Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

The Macrophage Inhibitor CNI-1493 Blocks Metastasis in a Mouse Model of Ewing Sarcoma through Inhibition of Extravasation

Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma.     

Evidence for a spectrin-like protein as a major component of the synaptosomal membrane cytoskeleton

After treatment of synaptosomes with Nonidet-containing buffers, a proportion of the proteins remained insoluble. The major component (50%) of the residue was identified as a spectrin-like protein by immunodetection after mono- and bi-dimensional gel electrophoresis and transfer to nitrocellulose paper. Actin was also present.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A.

Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.     

Eimeria vermiformis and E. mitis: inhibition of development in vivo by cyclosporin A

Treatment of the host (Mus musculus, Gallus domesticus) with cyclosporin A during infection with Eimeria vermiformis or E. mitis resulted in a reduction in the numbers of oocysts passed in the feces and/or a delay in patency. The general immunosuppressive effects of the treatment were confirmed in chickens by monitoring their antibody responses to human erythrocytes and lymphoproliferative responses to phytohemagglutinin. Nevertheless, mice and chickens treated with cyclosporin A during a primary infection with E. vermiformis or E. mitis, respectively, were immune to subsequent challenge with these organisms. Thus, cyclosporin A did not interfere with priming. The antiparasite effect of the drug did not allow an evaluation of its effect on established immunity to the coccidia when it was administered at the time of challenge. In an exceptional treated chicken, however, delayed patency of the challenge infection was followed by the production of a number of oocysts similar to that found in unprimed animals. This suggests that the mechanisms of immunity to challenge may be susceptible to disruption by cyclosporin A.     

Design and properties of a fluorescent indicator of intracellular free Na+ concentration.

We have recently described a cryptand structure, FCryp-1, with appropriate properties for an indicator of intracellular free Na+ concentration using the 19F-n.m.r. chemical shift of the incorporated 5FBAPTA [1,2-bis-(2-amino-5-fluorophenoxy)ethane-NNN'N'-tetra-acetic acid] reporter group to measure the free cytosolic Na+ concentration [( Na+]i) [Smith, Morris, Hesketh and Metcalfe (1986) Biochim. Biophys. Acta 889, 82-83]. FCryp-1 carries four carboxylate groups to confer aqueous solubility and the indicator is membrane-permeant when the carboxyls are esterified with acetoxymethyl ester groups. Here we describe the synthesis of FCryp-2 to provide a fluorescent indicator of [Na+]i. FCryp-2 retains the parent tribenzo (2:2:1) cryptand structure of FCryp-1, in which the benzenoid ring at C-21 in FCryp-1 is replaced by an indole derivative which acts as the fluorophor in FCryp-2. With excitation at 340 nm, FCryp-2 gives an emission maximum at 460 nm in the absence of Na+ which shifts to 395 nm when FCryp-2 is saturated with Na+, with an isosbestic point at 455 nm. The apparent dissociation constant of FCryp-2 in a buffer solution of 100 mM-KCl/20 mM-KH2PO4/K2HPO4, pH 7.0, at 37 degrees C is 6.0 mM and the free Na+ concentration can be measured either from the calibrated fluorescence intensity at 395 nm, which increases 25-fold when Na+ is bound to FCryp-2, or from the ratio of fluorescence intensities at 395 nm and 455 nm. The measurement of free [Na+] by either method is unaffected by K+, Ca2+ or Mg2+ in the normal intracellular concentration ranges. Free [Na+] measurements by the ratio method are unaffected by pH from 6.6 to 7.6.     

Phosphoinositide metabolism and the calcium response to concanavalin A in S49 T-lymphoma cells. A comparison with thymocytes.

Comparisons were made between transformed S49 T-lymphoma cells and normal murine thymocytes in their polyphosphoinositides, inositol polyphosphates and cytosolic free calcium concentrations ([Ca2+]i), and the effects of the T-cell mitogen concanavalin A (Con A) on these properties. 1. The ratios of the polyphosphoinositides to phosphatidylinositol in both exponential-phase S49 cells and mitogen-stimulated thymocytes (G1 phase) were greater than in quiescent (G0-phase) thymocytes. 2. In response to Con A, the amount of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in S49 cells decreased slightly (17% in 30 min), and this was sufficient to account for the small amounts of inositol phosphates that accumulated. In contrast, it has been shown previously that Con A stimulates a rapid resynthesis of PtdInsP2 in thymocytes and the amounts of inositol phosphates released rapidly exceed the steady-state amount of the PtdInsP2 precursor [Taylor, Metcalfe, Hesketh, Smith & Moore (1984) Nature (London) 312, 462-465]. 3. The [Ca2+]i did not differ significantly in S49 cells and thymocytes before the addition of Con A, and the increases in [Ca2+]i in response to Con A were similar in both types of cell. 4. The [Ca2+]i increase in response to Con A was inhibited by similar concentrations of intracellular cyclic AMP (2-10 microM) in S49 cells and thymocytes, suggesting that similar regulatory mechanisms act on this response in both types of cell. The data demonstrate that the basal [Ca2+]i and phosphoinositide metabolism is similar in both the normal cells and their transformed counterparts. In addition, they suggest that the activated Con A receptors generate very similar signals in the two cell types, and that any perturbations of primary signal transduction to the secondary phosphoinositide and [Ca2+]i responses in the S49 phenotype are quantitative rather than qualitative.     

Design of an indicator of intracellular free Na+ concentration using 19F-NMR

The development is described of an Na+ chelator with appropriate properties for an indicator of intracellular free Na+ concentration ([Na+]i). The new indicator, FCryp-1, is a tribenzo derivative of the parent (2:2:1) cryptand structure, incorporating the same F-substituted dibenzo 19F-NMR reporter group as the free [Ca2+] indicator, 5FBAPTA (Smith, G.A., Hesketh, T.R., Metcalfe, J.C., Feeney, J. and Morris, P.G. (1983) Proc. Natl. Acad. Sci., USA 80, 7178-7182). FCryp-1 has appropriate affinity for Na+ (KNa = 10(1.3) M-1) and selectivity over other intracellular cations (KK; KCa; K Mg less than 10(-1) M(-1)) for a [Na]i indicator. There is an 19F-NMR chemical shift of 2.00 ppm between free FCryp-1 and the Na-FCryp-1 complex which provides a direct read out of free [Na+]. FCryp-1 carries four carboxylate groups to confer aqueous solubility which can be esterified with acetoxymethyl groups to render the indicator membrane permeant. Experiments on pig lymphocytes loaded with FCryp-1 gave an indicated [Na+]i of 13.8 +/- 1.8 mM (n = 4). The FCryp-1 structure can also be readily modified to provide fluorescent [Na+]i indicators.