Novel redistribution of myosin-containing filaments in cultured keratinocytes identified by a human monoclonal autoantibody

Summary A time-dependent redistribution of microfilaments was observed in cultured human keratinocytes using a human monoclonal autoantibody specific for myosin. Immunofluorescent staining revealed that 5 days after plating keratinocytes in either 0.1 mM or 2.0 mM Ca⁺⁺, myosin was distributed uniformly throughout the cytoplasm. At day 6, parallel arrays of myosin-containing microfilaments were prominent in the cell peripheries. At day 7 the microfilaments formed circumferential rings. The distribution of the microfilaments was disrupted by cytochalasin but not by colchicine, indicating that this novel distribution of myosin was not dependent on colchicine-sensitive vimentin intermediate filaments. The time-dependent redistribution of myosin was not influenced by cell population density, cell shape or cell cycle phase, except for mitotic cells in which myosin was distributed diffusely through the cytoplasm. If, as suggested by Kolega (9), microfilaments align parallel to the direction of applied tension, the redistribution of myosin-containing microfilaments in cultured keratinocytes may reflect the increased tension between cells resulting from increasing strength of cell-cell junctions over time. In sectioned human skin, myosin was localized in the peripheral cytoplasm of stratified epidermal cells. Tensions arising from the numerous desmosomal junctions between cellsin vivo could account for this distribution of myosin. Supported by grant NS-23537 (V. A. L.) from the National Institutes of Health, Bethesda, MD, and by the Mayo Foundation. C. L. W. is recipient of the Kermit E. Osserman and Blanche McClure Fellowship, 1987, National Myasthenia Gravis Foundation.     

Linkage of a gene for macular corneal dystrophy to chromosome 16.

Autosomal recessive macular corneal dystrophy (MCD) is a heterogeneous disorder leading to visual impairment. Sixteen American and Icelandic families (11 type I and 5 type II) were analyzed for linkage, by use of 208 polymorphic microsatellite markers. A significant maximum LOD score Zmax of 7.82 at a maximum recombination fraction (thetamax) of .06 was found with the 16q22 locus D16S518 for MCD type I. In addition, a peak LOD score of 2.50 at a recombination fraction of .00 was obtained for the MCD type II families, by use of the identical marker. These findings raise the possibility that MCD type II may be due to the same genetic locus that is involved in MCD type I.     

Hemoglobin affinity for 2,3-bisphosphoglycerate in solutions and intact erythrocytes: studies using pulsed-field gradient nuclear magnetic resonance and Monte Carlo simulations.

The diffusion coefficient (D) of 2,3-bisphosphoglycerate (DPG) was measured using pulsed-field gradient (PFG)-31P nuclear magnetic resonance spectroscopy in solutions containing 2.7-5.0 mM hemoglobin (Hb) and a range of DPG concentrations. The dependence of the measured values of D on the fraction of the total DPG in the sample that is bound to Hb enabled the estimation of the dissociation constants (Kd) of complexes of DPG with carbonmonoxygenated, oxygenated, and deoxygenated Hb; the values of Kd (mM), measured at 25 degrees C, pH 6.9 and in 100 mM bis Tris/50 mM KCl, were 1.98 +/- 0.26, 1.8 +/- 0.5 and 0.39 +/- 0.26, respectively. In intact erythrocytes the apparent diffusion coefficient, Dapp, of DPG was larger in oxygenated and carbonmonoxygenated cells (6.17 +/- 0.20 x 10(-11) m2s-1) than in deoxygenated cells (4.10 +/- 0.23 x 10(-11) m2s-1). Changes in intracellular DPG concentration (5-55 mM) in erythrocytes, brought about by incubation in a medium containing inosine and pyruvate, did not result in significant changes in the value of Dapp; this result supports the hypothesis that DPG binds to other sites in the erythrocyte. Monte Carlo simulations of diffusion in biconcave discs were used to test the adequacy of the values of Kd estimated in solution to describe the binding of DPG to Hb in oxygenated and deoxygenated erythrocytes. The results of the simulations implied that the value of Kd estimated for deoxygenated Hb-DPG was greater than expected from the experiments involving intact erythrocytes. This difference is surmised to be at least partly due to the difficulty of measuring D at low-ligand concentrations. Notwithstanding this shortcoming, the PFG method appears to be suitable for probing interactions between macromolecules and ligands when the Kd is in the millimolar range. It is one of the few techniques available in which these interactions can be studied in intact cells. In addition, the Monte Carlo simulations of the diffusion experiments highlighted important differences between theory and experiment relating to the nature of molecular motion inside the cells.     

Human and animal mesenchymal progenitor cells from bone marrow: Identification of serum for optimal selection and proliferation

Summary An undifferentiated subset of cells within the stromal cell population of bone marrow in postnatal mammals retains the capacity to differentiate along osteogenic, adipogenic, fibroblastic, and chondrogenic lines. These cells, which are referred to as mesenchymal stem cells (MSCs), can be maintainedin vitro and expanded in number through a process of subculturing. MSCs are maintained in culture in medium supplemented with 10% fetal bovine serum (FBS). It is believed that certain, as yet unidentified, serum components play critical roles in the attachment and proliferation of MSCs. Commercially available FBS is poorly characterized and may vary in composition and quality from lot to lot. This study describes a method for the selection of lots of FBS that best support maintenance of the undifferentiated state, mitotic expansion of MSCsin vitro, and retention of multilineage developmental potential in response to appropriate cues.     

Direct sequence analysis of proteins by in-source fragmentation during delayed ion extraction.

Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix-assisted laser desorption/ionization-generated ion signals dominated by Cn fragmentation within the ion source of a linear time-of-flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data.     

Periodic structurally similar oligomers are found on one side of the axes of symmetry in the lac, trp, and gal operators

Three well-defined E. coli operator regions were examined for recurring conformational deviation from a regular B-DNA helix. All three, the lac, trp, and gal, show repeats of the same set of neighboring helical twist angles. These angles recur with a periodicity equal to the helix periodicity on one side of the operator's axes of symmetry. The probability that their occurrence is random was found to be extremely small. Therefore, we propose that in addition to specific bases, repeating twist angles patterns are likely to be among the local parameters involved in repressor-operator recognition.     

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.