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.     

A Z-DNA binding protein isolated from D. radiodurans

A DNA binding protein isolated from D. radiodurans changes CD-spectrum of Z-form poly(dG-dC) X poly(dG-dC). We have found that a positive band at 268 nm is converted close to that of B-form in the presence of the protein. Concomitantly, a negative band at 295 nm shown by Z-form poly(dG-dC) X poly (dG-dC) was weakened by the protein but not by albumin. Such changes in the CD-spectra were not induced by the protein and by albumin when they were mixed with Z- or B-form poly(dG-me5dC) X poly(dG-me5dC) or with B-form poly(dG-dC) X poly(dG-dC). The protein formed a complex preferentially with Z-form poly(dG-dC) X poly(dG-dC).     

Postnatal development of the anulospiral endings of Ia fibers in muscle spindles of mice

The formation of the anulospiral ending of Ia fibers in muscle spindles was investigated in the masseter muscle of developing mice. Before 15 days after birth, the complete anulospiral ending was not observed in almost all of the muscle spindles examined. With the growth of mice, the Ia fiber began to construct the spiral ending, and by the 40th postnatal day after weaning, almost all of the Ia fibers of the muscle spindles had complete coiled endings, though the formation still continued in some spindles. The continuous formation of anulospiral endings for a long period after weaning indicates that muscle spindle morphogenesis may be affected by muscle tension in the masseter muscle due to the movement activated after weaning.     

Defective endocytosis of low-density lipoprotein in monensin-resistant mutants of the mouse Balb/3T3 cell line

Two monensin-resistant clones show similar low-density lipoprotein binding activity but less internalization or degradation of low-density lipoprotein than the parental Balb/3T3 or other resistant clone. Sterol synthesis from radioactive acetate in the resistant mutant, MO-5, is inhibited by more than 70% of control in the presence of tenfold higher amounts of low-density lipoprotein than the dose that inhibits the parental Balb/3T3 to similar level. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity of Balb/3T3 and MO-5 is inhibited by 48% and 27% of control, respectively, in the presence of 10 micrograms/ml of low-density lipoprotein. Colloidal silica gradient centrifugation shows that transport of low-density lipoprotein from the surface membrane to the lysosome is much slower in MO-5 cells than in Balb/3T3 cells. Down regulation of low-density lipoprotein receptors on the cell surface in Balb/3T3 is observed by exposing the cells to 5-15 micrograms/ml low-density lipoprotein, whereas only slight if any down regulation is observed when MO-5 cells are treated with low-density lipoprotein. The altered endocytosis of low-density lipoprotein behaves as a dominant trait in hybrids of MO-5 and THO2-2, a derivative of Balb/3T3 resistant to both ouabain and 6-thioguanine.     

Induction of 3-Hydroxy-3-methylglutaryl CoA Reductase in Potato Tubers after Slicing, Fungal Infection or Chemical Treatment, and Some Properties of the Enzyme

Intact tubers of potato (Solanum tuberosum L. cv. Irish Cobblerand an interspecific hybrid between S. tuberosum and S. demissumcv. Rishiri) contain a very low activity of 3-hydroxy-3-methylglutaryl(HMG)-CoA reductase. The activity increased first in responseto slicing, and again in response to additional treatments suchas inoculation with an incompatible race of Phytophthora infestans,application of a hyphal wall component of the fungus or HgCl₂solution, and then decreased. Both the first and the secondincreases in activity in response to slicing and additionaltreatment with a hyphal wall component to elicit phytoalexinproduction were inhibited by blasticidin S. Properties of HMG-CoAreductase induced by slicing and by additional treatment withHgCl₂ or fungal inoculation were investigated. ² Present address: Faculty of Home Economics, Nagoya Women'sUniversity, Shioji-cho, Mizuho, Nagoya 467, Japan.     

Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.     

Growth Regulation in Dwarf Barley Coleoptiles by the Minor Cell Wall Components, Galactose and Mannose

Sugar compositions of cell walls of dark-grown coleoptiles from12 barley strains, 11 of which were coleoptilar semi-dwarf strains,were analyzed on days 2 and 3 after germination. Major wallcomponents of the 12 strains were arabinose, xylose, and glucosein hemicellulose and cellulose; minor components were galactoseand mannose. The sugar content of each wall component per unit length wasnot correlated to any growth parameters calculated from a logisticequation simulating coleoptile growth, but the relative contentsof galactose and mannose in relation to the total wall sugarcontent was correlated to the growth rate on day 3 and the growthcontinuing period. These facts suggest that growth of these12 barley strains in the late growth stage is regulated by theminor wall components, galactose and mannose. ¹ Dedicated to the late Professor Joji Ashida. (Received October 12, 1982; Accepted January 12, 1983)     

Auxin-Induced Changes in the Cell Wall Xyloglucans: Effects of Auxin on the Two Different Subtractions of Xyloglucans in the Epicotyl Cell Wall of Vigna angularis

In order to study the IAA-induced modifications of the cellwall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara)epicotyl segments, the xyloglucans were subfractionated intotwo components, i.e., 4K-U and 24K xyloglucans, which were obtainedby extraction with 4% KOH solution containing 8 M urea and 24%KOH solution, respectively. The weight-average molecular weightsof 4K-U and 24K xyloglucans were estimated to be 40 x 10⁴ and106 x 10⁴, respectively. Complete acid hydrolysis of 4K-U and24K xyloglucans gave glucose, xylose, galactose and fucose inmole % 48.3 : 33.5 : 13.8 : 4.4 and 45.3 : 30.9 : 19.6 : 4.3,respectively. Treatment of epicotyl segments with IAA (0.1 mM) caused a decreasein the amount of 24K xyloglucans and an increase in 4K-U xyloglucans,whereas the total amount of the two xyloglucans remained constant.Furthermore, IAA treatment caused a decrease in the molecularweight of 24K xyloglucans from 106 x 10⁴ to 78 x 10⁴ withoutcausing changes in their sugar compositions. With 4K-U xyloglucans,IAA caused an increase in the mole % of xylose and a decreasein the mole % of galactose and fucose. ¹ This paper is dedicated to the late Professor Joji Ashida. (Received November 26, 1982; Accepted February 7, 1983)     

Nucleic Acid Metabolism Involved in Auxin-induced Elongation of Yeast Cells

The following results were obtained using a variant yeast strain, N55, which can respond to the cell-elongating action of auxin. Base analogs of nucleic acids (2-thiouracil, 8-azaguanine, and 5-fluorouracil) inhibited the auxin-induced elongation of yeast cells only when they were added to the preculture prior to auxin treatment. The inhibitory effect of 2-thiouracil and 5-fluorouracil was reversed by uracil and that of 8-azaguanine by guanine. Actino-mycin D inhibited the auxin-induced elongation when given to the culture containing auxin, but not when given to the preculture. The similarity in these respects between yeast and tissues of higher plants is discussed.