Cytoplasmic elongation and rupture in megakaryoblastic leukemia cells via activation of adhesion and motility by staurosporine on fibronectin-bound substratum.

Human megakaryoblastic leukemia Meg-01 cells were attached to fibronectin (FN)-coated substratum, on which remarkable spreading and cytoplasmic elongation was induced by treatment with a protein kinase inhibitor, staurosporine (stp). This effect was inhibited by RGDS and was also not seen on FN-lacking substratum. The extended cytoplasm had swollen terminals and nodes, which contained GpIIb and beta-thromboglobulin, occasionally included alpha granules, and tended to form particles (2-5 microm) after rupture of the narrowed cytoplasm. Among other protein kinase modulators tested, only K252a promoted the elongation, while calphostin, herbimycin, TPA, and calyculin suppressed it. The cells began to migrate soon after addition of stp, with attachment to the substratum held at some sites during the migration. This tethered movement seemed to cause the cytoplasmic elongation and the rupture into particles. The elongation was retarded by pretreating the cells with cytochalasin A and Clostridium C3 toxin but not with demecolcine. Actin microfilaments in the stp-treated Meg-01 cells accumulated in the filopodia and periphery of the extended cytoplasm, in which vinculin was colocalized as adhesion plaques. The microtubules were longitudinally oriented through the cytoplasmic extension and showed no ring profile in the nodes and particles. Thus, stp in the presence of FN appears to stimulate reorganization of actin-based cytoskeleton and formation of focal contacts in Meg-01 cells. This leads to the activation of cell adhesion and motility, and then cytoplasmic elongation and rupture into particles.

N-Shc and Sck, two neuronally expressed Shc adapter homologs. Their differential regional expression in the brain and roles in neurotrophin and Src signaling.

The Shc adapter protein is ubiquitously expressed and has been implicated in phosphotyrosine signalings following a variety of extracellular stimulation, e.g. growth factor stimulation, Ca2+ elevation, and G-protein-coupled receptor stimulation. In neuronal cells such as PC12, Shc was demonstrated to be involved in vitro in Ras-dependent mitogen-activated protein kinase activation following nerve growth factor stimulation and Ca2+ entry. However, Shc mRNA was hardly detectable in the brain, and therefore, Shc is unlikely to participate in phosphotyrosine signaling in the central nervous system. Two recently isolated Shc homologs, N-Shc and Sck, have been shown to be expressed in the brain and are expected to function as neuronal adapters instead of Shc. In this study, the neuronal distribution and function of these novel Shc members were investigated. In human and rat central nervous systems, the expression profiles of N-Shc and Sck mRNAs considerably overlapped, although some distinct localization between them was observed: in the adult rat brain, the level of N-Shc mRNA was the highest in the thalamus, whereas that of Sck mRNA was the highest in the hippocampus. In the peripheral nervous system, transcripts of Shc and Sck, but not of N-Shc, were detected. Immunoprecipitation experiments demonstrated functional differences between N-Shc and Sck: (i) N-Shc was a higher affinity adapter molecule than Sck in nerve growth factor and brain-derived neurotrophic factor signaling; and (ii) N-Shc, but not Sck, was efficiently phosphorylated by activated Src tyrosine kinase, whereas Sck, but not N-Shc, formed a complex with pp135, a protein highly phosphorylated by v-Src. These results suggest that neurally expressed N-Shc and Sck may have distinct roles in neuronal signaling in the brain.

Is structural flexibility of antigen-binding loops involved in the affinity maturation of anti-DNA antibodies?

Effects of somatic mutations in Ig variable region genes on the affinity maturation of autoantibodies were investigated using single precursor B cell-derived anti-double-stranded DNA mAb generated from an autoimmune disease-prone (NZB x NZW)F1 mouse. Analyses of DNA sequences, homology modeling on a graphic computer and molecular dynamics simulation of antigen-binding sites showed that any single site of mutation and changes in the electrostatic or hydrogen-bonding potential of the residues and in the three-dimensional structure could not solely explain the difference in DNA-binding activities. However, a significant increase in the flexibility of antigen-binding Fv loops, particularly VL CDR1 and VH CDR3, was associated with affinity-maturated anti-DNA antibodies. Such high flexibility of the FV loops may provide the environment where the antibodies could effectively interact with antigen DNA, a model consistent with the 'induced-fit' hypothesis of antigen-antibody interactions.

N-Shc: a neural-specific adapter molecule that mediates signaling from neurotrophin/Trk to Ras/MAPK pathway.

Shc has been implicated in a variety of growth factor- and cytokine receptor-signaling through its specific binding to phosphotyrosine residues of the activated receptors. In neuronal cells, such as PC12, Shc has been shown to be involved in Ras-dependent MAP kinase activation following Trk receptor stimulation with NGF. While the ubiquitous role of Shc as an adaptor molecule in signal transduction is increasing in both neuronal and non-neuronal cells and tissues, the expression level of Shc is surprisingly low in the brain. We demonstrated here the isolation of a neural-specific member of the Shc family. This novel protein, named N-Shc (neuronal Shc), contains two potential phosphotyrosine-binding domains, PTB and SH2, and is expressed exclusively in the brain; whereas Shc is present in all other non-neuronal tissues. As in Shc, N-Shc can bind activated EGF receptor, become tyrosine phosphorylated, and form a complex with Grb2 adapter protein following EGF stimulation. Furthermore, N-Shc can bind activated TrkB receptor following the stimulation with brain-derived neurotrophic factor (BDNF), which is the most abundant neurotrophin in the brain. These data suggest that N-Shc, rather than Shc, mediates neurotrophin and other neuronal signalings in the central nervous system.

Cyclin I: a new cyclin encoded by a gene isolated from human brain.

A new member of the cyclin family has been isolated from an equalized cDNA library derived from human forebrain cortex. This putative cyclin, designated cyclin I, contains a typical cyclin box near the N-terminus and a PEST sequence near the C-terminus. Cyclin I shows the highest sequence similarity in the cyclin box to cyclins G and E, while the similarity between cyclins I and G also extends toward the C-terminus from the cyclin box. Cyclin I mRNA was expressed at high levels in postmitotic tissues, including skeletal muscle, heart, and brain, and was expressed constantly during cell cycle progression. The expression of cyclin I mRNA does not correlate directly to the cell cycle, and therefore cyclin I may be a novel cyclin member that functions independently of the cell cycle control.

Batchwise purification of specific tRNAs by a solid-phase DNA probe.

A simple and efficient method for purifying a specific tRNA in a single microcentrifuge tube was developed. Oligodeoxyribonucleotides (about 30 mer) with sequences complementary to the 3' side of target tRNAs were synthesized with an aminohexyl linker at the 5' end, immobilized on a silica gel at a high concentration, and used as a solid-phase probe. A mixture of tRNAs was added to a suspension of the solid-phase probe in 2.4 M tetraethylammonium chloride and incubated for 10-30 min. Only a target tRNA hybridized with the immobilized probe at appropriate temperatures and was eluted out by heating. The solid-phase probe showed a large hybridization capacity (up to 17 A260 units/g dry gel) and specific and quantitative recovery of the target tRNA. The intactness of recovered tRNAs was ascertained by both Donis-Keller sequencing and aminoacylation experiments. These features show the usefulness of the solid-phase probe method as a reliable tool for purifying tRNAs whose gene sequences are known.

Somatic diversification and affinity maturation of IgM and IgG anti-DNA antibodies in murine lupus.

Molecular events occurring during the process of generation of pathogenic immunoglobulin (Ig)G anti-DNA antibodies in systemic lupus erythematosus (SLE) were studied using a newly established method. We analyzed the Ig variable (V) region gene sequence and DNA-binding activity of IgM and IgG anti-DNA monoclonal antibodies (mAb) from individual SLE-prone (NZB x NZW) F1 mice. The first event appeared to be clonal selection and expansion of IgM anti-DNA clones, in which several clones had intraclonal V gene mutations. Although the number of mutations was small, the mutated IgM clones were associated with an increase in DNA-binding activity. The somatic mutations located in complementarity-determining regions (CDR) and in framework regions (FR) of V genes were apparently related to changes in DNA-binding activity. IgG anti-DNA clones that progressively increased in number with aging had numerous somatic mutations in the V region genes and there was a pair of clones which showed an intraclonal accumulation of mutations, in association with increase in the DNA-binding activity. All these findings show that somatic mutations associated with affinity maturation of the V region begin immediately before isotype-switching from IgM to IgG of the clones that have been selected and expanded, in an antigen-driven manner and/or by other forces. We propose that further accumulations of intraclonal somatic hypermutation, in association with selection and expansion of high affinity IgG clones, may lead to formation of highly pathogenic anti-DNA antibodies.

Sequence specific purification of a particular tRNA by solid phase DNA probe.

A novel method for the purification of a specific tRNA using solid phase DNA probe is developed. With this method, the probe DNA immobilized on HPLC gel hybridized with target tRNA within a minute at room temperature. The hybridizing capacity of the solid phase probe was about 20 O.D. per gram dry gel when yeast phenylalanine tRNA was used. The specificity of this method was extremely high and the recovery rate was about 90%.

Precise discrimination among homologous sequences by DNA probe column chromatography.

Synthesized oligo probes were immobilized to a HPLC gel in high concentrations (30-40 O.D. per gram dry gel), packed in a column (2mm x 10cm) and incorporated into a conventional HPLC system. The system was applied to the discrimination among homologous sequences. Two probes different in sequence and length (16mer and 24mer) were investigated under isothermal and isocratic conditions. For each probe, 4 oligos of similar sequences, one perfectly matched to the probe and others containing one mismatch in the center of the chain, were synthesized. The chromatogram obtained as the results of high performance liquid affinity chromatography (HPLAC) considerably varied with the column temperature and the type of mismatches. The dependency of the deformation of elution profile upon mismatch seemed to reflect the stability of the hybrid composed of samples and immobilized probe.