Synergistic interaction of a cytokine produced by embryonic fibroblasts and a lymphokine contained in Con A-stimulated spleen cell culture for murine macrophage differentiation: a model experiment using cell line cells, M1

The modulating activity of the culture supernatant of Con A-stimulated murine spleen cells for macrophages was investigated by using M-1 cells, which could differentiate into macrophage-like cells (referred to as M+1 hereafter), cocultured in a conditioned medium (CM) containing a differentiation factor (DF) obtained from the secondary culture of murine embryonic fibroblasts. DF induced Ia antigens on M-1 cells at a high rate in parallel with the appearance of Fc-receptor (FcR)-dependent phagocytic activity for erythrocytes coated with an antibody (EA). In contrast, Con A-sup alone had no modulating effect on M-1 cells. However, the Con A-sup stimulates synergistically M-1 cells with DF. Thus, coculture of M-1 cells with Con A-sup and DF generates M++1 cells which possess higher phagocytic activity than M+1 cells. These cells also exhibited stronger accessory cell activity than M+1 cells when tested for their promoting effect on IL-2 production by Sephadex G-10-passed spleen cells. The accessory cell activity of M++1 cells was further enhanced by culture with lymphocytes in the presence of indomethacin while that of M+1 cells did not change. These findings suggest that M-1 cells probably acquire potentiating, as well as inhibitory activity at the same time when cultured with DF and Con A-sup. The functional maturation caused by Con A-sup seemed to be associated with the expression of a receptor for a lymphokine, termed phagocytosis-augmenting factor (PAF) which is present in the Con A-sup. Such a receptor appeared to be common to macrophage lineage, since PAF in Con A-sup was absorbed out with splenic adherent cells and peritoneal exudate cells (PEC) in addition to M+1 cells, but not with nonadherent splenic lymphocytes or lymphoid cell line cells, such as EL-4 and L-1210. This fact suggests that PAF is different from interferon-gamma (IFN) which is known to modulate the function of lymphocytes. Inability of PAF to bind Cibacron Blue-Sepharose, unlike IFN, supports this notion. The molecular weight of PAF is approximately 2-3 X 10(4). Thus, the present studies suggest the requirement of at least two signals for the full maturation of macrophages, a cytokine represented by DF and a lymphokine, by PAF.     

Methyl cinnamate derivatives enhance UV-induced mutagenesis due to the inhibition of DNA excision repair in Escherichia coli B/r

UV-induced mutagenesis in Escherichia coli B/r WP2 was enhanced by certain derivatives of methyl cinnamate which themselves were not mutagenic. Methyl ferulate, methyl isoferulate and methyl sinapate showed this effect markedly. Such an enhancement effect was absent with the derivatives of cinnamic acid and ethyl cinnamate and was not observed in Escherichia coli WP2s uvrA. Methyl sinapate also enhanced 4NQO-induced mutation and suppressed liquid-holding recovery in the above repair-proficient strain. The presence of methyl sinapate in plating agar medium decreased the survival of UV-irradiated cells of a recombination-repair-deficient strain, CM571 recA. However, the effect was not observed with those of WP2s uvrA. In an in vitro experiment in which the removal rate of thymine dimers was measured, methyl sinapate clearly inhibited this repair event. From these results, we conclude that methyl sinapate inhibits DNA excision repair, thus enhancing UV mutagenicity.     

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.     

Physical effects of negative air ions in a wet sauna

 The physical effects of negative air ions on humans were determined in an experimental sauna room equipped with an ionizer. Thirteen healthy persons took a wet sauna bath (dry bulb temperature 42° C, relative humidity 100%, 10 min exposure) with or without negative air ions. The subjects were not told when they were being exposed to negative air ions. There were no differences in the moods of these persons or changes in their blood pressures between the two saunas. The surface temperatures of the foreheads, hands, and legs in the sauna with negative ions were significantly higher than those in the sauna without ions. The pulse rates and sweat produced in the sauna with ions were singificantly higher than those in the sauna without ions. The results suggest that negative ions may amplify the effects on humans of the sauna. Received: 31 March 1995 / Revised: 25 July 1995 / Accepted: 26 July 1996     

Involvement of intracellular caveolin‐1 distribution in the suppression of antigen‐induced mast cell activation by cationic liposomes

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross‐linking of high‐affinity immunoglobulin E receptor in a time‐ and dose‐dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the inhibition of store‐operated Ca²⁺ entry. Further, we revealed that the mechanism underlying an impaired [Ca²⁺]_i increase is the inhibition of the activation of the phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Yet, how cationic liposomes inhibit the PI3K‐Akt pathway is still unclear. Here, we focused on caveolin‐1, a major component of caveolae, which is reported to be involved in the activation of the PI3K‐Akt pathway in various cell lines. In this study, we showed that caveolin‐1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin‐1 to the plasma membrane and the colocalization of caveolin‐1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.     

Direct neurite-osteoblastic cell communication, as demonstrated by use of an in vitro co-culture system

Using an in vitro co-culture approach comprising cultured murine superior cervical ganglia and MC3T3-E1 osteoblast-like cells, we found that the addition of scorpion venom (SV) elicited neurite activation via intracellular Ca2+ mobilization and, after a lag period, osteoblastic Ca2+ mobilization. SV did not have any direct effect on the osteoblastic cells in the absence of neurites. The addition of an alpha1-adrenergic receptor (AR) antagonist, prazosin, dose-dependently prevented the osteoblastic activation that resulted as a consequence of neural activation by SV. These results demonstrate that osteoblastic activation occurred as a direct response to neuronal activation, which activation was mediated by alpha1-ARs in the osteoblastic cells.     

The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

A robust, target-driven, cell-based assay for checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a serine/threonine kinase, plays an important role in DNA damage checkpoint control and is an attractive target for cancer treatment. To develop a Chk1-specific cell-based assay, stable clones were established in which Chk1 kinase domain fused at its N-terminus with p53 through 4 tandem repeats of Gly-Gly-Gly-Gly-Ser was expressed in an inducible manner. Chk1 kinase specificity of the phosphorylation of fused p53 was confirmed by the experiments with a kinase-inactive Chk1. Only in the presence of an inducer molecule was phosphorylation of p53 at Ser-15 in the stable clones induced. Furthermore, its assay performance proved acceptable for high-throughput screening applications, judging from the Z' factor values (> 0.77). Finally, the cell-based assay thus established yielded structure-activity relationship data for a small set of test inhibitors of Chk1 within cells. Collectively, these results demonstrate that the established cell-based assay provides a novel and highly sensitive cellular platform for Chk1 inhibitor discovery.