Cell separation system studied by mixed culture of single wild strain with tetrads-forming mutant strain of Micrococcus luteus

Mixed culture study of singly occurring wild strain IFO 3333 of Micrococcus luteus and a tetrads-forming mutant strain MT, in the absence or presence of trypsin, supported our previous assumption that at least two kinds of separation systems were involved in cell separation of M. luteus, the one having a physiological role in cutting off the outermost layer of the cell wall (separation system-Om) and the other in cutting off the inner layer of the "proper" cell wall or the septum (separation system-In). The separation system-Om of IFO 3333 insensitive to trypsin substituted, freely from the cells, for that of MT sensitive to trypsin.     

Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 m in diameter) and less electron-dense than the D-granules (less than 0.6 m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.     

Mechanisms involved in the cellular uptake of hematoporphyrin by rat hepatoma cells

To clarify the mechanisms involved in the specific uptake of hematoporphyrin by cancer cells, we investigated the interaction of the heme- and/or hematoporphyrin-hemopexin complexes with rat hepatoma dRLh-84 cells. Hemopexin bound to the cells in a saturable, time- and temperature-dependent manner. The cells exhibited 0.55 nmol of binding sites/mg of protein for the heme-hemopexin complex and 0.38 nmol for the hematoporphyrin-hemopexin complex. The dissociation constants (Kd) for the heme-hemopexin and hematoporphyrin-hemopexin complexes were 0.57 and 0.54 microM, respectively. Specific binding of the labeled hemopexin was inhibited by the unlabeled heme- and hematoporphyrin-hemopexin complexes but was unaffected by albumin or neoglycoprotein. Hematoporphyrin bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that hematoporphyrin bound hemopexin was taken up by dRLh-84 cells, via the hemopexin receptors. When the hematoporphyrin-albumin complex was incubated with the cells, the hematoporphyrin-[125I]albumin complex bound to the cells in a time and temperature-dependent manner. Here the binding was not saturated up to 100 micrograms/ml of albumin. The binding of hematoporphyrin-[125I]albumin was partially inhibited by unlabeled albumin and hemopexin. Hematoporphyrin bound to albumin was taken up by the cells at 37 degrees C. Thus, the albumin-dependent uptake of hematoporphyrin by rat hepatoma dRL-84 cells could be differentiated from the hemopexin-mediated uptake of hematoporphyrin.     

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).     

Expression and phosphorylation of transferrin receptors in mitogen-activated peripheral blood lymphocytes

Expression of transferrin receptors of cultured human lymphocytes has been investigated by using monoclonal antibody (5E9) specific for human transferrin receptors. When isolated lymphocytes were cultured in a medium containing fetal calf serum, the biosynthesis of transferrin receptor was barely detectable. The addition of concanavalin A or human serum to the medium caused a slight stimulation of the biosynthesis. The addition of concanavalin A and human serum in combination caused the highest biosynthetic activity. Appearance of the receptor on the cell surface increased in parallel with the degree of the synthesis. Treatment of concanavalin A- and human serum-treated cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a marked stimulation of the phosphorylation of the receptor. Enhancement of phosphorylation occurred within 20 min after the addition of TPA. The density of the receptor on the cell surface slightly increased upon TPA treatment of cells, and the treatment was without effect on iron incorporation from transferrin into the cells. The density of newly synthesized receptor in TPA-treated cells was similar to that in non-treated cells. These results indicated that TPA treatment of mitogen-activated human lymphocytes stimulated the phosphorylation of transferrin receptors, but TPA had no effect on the expression of the receptors thereafter.     

Production of monozygotic twins following the transfer of bisected embryos in the goats

Embryos at the morula, blastocyst and hatched blastocyst stage were obtained from superovulated and naturally ovulated Japanese native goats. They were bisected into halves with a glass needle, and transferred immediately or after culture (for morula) to recipients. None of five does which received bisected morula became pregnant. Three of nine goats became pregnant after transfer of bisected hatched blastocysts, six of eleven recipients became pregnant. Four of them produced monozygotic twins and the remaining two produced singles. The present study demonstrated that the hatched blastocyst is suitable for bisection in the goat.     

Mapping of the lipoprotein signal peptidase gene (lsp)

A pBR322 plasmid which contains a fragment of Escherichia coli DNA encoding the lipoprotein signal peptidase gene was used to transform Hfr polA1 strains. Ampr transformants were used as donors in conjugation experiments, and the location of the plasmid amp gene adjacent to the chromosomal lsp gene was determined to be near the thr ara loci of the E. coli chromosome. P1 transduction experiments established that the location of the lsp gene is closely linked to that of dapB , at 0.5 to 0.6 min on the E. coli genetic map. The position of the lsp gene was further determined to be between ileS and dapB by complementation analysis of an E. coli mutant showing temperature-sensitive prolipoprotein signal peptidase activity.     

Monoclonal antibodies directed against mouse macrophages in different stages of activation for tumor cytotoxicity

Three rat monoclonal antibodies against mouse peritoneal macrophages in different stages of activation were produced and characterized. One of these (AcM.1) bound to activated macrophages induced by pyran and Corynebacterium parvum, but not to resident and thioglycollate medium- (TGC) or proteose peptone- (PP) elicited macrophages. On the contrary, the antigen identified by MM9 monoclonal antibody was expressed only on resident and TGC- or PP-elicited macrophages. WE15 monoclonal antibody, on the other hand, reacted with all of the macrophages described above. In the assay for function, AcM.1 and WE15 monoclonal antibodies in the presence of complement (C) abolished the capacity of activated macrophages induced by pyran or C. parvum but not the capacity of killer T cells and natural killer (NK) cells to kill tumor target cells. On the other hand, MM9 and anti-Thy-1.2 monoclonal antibodies in the presence of C, as expected, did not affect the cytotoxicity of activated macrophages. However, none of the four monoclonal antibodies in the absence of C had any blocking effect on macrophage-mediated cytotoxicity. AcM.1 antibody reacted with two polypeptides with m.w. of 70,000 and 45,000 on pyran-activated macrophages; however, the antigens recognized by WE15 and MM9 have not been determined yet. These results indicate that the three rat monoclonal antibodies define different antigens present on macrophages at different stages of activation for tumor cytotoxicity, and that these antibodies should prove to be useful probes for analyzing the mechanism of activation of macrophages for tumor cytotoxicity.     

Role of lymphokines in regulation of macrophage differentiation

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.     

Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule I. Mechanism of inhibition

Isotonic reabsorption by the rat kidney proximal tubule was drastically inhibited after less than 2 min intraluminal perfusion with fresh sera from rat (both homologous and autologous), cat, rabbit and human, but not with sera from mouse and guinea pig. The inhibitory factor in serum in a heat (56° C for 30 min) and storage (4°C for 2–5 days) labile macromolecule (mol. wt 50 000) and requires Ca²⁺ for its effect. The cellular electrical potential difference of the proximal tubular cells was irreversively destroyed and intraluminally perfused trypan blue dye incorporated into the tubular cells after the intraluminal perfusion with serum for 2 min. These observations suggest that lysis of the proximal tubular cells is the mechanism for serum-induced inhibition of proximal tubular isotonic reabsorption.