Effect of cytochalasin B on lymphocyte stimulation induced by concanavalin A or periodate

The effects of cytochalasin B on lymphocyte stimulation induced by concanavalin A (Con A) and by periodate were investigated. At low concentrations (0.1 – 1 μg/ml) cytochalosin B greatly potentiated the responses to these two mitogens. Cytochalasin B was most effective when added with the mitogens at the beginning of incubation. The action of cytochalasin B at low concentration was suggested to be on an early process of DNA synthesis induced by these mitogens.     

Both D factor/LIF and IL-6 inhibit the differentiation of mouse teratocarcinoma F9 cells

Differentiation-stimulating factor (D factor)/leukemia inhibitory factor (LIF) and IL-6 are reported to be cytokines having multifaced functions including the induction of differentiation in mouse myeloid leukemia M1 cells. We here report that both D factor/LIF and IL-6 inhibit the differentiation of mouse teratocarcinoma F9 cells induced by retinoic acid alone or combined with dibutyryl cAMP. From the microscopic observation as well as Northern blot analysis using cDNA probes encoding several marker proteins for differentiation of F9 cells, we concluded that D factor/LIF and IL-6 are functionally closely related in the induction of differentiation in M1 cells and in the inhibition of F9 differentiation.     

Selection of mouse macrophage-like sublines that differ in leukemogenic potential and characterization

The murine macrophage-like cell line (Mm-1), which is nonleukemogenic to syngeneic SL mice, was originally derived from spontaneously differentiated cells of a clonal line of mouse myeloid leukemia cells (M1). In the present experiment, variant cell lines with a high (Mm-A), moderate (Mm-P), and little or no (Mm-S1 and Mm-S2) leukemogenic potential were obtained from the Mm-1 cells. The mean survival times of syngeneic SL mice inoculated i.p. with 5 X 10(6) Mm-A and Mm-P cells were 17 and 33 days, respectively, whereas almost all the mice inoculated with Mm-S1 or Mm-S2 cells survived for more than 90 days. These variant cell lines did not lose their macrophage-like characteristics in vitro. These variant cell lines phagocytized latex beads and sensitized sheep erythrocytes, produced lysozyme, and adhered to culture dishes. The four variant cell lines showed no significant difference in proliferation rates in vitro in liquid medium containing 10% calf serum, but Mm-A cells could grow both in soft agar medium in the absence of ascitic fluid containing colony-stimulating factor (CSF) and in liquid medium containing 1% serum, whereas Mm-P cells could grow in the liquid medium but not in soft agar medium without ascitic fluid, and Mm-S1 and Mm-S2 cells could not grow in either medium. The ratio of the nuclear area to the cell area (NCR) of Mm-A cells was a high (51%) but those of Mm-S1 and Mm-S2 cells were low (40-41%), and that of Mm-P cells was intermediate (44%). The leukemogenicity of Mm-1 cell lines was roughly correlated with their NCR. The possibility that interactions between Mm-1 variant cells and host immune cells might be involved in the mechanisms of their different leukemogenicities was not supported by results on the in vitro susceptibilities of Mm-1 variant cells to the cytostatic actions by normal macrophages and spleen cells and on leukemogenicities of the Mm-1 variant cells in athymic nude mice. A possible method of control of the leukemogenicity of Mm-1 variant cells is discussed.     

Characterization of a factor inducing differentiation of mouse myeloid leukemic cells purified from conditioned medium of mouse Ehrlich ascites tumor cells

A factor inducing differentiation of mouse myeloid leukemic cells (MI) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was half-maximally active at 2 X 10(-11) M. The factor was analyzed by radioiodination, SDS-polyacrylamide gel electrophoresis and autoradiography. Its Mr was 40 000-50 000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo-beta-N-acetylglucosaminidase F, but not endo-beta-N-acetylglucosaminidase H, gave rise to a molecule of Mr 20 000-28 000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of Mr 50 000-70 000 from mouse fibroblast L929 cells.     

Structural aspects of skeletal muscle G-actin molecule as studied by proteolytic digestion: effect of nucleotide

Trypsin and chymotrypsin were used as probes of conformation of G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. G-actin is known to be nonrefractory to proteolysis [Jacobson, G.R., and Rosenbusch, J.P. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2742-2746]. It is really true that G-actin is cut easily into a 33-kDa fragment by trypsin or chymotrypsin, but only when free ATP is present in the medium. After the removal of free ATP from the medium, G-actin became more refractory to proteolysis. The amounts of degradation of G-actin depended on the ATP concentration in the medium with saturating at about 0.5 mM. epsilon-ADP also had the effect and its fluorescence spectrum was changed on the addition of G-actin. After the removal of free ATP, G-actin still bound 1 mol/mol of ATP. So, the present results suggest the presence of a second ATP interaction site on G-actin and that ATP interaction at this site induces conformational changes in G-actin molecule.     

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.     

Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear

Each of the sensory patches in the epithelium of the inner ear is a mosaic of hair cells and supporting cells. Notch signalling is thought to govern this pattern of differentiation through lateral inhibition. Recent experiments in the chick suggest, however, that Notch signalling also has a prior function - inductive rather than inhibitory - in defining the prosensory patches from which the differentiated cells arise. Several Notch ligands are expressed in each patch, but their individual roles in relation to the two functions of Notch signalling are unclear. We have used a Cre-LoxP approach to knock out two of these ligands, Delta1 (Dll1) and Jagged1 (Jag1), in the mouse ear. In the absence of Dll1, auditory hair cells develop early and in excess, in agreement with the lateral inhibition hypothesis. In the absence of Jag1, by contrast, the total number of these cells is strongly reduced, with complete loss of cochlear outer hair cells and some groups of vestibular hair cells, indicating that Jag1 is required for the prosensory inductive function of Notch. The number of cochlear inner hair cells, however, is almost doubled. This correlates with loss of expression of the cell cycle inhibitor p27(Kip1) (Cdkn1b), suggesting that signalling by Jag1 is also needed to limit proliferation of prosensory cells, and that there is a core part of this population whose prosensory character is established independently of Jag1-Notch signalling. Our findings confirm that Notch signalling in the ear has distinct prosensory and lateral-inhibitory functions, for which different ligands are primarily responsible.     

Cell adhesive peptide screening of the mouse laminin α1 chain G domain

Cell adhesive peptides have been widely applied for therapeutic drugs, drug delivery systems, and biomaterials. Previously, we identified various cell adhesive sequences in the G domains of four laminin α chains (α2-α5) by the systematic soluble peptide screening. We also identified five cell-binding sequences in the laminin α1 chain G domain using synthetic peptide-polystyrene beads. Here, we re-screened cell adhesive peptides in the laminin α1 chain G domain by the systematic soluble peptides screening. The 110 soluble peptides were evaluated for their cell adhesive activities using human fibrosarcoma HT1080 cells and human dermal fibroblasts. Fourteen peptides were newly identified as a cell adhesive. Additionally, four peptides (AG22: SSFHFDGSGYAM, AG42: TFDLLRNSYGVRK, AG76: HQNQMDYATLQLQ, AG86: LGGLPSHYRARNI) promoted integrin-mediated cell adhesion. Further, neurite outgrowth activity with rat pheochromocytoma PC12 cells was evaluated and two peptides (AG20: SIGLWNYIEREGK, AG26: SPNGLLFYLASNG) were newly identified for neurite outgrowth activity. These results suggested that the systematic soluble peptides screening approach is an accurate and powerful strategy for finding biologically active sequences. The active sequences newly identified here could be involved in the biological functions of this domain. The active peptides are useful for evaluating molecular mechanisms of laminin-receptor interactions and for developing cell adhesive biomaterials.