Inhibition of mycoplasma cell division by cytochalasin B

Mycoplasma gallisepticum has subcellular organelles which may function as a primitive "mitotic-like" apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time "mitotic-like" structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Amplification of the action of subthreshold doses of aldosterone by 19-hydroxyandrost-4-ene-3, 17-dione.

Various 1α-hydroxylated side chain analogs of vitamin D₃ have been studied for their ability to compete with 1α,25-dihydroxy[³H]vitamin D₃ for binding to the chick intestinal receptor. Of the analogs examined, 1α,24R-dihydroxyvitamin D₃ was found to be nearly equivalent to 1α,25-dihydroxyvitamin D₃ in its ability to compete for receptor binding. However, this near equivalence was not shared by its stereoisomer, 1α,24S-dihydroxyvitamin D₃, which was only 10% as effective a competitor. It is proposed that the ability of a 24R-hydroxyl group to mimic the 25-hydroxyl group is not due to a lack of side chain specificity on the part of the receptor, but is instead due to the similar orientation of the 25-hydroxyl and the 24R-hydroxyl such that they can be accommodated equivalently by the receptor.     

Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria.

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O₂^? at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H₂O₂ at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H₂O₂ was essentially produced by disproportionation of O₂^?, which constitutes the precursor of H₂O₂. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O₂^? and 0.63 mol of H₂O₂/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O₂ to O₂^?, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H₂O₂ generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H₂O₂ generation. The efficiency of added quinones as peroxide generators decreased in the order Q₁ > Q₀ > Q₂ > Q₆ = Q₁₀, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O₂^? and H₂O₂ during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q₀H₂), benzoquinol, duroquinol and menadiol generated O₂^? with k₃ values of 0.1 to 1.4 m^? · s^?1 and H₂O₂ with k₄ values of 0.009 to 4.3 m^?1 · s^?1.     

Analysis of Xenopus laevis ovary and somatic cell polyadenylated RNA by molecular hybridization

The number of sequences represented in the polyadenylated RNA population of the Xenopus oocyte and the degree to which these sequences are specific for early development have been determined by RNA-cDNA hybridization. Approximately 20,000 different sequences are found in the immature oocyte and the mature oocyte, and these sequences are the same in both types of cells. Approximately 5% of these sequences are present at a 15-fold higher concentration than the other 95%. The percentage of nonrepeated nuclear DNA homologous to the polyadenylated RNA was also determined and was in agreement with the number of sequences determined by the cDNA measurement. In addition, the number of sequences present in the Xenopus laevis kidney tissue culture cell and in the swimming tadpole was measured; approximately 20,000 different sequences were present in both the tissue culture cell and in the tadpole. The majority of the sequences present in the oocyte are not specific for early development since they are shared with both the tissue culture cell and the swimming tadpole; however, some sequences appear to be greatly increased in relative abundance in the tissue culture cell and the tadpole as compared to the oocyte.     

Diffusible factors are responsible for differences in nuclease sensitivity among chromatins originating from different cell types

We have examined the kinetics of nuclease digestion of chromatin from committed and uncommitted cells in experiments where the nuclei are mixed and co-digested. Cultures of the sea urchin, Arbacia punctulata, were grown to the 16-cell stage in either [3H]thymidine or [14C]thymidine and the macromere, mesomere, and micromere cell types separated. After isolation, sets of nuclei with two different blastomere types (each having different radionucleotide tagging) were mixed and co-digested with micrococcal nuclease or DNase. I. The extent of digestion was monitored by solubility in 5% perchloric acid (PCA). We find no significant differences in initial digestion rates or limit digests among the different cell types when co-digested with either nuclease. Differences in nuclease sensitivity observed when nuclei are digested separately are abolished when nuclei are probed in a mixing experiment. The results support the hypothesis that phenotypic differences in digestibility among different cell types in vitro reflect differences in chromatin-condensing factors which can diffuse between nuclei.     

Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence-activated cell sorting, and cell culture

We have used antibody and complement-mediated cell killing, fluorescence-activated cell sorting and tissue culture to study the development of rat oligodendrocytes. We show that (1) three ligands that bind to the majority of CNS neurons (the monoclonal antibodies A4 and A2B5 and tetanus toxin) also bind to immature oligodendrocytes and to precursor cells in 14-day embryonic rat brain that develop into oligodendrocytes in vitro; and (2) precursor cells in 17- to 18-day embryonic rat optic nerve can develop into oligodendrocytes in vitro in the absence of living neurons.     

A method for measuring anion transfer across red cell membranes by continuous monitoring of fluorescence

A method was developed for the identification of Ca²⁺-binding proteins after electrophoresis on polyacrylamide gels. The method involves equilibration of the gel with ⁴⁵Ca either during or after electrophoresis, followed by visualization of the ⁴⁵Ca-binding proteins by autoradiography.     

Modulation of human fetal hepatocyte survival and differentiation by interactions with a rat liver epithelial cell line

Our recent studies have shown that chick embryo epiphyseal cartilage synthesizes three distinct species of proteoglycan (PG-H, PG-Lb, and PG-Lt) which are analogous in having glycosaminoglycan side chains of the chondroitin (dermatan) sulfate type but different from one another in regard to the structure of core protein. In the present report, the expression of PG-H and PG-Lb has been studied in developing chick hind limbs (stages 19-33), using antibodies specific for these substances in indirect immunofluorescence. At the onset of cartilage morphogenesis (stage 24), PG-H became recognizable in the cartilage primordia, whereas a parallel section stained for PG-Lb showed no reaction. The first evidence of PG-Lb appearance was seen in a stage 28 cartilage (e.g., tibia) in which the cells in the middiaphysis became elongated in a direction perpendicular to the long axis of the cartilage. The PG-Lb fluorescence was confined to the zone of these flattened, disc-like cells, whereas the fluorescence for PG-H was uniformly distributed throughout the cartilage. With further development of cartilage (stage 29 approximately), the zone of flattened cells spread proximally and distally, and simultaneously large hypertrophied cells appeared at the diaphyseal region. During these zonal changes of cell morphology, the PG-Lb fluorescence remained restricted to the zone of flattened cells. Parallel sections stained for PG-H, in contrast, showed an evenly distributed pattern of the PG-H fluorescence throughout the cartilage. The results indicate that the appearance of PG-Lb is closely associated with the zonal changes of cell shape and orientation along the proximal-distal axis of the developing limb cartilage, and further suggest that the flattened chondrocytes in this particular zone have undergone additional changes in gene expression to form an extracellular matrix of still another chemical property.     

Cross-linking of fibronectin to sulfated proteoglycans at the cell surface.

Fibronectin is a major surface protein of normal animal cells but is absent from many transformed cells. Addition of fibronectin to transformed cells causes increased cell substrate adhesion and changes in the morphology and cytoskeleton of the cells. We have coupled fibronectin to photoactivable chemical cross-linkers and have added it to cells to identify those molecules to which it binds. In this way, fibronectin can be cross-linked to sulfated proteoglycans at the cell surface. The cross-linking is specific for fibronectin. The fibronectin-proteoglycan complex is sensitive to chondroitinase ABC and AC and to trypsin. Addition of fibronectin also affects binding of hyaluronic acid to the cells. These results suggest that fibronectin interacts with proteoglycans at the cell surface. The existence of such interactions may have implications for the role of fibronectin and proteoglycans in cell adhesion.     

Differentiation of fiber types in wing muscles during embryonic development: effect of neural tube removal

The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a sample muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.     

Enhanced chondrocytic differentiation in chick limb bud cell cultures by inhibitors of poly(ADP-ribose) synthetase

Inhibitors of poly(ADP-ribose) synthetase, namely nicotinamide, benzamide, m-methoxybenzamide and 3-aminobenzamide, augmented chondrocytic differentiation chick embryo limb bud mesenchymal cells, in culture. These inhibitors stimulated early appearance and massive formation of cartilage nodules in micromass cultures stage 23-24 chick embryos. They also induced nodule formation in micromass and cartilage colonies at micromass plating densities from stage 18-19 embryo Benzamide, however, did not prevent differentiated chondrocytes from undergoing a pleiotypic change in cell type. These results are compatible with the putative regulatory function of poly(ADP-ribose) on cell differentiation.     

Lack of correlation between agglutinability,the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines

Agglutinability by concanavalin A, distribution of surface-bound concanavalin A, and maximal cell density in monolayer culture were examined under similar conditions in parallel cultures of ten established cell lines. The degree of agglutinability of the cell lines did not correlate with the presence or absence of patching of concanavalin A bound to the cell surface, as determined with a hemocyanin marker. Agglutinability was also not always correlated with the loss of post-confluence inhibition of cell division. Two clones of mouse 3T3 fibroblasts that maintained post-confluence inhibition of cell division and low agglutinability differed substantially with respect to the surface distribution of concanavalin A. Patching of concanavalin A binding sites is neither necessary nor sufficient to explain differences in agglutinability between cell lines.     

The regulatory effect of macrophages on the antigen-induced lymph node cell proliferative response

Employing an antigen-induced T cell-dependent lymph node cell (LNC) proliferative assay in the mouse we observed differences in the capacity of unstimulated and thioglycollate-activated peritoneal exudate cells (PECs) to present antigen. Antigen-primed LNCs can be induced to proliferate by a brief (2 hr) antigen exposure and the addition of various numbers of thioglycollate PECs (thio-PECs) modifies the proliferative response depending on the ratio of $\text{PEC}\text{LNC}$ in cultures. With ratios of 3–12% both DNA and protein synthesis were enhanced, but at ratios greater than 12% suppression was significant. Treatment of thio-PECs with mitomycin C, irradiation (3000 rad), anti-Thy 1.2, or anti-Ia plus complement did not alter suppression, suggesting the possibility that the Ia negative macrophages present in the PECs were involved in the suppression. An enhancing effect on the proliferative response was noted following the addition of small numbers of thio-PECs. This was comparable to that seen with an equivalent concentration of supernatant from thio-PECs suggesting that soluble factors play an important role. Two enhancing fractions (separated on a G-75 column) which were themselves mitogenic were identified which eluted with approximate molecular weights of 15,000 and 60,000.     

Alterations in the cell cycle of Drosophila imaginal disc cells precede metamorphosis

Flow cytometric analyses of imaginal disc and brain nuclei of Drosophila melanogaster have been made throughout the third larval instar. In wing, haltere, and leg discs the proportion of cells in the G₂M phase of the cell cycle (tetraploid cells) increases with larval age. In contrast, in the eye disc and in brain the proportion of tetraploid cells, already low at the outset of the instar, declines further. Measurement of growth rates for disc and brain tissue during the same developmental period was carried out by the cell counting procedure of Martin (1982). Our results are consistent with the conclusion that imaginal discs grow exponentially with an apparent doubling time of 5–10 hr from the resumption of cell division (in the first or second larval instar) until about 95 hr, when the apparent doubling time increases. Cell numbers increase until at least 5 hr after formation of white prepupae (122 hr), but during the preceding 10 hr the rate of increase is low. Thus, for wing and leg discs, but not for the eye disc and brain, the declining growth rate is associated with an increase in the proportions of tetraploid cells. In conjunction with cell counts and flow cytometry, fluorometric determination of disc DNA content at 112 hr indicated that the diploid DNA content of imaginal disc nuclei is 0.45 pg.     

Studies on bacterial cell wall inhibitors: II. Inhibition of peptidoglycan synthesis in vivo and in vitro by amphomycin

Amphomycin has been reported by the present authors to be a selective inhibitor of cell wall peptidoglycan synthesis in Bacillus cereus T (ōmura, S., Tanaka, H., Shinohara, M., ōiwa, R. and Hata, T. (1975) Chemotherapy 5, 365–369). Investigations were carried out to clarify the target of amphomycin.Amphomycin (10 μg/ml) lysed growing cells of B. cereus T, and inhibited peptidoglycan synthesis, accompanied by accumulation of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc) peptides. The nucleotide precursors that accumulated in cells of Staphylococcus aureus FDA 209P in the presence of amphomycin were identified as UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala, UDP-MurNAc-L-Ala and UDP-MurNAc. In the experiments using a particulate enzyme system of Bacillus megaterium KM, amphomycin inhibited the polymerization of UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine, and also inhibited the formation of lipid intermediates, but did not inhibit the cross-linking, the last step of peptidoglycan synthesis. Unlike bacitracin, amphomycin did not lyse protoplasts of B. megaterium KM.We conclude that the site of action of amphomycin is the formation of MurNAc-(pentapeptide)-P-P-lipid from MurNAc-pentapeptide and undecaprenol (lipid) phosphate.