber types, type I, IIa, IIx fibers, and each fiber type has different characteristics. Typical characteristics are difference in force production, shortening velocity, and fatigue resistance. When the muscle is contract and stretched by a force that is greater than the force generated by the muscle, contraction-induced muscle damage frequently occurs. Several experimental models involving both human and animal have considered the susceptibility of different muscle fiber type and part of muscles to eccentric induced muscle damage. General consensus is a greater susceptibility of fast-twitch fiber or type II fiber to damage following eccentric contractions. However, the results from these previous efforts were not enough to conclude the susceptibility between each individual fiber types after eccentric contraction. Therefore, the purpose of this review is to explore detail limitation and interpretation of previous studies, and review the recent study that eliminated the prior limitations, such as strain magnitude, lengthening velocity, fiber type heterogeneity, and muscle architecture issue.

  16. Age dependence of myosin heavy chain transitions induced by creatine depletion in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Adams, Gregory R.; Baldwin, Kenneth M.

    1995-01-01

    This study was designed to test the hypothesis that myosin heavy chain (MHC) plasticity resulting from creatine depletion is an age-dependent process. At weaning (age 28 days), rat pups were placed on either standard rat chow (normal diet juvenile group) or the same chow supplemented with 1% wt/wt of the creatine analogue beta-guanidinopropionic acid (creatine depletion juvenile (CDJ) group). Two groups of adult rats (age approximately 8 wk) were placed on the same diet regimens (normal diet adult and creatine depletion adult (CDA) groups). After 40 days (CDJ and normal diet juvenile groups) and 60 days (CDA and normal diet adult groups), animals were killed and several skeletal muscles were removed for analysis of creatine content or MHC ditribution. In the CDJ group, creatine depletion (78%) was accompanied by significant shifts toward expression of slower MHC isoforms in two slow and three fast skeletal muscles. In contrast, creatine depletion in adult animals did not result in similar shifts toward slow MHC isoform expression in either muscle type. The results of this study indicate that there is a differential effect of creatine depletion on MHC tranitions that appears to be age dependent. These results strongly suggest that investigators contemplating experimental designs involving the use of the creatine analogue beta-guanidinopropionic acid should consider the age of the animals to be used.

  17. Myosin IIA Heavy Chain Phosphorylation Mediates Adhesion Maturation and Protrusion in Three Dimensions.

    PubMed

    Rai, Vandana; Thomas, Dustin G; Beach, Jordan R; Egelhoff, Thomas T

    2017-02-24

    Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.

  18. Electrophoretic separation of reptilian skeletal and cardiac muscle myosin heavy chain isoforms: dependence on gel format.

    PubMed

    Reiser, Peter J; Bicer, Sabahattin

    2014-09-01

    This report provides a comparison of multiple gel formats to study myosin heavy chain (MHC) isoforms that are expressed in reptilian skeletal and cardiac muscles of five turtle species, water monitor, and prehensile tailed skink. Three gel formats were tested. The results identify one format that is superior, for the overall extent of electrophoretic separation and for the assessment of the number of MHC isoforms in reptilian striated muscles. The same format was shown previously to separate MHC isoforms that are expressed in American alligator. The results also show that another gel format reveals the distinct electrophoretic mobility of MHC isoforms in atrial, ventricular, and jaw adductor samples, compared to those expressed in skeletal muscles in the limbs and elsewhere in the body. In addition, the results reveal that the electrophoretic mobility of specific MHC isoforms, relative to other isoforms, depends on the gel format, as shown previously for mammalian and avian species. The discovery of the expression of masticatory MHC, which is abundantly expressed in jaw adductors of members of Carnivora and several other vertebrate orders, in the homologous muscles of prehensile tailed skink, an herbivore, and the carnivorous water monitor, was made during the course of this study. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Regulation of neuronal ferritin heavy chain, a new player in opiate-induced chemokine dysfunction

    PubMed Central

    Abt, Anna Cook; Meucci, Olimpia

    2013-01-01

    The heavy chain subunit of ferritin (FHC), a ubiquitous protein best known for its iron-sequestering activity as part of the ferritin complex, has recently been described as a novel inhibitor of signaling through the chemokine receptor CXCR4. Levels of FHC as well as its effects on CXCR4 activation increase in cortical neurons exposed to mu-opioid receptor agonists such as morphine, an effect likely specific to neurons. Major actions of CXCR4 signaling in the mature brain include a promotion of neurogenesis, activation of pro-survival signals, and modulation of excitotoxic pathways; thus FHC up-regulation may contribute to the neuronal dysfunction often associated with opiate drug abuse. This review summarizes our knowledge of neuronal CXCR4 function, its regulation by opiates and the role of FHC in this process, and known mechanisms controlling FHC production. We speculate on the mechanism involved in FHC regulation by opiates, and offer FHC as a new target in opioid-induced neuropathology. PMID:21465240

  20. Neuronal ferritin heavy chain and drug abuse affect HIV-associated cognitive dysfunction

    PubMed Central

    Pitcher, Jonathan; Abt, Anna; Myers, Jaclyn; Han, Rachel; Snyder, Melissa; Graziano, Alessandro; Festa, Lindsay; Kutzler, Michele; Garcia, Fernando; Gao, Wen-Jun; Fischer-Smith, Tracy; Rappaport, Jay; Meucci, Olimpia

    2014-01-01

    Interaction of the chemokine CXCL12 with its receptor CXCR4 promotes neuronal function and survival during embryonic development and throughout adulthood. Previous studies indicated that μ-opioid agonists specifically elevate neuronal levels of the protein ferritin heavy chain (FHC), which negatively regulates CXCR4 signaling and affects the neuroprotective function of the CXCL12/CXCR4 axis. Here, we determined that CXCL12/CXCR4 activity increased dendritic spine density, and also examined FHC expression and CXCR4 status in opiate abusers and patients with HIV-associated neurocognitive disorders (HAND), which is typically exacerbated by illicit drug use. Drug abusers and HIV patients with HAND had increased levels of FHC, which correlated with reduced CXCR4 activation, within cortical neurons. We confirmed these findings in a nonhuman primate model of SIV infection with morphine administration. Transfection of a CXCR4-expressing human cell line with an iron-deficient FHC mutant confirmed that increased FHC expression deregulated CXCR4 signaling and that this function of FHC was independent of iron binding. Furthermore, examination of morphine-treated rodents and isolated neurons expressing FHC shRNA revealed that FHC contributed to morphine-induced dendritic spine loss. Together, these data implicate FHC-dependent deregulation of CXCL12/CXCR4 as a contributing factor to cognitive dysfunction in neuroAIDS. PMID:24401274

  1. Limited Expression of Slow Tonic Myosin Heavy Chain in Human Cranial Muscles

    PubMed Central

    Sokoloff, Alan J.; Li, Haiyan; Burkholder, Thomas J.

    2013-01-01

    Recent reports of slow tonic myosin heavy chain (MHCst) in human masticatory and laryngeal muscles suggest that MHCst may have a wider distribution in humans than previously thought. Because of the novelty of this finding, we sought to confirm the presence of MHCst in human masticatory and laryngeal muscles by reacting tissue from these muscles and controls from extraocular, intrafusal, cardiac, appendicular and developmental muscle with antibodies (Abs) ALD-58 and S46 considered highly specific for MHCst. At Ab dilutions producing minimal reaction to muscle fibers positive for MHCI, only extraocular, intrafusal and fetal tongue tissue reacted with Ab S46 had strong immunoreaction in an appreciable number of muscle fibers. In immunoblots Ab S46, but not Ab ALD-58, labeled adult extraocular muscles; no other muscles were labeled with either Ab. We conclude that, in humans, Ab S46 has greater specificity for MHCst than does Ab ALD-58. We suggest that reports of MHCst in human masticatory and laryngeal muscles reflect false-positive identification of MHCst due to cross-reactivity of Ab ALD-58 with another MHC isoform. PMID:17486578

  2. Unusual association of beta 2-microglobulin with certain class I heavy chains of the murine major histocompatibility complex.

    PubMed Central

    Bushkin, Y; Tung, J S; Pinter, A; Michaelson, J; Boyse, E A

    1986-01-01

    Class I products of the major histocompatibility complex (MHC) comprise a heavy chain of about 45 kDa noncovalently linked to a 12-kDa beta 2-microglobulin (beta 2m) light chain encoded on a different chromosome. We find that class I products of some mouse strains include an additional 62-kDa molecule which on the following evidence consists of a heavy chain linked covalently with beta 2m. Production of the 62-kDa protein invariably accorded with the occurrence of cysteine at position 121 of the heavy chain (Kb,Kbm1,Kbm3,Dd, and Ld). Substitution of arginine at position 121 invariably accorded with absence of the 62-kDa protein (Kbm6,Kbm7,Kbm9,Kd, and Db). On the basis of observed production versus nonproduction of the 62-kDa molecule, predictions are made regarding residue 121 in class I products for which this is not yet known; namely, Kk, Ks, and Dk, which produce the 62-kDa molecule, as compared with Kj, Qa-2, and TL, which do not. Reported differences in immunologic reactivity between Kb mutant strains with Arg-121 in place of Cys-121 imply that the occurrence of 62-kDa class I products in mice of Cys-121 genotype has functional consequences. Images PMID:3510435

  3. Expression and secretion of immunoglobulin alpha heavy chain with diverse VDJ recombinations by human epithelial cancer cells.

    PubMed

    Zheng, Hui; Li, Ming; Ren, Wei; Zeng, Liang; Liu, Hai-dan; Hu, Duosha; Deng, Xiyun; Tang, Min; Shi, Ying; Gong, Jianping; Cao, Ya

    2007-03-01

    Generally, only B lymphocytes express immunoglobulin. Recently, we found the expression of Ig alpha heavy chain in human epithelial cancer cells unexpectedly. We first detected Ig VDJ-Calpha and Ialpha-Calpha transcripts in multiple cancer cell lines. Further, the configuration of the Ig heavy chain genomic locus was analyzed in human cancer cells. We found that cancer cells have the recombination VDJ region, but bear Ig Salpha region in germline configuration, which is different from Ig expression pattern in B cells. And human epithelial cancers possess the essential effectors including RAG-1 and RAG-2, but not activation induced cytidine deaminase (AID) protein. These provide further proofs for Ig alpha expression. In addition, we found that human cancer cells not only express the protein of Ig alpha chain, but also secrete the protein in secretory IgA (SIgA) pattern. Importantly, diverse CDR3 recombinations were found in human cancer cells of different epithelial origin. Since IgA is the key immunoglobulin which contributes to local immunity of mucous membrane, the aberrant expression of Ig alpha heavy chain might increase our further comprehension to development and immunity of cancers.

  4. Thyroid hormone stimulates synthesis of a cardiac myosin isozyme. Comparison of the two-two-dimensional electrophoretic patterns of the cyanogen bromide peptides of cardiac myosin heavy chains from euthyroid and thyrotoxic rabbits.

    PubMed

    Flink, I L; Rader, J H; Morkin, E

    1979-04-25

    The CNBr peptides of [14C]carboxymethylated cardiac myosin heavy chains from euthyroid and thyrotoxic rabbits have been compared using a two-dimensional electrophoretic system. The results indicated that there were extensive differences in the peptide "maps" of these heavy chains, which included differences in the distribution of radiolabeled thiol peptides. Also, the patterns of heavy chain peptides from the cardiac myosins have been compared with those produced by the heavy chain myosin isozymes from skeletal muscles. Peptide maps of heavy chains from red skeletal muscle myosin closely resembled the pattern of peptides found with cardiac myosin heavy chains from euthyroid rabbits. However, peptide maps of heavy chains from white skeletal muscle myosin were dissimilar to those of the cardiac myosin isozymes. We conclude that thyroxine administration stimulates the synthesis of a cardiac myosin isozyme with a heavy chain primary structure which is different from either of the skeletal muscle myosin isozymes.

  5. Large-scale chromatin remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene regulation.

    PubMed

    Bolland, Daniel J; Wood, Andrew L; Corcoran, Anne E

    2009-01-01

    V(D)J recombination in lymphocytes is the cutting and pasting together of antigen receptor genes in cis to generate the enormous variety of coding sequences required to produce diverse antigen receptor proteins. It is the key role of the adaptive immune response, which must potentially combat millions of different foreign antigens. Most antigen receptor loci have evolved to be extremely large and contain multiple individual V, D and J genes. The immunoglobulin heavy chain (Igh) and immunoglobulin kappa light chain (Igk) loci are the largest multigene loci in the mammalian genome and V(D)J recombination is one of the most complicated genetic processes in the nucleus. The challenge for the appropriate lymphocyte is one of macro-management-to make all of the antigen receptor genes in a particular locus available for recombination at the appropriate developmental time-point. Conversely, these large loci must be kept closed in lymphocytes in which they do not normally recombine, to guard against genomic instability generated by the DNA double strand breaks inherent to the V(D)J recombination process. To manage all of these demanding criteria, V(D)J recombination is regulated at numerous levels. It is restricted to lymphocytes since the Rag genes which control the DNA double-strand break step of recombination are only expressed in these cells. Within the lymphocyte lineage, immunoglobulin recombination is restricted to B-lymphocytes and TCR recombination to T-lymphocytes by regulation of locus accessibility, which occurs at multiple levels. Accessibility of recombination signal sequences (RSSs) flanking individual V, D and J genes at the nucleosomal level is the key micro-management mechanism, which is discussed in greater detail in other chapters. This chapter will explore how the antigen receptor loci are regulated as a whole, focussing on the Igh locus as a paradigm for the mechanisms involved. Numerous recent studies have begun to unravel the complex and

  6. 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.

    PubMed

    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.

  7. Interferon-γ causes cardiac myocyte atrophy via selective degradation of myosin heavy chain in a model of chronic myocarditis.

    PubMed

    Cosper, Pippa F; Harvey, Pamela A; Leinwand, Leslie A

    2012-12-01

    Interferon-γ (IFN-γ), a proinflammatory cytokine, has been implicated in the pathogenesis of a number of forms of heart disease including myocarditis and congestive heart failure. In fact, overexpression of IFN-γ in mice causes dilated cardiomyopathy. However, the direct effects of IFN-γ on cardiac myocytes and the mechanism by which it causes cardiac dysfunction have not been described. Here, we present the molecular pathology of IFN-γ exposure and its effect on myofibrillar proteins in isolated neonatal rat ventricular myocytes. Treatment with IFN-γ caused cardiac myocyte atrophy attributable to a specific decrease in myosin heavy chain protein. This selective degradation of myosin heavy chain was not accompanied by a decrease in total protein synthesis or by an increase in total protein degradation. IFN-γ increased both proteasome and immunoproteasome activity in cardiac myocytes and their inhibition blocked myosin heavy chain loss and myocyte atrophy, whereas inhibition of the lysosome or autophagosome did not. Collectively, these results provide a mechanism by which IFN-γ causes cardiac pathology in the setting of chronic inflammatory diseases. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Interferon-γ Causes Cardiac Myocyte Atrophy via Selective Degradation of Myosin Heavy Chain in a Model of Chronic Myocarditis

    PubMed Central

    Cosper, Pippa F.; Harvey, Pamela A.; Leinwand, Leslie A.

    2013-01-01

    Interferon-γ (IFN-γ), a proinflammatory cytokine, has been implicated in the pathogenesis of a number of forms of heart disease including myocarditis and congestive heart failure. In fact, overexpression of IFN-γ in mice causes dilated cardiomyopathy. However, the direct effects of IFN-γ on cardiac myocytes and the mechanism by which it causes cardiac dysfunction have not been described. Here, we present the molecular pathology of IFN-γ exposure and its effect on myofibrillar proteins in isolated neonatal rat ventricular myocytes. Treatment with IFN-γ caused cardiac myocyte atrophy attributable to a specific decrease in myosin heavy chain protein. This selective degradation of myosin heavy chain was not accompanied by a decrease in total protein synthesis or by an increase in total protein degradation. IFN-γ increased both proteasome and immunoproteasome activity in cardiac myocytes and their inhibition blocked myosin heavy chain loss and myocyte atrophy, whereas inhibition of the lysosome or autophagosome did not. Collectively, these results provide a mechanism by which IFN-γ causes cardiac pathology in the setting of chronic inflammatory diseases. PMID:23058369

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

    PubMed

    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.

  10. Sequence of the 20-kilodalton heavy chain peptide from the carboxyl-terminus of bovine cardiac myosin subfragment-1.

    PubMed Central

    Flink, I L; Morkin, E

    1984-01-01

    An almost complete amino acid sequence of the carboxyl-terminal 20-kD tryptic heavy chain peptide from bovine cardiac myosin Subfragment-1 (S-1) has been determined by automated sequential degradation of the undigested peptide and subfragments derived by chemical and enzymatic digestion. The fragment contains 169 residues, including two reactive cysteinyl residues which are located nine residues apart. At six positions in the sequence, two amino acid residues were present and two different versions of a chymotryptic peptide were isolated in approximately 53 and 24% yields, suggesting that there are two cardiac myosin beta-type heavy chains in this species. Analysis of the secondary structure of the 20-kD peptide predicts that there are two distinct regions within the fragment. The first region (residues 1-121) contains 12% alpha-helix, 25% beta-sheet, 40% beta-bends, and 19% coil; the second region (residues 122-169) may form an extended alpha-helix. Comparison of the bovine sequence with the deduced amino acid sequence of a recombinant plasmid containing DNA sequences coding for the beta-heavy chain of rabbit cardiac myosin (pMHC beta 174) reveals approximately 86% homology. Images PMID:6746911

  11. [Cloning and expression of a single human immunoglobulin heavy-chain variable domain with vascular endothelial growth factor binding activity].

    PubMed

    Liu, Heng; Liu, Siguo; Wu, Yi; Zili, M; Liu, Yu; Zhang, Aimin; Chen, Jianquan; Cheng, Guoxiang

    2010-11-01

    In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.

  12. Myosin heavy chain 15 is associated with bovine pulmonary arterial pressure

    PubMed Central

    Neary, Joseph M.; Lund, Gretchen K.; Holt, Timothy N.; Garry, Franklyn B.; Mohun, Timothy J.; Breckenridge, Ross A.

    2014-01-01

    Abstract Bovine pulmonary hypertension, brisket disease, causes significant morbidity and mortality at elevations above 2,000 m. Mean pulmonary arterial pressure (mPAP) is moderately heritable, with inheritance estimated to lie within a few major genes. Invasive mPAP measurement is currently the only tool available to identify cattle at risk of hypoxia-induced pulmonary hypertension. A genetic test could allow selection of cattle suitable for high altitude without the need for invasive testing. In this study we evaluated three candidate genes (myosin heavy chain 15 [MYH15], NADH dehydrogenase flavoprotein 2, and FK binding protein 1A) for association with mPAP in 166 yearling Angus bulls grazing at 2,182 m. The T allele (rs29016420) of MYH15 was linked to lower mPAP in a dominant manner (CC 47.2 ± 1.6 mmHg [mean ± standard error of the mean]; CT/TT 42.8 ± 0.7 mmHg; P = 0.02). The proportions of cattle with MYH15 CC, CT, and TT genotypes were 55%, 41%, and 4%, respectively. Given the high frequency of the deleterious allele, it is likely that the relative contribution of MYH15 polymorphisms to pulmonary hypertension is small, supporting previous predictions that the disease is polygenic. We evaluated allelic frequency of MYH15 in the Himalayan yak (Bos grunniens), a closely related species adapted to high altitude, and found 100% prevalence of T allele homozygosity. In summary, we identified a polymorphism in MYH15 significantly associated with mPAP. This finding may aid selection of cattle suitable for high altitude and contribute to understanding human hypoxia-induced pulmonary hypertension. PMID:25621163

  13. Clathrin Heavy Chain Subunits Coordinate Endo- and Exocytic Traffic and Affect Stomatal Movement.

    PubMed

    Larson, Emily R; Van Zelm, Eva; Roux, Camille; Marion-Poll, Annie; Blatt, Michael R

    2017-10-01

    The current model for vesicular traffic to and from the plasma membrane is accepted, but the molecular requirements for this coordination are not well defined. We have identified the hot ABA-deficiency suppressor1 mutant, which has a stomatal function defect, as a clathrin heavy chain1 (CHC1) mutant allele and show that it has a decreased rate of endocytosis and growth defects that are shared with other chc1 mutant alleles. We used chc1 alleles and the related chc2 mutant as tools to investigate the effects that clathrin defects have on secretion pathways and plant growth. We show that secretion and endocytosis at the plasma membrane are sensitive to CHC1 and CHC2 function in seedling roots and that chc mutants have physiological defects in stomatal function and plant growth that have not been described previously. These findings suggest that clathrin supports specific functions in multiple cell types. Stomata movement and gas exchange are altered in chc mutants, indicating that clathrin is important for stomatal regulation. The aberrant function of chc mutant stomata is consistent with the growth phenotypes observed under different water and light conditions, which also are similar to those of the secretory SNARE mutant, syp121 The syp121 and chc mutants have impaired endocytosis and exocytosis compared with the wild type, indicating a link between SYP121-dependent secretion and clathrin-dependent endocytosis at the plasma membrane. Our findings provide evidence that clathrin and SYP121 functions are important for the coordination of endocytosis and exocytosis and have an impact on stomatal function, gas exchange, and vegetative growth in Arabidopsis (Arabidopsis thaliana). © 2017 The author(s). All Rights Reserved.

  14. Neurofilament heavy chain as a marker of neuroaxonal pathology and prognosis in acute encephalitis.

    PubMed

    Sellner, J; Davies, N W; Howard, R S; Petzold, A

    2014-06-01

    The neurological outcome of acute encephalitis can be devastating and early prognosis remains difficult. Biomarkers that quantify the extent of early brain injury are needed to improve the prognostic accuracy and aid patient management. Our objective was to assess whether cerebrospinal fluid (CSF) protein biomarkers of neuroaxonal and glial cell injury are elevated in distinct forms of acute encephalitis and predictive of poor outcome. This was a prospective study of patients presenting with acute encephalitis to three teaching hospitals in London, UK. Levels of neurofilament heavy chain (NfH, SMI35) and S100B were quantified in CSF using enzyme-linked immunosorbent assay. The outcome was assessed by the Glasgow Outcome Scale (GOS). Fifty-six patients with acute encephalitis were recruited and classified into the following diagnostic categories: infectious (n = 20), inflammatory (n = 14) and unknown etiology (n = 22). Pathological levels of NfH and S100B were observed in 24/56 (43%) and 54/56 (96%), respectively. Patients with infectious encephalitis had significantly higher NfH levels compared with the other two groups (P < 0.05). A poor outcome (GOS < 5) was associated with significantly higher CSF NfH levels within samples taken 2 weeks after symptom onset. This study suggests that longitudinal CSF NfH levels are of superior prognostic value compared with CSF S100B levels. Prolonged release of NfH, a marker of neuroaxonal damage, was associated with poor outcome. Potentially there is a window of opportunity for future neuroprotective treatment strategies in encephalitis. © 2014 The Author(s) European Journal of Neurology © 2014 EAN.

  15. Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.

    PubMed Central

    Bottinelli, R; Canepari, M; Pellegrino, M A; Reggiani, C

    1996-01-01

    1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force-velocity curves of sixty-seven fibres were obtained by load-clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross-sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack-test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I-IIA and IIA-IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty-one fibres in the range 12-22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol-1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12-17 degrees C range, but less dependent between 17 and 22 degrees C. Images Figure 1 Figure 3 Figure 6 PMID:8887767

  16. Regulation of naturally occurring antisense RNA of myosin heavy chain (MyHC) in neonatal cardiomyocytes.

    PubMed

    Luther, H P; Bartsch, H; Morano, I; Podlowski, S; Baumann, G

    2005-03-01

    Naturally occurring antisense RNA has been detected for a range of eukaryotic genes. Its abundance compared to levels of its complementary sense mRNA appears to be a factor indicating its possible regulatory function. In previous studies, we detected appreciable levels of antisense RNA against the two isoforms (alpha and beta) of the heavy myosin-chain (MyHC) in the myocardium of rats. If this is to play a significant role in gene expression antisense levels should vary in response to external and internal cellular influences. Recently, a bidirectional promoter located in the alpha/beta MyHC intergenic region was described, which was proposed to regulate coordinated transcription of alpha-MyHC sense and beta-MyHC antisense. To study MyHC antisense regulation in neonatal heart, we investigated cultivated myocytes stimulated with either trijodthyronin (T3) as an inductor of alpha-MyHC or phenylephrine with stimulation of beta-MyHC. RNA-quantification of sense and antisense transcripts of both isoforms was performed by real-time RT-PCR. Stimulation by T3 led to an induction of both sense and antisense of alpha-MyHC and to a decrease of beta-MyHC sense and antisense. Phenylephrine increased sense and antisense beta-MyHC but reduced antisense alpha-MyHC. The sense/antisense of alpha- and beta-MyHC ratio was unchanged compared to control. Results indicate a coregulation of sense and antisense MyHC RNA under stimulation of T3 and phenylephrine in neonatal cardiomyocytes.

  17. Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

    SciTech Connect

    Crew, Jennifer R.; Falzari, Kanakeshwari; DiMario, Joseph X.

    2010-04-01

    Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-{gamma} co-activator-1 (PGC-1{alpha}) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.

  18. Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Herrick, R. E.; Adams, G. R.; Baldwin, K. M.

    1993-01-01

    This study ascertained the effects of 9 days of zero gravity on the relative (percentage of total) and calculated absolute (mg/muscle) content of isomyosin expressed in both antigravity and locomotor skeletal muscle of ground control (CON) and flight-exposed (FL) rats. Results showed that although there were no differences in body weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) but not in the vastus lateralis (VL) or the tibialis anterior. Both total muscle protein and myofibril protein content were not different between the muscle regions examined in the FL and CON groups. In the VI, there were trends for reductions in the relative content of type I and IIa myosin heavy chains (MHCs) that were offset by increases in the relative content of both type IIb and possibly type IIx MHC protein (P > 0.05). mRNA levels were consistent with this pattern (P < 0.05). The same pattern held true for the red region of the VL as examined at both the protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute content of both type I and IIa MHCs that were offset by calculated increases in type IIb MHC in both VI and red VL. Collectively, these findings suggest that there are both absolute and relative changes occurring in MHC expression in the "red" regions of antigravity skeletal muscle during exposure to zero gravity that could affect muscle function.

  19. Analysis of Heavy-Chain Antibody Responses and Resistance to Parelaphostrongylus tenuis in Experimentally Infected Alpacas

    PubMed Central

    Purdy, S. R.; Gagliardo, L. F.; Lefman, S.; Hamel, P. J. S.; Ku, S.; Mainini, T.; Hoyt, G.; Justus, K.; Daley-Bauer, L. P.; Duffy, M. S.

    2012-01-01

    The parasitic nematode Parelaphostrongylus tenuis is an important cause of neurologic disease of camelids in central and eastern North America. The aim of this study was to determine whether alpacas develop resistance to disease caused by P. tenuis in response to a previous infection or a combination of controlled infection and immunization. Alpacas were immunized with a homogenate of third-stage larvae (L3) and simultaneously implanted subcutaneously with diffusion chambers containing 20 live L3. Sham-treated animals received adjuvant alone and empty chambers. The protocol was not effective in inducing resistance to oral challenge with 10 L3, and disease developed between 60 and 71 days following infection. Immediately following the onset of neurologic disease, affected animals were treated with a regimen of anthelmintic and anti-inflammatory drugs, and all recovered. One year later, a subset of alpacas from this experiment was challenged with 20 L3 and the results showed that prior infection induced resistance to disease. Primary and secondary infections induced production of conventional and heavy-chain IgGs that reacted with soluble antigens in L3 homogenates but did not consistently recognize a recombinant form of a parasite-derived aspartyl protease inhibitor. Thus, the latter antigen may not be a good candidate for serology-based diagnostic tests. Antibody responses to parasite antigens occurred in the absence of overt disease, demonstrating that P. tenuis infection can be subclinical in a host that has been considered to be highly susceptible to disease. The potential for immunoprophylaxis to be effective in preventing disease caused by P. tenuis was supported by evidence of resistance to reinfection. PMID:22593238

  20. Capillary supply in relation to myosin heavy chain fibre composition of human intrinsic tongue muscles.

    PubMed

    Granberg, I; Lindell, B; Eriksson, P-O; Pedrosa-Domellöf, F; Stål, P

    2010-01-01

    The capillary supply and myosin heavy chain (MyHC) composition of three different intrinsic tongue muscles was analysed in the anterior and posterior regions of the human tongue with biochemical and immunohistochemical techniques. Mean capillary density for the whole tongue was 796 ± 82 cap/mm², without regional differences. The overall number of capillaries around each fibre (CAF) was higher in the posterior than in the anterior region (2.5 vs. 2.1, p = 0.009). However, correcting for regional differences in fibre size, CAF per fibre area was higher in the anterior region (4.3 vs. 3.0, p < 0.001). Muscle fibres containing fast MyHCs predominated in the anterior region (78.7%), consisting of MyHCIIa (58.5%), MyHCIIx (1.0%), MyHCIIa+MyHCIIx (11.3%) and MyHCI+MyHCIIa (7.9%). Fibres containing slow MyHC predominated in the posterior region (65.2%), consisting of MyHCI (45.5%) and MyHCI+MyHCIIa (19.7%). A minor fibre population (<2%) contained unusual MyHC isoforms, namely MyHC foetal, MyHC slow-tonic, MyHC α-cardiac or MyHC embryonic. The microvascularization of the human tongue was twice as high as in human limb muscles. Regional similarities in capillary supply, but differences in fibre phenotype composition, suggest that human tongue muscle fibres are fatigue resistant independently of MyHC content. High frequency of hybrid fibres, that is fibres co-expressing two or more MyHC isoforms, indicates a wider spectrum of fibre contractile properties than in limb muscles. In conclusion, human intrinsic tongue muscles showed internal specialization in distribution of MyHC isoforms and capillary supply, but not in the expression of unusual MyHCs. Copyright © 2010 S. Karger AG, Basel.

  1. A highly sensitive electrochemiluminescence immunoassay for the neurofilament heavy chain protein.

    PubMed

    Kuhle, Jens; Regeniter, Axel; Leppert, David; Mehling, Matthias; Kappos, Ludwig; Lindberg, Raija L P; Petzold, Axel

    2010-03-30

    The loss of neurological function is closely related to axonal damage. Neurofilament subunits are concentrated in neurons and axons and have emerged as promising biomarkers for neurodegeneration. Electrochemiluminescence (ECL) based assays are known to be of superior sensitivity and require less sample volume than conventional ELISAs. We developed an ECL based solid-phase sandwich immunoassay to measure the neurofilament heavy chain protein (NfH(SMI35)) in CSF. We employed commercially available antibodies as previously used in a conventional ELISA (Petzold et al., 2003; Petzold and Shaw, 2007). The optimised and validated assay was applied in a reference cohort and defined patient groups. Analytical sensitivity (background plus three SD) of our assay was 2.4 pg/ml. The mean intra-assay coefficient of variation (CV) was 4.8% and the inter-assay CV 8.4%. All measured control and patient samples produced signals well above background. Patients with multiple sclerosis (MS) (median 46.2 pg/ml, n=95), amyotrophic lateral sclerosis (ALS) (160.1 pg/ml, n=50), mild cognitive impairment/Alzheimer's disease (MCI/AD) (65.6 pg/ml, n=20), Guillain-Barre syndrome (GBS) (91.0 pg/ml, n=20) or subarachnoid hemorrhage (SAH) (345.0 pg/ml, n=20) had higher CSF NfH(SMI35) values than the reference cohort (27.1 pg/ml, n=73, p<0.0001 for each comparison). The new ECL based assay for NfH(SMI35) in CSF is superior in terms of sensitivity, precision and accuracy to previously published methods (Petzold et al., 2003; Shaw et al., 2005; Teunissen et al., 2009). The improved performance and small sample volume requirement qualify this method in experimental settings and clinical trials designed to perform a number of tests on limited amounts of material. Copyright 2010 Elsevier B.V. All rights reserved.

  2. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches

    PubMed Central

    Welch, Kenneth C.

    2014-01-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25–60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. PMID:24671242

  3. Signal peptide-dependent inhibition of MHC class I heavy chain translation by rhesus cytomegalovirus.

    PubMed

    Powers, Colin J; Früh, Klaus

    2008-10-03

    The US2-11 region of human and rhesus cytomegalovirus encodes a conserved family of glycoproteins that inhibit MHC-I assembly with viral peptides, thus preventing cytotoxic T cell recognition. Since HCMV lacking US2-11 is no longer able to block assembly and transport of MHC-I, we examined whether this is also observed for RhCMV lacking the corresponding region. Unexpectedly, recombinant RhCMV lacking US2-11 was still able to inhibit MHC-I expression in infected fibroblasts, suggesting the presence of an additional MHC-I evasion mechanism. Progressive deletion analysis of RhCMV-specific genomic regions revealed that MHC-I expression is fully restored upon additional deletion of rh178. The protein encoded by this RhCMV-specific open reading frame is anchored in the endoplasmic reticulum membrane. In the presence of rh178, RhCMV prevented MHC-I heavy chain (HC) expression, but did not inhibit mRNA transcription or association of HC mRNA with translating ribosomes. Proteasome inhibitors stabilized a HC degradation intermediate in the absence of rh178, but not in its presence, suggesting that rh178 prevents completion of HC translation. This interference was signal sequence-dependent since replacing the signal peptide with that of CD4 or murine HC rendered human HCs resistant to rh178. We have identified an inhibitor of antigen presentation encoded by rhesus cytomegalovirus unique in both its lack of homology to any other known protein and in its mechanism of action. By preventing signal sequence-dependent HC translocation, rh178 acts prior to US2, US3 and US11 which attack MHC-I proteins after protein synthesis is completed. Rh178 is the first viral protein known to interfere at this step of the MHC-I pathway, thus taking advantage of the conserved nature of HC leader peptides, and represents a new mechanism of translational interference.

  4. Human heavy-chain variable region gene family nonrandomly rearranged in familial chronic lymphocytic leukemia

    SciTech Connect

    Shen, A.; Humphries, C.; Tucker, P.; Blattner, F.

    1987-12-01

    The authors have identified a family of human immunoglobulin heavy-chain variable-region (V/sub H/) genes, one member of which is rearranged in two affected members of a family in which the father and four of five siblings developed chronic lymphocytic leukemia. Cloning and sequencing of the rearranged V/sub H/ genes from leukemic lymphocytes of three affected siblings showed that two siblings had rearranged V/sub H/ genes (V/sub H/TS1 and V/sub H/WS1) that were 90% homologous. The corresponding germ-line gene, V/sub H/251, was found to part of a small (four gene) V/sub H/ gene family, which they term V/sub H/V. The DNA sequence homology to V/sub H/WS1 (95%) and V/sub H/TS1 (88%) and identical restriction sites on the 5' side of V/sub H/ confirm that rearrangement of V/sub H/251 followed by somatic mutation produced the identical V/sub H/ gene rearrangements in the two siblings. V/sub H/TS1 is not a functional V/sub H/ gene; a functional V/sub H/ rearrangement was found on the other chromosome of this patient. The other two siblings had different V/sub H/ gene rearrangements. All used different diversity genes. Mechanisms proposed for nonrandom selection of a single V/sub H/ gene include developmental regulation of this V/sub H/ gene rearrangement or selection of a subpopulation of B cells in which this V/sub H/ has been rearranged.

  5. Characterization of unique heavy chain fibroin filaments spun underwater by the caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae).

    PubMed

    Wang, Yujun; Sanai, Kazumi; Wen, Hongxiu; Zhao, Tianfu; Nakagaki, Masao

    2010-07-01

    The silks of both Lepidoptera and its sister order Trichoptera contain a homologue of heavy chain (H-fibroin), which is assumed to determine the physical properties of the fiber, such as elasticity and toughness. The long repetitive region of the H-fibroin caddisfly Stenopsyche marmorata shows a conspicuous hierarchical structure that is composed of huge units, which are mainly constructed from four large blocks (SA, SB, SC and SD) arranged in an orderly fashion. Each block contains short, distinct motifs such as SXSXSX(SX), GPXG(X)(1-3) or triplet GGX, which also occur in lepidopteran and spider filaments. The SA, SB and SC blocks have nearly fixed amino acid numbers, while the length of the SD block varies, usually due to a variable number of GPXGXXX repeats. The multiple sandwich structure that occurs in the SB block is assumed to be unique to the caddisfly and may be related to the use of silk in an aqueous environment. The overall average of hydrophilicity in the repetitive H-fibroin region of S. marmorata is -0.609, whereas hydrophobicity prevails in most lepidopteran H-fibroins. Gly (29.51%), Pro (11.28%) and Ser (10.90%) are the three predominant amino acids of H-fibroin, and the high content of essential amino acids reflects the energy-rich food resources of the caddisfly. The H-fibroin of S. marmorata is about 400-500 kDa and expressed in both the middle and posterior silk glands, which is different from the expression pattern in Lepidoptera species.

  6. Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients.

    PubMed

    Gresle, M M; Liu, Y; Dagley, L F; Haartsen, J; Pearson, F; Purcell, A W; Laverick, L; Petzold, A; Lucas, R M; Van der Walt, A; Prime, H; Morris, D R; Taylor, B V; Shaw, G; Butzkueven, H

    2014-11-01

    We evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS). Serum pNF-H concentrations were measured by ELISA in cases with relapsing-remitting (RR)-MS (n=81), secondary progressive (SP) MS (n=13) and primary progressive (PP)-MS; n=6) MS; first demyelinating event (FDE; n=82); and unaffected controls (n=135). A subset of MS cases (n=45) were re-sampled on one or multiple occasions. The Multiple Sclerosis Severity Score (MSSS) and MRI measures were used to evaluate associations between serum pNF-H status, disease severity and cerebral lesion load and activity. We confirmed the presence of pNF-H peptides in serum by ELISA. We showed that a high serum pNF-H titre was detectable in 9% of RR-MS and FDE cases, and 38.5% of SP-MS cases. Patients with a high serum pNF-H titre had higher average MSSS scores and T2 lesion volumes than patients with a low serum pNF-H titre. Repeated sampling of a subset of MS cases showed that pNF-H levels can fluctuate over time, likely reflecting temporal dynamics of axonal injury in MS. A subset of FDE/MS cases was found to have a high serum pNF-H titre, and this was associated with changes in clinical outcome measures. We propose that routine measurement of serum pNF-H should be further investigated for monitoring axonal injury in MS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  7. Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth

    PubMed Central

    Štrbenc, Malan; Smerdu, Vika; Pogačnik, Azra; Fazarinc, Gregor

    2006-01-01

    To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and α-glycerophosphate dehydrogenase (α-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4–6 weeks of age, which coincides with the increased physical activity of puppies. PMID:16879596

  8. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

    PubMed

    Velten, Brandy P; Welch, Kenneth C

    2014-06-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. Copyright © 2014 the American Physiological Society.

  9. Effect of Fetal Hypothyroidism on Cardiac Myosin Heavy Chain Expression in Male Rats

    PubMed Central

    Yousefzadeh, Nasibeh; Jeddi, Sajad; Alipour, Mohammad Reza

    2016-01-01

    Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05) were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, β-MHC expression was higher (201%) and α- MHC expression was lower (47%) than control. Conclusion: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of β-MHC to α- MHC ratio in the heart. PMID:27411095

  10. Myosin Heavy Chain Expression Can Vary over the Length of Jaw and Leg Muscles

    PubMed Central

    Korfage, J.A.M.; Kwee, K.E.; Everts, V.; Langenbach, G.E.J.

    2016-01-01

    Muscle fiber type classification can be determined by its myosin heavy chain (MyHC) composition based on a few consecutive sections. It is generally assumed that the MyHC expression of a muscle fiber is the same over its length since neural stimulation and systemic influences are supposed to be the same over its length. We analyzed this in detail in three muscle types: the temporalis (closer) and digastricus (opener; both first brachial arch), and the medial gastrocnemius (somite). Sections of the muscles were incubated with monoclonal antibodies against various MyHC isoforms, and the distribution of these isoforms within individual fibers was followed over a distance of approximately 1 mm. The staining intensity of a fiber was measured and compared with the other fibers in the section. In the temporalis, digastricus, and gastrocnemius, 46, 11, and 15%, respectively, of their MyHC-I fibers showed a variation in the staining intensity over the length of their fibers, as well as 47, 87, and 22%, respectively, of their MyHC-IIA fibers. Most variable fibers were found amongst those with an overall relative intermediate staining intensity, which are presumably hybrid fibers. We conclude that different parts of a muscle fiber can have different fiber type compositions and, thus, contractile properties. Some muscle parts might reach their maximum contraction peak sooner or later than a muscle part a few microns further away. Next to stimulation by the nerve and systemic influences, local influences might also have an impact on the MyHC expression of the fiber. PMID:26950765

  11. Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Herrick, R. E.; Adams, G. R.; Baldwin, K. M.

    1993-01-01

    This study ascertained the effects of 9 days of zero gravity on the relative (percentage of total) and calculated absolute (mg/muscle) content of isomyosin expressed in both antigravity and locomotor skeletal muscle of ground control (CON) and flight-exposed (FL) rats. Results showed that although there were no differences in body weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) but not in the vastus lateralis (VL) or the tibialis anterior. Both total muscle protein and myofibril protein content were not different between the muscle regions examined in the FL and CON groups. In the VI, there were trends for reductions in the relative content of type I and IIa myosin heavy chains (MHCs) that were offset by increases in the relative content of both type IIb and possibly type IIx MHC protein (P > 0.05). mRNA levels were consistent with this pattern (P < 0.05). The same pattern held true for the red region of the VL as examined at both the protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute content of both type I and IIa MHCs that were offset by calculated increases in type IIb MHC in both VI and red VL. Collectively, these findings suggest that there are both absolute and relative changes occurring in MHC expression in the "red" regions of antigravity skeletal muscle during exposure to zero gravity that could affect muscle function.

  12. Distinct functions for the glycans of tapasin and heavy chains in the assembly of MHC class I molecules.

    PubMed

    Rizvi, Syed Monem; Del Cid, Natasha; Lybarger, Lonnie; Raghavan, Malini

    2011-02-15

    Complexes of specific assembly factors and generic endoplasmic reticulum (ER) chaperones, collectively called the MHC class I peptide-loading complex (PLC), function in the folding and assembly of MHC class I molecules. The glycan-binding chaperone calreticulin (CRT) and partner oxidoreductase ERp57 are important in MHC class I assembly, but the sequence of assembly events and specific interactions involved remain incompletely understood. We show that the recruitments of CRT and ERp57 to the PLC are codependent and also dependent upon the ERp57 binding site and the glycan of the assembly factor tapasin. Furthermore, the ERp57 binding site and the glycan of tapasin enhance β(2)m and MHC class I heavy (H) chain recruitment to the PLC, with the ERp57 binding site having the dominant effect. In contrast, the conserved MHC class I H chain glycan played a minor role in CRT recruitment into the PLC, but impacted the recruitment of H chains into the PLC, and glycan-deficient H chains were impaired for tapasin-independent and tapasin-assisted assembly. The conserved MHC class I glycan and tapasin facilitated an early step in the assembly of H chain-β(2)m heterodimers, for which tapasin-ERp57 or tapasin-CRT complexes were not required. Together, these studies provide insights into how PLCs are constructed, demonstrate two distinct mechanisms by which PLCs can be stabilized, and suggest the presence of intermediate H chain-deficient PLCs.

  13. Human major histocompatibility complex class I antigens: residues 61-83 of the HLA-B7 heavy chain specify an alloreactive site.

    PubMed Central

    Walker, L E; Ketler, T A; Houghten, R A; Schulz, G; Chersi, A; Reisfeld, R A

    1985-01-01

    A chemically synthesized peptide (sequence in text) homologous to residues 61-83 of the HLA-B7 heavy chain, induced antibodies that specifically recognized the HLA heavy chain-beta 2-microglobulin complex and the free heavy chain of the HLA-B7 antigen. These antibodies specifically immunoprecipitated the HLA-B7 beta 2-microglobulin complex solubilized from human lymphoblastoid cells by nonionic detergents and reacted with free HLA-B7 heavy chains in blots on nitrocellulose. These observations suggest that the antigenic conformation of this region of the HLA-B7 molecule is independent of the presence of beta 2-microglobulin and that amino acid residues 61-83 mimic an alloreactive site expressed by the HLA-B7 antigen. Images PMID:3881768

  14. Homology of the NH2-terminal amino acid sequences of the heavy and light chains of human monoclonal lupus autoantibodies containing the dominant 16/6 idiotype.

    PubMed Central

    Atkinson, P M; Lampman, G W; Furie, B C; Naparstek, Y; Schwartz, R S; Stollar, B D; Furie, B

    1985-01-01

    The NH2-terminal amino acid sequences have been determined by automated Edman degradation for the heavy and light chains of five monoclonal IgM anti-DNA autoantibodies that were produced by human-human hybridomas derived from lymphocytes of two patients with systemic lupus erythematosus. Four of the antibodies were closely related to the idiotype system 16/6, whereas the fifth antibody was unrelated idiotypically. The light chains of the 16/6 idiotype-positive autoantibodies (HF2-1/13b, HF2-1/17, HF2-18/2, and HF3-16/6) had identical amino acid sequences from residues 1 to 40. Their framework structures were characteristic of VKI light chains. The light chain of the 16/6 idiotype-negative autoantibody HF6-21/28 was characteristic of the VKII subgroup. The heavy chains of the 16/6 idiotype-positive autoantibodies had nearly identical amino acid sequences from residues 1 to 40. The framework structures were characteristic of the VHIII subgroup. In contrast, the GM4672 fusion partner of the hybridoma produced small quantities of an IgG with a VHI heavy chain and a VKI light chain. The heavy chains of the lupus autoantibodies and the light chains of those autoantibodies that were idiotypically related to the 16/6 system had marked sequence homology with WEA, a Waldenstrom IgM that binds to Klebsiella polysaccharides and expresses the 16/6 idiotype. These results indicate a striking homology in the amino termini of the heavy and light chains of the lupus autoantibodies studied and suggest that the V regions of the heavy and light chains of the 16/6 idiotype-positive DNA-binding lupus auto-antibodies are each encoded by a single germ line gene. PMID:3921567

  15. Translocation of peptides through microsomal membranes is a rapid process and promotes assembly of HLA-B27 heavy chain and beta 2- microglobulin translated in vitro

    PubMed Central

    1991-01-01

    We have translated major histocompatibility complex (MHC) class I heavy chains and human beta 2-microglobulin in vitro in the presence of microsomal membranes and a peptide from the nucleoprotein of influenza A. This peptide stimulates assembly of HLA-B27 heavy chain and beta 2- microglobulin about fivefold. By modifying this peptide to contain biotin at its amino terminus, we could precipitate HLA-B27 heavy chains with immobilized streptavidin, thereby directly demonstrating class I heavy chain-peptide association under close to physiological conditions. The biotin-modified peptide stimulates assembly to the same extent as the unmodified peptide. Both peptides bind to the same site on the HLA-B27 molecule. Immediately after synthesis of the HLA-B27 heavy chain has been completed, it assembles with beta 2-microglobulin and peptide. These interactions occur in the lumen of the microsomes (endoplasmic reticulum), demonstrating that the peptide must cross the microsomal membrane in order to promote assembly. The transfer of peptide across the microsomal membrane is a rapid process, as peptide binding to heavy chain-beta 2-microglobulin complexes is observed in less than 1 min after addition of peptide. By using microsomes deficient of beta 2-microglobulin (from Daudi cells), we find a strict requirement of beta 2-microglobulin for detection of peptide interaction with the MHC class I heavy chain. Furthermore, we show that heavy chain interaction with beta 2-microglobulin is likely to precede peptide binding. Biotin-modified peptides are likely to become a valuable tool in studying MHC antigen interaction and assembly. PMID:1955465

  16. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  17. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  18. Phosphorylated neurofilament heavy chain is a marker of neurodegeneration in Leber hereditary optic neuropathy (LHON)

    PubMed Central

    Shaw, Gerry; Ross-Cisneros, Fred N.; Quiros, Peter; Salomao, Solange R.; Berezovsky, Adriana; Carelli, Valerio; Feuer, William J.; Sadun, Alfredo A.

    2008-01-01

    Purpose To determine the profile of neurodegeneration in Leber hereditary optic neuropathy (LHON). Methods We quantitated serum levels of phosphorylated neurofilament heavy chain (pNF-H) in a Brazilian pedigree of 16 affected patients and 59 carriers with LHON, both molecularly characterized as harboring the G to A mutation at nucleotide 11,778 of the mitochondrial genome. The association of subject characteristics to pNF-H levels was studied with multiple regression; pNF-H data were square-root transformed to effect normality of distribution of residuals. Relationships between the square-root of pNF-H and age and sex were investigated within groups with Pearson correlation and the two-sample t-test. Linear regression was used to assess the difference between groups and to determine if the relationship of age was different between affected individuals and carriers. Results of plotting pNF-H levels by age suggested a nonlinear, quadratic association so age squared was used in the statistical analysis. ANCOVA was used to assess the influence of age and group on pNF-H levels. Results In the carrier group, there was a significant correlation of square-root pNF-H (mean=0.24 ng/ml2) with age (r=0.30, p=0.022) and a stronger correlation with quadratic age (r=0.37, p=0.003). With a higher mean pNF-H (0.33 ng/ml2) for the affected group, correlations were of similar magnitude, although they were not statistically significant: age (r=0.22, p=0.42), quadratic age (r=0.22, p=0.45). There was no correlation between age and pNF-H levels (mean=0.34 ng/ml2) in the off-pedigree group: age (r=0.03, p=0.87), quadratic age (r=0.04, p=0.84). There was no difference between sexes and pNF-H levels in any of the groups (affected, p=0.65; carriers, p=0.19; off-pedigree, p=0.93). Conclusions Elevated pNF-H released into the serum of some affected LHON patients may suggest that axonal degeneration occurs at some point after loss of visual function. Increases in pNF-H levels of carriers with

  19. Redox reactivity of animal apoferritins and apoheteropolymers assembled from recombinant heavy and light human chain ferritins.

    PubMed

    Johnson, J L; Norcross, D C; Arosio, P; Frankel, R B; Watt, G D

    1999-03-30

    The redox reactivities of air-oxidized apo horse spleen ferritin (HoSF) and apo rat liver ferritin (RaF) were examined by microcoulometry and reductive optical titrations. Microcoulometry on several independent lots of commercial HoSF revealed two distinct types of redox activity: one requiring 3-4 electrons and one requiring 6-7 electrons for full reduction of the protein shell. ApoRaF required 8-9 electrons to fully reduce the oxidized form. Reductive optical titrations confirmed the microcoulometric reduction stoichiometry and, in addition, showed that the spectra of both oxidized and reduced apoHoSF were distinct and possessed absorbances tailing into the visible region. The redox reactivity of both apoRaF and apoHoSF correlated with their H-subunit composition. Identical microcoulometric and optical experiments were conducted with recombinant apo human liver heavy (rHuHF) and light (rHuLF) ferritins, but neither was redox-active. These results suggest that the redox reactivity of native ferritins is due to their heteropolymeric nature. This was confirmed by mixing various proportions of rHuHF and rHuLF, dissociating the 24-mers into individual subunits with guanidine hydrochloride at pH 3.5, and renaturing to form heteropolymeric 24-mers. Microcoulometric measurements of these apoheteropolymers reassembled in vitro showed that they were redox-active like their native apoheteropolymer counterparts. The redox activity of these apoheteropolymers increased with H-subunit composition, reached a maximum near 12 H- and 12 L-subunits, and then declined to zero with increasing L-subunit composition. The decline in redox reactivity at high L-subunit concentrations indicates that both H- and L-subunits are involved in forming the observed redox centers. Apoheteropolymers formed from rHuLF and W93F (an H-chain mutant) were redox-inactive, suggesting that the conserved tryptophan is necessary for redox center formation.

  20. Sarcomere length dependence of rat skinned cardiac myocyte mechanical properties: dependence on myosin heavy chain

    PubMed Central

    Korte, F Steven; McDonald, Kerry S

    2007-01-01

    The effects of sarcomere length (SL) on sarcomeric loaded shortening velocity, power output and rates of force development were examined in rat skinned cardiac myocytes that contained either α-myosin heavy chain (α-MyHC) or β-MyHC at 12 ± 1°C. When SL was decreased from 2.3 μm to 2.0 μm submaximal isometric force decreased ∼40% in both α-MyHC and β-MyHC myocytes while peak absolute power output decreased 55% in α-MyHC myocytes and 70% in β-MyHC myocytes. After normalization for the fall in force, peak power output decreased about twice as much in β-MyHC as in α-MyHC myocytes (41%versus 20%). To determine whether the fall in normalized power was due to the lower force levels, [Ca2+] was increased at short SL to match force at long SL. Surprisingly, this led to a 32% greater peak normalized power output at short SL compared to long SL in α-MyHC myocytes, whereas in β-MyHC myocytes peak normalized power output remained depressed at short SL. The role that interfilament spacing plays in determining SL dependence of power was tested by myocyte compression at short SL. Addition of 2% dextran at short SL decreased myocyte width and increased force to levels obtained at long SL, and increased peak normalized power output to values greater than at long SL in both α-MyHC and β-MyHC myocytes. The rate constant of force development (ktr) was also measured and was not different between long and short SL at the same [Ca2+] in α-MyHC myocytes but was greater at short SL in β-MyHC myocytes. At short SL with matched force by either dextran or [Ca2+], ktr was greater than at long SL in both α-MyHC and β-MyHC myocytes. Overall, these results are consistent with the idea that an intrinsic length component increases loaded crossbridge cycling rates at short SL and β-MyHC myocytes exhibit a greater sarcomere length dependence of power output. PMID:17347271

  1. Distinct functions for the glycans of tapasin and heavy chains in the assembly of major histocompatibility complex (MHC) class I molecules

    PubMed Central

    Rizvi, Syed Monem; Cid, Natasha Del; Lybarger, Lonnie; Raghavan, Malini

    2011-01-01

    Complexes of specific assembly factors and generic endoplasmic reticulum (ER) chaperones collectively called the major histocompatibility complex (MHC) class I peptide loading complex (PLC) function in the folding and assembly of MHC class I molecules. The glycan binding chaperone calreticulin and partner oxidoreductase ERp57 are important in MHC class I assembly, but the sequence of assembly events and specific interactions involved remain incompletely understood. We show that the recruitments of calreticulin and ERp57 to the PLC are co-dependent and also dependent upon the ERp57 binding site and the glycan of the assembly factor tapasin. Furthermore, the ERp57 binding site and the glycan of tapasin enhance β2m and MHC class I heavy chain recruitment to the PLC, with the ERp57 binding site having the dominant effect. On the other hand, the conserved MHC class I heavy chain glycan played a minor role in calreticulin recruitment into the PLC, but impacted the recruitment of heavy chains into the PLC, and glycan-deficient heavy chains were impaired for tapasin-independent and tapasin-assisted assembly. The conserved MHC class I glycan and tapasin facilitated an early step in the assembly of heavy chain-β2m heterodimers, for which tapasin-ERp57 or tapasin-calreticulin complexes were not required. Together, these studies provide insights into how PLCs are constructed, demonstrate two distinct mechanisms by which PLCs can be stabilized, and suggest the presence of intermediate heavy chain-deficient PLCs. PMID:21263072

  2. Abnormal heavy/light chain ratio after treatment is associated with shorter survival in patients with IgA myeloma.

    PubMed

    Suehara, Yasuhito; Takamatsu, Hiroyuki; Fukumoto, Kota; Fujisawa, Manabu; Narita, Kentaro; Usui, Yoshiaki; Takeuchi, Masami; Endean, Kelly; Matsue, Kosei

    2017-02-01

    Immunoglobulin (Ig) heavy/light chain (HLC) assays enable the separate quantification of the different light chain types of each Ig class. We retrospectively analyzed the correlation of heavy/light chain ratio (HLCR) with clinical status and its impact on outcome in 120 patients with multiple myeloma (MM). Abnormal HLCR was seen more frequently in patients with poorer myeloma response, and it appeared to be more sensitive for detecting clonality in IgA myeloma compared to IgG myeloma after treatment. Among the 85 patients who achieved ≥VGPR, the patients remained HLCR abnormal were showed significantly shorter overall survival (OS) compared to those achieving a normal HLCR (not reached vs 55.5 months, P = 0.032). This correlation was seen in IgA myeloma patients (not reached vs 30.1 months, P = 0.014), but not in IgG myeloma patients when patients were analyzed separately. Univariate and multivariate analysis of factors that may affect survival identified abnormal HLCR at the best response as the only independent risk factor (hazard ratio, 4.7; 95% confidence interval, 1.4 - 15.26; P = 0.012) for shorter OS in this subset of patients. This study highlighted the HLC assay as a prognostic predictor in patients with IgA myeloma.

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

    PubMed Central

    Shaffer, Justin F.; Kier, William M.

    2015-01-01

    The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities. PMID:26997860

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

    PubMed

    Shaffer, Justin F; Kier, William M

    2016-03-01

    The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities.

  5. Expression and production of llama variable heavy-chain antibody fragments (V(HH)s) by Aspergillus awamori.

    PubMed

    Joosten, Vivi; Gouka, Robin J; van den Hondel, Cees A M J J; Verrips, C Theo; Lokman, B Christien

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (V(HH)s) by Aspergillus awamori. Fragments encoding V(HH)s were cloned in a suitable Aspergillus expression vector and transformants secreting V(HH) fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V(HH)s were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted V(HH)s were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.

  6. Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin.

    PubMed

    Yamashita, Makiko; Harada, Gakuro; Matsumoto, Shin-ei; Aiba, Yoshihiro; Ichikawa, Akira; Fujiki, Tsukasa; Udono, Miyako; Kabayama, Shigeru; Yoshida, Tadashi; Zhang, Pingbo; Fujii, Hiroshi; Shirahata, Sanetaka; Katakura, Yoshinori

    2014-02-01

    In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14(+) monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14(+) monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.

  7. A unique description of stage IV extranodal marginal zone lymphoma (EMZL) in an adolescent associated with gamma heavy chain disease.

    PubMed

    Mittal, Nupur; Zhu, Bing; Gaitonde, Sujata; Lu, Yang; Schmidt, Mary Lou

    2015-05-01

    Extranodal Marginal zone lymphoma (EMZL) is a rare, usually localized disease in children. Advanced stage EMZL in adults is considered incurable, with prolonged remissions after chemotherapy. Gamma heavy chain disease (γHCD) is a rare disease of adults associated with lympho-proliferative processes with no comparable reports in children. A case of stage-IV EMZL with γHCD in an adolescent is discussed including treatment with Bendamustine plus Rituximab. The patient remains disease free 18 months from diagnosis. This case highlights necessity for careful diagnostic work-up to identify indolent lymphomas in children which may respond to less toxic chemotherapy than used for common pediatric lymphomas.

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2005-03-01

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

  10. High rate of somatic point mutation in vitro in and near the variable-region segment of an immunoglobulin heavy chain gene.

    PubMed Central

    Meyer, J; Jäck, H M; Ellis, N; Wabl, M

    1986-01-01

    The "silent" allele at the immunoglobulin heavy-chain locus in the pre-B-lymphocyte line 18-81 contains a correctly assembled gene. However, an amber termination codon within the variable-region gene segment prematurely terminates translation into complete heavy chain. Revertants that do produce heavy chain are generated at a high rate, which is termed hypermutation. By DNA sequencing of subclones, we have confirmed that whenever mu chain is produced by the usually silent allele, a true reversion is found in the DNA. Mutations are not confined to the position of the amber termination codon but are also found at other sites in and near the variable-region gene segment. Images PMID:3092221

  11. Calcium-mediated regulation of recombinant hybrids of full-length Physarum myosin heavy chain with Physarum/scallop myosin light chains

    PubMed Central

    Zhang, Ying; Kawamichi, Hozumi; Kohama, Kazuhiro; Nakamura, Akio

    2016-01-01

    Physarum myosin is a Ca2+-binding protein and its activity is inhibited by Ca2+. In the present study, to clarify the light chains (LCs) from the different species (Physarum and scallop) and to determine the specific Ca2+-regulated effects, we constructed hybrid myosins with a Physarum myosin heavy chain (Ph·HC) and Physarum and/or scallop myosin LCs, and examined Ca2+-mediated regulation of ATPases and motor activities. In these experiments, it was found that Ca2+ inhibited motilities and ATPase activities of Physarum hybrid myosin with scallop regulatory light chain (ScRLC) and Physarum essential light chain (PhELC) but could not inhibit those of the Physarum hybrid myosin mutant Ph·HC/ScRLC/PhELC-3A which lacks Ca2+-binding ability, indicating that PhELC plays a critical role in Ca2+-mediated regulation of Physarum myosin. Furthermore, the effects of Ca2+ on ATPase activities of Physarum myosin constructs are in the following order: Ph·HC/PhRLC/PhELC > Ph·HC/ScRLC/PhELC > Ph·HC/PhRLC/ScELC > Ph·HC/ScRLC/ScELC, suggesting that the presence of PhRLC and PhELC leads to the greatest Ca2+ sensitivity of Physarum myosin. Although we did not observe the motilities of Physarum hybrid myosin Ph·HC/PhRLC/ScELC and Ph·HC/ScRLC/ScELC, our results suggest that Ca2+-binding to the PhELC may alter the flexibility of the regulatory domain and induce a ’closed’ state, which may consequently prevent full activity and force generation. PMID:27125976

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

    PubMed

    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.

  13. Heavy-light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation.

    PubMed

    Lomakin, Yakov A; Zakharova, Maria Yu; Stepanov, Alexey V; Dronina, Maria A; Smirnov, Ivan V; Bobik, Tatyana V; Pyrkov, Andrey Yu; Tikunova, Nina V; Sharanova, Svetlana N; Boitsov, Vitali M; Vyazmin, Sergey Yu; Kabilov, Marsel R; Tupikin, Alexey E; Krasnov, Alexey N; Bykova, Nadezda A; Medvedeva, Yulia A; Fridman, Marina V; Favorov, Alexander V; Ponomarenko, Natalia A; Dubina, Michael V; Boyko, Alexey N; Vlassov, Valentin V; Belogurov, Alexey A; Gabibov, Alexander G

    2014-12-01

    The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.

  14. Neuregulin induces the expression of transcription factors and myosin heavy chains typical of muscle spindles in cultured human muscle.

    PubMed

    Jacobson, Christian; Duggan, David; Fischbach, Gerald

    2004-08-17

    Neuregulin (NRG) (also known as ARIA, GGF, and other names) is a heparin sulfate proteoglycan secreted into the neuromuscular junction by innervating motor and sensory neurons. An integral part of synapse formation, we have analyzed NRG-induced changes in gene expression over 48 h in primary human myotubes. We show that in addition to increasing the expression of acetylcholine receptors on the myotube surface, NRG treatment results in a transient increase of several members of the early growth response (Egr) family of transcription factors. Three Egrs, Egr1, -2, and -3, are induced within the first hour of NRG treatment, with Egr1 and -3 RNA levels showing the most significant increases of approximately 9- and 16-fold, respectively. Also noted was a corresponding increase in protein levels for both of these transcription factors. Previous literature indicates that Egr3 expression is required for the formation of muscle spindle fibers, sensory organs that are distinct from skeletal muscle contractile fibers. At the molecular level, muscle spindle fibers express a unique subset of myosin heavy chains. Two isoforms of the myosin heavy chain, the slow development and neonatal, were found to be increased in our myotube cultures after 48 h of treatment with NRG. Taken together, these results indicate that not only can NRG induce the expression of a transcription factor key to spindle fiber development (Egr3), but that a portion of this developmental process can be replicated in vitro.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    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.

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

    PubMed

    Shaffer, Justin F; Kier, William M

    2012-01-15

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

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

    PubMed Central

    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. PMID:26284622

  19. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

    PubMed

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-10-01

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

  4. Interaction between T cell receptor beta chain and immunoglobulin heavy chain region genes in susceptibility to insulin-dependent diabetes mellitus.

    PubMed

    Field, L L; Stephure, D K; McArthur, R G

    1991-09-01

    Despite some reports of an association between insulin-dependent diabetes mellitus (IDDM) and a BglII RFLP in the T cell receptor beta chain (TCRB) constant region, results of several recent studies, including our own, have failed to support such an association. However, we here report evidence for an IDDM-TCRB relationship which is dependent on immunoglobulin heavy-chain-region genes. We analyzed 198 unrelated diabetics and 84 normal siblings (maximum one sibling per diabetic) typed for the BglII TCRB RFLP and Gm immunoglobulin allotypes Glm(1), Glm(2), G2m(23), and G3m(5), which identify the four common Gm haplotypes. The BglIII TCRB genotype frequencies were significantly different between diabetics positive and negative for G2m(23) (P = .017) and G3m(5) (P = .021) but were not different between normal siblings positive and negative for those allotypes (P = .94 and P = .77, respectively). Thus, there were significant interactions between TCRB, Gm, and IDDM for two of the four immunoglobulin allotypes examined. We have previously reported interactions between HLA, Gm (particularly G2m(23)), and IDDM and postulate that the TCRB-Gm-IDDM and HLA-Gm-IDDM interaction effects may be functionally related.

  5. Caffeine Positively Modulates Ferritin Heavy Chain Expression in H460 Cells: Effects on Cell Proliferation.

    PubMed

    Zolea, Fabiana; Biamonte, Flavia; Battaglia, Anna Martina; Faniello, Maria Concetta; Cuda, Giovanni; Costanzo, Francesco

    Both the methylxanthine caffeine and the heavy subunit of ferritin molecule (FHC) are able to control the proliferation rate of several cancer cell lines. While caffeine acts exclusively as a negative modulator of cell proliferation, FHC might reduce or enhance cell viability depending upon the different cell type. In this work we have demonstrated that physiological concentrations of caffeine reduce the proliferation rate of H460 cells: along with the modulation of p53, pAKT and Cyclin D1, caffeine also determines a significant FHC up-regulation through the activation of its transcriptional efficiency. FHC plays a central role in the molecular pathways modulated by caffeine, ending in a reduced cell growth, since its specific silencing by siRNA almost completely abolishes caffeine effects on H460 cell proliferation. These results allow the inclusion of ferritin heavy subunits among the multiple molecular targets of caffeine and open the way for studying the relationship between caffeine and intracellular iron metabolism.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    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.

  8. Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain.

    PubMed

    Bernstein, S I; Hansen, C J; Becker, K D; Wassenberg, D R; Roche, E S; Donady, J J; Emerson, C P

    1986-07-01

    Genomic and cDNA sequencing studies show that transcripts from the muscle myosin heavy-chain (MHC) gene of Drosophila melanogaster are alternatively spliced, producing RNAs that encode at least two MHC isoforms with different C termini. Transcripts encoding an MHC isoform with 27 unique C-terminal amino acids accumulate during both larval and adult muscle differentiation. Transcripts for the second isoform encode one unique C-terminal amino acid and accumulate almost exclusively in pupal and adult thoracic segments, the location of the indirect flight muscles. The 3' splice acceptor site preceding the thorax-specific exon is unusually purine rich and thus may serve as a thorax-specific splicing signal. We suggest that the alternative C termini of these two MHC isoforms control myofilament assembly and may play a role in generating the distinctive myofilament organizations of flight muscle and other muscle types.

  9. Repeat organization and epigenetic regulation of the DH-Cmu domain of the immunoglobulin heavy-chain gene locus.

    PubMed

    Chakraborty, Tirtha; Chowdhury, Dipanjan; Keyes, Amanda; Jani, Anant; Subrahmanyam, Ramesh; Ivanova, Irina; Sen, Ranjan

    2007-09-07

    The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.

  10. Recombinant human cytoplasmic dynein heavy chain 1 and 2: observation of dynein-2 motor activity in vitro.

    PubMed

    Ichikawa, Muneyoshi; Watanabe, Yuta; Murayama, Takashi; Toyoshima, Yoko Yano

    2011-08-04

    Cytoplasmic dynein is a microtubule (MT) motor protein comprising two classes: dynein-1 and dynein-2. We purified recombinant human dynein-1 and dynein-2 from HEK-293 cells by expressing the streptavidin-binding peptide-tagged human cytoplasmic dynein-1 and dynein-2 heavy chains (HCs), respectively. Electron microscopy of the purified molecules revealed a two-headed structure composed of characteristic dynein motor domains. In an in vitro MT gliding assay, both dynein-1 and dynein-2 showed minus-end-directed motor activities. This is the first demonstration of dynein-2 motor activity, which supports the retrograde intraflagellar transport role of dynein-2. Our expression system of dynein HCs provides a useful means to investigate dynein functions.

  11. Effects of opiates and HIV proteins on neurons: the role of ferritin heavy chain and a potential for synergism.

    PubMed

    Festa, Lindsay; Meucci, Olimpia

    2012-07-01

    Human immunodeficiency virus 1 (HIV-1) and its associated proteins can have a profound impact on the central nervous system. Co-morbid abuse of opiates, such as morphine and heroin, is often associated with rapid disease progression and greater neurological dysfunction. The mechanisms by which HIV proteins and opiates cause neuronal damage on their own and together are unclear. The emergence of ferritin heavy chain (FHC) as a negative regulator of the chemokine receptor CXCR4, a co-receptor for HIV, may prove to be important in elucidating the interaction between HIV proteins and opiates. This review summarizes our current knowledge of central nervous system damage inflicted by HIV and opiates, as well as the regulation of CXCR4 by opiate-induced changes in FHC protein levels. We propose that HIV proteins and opiates exhibit an additive or synergistic effect on FHC/CXCR4, thereby decreasing neuronal signaling and function.

  12. Myosin heavy chain isoform expression in human extraocular muscles: longitudinal variation and patterns of expression in global and orbital layers.

    PubMed

    Park, Kyung-Ah; Lim, Jeonghee; Sohn, Seongsoo; Oh, Sei Yeul

    2012-05-01

    We investigated the distribution of myosin heavy chain (MyHC) isoforms along the length of the global and orbital layers of human extraocular muscles (EOMs). Whole muscle tissue extracts of human EOMs were cross-sectioned consecutively and separated into orbital and global layers. The extracts from these layers were subjected to electrophoretic analysis, followed by quantification with scanning densitometry. MyHC isoforms displayed different distributions along the lengths of EOMs. In the orbital and global layers of all EOMs except for the superior oblique muscle, MyHCeom was enriched in the central regions. MyHCIIa and MyHCI were most abundant in the proximal and distal ends. A variation in MyHC isoform expression was apparent along the lengths of human EOMs. These results provide a basis for understanding the molecular mechanisms underlying the functional diversity of EOMs. Copyright © 2012 Wiley Periodicals, Inc.

  13. Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

    1999-01-01

    The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

  14. Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

    1999-01-01

    The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

  15. Cloning of size-selected human immunoglobulin heavy-chain rearrangements from third complementarity-determining region fingerprint profiles.

    PubMed

    Raaphorst, F M; Tami, J; Sanz, I E

    1996-01-01

    Methods have been developed to rapidly visualize the size distribution of third complementarity-determining regions (CDR3) in immunoglobulin (Ig) and T-cell receptor (TCR) molecules. DNA fragments spanning the Ig or TCR CDR3 are generated by PCR using primers at fixed positions in the variable and constant segments. These fragments differ in length due to size variation of the CDR3s. Visualization of the amplification products in polyacrylamide gels as a "CDR3 fingerprint profile" is a rough measure for the complexity of the Ig and TCR antigen-binding specificities. We report an adaptation of this method for the analysis of human Ig heavy-chain genes that incorporates silver staining, which allows for the fine analysis of specific regions of the profiles. This is especially useful for the study of low-abundant transcripts.

  16. Caffeine Positively Modulates Ferritin Heavy Chain Expression in H460 Cells: Effects on Cell Proliferation

    PubMed Central

    Battaglia, Anna Martina; Faniello, Maria Concetta; Cuda, Giovanni; Costanzo, Francesco

    2016-01-01

    Both the methylxanthine caffeine and the heavy subunit of ferritin molecule (FHC) are able to control the proliferation rate of several cancer cell lines. While caffeine acts exclusively as a negative modulator of cell proliferation, FHC might reduce or enhance cell viability depending upon the different cell type. In this work we have demonstrated that physiological concentrations of caffeine reduce the proliferation rate of H460 cells: along with the modulation of p53, pAKT and Cyclin D1, caffeine also determines a significant FHC up-regulation through the activation of its transcriptional efficiency. FHC plays a central role in the molecular pathways modulated by caffeine, ending in a reduced cell growth, since its specific silencing by siRNA almost completely abolishes caffeine effects on H460 cell proliferation. These results allow the inclusion of ferritin heavy subunits among the multiple molecular targets of caffeine and open the way for studying the relationship between caffeine and intracellular iron metabolism. PMID:27657916

  17. The biochemistry of environmental heavy metal uptake by plants: implications for the food chain.

    PubMed

    Peralta-Videa, Jose R; Lopez, Martha Laura; Narayan, Mahesh; Saupe, Geoffrey; Gardea-Torresdey, Jorge

    2009-01-01

    Plants absorb a number of elements from soil, some of which have no known biological function and some are known to be toxic at low concentrations. As plants constitute the foundation of the food chain, some concerns have been raised about the possibility of toxic concentrations of certain elements being transported from plants to higher strata of the food chain. Special attention has been given to the uptake and biotransformation mechanisms occurring in plants and its role in bioaccumulation and impact on consumers, especially human beings. While this review draws particular attention to metal accumulation in edible plants, researched studies of certain wild plants and their consumers are included. Furthermore, this review focuses on plant uptake of the toxic elements arsenic, cadmium, chromium, mercury, and lead and their possible transfer to the food chain. These elements were selected because they are well-established as being toxic for living systems and their effects in humans have been widely documented. Arsenic is known to promote cancer of the bladder, lung, and skin and can be acquired, for example, through the consumption of As-contaminated rice. Cadmium can attack kidney, liver, bone, and it also affects the female reproduction system. Cadmium also can be found in rice. Chromium can produce cancer, and humans can be exposed through smoking and eating Cr-laden vegetables. Lead and mercury are well known neurotoxins that can be consumed via seafood, vegetables and rice.

  18. Thrombin Cleavage of Inter-α-inhibitor Heavy Chain 1 Regulates Leukocyte Binding to an Inflammatory Hyaluronan Matrix.

    PubMed

    Petrey, Aaron C; de la Motte, Carol A

    2016-11-18

    Dynamic alterations of the extracellular matrix in response to injury directly modulate inflammation and consequently the promotion and resolution of disease. During inflammation, hyaluronan (HA) is increased at sites of inflammation where it may be covalently modified with the heavy chains (HC) of inter-α-trypsin inhibitor. Deposition of this unique, pathological form of HA (HC-HA) leads to the formation of cable-like structures that promote adhesion of leukocytes. Naive mononuclear leukocytes bind specifically to inflammation-associated HA matrices but do not adhere to HA constitutively expressed under homeostatic conditions. In this study, we have directly investigated a role for the blood-coagulation protease thrombin in regulating the adhesion of monocytic cells to smooth muscle cells producing an inflammatory matrix. Our data demonstrate that the proteolytic activity of thrombin negatively regulates the adhesion of monocytes to an inflammatory HC-HA complex. This effect is independent of protease-activated receptor activation but requires proteolytic activity toward a novel substrate. Components of HC-HA complexes were predicted to contain conserved thrombin-susceptible cleavage sites based on sequence analysis, and heavy chain 1 (HC1) was confirmed to be a substrate of thrombin. Thrombin treatment is sufficient to cleave HC1 associated with either cell-surface HA or serum inter-α-trypsin inhibitor. Furthermore, thrombin treatment of the inflammatory matrix leads to dissolution of HC-HA cable structures and abolishes leukocyte adhesion. These data establish a novel mechanism whereby thrombin cleavage of HC1 regulates the adhesive properties of an inflammatory HA matrix.

  19. Heavy chain of Acanthamoeba myosine IB is a fusion of myosin-like and non-myosin-like sequences

    SciTech Connect

    Jung, G.; Korn, E.D.; Hammer, J.A. III

    1987-10-01

    Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. The authors present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which they deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approx. 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approx. 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an ..cap alpha..-helical, coiled-coil structure. They conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, they find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. They discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure.

  20. Immunoglobulin Tau Heavy Chain (IgT) in Flounder, Paralichthys olivaceus: Molecular Cloning, Characterization, and Expression Analyses

    PubMed Central

    Du, Yang; Tang, Xiaoqian; Zhan, Wenbin; Xing, Jing; Sheng, Xiuzhen

    2016-01-01

    Immunoglobulin tau (IgT) is a new teleost immunoglobulin isotype, and its potential function in adaptive immunity is not very clear. In the present study, the membrane-bound and secreted IgT (mIgT and sIgT) heavy chain genes were cloned for the first time and characterized in flounder (Paralichthys olivaceus), and found the nucleic acid sequence were exactly same in the Cτ1–Cτ4 constant domains of mIgT and sIgT, but different in variable regions and the C-terminus. The amino acid sequence of mIgT shared higher similarity with Bovichtus diacanthus (51.2%) and Dicentrarchus labrax (45.0%). Amino acid of flounder IgT, IgM, and IgD heavy chain was compared and the highest similarity was found between IgT Cτ1 and IgM Cμ1 (38%). In healthy flounder, the transcript levels of IgT mRNA were the highest in gill, spleen, and liver, and higher in peripheral blood leucocytes, skin, and hindgut. After infection and vaccination with Edwardsiella tarda via intraperitoneal injection and immersion, the qRT-PCR analysis demonstrated that the IgT mRNA level was significantly upregulated in all tested tissues, with similar dynamic tendency that increased firstly and then decreased, and higher in gill, skin, hindgut, liver, and stomach in immersion than in the injection group, but no significant difference existed in spleen and head kidney between immersion and injection groups. These results revealed that IgT responses could be simultaneously induced in both mucosal and systemic tissues after infection/vaccination via injection and immersion route, but IgT might play a more important role in mucosal immunity than in systemic immunity. PMID:27649168

  1. Effects of prolonged strenuous endurance exercise on plasma myosin heavy chain fragments and other muscular proteins. Cycling vs running.

    PubMed

    Koller, A; Mair, J; Schobersberger, W; Wohlfarter, T; Haid, C; Mayr, M; Villiger, B; Frey, W; Puschendorf, B

    1998-03-01

    This study evaluates creatine kinase, myosin heavy chain, and cardiac troponin blood levels following three types of exercise: 1) short-distance uphill or downhill running; 2) alpine ultramarathon; and 3) alpine long-distance cycling. Comparative field study; follow-up up to 10 days. Department of Sports Medicine. All biochemical markers were analysed at the Department of Medical Chemistry and Biochemistry. Subjects included healthy, trained males (N = 53). All subjects were nonsmokers and free from medication prior to and during the study. Each volunteer was an experienced runner or cyclist, who had at least once successfully finished the Swiss Alpine Marathon of Davos or the Otztal-Radmarathon before. Running or cycling. Plasma concentrations of creatine kinase, myosin heavy chain fragments and cardiac troponins were measured to diagnose skeletal and cardiac muscle damage, respectively. Skeletal muscle protein release is markedly different between uphill and downhill running, with very little evidence for muscle damage in the uphill runners. There is considerable muscle protein leakage in the ultramarathoners (67 km distance; 30 km downhill running). In contrast, only modest amounts of skeletal muscle damage are found after alpine long-distance cycling (230 km distance). This study proves that there is slow-twitch skeletal muscle fiber damage after prolonged strenuous endurance exercise and short-distance downhill running. Exhaustive endurance exercise involving downhill running and short-distance downhill running lead to more pronounced injury than strenuous endurance exercise involving concentric actions. From our results there is no reason for suggesting that prolonged intense exercise may induce myocardial injury in symptom-less athletes without cardiac deseases.

  2. Effect of glucocorticoïd receptor ligands on myosin heavy chains expression in rat skeletal muscles during controllable stress.

    PubMed

    Martrette, J M; Hartmann, N; Westphal, A; Favot, L

    2004-01-01

    The influence of agonist (dexamethasone) and antagonist (mifepristone) of glucocorticoïd receptor during controllable painless stress was evaluated on myosin heavy chains expression in three masticatory and two nape rat muscles: anterior digastric (AD), anterior temporalis (AT), masseter superficialis (MS), longissimus capitis (L) and rectus capitis dorsalis major (R). The relative amounts of myosin heavy chain (MHC) protein isoform contained were significantly affected in four muscles studied by dexamethasone and in three muscles studied under mifepristone, versus control during the stress procedure, after only 1 week of treatment. The control group AT muscles contained respectively 18.2% of MHC 2A, 34.5% of MHC 2X and 47.4% of MHC 2B. The effects of dexamethasone and mifepristone were opposite in this muscle: under dexamethasone, the relative proportions of the three isoforms were 14.2, 31.0 and 54.8%: consequently, MHC 2A and 2X decreased with the profit of 2B. Under mifepristone, the relative proportions were 21.1, 36.6 and 42.3% (MHC 2A and 2X increased to the detriment of 2B). The L muscle was not affected by the two treatments and MS muscle was only affected by dexamethasone. Dexamethasone increased MHC 2B to the detriment of MHC 2A in MS, AD and R. Mifepristone and dexamethasone induced the same changes in AD. The mifepristone treatment decreased the MHC 2X profile in R. Under dexamethasone, four muscles exhibited a significantly higher proportion of the more rapid isoforms than under mifepristone. A previous work showed that controllable stress induced a marked increase in the relative expression of MHC 2B in the same skeletal muscles (Martrette et al. , 1998). Our results confirm then a significant participation of glucocorticoïd in MHC isoform expression during controllable stress.

  3. Multiple myeloma and multiple plasmacytomas associated with free gamma heavy chain, free kappa light chain and IgGk paraproteins: an unusual triple gammopathy.

    PubMed

    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.

  4. Bioaccumulation of heavy metals both in wild and mariculture food chains in Daya Bay, South China

    NASA Astrophysics Data System (ADS)

    Qiu, Yao-Wen

    2015-09-01

    Bioaccumulation and trophic transfer of heavy metals both in the natural marine ecosystem (seawater, sediment, coral reef, phytoplankton, macrophyte, shrimp, crab, shellfish, planktivorous and carnivorous fish) and in the mariculture ecosystem (compound feed, trash fish, farmed pompano and snapper) were studied at Daya Bay, a typical subtropical bay in Southern China. The levels of Cu, Zn, Pb and Cd in sediment were 11.7, 10.2, 53.8 and 2.8 times than those in coral reef, respectively. Pb and Zn levels were markedly higher in phytoplankton than in macrophyte, probably caused by the larger specific surface area in phytoplankton. The highest levels of Zn (98.1), Pb (1.87) and Cd (5.11 μg g-1 dw) in wild organisms were all found in clam (Veremolpa scabra), indicating that these metals were apt to bioaccumulate in shellfish. The average concentrations of Cu, Zn, Pb and Cd in wild fish were 3.7, 2.1, 0.4 and 22.2 times than those in farmed fish, confirming the "growth dilution" hypothesis in farmed fish. Heavy metal bioconcentration factors (BCFs) in algae, bioaccumulation factors (BAFs) in wild species and transfer factors (TFs) in organism were calculated and discussed. The results suggested that biologically essential Cu and Zn were easier to accumulate in fish than non-essential Cd. Concentrations of Cu, Zn and Cd were several times higher in wild fish than in farmed fish whereas the opposite was observed for Pb. This metal also showed the highest transfer factor from food, which means that special attention must be given to fish feed production in relation to metal contamination.

  5. Monoclonal antibodies to kinesin heavy and light chains stain vesicle- like structures, but not microtubules, in cultured cells

    PubMed Central

    1989-01-01

    Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane- bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface. PMID:2522455

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

    PubMed

    Paolini, Lucia

    2016-06-01

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

  7. N332-Directed Broadly Neutralizing Antibodies Use Diverse Modes of HIV-1 Recognition: Inferences from Heavy-Light Chain Complementation of Function

    PubMed Central

    Louder, Mark K.; Gorman, Jason; Lu, Gabriel; McLellan, Jason S.; Stuckey, Jonathan; Zhu, Jiang; Burton, Dennis R.; Koff, Wayne C.; Mascola, John R.; Kwong, Peter D.

    2013-01-01

    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. PMID:23431362

  8. Allelic polymorphism of murine IgE controlled by the seventh immunoglobulin heavy chain allotype locus.

    PubMed

    Borges, M S; Kumagai, Y; Okumura, K; Hirayama, N; Ovary, Z; Tada, T

    1981-01-01

    Two alloantisera against hybridoma-derived IgE detected allotypic determinants expressed on the murine epsilon chain. An antiserum raised in BALB/c mice against monoclonal IgE of C57BL/6 origin reacted exclusively with IgE of strains having Igh-1b (IgG2 a) allotype. The second antiserum, C57BL/6 anti-BALB/c monoclonal IgE, reacted with IgE of strains having Igh-1a, Igh-1d, Igh-1e and Igh-1j allotypes. The genetic studies of (BALB/c x C57BL/6)F1 and backcross F2 animals indicated that the locus controlling the IgE allotype is linked to the Igh-1 locus. This was further confirmed by the possession of respective IgE allotypes by Igh-C congenic mice, BALB/c and BAB-14, C3H.SW/Hz and CWB/Hz. Thus, the allotype detected on the epsilon chain is controlled by the seventh murine immunoglobulin allotype locus, and should be designated as the Igh-7 allotype.

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

    PubMed

    Okuneva, A D; Vikhliantsev, I M; Shpagina, M D; Rogachevskiĭ, V V; Khutsian, S S; Poddubnaia, Z A; Grigor'ev, A I

    2012-01-01

    Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus ) were investigated after 12-day spaceflight on board of Russian space vehicle "Foton-M3". In m. psoas and m. soleus in the gerbils from "Flight" group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in "Flight" group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from "Flight" group were observed. In skeletal muscles of the gerbils from "Flight" group the relative content of titin N2A-isoform was reduced (by 1,2-1,7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from "Flight" group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.

  10. High fat/low carbohydrate diet attenuates left ventricular hypertrophy and prevents myosin heavy chain isoform switching induced by chronic hypertenstion

    USDA-ARS?s Scientific Manuscript database

    A switch in the expression of myosin heavy chain isoform (MHC) alpha to beta is observed with left ventricular hypertrophy (LVH) and heart failure. This switch is associated with a defect in myocardial energy production and contractile dysfunction. Similar MHC isoform profile is observed in the fe...

  11. Familial hypertrophic cardiomyopathy. Microsatellite haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain gene.

    PubMed

    Dausse, E; Komajda, M; Fetler, L; Dubourg, O; Dufour, C; Carrier, L; Wisnewsky, C; Bercovici, J; Hengstenberg, C; al-Mahdawi, S

    1993-12-01

    Familial hypertrophic cardiomyopathy (FHC) is a clinically and genetically heterogeneous disease. The first identified disease gene, located on chromosome 14q11-q12, encodes the beta-myosin heavy chain. We have performed linkage analysis of two French FHC pedigrees, 720 and 730, with two microsatellite markers located in the beta-myosin heavy chain gene (MYO I and MYO II) and with four highly informative markers, recently mapped to chromosome 14q11-q12. Significant linkage was found with MYO I and MYO II in pedigree 720, but results were not conclusive for pedigree 730. Haplotype analysis of the six markers allowed identification of affected individuals and of some unaffected subjects carrying the disease gene. Two novel missense mutations were identified in exon 13 by direct sequencing, 403Arg-->Leu and 403Arg-->Trp in families 720 and 730, respectively. The 403Arg-->Leu mutation was associated with incomplete penetrance, a high incidence of sudden deaths and severe cardiac events, whereas the consequences of the 403Arg-->Trp mutation appeared less severe. Haplotyping of polymorphic markers in close linkage to the beta-myosin heavy chain gene can, thus, provide rapid analysis of non informative pedigrees and rapid detection of carrier status. Our results also indicate that codon 403 of the beta-myosin heavy chain gene is a hot spot for mutations causing FHC.

  12. Mining the antibodyome for HIV-1-neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains.

    PubMed

    Zhu, Jiang; Ofek, Gilad; Yang, Yongping; Zhang, Baoshan; Louder, Mark K; Lu, Gabriel; McKee, Krisha; Pancera, Marie; Skinner, Jeff; Zhang, Zhenhai; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E; Blinn, Julie; Alam, S Munir; Haynes, Barton F; Simek, Melissa; Burton, Dennis R; Koff, Wayne C; Mullikin, James C; Mascola, John R; Shapiro, Lawrence; Kwong, Peter D

    2013-04-16

    Next-generation sequencing of antibody transcripts from HIV-1-infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy- and light-chain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy- and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141-145. Heavy- and light-chain phylogenetic trees of PGT141-145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141-145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings.

  13. Mining the antibodyome for HIV-1–neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains

    PubMed Central

    Zhu, Jiang; Ofek, Gilad; Yang, Yongping; Zhang, Baoshan; Louder, Mark K.; McKee, Krisha; Pancera, Marie; Skinner, Jeff; Zhang, Zhenhai; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Blinn, Julie; Alam, S. Munir; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Mullikin, James C.; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.; Becker, Jesse; Benjamin, Betty; Blakesley, Robert; Bouffard, Gerry; Brooks, Shelise; Coleman, Holly; Dekhtyar, Mila; Gregory, Michael; Guan, Xiaobin; Gupta, Jyoti; Han, Joel; Hargrove, April; Ho, Shi-ling; Johnson, Taccara; Legaspi, Richelle; Lovett, Sean; Maduro, Quino; Masiello, Cathy; Maskeri, Baishali; McDowell, Jenny; Montemayor, Casandra; Mullikin, James; Park, Morgan; Riebow, Nancy; Schandler, Karen; Schmidt, Brian; Sison, Christina; Stantripop, Mal; Thomas, James; Thomas, Pam; Vemulapalli, Meg; Young, Alice

    2013-01-01

    Next-generation sequencing of antibody transcripts from HIV-1–infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy- and light-chain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy- and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141–145. Heavy- and light-chain phylogenetic trees of PGT141–145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141–145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings. PMID:23536288

  14. The evolution of multiple isotypic IgM heavy chain genes in the shark.

    PubMed

    Lee, Victor; Huang, Jing Li; Lui, Ming Fai; Malecek, Karolina; Ohta, Yuko; Mooers, Arne; Hsu, Ellen

    2008-06-01

    The IgM H chain gene organization of cartilaginous fishes consists of 15-200 miniloci, each with a few gene segments (V(H)-D1-D2-J(H)) and one C gene. This is a gene arrangement ancestral to the complex IgH locus that exists in all other vertebrate classes. To understand the molecular evolution of this system, we studied the nurse shark, which has relatively fewer loci, and characterized the IgH isotypes for organization, functionality, and the somatic diversification mechanisms that act upon them. Gene numbers differ slightly between individuals ( approximately 15), but five active IgM subclasses are always present. Each gene undergoes rearrangement that is strictly confined within the minilocus; in B cells there is no interaction between adjacent loci located > or =120 kb apart. Without combinatorial events, the shark IgM H chain repertoire is based on junctional diversity and, subsequently, somatic hypermutation. We suggest that the significant contribution by junctional diversification reflects the selected novelty introduced by RAG in the early vertebrate ancestor, whereas combinatorial diversity coevolved with the complex translocon organization. Moreover, unlike other cartilaginous fishes, there are no germline-joined VDJ at any nurse shark mu locus, and we suggest that such genes, when functional, are species-specific and may have specialized roles. With an entire complement of IgM genes available for the first time, phylogenetic analyses were performed to examine how the multiple Ig loci evolved. We found that all domains changed at comparable rates, but V(H) appears to be under strong positive selection for increased amino acid sequence diversity, and surprisingly, so does Cmicro2.

  15. Estimating Copy Number and Allelic Variation at the Immunoglobulin Heavy Chain Locus Using Short Reads.

    PubMed

    Luo, Shishi; Yu, Jane A; Song, Yun S

    2016-09-01

    The study of genomic regions that contain gene copies and structural variation is a major challenge in modern genomics. Unlike variation involving single nucleotide changes, data on the variation of copy number is difficult to collect and few tools exist for analyzing the variation between individuals. The immunoglobulin heavy variable (IGHV) locus, which plays an integral role in the adaptive immune response, is an example of a complex genomic region that varies in gene copy number. Lack of standard methods to genotype this region prevents it from being included in association studies and is holding back the growing field of antibody repertoire analysis. Here we develop a method that takes short reads from high-throughput sequencing and outputs a genetic profile of the IGHV locus with the read coverage depth and a putative nucleotide sequence for each operationally defined gene cluster. Our operationally defined gene clusters aim to address a major challenge in studying the IGHV locus: the high sequence similarity between gene segments in different genomic locations. Tests on simulated data demonstrate that our approach can accurately determine the presence or absence of a gene cluster from reads as short as 70 bp. More detailed resolution on the copy number of gene clusters can be obtained from read coverage depth using longer reads (e.g., ≥ 100 bp). Detail at the nucleotide resolution of single copy genes (genes present in one copy per haplotype) can be determined with 250 bp reads. For IGHV genes with more than one copy, accurate nucleotide-resolution reconstruction is currently beyond the means of our approach. When applied to a family of European ancestry, our pipeline outputs genotypes that are consistent with the family pedigree, confirms existing multigene variants and suggests new copy number variants. This study paves the way for analyzing population-level patterns of variation in IGHV gene clusters in larger diverse datasets and for quantitatively

  16. Layered inorganic/organic mercaptopropyl pendant chain hybrid for chelating heavy cations

    SciTech Connect

    Macedo, Thais R.; Petrucelli, Giovanni C.; Pinto, Alane A.; Airoldi, Claudio

    2011-12-15

    Graphical abstract: Crystalline lamellar silicate RUB-18 was immobilized with mercaptopropyl groups at the surface and then used as support for cadmium and lead removal from aqueous solutions. Highlights: Black-Right-Pointing-Pointer Synthetic methodology requires intercalation. Black-Right-Pointing-Pointer Organofunctionalized ilerite compound as sorbent. Black-Right-Pointing-Pointer Active mercaptopropyl groups remove cations. Black-Right-Pointing-Pointer High maximum sorption capacity for cadmium. -- Abstract: Heavy metal sorbents with uptake capacities for divalent cadmium and lead cation removal from aqueous solutions have been synthesized by grafting mercaptopropyltrimethoxysilane onto the surface of two different precursors obtained from lamellar ilerite, its acidic and the cetyltrimethylammonium exchanged forms. The organofunctionalization was carried out by two different procedures: reflux and solvent evaporation methodologies. Elemental analysis data based on carbon content gave 1.37 and 3.53 mmol of organic pendant groups per gram of hybrid by the reflux method, when starting from acidic ilerite and the surfactant form. X-ray diffraction corroborated the maintenance of the original crystallinity. Infrared spectroscopy and nuclear magnetic resonance for {sup 29}Si and {sup 13}C nuclei are in agreement with the success of the proposed method. The sulfur basic centers attached to the lamellar structure are used to coordinate both cations at the solid/liquid interface. The isotherms were obtained through the batchwise process and the experimental data were adjusted to the Freundlich model. The maximum sorption capacities of 5.55 and 5.12 mmol g{sup -1} for lead and 6.10 and 7.10 mmol g{sup -1} for cadmium were obtained for organofunctionalized ilerite and its surfactant form, synthesized by reflux methodology. This behavior suggested that these hybrids could be employed as promising sorbents with a polluted system.

  17. A two-in-one antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions.

    PubMed

    Lee, Chingwei V; Koenig, Patrick; Fuh, Germaine

    2014-01-01

    A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.

  18. Variable regions of Ig heavy chain genes encoding antithyrotropin receptor antibodies of patients with Graves' disease

    SciTech Connect

    Shin, Euy Kyun; Akamizu, Takashi; Matsuda, Fumihiko; Sugawa, Hideo; Fujikura, Junji; Mori, Toru; Honjo, Tasuku )

    1994-02-01

    The authors have established EBV-transformed human B cell clones producing monoclonal antithyrotropin receptor antibodies from two patients with Graves' disease. They then isolated and characterized Ig H chain genes of 5 B cell clones with thyrotropin-binding inhibitor Ig (TBII) activity and 4 B cell clones with thyroid-stimulating antibody (TSAb) activity. They found that V[sub H] gene families used in the 5 TBII clones were diverse, including V[sub H-II, -III, -IV,] and [sub -V]. Most of V[sub H] segments used in TBII and TSAb are commonly used in other autoantibodies and fetal liver repertoire. The frequency of somatic mutations in TBII was higher than that in TSAb. In as much as the same germline V[sub H] segment (V3-23) was used for both TBII and TSAb, the frequency and position of somatic mutations may be important for generation of TBII and TSAb. 56 refs., 2 figs., 2 tabs.

  19. Peptide chain dynamics in light and heavy water: zooming in on internal friction.

    PubMed

    Schulz, Julius C F; Schmidt, Lennart; Best, Robert B; Dzubiella, Joachim; Netz, Roland R

    2012-04-11

    Frictional effects due to the chain itself, rather than the solvent, may have a significant effect on protein dynamics. Experimentally, such "internal friction" has been investigated by studying folding or binding kinetics at varying solvent viscosity; however, the molecular origin of these effects is hard to pinpoint. We consider the kinetics of disordered glycine-serine and α-helix forming alanine peptides and a coarse-grained protein folding model in explicit-solvent molecular dynamics simulations. By varying the solvent mass over more than two orders of magnitude, we alter only the solvent viscosity and not the folding free energy. Folding dynamics at the near-vanishing solvent viscosities accessible by this approach suggests that solvent and internal friction effects are intrinsically entangled. This finding is rationalized by calculation of the polymer end-to-end distance dynamics from a Rouse model that includes internal friction. An analysis of the friction profile along different reaction coordinates, extracted from the simulation data, demonstrates that internal as well as solvent friction varies substantially along the folding pathways and furthermore suggests a connection between friction and the formation of hydrogen bonds upon folding.

  20. Contributions of Conventional and Heavy-Chain IgG to Immunity in Fetal, Neonatal, and Adult Alpacas▿

    PubMed Central

    Daley-Bauer, L. P.; Purdy, S. R.; Smith, M. C.; Gagliardo, L. F.; Davis, W. C.; Appleton, J. A.

    2010-01-01

    In addition to conventional immunoglobulins, camelids produce antibodies that do not incorporate light chains into their structures. These so-called heavy-chain (HC) antibodies have incited great interest in the biomedical community, as they have considerable potential for biotechnological and therapeutic application. Recently, we have begun to elucidate the immunological functions of HC antibodies, yet little is known about their significance in maternal immunity or about the B lymphocytes that produce them. This study describes the application of isotype-specific reagents toward physiological assessments of camelid IgGs and the B cells that produce them. We document the specificities of monoclonal antibodies that distinguish two conventional IgG1 isotypes and two HC IgG3 variants produced by alpacas. Next, we report that the relative concentrations of five isotypes are similar in serum, milk, and colostrum; however, following passive transfer, the concentrations of HC IgG2 and IgG3 declined more rapidly than the concentration of conventional IgG1 in the sera of neonates. Finally, we assessed the distribution of B cells of distinct isotypes within lymphoid tissues during fetal and adult life. We detected IgG1, IgG2, and IgG3 in lymphocytes located in lymph node follicles, suggesting that HC B cells affinity mature and/or class switch. One IgG3 isotype was present in B cells located in ileal Peyer's patches, and one conventional IgG1 isotype was detected in splenic marginal zone B cells. Our findings contribute to the growing body of knowledge pertaining to HC antibodies and are compatible with functional specialization among conventional and HC IgGs in the alpaca. PMID:20926693

  1. Contributions of conventional and heavy-chain IgG to immunity in fetal, neonatal, and adult alpacas.

    PubMed

    Daley-Bauer, L P; Purdy, S R; Smith, M C; Gagliardo, L F; Davis, W C; Appleton, J A

    2010-12-01

    In addition to conventional immunoglobulins, camelids produce antibodies that do not incorporate light chains into their structures. These so-called heavy-chain (HC) antibodies have incited great interest in the biomedical community, as they have considerable potential for biotechnological and therapeutic application. Recently, we have begun to elucidate the immunological functions of HC antibodies, yet little is known about their significance in maternal immunity or about the B lymphocytes that produce them. This study describes the application of isotype-specific reagents toward physiological assessments of camelid IgGs and the B cells that produce them. We document the specificities of monoclonal antibodies that distinguish two conventional IgG1 isotypes and two HC IgG3 variants produced by alpacas. Next, we report that the relative concentrations of five isotypes are similar in serum, milk, and colostrum; however, following passive transfer, the concentrations of HC IgG2 and IgG3 declined more rapidly than the concentration of conventional IgG1 in the sera of neonates. Finally, we assessed the distribution of B cells of distinct isotypes within lymphoid tissues during fetal and adult life. We detected IgG1, IgG2, and IgG3 in lymphocytes located in lymph node follicles, suggesting that HC B cells affinity mature and/or class switch. One IgG3 isotype was present in B cells located in ileal Peyer's patches, and one conventional IgG1 isotype was detected in splenic marginal zone B cells. Our findings contribute to the growing body of knowledge pertaining to HC antibodies and are compatible with functional specialization among conventional and HC IgGs in the alpaca.

  2. Myosin heavy chain expression and atrophy in rat skeletal muscle during transition from cardiac hypertrophy to heart failure.

    PubMed

    Carvalho, Robson Francisco; Cicogna, Antonio Carlos; Campos, Gerson Eduardo Rocha; De Assis, Jeane Marlene Fogaça; Padovani, Carlos Roberto; Okoshi, Marina Politi; Pai-Silva, Maeli Dal

    2003-08-01

    The purpose of this investigation was to determine whether changes in myosin heavy chain (MHC) expression and atrophy in rat skeletal muscle are observed during transition from cardiac hypertrophy to chronic heart failure (CHF) induced by aortic stenosis (AS). AS and control animals were studied 12 and 18 weeks after surgery and when overt CHF had developed in AS animals, 28 weeks after the surgery. The following parameters were studied in the soleus muscle: muscle atrophy index (soleus weight/body weight), muscle fibre diameter and frequency and MHC expression. AS animals presented decreases in both MHC1 and type I fibres and increases in both MHC2a and type IIa fibres during late cardiac hypertrophy and CHF. Type IIa fibre atrophy occurred during CHF. In conclusion, our data demonstrate that skeletal muscle phenotype changes occur in both late cardiac hypertrophy and heart failure; this suggests that attention should be given to the fact that skeletal muscle phenotype changes occur prior to overt heart failure symptoms.

  3. Allelic exclusion of the immunoglobulin heavy chain locus is independent of its nuclear localization in mature B cells

    PubMed Central

    Holwerda, Sjoerd J. B.; van de Werken, Harmen J. G.; Ribeiro de Almeida, Claudia; Bergen, Ingrid M.; de Bruijn, Marjolein J. W.; Verstegen, Marjon J. A. M.; Simonis, Marieke; Splinter, Erik; Wijchers, Patrick J.; Hendriks, Rudi W.; de Laat, Wouter

    2013-01-01

    In developing B cells, the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here, we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is, however, unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells—but not in T cells—the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element. PMID:23748562

  4. Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies

    PubMed Central

    Stanfield, Robyn L.; Wilson, Ian A.; Smider, Vaughn V.

    2016-01-01

    A subset of bovine antibodies have an exceptionally long third heavy-chain complementarity determining region (CDR H3) that is highly variable in sequence and includes multiple cysteines. These long CDR H3s (up to 69 residues) fold into a long stalk atop which sits a knob domain that is located far from the antibody surface. Three new bovine Fab crystal structures have been determined to decipher the conserved and variable features of ultralong CDR H3s that lead to diversity in antigen recognition. Despite high sequence variability, the stalks adopt a conserved β-ribbon structure, while the knob regions share a conserved β-sheet that serves as a scaffold for two connecting loops of variable length and conformation, as well as one conserved disulfide. Variation in patterns and connectivity of the remaining disulfides contribute to the knob structural diversity. The unusual architecture of these ultralong bovine CDR H3s for generating diversity is unique in adaptive immune systems. PMID:27574710

  5. Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.

    PubMed

    Fong, Y; Rosenbaum, M; Tracey, K J; Raman, G; Hesse, D G; Matthews, D E; Leibel, R L; Gertner, J M; Fischman, D A; Lowry, S F

    1989-05-01

    A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous refeeding. A 6-hr methionyl growth hormone infusion achieved steady-state serum levels comparable to normal physiologic peaks and was associated with a significant increase in limb amino acid uptake, without a change in body amino acid oxidation. Myosin heavy-chain mRNA levels, measured by quantitative dot blot hybridization, were also significantly elevated after growth hormone administration. The data indicate that methionyl growth hormone can induce intracellular amino acid accrual and increased levels of myofibrillar protein mRNA during hospitalized nutritional support and suggest growth hormone to be a potential therapy of lean body wasting.

  6. Variable N-terminal regions of muscle myosin heavy chain modulate ATPase rate and actin sliding velocity.

    PubMed

    Swank, Douglas M; Knowles, Aileen F; Kronert, William A; Suggs, Jennifer A; Morrill, George E; Nikkhoy, Massoud; Manipon, Gracielle G; Bernstein, Sanford I

    2003-05-09

    We integratively assessed the function of alternative versions of a region near the N terminus of Drosophila muscle myosin heavy chain (encoded by exon 3a or 3b). We exchanged the alternative exon 3 regions between an embryonic isoform and the indirect flight muscle isoform. Each chimeric myosin was expressed in Drosophila indirect flight muscle, in the absence of other myosin isoforms, allowing for purified protein analysis and whole organism locomotory studies. The flight muscle isoform generates higher in vitro actin sliding velocity and solution ATPase rates than the embryonic isoform. Exchanging the embryonic exon 3 region into the flight muscle isoform decreased ATPase rates to embryonic levels but did not affect actin sliding velocity or flight muscle ultrastructure. Interestingly, this swap only slightly impaired flight ability. Exchanging the flight muscle-specific exon 3 region into the embryonic isoform increased actin sliding velocity 3-fold and improved indirect flight muscle ultrastructure integrity but failed to rescue the flightless phenotype of flies expressing embryonic myosin. These results suggest that the two structural versions of the exon 3 domain independently influence the kinetics of at least two steps of the actomyosin cross-bridge cycle.

  7. Comparison of Characteristics of Myosin Heavy Chain-based Fiber and Meat Quality among Four Bovine Skeletal Muscles

    PubMed Central

    Kim, Gap-Don; Yang, Han-Sul; Jeong, Jin-Yeon

    2016-01-01

    Muscle fiber characteristics account for meat quality and muscle fibers are mainly classified into three or more types according to their contractile and metabolic properties. However, the majority of previous studies on bovine skeletal muscle are based on myosin ATPase activity. In the present study, the differences in the characteristics of muscle fibers classified by the expression of myosin heavy chain (MHC) among four bovine skeletal muscles such as longissimus thoracis (LT), psoas major (PM), semimembranosus (SM) and semitendinosus (ST) and their relationships to beef quality were investigated. MHCs 2x, 2a and slow were identified by LC-MS/MS and IIX, IIA and I fiber types were classified. PM, which had the smallest size and highest density of fibers regardless of type, showed the highest myoglobin content, CIE L*, a*, b* and sarcomere length (p<0.05), whereas ST with the highest composition of IIX, showed high shear force and low sarcomere length (p<0.05). The correlation coefficients between muscle fiber characteristics and meat quality showed that type IIX is closely related to poor beef quality and that a high density of small-sized fibers is related to redness and tenderness. Therefore, the differences in meat quality between muscles can be explained by the differences in muscle fiber characteristics, and especially, the muscles with good quality are composed of more small-sized fibers regardless of fiber type. PMID:28115894

  8. ETS-Mediated Cooperation between Basic Helix-Loop-Helix Motifs of the Immunoglobulin μ Heavy-Chain Gene Enhancer

    PubMed Central

    Dang, Wei; Sun, Xiao-hong; Sen, Ranjan

    1998-01-01

    The μE motifs of the immunoglobulin μ heavy-chain gene enhancer bind ubiquitously expressed proteins of the basic helix-loop-helix (bHLH) family. These elements work together with other, more tissue-restricted elements to produce B-cell-specific enhancer activity by presently undefined combinatorial mechanisms. We found that μE2 contributed to transcription activation in B cells only when the μE3 site was intact, providing the first evidence for functional interactions between bHLH proteins. In vitro assays showed that bHLH zipper proteins binding to μE3 enhanced Ets-1 binding to μA. One of the consequences of this protein-protein interaction was to facilitate binding of a second bHLH protein, E47, to the μE2 site, thereby generating a three-protein–DNA complex. Furthermore, transcriptional synergy between bHLH and bHLH zipper factors also required an intermediate ETS protein, which may bridge the transcription activation domains of the bHLH factors. Our observations define an unusual form of cooperation between bHLH and ETS proteins and suggest mechanisms by which tissue-restricted and ubiquitous factors combine to generate tissue-specific enhancer activity. PMID:9488464

  9. Synergistic ablation does not affect atrophy or altered myosin heavy chain expression in the non-weight bearing soleus muscle

    NASA Technical Reports Server (NTRS)

    Linderman, J. K.; Talmadge, R. J.; Gosselink, K. L.; Tri, P. N.; Roy, R. R.; Grindeland, R. E.

    1996-01-01

    The purpose of this study was to investigate whether the soleus muscle undergoes atrophy and alterations in myosin heavy chain (MHC) composition during non-weight bearing in the absence of synergists. Thirty-two female rats were randomly assigned to four groups: control (C), synergistic ablation (ABL) of the gastrocnemius and plantaris muscles to overload the soleus muscle, hindlimb suspension (HLS), or a combination of synergistic ablation and hindlimb suspension (HLS-ABL). After 28 days of hindlimb suspension, soleus atrophy was more pronounced in HLS (58%) than in HLS-ABL (43%) rats. Compared to C rats, non-weight bearing decreased mixed and myofibrillar protein contents and Type I MHC 49%, 45%, and 7%, respectively, in HLS animals. In addition, de novo expression of fast Type IIx and Type IIb MHC (5% and 2%, respectively) was observed in HLS animals. Similarly, when compared to C rats, mixed and myofibrillar protein contents and Type I MHC decreased 43%, 46%, and 4%, respectively, in HLS-ABL animals. Also, de novo expression of Type IIx (4%) and IIb (1%) MHC was observed. Collectively, these data indicate that the loss of muscle protein and Type I MHC, and the de novo expression of Type IIx and Type IIb MHC in the rat soleus occur independently of the presence of synergists during non-weight bearing. Furthermore, these results confirm the contention that soleus mass and MHC expression are highly sensitive to alterations in mechanical load.

  10. Motor domain-based motility system and motile properties of alpha heavy chain in Tetrahymena outer arm dynein.

    PubMed

    Edamatsu, Masaki

    2014-10-24

    Axonemal dynein plays an essential role in ciliary motility, and impaired ciliary motility causes human diseases such as primary ciliary dyskinesia (PCD). The motor domain of axonemal dynein powers ciliary motility and its function is regulated by several accessary proteins bound to the tail region. Therefore, to understand the essential properties of dynein motility, examining the motile properties of the motor domain without the tail is necessary. In this study, the functional motor domain of the alpha heavy chain in Tetrahymena outer arm dynein was purified, and its motile properties were examined using an in vitro motility system. The purified protein caused microtubules to glide at a velocity of 5.0μm/s with their minus-end trailing, and motility was inhibited in an ATP concentration-dependent manner, which is in contrast with kinesin1. This method could be applicable to other axonemal dyneins and will enable further molecular studies on diverse axonemal dyneins and ciliary motility. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. The inv(16) Fusion Protein Associates with Corepressors via a Smooth Muscle Myosin Heavy-Chain Domain

    PubMed Central

    Durst, Kristie L.; Lutterbach, Bart; Kummalue, Tanawan; Friedman, Alan D.; Hiebert, Scott W.

    2003-01-01

    Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor β to the coiled-coil region of a smooth muscle myosin heavy chain (CBFβ/SMMHC). CBFβ interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBFβ/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBFβ/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBFβ/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor. PMID:12509458

  12. Fiber size, type, and myosin heavy chain content in rhesus hindlimb muscles after 2 weeks at 2 G

    NASA Technical Reports Server (NTRS)

    Tavakol, Morteza; Roy, Roland R.; Kim, Jung A.; Zhong, Hui; Hodgson, John A.; Hoban-Higgins, Tana M.; Fuller, Charles A.; Edgerton, V. Reggie

    2002-01-01

    BACKGROUND: Fiber atrophy and an increase in the percentage of fast fibers have been observed in Rhesus leg muscles after spaceflight. Hypothesis: Hypergravity will result in muscle fiber hypertrophy and an increase in the percentage of slow fibers. METHODS: Open muscle biopsies were obtained from Rhesus soleus, medial gastrocnemius (MG), and tibialis anterior (TA) muscles before and after 14 d of centrifugation (2 G) and in time-matched controls. Cage activity levels were measured by telemetry. RESULTS: Based on monoclonal antibody binding for myosin heavy chains (MHC), the fastest region of soleus contained a higher proportion of type I+II (27 vs. 13%) and had a tendency for a lower proportion of type I (38 vs. 61%, p = 0.10) fibers after than before centrifugation. There was a higher proportion of type I+II fibers in post- vs. pre-2 G (10 vs. 0.6%) MG biopsies. Fiber type distribution and MHC composition were unaffected in the TA. Overall, mean fiber sizes were unaffected by centrifugation. Average cage activity levels were 36% lower during than before 2 G. CONCLUSIONS: Our hypothesis was rejected. The changes in the proportion of fibers expressing type I MHC are the reverse of that expected with chronic loading of extensors and, paradoxically, are similar to changes observed with chronic unloading, such as occurs during spaceflight, in this primate model. The data are consistent with the observed decrease in total daily activity levels.

  13. Structural abnormalities develop in the brain after ablation of the gene encoding nonmuscle myosin II-B heavy chain.

    PubMed

    Tullio, A N; Bridgman, P C; Tresser, N J; Chan, C C; Conti, M A; Adelstein, R S; Hara, Y

    2001-04-23

    Ablation of nonmuscle myosin heavy chain II-B (NMHC-B) in mice results in severe hydrocephalus with enlargement of the lateral and third ventricles. All B(-)/B(-) mice died either during embryonic development or on the day of birth (PO). Neurons cultured from superior cervical ganglia of B(-)/B(-) mice between embryonic day (E) 18 and P0 showed decreased rates of neurite outgrowth, and their growth cones had a distinctive narrow morphology compared with those from normal mice. Serial sections of E12.5, E13.5, and E15 mouse brains identified developmental defects in the ventricular neuroepithelium. On E12.5, disruption of the coherent ventricular surface and disordered cell migration of neuroepithelial and differentiated cells were seen at various points in the ventricular walls. These abnormalities resulted in the formation of rosettes in various regions of the brain and spinal cord. On E13.5 and E15, disruption of the ventricular surface and aberrant protrusions of neural cells into the ventricles became more prominent. By E18.5 and P0, the defects in cells lining the ventricular wall resulted in an obstructive hydrocephalus due to stenosis or occlusion of the third ventricle and cerebral aqueduct. These defects may be caused by abnormalities in the cell adhesive properties of neuroepithelial cells and suggest that NMHC-B is essential for both early and late developmental processes in the mammalian brain.

  14. Ferritin heavy chain is a negative regulator of ovarian cancer stem cell expansion and epithelial to mesenchymal transition

    PubMed Central

    Pisanu, Maria Elena; Faniello, Maria Concetta; Jakopin, Žiga; Chiarella, Emanuela; Giovannone, Emilia Dora; Mancini, Rita; Ciliberto, Gennaro

    2016-01-01

    Objectives Ferritin is the major intracellular iron storage protein essential for maintaining the cellular redox status. In recent years ferritin heavy chain (FHC) has been shown to be involved also in the control of cancer cell growth. Analysis of public microarray databases in ovarian cancer revealed a correlation between low FHC expression levels and shorter survival. To better understand the role of FHC in cancer, we have silenced the FHC gene in SKOV3 cells. Results FHC-KO significantly enhanced cell viability and induced a more aggressive behaviour. FHC-silenced cells showed increased ability to form 3D spheroids and enhanced expression of NANOG, OCT4, ALDH and Vimentin. These features were accompanied by augmented expression of SCD1, a major lipid metabolism enzyme. FHC apparently orchestrates part of these changes by regulating a network of miRNAs. Methods FHC-silenced and control shScr SKOV3 cells were monitored for changes in proliferation, migration, ability to propagate as 3D spheroids and for the expression of stem cell and epithelial-to-mesenchymal-transition (EMT) markers. The expression of three miRNAs relevant to spheroid formation or EMT was assessed by q-PCR. Conclusions In this paper we uncover a new function of FHC in the control of cancer stem cells. PMID:27566559

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

    PubMed

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

    2017-01-25

    Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent Afrotheria. Manatee health and resistance to infectious disease is of great concern to conservation groups, but little is known about their immune system. To develop manatee-specific tools for monitoring health, we first must have a general knowledge of how the immunoglobulin heavy (IgH) chain locus is organized and transcriptionally expressed. Using the genomic scaffolds of the Florida manatee (Trichechus manatus latirostris), we characterized the potential IgH segmental diversity and constant region isotypic diversity and performed the first Afrotherian repertoire analysis. The Florida manatee has low V(D)J combinatorial diversity (3744 potential combinations) and few constant region isotypes. They also lack clan III V segments, which may have caused reduced VH segment numbers. However, we found productive somatic hypermutation concentrated in the complementarity determining regions. In conclusion, manatees have limited IGHV clan and combinatorial diversity. This suggests that clan III V segments are essential for maintaining IgH locus diversity.

  16. A catalytic antibody heavy chain HpU-2 degrading its epitope peptide and H. pylori urease.

    PubMed

    Hifumi, Emi; Yamada, Yukiko; Uda, Taizo

    2006-02-28

    The HpU-2 monoclonal antibody (mAb) raised against Helicobacter pylori urease mainly recognized the alpha-subunit of the urease. On the other hand, the heavy chain of HpU-2 mAb (HpU-2-H) isolated from the parent mAb recognized both the alpha- and beta-subunit, in which the beta-subunit was recognized more strongly than the beta-subunit. HpU-2-H cleaved a peptide, SVELIDIGGNRRIFGFNALVD, which is the epitope sequence recognized by HpU-2 mAb, showing a double-phase reaction profile at 25 degrees C in a phosphate buffer. After an induction time of 24h, the cleavage of the peptide was initiated by HpU-2-H at a high rate and it was completed at 80 h of incubation. By mass spectroscopy, two main fragmented peptides, SVELIDIGGNRR and SVELIDIGGNRRIFG, were identified. In addition, many small peptide fragments were produced by successive cleavage of the fragmented peptides. Cleavage tests for H. pylori urease by HpU-2-H revealed that the beta-subunit of the urease was cleaved first and completely decomposed at 20 h of incubation. Cleavage of the alpha-subunit started after the complete decomposition of the beta-subunit. These cleavage results were in good agreement with the immunological features of HpU-2-H. The irrelevant proteins, BSA and HSA, were hardly cleaved by HpU-2-H.

  17. Src-dependent Tyrosine Phosphorylation of Non-muscle Myosin Heavy Chain-IIA Restricts Listeria monocytogenes Cellular Infection*

    PubMed Central

    Almeida, Maria Teresa; Mesquita, Francisco S.; Cruz, Rui; Osório, Hugo; Custódio, Rafael; Brito, Cláudia; Vingadassalom, Didier; Martins, Mariana; Leong, John M.; Holden, David W.; Cabanes, Didier; Sousa, Sandra

    2015-01-01

    Bacterial pathogens often interfere with host tyrosine phosphorylation cascades to control host responses and cause infection. Given the role of tyrosine phosphorylation events in different human infections and our previous results showing the activation of the tyrosine kinase Src upon incubation of cells with Listeria monocytogenes, we searched for novel host proteins undergoing tyrosine phosphorylation upon L. monocytogenes infection. We identify the heavy chain of the non-muscle myosin IIA (NMHC-IIA) as being phosphorylated in a specific tyrosine residue in response to L. monocytogenes infection. We characterize this novel post-translational modification event and show that, upon L. monocytogenes infection, Src phosphorylates NMHC-IIA in a previously uncharacterized tyrosine residue (Tyr-158) located in its motor domain near the ATP-binding site. In addition, we found that other intracellular and extracellular bacterial pathogens trigger NMHC-IIA tyrosine phosphorylation. We demonstrate that NMHC-IIA limits intracellular levels of L. monocytogenes, and this is dependent on the phosphorylation of Tyr-158. Our data suggest a novel mechanism of regulation of NMHC-IIA activity relying on the phosphorylation of Tyr-158 by Src. PMID:25635050

  18. Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama.

    PubMed

    Ledeboer, A M; Bezemer, S; de Hiaard, J J W; Schaffers, I M; Verrips, C T; van Vliet, C; Düsterhöft, E M; Zoon, P; Moineau, S; Frenken, L G J

    2002-06-01

    Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.

  19. Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.

    PubMed Central

    Rindt, H; Knotts, S; Robbins, J

    1995-01-01

    The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7878016

  20. Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.

    PubMed

    Rindt, H; Knotts, S; Robbins, J

    1995-02-28

    The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter.

  1. Transgenic analysis of the thyroid-responsive elements in the alpha-cardiac myosin heavy chain gene promoter.

    PubMed

    Subramaniam, A; Gulick, J; Neumann, J; Knotts, S; Robbins, J

    1993-02-25

    The role of two putative, cis-acting thyroid hormone-responsive elements, TRE1 and TRE2, located at -129 to -149 and -102 to -120, respectively, on the murine alpha-myosin heavy chain (MHC) gene, has been investigated in transgenic mice. These motifs are present in a 4.5-kilobase fragment lying upstream of the transcriptional start site of the mouse alpha-MHC gene: this fragment directs appropriate expression of a reporter gene in transgenic mice (Subramaniam, A., Jones, W. K., Gulick, J., Wert, S., Neumann, J., and Robbins, J. (1991) J. Biol. Chem. 266, 24613-24620). Here, we independently mutate the TRE1 and TRE2 elements by base substitution. The mice were analyzed for transgene expression in different muscle and non-muscle tissues including the atria and ventricles. Normal levels of transgene expression were observed in euthyroid mice carrying a mutation in TRE1. In contrast to these results, mice in which TRE2 was mutated showed reduced levels of CAT activity in both the atria and ventricles, suggesting a previously undefined role for this element in the constitutive up-regulation of the alpha-MHC gene. In hypothyroid mice carrying either of these mutations, the complete cessation of ventricular expression of the chloramphenicol acetyltransferase transcripts that takes place in the alpha-5.5 (wild type) animals did not occur.

  2. Fiber size, type, and myosin heavy chain content in rhesus hindlimb muscles after 2 weeks at 2 G

    NASA Technical Reports Server (NTRS)

    Tavakol, Morteza; Roy, Roland R.; Kim, Jung A.; Zhong, Hui; Hodgson, John A.; Hoban-Higgins, Tana M.; Fuller, Charles A.; Edgerton, V. Reggie

    2002-01-01

    BACKGROUND: Fiber atrophy and an increase in the percentage of fast fibers have been observed in Rhesus leg muscles after spaceflight. Hypothesis: Hypergravity will result in muscle fiber hypertrophy and an increase in the percentage of slow fibers. METHODS: Open muscle biopsies were obtained from Rhesus soleus, medial gastrocnemius (MG), and tibialis anterior (TA) muscles before and after 14 d of centrifugation (2 G) and in time-matched controls. Cage activity levels were measured by telemetry. RESULTS: Based on monoclonal antibody binding for myosin heavy chains (MHC), the fastest region of soleus contained a higher proportion of type I+II (27 vs. 13%) and had a tendency for a lower proportion of type I (38 vs. 61%, p = 0.10) fibers after than before centrifugation. There was a higher proportion of type I+II fibers in post- vs. pre-2 G (10 vs. 0.6%) MG biopsies. Fiber type distribution and MHC composition were unaffected in the TA. Overall, mean fiber sizes were unaffected by centrifugation. Average cage activity levels were 36% lower during than before 2 G. CONCLUSIONS: Our hypothesis was rejected. The changes in the proportion of fibers expressing type I MHC are the reverse of that expected with chronic loading of extensors and, paradoxically, are similar to changes observed with chronic unloading, such as occurs during spaceflight, in this primate model. The data are consistent with the observed decrease in total daily activity levels.

  3. Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignitus.

    PubMed

    Wang, Dong; Kim, Bo Yeon; Lee, Kwang Sik; Yoon, Hyung Joo; Cui, Zheng; Lu, Wei; Jia, Jing Ming; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2009-01-01

    Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5'UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.

  4. In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector

    SciTech Connect

    Wang Shengpeng; Guo Tingqing; Guo Xiuyang; Huang Junting; Lu Changde . E-mail: cdlu@sibs.ac.cn

    2006-03-24

    In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.

  5. Sexually Dimorphic Expression of a Laryngeal-Specific, Androgen-Regulated Myosin Heavy Chain Gene during Xenopus laevis Development

    PubMed Central

    Catz, Diana S.; Fischer, Leslie M.; Moschella, Maria C.; Tobias, Martha L.

    2012-01-01

    Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue. PMID:1426643

  6. Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

    PubMed

    Eizema, Karin; van den Burg, Maarten; Kiri, Arpna; Dingboom, Elizabeth G; van Oudheusden, Hans; Goldspink, Geoffrey; Weijs, Wim A

    2003-09-01

    The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

  7. Ubiquitin Ligase, MuRF-1 regulates myosin heavy chain type IIa transcripts during muscle atrophy under microgravity conditions

    NASA Astrophysics Data System (ADS)

    Kagawa, Sachiko

    Skeletal muscles are vulnerable to marked atrophy under microgravity conditions. We previously reported that gastrocnemius muscle atrophy by spaceflight was specifically sensitive to the ubiquitin-proteasome proteolytic pathway. We also screened more over 26,000 skeletal muscle genes in rats exposed to real weightlessness and found that the expression of Ubiquitin Ligase, Muscle specific Ring Finger-1 (MuRF-1) upregulated under microgravity. In the present study, we examined the role of MuRF-1 in microgravity-induced muscle atrophy. The amounts of MuRF-1 transcripts significantly increased in skeletal muscle after denervation, an in vivo model of microgravity-induced unloading. MuRF-1 deficient (MuRF-1-/-) mice significantly inhibited reduction of muscle weight for muscle atrophy, compared with wild type mice. Interestingly, MuRF-1-/- mice significantly inhibited upregulation of myosin heavy chain (MyHC) type IIa transcrips, while wild type mice significantly increased expression of MyHC type IIa transcripts in denervated skeletal muscle. Our present results suggest that MuRF-1 may play an important role in regulation of MyHC type IIa during muscle atrophy under microgravity conditions.

  8. Role of PTIP in class switch recombination and long-range chromatin interactions at the immunoglobulin heavy chain locus.

    PubMed

    Schwab, Kristopher R; Patel, Sanjeevkumar R; Dressler, Gregory R

    2011-04-01

    How distal transcriptional enhancer sequences interact with proximal promoters is poorly understood within the context of chromatin. In this report, we have used the immunoglobulin heavy chain locus to address the role of the PTIP protein in transcription regulation and class switch recombination in B cells, a process that depends on regulated transcription and DNA recombination via Pax5 and distal 3' enhancer sequences. We first show that PTIP is recruited to a Pax5 binding site to promote histone H3 lysine 4 (H3K4) methylation. Using a CD19-Cre driver strain, we deleted PTIP in mature B cells. Loss of PTIP inhibited class switch recombination by suppressing transcription and histone H3K4 methylation at the germ line transcript promoters. In the absence of PTIP, Pax5 binding to the promoter regions is reduced and long-range chromatin interactions between the distal enhancer at the 3' regulatory region and the germ line transcript promoters are not detected. We propose a model whereby PTIP stabilizes the Pax5 DNA interactions that promote chromatin looping and regulate transcriptional responses needed for class switch recombination.

  9. Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus.

    PubMed

    Zhu, Limin; Vranckx, Roger; Khau Van Kien, Philippe; Lalande, Alain; Boisset, Nicolas; Mathieu, Flavie; Wegman, Mark; Glancy, Luke; Gasc, Jean-Marie; Brunotte, François; Bruneval, Patrick; Wolf, Jean-Eric; Michel, Jean-Baptiste; Jeunemaitre, Xavier

    2006-03-01

    We have recently described two kindreds presenting thoracic aortic aneurysm and/or aortic dissection (TAAD) and patent ductus arteriosus (PDA) and mapped the disease locus to 16p12.2-p13.13 (ref. 3). We now demonstrate that the disease is caused by mutations in the MYH11 gene affecting the C-terminal coiled-coil region of the smooth muscle myosin heavy chain, a specific contractile protein of smooth muscle cells (SMC). All individuals bearing the heterozygous mutations, even if asymptomatic, showed marked aortic stiffness. Examination of pathological aortas showed large areas of medial degeneration with very low SMC content. Abnormal immunological recognition of SM-MHC and the colocalization of wild-type and mutant rod proteins in SMC, in conjunction with differences in their coimmunoprecipitation capacities, strongly suggest a dominant-negative effect. Human MYH11 gene mutations provide the first example of a direct change in a specific SMC protein leading to an inherited arterial disease.

  10. Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles

    PubMed Central

    Xu, Jin; Gao, Jie; Li, Junling; Xue, Liangyi; Clark, Karl J.; Ekker, Stephen C.; Du, Shao Jun

    2014-01-01

    Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles. PMID:22361506

  11. Evolution of the recombination signal sequences in the Ig heavy-chain variable region locus of mammals

    PubMed Central

    Hassanin, Alexandre; Golub, Rachel; Lewis, Susanna M.; Wu, Gillian E.

    2000-01-01

    The Ig and T cell receptor (TCR) loci have an exceptionally dynamic evolutionary history, but the mechanisms responsible remain a subject of speculation. Ig and TCR genes are unique in vertebrates in that they are assembled from V, D, and J segments by site-specific recombination in developing lymphocytes. Here we examine the extent to which the V(D)J recombination in germline cells may have been responsible for remodeling Ig and TCR loci in mammals by asking whether gene segments have evolved as a unit, or whether, instead, recombination signal sequences (RSSs) and coding sequences have different phylogenies. Four distinct types of RSS have been defined in the human Ig heavy-chain variable region (Vh) locus, namely H1, H2, H3, and H5, and no other RSS type has been detected in other mammalian species. There is a well-supported discrepancy between the evolutionary history of the RSSs as compared with the Vh coding sequences: the RSS type H2 of one Vh gene segment has clearly become replaced by a RSS type H3 during mammalian evolution, between 115 and 65 million years ago. Two general models might explain the RSS swap: the first involves an unequal crossing over, and the second implicates germline activation of V(D)J recombination. The Vh-H2/RSS-H3 recombination product has likely been selected during the evolution of mammals because it provides better V(D)J recombination efficiency. PMID:11027341

  12. Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function.

    PubMed Central

    Mogami, K; O'Donnell, P T; Bernstein, S I; Wright, T R; Emerson, C P

    1986-01-01

    Mutations of the myosin heavy-chain (MHC) gene of Drosophila melanogaster were identified among a group of dominant flightless and recessive lethal mutants (map position 2-52, 36A8-B1,2). One mutation is a 0.1-kilobase deletion in the 5' region of the MHC gene and reduces MHC protein in the leg and thoracic muscles of heterozygotes to levels found in 36AC haploids. Three mutations are insertions of 8-to 10-kilobase DNA elements within the MHC gene and produce truncated MHC transcripts. Heterozygotes of these insertional mutations possess levels of MHC intermediate between those of haploids and diploids. An additional mutation has no gross alteration of the MHC gene or its RNA transcripts. Although leg and larval muscles function normally in each mutant heterozygote, indirect flight muscles are defective and possess disorganized myofibrils. Homozygous mutants die during embryonic or larval development and display abnormal muscle function prior to death. These findings provide direct genetic evidence that the MHC gene at 36B (2L) is essential for both larval and adult muscle development and function. The results are consistent with the previous molecular evidence that Drosophila, unlike other organisms, has only a single muscle MHC gene per haploid genome. Quantitative expression of both copies of the MHC gene is required for function of indirect flight muscle, whereas expression of a single MHC gene is sufficient for function of larval muscles and adult tubular muscles. Images PMID:3006049

  13. Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation

    PubMed Central

    Sabbir, Mohammad G.; Dillon, Rachelle; Mowat, Michael R. A.

    2016-01-01

    ABSTRACT The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype. PMID:26977077

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

    USGS Publications Warehouse

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

    2017-01-01

    Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent Afrotheria. Manatee health and resistance to infectious disease is of great concern to conservation groups, but little is known about their immune system. To develop manatee-specific tools for monitoring health, we first must have a general knowledge of how the immunoglobulin heavy (IgH) chain locus is organized and transcriptionally expressed. Using the genomic scaffolds of the Florida manatee (Trichechus manatus latirostris), we characterized the potential IgH segmental diversity and constant region isotypic diversity and performed the first Afrotherian repertoire analysis. The Florida manatee has low V(D)J combinatorial diversity (3744 potential combinations) and few constant region isotypes. They also lack clan III V segments, which may have caused reduced VH segment numbers. However, we found productive somatic hypermutation concentrated in the complementarity determining regions. In conclusion, manatees have limited IGHV clan and combinatorial diversity. This suggests that clan III V segments are essential for maintaining IgH locus diversity.

  15. Synergistic ablation does not affect atrophy or altered myosin heavy chain expression in the non-weight bearing soleus muscle

    NASA Technical Reports Server (NTRS)

    Linderman, J. K.; Talmadge, R. J.; Gosselink, K. L.; Tri, P. N.; Roy, R. R.; Grindeland, R. E.

    1996-01-01

    The purpose of this study was to investigate whether the soleus muscle undergoes atrophy and alterations in myosin heavy chain (MHC) composition during non-weight bearing in the absence of synergists. Thirty-two female rats were randomly assigned to four groups: control (C), synergistic ablation (ABL) of the gastrocnemius and plantaris muscles to overload the soleus muscle, hindlimb suspension (HLS), or a combination of synergistic ablation and hindlimb suspension (HLS-ABL). After 28 days of hindlimb suspension, soleus atrophy was more pronounced in HLS (58%) than in HLS-ABL (43%) rats. Compared to C rats, non-weight bearing decreased mixed and myofibrillar protein contents and Type I MHC 49%, 45%, and 7%, respectively, in HLS animals. In addition, de novo expression of fast Type IIx and Type IIb MHC (5% and 2%, respectively) was observed in HLS animals. Similarly, when compared to C rats, mixed and myofibrillar protein contents and Type I MHC decreased 43%, 46%, and 4%, respectively, in HLS-ABL animals. Also, de novo expression of Type IIx (4%) and IIb (1%) MHC was observed. Collectively, these data indicate that the loss of muscle protein and Type I MHC, and the de novo expression of Type IIx and Type IIb MHC in the rat soleus occur independently of the presence of synergists during non-weight bearing. Furthermore, these results confirm the contention that soleus mass and MHC expression are highly sensitive to alterations in mechanical load.

  16. Development and evaluation of polyclonal antisera for detection of the IgM heavy chain of multiple fish species.

    PubMed

    Jirapongpairoj, Walissara; Hirono, Ikuo; Kondo, Hidehiro

    2017-10-01

    Antibodies are widely considered to be essential tools for detection of immune responses in various fish species. Here we produced the peptide polyclonal antisera (anti-fish IgH-1 and anti-fish IgH-2) to detect IgM of various fish species. The peptides were designed based on the conserved sequence of the fish immunoglobulin heavy chains of seven fish species (Japanese flounder, seabream, yellowtail, carp, rainbow trout, hybrid sturgeon and banded houndshark). By Western blotting, anti-fish IgH-1 antiserum detected the IgMs of all fish species except banded houndshark. Anti-fish IgH-2 antiserum clearly reacted with the IgMs of only three of the fish species (seabream, yellowtail and rainbow trout). Attempts to use the antisera to measure fish antibody titer by ELISA were unsuccessful. These results demonstrate that anti-fish IgH-1 peptide polyclonal antiserum is a potentially applicable tool for detecting immunoglobulins in various fish species by Western blotting. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Structure and expression of mouse germline immunoglobulin gamma 3 heavy chain transcripts induced by the mitogen lipopolysaccharide.

    PubMed

    Gerondakis, S; Gaff, C; Goodman, D J; Grumont, R J

    1991-01-01

    Germline immunoglobulin heavy chain gene transcription is though to direct isotype switching by modulating the accessibility of specific switch regions to a recombinase. In this study, cloned cDNA copies of mouse germline Igh-8 RNAs have been used to characterize the Igh-8 transcription unit. The 5' end of these transcripts are derived from an exon denoted Ig3, located 1 kilobase 5' of the Igh-8 switch region. Sequence analysis of cDNA and genomic clones reveals that these RNAs are noncoding. In splenic B cell cultures treated with lipopolysaccharide (LPS), germline Igh-8 transcript levels are upregulated after 8 h due to increased transcription. This induction is consistent with the identification of a putative binding site for the LPS inducible transcription factor NF-kappa B approximately 150 nucleotides upstream of the sites of transcript initiation. Furthermore, nucleotide sequence comparisons reveal that the region encompassing the site of germline Igh-8 transcription initiation is highly homologous to part of the Ig2b exon, and is also conserved upstream of the Igh-1 switch region. The implications of these findings for the control of germline Igh-8 transcription is discussed.

  18. Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles.

    PubMed

    Xu, Jin; Gao, Jie; Li, Junling; Xue, Liangyi; Clark, Karl J; Ekker, Stephen C; Du, Shao Jun

    2012-02-01

    Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.

  19. bHLH transcription factor MyoD affects myosin heavy chain expression pattern in a muscle-specific fashion.

    PubMed

    Seward, D J; Haney, J C; Rudnicki, M A; Swoap, S J

    2001-02-01

    A strong correlative pattern between MyoD gene expression and myosin heavy chain IIB (MHC IIB) gene expression exists. To test whether this correlative relationship is causative, MHC gene expression in muscles from MyoD(-/-) mice was analyzed. The MHC IIB gene was not detectable in the MyoD(-/-) diaphragm, whereas the MHC IIB protein made up 10.0 +/- 1.7% of the MHC protein pool in the wild-type (WT) mouse diaphragm. Furthermore, the MHC IIA protein was not detectable in the MyoD(-/-) biceps brachii, and the MHC IIB protein was overexpressed in the masseter. To examine whether MyoD is required for the upregulation of the MHC IIB gene within slow muscle after disuse, MyoD(-/-) and WT hindlimb musculature was unweighted. MyoD(-/-) exhibited a diminished response in the upregulation of the MHC IIB mRNA within the soleus muscle as a result of the hindlimb unweighting. Collectively, these data suggest that MyoD plays a role in the MHC profile in a muscle-specific fashion.

  20. Automated muscle fiber type population analysis with ImageJ of whole rat muscles using rapid myosin heavy chain immunohistochemistry.

    PubMed

    Bergmeister, Konstantin D; Gröger, Marion; Aman, Martin; Willensdorfer, Anna; Manzano-Szalai, Krisztina; Salminger, Stefan; Aszmann, Oskar C

    2016-08-01

    Skeletal muscle consists of different fiber types which adapt to exercise, aging, disease, or trauma. Here we present a protocol for fast staining, automatic acquisition, and quantification of fiber populations with ImageJ. Biceps and lumbrical muscles were harvested from Sprague-Dawley rats. Quadruple immunohistochemical staining was performed on single sections using antibodies against myosin heavy chains and secondary fluorescent antibodies. Slides were scanned automatically with a slide scanner. Manual and automatic analyses were performed and compared statistically. The protocol provided rapid and reliable staining for automated image acquisition. Analyses between manual and automatic data indicated Pearson correlation coefficients for biceps of 0.645-0.841 and 0.564-0.673 for lumbrical muscles. Relative fiber populations were accurate to a degree of ± 4%. This protocol provides a reliable tool for quantification of muscle fiber populations. Using freely available software, it decreases the required time to analyze whole muscle sections. Muscle Nerve 54: 292-299, 2016. © 2016 Wiley Periodicals, Inc.

  1. Effects of myosin heavy chain (MHC) plasticity induced by HMGCoA-reductase inhibition on skeletal muscle functions.

    PubMed

    Trapani, Laura; Melli, Luca; Segatto, Marco; Trezza, Viviana; Campolongo, Patrizia; Jozwiak, Adam; Swiezewska, Ewa; Pucillo, Leopoldo Paolo; Moreno, Sandra; Fanelli, Francesca; Linari, Marco; Pallottini, Valentina

    2011-11-01

    The rate-limiting step of cholesterol biosynthetic pathway is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme reductase (HGMR), whose inhibitors, the statins, widely used in clinical practice to treat hypercholesterolemia, often cause myopathy, and rarely rhabdomyolysis. All studies to date are limited to the definition of statin-induced myotoxicity omitting to investigate whether and how HMGR inhibition influences muscle functions. To this end, 3-mo-old male rats (Rattus norvegicus) were treated for 3 wk with a daily intraperitoneal injection of simvastatin (1.5 mg/kg/d), and biochemical, morphological, mechanical, and functional analysis were performed on extensor digitorum longus (EDL) muscle. Our results show that EDL muscles from simvastatin-treated rats exhibited reduced HMGR activity; a 15% shift from the fastest myosin heavy-chain (MHC) isoform IIb to the slower IIa/x; and reduced power output and unloaded shortening velocity, by 41 and 23%, respectively, without any change in isometric force and endurance. Moreover, simvastatin-treated rats showed a decrease of maximum speed reached and the latency to fall off the rotaroad (∼-30%). These results indicate that the molecular mechanism of the impaired muscle function following statin treatment could be related to the plasticity of fast MHC isoform expression.

  2. Characterization of a complete immunoglobulin heavy-chain variable region germ-line gene of rainbow trout.

    PubMed

    Matsunaga, T; Chen, T; Törmänen, V

    1990-10-01

    A germ-line heavy-chain variable region (VH) gene (RTVH431) has been isolated from a rainbow trout (Salmo gairdneri) and characterized by complete nucleotide sequencing. It is characteristic of VH, as shown by the conserved octamer and TATA motif in the 5' region, the heptamernonamer recombination signal sequence in the 3' region, and the 18-amino-acid-long hydrophobic leader interrupted by an intron. The 98-amino-acid-long VH coding region has 50-70% nucleotide sequence homology and 40-60% amino acid sequence homology with VHS of various vertebrate species. We have also found unique or species-specific amino acid residues in the VHS of rainbow trout, amphibia (Xenopus), reptile (Caiman), and shark (Heterodontus) in our sequence analyses. The RTVH431 has an unusual amino acid in the conserved 34th position in complementarity-determining region 1 of VH. Southern hybridization results suggest the presence of a large gene family related to RTVH431 in the trout genome. The complex evolution of antibody V genes is discussed.

  3. Somatic variation precedes extensive diversification of germline sequences and combinatorial joining in the evolution of immunoglobulin heavy chain diversity

    PubMed Central

    1993-01-01

    In Heterodontus, a phylogenetically primitive shark species, the variable (VH), diversity (DH), joining (JH) segments, and constant (CH) exons are organized in individual approximately 18-20-kb "clusters." A single large VH family with > 90% nucleic acid homology and a monotypic second gene family are identified by extensive screening of a genomic DNA library. Little variation in the nucleotide sequences of DH segments from different germline gene clusters is evident, suggesting that the early role for DH was in promoting junctional diversity rather than contributing unique coding specificities. A gene-specific oligodeoxynucleotide screening method was used to relate specific transcription products (cDNAs) to individual gene clusters and showed that gene rearrangements are intra- rather than intercluster. This provides further evidence for restricted diversity in the immunoglobulin heavy chain of Heterodontus, from which it is inferred that combinatorial diversity is a more recently acquired means for generating diversity. The observed differences between cDNA sequences selected and the sequences of segmental elements derived from conventional genomic libraries as well as from VH segment-specific libraries generated by direct PCR amplification of genomic DNA indicate that the VH repertoire is diversified by both junctional diversity and somatic mutation. Taken together, these findings suggest a heretofore unrecognized contribution of somatic variation that preceded both extensive diversification of the germline repertoire and the combinatorial joining process in the evolution of humoral immunity. PMID:8350055

  4. Somatic variation precedes extensive diversification of germline sequences and combinatorial joining in the evolution of immunoglobulin heavy chain diversity.

    PubMed

    Hinds-Frey, K R; Nishikata, H; Litman, R T; Litman, G W

    1993-09-01

    In Heterodontus, a phylogenetically primitive shark species, the variable (VH), diversity (DH), joining (JH) segments, and constant (CH) exons are organized in individual approximately 18-20-kb "clusters." A single large VH family with > 90% nucleic acid homology and a monotypic second gene family are identified by extensive screening of a genomic DNA library. Little variation in the nucleotide sequences of DH segments from different germline gene clusters is evident, suggesting that the early role for DH was in promoting junctional diversity rather than contributing unique coding specificities. A gene-specific oligodeoxynucleotide screening method was used to relate specific transcription products (cDNAs) to individual gene clusters and showed that gene rearrangements are intra- rather than intercluster. This provides further evidence for restricted diversity in the immunoglobulin heavy chain of Heterodontus, from which it is inferred that combinatorial diversity is a more recently acquired means for generating diversity. The observed differences between cDNA sequences selected and the sequences of segmental elements derived from conventional genomic libraries as well as from VH segment-specific libraries generated by direct PCR amplification of genomic DNA indicate that the VH repertoire is diversified by both junctional diversity and somatic mutation. Taken together, these findings suggest a heretofore unrecognized contribution of somatic variation that preceded both extensive diversification of the germline repertoire and the combinatorial joining process in the evolution of humoral immunity.

  5. Characterization of a complete immunoglobulin heavy-chain variable region germ-line gene of rainbow trout.

    PubMed Central

    Matsunaga, T; Chen, T; Törmänen, V

    1990-01-01

    A germ-line heavy-chain variable region (VH) gene (RTVH431) has been isolated from a rainbow trout (Salmo gairdneri) and characterized by complete nucleotide sequencing. It is characteristic of VH, as shown by the conserved octamer and TATA motif in the 5' region, the heptamernonamer recombination signal sequence in the 3' region, and the 18-amino-acid-long hydrophobic leader interrupted by an intron. The 98-amino-acid-long VH coding region has 50-70% nucleotide sequence homology and 40-60% amino acid sequence homology with VHS of various vertebrate species. We have also found unique or species-specific amino acid residues in the VHS of rainbow trout, amphibia (Xenopus), reptile (Caiman), and shark (Heterodontus) in our sequence analyses. The RTVH431 has an unusual amino acid in the conserved 34th position in complementarity-determining region 1 of VH. Southern hybridization results suggest the presence of a large gene family related to RTVH431 in the trout genome. The complex evolution of antibody V genes is discussed. Images PMID:2120708

  6. Sulfasalazine Treatment Suppresses the Formation of HLA-B27 Heavy Chain Homodimer in Patients with Ankylosing Spondylitis.

    PubMed

    Yu, Hui-Chun; Lu, Ming-Chi; Huang, Kuang-Yung; Huang, Hsien-Lu; Liu, Su-Qin; Huang, Hsien-Bin; Lai, Ning-Sheng

    2015-12-29

    Human leukocytic antigen-B27 heavy chain (HLA-B27 HC) has the tendency to fold slowly, in turn gradually forming a homodimer, (B27-HC)₂ via a disulfide linkage to activate killer cells and T-helper 17 cells and inducing endoplasmic reticulum (ER) stress to trigger the IL-23/IL-17 axis for pro-inflammatory reactions. All these consequences lead to the pathogenesis of ankylosing spondylitis (AS). Sulfasalazine (SSA) is a common medication used for treatment of patients with AS. However, the effects of SSA treatment on (B27-HC)₂ formation and on suppression of IL-23/IL-17 axis of AS patients remain to be determined. In the current study, we examine the (B27-HC)₂ of peripheral blood mononuclear cells (PBMC), the mean grade of sarcoiliitis and lumbar spine Bath Ankylosing Spondylitis Radiology Index (BASRI) scores of 23 AS patients. The results indicated that AS patients without (B27-HC)₂ on PBMC showed the lower levels of mean grade of sarcoiliitis and the lumbar spine BASRI scores. In addition, after treatment with SSA for four months, the levels of (B27-HC)₂ on PBMCs were significantly reduced. Cytokines mRNA levels, including TNFα, IL-17A, IL-17F and IFNγ, were also significantly down-regulated in PBMCs. However, SSA treatment did not affect the levels of IL-23 and IL-23R mRNAs.

  7. Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) MHC class I heavy chain and β2-microglobulin.

    PubMed

    Pinto, Rute D; Randelli, Elisa; Buonocore, Francesco; Pereira, Pedro J B; dos Santos, Nuno M S

    2013-03-01

    In this work, the gene and cDNA of sea bass (Dicentrarchus labrax) β2-microglobulin (Dila-β2m) and several cDNAs of MHC class I heavy chain (Dila-UA) were characterized. While Dila-β2m is single-copy, numerous Dila-UA transcripts were identified per individual with variability at the peptide-binding domain (PBD), but also with unexpected diversity from the connective peptide (CP) through the 3' untranslated region (UTR). Phylogenetic analysis segregates Dila-β2m and Dila-UA into each subfamily cluster, placing them in the fish class and branching Dila-MHC-I with lineage U. The α1 domains resemble those of the recently proposed L1 trans-species lineage. Although no Dila-specific α1, α2 or α3 sub-lineages could be observed, two highly distinct sub-lineages were identified at the CP/TM/CYT regions. The three-dimensional homology model of sea bass MHC-I complex is consistent with other characterized vertebrate structures. Furthermore, basal tissue-specific expression profiles were determined for both molecules, and expression of β2m was evaluated after poly I:C stimulus. Results suggest these molecules are orthologues of other β2m and teleost classical MHC-I and their basic structure is evolutionarily conserved, providing relevant information for further studies on antigen presentation in this fish species.

  8. Heat-Stress effects on the myosin heavy chain phenotype of rat soleus fibers during the early stages of regeneration.

    PubMed

    Oishi, Yasuharu; Roy, Roland R; Ogata, Tomonori; Ohira, Yoshinobu

    2015-12-01

    We investigated heat-stress effects on the adult myosin heavy chain (MyHC) profile of soleus muscle fibers at an early stage of regeneration. Regenerating fibers in adult rats were analyzed 2, 4, or 6 days after bupivacaine injection. Rats were heat stressed by immersion in water (42 ± 1°C) for 30 minutes 24 hours after bupivacaine injection and every other day thereafter. No adult MyHC isoforms were observed after 2 days, whereas some fibers expressed only fast MyHC after 4 days. Heat stress increased fast and slow MyHC in regenerating fibers after 6 days. Regenerating fibers expressing only slow MyHC were observed only in heat-stressed muscles. Bupivacaine injection increased the number of Pax7(+) and MyoD(+) satellite cells in regenerating fibers, more so in heat-stressed rats. The results indicate that heat stress accelerates fast-to-slow MyHC phenotype conversion in regenerating fibers via activation of satellite cells. © 2015 Wiley Periodicals, Inc.

  9. The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain.

    PubMed

    Ducret, Axel; Nguyen, Mai; Breckenridge, David G; Shore, Gordon C

    2003-01-01

    BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase-8. Recently, we reported that the caspase-resistant BAP31 inhibited Fas-mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731-6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle gamma-actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis.

  10. High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

    PubMed

    Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

    2013-01-01

    Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

  11. Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses.

    PubMed

    Ferrari, A; Weill, F S; Paz, M L; Cela, E M; González Maglio, D H; Leoni, J

    2012-03-01

    Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a β-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.

  12. Multiple amino acid substitutions between murine gamma 2a heavy chain Fc regions of Ig1a and Ig1b allotypic forms.

    PubMed Central

    Dognin, M J; Lauwereys, M; Strosberg, A D

    1981-01-01

    The complete amino acid sequence of the murine gamma 2a heavy chain CBPC-101 Fc region of allotype Ig1b was determined by automated and manual Edman degradation procedures. Both chemical and enzymatic cleavages were used to obtain peptides which were purified by gel filtration followed by high-pressure liquid chromatography. The sequence was in good agreement with that predicted from the nucleotide sequence of a gamma 2a gene of allotype Ig1b except for two positions. Comparison with published data on MOPC 173 gamma 2a heavy chain of allotype Ig1a revealed 8 amino acid substitutions in the CH2 domain (7% differences) and 18 substitutions (27%) in the CH3 domain. Many of the observed interchanges occur at positions at which murine heavy chains of other classes also differ from the gamma 2a chains. Our data suggest that the divergence of the gamma 2a Ig1a gene found in BALB/c mice and of the gamma 2a Ig1b gene found in CB 20 mice must have occurred long ago and that the CH2 domain was much more conserved than the CH3 domain. PMID:6794027

  13. Identification and characterization of a novel member of the heterodimeric amino acid transporter family presumed to be associated with an unknown heavy chain.

    PubMed

    Chairoungdua, A; Kanai, Y; Matsuo, H; Inatomi, J; Kim, D K; Endou, H

    2001-12-28

    We identified a novel amino acid transporter designated Asc-2 (for asc-type amino acid transporter 2). Asc-2 exhibited relatively low but significant sequence similarity to the members of the heterodimeric amino acid transporters. The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits in the heterodimeric amino acid transporters is conserved for Asc-2. Asc-2 is, however, not colocalized with the already known heavy chains such as 4F2 heavy chain (4F2hc) or related to b(0,+) amino acid transporter (rBAT) in mouse kidney. Because Asc-2 solely expressed or coexpressed with 4F2hc or rBAT did not induce functional activity, we generated fusion proteins in which Asc-2 is connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the function corresponding to the Na(+)-independent small neutral amino acid transport system asc. Distinct from the already identified system asc transporter Asc-1 which is associated with 4F2hc, Asc-2-mediated transport is less stereoselective and did not accept some of the high affinity substrates of Asc-1 such as alpha-aminoisobutyric acid and beta-alanine. Asc-2 message was detected in kidney, placenta, spleen, lung, and skeletal muscle. In kidney, Asc-2 protein was present in the epithelial cells lining collecting ducts. In the Western blot analysis on mouse erythrocytes and kidney, Asc-2 was detected as multiple bands in the nonreducing condition, whereas the bands shifted to a single band at lower molecular weight, suggesting the association of Asc-2 with other protein(s) via a disulfide bond. The finding of Asc-2 would lead to the establishment of a new subgroup of heterodimeric amino acid transporter family which includes transporters associated not with 4F2hc or rBAT but with other unknown heavy chains.

  14. Tumor necrosis factor and immune interferon synergistically increase transcription of HLA class I heavy- and light-chain genes in vascular endothelium

    SciTech Connect

    Johnson, D.R.; Pober, J.S. )

    1990-07-01

    Tumor necrosis factor and immune interferon synergistically increase cell-surface expression of class I major histocompatibility complex molecules in cultured human endothelial cells. The authors report that tumor necrosis factor and interferon {gamma} each independently increase mRNA levels and together cause a greater-than-additive (i.e., synergistic) increase in steady-state mRNA levels and transcriptional rates of the class I heavy- and light-chain genes. HLA heavy-chain mRNA is equally stable in cytokine-treated and -untreated endothelial cells. Interferon {gamma} does not increase tumor necrosis factor receptor number or affinity on human endothelial cells. They conclude that the synergistic increase in class I major histocompatibility complex cell-surface expression results principally from the synergistic increase in transcriptional rates. They propose that this increase is caused by the cooperative binding of independently activated transcription factors to the promoter/enhancer sequences of class I genes.

  15. Sequence analysis of cDNAs encoding the heavy and light chain variable regions of a WSSV-neutralizing monoclonal antibody.

    PubMed

    Natividad, Karlo Dante T; Nomura, Nakao; Matsumura, Masatoshi

    2008-12-01

    Antibodies raised against individual viral envelope proteins have been used in white spot syndrome virus (WSSV) neutralization assays. We report here the sequence analysis of cDNAs encoding the variable regions of a novel monoclonal antibody that binds to the viral envelope protein and neutralizes WSSV infection. The heavy and light variable chains are most homologous to VH7183 germline gene (AF120472) and IgVk RF germline gene (AJ235936), respectively. Database searches using the derived sequences predicted residues comprising CDR loops. The 12 amino acid residue of the heavy chain CDR3 is rich in negatively charged aspartic acid (25%) and did not show significant homology to any murine V gene available on the database. This study provides insights on the paratope-epitope interaction and can be used to identify compounds with comparable properties as the paratope leading to future development of drugs and vaccines for WSSV infection.

  16. The third P-loop domain in cytoplasmic dynein heavy chain is essential for dynein motor function and ATP-sensitive microtubule binding.

    PubMed

    Silvanovich, Andre; Li, Min-Gang; Serr, Madeline; Mische, Sarah; Hays, Thomas S

    2003-04-01

    Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.

  17. Development of a Bioinformatics Framework for the Detection of Gene Conversion and the Analysis of Combinatorial Diversity in Immunoglobulin Heavy Chains in Four Cattle Breeds

    PubMed Central

    Czerny, Claus-Peter; König, Sven; Diesterbeck, Ulrike S.

    2016-01-01

    We have developed a new bioinformatics framework for the analysis of rearranged bovine heavy chain immunoglobulin (Ig) variable regions by combining and refining widely used alignment algorithms. This bioinformatics framework allowed us to investigate alignments of heavy chain framework regions (FRHs) and the separate alignments of FRHs and heavy chain complementarity determining regions (CDRHs) to determine their germline origin in the four cattle breeds Aubrac, German Black Pied, German Simmental, and Holstein Friesian. Now it is also possible to specifically analyze Ig heavy chains possessing exceptionally long CDR3Hs. In order to gain more insight into breed specific differences in Ig combinatorial diversity, somatic hypermutations and putative gene conversions of IgG, we compared the dominantly transcribed variable (IGHV), diversity (IGHD), and joining (IGHJ) segments and their recombination in the four cattle breeds. The analysis revealed the use of 15 different IGHV segments, 21 IGHD segments, and two IGHJ segments with significant different transcription levels within the breeds. Furthermore, there are preferred rearrangements within the three groups of CDR3H lengths. In the sequences of group 2 (CDR3H lengths (L) of 11–47 amino acid residues (aa)) a higher number of recombination was observed than in sequences of group 1 (L≤10 aa) and 3 (L≥48 aa). The combinatorial diversity of germline IGHV, IGHD, and IGHJ-segments revealed 162 rearrangements that were significantly different. The few preferably rearranged gene segments within group 3 CDR3H regions may indicate specialized antibodies because this length is unique in cattle. The most important finding of this study, which was enabled by using the bioinformatics framework, is the discovery of strong evidence for gene conversion as a rare event using pseudogenes fulfilling all definitions for this particular diversification mechanism. PMID:27828971

  18. Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail

    PubMed Central

    1987-01-01

    We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity. PMID:3040773

  19. Isolation and Characterization of the Promoter and Partial Enhancer Region of the Porcine Inter-α-Trypsin Inhibitor Heavy Chain 4 Gene

    PubMed Central

    Harraghy, Niamh; Mitchell, Timothy J.

    2005-01-01

    A porcine genomic library was screened for clones containing the promoter of the major acute-phase protein in pigs, inter-α-trypsin heavy chain 4 (ITIH4). Following isolation of the promoter, a functional analysis was performed with Hep3B cells. The promoter was induced by interleukin-6 (IL-6) but not by IL-1β. However, IL-1β was shown to inhibit the IL-6-induced activation of the porcine ITIH4 promoter. PMID:16275952

  20. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    PubMed

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  1. The T suppressor cell alloantigen Tsud maps near immunoglobulin allotype genes and may be an heavy chain constant-region marker on a T cell receptor

    PubMed Central

    1981-01-01

    The mouse T cell alloantigen, Tsud, is expressed on a minority of mature, Lyt-2+ cells, and its expression is controlled by a gene linked to the immunoglobulin heavy chain gene cluster, Igh. Tsud can be assayed by immunofluorescence staining with an antiserum made in BALB/c mice against C.AL-20 concanavalin A blasts. This antiserum can also be used to induced T suppressor cells in mice expressing Tsu(d). Both of these assays were used to type several panels of recombinant inbred strains and Igh recombinant strains to accurately map the Tsu(d) locus. The Tsu(d) gene is located very near the heavy chain constant-region genes, Igh-C, on the side toward the prealbumin gene, Pre-1. Tsu(d) is not among the heavy chain variable-region genes, Igh-V, and thus is not a variable-region framework allotype, subgroup determinant, or idiotype. The map position suggest that the Tsu(d) antigen is a constant region allotypic determinant on the as yet uncharacterized T cell receptor. PMID:6454740

  2. Aberrant splicing and expression of the non muscle myosin heavy-chain gene MYH14 in DM1 muscle tissues.

    PubMed

    Rinaldi, F; Terracciano, C; Pisani, V; Massa, R; Loro, E; Vergani, L; Di Girolamo, S; Angelini, C; Gourdon, G; Novelli, G; Botta, A

    2012-01-01

    Myotonic dystrophy type 1 (DM1) is a complex multisystemic disorder caused by an expansion of a CTG repeat located at the 3' untranslated region (UTR) of DMPK on chromosome 19q13.3. Aberrant messenger RNA (mRNA) splicing of several genes has been reported to explain some of the symptoms of DM1 including insulin resistance, muscle wasting and myotonia. In this paper we analyzed the expression of the MYH14 mRNA and protein in the muscle of DM1 patients (n=12) with different expansion lengths and normal subjects (n=7). The MYH14 gene is located on chromosome 19q13.3 and encodes for one of the heavy chains of the so called class II "nonmuscle" myosins (NMHCII). MYH14 has two alternative spliced isoforms: the inserted isoform (NMHCII-C1) which includes 8 amino acids located in the globular head of the protein, not encoded by the non inserted isoform (NMHCII-C0). Results showed a splicing unbalance of the MYH14 gene in DM1 muscle, with a prevalent expression of the NMHCII-C0 isoform more marked in DM1 patients harboring large CTG expansions. Minigene assay indicated that levels of the MBNL1 protein positively regulates the inclusion of the MYH14 exon 6. Quantitative analysis of the MYH14 expression revealed a significant reduction in the DM1 muscle samples, both at mRNA and protein level. No differences were found between DM1 and controls in the skeletal muscle localization of MYH14, obtained through immunofluorescence analysis. In line with the thesis of an "RNA gain of function" hypothesis described for the CTG mutation, we conclude that the alterations of the MYH14 gene may contribute to the DM1 molecular pathogenesis.

  3. Effects of spaceflight on myosin heavy-chain content, fibre morphology and succinate dehydrogenase activity in rat diaphragm.

    PubMed

    Hansen, Gregory; Martinuk, Karen J B; Bell, Gordon J; MacLean, Ian M; Martin, Thomas P; Putman, Charles T

    2004-05-01

    The present study examined the effect of 14 days of exposure to microgravity during the Spacelab Life Sciences-2 (SLS-2) space shuttle mission on the myosin heavy-chain (MHC) content, fibre size and type distributions and metabolic properties of rat diaphragm. Five adult male Sprague-Dawley rats were exposed to 14 days of microgravity (SF, spaceflight) and compared to five ground-based controls (C). Immunohistochemical analyses using isoform-specific anti-MHC monoclonal antibodies revealed that 14 days of SF did not alter the proportions of type-I, -IIA, -IID/X or -IIB fibres within the crural, sternal or lateral costal regions of the diaphragm; the electrophoretically quantified MHC-isoform contents also remained unchanged. In contrast, the medial gastrocnemius (MG) and tibialis anterior (TA) muscles displayed slow-to-fast fibre type transitions: within the MG the proportion of type-IID/X fibres was reduced by 59% ( P<0.04) and corresponded to a 51% increase ( P<0.03) in type-IIB fibres. Within the TA, the sum of type-IID/X+IIB fibres was elevated by 24% ( P<0.02) at the expense of the slower type-IIA fibres, which decreased by 33% ( P<0.04). Electrophoretic analyses yielded qualitatively similar patterns of transformation. SF did not induce atrophic changes within the diaphragm, MG or TA. Succinate dehydrogenase activity remained unchanged in the crural diaphragm ( P>0.96) but was 34% lower ( P<0.0001) in the TA. We conclude that 14 days of SF did not alter structural or metabolic factors that are known to underlie functional properties of the diaphragm. The findings of the present study show that 14 days of SF does not induce deleterious adaptive changes in the rat diaphragm that occur in hindlimb muscles.

  4. Cold exposure increases slow-type myosin heavy chain 1 (MyHC1) composition of soleus muscle in rats.

    PubMed

    Mizunoya, Wataru; Iwamoto, Yohei; Sato, Yusuke; Tatsumi, Ryuichi; Ikeuchi, Yoshihide

    2014-03-01

    The aim of this study was to examine the effects of cold exposure on rat skeletal muscle fiber type, according to myosin heavy chain (MyHC) isoform and metabolism-related factors. Male Wistar rats (7 weeks old) were housed individually at 4 ± 2°C as a cold-exposed group or at room temperature (22 ± 2°C) as a control group for 4 weeks. We found that cold exposure significantly increased the slow-type MyHC1 content in the soleus muscle (a typical slow-type fiber), while the intermediate-type MyHC2A content was significantly decreased. In contrast to soleus, MyHC composition of extensor digitorum longus (EDL, a typical fast-type fiber) and gastrocnemius (a mix of slow-type and fast-type fibers) muscle did not change from cold exposure. Cold exposure increased mRNA expression of mitochondrial uncoupling protein 3 (UCP3) in both the soleus and EDL. Cold exposure also increased mRNA expression of myoglobin, peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and forkhead box O1 (FOXO1) in the soleus. Upregulation of UCP3 and PGC1α proteins were observed with Western blotting in the gastrocnemius. Thus, cold exposure increased metabolism-related factors in all muscle types that were tested, but MyHC isoforms changed only in the soleus. © 2013 Japanese Society of Animal Science.

  5. Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila.

    PubMed

    Swank, D M; Wells, L; Kronert, W A; Morrill, G E; Bernstein, S I

    2000-09-15

    Drosophila melanogaster is an excellent system for examining the structure/function relationships of myosin. It yields insights into the roles of myosin in assembly and stability of myofibrils, in defining the mechanical properties of muscle fibers, and in dictating locomotory abilities. Drosophila has a single gene encoding muscle myosin heavy chain (MHC), with alternative RNA splicing resulting in stage- and tissue-specific isoform production. Localization of the alternative domains of Drosophila MHC on a three-dimensional molecular model suggests how they may determine functional differences between isoforms. We are testing these predictions directly by using biophysical and biochemical techniques to characterize myosin isolated from transgenic organisms. Null and missense mutations help define specific amino acid residues important in actin binding and ATP hydrolysis and the function of MHC in thick filament and myofibril assembly. Insights into the interaction of thick and thin filaments result from studying mutations in MHC that suppress ultrastructural defects induced by a troponin I mutation. Analysis of transgenic organisms expressing engineered versions of MHC shows that the native isoform of myosin is not critical for myofibril assembly but is essential for muscle function and maintenance of muscle integrity. We show that the C-terminus of MHC plays a pivotal role in the maintenance of muscle integrity. Transgenic studies using headless myosin reveal that the head is important for some, but not all, aspects of myofibril assembly. The integrative approach described here provides a multi-level understanding of the function of the myosin molecular motor. Copyright 2000 Wiley-Liss, Inc.

  6. Severe atrophy of slow myofibers in aging muscle is concealed by myosin heavy chain co-expression.

    PubMed

    Purves-Smith, Fennigje M; Solbak, Nathan M; Rowan, Sharon L; Hepple, Russell T

    2012-12-01

    Although slow myofibers are considered less susceptible to atrophy with aging, slow fiber atrophy may have been underestimated previously. First, the marked atrophy of the aging rat soleus (Sol) muscle cannot be explained by the atrophy of only the fast fibers, due to their low abundance. Second, the increase in small fibers co-expressing both fast and slow myosin heavy chains (MHC) in the aging rat Sol is proportional to a decline in pure MHC slow fibers (Snow et al., 2005), suggesting that these MHC co-expressing fibers represent formerly pure slow fibers. Thus, we examined the size and proportion of MHC slow, MHC fast, and MHC fast-slow co-expressing fibers in the Sol and mixed region of the gastrocnemius (Gas) muscle in young adult (YA) and senescent (SEN) rats. Our results suggest that formerly pure MHC slow fibers are the source of MHC co-expressing fibers with aging in both muscle regions. Accounting for the atrophy of these fibers in calculating MHC slow fiber atrophy with aging revealed that MHC slow fibers atrophy on average by 40% in the Sol and by 38% in the mixed Gas, values which are similar to the 60% and 31% atrophy of pure MHC fast fibers in the Sol and mixed Gas, respectively. Probing for the atrophy-dependent ubiquitin ligase, MAFbx (atrogin 1), it was suggested that former slow fibers acquire atrophy potential via the up-regulation of MAFbx coincident with the co-expression of fast MHC. These results redefine the impact of aging on slow fiber atrophy, and emphasize the necessity of addressing the atrophy of fast and slow fibers in seeking treatments for aging muscle atrophy.

  7. Inhibition of Proliferation and Epithelial Mesenchymal Transition in Retinal Pigment Epithelial Cells by Heavy Chain-Hyaluronan/Pentraxin 3

    PubMed Central

    He, Hua; Kuriyan, Ajay E.; Su, Chen-Wei; Mahabole, Megha; Zhang, Yuan; Zhu, Ying-Ting; Flynn, Harry W.; Parel, Jean-Marie; Tseng, Scheffer C. G.

    2017-01-01

    Proliferative vitreoretinopathy (PVR) is mediated by proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelium (RPE). Because heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) purified from human amniotic membrane exerts anti-inflammatory and anti-scarring actions, we hypothesized that HC-HA/PTX3 could inhibit these PVR-related processes in vitro. In this study, we first optimized an ARPE-19 cell culture model to mimic PVR by defining cell density, growth factors, and cultivation time. Using this low cell density culture model and HA as a control, we tested effects of HC-HA/PTX3 on the cell viability (cytotoxicity), proliferation (EGF + FGF-2) and EMT (TGF-β1). Furthermore, we determined effects of HC-HA/PTX3 on cell migration (EGF + FGF-2 + TGF-β1) and collagen gel contraction (TGF-β1). We found both HA and HC-HA/PTX3 were not toxic to unstimulated RPE cells. Only HC-HA/PTX3 dose-dependently inhibited proliferation and EMT of stimulated RPE cells by down-regulating Wnt (β-catenin, LEF1) and TGF-β (Smad2/3, collagen type I, α-SMA) signaling, respectively. Additionally, HA and HC-HA/PTX3 inhibited migration but only HC-HA/PTX3 inhibited collagen gel contraction. These results suggest HC-HA/PTX3 is a non-toxic, potent inhibitor of proliferation and EMT of RPE in vitro, and HC-HA/PTX3’s ability to inhibit PVR formation warrants evaluation in an animal model. PMID:28252047

  8. Shared Gene Structures and Clusters of Mutually Exclusive Spliced Exons within the Metazoan Muscle Myosin Heavy Chain Genes

    PubMed Central

    Kollmar, Martin; Hatje, Klas

    2014-01-01

    Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times

  9. Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression.

    PubMed

    Clause, Kelly C; Tchao, Jason; Powell, Mary C; Liu, Li J; Huard, Johnny; Keller, Bradley B; Tobita, Kimimasa

    2012-01-01

    Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle.

  10. Serum Phosphorylated Neurofilament-Heavy Chain, a Potential Biomarker, is Associated With Peripheral Neuropathy in Patients With Type 2 Diabetes

    PubMed Central

    Qiao, Xiaona; Zhang, Shuo; Zhao, Weiwei; Ye, Hongying; Yang, Yehong; Zhang, Zhaoyun; Miao, Qing; Hu, Renming; Li, Yiming; Lu, Bin

    2015-01-01

    Abstract Neurofilament (NF), one of the major axonal cytoskeletal proteins, plays a critical role in degenerative diseases in both the central and the peripheral nervous systems. The aim of this study is to explore the relationship between serum phosphorylated neurofilament-heavy chain (pNF-H) and diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes. Serum pNF-H concentrations were measured by ELISA in hospitalized patients with and without DPN (n = 118). DPN was assessed by clinical symptoms, signs, and electromyography. Compared with the non-DPN group (311.98 [189.59–634.12] pg/mL), the confirmed group (605.99 [281.17–1332.78] pg/mL) patients had the higher serum pNF-H levels (P = 0.007). DPN was significantly correlated with C-peptide (r = −0.269), total cholesterol (TC) (r = 0.185), and pNF-H (r = 0.258). Serum pNF-H levels were independently associated with DPN (P = 0.004), even after adjusting for age, sex, duration of diabetes, fasting plasma glucose, glycosylated hemoglobin A1c, TC, C-peptide, urinary albuminto/creatinine ratio, and estimated glomerular filtration rate. Compared with pNF-H quartile 1 (referent), patients in quartile 3 (odds ratio [OR], 3.977; 95% confidence interval [CI], 1.243–12.728; P = 0.021) and quartile 4 (OR, 10.488; 95% CI, 3.020–34.429; P = 0.000) had the higher risk of DPN after adjusting for the confounders. Serum pNF-H levels might be associated with the DPN, and the correlationship between serum pNF-H and DPN should be further studied. PMID:26554790

  11. Increased myocardial short-range forces in a rodent model of diabetes reflect elevated content of β myosin heavy chain.

    PubMed

    Chung, Charles S; Mitov, Mihail I; Callahan, Leigh Ann; Campbell, Kenneth S

    2014-06-15

    Diastolic dysfunction is a clinically significant problem for patients with diabetes and often reflects increased ventricular stiffness. Attached cross-bridges contribute to myocardial stiffness and produce short-range forces, but it is not yet known whether these forces are altered in diabetes. In this study, we tested the hypothesis that cross-bridge-based short-range forces are increased in the streptozotocin (STZ) induced rat model of type 1 diabetes. Chemically permeabilized myocardial preparations were obtained from 12week old rats that had been injected with STZ or vehicle 4weeks earlier, and activated in solutions with pCa (=-log10[Ca(2+)]) values ranging from 9.0 to 4.5. The short-range forces elicited by controlled length changes were ∼67% greater in the samples from the diabetic rats than in the control preparations. This change was mostly due to an increased elastic limit (the length change at the peak short-range force) as opposed to increased passive muscle stiffness. The STZ-induced increase in short-ranges forces is thus unlikely to reflect changes to titin and/or collagen filaments. Gel electrophoresis showed that STZ increased the relative expression of β myosin heavy chain. This molecular mechanism can explain the increased short-ranges forces observed in the diabetic tissue if β myosin molecules remain bound between the filaments for longer durations than α molecules during imposed movements. These results suggest that interventions that decrease myosin attachment times may be useful treatments for diastolic dysfunction associated with diabetes.

  12. Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries.

    PubMed

    Hubbell, Margaret C; Semotiuk, Andrew J; Thorpe, Richard B; Adeoye, Olayemi O; Butler, Stacy M; Williams, James M; Khorram, Omid; Pearce, William J

    2012-11-15

    Chronic hypoxia increases vascular endothelial growth factor (VEGF) and thereby promotes angiogenesis. The present study explores the hypothesis that hypoxic increases in VEGF also remodel artery wall structure and contractility through phenotypic transformation of smooth muscle. Pregnant and nonpregnant ewes were maintained at sea level (normoxia) or 3,820 m (hypoxia) for the final 110 days of gestation. Common carotid arteries harvested from term fetal lambs and nonpregnant adults were denuded of endothelium and studied in vitro. Stretch-dependent contractile stresses were 32 and 77% of normoxic values in hypoxic fetal and adult arteries. Hypoxic hypocontractility was coupled with increased abundance of nonmuscle myosin heavy chain (NM-MHC) in fetal (+37%) and adult (+119%) arteries. Conversely, hypoxia decreased smooth muscle MHC (SM-MHC) abundance by 40% in fetal arteries but increased it 123% in adult arteries. Hypoxia decreased colocalization of NM-MHC with smooth muscle α-actin (SM-αA) in fetal arteries and decreased colocalization of SM-MHC with SM-αA in adult arteries. Organ culture with physiological concentrations (3 ng/ml) of VEGF-A(165) similarly depressed stretch-dependent stresses to 37 and 49% of control fetal and adult values. The VEGF receptor antagonist vatalanib ablated VEGF's effects in adult but not fetal arteries, suggesting age-dependent VEGF receptor signaling. VEGF replicated hypoxic decreases in colocalization of NM-MHC with SM-αA in fetal arteries and decreases in colocalization of SM-MHC with SM-αA in adult arteries. These results suggest that hypoxic increases in VEGF not only promote angiogenesis but may also help mediate hypoxic arterial remodeling through age-dependent changes in smooth muscle phenotype and contractility.

  13. Correlation between histochemically assessed fiber type distribution and isomyosin and myosin heavy chain content in porcine skeletal muscles.

    PubMed

    Bee, G; Solomon, M B; Czerwinski, S M; Long, C; Pursel, V G

    1999-08-01

    Highly sensitive enzyme assays developed to differentiate skeletal muscle fibers allow the recognition of three main fiber types: slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG). Myosin, the predominant contractile protein in mammalian skeletal muscle, can be separated based on the electrophoretic mobility under nondissociating conditions into SM2, SM1, IM, FM3, and FM2 isoforms, or under dissociating conditions into myosin heavy chain (MHC) I, IIb, IIx/d, and IIa. The purpose of the present study was to determine whether the histochemical method of differentiation of fiber types is consistent with the electrophoretically identified isomyosin and MHC isoforms. These comparisons were made using serratus ventralis (SV), gluteus medius (GM), and longissimus muscles (LM) from 13 pigs. Two calculation methods for the histochemical assessed fiber type distribution were adopted. The first method incorporated the number of fibers counted for each fiber type and calculated a percentage of the total fiber number (fiber number percentage: FNP). The second method expressed the cross-sectional area of each fiber type as a percentage of the total fiber area measured per muscle (fiber area percentage: FAP). Independent of the calculation methods, correlation analyses revealed in all muscles a strong relation between SO fibers, the slow isomyosin (SM1 and SM2), and MHCI, as well as between the FG fibers, the fast isomyosin (FM3 and FM2), and MHCIIx/b content (P<.05). There were no correlations between FOG fiber population assessed by histochemical analysis and intermediate isoform (IM) or MHCIIa content. The present results did not provide conclusive evidence as to which of the calculation methods (FNP or FAP) was more closely related to myosin composition of skeletal muscles. Despite some incompatibility between the methods, the present study shows that histochemical as well as electrophoretic analyses yielded important

  14. Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries

    PubMed Central

    Hubbell, Margaret C.; Semotiuk, Andrew J.; Thorpe, Richard B.; Adeoye, Olayemi O.; Butler, Stacy M.; Williams, James M.; Khorram, Omid

    2012-01-01

    Chronic hypoxia increases vascular endothelial growth factor (VEGF) and thereby promotes angiogenesis. The present study explores the hypothesis that hypoxic increases in VEGF also remodel artery wall structure and contractility through phenotypic transformation of smooth muscle. Pregnant and nonpregnant ewes were maintained at sea level (normoxia) or 3,820 m (hypoxia) for the final 110 days of gestation. Common carotid arteries harvested from term fetal lambs and nonpregnant adults were denuded of endothelium and studied in vitro. Stretch-dependent contractile stresses were 32 and 77% of normoxic values in hypoxic fetal and adult arteries. Hypoxic hypocontractility was coupled with increased abundance of nonmuscle myosin heavy chain (NM-MHC) in fetal (+37%) and adult (+119%) arteries. Conversely, hypoxia decreased smooth muscle MHC (SM-MHC) abundance by 40% in fetal arteries but increased it 123% in adult arteries. Hypoxia decreased colocalization of NM-MHC with smooth muscle α-actin (SM-αA) in fetal arteries and decreased colocalization of SM-MHC with SM-αA in adult arteries. Organ culture with physiological concentrations (3 ng/ml) of VEGF-A165 similarly depressed stretch-dependent stresses to 37 and 49% of control fetal and adult values. The VEGF receptor antagonist vatalanib ablated VEGF's effects in adult but not fetal arteries, suggesting age-dependent VEGF receptor signaling. VEGF replicated hypoxic decreases in colocalization of NM-MHC with SM-αA in fetal arteries and decreases in colocalization of SM-MHC with SM-αA in adult arteries. These results suggest that hypoxic increases in VEGF not only promote angiogenesis but may also help mediate hypoxic arterial remodeling through age-dependent changes in smooth muscle phenotype and contractility. PMID:22992677

  15. HDAC3-dependent reversible lysine acetylation of cardiac myosin heavy chain isoforms modulates their enzymatic and motor activity.

    PubMed

    Samant, Sadhana A; Courson, David S; Sundaresan, Nagalingam R; Pillai, Vinodkumar B; Tan, Minjia; Zhao, Yingming; Shroff, Sanjeev G; Rock, Ronald S; Gupta, Mahesh P

    2011-02-18

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the K(m) for the actin-activated ATPase activity of both α- and β-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and β-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

  16. Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix*

    PubMed Central

    Briggs, David C.; Birchenough, Holly L.; Ali, Tariq; Rugg, Marilyn S.; Waltho, Jon P.; Ievoli, Elena; Jowitt, Thomas A.; Enghild, Jan J.; Richter, Ralf P.; Salustri, Antonietta; Milner, Caroline M.; Day, Anthony J.

    2015-01-01

    The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix. PMID:26468290

  17. Effects of low-level α-myosin heavy chain expression on contractile kinetics in porcine myocardium

    PubMed Central

    Razumova, Maria V.; Stelzer, Julian E.; Norman, Holly S.; Moss, Richard L.

    2011-01-01

    Myosin heavy chain (MHC) isoforms are principal determinants of work capacity in mammalian ventricular myocardium. The ventricles of large mammals including humans normally express ∼10% α-MHC on a predominantly β-MHC background, while in failing human ventricles α-MHC is virtually eliminated, suggesting that low-level α-MHC expression in normal myocardium can accelerate the kinetics of contraction and augment systolic function. To test this hypothesis in a model similar to human myocardium we determined composite rate constants of cross-bridge attachment (fapp) and detachment (gapp) in porcine myocardium expressing either 100% α-MHC or 100% β-MHC in order to predict the MHC isoform-specific effect on twitch kinetics. Right atrial (∼100% α-MHC) and left ventricular (∼100% β-MHC) tissue was used to measure myosin ATPase activity, isometric force, and the rate constant of force redevelopment (ktr) in solutions of varying Ca2+ concentration. The rate of ATP utilization and ktr were approximately ninefold higher in atrial compared with ventricular myocardium, while tension cost was approximately eightfold greater in atrial myocardium. From these values, we calculated fapp to be ∼10-fold higher in α- compared with β-MHC, while gapp was 8-fold higher in α-MHC. Mathematical modeling of an isometric twitch using these rate constants predicts that the expression of 10% α-MHC increases the maximal rate of rise of force (dF/dtmax) by 92% compared with 0% α-MHC. These results suggest that low-level expression of α-MHC significantly accelerates myocardial twitch kinetics, thereby enhancing systolic function in large mammalian myocardium. PMID:21217059

  18. Relationship between pork quality and characteristics of muscle fibers classified by the distribution of myosin heavy chain isoforms.

    PubMed

    Kim, Gap-Don; Ryu, Youn-Chul; Jeong, Jin-Yeon; Yang, Han-Sul; Joo, Seon-Tea

    2013-11-01

    A total of six fiber types, including four pure types (type I, IIA, IIX, and IIB) and two hybrid types (type IIAX and IIXB), were classified according to the expression of myosin heavy chain (MHC) isoforms by immunohistochemistry with MHC specific monoclonal antibodies. The comparison of the muscle fiber characteristics and pork quality between pork quality groups (DFD: dark, firm, and dry; PSE: pale, soft, and exudative; RFN: reddish pink, firm, and nonexudative; and RSE: reddish pink, soft, and exudative) classified by muscle pH, drip loss, and lightness was conducted and the relationship of myofiber characteristics to pork quality was investigated. The DFD group had the highest value of IIAX fiber density (P<0.05). The DFD group also showed the greatest fiber relative area of type I, IIA, and IIAX (P<0.05) whereas there were no significant differences in area composition for types I, IIA, and IIAX among the other groups including PSE, RFN, and RSE (P>0.05). The DFD group had the highest cross-sectional area (CSA) in types I, IIA, and IIX among the groups. The increase in density of type IIAX was related with the higher pH and the lower hue and drip loss. An increase in the fiber number composition of hybrid type IIXB increased the lightness and cooking loss and decreased sarcoplasmic protein solubility (SPS). Regarding fiber relative area, pure type I and IIA and hybrid type IIAX were greater in the DFD group and had lower lightness and drip loss. Hybrid type IIAX influences the desirability of the pork due to its association with low lightness and high pH and water-holding capacity (WHC). In contrast, type IIXB was related to poor quality pork, including pale color, low WHC in cooked meat, and low SPS.

  19. HDAC3-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity*

    PubMed Central

    Samant, Sadhana A.; Courson, David S.; Sundaresan, Nagalingam R.; Pillai, Vinodkumar B.; Tan, Minjia; Zhao, Yingming; Shroff, Sanjeev G.; Rock, Ronald S.; Gupta, Mahesh P.

    2011-01-01

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of both α- and β-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and β-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms. PMID:21177250

  20. Pulsed-field electrophoresis screening for immunoglobulin heavy-chain constant-region (IGHC) multigene deletions and duplications.

    PubMed Central

    Bottaro, A; Cariota, U; DeMarchi, M; Carbonara, A O

    1991-01-01

    Genome regions containing multiple copies of homologous genes, such as the immunoglobulin (Ig) heavy-chain constant-region (IGHC) locus, are often unstable and give rise to duplicated and deleted haplotypes. Analysis of such processes is fundamental to understanding the mechanisms of evolution of multigene families. In the IGHC region, a number of single and multiple gene deletions, derived from either unequal crossing-over or looping-out excision, have been described. To study these haplotypes at the population level, a simple and efficient method for preparing large numbers of DNA samples suitable for pulsed-field gel electrophoresis (PFGE) analysis was set up, and a sample of 110 blood donors was screened. Deletions were found to be frequent, as expected on the basis of previous serological surveys for homozygotes. Furthermore, a number of multigene duplications, never identified before, were detected. The total frequency of individuals bearing rearranged IGHC haplotypes was 10%. The genes involved in these deletions and duplications were assessed by densitometric analysis of standard Southern blots hybridized with several IGHC probes; two types of deletion and two types of duplication could thus be characterized. These data provide further evidence of the instability of the IGHC locus and demonstrate that unequal crossing-over is the most likely origin of rearranged IGHC haplotypes; they also suggest that such recombination events may be relatively frequent. Moreover, the simplicity and effectiveness of the large-scale PFGE screening approach will be of great help in the study of multigene families and of other loci involved in aberrant recombinations. Images p[750]-a Figure 2 Figure 3 Figure 5 Figure 6 PMID:1901690

  1. Clonorchis sinensis ferritin heavy chain triggers free radicals and mediates inflammation signaling in human hepatic stellate cells.

    PubMed

    Mao, Qiang; Xie, Zhizhi; Wang, Xiaoyun; Chen, Wenjun; Ren, Mengyu; Shang, Mei; Lei, Huali; Tian, Yanli; Li, Shan; Liang, Pei; Chen, Tingjin; Liang, Chi; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing

    2015-02-01

    Clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis, is associated with hepatobiliary damage, inflammation, periductal fibrosis, and the development of cholangiocarcinoma. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators which drive fibrogenesis; however, their endogenous sources and pathophysiological roles in host cells were not determined. C. sinensis ferritin heavy chain (CsFHC) was previously confirmed as a component of excretory/secretory products and exhibited a number of extrahepatic immunomodulatory properties in various diseases. In this study, we investigated the expression pattern and biological role of CsFHC in C. sinensis. CsFHC was expressed throughout life stages of C. sinensis. More importantly, we found that treatment of human hepatic stellate cell line LX-2 with CsFHC triggered the production of free radicals via time-dependent activation of NADPH oxidase, xanthine oxidase, and inducible nitric oxide synthase. The increase in free radicals substantially promoted the degradation of cytosolic IκBα and nuclear translocation of NF-κB subunits (p65 and p50). CsFHC-induced NF-κB activation was markedly attenuated by preincubation with specific inhibitors of corresponding free radical-producing enzyme or the antioxidant. In addition, CsFHC induced an increased expression level of proinflammatory cytokines, IL-1β and IL-6, in NF-κB-dependent manner. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

  2. Sexually differentiated, androgen-regulated, larynx-specific myosin heavy-chain isoforms in Xenopus tropicalis; comparison to Xenopus laevis

    PubMed Central

    Baur, Laura A.; Nasipak, Brian T.; Kelley, Darcy B.

    2010-01-01

    We have shown that the sarcoplasmic myosin heavy-chain (MyHC) isoform xtMyHC-101d is highly and specifically expressed in the larynx of the aquatic anuran, Xenopus tropicalis. In male larynges, the predominant MyHC isoform is xtMyHC-101d, while in females, another isoform, xtMyHC-270c, predominates. The X. tropicalis genome has been sequenced in its entirety, and xtMyHC-101d is part of a specific array of xtMyHC genes expressed otherwise in embryonic muscles. The administration of the androgen dihydrotestosterone increases the expression of xtMyHC-101d in juvenile larynges of both sexes. Using ATPase histochemistry, we found that in adults, X. tropicalis male laryngeal muscle contains only fast-twitch fibers, while the female laryngeal muscle contains a mix of fast- and slow-twitch fibers. Juvenile larynges are female-like in fiber type composition (44% slow twitch, 56% fast twitch); androgen treatment increases the percentage of fast-twitch fibers to 86%. xtMyHC-101d predominates in larynges of dihydrotestosterone-treated juveniles but not in larynges of untreated juveniles. We compared the larynx-specific expression of xtMyHC genes in X. tropicalis to the MyHC gene expressed in X. laevis larynx (xlMyHC-LM) by sequencing the entire xlMyHC-LM gene. The androgen-regulated xtMyHC that predominates in the male larynx of X. tropicalis is not the gene phylogenetically most similar to xlMyHC-LM at the nucleotide level but is instead a similar isoform found in the same MyHC array and expressed in the embryonic muscle. PMID:18551305

  3. A continuum of myofibers in adult rabbit extraocular muscle: force, shortening velocity, and patterns of myosin heavy chain colocalization.

    PubMed

    McLoon, Linda K; Park, Han Na; Kim, Jong-Hee; Pedrosa-Domellöf, Fatima; Thompson, Ladora V

    2011-10-01

    Extraocular muscle (EOM) myofibers do not fit the traditional fiber typing classifications normally used in noncranial skeletal muscle, in part, due to the complexity of their individual myofibers. With single skinned myofibers isolated from rectus muscles of normal adult rabbits, force and shortening velocity were determined for 220 fibers. Each fiber was examined for myosin heavy chain (MyHC) isoform composition by densitometric analysis of electrophoresis gels. Rectus muscle serial sections were examined for coexpression of eight MyHC isoforms. A continuum was seen in single myofiber shortening velocities as well as force generation, both in absolute force (g) and specific tension (kN/m(2)). Shortening velocity correlated with MyHCIIB, IIA, and I content, the more abundant MyHC isoforms expressed within individual myofibers. Importantly, single fibers with similar or identical shortening velocities expressed significantly different ratios of MyHC isoforms. The vast majority of myofibers in both the orbital and global layers expressed more than one MyHC isoform, with up to six isoforms in single fiber segments. MyHC expression varied significantly and unpredictably along the length of single myofibers. Thus EOM myofibers represent a continuum in their histological and physiological characteristics. This continuum would facilitate fine motor control of eye position, speed, and direction of movement in all positions of gaze and with all types of eye movements-from slow vergence movements to fast saccades. To fully understand how the brain controls eye position and movements, it is critical that this significant EOM myofiber heterogeneity be integrated into hypotheses of oculomotor control.

  4. Thyroid hormone partially corrects the effect of diabetes on mysoin heavy chain RNAs in the rat ventricle

    SciTech Connect

    Barrieux, A.; Dillmann, W.H.

    1986-05-01

    The relative abundance of the 2 cardiac myosin heavy chains (MHH-..cap alpha.. and MHC-..beta..) and their corresponding RNAs is similarly affected by hypothyroidism and diabetes in the rat ventricle. Since circulating levels of thyroid hormone (T/sub 3/) are significantly decreased in diabetes the decreased RNA-..cap alpha.. and increased RNA-..beta.. associated with diabetes may be related to T/sub 3/ deficiency. Chronically diabetic rats were injected with T/sub 3/, insulin (I), or both and sacrificed between 1 and 12 hrs after injection, MHC RNA was quantified by hybridization of total RNA to a (/sup 32/P)-cDNA MHC-..cap alpha.. probe. Total MHC RNA was measured by retention of S1-resistant label on DE-81 while RNA-..cap alpha.. and RNA-..beta.. were quantified by separation of intact (..cap alpha..) and partially digested (..beta..) probe by gel electrophoresis. Total MHC RNA was not changed by T/sub 3/ or I (.9-1.2 ng/..mu..g of cellular RNA). T/sub 3/ and I elicited a very rapid increase in RNA-..cap alpha.. (18% in untreated to 38% (I) and 47% (T/sub 3/ within 1 hr). I administered either 7 hr before or 1 hr after T/sub 3/ did not modify the RNA-..cap alpha.. increase observed after T/sub 3/ alone. However, the response of diabetic rats to T/sub 3/ was markedly different from that of hypothyroid rats. Conclusions: 1) T/sub 3/ in diabetic rats does not mimick the effect of T/sub 3/ in hypothyroid rats; it may simply impose a hyperthyroid state on the existing diabetes and 2) since neither T/sub 3/ nor I are able to increase RNA-..cap alpha.. to control levels within 12 hrs, neither hormone is sufficient to regulate the expression of MHC RNAs.

  5. Heavy Chain Diseases

    MedlinePlus

    ... and Metabolic Disorders Immune Disorders Infections Injuries and Poisoning Kidney and Urinary Tract Disorders Liver and Gallbladder ... and Metabolic Disorders Immune Disorders Infections Injuries and Poisoning Kidney and Urinary Tract Disorders Liver and Gallbladder ...

  6. Photosensitized cleavage of dynein heavy chains. Cleavage at the "V1 site" by irradiation at 365 nm in the presence of ATP and vanadate.

    PubMed

    Gibbons, I R; Lee-Eiford, A; Mocz, G; Phillipson, C A; Tang, W J; Gibbons, B H

    1987-02-25

    Irradiation of soluble dynein 1 from sea urchin sperm flagella at 365 nm in the presence of MgATP and 0.05-50 microM vanadate (Vi) cleaves the alpha and beta heavy chains (Mr 428,000) at their V1 sites to give peptides of Mr 228,000 and 200,000, without the nonspecific side effects produced by irradiation at 254 nm as described earlier (Lee-Eiford, A., Ow, R. A., and Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342). The decrease in intact heavy chain material is biphasic; in 10 microM Vi, approximately 80% occurs with a half-time of 7 min and the remainder with a half-time of about 90 min, and the yield of cleavage peptides is better than 90%. Loss of dynein ATPase activity appears to be a direct result of the cleavage process and is not significantly affected by the presence of up to 0.1 M cysteamine (CA, 60-23-1) or 2-aminoethyl carbamimidothioic acid dihydrobromide (CA, 56-10-0) as free radical trapping agents. The concentration of Vi required for 50% maximal initial cleavage rate is 4.5 microM, while that for 50% ATPase inhibition is 0.8 microM, both in a 0.6 M NaCl medium. In the presence of 20 microM Vi, CTP and UTP support cleavage at about half the rate of ATP, whereas GTP and ITP support cleavage only if the Vi concentration is raised to about 200 microM. Substitution of any of the transition metal cations Cr2+, Mn2+, Fe2+, or Co2+ for the usual Mg2+ suppresses the photocleavage, presumably by quenching the excited chromophore prior to scission of the heavy chain. The photocleaved dynein 1 binds to dynein-depleted flagella similarly to intact dynein 1, but upon reactivation of the flagella with 1 mM ATP their motility is partially inhibited, rather than being augmented as with intact dynein. These results indicate that Vi acts as a photosensitizing catalyst and suggest that the cleavage proceeds through excitation of Vi bound to dynein at the hydrolytic ATP binding site on each heavy chain, probably in a dynein X MgADP X Vi complex. The exquisite

  7. Abolition of ATPase activities of skeletal myosin subfragment 1 by a new selective proteolytic cleavage within the 50-kilodalton heavy chain segment.

    PubMed

    Chaussepied, P; Mornet, D; Audemard, E; Derancourt, J; Kassab, R

    1986-03-11

    We have isolated and chemically characterized several 5-thio-2-nitrobenzoate-subfragment 1 derivatives (TNB-S-1) generated by the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB, up to 10-fold molar excess) with native S-1, N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-S-1 (AEDANS-S-1), and N,N'-p-phenylenedimaleimide-S-1 (pPDM-S-1) at 4 degrees C, pH 8.0. The reaction of the reagent with AEDANS-S-1, which has a blocked -SH1 group, induced the formation of an intramolecular cystine disulfide between two vicinal -SH groups in S-1; in contrast, the treatment of pPDM-S-1 with DTNB resulted in the formation of TNB mixed disulfides only. The incorporation of the TNB groups (up to 3 mol/mol of S-1) into the native or premodified S-1 led to a local conformational change in the 50K heavy chain region that was fully reversed upon disulfide reduction. Exploiting this peculiarity of the DTNB-modified S-1's, we have realized a highly selective proteolysis of the S-1 heavy chain by thrombin and chymotrypsin, which do not act at all on the normal S-1. The 95K heavy chain was cut by thrombin into two fragments with apparent masses of 68K and 30K, whereas the "connector segments" and the light chains were unaffected. The two new fragments were issued from a primary peptide-bound cleavage between Lys-560 and Ser-561 within the amino acid sequence of the 50K region (M. Elzinga, personal communication).(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Expression profiles of myostatin, myogenin, and Myosin heavy chain in skeletal muscles of two rabbit breeds differing in growth rate.

    PubMed

    Kuang, Liangde; Xie, Xiaohong; Zhang, Xiangyu; Lei, Min; Li, Congyan; Ren, Yongjun; Zheng, Jie; Guo, Zhiqiang; Zhang, Cuixia; Yang, Chao; Zheng, Yucai

    2014-01-01

    The purpose of the present study was to compare mRNA levels of myostatin (MSTN), myogenin (MyoG), and fiber type compositions in terms of myosin heavy chain (MyHC) in skeletal muscles of two rabbit breeds with different body sizes and growth rates. Longissimus dorsi and biceps femoris muscles of 16 Californian rabbits (CW) and 16 Germany great line of ZIKA rabbits (GZ) were collected at the ages of 35d and 84d (slaughter age). The results showed that the live weights of GZ rabbits of 35d and 84d old were approximately 36% and 26% greater than those of CW rabbits, respectively. Quantitative real-time PCR analysis revealed that at the age of 84d GZ rabbits contained significantly lower MSTN mRNA level and higher MyoG mRNA level in both longissimus dorsi and biceps femoris muscles than CW rabbits, and mRNA levels of MSTN and MyoG exhibited opposite changes from the age of 35d to 84d, suggesting that GZ rabbits were subjected to less growth inhibition from MSTN at slaughter age, which occurred most possibly in skeletal muscles. Four types of fiber were identified by real-time PCR in rabbit muscles, with MyHC-1 and MyHC-2D, MyHC-2B were the major types in biceps femoris and longissimus dorsi muscles, respectively. At the age of 84d, GZ rabbits contained greater proportion of MyHC-1 and decreased proportion of MyHC-2D and decreased lactate dehydrogenase activity in biceps femoris than CW rabbits, and the results were exactly opposite in longissimus dorsi, suggesting that GZ rabbits show higher oxidative capacity in biceps femoris muscle than CW rabbits. In conclusion, the trends of mRNA levels of MSTN and fiber types in GZ rabbits' skeletal muscles might be consistent with the putative fast growth characteristic of GZ rabbits compared to CW rabbits.

  9. Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development.

    PubMed

    Cho, M; Webster, S G; Blau, H M

    1993-05-01

    Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or

  10. Divergent effects of α- and β-myosin heavy chain isoforms on the N terminus of rat cardiac troponin T

    PubMed Central

    Mamidi, Ranganath

    2013-01-01

    Divergent effects of α– and β–myosin heavy chain (MHC) isoforms on contractile behavior arise mainly because of their impact on thin filament cooperativity. The N terminus of cardiac troponin T (cTnT) also modulates thin filament cooperativity. Our hypothesis is that the impact of the N terminus of cTnT on thin filament activation is modulated by a shift from α- to β-MHC isoform. We engineered two recombinant proteins by deleting residues 1–43 and 44–73 in rat cTnT (RcTnT): RcTnT1–43Δ and RcTnT44–73Δ, respectively. Dynamic and steady-state contractile parameters were measured at sarcomere length of 2.3 µm after reconstituting proteins into detergent-skinned muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rat hearts. α-MHC attenuated Ca2+-activated maximal tension (∼46%) in RcTnT1–43Δ fibers. In contrast, β-MHC decreased tension only by 19% in RcTnT1–43Δ fibers. Both α- and β-MHC did not affect tension in RcTnT44–73Δ fibers. The instantaneous muscle fiber stiffness measurements corroborated the divergent impact of α- and β-MHC on tension in RcTnT1–43Δ fibers. pCa50 (-log of [Ca2+]free required for half-maximal activation) decreased significantly by 0.13 pCa units in α-MHC + RcTnT1–43Δ fibers but remained unaltered in β-MHC + RcTnT1–43Δ fibers, demonstrating that β-MHC counteracted the attenuating effect of RcTnT1–43Δ on myofilament Ca2+ sensitivity. β-MHC did not alter the sudden stretch–mediated recruitment of new cross-bridges (ER) in RcTnT1–43Δ fibers, but α-MHC attenuated ER by 36% in RcTnT1–43Δ fibers. The divergent impact of α- and β-MHC on how the N terminus of cTnT modulates contractile dynamics has implications for heart disease; alterations in cTnT and MHC are known to occur via changes in isoform expression or mutations. PMID:24043862

  11. Association between myosin heavy chain protein isoforms and intramuscular anabolic signaling following resistance exercise in trained men

    PubMed Central

    Gonzalez, Adam M.; Hoffman, Jay R.; Townsend, Jeremy R.; Jajtner, Adam R.; Wells, Adam J.; Beyer, Kyle S.; Willoughby, Darryn S.; Oliveira, Leonardo P.; Fukuda, David H.; Fragala, Maren S.; Stout, Jeffrey R.

    2015-01-01

    Abstract Resistance exercise stimulates an increase in muscle protein synthesis regulated by intracellular anabolic signaling molecules in a mammalian/mechanistic target of rapamycin (mTOR)‐dependent pathway. The purpose of this study was to investigate acute anabolic signaling responses in experienced, resistance‐trained men, and to examine the association between myosin heavy chain (MHC) isoform composition and the magnitude of anabolic signaling. Eight resistance‐trained men (24.9 ± 4.3 years; 91.2 ± 12.4 kg; 176.7 ± 8.0 cm; 13.3 ± 3.9 body fat %) performed a whole body, high‐volume resistance exercise protocol (REX) and a control protocol (CTL) in a balanced, randomized order. Participants were provided a standardized breakfast, recovery drink, and meal during each protocol. Fine needle muscle biopsies were completed at baseline (BL), 2 h (2H) and 6 h post‐exercise (6H). BL biopsies were analyzed for MHC isoform composition. Phosphorylation of proteins specific to the Akt/mTOR signaling pathway and MHC mRNA expression was quantified. Phosphorylation of p70S6k was significantly greater in REX compared to CTL at 2H (P = 0.04). MHC mRNA expression and other targets in the Akt/mTOR pathway were not significantly influenced by REX. The percentage of type IIX isoform was inversely correlated (P < 0.05) with type I and type IIA MHC mRNA expression (r = −0.69 to −0.93). Maximal strength was also observed to be inversely correlated (P < 0.05) with Type I and Type IIA MHC mRNA expression (r = −0.75 to −0.77) and p70S6k phosphorylation (r = −0.75). Results indicate that activation of p70S6k occurs within 2‐h following REX in experienced, resistance‐trained men. Further, results also suggest that highly trained, stronger individuals have an attenuated acute anabolic response. PMID:25626869

  12. Responses of Myosin Heavy Chain Phenotypes and Gene Expressions in Neck Muscle to Micro- an Hyper-Gravity in Mice

    NASA Astrophysics Data System (ADS)

    Ohira, Tomotaka; Ohira, Takashi; Kawano, F.; Shibaguchi, T.; Okabe, H.; Ohno, Y.; Nakai, N.; Ochiai, T.; Goto, K.; Ohira, Y.

    2013-02-01

    Neck muscles are known to play important roles in the maintenance of head posture against gravity. However, it is not known how the properties of neck muscle are influenced by gravity. Therefore, the current study was performed to investigate the responses of neck muscle (rhomboideus capitis) in mice to inhibition of gravity and/or increase to 2-G for 3 months to test the hypothesis that the properties of neck muscles are regulated in response to the level of mechanical load applied by the gravitational load. Three male wild type C57BL/10J mice (8 weeks old) were launched by space shuttle Discovery (STS-128) and housed in Japanese Experimental Module “KIBO” on the International Space Station in mouse drawer system (MDS) project, which was organized by Italian Space Agency. Only 1 mouse returned to the Earth alive after 3 months by space shuttle Atlantis (STS-129). Neck muscles were sampled from both sides within 3 hours after landing. Cage and laboratory control experiments were also performed on the ground. Further, 3-month ground-based control experiments were performed with 6 groups, i.e. pre-experiment, 3-month hindlimb suspension, 2-G exposure by using animal centrifuge, and vivarium control (n=5 each group). Five mice were allowed to recover from hindlimb suspension (including 5 cage control) for 3 months in the cage. Neck muscles were sampled bilaterally before and after 3-month suspension and 2-G exposure, and at the end of 3-month ambulation recovery. Spaceflight-associated shift of myosin heavy chain phenotype from type I to II and atrophy of type I fibers were observed. In response to spaceflight, 17 genes were up-regulated and 13 genes were down-regulated vs. those in the laboratory control. Expression of 6 genes were up-regulated and that of 88 genes were down-regulated by 3-month exposure to 2-G vs. the age-matched cage control. In response to chronic hindlimb suspension, 4 and 20 genes were up- or down-regulated. Further, 98 genes responded

  13. Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression.

    PubMed

    Miller, J B; Teal, S B; Stockdale, F E

    1989-08-05

    A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that

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

    PubMed Central

    2012-01-01

    Background The aim of the paper is to assess by the principal components analysis (PCA) the heavy metal contamination of soil and vegetables widely used as food for people who live in areas contaminated by heavy metals (HMs) due to long-lasting mining activities. This chemometric technique allowed us to select the best model for determining the risk of HMs on the food chain as well as on people's health. Results Many PCA models were computed with different variables: heavy metals contents and some agro-chemical parameters which characterize the soil samples from contaminated and uncontaminated areas, HMs contents of different types of vegetables grown and consumed in these areas, and the complex parameter target hazard quotients (THQ). Results were discussed in terms of principal component analysis. Conclusion There were two major benefits in processing the data PCA: firstly, it helped in optimizing the number and type of data that are best in rendering the HMs contamination of the soil and vegetables. Secondly, it was valuable for selecting the vegetable species which present the highest/minimum risk of a negative impact on the food chain and human health. PMID:23234365

  15. Semiquantitative evaluation of mRNAs for the membranous form of immunoglobulin heavy chain is useful for investigating the etiology in CVID.

    PubMed

    Terada, T; Kaneko, H; Fukao, T; Teramoto, T; Asano, T; Li, A L; Kasahara, K; Kondo, N

    2003-12-01

    Common variable immunodeficiency (CVID) is a primary antibody deficiency syndrome characterized by defective B-cell maturation and antibody formation resulting in low serum antibody levels of all immunoglobulin (Ig) isotypes. To investigate the pathogenesis of CVID, we developed a set of competitive polymerase chain reaction for membrane-bound Ig heavy chain (mHC) mRNAs for IgM, IgG and IgA. Data on three children with CVID in group A of Bryant's classification were analysed. All the three mHC mRNA levels in Patient 1 were almost same as those in healthy controls. In Patient 2, mHC mRNA for IgM was detected at a level similar to that in controls, but mHC mRNAs for IgG and IgA heavy chains were not detected. In Patient 3, all the three mHC mRNAs were undetectable. Our data suggest that a different molecular basis exists in these patients with CVID even though all belong to group A of Bryant's classification. Use of our method facilitates a better understanding of molecular events in CVID patients and may be useful for precise classifications of CVID.

  16. Photosensitized cleavage of dynein heavy chains. Cleavage at the V1 site by irradiation at 365 nm in the presence of ATP and vanadate

    SciTech Connect

    Gibbons, I.R.; Lee-Eiford, A.; Mocz, G.; Phillipson, C.A.; Tang, W.J.; Gibbons, B.H.

    1987-02-25

    Irradiation of soluble dynein 1 from sea urchin sperm flagella at 365 nm in the presence of MgATP and 0.05-50 microM vanadate (Vi) cleaves the alpha and beta heavy chains (Mr 428,000) at their V1 sites to give peptides of Mr 228,000 and 200,000, without the nonspecific side effects produced by irradiation at 254 nm as described earlier. The decrease in intact heavy chain material is biphasic; in 10 microM Vi, approximately 80% occurs with a half-time of 7 min and the remainder with a half-time of about 90 min, and the yield of cleavage peptides is better than 90%. Loss of dynein ATPase activity appears to be a direct result of the cleavage process and is not significantly affected by the presence of up to 0.1 M cysteamine (CA, 60-23-1) or 2-aminoethyl carbamimidothioic acid dihydrobromide (CA, 56-10-0) as free radical trapping agents. The concentration of Vi required for 50% maximal initial cleavage rate is 4.5 microM, while that for 50% ATPase inhibition is 0.8 microM, both in a 0.6 M NaCl medium. In the presence of 20 microM Vi, CTP and UTP support cleavage at about half the rate of ATP, whereas GTP and ITP support cleavage only if the Vi concentration is raised to about 200 microM. Substitution of any of the transition metal cations Cr2+, Mn2+, Fe2+, or Co2+ for the usual Mg2+ suppresses the photocleavage, presumably by quenching the excited chromophore prior to scission of the heavy chain. The photocleaved dynein 1 binds to dynein-depleted flagella similarly to intact dynein 1, but upon reactivation of the flagella with 1 mM ATP their motility is partially inhibited, rather than being augmented as with intact dynein.

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

    SciTech Connect

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  18. Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility

    SciTech Connect

    Gibbons, B.H.; Gibbons, I.R.

    1987-06-15

    Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein. The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility.

  19. Designing Optimal LNG Station Network for U.S. Heavy-Duty Freight Trucks using Temporally and Spatially Explicit Supply Chain Optimization

    NASA Astrophysics Data System (ADS)

    Lee, Allen

    The recent natural gas boom has opened much discussion about the potential of natural gas and specifically Liquefied Natural Gas (LNG) in the United States transportation sector. The switch from diesel to natural gas vehicles would reduce foreign dependence on oil, spur domestic economic growth, and potentially reduce greenhouse gas emissions. LNG provides the most potential for the medium to heavy-duty vehicle market partially due to unstable oil prices and stagnant natural gas prices. As long as the abundance of unconventional gas in the United States remains cheap, fuel switching to natural gas could provide significant cost savings for long haul freight industry. Amid a growing LNG station network and ever increasing demand for freight movement, LNG heavy-duty truck sales are less than anticipated and the industry as a whole is less economic than expected. In spite of much existing and mature natural gas infrastructure, the supply chain for LNG is different and requires explicit and careful planning. This thesis proposes research to explore the claim that the largest obstacle to widespread LNG market penetration is sub-optimal infrastructure planning. No other study we are aware of has explicitly explored the LNG transportation fuel supply chain for heavy-duty freight trucks. This thesis presents a novel methodology that links a network infrastructure optimization model (represents supply side) with a vehicle stock and economic payback model (represents demand side). The model characterizes both a temporal and spatial optimization model of future LNG transportation fuel supply chains in the United States. The principal research goal is to assess the economic feasibility of the current LNG transportation fuel industry and to determine an optimal pathway to achieve ubiquitous commercialization of LNG vehicles in the heavy-duty transport sector. The results indicate that LNG is not economic as a heavy-duty truck fuel until 2030 under current market conditions

  20. Nucleotide sequence of the variable region of the heavy and light chains of a monoclonal IgG antibody reactive to herbicides, terbutryn and prometryn.

    PubMed

    Kreissig, S B; Ward, V K; Hammock, B D; Choudary, P V

    1995-01-01

    Members of the triazine family of herbicides are reliable indicators of contamination of the ground water or soil with pesticide residues. To facilitate better detection of the chemical residues using improved immunoassay procedures, several monoclonal antibodies against triazine herbicides have been developed. K1F4 is a hybridoma secreting monoclonal (IgG) antibody reactive to terbutryn and prometryn, two members of the triazine family. We have cloned the genes encoding the variable regions of the heavy and light chains of this monoclonal antibody and report the nucleotide sequence here.

  1. Characteristics Peripheral Blood IgG and IgM Heavy Chain Complementarity Determining Region 3 Repertoire before and after Immunization with Recombinant HBV Vaccine

    PubMed Central

    Yang, Jiezuan; Shi, Bin; He, Xiaoyan; Ma, Rui; Ma, Qingqing; Yao, Xinsheng

    2017-01-01

    Immunization with recombinant HBV vaccine induces specific immune responses in human causing B lymphocytes to produce protective HBsAb, and to form memory B lymphocytes, thereby facilitating HBV immunity in the body. However, B lymphocytes heterogeneity and characteristics are not fully elucidated. In this study, we conducted high-throughput sequencing of BCR heavy chain CDR3 repertoires in 3 healthy volunteers before and after the third immunization with recombinant HBV vaccine. We used Roche 454 Genome Sequencer FLX system to perform a comparative analysis of IgM and IgG H chain CDR3 repertoires. First, we found that the diversity of IgG H chain CDR3 repertoires was 1/6 of IgM on average. Moreover, after the third immunization with HBV vaccine, the diversity of IgG H chain CDR3 repertoires was 1/26 of IgM on average. Second, we detected relatively high levels of HBsAbs in all the healthy volunteers after immunization with HBV vaccine. The volunteers shared a small number of CDR3 sequences before and after immunization, and among each other. However, we did not find completely identical BCR H chain CDR3 amino acid sequences in these volunteers. Lastly, after immunization with recombinant HBV vaccine, the volunteers showed high frequency of IgG H chain CDR3 amino acid sequences mostly resulting from rearrangements of IGHV, IGHD and IGHJ, suggesting that the mechanism of high frequency CDR3 generation might be associated with the maturation of IgG affinity (somatic hypermutation) during the recombinant HBV vaccine-induced B lymphocyte responses. This study identified the characteristics and changes of BCR CDR3 repertoires before and after immunization with HBV vaccine, and evaluated the performance of the sequencing technology for this application. Our findings provide a basis for further research in B lymphocyte generated HBsAb heterogeneity and monitoring the maintenance of memory B lymphocytes. PMID:28114326

  2. Triggering of monoclonal human lymphoma B cells with antibodies to IgM heavy chains: differences of response obtained with monoclonal as compared to polyclonal antibodies.

    PubMed Central

    Godal, T; Ruud, E; Heikkilä, R; Funderud, S; Michaelsen, T; Jefferis, R; Ling, N R; Hildrum, K

    1983-01-01

    A comparative study of human B lymphoma cells activation by monoclonal (murine hybridoma) antibodies to mu heavy chains (Ma-mu) as compared to polyclonal (rabbit) antibodies to mu heavy chains (Ra-mu) has been carried out. Early events related to calmodulin activation such as 86Rb influx and changes in cell volume at 4 h could be induced by Ma-mu. One antibody (AF6) approached Ra-mu with regard to the strength of response obtained. However, Ma-mus including AF6 were deficient in inducing DNA synthesis under conditions where this was achieved with Ra-mu. Studies in one lymphoma, where stimulation of re-expressed surface IgM could be studied, revealed that Ma-mu was deficient in stimulating re-expressed sIgM. These findings raise questions with regard to polyclonal antibody to surface Ig as a model for B cell triggering by antigen and suggest that antigen-induced B cell triggering may be more complex than indicated by previous studies with polyclonal antibody. PMID:6418424

  3. MicroRNA-23a reduces slow myosin heavy chain isoforms composition through myocyte enhancer factor 2C (MEF2C) and potentially influences meat quality.

    PubMed

    Shen, Linyuan; Chen, Lei; Zhang, Shunhua; Zhang, Yi; Wang, Jingyong; Zhu, Li

    2016-06-01

    MicroRNAs (miRNAs) are non-coding small RNAs that participate in the regulation of a variety of biological processes. Muscle fiber types were very important to meat quality traits, however, the molecular mechanism by which miRNAs regulate the muscle fiber type composition is not fully understood. The aim of this study was to investigate whether miRNA-23a can affect muscle fiber type composition. Luciferase reporter assays proved that miRNA-23a directly targets the 3' untranslated region (UTRs) of MEF2c. Overexpression of miRNA-23a significantly suppressed the expression of MEF2c both in mRNA and protein levels, thus caused down-regulation of the expression of some key downstream genes of MEF2c (PGC1-α, NRF1 and mtTFA). More interestingly, overexpression of miRNA-23a significantly restrained the myogenic differentiation and decreased the ratio of slow myosin heavy chain in myoblasts (p<0.05). Our findings hinted a novel role of miRNA-23a in the epigenetic regulation of meat quality via decreasing the ratio of slow myosin heavy chain isoforms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardio-facial syndrome.

    PubMed

    Sirotkin, H; Morrow, B; DasGupta, R; Goldberg, R; Patanjali, S R; Shi, G; Cannizzaro, L; Shprintzen, R; Weissman, S M; Kucherlapati, R

    1996-05-01

    Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.

  5. Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Peggion, Caterina; Massimino, Maria Lina; Biancotto, Giancarlo; Angeletti, Roberto; Reggiani, Carlo; Sorgato, Maria Catia; Bertoli, Alessandro; Stella, Roberto

    2017-03-01

    Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

  6. A monoclonal antibody distinguishes between two IgM heavy chain isotypes in Atlantic salmon and brown trout: protein characterization, 3D modeling and epitope mapping.

    PubMed

    Kamil, Atif; Falk, Knut; Sharma, Animesh; Raae, Arnt; Berven, Frode; Koppang, Erling Olaf; Hordvik, Ivar

    2011-09-01

    Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) possess two distinct subpopulations of IgM which can be separated by anion exchange chromatography. Accordingly, there are two isotypic μ genes in these species, related to ancestral tetraploidy. In the present work it was verified by mass spectrometry that IgM of peak 1 (subpopulation 1) have heavy chains previously designated as μB type whereas IgM of peak 2 (subpopulation 2) have heavy chains of μA type. Two adjacent cysteine residues are present near the C-terminal part of μB, in contrast to one cysteine residue in μA. Salmon IgM of both peak 1 and peak 2 contain light chains of the two most common isotypes: IgL1 and IgL3. In contrast to salmon and brown trout, IgM of rainbow trout (Oncorhynchus mykiss) is eluted in a single peak when subjected to anion exchange chromatography. Surprisingly, a monoclonal antibody MAb4C10 against rainbow trout IgM, reacted with μA in salmon, whereas in brown trout it reacted with μB. It is plausible to assume that DNA has been exchanged between the paralogous A and B loci during evolution while maintaining the two sub-variants, with and without the extra cysteine. MAb4C10 was conjugated to magnetic beads and used to separate cells, demonstrating that μ transcripts residing from captured cells were primarily of A type in salmon and B type in brown trout. An analysis of amino acid substitutions in μA and μB of salmon and brown trout indicated that the third constant domain is essential for MAb4C10 binding. This was supported by 3D modeling and was finally verified by studies of MAb4C10 reactivity with a series of recombinant μ3 constructs. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

  8. Zebrafish immunoglobulin IgD: unusual exon usage and quantitative expression profiles with IgM and IgZ/T heavy chain isotypes.

    PubMed

    Zimmerman, Anastasia M; Moustafa, Farah M; Romanowski, Kryzstof E; Steiner, Lisa A

    2011-09-01

    The zebrafish is an emerging model for comparative immunology and biomedical research. In contrast to the five heavy chain isotype system of mice and human (IgD, IgM, IgA, IgG, IgE), zebrafish harbor gene segments for IgD, IgM, and novel heavy chain isotype called IgZ/T which appears restricted to bony fishes. The purpose of this study was to design and validate a suite of quantitative real time RT-PCR protocols to measure IgH expression in a vertebrate model which has considerable promise for modeling both pathogenic infection and chronic conditions leading to immune dysfunction. Specific primers were designed and following verification of their specificty, relative expression levels of IgD, IgM, and IgZ/T were measured in triplicate for zebrafish raised under standard laboratory conditions. During embryonic stages, low levels of each heavy chain isotype (IgH) were detected with each increasing steadily between 2 and 17 weeks post fertilization. Overall IgM>IgZ>IgD throughout zebrafish development with the copy number of IgM being several fold higher than that of IgD or IgZ/T. IgD exon usage was also characterized, as its extremely long size and presence of a stop codon in the second IgD exon in zebrafish, raised questions as to how this antibody might be expressed. Zebrafish IgD was found to be a chimeric immunoglobulin, with the third IgD exon spliced to the first IgM constant exon thereby circumventing the first and second IgD exons. Collectively, the qRT-PCR results represent the first comparative profile of IgD, IgM, IgZ/T expression over the lifespan of any fish species and the primers and assay parameters reported should prove useful in enabling researchers to rapidly quantify changes in IgH expression in zebrafish models of disease where altered IgH expression is manifested.

  9. Zebrafish immunoglobulin IgD: unusual exon usage and quantitative expression profiles with IgM and IgZ/T heavy chain isotypes

    PubMed Central

    Zimmerman, Anastasia M.; Moustafa, Farah M.; Romanowski, Kryzstof E.; Steiner, Lisa A.

    2011-01-01

    The zebrafish is an emerging model for comparative immunology and biomedical research. In contrast to the five heavy chain isotype system of mice and human (IgD, IgM, IgA, IgG, IgE), zebrafish harbor gene segments for IgD, IgM, and novel heavy chain isotype called IgZ/T which appears restricted to bony fishes. The purpose of this study was to design and validate a suite of quantitative real time RT-PCR protocols to measure IgH expression in a vertebrate model which has considerable promise for modelling both pathogenic infection and chronic conditions leading to immune dysfunction. Specific primers were designed and following verification of their specificty, relative expression levels of IgD, IgM, and IgZ/T were measured in triplicate for zebrafish raised under standard laboratory conditions. During embryonic stages, low levels of each heavy chain isotype (IgH) were detected with each increasing steadily between 2 and 17 weeks post fertilization. Overall IgM>IgZ>IgD throughout zebrafish development with the copy number of IgM being several fold higher than that of IgD or IgZ/T. IgD exon usage was also characterized, as its extremely long size and presence of a stop codon in the second IgD exon in zebrafish, raised questions as to how this antibody might be expressed. Zebrafish IgD was found to be a chimeric immunoglobulin, with the third IgD exon spliced to the first IgM constant exon thereby circumventing the first and second IgD exons. Collectively, the qRT-PCR results represent the first comparative profile of IgD, IgM, IgZ/T expression over the lifespan of any fish species and the primers and assay parameters reported should prove useful in enabling researchers to rapidly quantify changes in IgH expression in zebrafish models of disease where altered IgH expression is manifested. PMID:21820179

  10. Akirin2 promotes slow myosin heavy chain expression by CaN/NFATc1 signaling in porcine skeletal muscle satellite cells.

    PubMed

    Chen, Xiaoling; Luo, Yanliu; Huang, Zhiqing; Liu, Guangmang; Zhao, Hua

    2017-02-16

    The objective of this study was to evaluate the effect of Akirin2 on slow myosin heavy chain (slow MyHC, MyHC I) gene expression and its molecular mechanisms. In this study, we showed that the protein expression of Akirin2 in pig slow oxidative Psoas major muscle is higher than that in fast glycolytic tibialis anterior muscle, suggesting that Akirin2 may play a role in myofiber typing. Knockdown of Akirin2 decreased the MyHC I expression and the calcineurin (CaN) activity, and also decreased the expressions of NFATc1 and MCIP1.4. Conversely, overexpression of Akirin2 got the opposite results. Furthermore, inhibition of CaN or knockdown of NFATc1 attenuated Akirin2 overexpression-induced upregulation of MyHC I. Together, these results demonstrate that Akirin2 promotes MyHC I expression via CaN/NFATc1 signaling pathway in porcine skeletal muscle satellite cells.

  11. Lamellipodial localization of Dictyostelium myosin heavy chain kinase A is mediated via F-actin binding by the coiled-coil domain.

    PubMed

    Steimle, Paul A; Licate, Lucila; Côté, Graham P; Egelhoff, Thomas T

    2002-04-10

    Myosin heavy chain kinase A (MHCK A) modulates myosin II filament assembly in the amoeba Dictyostelium discoideum. MHCK A localization in vivo is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events, with preferential recruitment into anterior filamentous actin (F-actin)-rich structures. The current work reveals that an amino-terminal segment of MHCK A, previously identified as forming a coiled-coil, mediates anterior localization. MHCK A co-sediments with F-actin, and deletion of the amino-terminal domain eliminated actin binding. These results indicate that the anterior localization of MHCK A is mediated via direct binding to F-actin, and reveal the presence of an actin-binding function not previously detected by primary sequence evaluation of the coiled-coil domain.

  12. Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

    PubMed

    Knotts, S; Rindt, H; Robbins, J

    1995-08-25

    Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.

  13. Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

    PubMed Central

    Knotts, S; Rindt, H; Robbins, J

    1995-01-01

    Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms. Images PMID:7667107

  14. Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR.

    PubMed

    Okazaki, Fumiyoshi; Aoki, Jun-ichi; Tabuchi, Soichiro; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

    2012-10-01

    We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V(HH)) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V(HH) with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V(HH) reached 73.8 mg/1 in a Sakaguchi flask. The V(HH) fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V(HH) fragment was specifically recognized and could bind to the EGFR with a high affinity.

  15. 1A.02: MICRORNA-208A AND ITS HOST GENE CARDIAC MYOSIN HEAVY CHAIN MYH6 ARE INVOLVED IN HYPERTROPHIC HEART DYSFUNCTION.

    PubMed

    Dóka, G; Radik, M; Krenek, P; Kyselovic, J; Klimas, J

    2015-06-01

    Circulating microRNAs may be markers of cardiac damage or dysfunction. miR-208a has heart-specific expression since its host gene - myosin heavy chain MYH6, dominant cardiac myosin motor is also heart-specific and its expression maintains proper cardiac output. Our aim was to evaluate miR-208a and MYH6 as key contributing factors involved in hypertrophic heart dysfunction. 18-20 weeks old male Wistar rats were treated for 8 days with isoproterenol (ISO; N=12; 5 mg/kg intraperitoneally) or vehicle (CON; N=12). Relative expressions of cardiac myosin heavy chains (MYH6, MYH7, MYH7B), markers of cardiac damage (natriuretic peptides ANP, BNP) and heart-related microRNAs miR-1, miR-133a, miR-208a, miR-499 were analyzed using quantitative real-time PCR in samples from left ventricle, microRNAs also in venous blood. Cardiac hypertrophy and dysfunction was quantified with heart gravimetry and left heart catheterization, respectively. Treatment with isoproterenol induced cardiac hypertrophy (heart mass increased by +36% vs. CON (P<0.01) and 53% mortality associated with cardiovascular dysfunction characterized by a deteriorated peak left ventricular pressure and rate of isovolumetric pressure change during contraction (+dP/dt) compared to CON (-8% and -27% resp., P < 0.01). Gene expression of cardiac myosin heavy chain MYH6 in left ventricles was decreased by -61% indicating myosin switching in contractile apparatus. Cardiac dysfunction was further confirmed by 10-fold increase of atrial natriuretic peptide (ANP; P<0.01). Cardiac levels of microRNAs miR-1, miR-133a, miR-208a, miR-499 were strongly decreased (-71%, -65%, -59%, -75% resp., P < 0.01). Plasma levels of the same microRNAs were unchanged except for the cardio-specific miR-208a that showed a significant, 56-fold increase (P<0.01). ROC curve for detection of cardiac hypertrophy based on plasma miR-208a had a corresponding AUC = 98.8% (95% confidence interval, 85.3% - 100%). Cardiac hypertrophy was

  16. Cardiac beta-myosin heavy chain defects in two families with non-compaction cardiomyopathy: linking non-compaction to hypertrophic, restrictive, and dilated cardiomyopathies.

    PubMed

    Hoedemaekers, Yvonne M; Caliskan, Kadir; Majoor-Krakauer, Danielle; van de Laar, Ingrid; Michels, Michelle; Witsenburg, Maarten; ten Cate, Folkert J; Simoons, Maarten L; Dooijes, Dennis

    2007-11-01

    Cardiomyopathies are classified according to distinct morphological characteristics. They occur relatively frequent and are an important cause of mortality and morbidity. Isolated ventricular non-compaction or non-compaction cardiomyopathy (NCCM) is characterized by an excessively thickened endocardial layer with deep intertrabecular recesses, reminiscent of the myocardium during early embryogenesis. Aims Autosomal-dominant as well as X-linked inheritance for NCCM has been described and several loci have been associated with the disease. Nevertheless, a major genetic cause for familial NCCM remains to be identified. Methods and Results We describe, in two separate autosomal-dominant NCCM families, the identification of mutations in the sarcomeric cardiac beta-myosin heavy chain gene (MYH7), known to be associated with hypertrophic cardiomyopathy (HCM), restricted cardiomyopathy (RCM), and dilated cardiomyopathy (DCM). Conclusion These results confirm the genetic heterogeneity of NCCM and suggest that the molecular classification of cardiomyopathies includes an MYH7-associated spectrum of NCCM with HCM, RCM, and DCM.

  17. Immunoglobulin heavy-chain enhancer is required to maintain transfected gamma 2A gene expression in a pre-B-cell line.

    PubMed Central

    Porton, B; Zaller, D M; Lieberson, R; Eckhardt, L A

    1990-01-01

    The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed. Images PMID:2106067

  18. Recombination events near the immunoglobulin Cmu gene join variable and constant region genes, switch heavy-chain expression, or inactivate the locus.

    PubMed

    Cory, S; Webb, E; Gough, J; Adams, J M

    1981-04-28

    Immunoglobulin heavy-chain expression is initiated by recombination between a variable region (VH) gene and one of several joining region (JH) genes located near the mu constant region (Cmu) gene, and the active VH gene can subsequently switch to another CH gene. That the general mechanism for CH switching involves recombination between sites within the JH-Cmu intervening sequence and the 5' flanking region of another CH gene is supported here by Southern blot hybridization analysis of eight IgG- and IgA-secreting plasmacytomas. An alternative model requiring successive VH linkage to similar JH clusters near each CH gene is shown to be very unlikely since the mouse genome appears to contain only one complement of the JH locus and no JH gene was detectable within large cloned sequences flanking germline C gamma 3 and C gamma 1 genes. Thus, VH-JH joining and CH switching are mediated by separate regions of "the joining-switch" or J-S element. In each plasmacytoma examined, the J-S element had undergone recombination within both the JH locus and the switch region and was shown to be linked to the functional CH gene in an IgG3, and IgG1, and three IgA secretors. Both JH joining and CH switching occurred by deletion of DNA. Switch recombination occurred at more than one site within the J-S element in different lines, even for recombination with the same CH gene. Significantly, although heavy-chain expression is restricted to one allele ("allelic exclusion"), all rearranged in each plasmacytoma. Some rearrangements were aberrant, involving, for example, deletion of all JH genes from the allele. Hence, an error-prone recombination machinery may account for allelic exclusion in many plasmacytomas.

  19. Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA

    PubMed Central

    Badyal, Sandip K.; Basran, Jaswir; Bhanji, Nina; Kim, Ju Hwan; Chavda, Alap P.; Jung, Hyun Suk; Craig, Roger; Elliott, Paul R.; Irvine, Andrew F.; Barsukov, Igor L.; Kriajevska, Marina; Bagshaw, Clive R.

    2011-01-01

    The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca2+ binds to the EF2 domain of S100A4 with micromolar affinity and that the Kd value for Ca2+ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in Kd results from a reduced dissociation rate constant (from 16 s− 1 to 0.3 s− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca2+]. However, for Ca2+ transients to be effective in further promoting dissociation, the elevated Ca2+ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein–myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data. PMID:21110983

  20. Differential effects of docoosahexaenoic and arachidonic acid on fatty acid composition and myosin heavy chain-related genes of slow- and fast-twitch skeletal muscle tissues.

    PubMed

    Hashimoto, Michio; Inoue, Takayuki; Katakura, Masanori; Hossain, Shahdat; Mamun, Abdullah Al; Matsuzaki, Kentaro; Arai, Hiroyuki; Shido, Osamu

    2016-04-01

    Myosin heavy chain (MHC) mediates the metabolic and contractile responses of skeletal muscles. MHC displays different isoforms, each of which has different characteristics. To better understand the effect of polyunsaturated fatty acids in skeletal muscles, rats were fed with control-, docosahexaenoic acid (DHA)-, and arachidonic acid (ARA)-oil, and the effects on plasma and muscular fatty acid profile, oxidative stress, mRNA levels of myosin heavy chain isoforms MHC1 of slow-twitch muscle (SO) and MHC2A, MHC2X, and MHCB isoforms of extensor digitorum longus (EDL) of fast-twitch muscle were evaluated. Concomitantly, mRNA levels of anti-oxidative enzymes, such as, catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD were determined. The expressions of MHC1, MHC2A, MHC2X, and MHC2B were lower in the SO of the DHA-fed rats. In the EDL muscles of DHA-fed rats, the expressions of MHC1 and MHC2A increased; however, the expressions of MHC2X increased and that of the MHC2 were not altered. Oxidative stress, as indicated by the levels of LPO, was significantly higher in the plasma of the ARA-fed rats, when compared with that of the DHA-fed rats. The LPO levels were higher both in the SO and EDL muscles of ARA-fed rats. Compared with ARA oil intake, DHA oil showed higher mRNA levels of GPx and SOD. Catalase expression was higher only in the EDL but not in the SO-type muscles. Our studies finally indicate that DHA and ARA differentially affect the regulation of contractile and metabolic properties of slow- and fast-twitch skeletal muscles.

  1. Groove-type Recognition of Chlamydiaceae-specific Lipopolysaccharide Antigen by a Family of Antibodies Possessing an Unusual Variable Heavy Chain N-Linked Glycan*

    PubMed Central

    Haji-Ghassemi, Omid; Müller-Loennies, Sven; Saldova, Radka; Muniyappa, Mohankumar; Brade, Lore; Rudd, Pauline M.; Harvey, David J.; Kosma, Paul; Brade, Helmut; Evans, Stephen V.

    2014-01-01

    The structure of the antigen binding fragment of mAb S25-26, determined to 1.95 Å resolution in complex with the Chlamydiaceae family-specific trisaccharide antigen Kdo(2→8)Kdo(2→4)Kdo (Kdo = 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid), displays a germ-line-coded paratope that differs significantly from previously characterized Chlamydiaceae-specific mAbs despite being raised against the identical immunogen. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the entire trisaccharide antigen and so confer strict specificity. Interest in S25-23 was sparked by its rare high μm affinity and strict specificity for the family-specific trisaccharide antigen; however, only the related antibody S25-26 proved amenable to crystallization. The structures of three unliganded forms of S25-26 have a labile complementary-determining region H3 adjacent to significant glycosylation of the variable heavy chain on asparagine 85 in Framework Region 3. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the few reported structures of glycosylated mAbs containing these epitopes is the therapeutic antibody Cetuximab; however, unlike Cetuximab, one of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of an αGal-containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic response in patients receiving therapeutic antibodies. PMID:24682362

  2. Identification of a stimulus-dependent DNase I hypersensitive site between the Ialpha and Calpha exons during immunoglobulin heavy chain class switch recombination.

    PubMed

    Ono, S J; Zhou, G; Tai, A K; Inaba, M; Kinoshita, K; Honjo, T

    2000-02-11

    The complete humoral response to foreign antigen depends upon two distinct recombination events within the heavy chain locus of immunoglobulin. The first recombination event takes place in what will become the antigen combining site of the antibody molecule, encoded by V, D and J segments. The second recombination event involves the looping-out of large spans of DNA which separate the various clusters of heavy chain exons which define the different immunoglobulin isotypes, or classes. While a great deal has been learned about the nature of the VDJ recombinase, very little is known about the nature of the class-switch recombinase. Using a cell system where class-switch recombination occurs primarily to the IgA locus, we have looked for stimulus-dependent changes in the chromatin structure of the IgA locus which might result from interactions between components of the recombinase and cis-elements within the region. We present evidence that strongly suggests that the class-switch recombinase interacts between the Ialpha and Calpha exons of IgA, just upstream of the highly reiterated DR1 and DR2 elements. However, although multiple potential SMAD-4 sites are located precisely within the DNase I hypersensitive site and 160 bp upstream of that site, we failed to detect any evidence of DNA/protein interactions near the hypersensitive site. Moreover, recombinant SMAD-3/4 proteins fail to interact with these sites with appreciable affinity in vitro. These data suggest that some other structural alteration at this site (e.g. RNA/DNA hybrid) may mediate the nuclease sensitivity.

  3. Dynamics of Inter-heavy Chain Interactions in Human Immunoglobulin G (IgG) Subclasses Studied by Kinetic Fab Arm Exchange

    PubMed Central

    Rispens, Theo; Davies, Anna M.; Ooijevaar-de Heer, Pleuni; Absalah, Samira; Bende, Onno; Sutton, Brian J.; Vidarsson, Gestur; Aalberse, Rob C.

    2014-01-01

    Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange (“Fab arm exchange”). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4. PMID:24425871

  4. Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter-alpha-trypsin inhibitor and saposin B but increased expression of an inter-alpha-trypsin inhibitor heavy chain 4 fragment.

    PubMed

    Jayapalan, Jaime J; Ng, Keng L; Shuib, Adawiyah S; Razack, Azad H A; Hashim, Onn H

    2013-06-01

    The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required.

  5. Immunoglobulin heavy and light chains and T-cell receptor beta and gamma chains PCR assessment on cytological samples. A study comparing FTA cards and cryopreserved lymph node fine-needle cytology.

    PubMed

    Peluso, A L; Cozzolino, I; Bottiglieri, A; Lucchese, L; Di Crescenzo, R M; Langella, M; Selleri, C; Zeppa, P

    2017-06-01

    To evaluate and compare the DNA yield and quality extracted from lymph node fine needle cytology (FNC) samples stored on FTA cards to those cryopreserved, and to assess the immunoglobulin heavy and light chains (IGHK) and T-Cell receptor beta and gamma chains (TCRBG) PCR tests. DNA extractions were performed on FNC of 80 non-Hodgkin lymphomas (NHL), four myelomas and 56 benign reactive hyperplasias (BRH) cryopreserved and stored on FTA cards. The JAK2 gene was amplified to assess the DNA integrity and the IGHK/TCRBG clonality status was tested. IGHK monoclonality was found in 99% of B-cell NHL and 100% of myeloma. TCRBG monoclonality was found in 100% of T-cell NHL. TCRBG polyclonality was detected in 97% of B-cell NHL, 100% of myeloma and 96% of BRH. IGHK/TCRBG PCR data were confirmed by histological and/or follow-up controls. No differences were found in the DNA quality between cryopreservation and FTA cards storage methods. IGHK/TCRBG PCR of the lymphoproliferative process on FTA cards is comparable to those cryopreserved. FTA cards can be used to store lymph node FNC for further molecular investigations. © 2016 John Wiley & Sons Ltd.

  6. Plasma cell infiltration of the small bowel: lack of evidence for a non-secretory form of alpha-heavy chain disease.

    PubMed Central

    Gilinsky, N H; Mee, A S; Beatty, D W; Novis, B H; Young, G; Price, S; Purves, L R; Marks, I N

    1985-01-01

    Eight patients with diffuse plasma cell infiltration of the small bowel who had the clinical features of immunoproliferative small intestinal disease (IPSID), but whose serum was negative for free alpha-heavy chains, were investigated for evidence of a non-secretory form of alpha-chain disease (alpha-CD). Molecular sieving and immunoblotting of serum, immunoperoxidase staining of biopsy specimens, and in vitro protein synthesis studies utilising an immunoprecipitation technique and polyacrylamide gel electrophoresis, failed to detect any new cases of alpha-CD. Four of the eight cases were found to have diffuse intestinal lymphoma. The remaining four patients, who were unsuccessfully investigated for evidence of a significant abnormality in cellular immunity, have not developed detectable alpha-CD protein or lymphoma over a mean of 143 months. Despite continuing exposure to possible environmental stimuli, it is concluded that not all cases of IPSID elaborate detectable alpha-CD protein or evolve to lymphoma. Images Fig. 2 Fig. 3 PMID:3928450

  7. Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E.

    PubMed

    Mansour, Abdoulreza Agheli; Mousavi, Seyed Latif; Rasooli, Iraj; Nazarian, Shahram; Amani, Jafar; Farhadi, Nima

    2010-03-01

    Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain and a heavy chain (HC) linked through a disulfide bond. In the present study we explored the immunogenicity and protective capability of the most effective part corresponding to 1163-1256 residues of botulinum type E neurotoxin HC gene. DNA encoding the 93 C-terminal amino acid of HC residues was synthesized with optimal codon usage for expression. These DNA fragments were ligated into a pLivSelect vector and subcloned into expression vector pET32a. Recombinant plasmids were then transformed into Escherichia coli strain BL21 DE3. The recombinant protein was purified by nickel affinity gel column chromatography. The HC1163-1256 was identified by antibodies raised against BoNT/E. HC1163-1256 was shown to bind with synaptotagmin and gangliosides, indicating that the expressed and purified HC1163-1256 protein retains a functionally active conformation. The immunization with recombinant protein induced a protection level in mice. The immunization with 2mug of the recombinant protein induced a significant protection level in mice. In conclusion, availability of the recombinant protein provides an effective system to study the biochemical and physical interactions involved during BoNT binding to nerve cells and protection against its toxicity.

  8. The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

    PubMed Central

    Myster, S H; Knott, J A; O'Toole, E; Porter, M E

    1997-01-01

    Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex. Images PMID:9247642

  9. Mutated recombinant human heavy-chain ferritins and myelosuppression in vitro and in vivo: a link between ferritin ferroxidase activity and biological function.

    PubMed Central

    Broxmeyer, H E; Cooper, S; Levi, S; Arosio, P

    1991-01-01

    Human heavy-chain (H-) ferritin muteins obtained by oligonucleotide site-directed mutagenesis, together with wild-type recombinant human H- and light-chain (L-) ferritins, were evaluated for in vitro effects on the suppression of human bone marrow myeloid progenitor cells and for in vivo effects on marrow and splenic myelopoiesis in C3H/HeJ mice. The 10 H-ferritin muteins exhibited alterations of various regions of the molecule, including ones exposed on the outer surface, on the inner cavity, and on the hydrophilic and hydrophobic channels and of the four-alpha-helix bundle forming the subunit structure. They were stable and were electrophoretically analogous to wild-type H-ferritin. The muteins showed in vitro and in vivo myelosuppressive activity analogous to wild type, except for mutein 222, which was totally inactive and which lacked ferroxidase activity. Recombinant human L-ferritin, devoid of ferroxidase activity, was also inactive as a suppressor. The results demonstrate that H-ferritin myelosuppressive and ferroxidase activities are linked. One possibility is that ferroxidase activity may interfere with the cellular uptake of transferrin iron that is needed for cell proliferation, an interpretation consistent with the presently described ability of hemin to overcome H-ferritin suppressive effects. PMID:1992468

  10. Insights into the Structural Organization of the I1 Inner Arm Dynein from a Domain Analysis of the 1β Dynein Heavy Chain

    PubMed Central

    Perrone, Catherine A.; Myster, Steven H.; Bower, Raqual; O'Toole, Eileen T.; Porter, Mary E.

    2000-01-01

    To identify domains in the dynein heavy chain (Dhc) required for the assembly of an inner arm dynein, we characterized a new motility mutant (ida2-6) obtained by insertional mutagenesis. ida2-6 axonemes lack the polypeptides associated with the I1 inner arm complex. Recovery of genomic DNA flanking the mutation indicates that the defects are caused by plasmid insertion into the Dhc10 transcription unit, which encodes the 1β Dhc of the I1 complex. Transformation with Dhc10 constructs encoding <20% of the Dhc can partially rescue the motility defects by reassembly of an I1 complex containing an N-terminal 1β Dhc fragment and a full-length 1α Dhc. Electron microscopic analysis reveals the location of the missing 1β Dhc motor domain within the axoneme structure. These observations, together with recent studies on the 1α Dhc, identify a Dhc domain required for complex assembly and further demonstrate that the intermediate and light chains are associated with the stem regions of the Dhcs in a distinct structural location. The positioning of these subunits within the I1 structure has significant implications for the pathways that target the assembly of the I1 complex into the axoneme and modify the activity of the I1 dynein during flagellar motility. PMID:10888669

  11. The serum heavy/light chain immunoassay: A valuable tool for sensitive paraprotein assessment, risk, and disease monitoring in monoclonal gammopathies.

    PubMed

    Greil, Christine; Ihorst, Gabriele; Gaiser, Felix; Salzer, Ulrich; Bisse, Emanuel; Kastritis, Efstathios; Ludwig, Heinz; Wäsch, Ralph; Engelhardt, Monika

    2017-09-08

    The heavy/light chain (HLC)-immunoassay quantifies light chain types of each immunoglobulin class in patients with monoclonal gammopathies. We assessed 147 consecutive patients with different forms and stages of plasma cell dyscrasias (PCD) who received standard tests (serum and urine protein electrophoresis [SPEP, UPEP], immunofixation [IFE], serum-free light chain [SFLC]), and HLC-immunoassay. Patients with multiple myeloma (MM, n = 102), smoldering MM (SMM, n = 5), monoclonal gammopathy of undetermined significance (MGUS, n = 28), and Waldenström's macroglobulinemia (WM, n = 12) were included. We verified a significant correlation between HLC- and standard monoclonal protein (mp)-parameters, and HLC-increases with higher disease stage and unfavorable remission status. In patients with difficult to quantify mp, more abnormal HLC- than SPEP-, immunoglobulin-, or SFLC-results were found. In WM, a pathological HLC κ/λ-ratio and M-component were observed in 95% and 58%, respectively. In 21/28 MGUS and 5/5 SMM patients, HLC κ/λ-ratios were abnormal. Testing different HLC cutoffs, patients with extreme HLC values showed impaired progression-free survival (PFS). Despite the fact that different PCD patients were included, the assessment of the HLC-immunoassay in MGUS, SMM, MM, and WM, our comparison with standard mp-assays, and relevant PFS differences may excite future applications, which should be confirmed in prospective multicenter trials. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Heavy metals in the habitat and throughout the food chain of the Neotropical otter, Lontra longicaudis, in protected Mexican wetlands.

    PubMed

    Ramos-Rosas, Nadia N; Valdespino, Carolina; García-Hernández, Jaqueline; Gallo-Reynoso, Juan P; Olguín, Eugenia J

    2013-02-01

    Top predators like the Neotropical otter, Lontra longicaudis annectens, are usually considered good bioindicators of habitat quality. In this study, we evaluated heavy metal contamination (Hg(tot), Pb, Cd) in the riverine habitat, prey (crustaceans and fish), and otter feces in two Ramsar wetlands with contrasting upstream contamination discharges: Río Blanco and Río Caño Grande in Veracruz, Mexico, during the dry, the wet, and the nortes seasons. Most comparisons revealed no differences between sites while seasonal differences were repeatedly detected for all of the compartments. Higher concentrations of Pb during the dry season and of Cd during the wet season in otter feces mirrored differences detected in the most seasonally consumed prey. Compared with fecal methylmercury values reported for the European otter (0.25-0.75 mg kg(-1)) in unprotected areas, the Hg(tot) levels that we measured were lower (0.02-0.17 mg kg(-1)). However, Pb (117.87 mg kg(-1)) and Cd (9.14 mg kg(-1)) concentrations were higher (Pb, 38.15 mg kg(-1) and Cd, 4.72 mg kg(-1)) in the two Ramsar wetlands. Protected areas may shelter species, but those with water-linked diets may suffer the effect of chemicals used upstream.

  13. Anti-idiotypic antibody D12 and superantigen SPA both interact with human VH3-encoded antibodies on the external face of the heavy chain involving FR1, CDR2 and FR3.

    PubMed

    Potter, K N; Li, Y; Mageed, R A; Jefferis, R; Capra, J D

    1998-12-01

    The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V(H)3 encoded antibodies. We used mutational analysis to determine the subregions of a V(H)3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FRI, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V(H)3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.

  14. The influence of genetic factors associated with the immunoglobulin heavy chain locus on the development of benign monoclonal gammapathy in ageing IgH-congenic mice.

    PubMed

    van den Akker, T W; de Glopper-van der Veer, E; Radl, J; Benner, R

    1988-09-01

    The role of genetic factors associated with the immunoglobulin heavy chain locus (Igh) in the development of benign monoclonal gammapathy (BMG), a benign B-cell proliferative disorder, was investigated in six Igh congenic mouse strains during ageing. The strains used had a C57BL or BALB background: C57BL/6, BALB.Igb and CB-20 carrying the C57BL Igh (Ighb allotype), BALB/c and C57BL/6.Iga carrying the BALB/c Igh (Igha allotype) and BAB-14, that is of BALB/c origin with the exception of the constant part of the Igh, which is of C57BL origin. The frequency of homogeneous immunoglobulins (H-Ig), both single and multiple, was the highest in C57BL/6 mice, followed by C57BL/6.Iga. The frequencies of H-Ig in BALB.Igb and CB-20 mice were higher than those of BALB/c and BAB-14, although somewhat lower than in C57BL/6.Iga mice. Multiple H-Ig were found especially in the sera of C57BL/6 mice. Categorization of the monoclonal gammapathies (MG) on the basis of their origin showed a single transient monoclonal B-cell proliferation in 0-8% of the mice of all strains. Persistent, non-progressive MG, presumably BMG, were detected in 64% of C57BL/6, 30% of C57BL/6.Iga, 22% of BALB.Igb, 17% of CB-20, 13% of BAB-14 and 6% of BALB/c mice. Multiple myeloma or Waldenström-like B-cell lymphoma were found to be responsible for 2-4% of the paraproteinemias in all strains. The remaining H-Ig, varying from 11% of the C57BL/6 to 70% of the BAB-14 mice, could not be evaluated in time. The most frequent isotypes of the BMG within C57BL/6 and C57BL/6.Iga were IgG2a and IgG2b, respectively; IgM was the most frequent isotype within the four BALB congenic strains. The immunoglobulin heavy chain allotypes under investigation appeared to be only partly related to the onset, occurrence, multiplicity and persistence of the BMG developing in these Igh congenic C57BL and BALB strains during ageing. The immunoglobulin heavy chain allotypes, however, were not related to the major isotype of the BMG. The

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

    PubMed

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

    2015-04-01

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

  16. Induction of Interleukin-1β by Human Immunodeficiency Virus-1 Viral Proteins Leads to Increased Levels of Neuronal Ferritin Heavy Chain, Synaptic Injury, and Deficits in Flexible Attention

    PubMed Central

    Festa, Lindsay; Gutoskey, Christopher J.; Graziano, Alessandro; Waterhouse, Barry D.

    2015-01-01

    Synaptodendritic pruning and alterations in neurotransmission are the main underlying causes of HIV-associated neurocognitive disorders (HAND). Our studies in humans and nonhuman primates indicated that the protein ferritin heavy chain (FHC) is a critical player in neuronal changes and ensuing cognitive deficit observed in these patients. Here we focus on the effect of HIV proteins and inflammatory cytokines implicated in HAND on neuronal FHC levels, dendritic changes, and neurocognitive behavior. In two well characterized models of HAND (HIV transgenic and gp120-treated rats), we report reductions in spine density and dendritic branches in prefrontal cortex pyramidal neurons compared with age-matched controls. FHC brain levels are elevated in these animals, which also show deficits in reversal learning. Moreover, IL-1β, TNF-α, and HIV gp120 upregulate FHC in rat cortical neurons. However, although the inflammatory cytokines directly altered neuronal FHC, gp120 only caused significant FHC upregulation in neuronal/glial cocultures, suggesting that glia are necessary for sustained elevation of neuronal FHC by the viral protein. Although the envelope protein induced secretion of IL-1β and TNF-α in cocultures, TNF-α blockade did not affect gp120-mediated induction of FHC. Conversely, studies with an IL-1β neutralizing antibody or specific IL-1 receptor antagonist revealed the primary involvement of IL-1β in gp120-induced FHC changes. Furthermore, silencing of neuronal FHC abrogates the effect of gp120 on spines, and spine density correlates negatively with FHC levels or cognitive deficit. These results demonstrate that viral and host components of HIV infection increase brain expression of FHC, leading to cellular and functional changes, and point to IL-1β-targeted strategies for prevention of these alterations. SIGNIFICANCE STATEMENT This work demonstrates the key role of the cytokine IL-1β in the regulation of a novel intracellular mediator [i.e., the

  17. Regeneration of the immunoglobulin heavy-chain repertoire after transient B-cell depletion with an anti-CD20 antibody

    PubMed Central

    Rouzière, Anne-Sophie; Kneitz, Christian; Palanichamy, Arumugam; Dörner, Thomas; Tony, Hans-Peter

    2005-01-01

    B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis. Nevertheless, the role of B cells in the pathogenesis of the disease is not clear. In particular, it is not known how the regeneration of the B-cell repertoire takes place. Two patients with active rheumatoid arthritis were treated with rituximab, and the rearranged immunoglobulin heavy-chain genes (Ig-VH) were analysed to follow the B-cell regeneration. Patient A was treated with two courses of rituximab, and B-cell regeneration was followed over 27 months by analysing more than 680 Ig-VH sequences. Peripheral B-cell depletion lasted 7 months and 10 months, respectively, and each time was accompanied by a clinical improvement. Patient B received one treatment course. B-cell depletion lasted 5 months and was accompanied by a good clinical response. B cells regenerated well in both patients, and the repopulated B-cell repertoire was characterised by a polyclonal and diverse use of Ig-VH genes, as expected in adult individuals. During the early phase of B-cell regeneration we observed the expansion and recirculation of a highly mutated B-cell population. These cells expressed very different Ig-VH genes. They were class-switched and could be detected for a short period only. Patient A was followed long term, whereby some characteristic changes in the VH2 family as well as in specific mini-genes like VH3–23, VH 4–34 or VH 1–69 were observed. In addition, rituximab therapy resulted in the loss of clonal B cells for the whole period. Our data show that therapeutic transient B-cell depletion by anti-CD20 antibodies results in the regeneration of a diverse and polyclonal heavy-chain repertoire. During the early phase of B-cell regeneration, highly mutated B cells recirculate for a short time period in both the patients analysed. The longitudinal observation of a single patient up to 27 months shows subtle intraindividual changes, which may indicate repertoire

  18. Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEmu enhancer and 3'Ealpha enhancers in human lymphoblastoid B cells.

    PubMed

    Komori, Atsumasa; Xu, Zhenming; Wu, Xiaoping; Zan, Hong; Casali, Paolo

    2006-04-01

    Somatic hypermutation (SHM) in immunoglobulin gene (Ig) variable (V) regions is critical for the maturation of the antibody response. It is dependent on the expression of activation-induced cytidine deaminase (AID) and translesion DNA polymerases in germinal center B cells as well as Ig V transcription, as regulated by the Ig heavy chain (H) intronic enhancer (iEmu) and the 3' enhancer (3'Ealpha) region. We analyzed the role of these cis elements in SHM by stably transfecting Ramos human lymphoblastoid B cells with a rearranged human IgH chain VD (diversity) J (joining) DNA construct containing a V(H) promoter at the 5' end and C(H)1 and C(H)2 exons of Cgamma1 at the 3' end. In this construct, mutations preferentially targeted dA/dT basepairs in the RGYW/WRCY hotspot. Most of the dA/dT mutations and accompanying dC/dG mutations were transitions. Deletion of iEmu resulted in decreased SHM which could be partially restored by insertion of the IgH hs1,2 enhancer. Other two 3'Ealpha enhancers, hs3-hs4, did not significantly increase the mutation frequency, but further strengthened the dA/dT bias. The frequency and spectrum of the mutations were independent of the genomic integration of the transgene or V gene transcription level. Thus, we have established a novel in vitro system to analyze SHM and identify the role of multiple cis-regulatory elements in regulating dA/dT biased SHM. This model system will be useful to further address the role of other cis-regulating elements and recruited trans-acting factors in expressing the modalities of SHM.

  19. Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: A systematic expression analysis

    PubMed Central

    Hamm, Alexander; Veeck, Juergen; Bektas, Nuran; Wild, Peter J; Hartmann, Arndt; Heindrichs, Uwe; Kristiansen, Glen; Werbowetski-Ogilvie, Tamra; Del Maestro, Rolando; Knuechel, Ruth; Dahl, Edgar

    2008-01-01

    Background The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. Methods We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry. Results We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule. Conclusion Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies. PMID:18226209

  20. Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain.

    PubMed

    Avril, Arnaud; Miethe, Sebastian; Popoff, Michel R; Mazuet, Christelle; Chahboun, Siham; Rasetti-Escargueil, Christine; Sesardic, Dorothea; Thullier, Philippe; Hust, Michael; Pelat, Thibaut

    2015-09-17

    Botulism is a naturally occurring disease, mainly caused by the ingestion of food contaminated by the botulinum neurotoxins (BoNTs). Botulinum neurotoxins are the most lethal. They are classified among the six major biological warfare agents by the Centers for Disease Control. BoNTs act on the cholinergic motoneurons, where they cleave proteins implicated in acetylcholine vesicle exocytosis. This exocytosis inhibition induces a flaccid paralysis progressively affecting all the muscles and generally engendering a respiratory distress. BoNTs are also utilized in medicine, mainly for the treatment of neuromuscular disorders, preventing large scale vaccination. Botulism specific treatment requires injections of antitoxins, usually of equine origin and thus poorly tolerated. Therefore, development of human or human-like neutralizing antibodies is of a major interest, and it is the subject of the European framework project called "AntiBotABE". In this study, starting from a macaque immunized with the recombinant heavy chain of BoNT/A1 (BoNT/A1-HC), an immune antibody phage-display library was generated and antibody fragments (single chain Fragment variable) with nanomolar affinity were isolated and further characterized. The neutralization capacities of these scFvs were analyzed in the mouse phrenic nerve-hemidiaphragm assay. After a three-round panning, 24 antibody fragments with affinity better than 10 nM were isolated. Three of them neutralized BoNT/A1 efficiently and two cross-neutralized BoNT/A1 and BoNT/A2 subtypes in the mouse phrenic nerve-hemidiaphragm assay. These are the first monoclonal human-like antibodies cross-neutralizing both BoNT/A1 and BoNT/A2. The antibody A1HC38 was selected for further development, and could be clinically developed for the prophylaxis and treatment of botulism.

  1. Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression.

    PubMed

    Ushmorov, Alexey; Ritz, Olga; Hummel, Michael; Leithäuser, Frank; Möller, Peter; Stein, Harald; Wirth, Thomas

    2004-11-15

    Immunoglobulin production is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) in spite of functional clonal rearrangements. The presence of "crippling" mutations in coding and regulatory regions, as well as down-regulation of B-cell-specific transcription factors, has been suggested as a potential reason for the lack of immunoglobulin (Ig) chain gene transcription. We have investigated the impact of epigenetic silencing in suppressing Ig heavy (H)-chain expression. Chromatin immunoprecipitation (ChIP) was used to analyze transcription factor binding to octamer motifs present in the IgH regulatory regions. Transcription factors were bound to these motifs in control cell lines, however, they were absent in the cHL-derived cell lines KMH2, L1236, and L428. Ectopic expression of octamer-binding transcription factor (Oct2) and/or B-cell Oct binding protein/Oct-binding factor (BOB.1/OBF.1) did not result in any measurable binding to these sites. Increased histone 3 Lysine 9 (H3-K9) methylation was observed in the promoter region of the IgH locus in L428 and L1236 cells. This is a typical feature of heterochromatic, transcriptionally silent regions. Treatment of cHL-derived cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) partially reactivated IgH transcription and affected chromatin modifications. Our results suggest an important role of epigenetic silencing in the inhibition of IgH transcription in HRS cells.

  2. Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

    PubMed Central

    Lian, Zhirui; Wu, Qindong; Wang, Tongtong

    2016-01-01

    ABSTRACT Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins. PMID:26652198

  3. Prognostic Impact of Serum Heavy/Light Chain Pairs in Patients With Monoclonal Gammopathy of Undetermined Significance and Smoldering Myeloma: Long-Term Results From a Single Institution.

    PubMed

    Magnano, Laura; Fernández de Larrea, Carlos; Elena, Montserrat; Cibeira, María Teresa; Tovar, Natalia; Aróstegui, Juan I; Pedrosa, Fabiola; Rosiñol, Laura; Filella, Xavier; Yagüe, Jordi; Bladé, Joan

    2016-06-01

    Asymptomatic monoclonal gammopathies, such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), are clinical conditions that usually precede symptomatic multiple myeloma. Therefore, risk stratification is crucial owing to the heterogeneous progression rate among these patients. In previous years, suppression of the uninvolved chain of specific heavy/light chain (HLC) pairs in serum has been identified as a new risk factor in MGUS. The aim of the present study was to investigate the prognostic effect of involved and uninvolved HLC pairs and HLC ratios on progression in a series of patients with MGUS and SMM. All specific serum HLC pairs were measured in 114 patients diagnosed with SMM (n = 27) and MGUS (n = 87) from 1983 to 2003. Also, the HLC ratios were calculated. Progression to symptomatic multiple myeloma was observed in 13 patients (8 with SMM and 5 with MGUS). The risk of progression was 6 times greater in those with SMM (P = .001) and 4 times greater for those with the IgA isotype (P = .01). The suppression of any IgM isotypes (IgMκ or IgMλ) in patients with IgA or IgG gammopathy or any IgA isotypes (IgAκ or IgAλ) in patients with IgG or IgM gammopathy was associated with a shorter time to progression to symptomatic gammopathy (P = .001 and P = .03, respectively). On multivariate analysis, the evolving pattern and suppression of any IgM HLC pair remained significant. HLC ratios could be a valuable tool in the risk stratification of patients with SMM and MGUS, especially patients with IgG isotypes. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. A single mutation in framework 2 of the heavy variable domain improves the properties of a diabody and a related single-chain antibody.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Ledezma-Candanoza, Luis M; Contreras-Ferrat, Luis Gabriel; Olamendi-Portugal, Timoteo; Possani, Lourival D; Becerril, Baltazar; Riaño-Umbarila, Lidia

    2012-10-26

    Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.

  5. Structure and implied functions of truncated B-cell receptor mRNAs in early embryo and adult mesenchymal stem cells: Cdelta replaces Cmu in mu heavy chain-deficient mice.

    PubMed

    Lapter, Smadar; Livnat, Idit; Faerman, Alexander; Zipori, Dov

    2007-03-01

    Stem cells exhibit a promiscuous gene expression pattern. We show herein that the early embryo and adult MSCs express B-cell receptor component mRNAs. To examine possible bearings of these genes on the expressing cells, we studied immunoglobulin mu chain-deficient mice. Pregnant mu chain-deficient females were found to produce a higher percentage of defective morulae compared with control females. Structure analysis indicated that the mu mRNA species found in embryos and in mesenchyme consist of the constant region of the mu heavy chain that encodes a recombinant 50-kDa protein. In situ hybridization localized the constant mu gene expression to loose mesenchymal tissues within the day-12.5 embryo proper and the yolk sac. In early embryo and in adult mesenchyme from mu-deficient mice, delta replaced mu chain, implying a possible requirement of these alternative molecules for embryo development and mesenchymal functions. Indeed, overexpression of the mesenchymal-truncated mu heavy chain in 293T cells resulted in specific subcellular localization and in G(1) growth arrest. The lack of such occurrence following overexpression of a complete, rearranged form of mu chain suggests that the mesenchymal version of this mRNA may possess unique functions.

  6. Analysis of tcRNA102 associated with myosin heavy chain-mRNPs in control and dystrophic chick pectoralis muscle.

    PubMed

    Zezza, D J; Heywood, S M

    1986-06-05

    Translational control RNA (tcRNA102) is closely associated with nonpolysomal myosin heavy chain-mRNA in mRNP particles. The nucleotide sequence of tcRNA102 has revealed a heterogeneity at the 3' end. This heterogeneity is mostly with regard to an ambiguity between adenine and guanine residues. tcRNA102 (obtained from pectoralis muscle) runs as a single band on denaturing acrylamide gels. When this band is extracted and rerun on a native gel at low voltage, two individual bands appear (A the slower moving and B the faster moving). From the partial RNase U2 sequence analysis and our previous sequence determinations (McCarthy, T. L., Siegel, E., Mrockowski, B., and Heywood, S. M. (1983) Biochemistry 22, 935-941), we may now assign tcRNA102 (A) the 3'-terminal sequence ... GGUUGGACGG-3' and tcRNA102(B) and 3' terminal sequence ... GAUUAAGCAA-3'. Analysis of the tcRNA102s indicates that dystrophic pectoralis muscle contains much less tcRNA102 than a similar preparation from control muscle. The tcRNA102 found in dystrophic pectoralis muscle is of the "A" type while normal pectoralis muscle contains predominantly the "B" type. In addition, control leg muscle from dystrophic chick contains predominantly "B" type. These results suggest that the differences observed at the DNA level (see accompanying paper, Zezza, D. J., and Heywood, S. M. (1986) J. Biol. Chem. 261, 7455-7460) may be reflected in the RNA transcripts.

  7. Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Chu, Wuying; Fu, Guihong; Bing, Shiyu; Meng, Tao; Zhou, Ruixue; Cheng, Jia; Zhao, Falan; Zhang, Hongfang; Zhang, Jianshe

    2010-03-01

    The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%-76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%-80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

  8. Effects of chronic Akt/mTOR inhibition by rapamycin on mechanical overload-induced hypertrophy and myosin heavy chain transition in masseter muscle.

    PubMed

    Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

    2013-01-01

    To examine the effects of the Akt/mammalian target of rapamycin (mTOR) pathway on masseter muscle hypertrophy and myosin heavy chain (MHC) transition in response to mechanical overload, we analyzed the effects of bite-opening (BO) on the hypertrophy and MHC composition of masseter muscle of BO-rats treated or not treated with rapamycin (RAPA), a selective mTOR inhibitor. The masseter muscle weight in BO-rats was significantly greater than that in controls, and this increase was attenuated by RAPA treatment. Expression of slow-twitch MHC isoforms was significantly increased in BO-rats with/without RAPA treatment, compared with controls, but the magnitude of the increase was much smaller in RAPA-treated BO-rats. Phosphorylation of p44/42 MAPK (ERK1/2), which preserves fast-twitch MHC isoforms in skeletal muscle, was significantly decreased in BO-rats, but the decrease was abrogated by RAPA treatment. Calcineurin signaling is known to be important for masseter muscle hypertrophy and fast-to-slow MHC isoform transition, but expression of known calcineurin activity modulators was unaffected by RAPA treatment. Taken together, these results indicate that the Akt/mTOR pathway is involved in both development of masseter muscle hypertrophy and fast-to-slow MHC isoform transition in response to mechanical overload with inhibition of the ERK1/2 pathway and operates independently of the calcineurin pathway.

  9. On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed.

    PubMed

    Lang, R B; Stanton, L W; Marcu, K B

    1982-01-22

    During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.

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

    SciTech Connect

    Demaison, C.; Chastagner, P.; Theze, J.; Zouali, M. )

    1994-01-18

    Monoclonal anti-DNA antibodies bearing a lupus nephritis-associated idiotype were derived from five patients with systemic lupus erythematosus (SLE). Genes encoding their heavy (H)-chain variable (V[sub H]) regions were cloned and sequenced. When compared with their closest V[sub h] germ-line gene relatives, these sequences exhibit a number of silent (S) and replacement (R) substitutions. The ratios of R/S mutations were much higher in the complementarity-determining regions (CDRs) of the antibodies than in the framework regions. Molecular amplification of genomic V[sub H] genes and Southern hybridization with somatic CDR2-specific oligonucleotide probes showed that the configuration of the V[sub H] genes corresponding to V[sub H] sequences in the nephritogenic antibodies is not present in the patient's own germ-line DNA, implying that the B-cell clones underwent somatic mutation in vivo. These findings, together with the characteristics of the diversity and junctional gene elements utilized to form the antibody, indicate that these autoantibodies have been driven through somatic selection processes reminiscent of those that govern antibody responses triggered by exogenous stimuli.

  11. Establishment of a mutation system in Tetrahymena outer arm dynein and P-loop functions of the alpha heavy chain (Dyh3p).

    PubMed

    Edamatsu, Masaki

    2017-01-29

    Axonemal dyneins are large AAA+ type motor proteins that exhibit unique motor properties during ciliary beating. This study established a mutation system for Tetrahymena outer arm dynein and characterized four nucleotide-binding loops (P-loops; P1-P4) in the alpha heavy chain (Dyh3p). Macronuclear transformation of the mutant DYH3 genes in DYH3-knockout (KO-DYH3) cells enabled P-loop mutations that abolish the ability of nucleotide binding to be stably maintained in the polyploid genome. This mutation system revealed that the P3 and P4 mutant dyneins rescued lethality in macronuclear KO-DYH3 cells and exhibited normal ciliary localization. Intriguingly, however, an in vitro motility assay showed that the P3 mutation abolished the motor activity of Dyh3p, whereas the P4 mutation did not affect the gliding velocity or gliding index of Dyh3p. In contrast, no P1 or P2 mutant cells were isolated from the KO-DYH3 cells, which suggests that nucleotide binding at the P1 and P2 sites is required for the intracellular function of Dyh3p. This mutation system will be useful for further molecular studies of diverse axonemal dyneins and ciliary motility. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Muscle-Specific Myosin Heavy Chain Shifts in Response to a Long-Term High Fat/High Sugar Diet and Resveratrol Treatment in Nonhuman Primates

    PubMed Central

    Hyatt, Jon-Philippe K.; Nguyen, Lisa; Hall, Allison E.; Huber, Ashley M.; Kocan, Jessica C.; Mattison, Julie A.; de Cabo, Rafael; LaRocque, Jeannine R.; Talmadge, Robert J.

    2016-01-01

    Shifts in myosin heavy chain (MHC) expression within skeletal muscle can be induced by a host of stimuli including, but not limited to, physical activity, alterations in neural activity, aging, and diet or obesity. Here, we hypothesized that both age and a long-term (2 year) high fat/high sugar diet (HFS) would induce a slow to fast MHC shift within the plantaris, soleus, and extensor digitorum longus (EDL) muscles from rhesus monkeys. Furthermore, we tested whether supplementation with resveratrol, a naturally occurring compound that has been attributed with augmenting aerobic potential through mitochondrial proliferation, would counteract any diet-induced MHC changes by promoting a fast to slow isoform switch. In general, we found that MHC isoforms were not altered by aging during mid-life. The HFS diet had the largest impact within the soleus muscle where the greatest slow to fast isoform shifts were observed in both mRNA and protein indicators. As expected, long-term resveratrol treatment counteracted, or blunted, these diet-induced shifts within the soleus muscle. The plantaris muscle also demonstrated a fast-to-slow phenotypic response to resveratrol treatment. In conclusion, diet or resveratrol treatment impacts skeletal muscle phenotype in a muscle-specific manner and resveratrol supplementation may be one approach for promoting the fatigue-resistant MHC (type I) isoform especially if its expression is blunted as a result of a long-term high fat/sugar diet. PMID:26973542

  13. Muscle-specific accumulation of Drosophila myosin heavy chains: a splicing mutation in an alternative exon results in an isoform substitution.

    PubMed

    Kronert, W A; Edwards, K A; Roche, E S; Wells, L; Bernstein, S I

    1991-09-01

    We show that the molecular lesions in two homozygousviable mutants of the Drosophila muscle myosin heavy chain gene affect an alternative exon (exon 9a) which encodes a portion of the myosin head that is highly conserved among both cytoplasmic and muscle myosins of all organisms. In situ hybridization and Northern blotting analysis in wild-type organisms indicates that exon 9a is used in indirect flight muscles whereas both exons 9a and 9b are utilized in jump muscles. Alternative exons 9b and 9c are used in other larval and adult muscles. One of the mutations in exon 9a is a nonsense allele that greatly reduces myosin RNA stability. It prevents thick filament accumulation in indirect flight muscles and severely reduces the number of thick filaments in a subset of cells of the jump muscles. The second mutation affects the 5' splice site of exon 9a. This results in production of an aberrantly spliced transcript in indirect flight muscles, which prevents thick filament accumulation. Jump muscles of this mutant substitute exon 9b for exon 9a and consequently have normal levels of thick filaments in this muscle type. This isoform substitution does not obviously affect the ultrastructure or function of the jump muscle. Analysis of this mutant illustrates that indirect flight muscles and jump muscles utilize different mechanisms for alternative RNA splicing.

  14. Changes in muscle fib