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T W Huiatt

Publications and source records attributed to T W Huiatt.

30 records · Page 2Linked to original sources
PubMed1992

Paraneoplastic IgG striational autoantibodies produced by clonal thymic B cells and in serum of patients with myasthenia gravis and thymoma react with titin.

Autoantibodies with heterogeneous specificities for contractile elements of striated muscle are found in 80 to 90% of patients who have myasthenia gravis (MG) with thymoma. The stimulus for production of these paraneoplastic striational autoantibodies (StrAb) is unknown. One approach to understanding their association with MG and thymoma is to define the antigenic specificities of monoclonal StrAb secreted by B cell lines established from patients who have MG and thymoma. Here, we report the isolation from a single patient of two independent thymic B cell clones secreting StrAb of IgG isotype. In immunoblots, both StrAb bound monospecifically to an antigen of human skeletal muscle that comigrated with the high molecular weight protein, titin. The pattern of immunofluorescence staining yielded by both antibodies in cultured human muscle cells was similar to that produced by rabbit polyclonal anti-titin antibodies. Each monoclonal antibody bound to a different region of the sarcomere in stretched myofibrils; these corresponded to sites previously reported to bind murine anti-titin monoclonal antibodies. The pattern of sarcomere immunostaining produced by combining the two human monoclonal antibodies was indistinguishable from that produced by serum IgG from the patient whose thymus yielded the B cell clones. Thus, the monoclonal antibodies appear to identify two epitopes of titin that are recognized by IgG StrAb in serum. The finding that IgG anti-titin autoantibodies are restricted to serum of MG patients who have thymoma suggests that titin is a major specificity of IgG StrAb. Our additional finding that anti-titin IgG binds to striated elements in medullary myoid cells of the human thymus supports the hypothesis that StrAb represent an intrathymic B cell immune response that is initiated by autoantigens that are rendered immunogenic for helper T cells in the course of noeplastic transformation to thymoma.

Adult↗
PubMed1990

Determination of the critical concentration required for desmin assembly.

The critical concentration required for filament assembly in vitro from highly purified desmin was determined by both turbidity and centrifugation assays. Assembly was done in the presence of 2 mM-Ca2+, 2 mM-Mg2+ or 150 mM-Na+ at 2, 22 and 37 degrees C. Similar values for critical concentration were obtained by both assays. As temperature increased, critical concentration decreased for each cation. The critical concentration was lowest in the presence of Ca2+ at 2, 22 and 37 degrees C, but was highest in the presence of 150 mM-Na+ at 2 degrees C. Negative staining showed that supernatants from the centrifugation assays contained protofilaments, protofibrils and short particles (less than 300 nm), but pellets contained long filaments (greater than 1 micron) with an average diameter of 10 nm. As the temperature increased, both the average diameter and average length of particles in the supernatant increased. Thermodynamic analysis indicated that hydrophobic interactions were dominant during desmin assembly, but that ionic interactions might also be involved. Our results demonstrated that the specific cation and temperature and temperature-cation interactions all are important in assembly of desmin intermediate filaments.

Actin Cytoskeleton↗
PubMed1987

Control of filament length by the regulatory light chains in skeletal and cardiac myosins.

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.

Actin Cytoskeleton↗
PubMed1984

Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines. Intrinsic differences in growth characteristics and insulin response.

Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant (P less than 0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10(-10)-10(-9) M. Slow cultures bound more [125I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.

Animals↗
PubMed1982

A bent monomeric conformation of myosin from smooth muscle.

Smooth muscle myosin filaments formed in 0.15 M KCl are depolymerized by MgATP to a 10S component, rather than to the 6S component typical of myosin monomer in high salt concentrations. This 10S species is also monomeric as determined by sedimentation equilibrium and calculated from the diffusion and sedimentation coefficients. The conformation of 10S myosin is, however, very different from that of 6S myosin, which has a flexible but extended rod. The Stokes radius and the viscosity of 10S myosin are less than those of 6S myosin, consistent with a structure in which the rod is bent. Electron microscopy of rotary-shadowed preparations confirmed that the light meromyosin region of the rod is bent back on subfragment 2, that region of the rod adjacent to the two globular heads. MgATP and dephosphorylation of the 20,000 molecular weight light chain increase the amount of 10S myosin present in 0.15 M KCl; addition of salt converts 10S myosin back to the typical 6S conformation. We conclude that smooth muscle myosin preferentially forms a bent or folded conformation instead of the extended shape usually associated with skeletal muscle myosin, provided that the salt concentration is kept sufficiently low.

Animals↗
PubMed1981

Purification of desmin from adult mammalian skeletal muscle.

A method has been developed for preparation of purified desmin from mature mammalian (porcine) skeletal muscle. A crude desmin-containing fraction was prepared by modification of procedures used for isolation of smooth-muscle intermediate-filament protein [Small & Sobieszek (1977) J. Cell Sci. 23, 243-268]. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4 degrees C for 15h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE-Sepharose CL-6B in 6M-urea yielded desmin that was routinely more than 97% 55 000-dalton protein and that had no detectable actin contamination. Removal of urea by dialysis against 10mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134 000 g (rav. 5.9 cm) for 1 h results in a clear desmin solution. Dialysis of purified desmin against 100 mM-NaCl/1 mM-MgCl2/10 mM-imidazole/HCl, pH 7.0, at 2 degrees C resulted in the formation of synthetic desmin filaments have an average diameter of 9-11.5 nm. The present studies demonstrate that the relatively small amount of desmin in mature skeletal muscle can be isolated in sufficient quantity and purity to permit detailed studies of its properties and function. Although 10nm filaments have not been unequivocally demonstrated in mature muscle in vivo, that the purified skeletal-muscle desmin will form 10 nm filaments in vitro lends support to their possible existence and cytoskeletal function in mature skeletal-muscle cells.

Actins↗
PubMed1981

Immunoelectron and immunofluorescence localization of desmin in mature avian muscles.

Antisera or affinity-purified antibodies shown to be specific for avian gizzard desmin antigen by double immunodiffusion, antigen-blocking, and immunoautoradiography experiments have been used in indirect immunofluorescence and indirect immunoelectron microscopy to demonstrate localization of desmin in myofibrils from mature avian muscles. The light microscope results agree with the work of others in that they suggest that desmin is primarily at or near the periphery of Z-lines of striated muscle myofibrils. Immunoperoxidase labelling more clearly shows that the reactive desmin antigen is located almost entirely between Z-lines of adjacent parallel myofibrils and that there is no obvious correlation between locations of T-tubules and desmin structures. The electron-dense reaction product often followed an approximately linear course between Z-lines of adjacent myofibrils and indicated the desmin antibodies had decorated a small number of filaments spanning this region. These results suggest that the desmin found in close association with myofibrils of mature striated muscle is in the aggregated form of 10-nm filaments.

Animals↗
PubMed1981

Disassembly of synthetic 10-nm desmin filaments from smooth muscle into protofilaments.

Synthetic 10-nm filaments formed from highly purified turkey gizzard desmin have a helically oriented substructure and disassemble into 2 to 2.5 nm protofilaments and 3.5 to 5 nm subfilaments after treatment with 1 or 2 M urea or with low-ionic-strength buffer (8 mM Tris, pH 8.2). SDS-gels of 10-nm filaments treated with these solutions show that a single 55 000-dalton band is present before and after all treatments and indicate that the newly revealed substructure is not caused by proteolysis. Antibodies to electrophoretically purified desmin react, in immunodiffusion, only with the antigen and decorate both the subfilamentous particles and the synthetic 10-nm filaments. These studies indicate that a synthetic 10-nm desmin filament is a rope-like structure constructed from the 2 to 2.5 nm diameter protofilaments.

Animals↗