DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

A nonendocytotic mechanism for the selective uptake of high density lipoprotein-associated cholesterol esters

We have previously described in rats the selective uptake of HDL-associated cholesterol esters (traced by [3H]cholesteryl oleyl ether) in excess of the uptake of HDL-associated apoA-I. In the present studies we show that the mechanism also exists in cultured cells of human and mouse origin as well. This selective uptake represents a net uptake of cholesterol esters and not an isotope exchange, as shown by mass flux studies in adrenal cells. Inhibitors of receptor recycling, chloroquine, monensin, and colchicine, inhibited uptake of apoA-I from HDL by Hep G-2 human hepatoma cells to about the same extent as a reference protein, asialofetuin, but inhibited uptake of the cholesteryl ether tracer much less. Levels of NaN3 which effectively inhibited sucrose pinocytosis inhibited uptake of apoA-I to about the same extent but did not inhibit uptake of the cholesteryl ether at all. Thus, not only receptor recycling, but endocytosis as well, appears not to be involved in selective uptake. This conclusion was supported by studies in which synthetic HDL particles were made to contain two neutral lipid core tracers; one of them, the [3H]cholesteryl ether previously used, was selectively taken up, whereas the other, [14C]sucrose octaoleate, was excluded from selective uptake. Thus, selective uptake cannot involve endocytosis of the entire lipid core, but may involve other specific transfer mechanisms.     

Activation of neutrophils by recombinant interleukin 6

The cytokine interleukin 6 (IL-6) has been shown to have multiple biological activities against many cellular targets. The present studies were designed to determine whether these activities extended to the neutrophil (PMN). Initially, we investigated the ability of IL-6 to modulate PMN-mediated antibody-dependent cellular cytotoxicity. The presence of IL-6 stimulated 51Cr release from labeled, opsonized targets by 67.1% (from 21.6 +/- 1.4% to 36.1 +/- 1.3% at 10 U of IL-6 (P less than 0.01)). IL-6 was not directly toxic to the target cells and stimulation of ADCC was shown to occur across a range of effector-to-target ratios. To investigate the basis of the capacity of IL-6 to stimulate PMN, we studied the effects of IL-6 on PMN chemotaxis, degranulation, and the respiratory burst. IL-6 was not chemotactic or chemokinetic for PMN. However, IL-6 stimulated lysozyme secretion from 14.1 +/- 2.5 to 23.7 +/- 3.6% at 100 U (P less than 0.01). IL-6 was a complete secretagogue, being able to induce the secretion of both the secretory granule marker lactoferrin (11.2 +/- 2.0 to 23.5 +/- 2.2%) and the primary granule marker beta-glucuronidase (5.0 +/- 1.0 to 18.2 +/- 4.0%). IL-6 was not able to directly stimulate the PMN respiratory burst. However, IL-6 did "prime" PMN, enhancing superoxide secretion by fMLP (10(-7) M)-treated PMN by 50.8% (5.9 +/- 1.0 to 8.9 +/- 1.5 nmol superoxide at 100 U of IL-6; P less than 0.01) and PMA (5.0 nM) by 54.3% (8.1 +/- 2.6 to 12.5 +/- 3.6 nmol; P less than 0.05). In conclusion, IL-6 is a PMN stimulant, enhancing the toxicity of PMN in an antibody-dependent cellular cytotoxicity assay. Enhanced cytotoxicity may have been mediated, at least in part, by the stimulation of secretion of toxic components from PMN targets and by the priming of stimulating respiratory burst activity.     

Demonstration of a basic fibroblast growth factor-like molecule in mouse hepatic endothelial cells

We have investigated whether isolated mouse hepatic sinusoidal endothelial cells (HSEC) synthesized basic FGF. HSEC lysate was fractionated by heparin-Sepharose chromatography. A peak of mitogenic activity for Balb/c 3T3 fibroblasts was eluted with 3M NaCl. Several arguments suggested that the mitogenic factor was related to bFGF: a) its affinity for heparin; b) the loss of its mitogenic activity by heating at 65 degrees C, which was prevented in the presence of heparin; c) the abolition of its mitogenic activity in the presence of protamine sulfate; d) finally, its mitogenic effect was reduced in the presence of antibody to bFGF. These data demonstrate the presence of a bFGF-like molecule in HSEC. This molecule could be involved in the regulation of the neighboring Ito cell proliferation.     

Replication of basal bodies and centrioles.

During the past year, studies on the centrioles and basal bodies of animal and algal cells, and the spindle pole bodies of yeast and other fungi, have added significantly to our knowledge of how these cell organelles form and how they function in initiating microtubule assembly throughout the cell cycle. Most of these studies have used antibodies to identify proteins within and around these organelles and, in some cases, to disrupt their ability to nucleate microtubules. Genetic methods have been used to identify specific proteins, including a new member of the tubulin superfamily, involved in the function and replication of spindle pole bodies and centrioles.     

Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

Adenosine deaminase activity in relation to the appearance of early and late Epstein-Barr virus antigens induced in lymphoblastoid cells

Summary Induction of Epstein-Barr virus (EBV) capsid antigen synthesis in 59.6% of P3HR-1 cells was followed by a decrease to 70% in adenosine deaminase (ADA) activity. In Daudi cells synthesizing EBV early antigen, ADA activity did not decrease.