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.     

Corticosteroid-induced abnormality in fetal mice and H-2 haplotype: Evidence of a cytoplasmic effect

Different strains of H-2 congenic mice have different susceptibilities to corticosteroid-induced fetal loss and cleft palate. Applying this knowledge, we tested the null hypothesis, which assumes that there are no statistically significant differences in the frequency of abnormality among various types of treated backcross offspring and, thus, no evidence of a cytoplasmic effect. In the present study this null hypothesis was frequently, but not consistently, rejected. Therefore, there was some evidence of a cytoplasmic effect. One possible explanation of these results is seen when one considers the phenotypic effects of “gene-gene interaction” between variant H-2 genotypes and an invariant mitochondrial genotype.     

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.     

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.     

The microbial ecology evaluation Device mycology spaceflight studies of Apollo 16

Four fungal species were selected as the test systems for the Microbial Ecology Evaluation Device (MEED) of Apollo 16. Fungal cells were exposed to various quantitative and qualitative spaceflight parameters then returned for postflight analyses. Survival rates and phenotype numbers varied according to exposure parameters. Additional studies underway will further identify the spaceflight changes.