Tumor-promoting phorbol esters inhibit the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

L-cell colony-stimulating factor (CSF-1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue-derived mononuclear phagocyte colony-forming cells (Stanley et al., 1978). CSF-1 interacts with target cells by direct and specific binding to membrane receptors (CSF-1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor-promoting phorbol esters on the binding of 125I-labeled CSF-1 (125I-CSF-1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12-O-tetradecanoyl phorbol-13-acetate) inhibits the binding of 125I-CSF-1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, maximum inhibition occurred at about 10(-7) M; inhibition was 50% at 5 X 10(-9) M. At 0 degrees C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I-CSF-1-binding activity whether TPA is later removed or not. The process of recovering CSF-1-binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF-1 receptors rather than a decrease in receptor affinity.     

Kinetics of dissociation of reduced nicotinamide adenine dinucleotide phosphate from its complexes with malic enzyme in relation to substrate inhibition and half-of-the-sites reactivity

Malic enzyme of pigeon liver binds NADPH at four equivalent enzyme sites and binds Mn2+ and malate each at two sets of "tight" and "weak" sites with negative cooperativity [Pry, T. A., & Hsu, R. Y. (1980) Biochemistry 19, 951-962]. Stopped-flow studies on the displacement of NADPH from the malate-enzyme complexes E4-NADPH4, E4-Mn2(2+)-NADPH4, E4-Mn2(2+)-NADPH4-dimalate, and E4-Mn2(2+)-NADPH4-tetramalate by large excess NADP+ or its analogue phosphoadenosine(2')diphospho(5')ribose show that NADPH dissociates from the binary complex rapidly with a first-order rate constant of 427 s-1. Dissociation from the ternary E4-Mn2(2+)-NADPH4 complex containing two tightly bound Mn2+ ions can be described by a single first-order process with a rate constant of 135 s-1, or more satisfactorily by two simultaneous first-order processes attributable to the reactions of Mn2+-deficient (k congruent to 427 s-1) and Mn2+-liganded (k = 96 s-1) subunits. The latter equals twice the maximum steady-state turnover rate of 53.2 + 3.0 s-1 assigned to dissociation of the reduced nucleotide from transient E-Mn2+-NADPH, and this 2:1 ratio strongly supports our proposed "half-of-the-sites" model [Hsu, R. Y., & Pry, T. A. (1980) Biochemistry 19, 962-968]. Dissociation from the E4-Mn2(2+)-NADPH4-dimalate complex (k = 100 s-1) follows only the slower process, suggesting that occupancy of malate at two sites tightens enzyme-bound NADPH on the adjacent sites. Binding of malate at two additional weak sites yields E4-Mn2(2+)-NADPH4-tetramalate and a NADPH dissociation rate constant of 2.69 s-1. The 97% decrease in NADPH dissociation parallels the observed 93% maximal inhibition by malate and is the cause of substrate inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cytochemical study of acid phosphatase,thiamine pyrophosphatase and horseradish peroxidase in the Golgi-GERL complex of hepatoma ascites cells

Data from studies of ascitic cells of Chang hepatoma have shown that acid phosphatase (ACPase) can be localized simultaneously within the trans portion of the Golgi apparatus and in tubules of the Golgi-endoplasmic reticulum-lysosome (GERL) system. Reaction products of thiamine pyrophosphatase (TPPase) were also present consistently within trans elements of the Golgi apparatus and within GERL tubules. These new findings indicate that a close physiological association may exist between the Golgi apparatus and GERL, a concept that is consistent with previous observations of fibroblasts. When horseradish peroxidase (PO) is injected intraperitoneally into ascites-bearing rats and the ascitic cells withdrawn at different time intervals, PO could be localized within vesicles and tubules in the GERL region but could not be detected within the Golgi apparatus. Bulk-phase endocytosis requires a long time and a high concentration of PO to occur. The presence of PO within GERL indicates that this organelle may play a role in transporting or processing of certain exogenous proteins.     

DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Summary Two active enzymes of the galactose operon of Escherichia coli, uridyl transferase and galactokinase have been synthesized with high yields in a DNA dependent system for protein synthesis. The unspecific blank values amount to less than two percent of the rate obtained under optimal conditions and permit the accurate determination of even a small fraction of the maximum synthesis rate. Therefore this system provides a sensitive assay for the biological activity of DNA that contains the intact galactose operon of Escherichia coli.The synthesis of these galactose enzymes is to a high extent dependent on the presence of cyclic adenosine-3:5-monophosphate.D-fucose, known as an inducer of the galactose operon in vivo, stimulates the synthesis of galactokinase, indicating that the repressor of the galactose operon in active under these conditions. This stimulation is not observed, if the bacterial extract is prepared from a strain defective for the galactose repressor or if the DNA carries an operator constitutive mutation in the galactose operon. Therefore the stimulation by D-fucose is true derepression.     

Phycobilisomes in Blue-Green Algae

Fifteen species of freshwater blue-green algae, including unicellular, filamentous, and colonial forms, were subjected to a variety of fixatives, fixation conditions, and stains for comparison of the preservation of phycobilisomes. Absorption spectra of the corresponding in vivo and released photosynthetic pigments, in 10 of the species that were maintained in culture, demonstrated the presence of phycocyanin in all 10 species and phycoerythrin in only 2 of them. Spectroscope and electron microscope evidence was obtained for localization of phycobiliproteins in phycobilisomes of Nostoc muscorum. Phycobilisomes were observed in all species examined in situ, strenghening the hypothesis that phycobilisomes are common to all phycobiliprotein-containing photosynthetic blue-green algae.     

Electron Microscope Analysis of Partial Denaturation of F Factor Deoxyribonucleic Acid

Partial denaturation pattern of sex factor deoxyribonucleic acid of Escherichia coli was studied by electron microscopy. Clustering of the adenine-plusthymine-rich regions in one part of the molecule was revealed. The positions of these regions were located on the physical map of F by analyzing the partial denaturation pattern of heteroduplexes between F and F-prime factors with various parts of F sequences deleted.