Intracellular transport of phosphatidylcholine to the plasma membrane

We have used pulse-chase labeling of Chinese hamster ovary cells with choline followed by plasma membrane isolation on cationic beads to study the transport of phosphatidylcholine from the endoplasmic reticulum to the plasma membrane. We have found that the process is rapid (t1/2 [25 degrees C] = 2 min) and not affected by energy poisons or by cytochalasin B, colchicine, monensin, or carbonyl cyanide p-chlorophenylhydrazone. Cooling cells to 0 degree C effectively stops the transport process. The intracellular transport of phosphatidylcholine is distinct in several ways from the intracellular transport of cholesterol (Kaplan, M. R., and R. D. Simoni, 1985, J. Cell. Biol., 101:446-453).     

A histamine-releasing factor from activated human mononuclear cells

Human mononuclear cells activated by streptokinase-streptodornase have been shown to elaborate a factor capable of releasing histamine from human basophils. We have developed reproducible methods for its production in large quantities by using cells obtained from leukapheresis packs, by detection utilizing donor basophils known to release well with anti IgE, and by quantitation of histamine by the radioenzyme method. Human histamine-releasing factor (HRF) gave a single peak upon gel filtration with an estimated m.w. of 32,000; SDS gel electrophoresis revealed a single major band as seen at m.w. 30,000. HRF can be resolved into at least two forms separable by ion-exchange chromatography on QAE Sephadex, and two peaks of activity were obtained by chromatofocusing or isoelectric focusing in gels at pH 6.9 and between 7.4 and 8.3. This factor represents an important potential link between cellular immunity and immediate hypersensitivity.     

Antigen presentation in the rat. II. An Ia+ radiosensitive T cell can present antigen to primed Ia- T cells

We demonstrated previously the presence of an Ia+ (OX-6+) antigen-presenting cell within the rat T cell fraction that is capable of presenting antigen to antigen-primed OX-6-T cells. This antigen-presenting cell (T-APC) reacted with the monoclonal antibodies W3/25 and W3/13, which is known to react mainly with rat T cells. Further characterization of the T-APC indicated that the cell also reacted with the monoclonal antibody OX-19, which is highly specific for rat T cells. Moreover, the antigen-presenting function of the T-APC was sensitive to treatment with mitomycin C or gamma-irradiation (2000 rad). Under similar conditions, antigen presentation by partially purified dendritic cells or macrophages was totally resistant to these treatments. The antigen-presenting activity of gamma-irradiated T-APC was not reconstituted by the addition of the lymphokines IL 1, IL 2, or Con A supernatants. Although unirradiated T-APC were able to stimulate an MLR response, this function was also sensitive to gamma-irradiation, whereas the MLR-stimulating ability of macrophages and dendritic cells was resistant to gamma-irradiation. These data indicate that Ia+ T cells from the rat are capable of presenting antigen to antigen-primed T lymphocytes and that, in contrast to antigen presentation by macrophages and dendritic cells, the function of T-APC is gamma-radiation sensitive.     

Induction of functional Fc receptors in P388 leukemia cells : Requirement for multiple differentiation signals

The development of functional Fc receptors (FcR) during induced differentiation with the tumor promoter, phorbol myristate acetate (PMA), was studied in the murine tumor cell line, P388. PMA induced the appearance of FcR on the membranes of P388 cells as indicated by the binding of IgG-coated sheep red blood cells (IgG-SRBC). Concentrations of PMA as low as 1 ng/ml were sufficient to induce the expression of FcR as well as to inhibit cellular division and to induce adherence in the P388 tumor cell line; however, optimal FcR induction occurred at PMA concentrations of 10-100 ng/ml. Immunofluorescent analysis with heat-aggregated myeloma proteins indicated that PMA induced FcR which were capable of binding IgG2a and IgG2b immunoglobulins, but not IgG1. Adherence to a substratum was determined to be a second required signal for expression of FcR, since PMA induction of P388 tumor cells in teflon dishes failed to fully develop FcR and adherence of P388 cells to poly-L-lysine-coated culture dishes in the absence of PMA was insufficient for FcR expression. FcR which appeared after PMA induction were non-functional in the sense that membrane-bound IgG-SRBC were not ingested to any significant extent by the tumor cells. However, if FcR induction occurred in the presence conA-induced rat spleen cell culture supernatants, phagocytosis of membrane-bound erythrocytes occurred. These findings suggest that for the expression of FcR which are capable of particle internalization, at least three identifiable membrane-transmitted signals are required during differentiation.     

Evolution and Extinction of Transposable Elements in Mendelian Populations

A model of the evolution of a transposable element family in a Mendelian host population is proposed that incorporates heritable phenotypic mutations in the elements. The temporal behavior of the numbers of mutant and wild-type elements is studied, and the expected extinction time of the transposable element family is examined. Our results indicate that, if the mutant can be transposed equally well in the presence of the wild type, then it can be expected to be found in preponderance, whereas elements, such as retroviruses, where the transposing genome and its phenotypic expression are coupled, may be characterized by a low mutant frequency.     

Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system.

The enzymes for luminescence in Vibrio fischeri are induced by the accumulation of a species-specific metabolite (autoinducer) in the culture medium. Tritium-labeled autoinducer was used to study the mechanism of autoinduction. When 3H-autoinducer was added to suspensions of V. fischeri or Escherichia coli, cellular concentrations equaled external concentrations. For V. fischeri, equilibration of 3H-autoinducer was rapid (within 20 s), and greater than 90% of the cellular tritium remained in unmodified autoinducer. When V. fischeri or E. coli cells containing 3H-autoinducer were transferred to autoinducer-free buffer, 85 to 99.5% of the radiotracer escaped from the cells, depending on the strain. Concentrations of autoinducer as low as 10 nM, which is equivalent to 1 or 2 molecules per cell, were sufficient for induction, and the maximal response to autoinducer occurred at about 200 nM. If external autoinducer concentrations were decreased to below 10 nM after induction had commenced, the induction response did not continue. Based on this study, a model for autoinduction is described wherein autoinducer association with cells is by simple diffusion and binding of autoinducer to its active site is reversible.     

Malformation uropathies and multiple malformation syndromes

The authors, from their experience emphasize the associated malformations' frequency in major congenital urinary tract malformations (26,9%). It is essential to recognize in these multiple defects some certified syndromes - inherited or not. The most associations are still unknown, nevertheless the genetic counselling require an accurate diagnosis.     

Leishmanial phosphatase blocks neutrophil O-2 production

Leishmania donovani, the causative agent in kala-azar or visceral leishmaniasis, infects cells of the macrophage system. We show that a purified preparation of the tartrate-resistant acid phosphatase, isolated from the external surface of L. donovani promastigotes, inhibits superoxide anion production by human neutrophils. Preincubation of neutrophils for 15-30 min at 37 degrees C with 240 units (1 unit equals 1 nmol of 4-methylumbelliferyl phosphate cleaved per h) of the acid phosphatase decreases both the rate and extent of superoxide generation by 90% upon stimulation with the chemoattractant peptide fMet-Leu-Phe. The ability of the phosphatase to suppress superoxide anion production is abolished by heat inactivation of the enzyme or by incorporation of an acid phosphatase inhibitor in the preincubation medium, indicating that the effect is dependent on the catalytic activity of the enzyme. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.     

Analysis of 31P NMR spectra of enzyme-bound reactants and products of adenylate kinase using density matrix theory of chemical exchange

31P NMR spectra of equilibrium mixtures of enzyme-bound reactants and products of the adenylate kinase reaction (formula; see text) were analyzed by using computer simulations based on density matrix theory of chemical exchange. Since adenylate kinase has the unique feature that the reactants in the reverse direction are both ADP molecules, which are indistinguishable off the enzyme, the density matrix equations are formulated for the ABC + D in equilibrium A'B' + A"B" exchange appropriate for the reaction, in which the interchange of A'B' and A"B" is explicitly introduced. It is shown that the consideration of this interchange is essential to explain the experimentally observed line shapes. By comparison of the computer-simulated spectra with various values for the rates of the exchange with the experimental spectra for porcine adenylate kinase at pH 7.0 and T = 4 degrees C, the following characteristic rates were determined: interconversion rates, 375 +/- 30 s-1 (ATP formation) and 600 +/- 50 s-1 (ADP formation); interchange rates of donor and acceptor ADP's, 100 +/- 30 s-1 (in the presence of optimal Mg2+ concentration), 1500 +/- 100 s-1 (in the absence of Mg2+). It is shown that under the conditions of the experiments the interchange rate is the lower limit of the dissociation rate of ADP (or MgADP from the acceptor site if Mg2+ was present) from the enzyme complexes. The significance of these interchange rates and their values relative to the interconversion rates is discussed with special reference to the role of the Mg2+ ion in the differentiation of the two nucleotide binding sites on adenylate kinase.