The biochemical and ultrastructural effects of tunicamycin and D-glucosamine in L1210 leukemic cells

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glyco-protein biosynthesis led to a cessation of cell growth which was reversible in a dose-dependent and time-dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP-N-acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP-sugars were observed in L1210 cells exposed to 5 mM D-glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine-linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D-glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin-treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.     

Cell-mediated lysis of tumor targets directed by murine monoclonal antibodies of IgM and all IgG isotypes

Seven murine monoclonal antibodies to antigens expressed on T lymphoma targets were tested for directing antibody-dependent cellular cytotoxicity (ADCC). Peptone-induced peritoneal exudate macrophages, the LPS-stimulated RAW264.10 cell line, and human blood nonadherent mononuclear leukocytes were used as effector cells. All six IgG monoclonals tested, representing the four murine IgG isotypes and directed against four antigens (Thy-1.2, H-2k, Ly-2.1, Ly-9.2), were all active in ADCC. In contrast, an IgM anti-Thy-1.2 showed no activity despite very high C-cytotoxic titers. Thus, there does not seem to be any restriction among IgG classes for directing ADCC to tumor targets mediated by murine macrophages or human K cells.     

Sequence interrelationships of the subunits of vicilin from pea seeds

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than M_r～50 000 (i.e., M_r 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within M_r～50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than M_r～50 000 arose by endoproteolytic cleavage of parent molecules within the M_r～50 000 group. Cleavage in different M_r 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than M_r～50 000, with the possible exception of the M_r34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the M_r～50 000 group.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

Expression of creatine kinase isoenzyme during oogenesis and embryogenesis in the mouse

Creatine kinase activity was discovered in the growing mouse oocyte and in the preimplantation embryo. Changes in the enzyme activity during the growth and maturation of the egg and during the development of the embryo up to the blastocyst stage were determined. Close similarity of the protein to the brain-type isoenzyme of creatine kinase was established immunochemically. The kinetic parameters of the brain-type isoenzyme (M. R. Iyengar, C. E. Fluellen, and C. W. L. Iyengar, 1982, J. Muscle Cell Motil. 3, 231–246) and the pattern of development-associated changes in activity suggest a possible role for creatine kinase in maintaining the reported high ATP/ADP ratio (L. Ginsberg and N. Hillman, 1975, J. Reprod. Fertil. 43, 83–90), which is essential for the biosynthetic activities of the embryo.     

Elution of low molecular weight solutes from viable cells of Saccharomyces bisporus

Previous work has shown that high molecular weight compounds were released from Saccharomyces bisporus by -mercaptoethanol, 2 M KCl, 0.5 M KCl and osmotic shock without affecting viability of the cells. In this current experiment, it was shown that low molecular weight compounds were also eluted when cells were treated in sequence with the same reagents. Alanine, glutamate, serine, an unidentified amino acid, glucose, glycerol, and arabitol were all eluted by each of the first three reagents. The osmotic shock eluate contained a larger number and quantity of amino acids than the first three eluates but, otherwise, the compounds in this eluate were the same. One hundred percent of the cellular glycerol and 65–70% of the total amounts of the other above mentioned solutes were released by the 4 eluting treatments. A hot water treatment was needed to extract the remainder of these solutes. The hot water extract also contained almost all the cellular proline. It was suggested that the elutable solutes are contained by cells in compartments (or vesicles) whose membranes are accessible to the eluting reagents without affecting the plasmalemma.     

Comparison by Gas-Liquid Chromatography of the Fatty Acids of Mycobacterium avium and Some Other Nonphotochromogenic Mycobacteria

Fifty-eight strains of nonphotochromogenic mycobacteria representing nine different serotypes were studied, including 38 strains of Mycobacterium avium and 20 strains of Battey bacilli. The lipids were extracted from whole cells, saponified with potassium hydroxide, and esterified with diazomethane. The fatty-acid profiles determined by gas-liquid chromatography included saturated fatty acids ranging from C(8) to C(24), plus some unsaturated analogues and a branched-chain acid. No consistent differences in the fatty-acid profiles were observed between strains of M. avium and Battey bacilli. Quantitative differences in the means (significant at P < 0.01) were observed in the relative amounts of 14:0, 16:1, 18:0, 18:1, and a branched-chain acid among strains of the same serotype. We were unable to differentiate the fatty-acid profiles of recently isolated strains or those maintained in culture for more than 2 years.     

Oxidation of Acetate by Various Strains of Bacillus popilliae

A number of strains of Bacillus popilliae were examined for their ability to oxidize acetate. Some of these would not sporulate in vitro, and some were oligosporogenous. The ability to oxidize acetate varied widely among the strains tested. A culture derived from spores of the parent strain produced in vivo and one of the asporogenous strains derived from it failed to produce any significant levels of (14)CO(2) from [(14)C]acetate. Oligosporogenous strains derived from the same parent culture all produced (14)CO(2) from both [1-(14)C] and [2-(14)C]acetate but at relatively low rates. The highest rates of acetate oxidation were observed with three strains which did not produce spores in vitro. When cultured under appropriate conditions, one of these strains displayed a secondary growth response concomitant with a decrease in the titratable acidity and an increase in the pH of the medium. The data indicate that B. popilliae has a complete citric acid cycle but that the activity of the cycle is strongly repressed in wild-type strains under the usual conditions used for in vitro cultivation.     

Chemical Detection of Microbial Prey by Bacterial Predators

A motile, predacious bacterium which degraded Pythium debaryanum was strongly attracted to substances released into the medium by the fungus. A nonpredacious bacterium was not attracted to these substances. The predator bacterium was specifically attracted to cellulose and its oligomers which are known to be components of the cell wall of Pythium. Ethanol inhibited chemotaxis of the bacterium without affecting either its motility or its ability to degrade cellulose. A second predacious bacterium was isolated for the alga, Skeletonema costatum. The role of chemoreception in the detection of microbial prey by bacterial predators in natural habitats is discussed.