Search PubMed⌕ Search

Biomedical subjects

D B Murphy

Publications and source records attributed to D B Murphy.

At least 73 records · Page 4Linked to original sources
PubMed1986

Serologic heterogeneity in I-J determinants associated with functionally distinct T cell regulatory factors.

Information transfer among regulatory T cell subsets is mediated by biologically active T cell factors. Many of these factors are comprised of two molecules: one that binds antigen, and another that is I-J+ and determines the self recognition capability of the factor (I-J molecule). In the in vitro response to sheep red blood cells, we used three functionally distinct I-J+ factors to study the relationship between polymorphic I-J determinants and the biological activity of these factors. Our study shows that several monoclonal I-J antibodies react with I-J molecules associated with T suppressor-inducer factor (TsiF) and T suppressor-effector factor (TseF), but not with T contrasuppressor inducer factor (TcsiF). In contrast, a different set of monoclonal I-J reagents reacts with TcsiF but not TsiF or TseF. Finally, some monoclonal I-J antibodies distinguish between I-J molecules associated with TsiF and TseF. Thus anti-I-J reagents differentially react with I-J determinants on regulatory factors, and this differential pattern of reactivity correlates with the functional activity of the factors. The possible relationship between I-J heterogeneity and the biological function of I-J molecules in regulation is discussed.

Animals↗
PubMed1986

Erythrocyte microtubule assembly in vitro. Tubulin oligomers limit the rate of microtubule self-assembly.

Chicken erythrocyte tubulin containing a unique beta tubulin variant polymerizes with greater efficiency (lower critical concentration) but at a slower rate than chicken brain tubulin. In a previous study we demonstrated that the low net rate of assembly is partly due to the presence of large oligomers and rings which reduce the initial rate of subunit elongation on microtubule seeds (Murphy, D.B., and Wallis, K.T. (1985) J. Biol. Chem. 260, 12293-12301). In this study we show that erythrocyte tubulin oligomers also retard the rate of microtubule nucleation and the net rate of self-assembly. The inhibitory effect is most likely to be due to the increased stability of erythrocyte tubulin oligomers, including a novel polymer of coiled rings that forms during the rapid phase of microtubule polymerization. The slow rate of dissociation of rings and coils into dimers and small oligomers appears to limit both the nucleation and elongation steps in the self-assembly of erythrocyte microtubules.

Animals↗
PubMed1986

Altered I-J phenotype in E alpha transgenic mice.

One of the more intriguing puzzles in immunology is the genetic basis for control of murine T-cell I-J determinants. Molecules bearing I-J determinants (I-J molecules) play a role in information trafficking among immunocompetent cells, probably serving as self-recognition molecules that channel regulatory factors to their appropriate target cells. Although it is clear that I-J polymorphism is influenced by the major histocompatibility complex (MHC), molecular genetic studies provide evidence that an MHC gene does not encode I-J molecules. A possible explanation for this paradox is that I-J molecules are a set of non-MHC-encoded T cell receptors that are directly or indirectly selected for by self-MHC products. One key to resolving the genetic and molecular basis for control of I-J determinants is the identification of the MHC gene(s) involved. Herein, data are presented which show that E alpha transgenic mice express an altered I-J phenotype, providing clear evidence that I region class II genes influence I-J polymorphism. Although further study is required to resolve how class II genes mediate this effect, this is a major piece to the I-J puzzle.

Animals↗
PubMed1986

End-to-end annealing of microtubules in vitro.

Mixtures of pre-formed microtubules, polymerized from chicken erythrocyte and brain tubulin, rapidly anneal end-to-end in vitro in standard microtubule assembly buffer. The erythrocyte tubulin segments in annealed heteropolymers can be distinguished by an immunoelectron microscopic assay that uses an antibody specific for chicken erythrocyte beta-tubulin. An annealing process is consistent with the following observations: (a) Microtubule number decreases while the polymer mass remains constant. (b) As the total number of microtubules declines, the number of heteropolymers, and the number of segments contained in each heteropolymer, increases. (c) The size of the segments determined after annealing and antibody labeling is the same as the original microtubule polymers. (d) Points of discontinuity in the annealing heteropolymers can be observed directly by electron microscopy, and correspond to type-specific polymer domains. The junctions probably represent initial contact points during the annealing process. Microtubule annealing occurs rapidly in vitro and may be significant for determining properties of microtubule dynamics in vivo.

Animals↗
PubMed1986

Immunofluorescence examination of beta tubulin expression and marginal band formation in developing chicken erythroblasts.

Chicken erythrocyte beta tubulin, a tubulin variant with unique biochemical and assembly properties, is found to be specifically contained in two chicken blood cell types--erythrocytes and thrombocytes. The beta tubulin variant is absent or present in low amounts in a variety of white blood cell types and other body tissues, as determined by immunofluorescence microscopy and a semi-quantitative immunoblotting procedure. During differentiation in the marrow the beta tubulin variant appears suddenly in mid-stage erythroblasts at the onset of hemoglobin synthesis, and forming marginal bands are seen in all subsequent polychromatophilic erythroblast stages. The developmental sequence of events in marginal band formation entails microtubule nucleation at the centrosome, followed by microtubule elongation, consolidation of loose parallel microtubules into a compact bundle, and microtubule association with the cell membrane.

Animals↗
PubMed1986

Identification of a 34-kD polypeptide as a light chain of microtubule-associated protein-1 (MAP-1) and its association with a MAP-1 peptide that binds to microtubules.

We examined the association of a 34-kD light chain component to the heavy chains of MAP-1 using a monoclonal antibody that specifically binds the 34-kD component and labels neuronal microtubules in a specific and saturable manner. Immunoprecipitation of MAP-1 heavy chains together with the 34-kD component by the antibody indicates that the 34-kD polypeptide forms a complex with MAP-1 heavy chains. Both major isoforms of MAP-1 heavy chains (MAP-1A and MAP-1B) were found in the immunoprecipitate. Digestion of MAP-1 with alpha-chymotrypsin and analysis of the chymotryptic peptides reveals a 120-kD fragment of the MAP-1 heavy chain that binds to microtubules and is precipitable with the 34-kD light chain antibody, suggesting that the 34-kD light chain also binds to this domain of the molecule. Since microtubules that contain the 120-kD fragment lack the long lateral projections characteristic of microtubules with intact MAP-1, the 34-kD light chains may be localized at or near the microtubule surface.

Animals↗
PubMed1985

Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly.

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.

Animals↗
PubMed1985

Different reactivities of brain and erythrocyte tubulins toward a sulfhydryl group-directed reagent that inhibits microtubule assembly.

There is considerable evidence that tubulin exists in multiple isotypes, differing in amino acid sequence and tissue distribution. Little is known, however, about the functional significance of these isotypes. Chicken erythrocyte beta-tubulin has been shown by peptide mapping to differ significantly from chicken brain beta-tubulin (Murphy, D. B., and Wallis, K. T. (1983) J. Biol. Chem. 258, 7870-7875). We now find that when the two tubulins, in their native states, are incubated with N,N'-ethylenebis(iodoacetamide) (EBI), a bifunctional sulfhydryl-directed reagent, microtubule assembly by brain tubulin is much more sensitive to inhibition by EBI than is erythrocyte tubulin assembly. The resistance of erythrocyte microtubule assembly to inhibition by EBI is correlated with a low reactivity of erythrocyte tubulin with [14C]EBI. This difference is most marked in the beta subunit which reacts 15 and 17% as well, respectively, with [14C]EBI as do the beta 1 and beta 2 subunits of brain tubulin. Also, erythrocyte beta reacts about 33% as well as does brain beta with iodo[14C]acetamide. These results suggest that a reactive sulfhydryl group, whose oxidation prevents microtubule assembly, is present in brain tubulin but absent or inaccessible in erythrocyte tubulin. Since purified erythrocyte tubulin self-aggregates much more readily than does brain tubulin, it is conceivable that erythrocyte and brain tubulin may differ in that the latter may have its assembly subject to a complex regulation, while erythrocyte tubulin assembly may be regulated by a simpler mechanism.

Animals↗
PubMed1985

Identification of a unique tumor-specific antigen as a novel class I major histocompatibility molecule.

Cancers induced by physical or chemical carcinogens express tumor-specific antigens that are uniquely specific for any given tumor; therefore, there is a seemingly endless variety of these unique antigens. We have studied a UV-induced fibrosarcoma, designated 1591, to elucidate the obscure molecular nature and genetic origins of unique tumor-specific antigens. A monoclonal antibody raised against syngeneic 1591 tumor cells has unique tumor specificity. This tumor-specific monoclonal antibody precipitated from the tumor a 45-kDa molecule associated with a 12-kDa molecule having the pI of beta2-microglobulin. This and other evidence indicated that the 1591 tumor expresses a novel class I molecule. A 1591 variant selected for the absence of binding to the monoclonal antibody lacked the novel class I MHC molecule as well as reactivity with cytotoxic T lymphocytes specific for the 1591 tumor. Furthermore, tumor cells bearing the antigen are rejected while variants that have lost the antigen grow progressively. Fourteen of 14 host-selected progressor tumor variants lost reactivity with the monoclonal antibody and provided further evidence that this novel class I molecule is a transplantation antigen on the parental 1591 tumor required for immune rejection. The identification of a unique tumor-specific antigen as a novel class I major histocompatibility complex gene product allows us to search for the possible genetic mechanisms involved and to explore further the role such molecules play in tumor immunity and malignancy.

Animals↗
PubMed1985

Direct observation of microtubule treadmilling by electron microscopy.

Using an immunoelectron microscopic procedure, we directly observed the concurrent addition and loss of chicken brain tubulin subunits from the opposite ends of microtubules containing erythrocyte tubulin domains. The polarity of growth of the brain tubulin on the ends of erythrocyte microtubules was determined to be similar to growth off the ends of Chlamydomonas axonemes. The flux rate for brain tubulin subunits in vitro was low, approximately 0.9 micron/h. Tubulin subunit flux did not continue through the entire microtubule as expected, but ceased when erythrocyte tubulin domains became exposed, resulting in a metastable configuration that persisted for at least several hours. We attribute this to differences in the critical concentrations of erythrocyte and brain tubulin. The exchange of tubulin subunits into the walls of preformed microtubules other than at their ends was also determined to be insignificant, the exchange rate being less than the sensitivity of the assay, or less than 0.2%/h.

Animals↗
PubMed1985

Analysis of the microtubule-binding domain of MAP-2.

We examined the microtubule-binding domain of the microtubule-associated protein (MAP), MAP-2, using rabbit antibodies that specifically bind to the microtubule-binding region ("stub") and the projection portion ("arm") of MAP-2. We found that (a) microtubules decorated with arm antibody look similar to those labeled with whole unfractionated MAP antibody, though microtubules are not labeled with stub antibody; (b) incubation of depolymerized microtubule protein with stub antibody prior to assembly partially inhibits the rate of microtubule elongation, presumably because MAPs that are complexed with antibody cannot bind to microtubules and stabilize elongating polymers; (c) the rate of appearance and amounts of 36- and 40-kD microtubule-binding peptides produced by digestion with chymotrypsin are distinct for MAPs associated with microtubules vs. MAPs free in solution. The enhanced stability of the 40-kD peptide when associated with microtubules suggests that this domain of the protein is closely associated with, or partially buried in, the microtubule surface; (d) MAP-2 is a slender, elongate molecule as determined by unidirectional platinum shadowing (90 +/- 30 nm), which is in approximate agreement with previous observations. Stub antibody labels MAP-2 in the terminal one-quarter of the extended protein, indicating an intrinsic asymmetry in the molecule.

Animals↗
PubMed1985

The putative I-Jk- strain AKR/J synthesizes I-Jk+ molecules: implications for Jt gene control of I-J expression.

At present, the genetic basis for control of murine I-J determinants is unknown. On one hand, it is clear that polymorphism in I-J molecules is controlled by genes mapping in the I region of the H-2 gene complex on chromosome 17. On the other hand, molecular genetic studies provide evidence that I-J molecules are not encoded by I region genes. Although formal proof of the latter must await isolation and characterization of I-J structural genes, these observations are compatible with the concept that I-J molecules are encoded by non-H-2 genes, but the expression of these non-H-2 genes is regulated or influenced by I region genes. Recent studies by Hayes et al. provide evidence for non-H-2 control of the cell surface expression of I-Jk determinants in strain AKR/J. This strain typed I-Jk-, as judged by complement dependent cytolysis with monoclonal I-Jk antibodies. Studies with recombinant inbred and congenic strains suggested that the I-Jk- phenotype in strain AKR/J was controlled by a gene (Jt) mapping on chromosome 4. Based on these observations and studies with F1 hybrids and numerous other strains, Hayes et al. concluded that interaction between the Jt gene and an H-2 gene on chromosome 17 (probably E beta or E alpha) regulates the production and expression of I-Jk molecules, and hypothesized that I-Jk epitopes may reside on Jt modified Class II molecules or on the Jt gene product.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

A monoclonal antibody that recognizes an Ly-6-linked antigen inhibits the generation of functionally active T cell subsets.

A monoclonal antibody (mAb) generated against the chemically-induced BALB/c Meth A sarcoma, designated HD42, reacts in cytotoxic tests with Meth A as well as with BALB/c peripheral lymph node cells and mitogen-activated spleen cells. The antigen was detected by FACS analysis on BALB/c spleen and lymph node cells, and by absorption assays on all normal lymphoid cells of BALB/c but not B6 mice. The expression of the antigen was not found on normal adult lung fibroblasts, on brain, nor on an extensive panel of tumors of BALB/c and B6 origin. Because the strain distribution of the antigen is reciprocal to that of Ly-6.2 and is not expressed in congenic C3H.Ly-6b mice, we have tentatively defined it as Ly-6.1 and referred to the mAb as alpha-Ly-6.1. The presence of alpha-Ly-6.1 abrogates both the Con A-induced and the IL 2-dependent proliferative response of normal T cells, whereas the response of normal B cells to LPS remains unaffected. alpha-Ly-6.1 is a potent suppressor of the primary in vitro plaque-forming cell (PFC) response to SRBC. Pretreatment of normal splenic T cells with alpha-Ly-6.1 and complement had no effect on the ability of these cells to generate in vitro either T helper cells (TH) or T suppressor cells (TS) to SRBC. However, addition of antibody in the absence of complement during the generation of TH or TS, or posttreatment of these T cell subsets with antibody and complement after in vitro education, completely removed the functional activity of these cell types. Addition of alpha-Ly-6.1 to MLC suppressed the MLR as well as the generation of cytotoxic lymphocytes (CTL), whereas the presence of the antibody during a cell-mediated lympholysis (CML) had no effect. Therefore, it appears that alpha-Ly-6.1 recognizes an antigen that is important for the generation of TH and TS cell subsets.

Animals↗
PubMed1984

Arrest of pigment granule motion in erythrophores by quick-freezing.

We report the use of quick-freezing, as an alternative to conventional chemical fixation, to arrest the movement of pigment granules at various stages of the dispersion-aggregation cycle in Holocentrus erythrophores. During pigment aggregation, the granules in these cells move at up to 20 microns/sec, hence the structural changes underlying the movement are likely to be too fleeting to be captured faithfully by conventional aldehyde fixation. On the other hand, quick-frozen cells, when examined by freeze-etch electron microscopy, provide novel views of certain cytoplasmic components which appear to be involved in pigment granule movement, namely, fine (2- to 6-nm diameter) fibrils which link the granules to each other and to the radial array of microtubules. These fine crosslinking fibrils can be distinguished from thicker (8- to 15-nm diameter) strands of coherent granular material which pervade the cytoplasm of pigment-dispersed as well as pigment-aggregated cells. This granular matrix is removed by detergent permeabilization, after which it becomes apparent that the fine fibrils are insoluble and are distributed both within and distal to the aggregated pigment mass. The diameter of the specific fibrils does not change during pigment motion, which indicates that they are not contractile.

Animals↗