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R Sullivan

Publications and source records attributed to R Sullivan.

At least 127 records · Page 7Linked to original sources
PubMed1987

Effects of recombinant human granulocyte and macrophage colony-stimulating factors on signal transduction pathways in human granulocytes.

We studied the ability of the recombinant human-active hemopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSFrh) and granulocyte colony-stimulating factor (G-CSFrh) to activate receptor-mediated transduction pathways which have been implicated in the stimulation of cytotoxic functions in granulocytes. With the use of a panel of fluorescent probes, we found that these two growth factors exerted no detectable immediate effect on the resting transmembrane electrical potential, the intracellular concentration of free calcium ions, or the cytosolic pH of isolated, mature granulocytes. However, when granulocytes were "primed" by preincubation for 90 min with GM-CSFrh or G-CSFrh, the rate of membrane depolarization induced by 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, but not the rate of rise in free calcium ions, was greatly accelerated. In examining potential mechanisms to account for the priming effect of these growth factors, we found that although they did not induce translocation of protein kinase C or stimulate significant degranulation, they each directly caused prompt release of arachidonic acid from plasma membrane phospholipids. Our data indicate that although GM-CSFrh and G-CSFrh do not activate the transduction signals that have most clearly been implicated in receptor-mediated activation of cytotoxic functions in granulocytes--namely, those coupled to membrane depolarization or release of intracellular calcium ions--they appear directly to induce the release of arachidonic acid esterified to membrane phospholipids, an event which may represent the receptor-mediated activation of membrane phospholipases and which may contribute to the "priming" of the cells for enhancement of their functional responsiveness.

Arachidonic Acid↗
PubMed1987

The effects of phorbol myristate acetate and chemotactic peptide on transmembrane potentials and cytosolic free calcium in mature granulocytes evolve sequentially as the cells differentiate.

We isolated myeloid precursors from human marrow and studied the effects of phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) upon transmembrane potentials and cytosolic calcium ([Ca2+]i) as the cells matured. Using a panel of fluorescent probes, we found that membrane depolarization induced by PMA and fMLP in granulocytes, and elevation in [Ca2+]i stimulated by fMLP, were absent in myeloblasts. When we induced differentiation with granulocyte-macrophage colony-stimulating factors, we found that both ionic responses appeared at approximately the promyelocyte stage. By using di-O-C5(3), we detected an initial phase of fMLP-induced hyperpolarization which appeared ontogenetically earlier than depolarization and which could be evoked in mature granulocytes with lower concentrations of the ligand. Hyperpolarization was partially dependent on extracellular Na+, was abrogated by increasing the external K+ concentration, and was accompanied by mild acidification of the cytoplasm. Bordetella pertussis toxin abolished both hyperpolarization and depolarization. Our findings indicate that shifts in [Ca2+]i and membrane potential changes in response to PMA and fMLP evolve as granulocytes mature. In addition, transmembrane ionic fluxes induced by fMLP appear to be more complex than previously considered, involving at least two separable phases of membrane potential change.

Aminoquinolines↗
PubMed1987

Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet-derived growth factor-like protein which is secreted.

Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin-Sepharose at 1.4-1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross-reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin-Sepharose at 0.4-0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet-derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF-like, but secrete little if any basic FGF. It is suggested that endothelial cell-associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell-secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall.

Animals↗
PubMed1987

Multiple forms of basic fibroblast growth factor: amino-terminal cleavages by tumor cell- and brain cell-derived acid proteinases.

Basic fibroblast growth factor (FGF) was purified by heparin-Sepharose chromatography from two sources, brain and hepatoma cells. Brain cell-derived basic FGF (brFGF) and hepatoma cell-derived basic FGF (heFGF) were found to exist in multiple forms whose molecular weights depended on whether they were extracted from their respective tissue or cells at neutral or acid pH. When extracted at pH 7.0 brFGF and heFGF comigrated on NaDodSO4/PAGE with a Mr of approximately 18,400. When extracted at pHs 3.5-4.5, acid proteinases cleaved brFGF and heFGF to lower molecular weight forms but to different extents. brFGF was cleaved to a Mr 18,000 form at acid pH by a brain-derived acid proteinase that could be inhibited by pepstatin. heFGF was cleaved mostly to a Mr 16,500 form at acid pH by a hepatoma cell-derived acid proteinase that was inhibited by leupeptin. Electrophoretic transfer blot analysis using site-specific anti-FGF antibodies suggested that the cleavages occurred at the amino-terminal ends of brFGF and heFGF. Cleavage to lower molecular weight forms of brFGF and heFGF did not affect growth factor activity or chromatographic behavior on heparin-Sepharose columns.

Amino Acid Sequence↗
PubMed1987

Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix.

Bovine aortic and corneal endothelial cells synthesize a growth factor that remains mostly cell-associated but can also be extracted from the subendothelial extracellular matrix (ECM) deposited by these cells. The endothelial cell-derived growth factors extracted from cell lysates and from the extracellular matrix appear to be structurally related to basic fibroblast growth factor by the criteria that they bind to heparin-Sepharose and are eluted at 1.4-1.6 M NaCl, have a molecular weight of about 18,400, cross-react with anti-basic fibroblast growth factor antibodies when analyzed by electrophoretic blotting and immunoprecipitation, and are potent mitogens for bovine aortic and capillary endothelial cells. It is suggested that endothelium can store growth factors capable of autocrine growth promotion in two ways: by sequestering growth factor within the cell and by incorporating it into the underlying extracellular matrix.

Animals↗
PubMed1987

Seroepidemiologic study of giardiasis patients and high-risk groups in a midwestern city in the United States.

Serum antibodies to Giardia lamblia were measured in giardiasis patients, in groups at high risk for intestinal parasite infection, and in controls by an indirect fluorescent antibody (IFA) technique. Symptomatic patients had the highest antibody titers, and antibodies remained present for up to 18 months in persons with chronic infection. Indochinese refugees and male homosexuals with the acquired immunodeficiency syndrome (AIDS) and pre-AIDS had higher mean antibody levels than did healthy controls, whereas sewer and highway maintenance workers had levels similar to those of controls. Serum antibodies to Entamoeba histolytica measured by an indirect hemagglutination antibody technique were detected in only a few Indochinese refugees. We conclude that serology is a promising tool in G. lamblia epidemiology and that further population studies would be of interest.

Entamoeba histolytica↗
PubMed1987

Acquisition of formyl peptide receptors during normal human myeloid differentiation.

By using a newly developed immune rosetting technique to isolate highly purified populations of myeloid precursor cells from normal human bone marrow and then inducing their differentiation with granulocyte and macrophage colony-stimulating factors (G/M-CSFs) in vitro, we studied the surface expression of chemotactic peptide receptors as the cells matured from the stage of the myeloblast to that of the mature, segmented neutrophil. We used ethylene glycol bis(succinimidyl succinate) to link N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys to chemotactic peptide receptors on the surface of myeloid cells at sequential stages of maturation and then determined the density of receptor-radioligand complexes by autoradiography after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Specific, saturable formyl peptide receptors were not detectable at the level of the myeloblast but gradually emerged through progressive stages of neutrophil maturation. The specific receptors for formyl peptide that appeared during cellular maturation had a mol wt of 55 to 70 kiloDalton (kD), corresponding to those present on the surface of peripheral blood neutrophils, and binding of the radioligand was highly specific in that it was completely inhibited by a 1,000-fold excess of F-Met Leu Phe. These data correlate with and provide insight into our recent observation that F-Met Leu Phe-induced membrane depolarization and transient increases in cytosolic free calcium are gradually acquired as neutrophils mature. This report represents to our knowledge the first description of the maturational development of chemotactic peptide receptor expression in normal human myeloid cells.

Bone Marrow↗
PubMed1986

Purification and characterization of heparin-binding endothelial cell growth factors.

Thirteen endothelial cell growth factors have been purified to homogeneity by heparin affinity and reversed-phase high performance liquid chromatography, and their chromatographic and electrophoretic properties were compared. The amino acid compositions of 10 of these mitogens have also been determined. The results indicate that these heparin-binding growth factors (HBGFs) can be subdivided into two classes. Class 1 HBGFs are anionic mitogens of molecular weight 15,000-17,000 found in high levels in neural tissue and include acidic brain fibroblast growth factor and retina-derived growth factor. Class 2 HBGFs are cationic mitogens of molecular weight 18,000-20,000 found in a variety of normal tissues and are typified by pituitary fibroblast growth factor and cartilage-derived growth factor. Typical class 2 HBGFs have also been isolated from a rat chondrosarcoma, a human melanoma, and a human hepatoma, suggesting that tumors do not make a structurally distinct HBGF class. These results provide a sound basis for the evaluation of the HBGFs purified from a variety of tissues and species and for the delineation of their normal and pathological functions in vivo.

Amino Acids↗
PubMed1986

Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast growth factor.

A human hepatoma cell line synthesizes, as evidenced by metabolic labeling, an endothelial cell mitogen that is found to be mostly cell associated. The hepatoma-derived growth factor (HDGF) has been purified to homogeneity by a combination of Bio-Rex 70, heparin-Sepharose, and reverse-phase chromatography; it is a cationic polypeptide with a molecular weight of about 18,500-19,000. HDGF is structurally related to basic fibroblast growth factor (FGF). Immunological analysis demonstrates that antiserum prepared against a synthetic peptide corresponding to the amino-terminal sequence of basic FGF cross-reacts with HDGF when analyzed by electrophoretic blotting and by immunoprecipitation. Sequence analysis of tryptic fragments demonstrates that HDGF contains sequences that are homologous to both amino-terminal and carboxyl-terminal sequences of basic FGF.

Amino Acid Sequence↗
PubMed1986

Production of erythroid burst-promoting activity (BPA) and granulocyte-monocyte colony-stimulating activity (GM-CSA) by isolated human T-lymphocyte subpopulations.

T-lymphocytes and monocytes are prominent among the classes of normal human cells that have been implicated in the production of the hemopoietic growth factors granulocyte-macrophage colony-stimulating activity (GM-CSA) and erythroid burst-promoting activity (BPA). To investigate the nature of the cooperativity that occurs during the elaboration of these growth factors by activated T-lymphocytes and monocytes in vitro, and to define the subsets of T cells involved in this response, we studied the production of GM-CSA and BPA by populations of T-lymphocytes isolated by fluorescence-activated cell sorting, using the monoclonal antibodies OKT3, OKT4, and OKT8. When OKT3+, OKT4+, or OKT8+ cells were incubated for five days in liquid suspension cultures, their production of GM-CSA and BPA was undetectably low. When 5% autologous monocytes were added to the cultures, no increase in the secretion of either of these classes of growth factors was noted. In the presence of concanavalin A (Con A), measurable quantities of both GM-CSA and BPA were elaborated by all three populations of T cells in the absence of monocytes; however, when autologous monocytes were added to the Con A-stimulated T cells, the secretion of both GM-CSA and BPA was markedly enhanced. In addition, we found that supernates of unfractionated T cells incubated with Con A contained not only GM-CSA and BPA but also a potent inhibitor(s) of BPA that could be demonstrated by dilution of the media and removed by gel filtration. In contrast, no inhibitor of GM-CSA was found. By molecular sieve chromatography of the supernates, GM-CSA and BPA coeluted as a single peak. However, the two biologic activities could be separated on the basis of heat stability, since GM-CSA proved to be heat labile whereas BPA did not. Our data indicate that GM-CSA and BPA derived from human T cells are similar in their apparent molecular weights and in the pattern of their production in suspension cultures in response to lectin stimulation. The secretion of both GM-CSA and BPA by Con A-stimulated T cells is facilitated by the presence of autologous monocytes, and is not restricted to either the OKT4- or the OKT8-defined subset.

Antibodies, Monoclonal↗
PubMed1985

Effect of bacterial endotoxin on the transmembrane electrical potential and plasma membrane fluidity of human monocytes.

In order to gain insight into the physical interaction between bacterial endotoxins and the surface of human monocytes, we investigated the effects of Salmonella typhi endotoxin and lipid A on two functional properties of the plasma membrane of these cells: (1) the transmembrane electrical potential and (2) the fluidity of the lipid bilayer. Using the fluorescent lipophilic cationic probe 3,3'-dipropylthiodicarbocyanine (di-S-C3(5] to monitor the transmembrane electrical potential, we found that neither endotoxin nor lipid A induced depolarization of the monocyte's plasma membrane or impeded its ability to undergo depolarization in response to phorbol myristate acetate. When the resting transmembrane potential of the monocyte was analyzed by exposing di-S-C3(5)-labeled cells suspended in media containing incremental concentrations of potassium ion (K+) to valinomycin, no difference between the response of control cells and cells pretreated with endotoxin was noted. We next examined the effect of endotoxin and lipid A on the fluidity of the monocyte's plasma membrane by monitoring the intensity of the fluorescence of 1,6-diphenyl-1,3,5-hexatriene. By quantifying the intensity of parallel and perpendicular polarized light emitted by this membrane-embedded probe between 8 and 56 degrees C, measurements of molecular anisotropy were used to identify temperature-dependent phase transitions within the hydrocarbon region of the plasma membrane and to estimate the relative microviscosity of the lipid bilayer before and after exposing the cells to endotoxin or lipid A. Although the temperature at which phase transitions occurred was the same in all experimental groups of cells, preincubation of monocytes with either endotoxin or lipid A appeared to increase both the apparent microviscosity of the cell membrane and the order of the lipid bilayer as reflected by a decrease in its flow-activation energy. Our data indicate that when endotoxin molecules contact the surface of the monocyte, the lipid A moiety appears to become incorporated into the plasma membrane, increasing the microviscosity of the lipid bilayer without significantly altering its ionic permeability. We therefore conclude that the metabolic activation of monocytes by endotoxin is not coupled to, or initiated by, membrane depolarization.

Endotoxins↗
PubMed1985

Purification of cartilage-derived growth factor by heparin affinity chromatography.

Cartilage-derived growth factor (CDGF) was found to bind tightly to columns of immobilized heparin and could be eluted with concentrations of salt in the order of 1.6-1.8 M NaCl. The molecular weight of CDGF was estimated to be 18,000-20,000 by high performance liquid-size exclusion chromatography. The affinity of CDGF for heparin greatly facilitated its purification. Highly purified CDGF active at about 1-2 ng/ml was obtained when crude cartilage extract was applied to heparin-Sepharose and the growth factor activity was recycled over heparin-Sepharose two more times. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain visualization of highly purified CDGF showed one major polypeptide band with a molecular weight of about 19,000 containing over 95% of the protein and one minor polypeptide band containing the rest of the protein. Only the Mr 19,000 polypeptide was active after elution from the polyacrylamide gel. Although CDGF bound tightly to immobilized heparin, it did not bind to immobilized chondroitin sulfate or hyaluronic acid. In addition, CDGF bound to heparin much more tightly than did platelet-derived growth factor even though these two growth factors had similar isoelectric points of about 10. These results suggest that the binding of CDGF to heparin was due to a specific affinity of the 2 molecules for each other.

Animals↗
PubMed1985

Accelerated wound repair, cell proliferation, and collagen accumulation are produced by a cartilage-derived growth factor.

Cartilage-derived growth factor (CDGF), a cationic polypeptide of approximately 18,000 mol wt, was prepared from bovine articular cartilage; other sources were bovine and human scapular and costal cartilage. Previous studies have shown that CDGF stimulates the proliferation of cultured mouse fibroblasts as well as chondrocytes and endothelial cells from various sources. In this study, CDGF was shown to stimulate dose-dependently the accumulation of DNA and collagen by rat embryo fibroblasts and a population of fibroblasts derived from granulation tissue. CDGF also stimulated the proliferation of cultured bovine capillary endothelial cells dose-dependently. To evaluate the effects of CDGF in vivo, we implanted polyvinyl alcohol sponges subcutaneously in rats. 6 d postimplantation, sponges were injected with 300 micrograms of partially purified CDGF, a dose which takes into account the cell numbers in the sponges as compared with cell cultures. CDGF rapidly disappeared from the sponges and only approximately 10% of the initial dose was present at 4 h. Despite its transient presence, CDGF caused a relative increase in sponge DNA content of 2.6-fold at 48 h and 2.4-fold at 72 h. We repeated the sponge experiment by using 500-ng injections of CDGF purified to near homogeneity by heparin-Sepharose chromatography. Purified CDGF caused significant increases in sponge collagen, protein, and DNA content at 48 and 72 h after a single injection. The effects of CDGF were abolished by heat and unaffected by reduction of disulfide linkages. Morphologically, CDGF did not evoke an inflammatory response, and its effect on proliferating endothelial cells and fibroblasts was, therefore, probably direct. However, increases in DNA content of sponges could not be fully accounted for by increased DNA synthesis, which suggests that recruitment may be an important component of the in vivo response. Taken together, the effects of CDGF on cultured cells and granulation tissue suggest that the sustained presence of CDGF in vivo may greatly enhance its effects upon wound repair.

Animals↗
PubMed1985

Compliance and policy issues and recommendations related to revision of the National Interim Primary Drinking Water Regulations for Radionuclides.

This paper summarizes the deliberations and conclusions of the Compliance and Policy Committee of the National Workshop for Radioactivity in Drinking Water. Prior to and during the workshop the committee considered a total of 32 possible compliance and policy issues and determined that 22 were valid. The committee developed positions on seven of these and these positions are presented herein. The remaining 25 issues are also listed with the committee's evaluation of each.

Legislation as Topic↗
PubMed1985

Clinical trial of the efficacy and safety of ticarcillin and clavulanic acid.

Forty-three hospitalized patients were treated with a new antibiotic combination containing ticarcillin plus the beta-lactamase inhibitor, clavulanic acid, in a fixed combination for intravenous use. A variety of infections were treated, including pneumonia, bacteremia, urinary tract infection, and osteomyelitis. Of 50 episodes of infection in 43 patients, 44 clinical cures were obtained, with 5 patients improving and 1 patient failing to respond to treatment. In vitro susceptibility testing of 101 clinical isolates was notable for the rarity of resistance to the combination antibiotic. Of specific interest, all 14 isolates of Staphylococcus aureus were susceptible to ticarcillin plus clavulanic acid, whereas only 2 of the 14 isolates were susceptible to ticarcillin alone. Adverse reactions to the study drug were minimal; eosinophilia, unaccompanied by other allergic phenomena, and oral candidiasis were most frequent. Overall, the combination of ticarcillin with the beta-lactamase inhibitor, clavulanic acid, appears to be a safe and effective drug for the treatment of infections caused by susceptible organisms.

Aged↗
PubMed1985

Antimalarial activities and subacute toxicity of RC-12, a 4-amino-substituted pyrocatechol.

RC-12 [1,2-dimethoxy-4-(bis-diethylaminoethyl)-amino-5-bromobenzene] was evaluated for prophylactic, radical curative, and suppressive activities against infections with Plasmodium cynomolgi and subacute toxicity in rhesus monkeys. Applied as a prophylactic agent, RC-12, administered in doses of 6.25 to 25.0 mg/kg daily throughout the incubation period, provided near-complete to complete protection against 10(5) to 10(6) times the minimum infective dose of sporozoites. Applied as a suppressive agent, daily doses of 100.0 mg of RC-12 per kg did not eradicate blood schizonts regularly; hence, the need for concomitant administration of a blood schizonticide, such as chloroquine, in assessments of radical curative activity. In such appraisals, daily doses of 6.25 to 25.0 mg of RC-12 per kg for 14 days, in combination with 2.5 mg of chloroquine per kg daily for 7 days, effected cure of 69 and 93% of established infections, respectively. The curative activity of RC-12 was related to the total dose and could be achieved with a regimen as brief as 4 days. With respect to outward expressions of toxicity, daily doses of 50.0 mg/kg or lower for 15 to 225 days evoked no reactions. Doses of 100.0 or 200.0 mg/kg, scheduled for 15 days, evoked convulsions and depression and were, respectively, lethal to 4 of 17 and 7 of 7 recipients. Doses of 25.0 mg/kg or lower evoked no discrete reactions. Doses of 50.0 mg/kg and higher evoked hepatomegaly, vacuolation of hepatocytes, and elevations of glutamic oxalacetic and glutamic pyruvic transferase activities in serum, reactions related in intensity to dose but not duration of dosage.

Animals↗