Search PubMed⌕ Search

Biomedical subjects

E Pick

Publications and source records attributed to E Pick.

At least 73 records · Page 4Linked to original sources
PubMed1981

Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader.

Two simple semiautomated microassays for the measurement of superoxide (O-2) and hydrogen peroxide (H2O2) production by cultured macrophages (MPs) are described. The measurement of O-2 is based on the reduction of ferricytochrome c as assayed by the increase in its absorbance at 550 nm. Quantitation of H2O2 is based on the horseradish peroxidase (HRPO)-dependent oxidation of phenol red which is assayed by its increased absorbance at 600 nm. MPs are cultured in monolayers in 96-well flat-bottom tissue culture plates and covered with 100 mul amounts per well of either a ferricytochrome c solution containing phenol red and HRPO. Following the addition of an agent eliciting an oxidative burst (OB) and incubation of the plates at 37 degrees C for various time intervals, the changes in the absorbance of ferricytochrome c and phenol red, respectively, are measured directly in the wells of the tissue culture plates with the cells in situ, by using an automatic 8-channel photometer which reads absorbances vertically through individual wells. This instrument, which was originally designed for reading enzyme immunoassays in microtitration plates, can be easily adapted for use in the above test, when fitted with interference filters with wave lengths of 550 nm (for the assay of O-2) and 600 nm (for the assay of H2O2). The principal advantages of this techniques are: the ability to perform the assays directly in the culture plates with cells in situ; the small amounts of cells and reagents needed; its sensitivity and reproducibility; the ease with which kinetic experiments can be done; the large number of samples which can be tested in parallel, and especially the speed and convenience offered by the automated reading and printout of absorbance values.

Animals↗
PubMed1980

A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture.

A simple, rapid and inexpensive method for the measurement of hydrogen peroxide (H2O2) produced by cells in culture is described. The assay is based on the horseradish peroxidase (HRPO)-mediated oxidation of phenol red by H2O2 which results in the formation of a compound demonstrating increased absorbance at 610 nm. A linear relationship between absorbance at 610 nm and concentration of H2O2 was found in the 1--60 microM (1--60 nmoles/ml) range. Due to the non-toxic character of phenol red and HRPO, the assay permits measurement of H2O2 production and release by macrophages for time intervals of 5--60 min under regular tissue culture conditions. Using this assay, the ability of a number of agents to induce H2O2 release by guinea pig peritoneal macrophages was demonstrated. These agents were: phorbol myristate acetate (PMA), opsonized zymosan, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and A23187.

Animals↗
PubMed1979

The mechanism of action of soluble lymphocyte mediators. VI. Effect of migration inhibitory factor (MIF) on macrophage microtubules.

The effect of migration inhibitory factor (MIF) on macrophage microtubules was examined by functional, biochemical and morphological methods. It was found that: (1) the microtubule-stabilizing agent deuterium oxide (D2O) inhibits spontaneous macrophage migration from capillaries and enhances the migration blocking effect of MIF; (2) MIF does not modify the amount of total tubulin in macrophages, as determined by an 3H-colchicine binding assay, but increases significantly the proportion of tubulin present in polymeric form; (3) macrophages exposed to MIF and examined by immunofluorescence with specific antitubulin antibody demonstrate a striking increase in the percentage of cells with a well-organized microtubular network, characterized by a high density of thick fibrils and prominent paranuclear microtubule organizing centers. It is concluded that MIF inhibits cellular motility by inducing the formation of numerous microtubules, probably by enhancing tubulin polymerization.

Animals↗
PubMed1979

Antigen and mitogen induced production of macrophage migration inhibitory factor in the mouse.

Spleen cells of C57B1/6J mice immunized with complete Freund's adjuvant produced macrophage migration inhibitory factor (MIF) when incubated in vitro with tuberculin purified protein derivative (PPD). For optimal MIF production spleen cells were cultured for 48 h in a serum-free medium, at a concentration of 2 x 10(7) cells/ml. MIF was assayed in a xenogenic system, using oil-induced guinea pig peritoneal exudate cells as targets. MIF synthesis could also be induced by pulsing spleen cells for 2 h with concanavalin A, phytohemagglutinin, pokeweed mitogen or lipopolysaccharide, followed by culture in plain medium. No MIF secretion was induced by incubation of spleen cells with anti-theta or rabbit anti-mouse IgG sera. Cells producing MIF in response to PPD were characterized as B cells by virtue of being insensitive to anti-theta serum and complement, by being retained on nylon wool, glass bead and anti-Ig colums and by the presence of Fc receptors. PPD-stimulated T cells did not produce MIF. PPD-induced mouse spleen cell MIF demonstrated a moderate loss of activity by heating at 56 and 80 degrees C and was completely inactivated after digestion with chymotrypsin. By fractionation on Sephadex G-200, migration inhibitory activity was recovered in a molecular range of 100,000-12,400 daltons.

Animals↗
PubMed1979

Studies on guinea-pig macrophage migration inhibitory factor (MIF). I. Glycoprotein nature and net charge.

Guinea-pig macrophage migration inhibitory factor (MIF), obtained by the stimulation of sensitized lymph node cells with tuberculin PPD, was characterized as a glycoprotein by the following criteria: (a) its activity is destroyed by 0.02 M sodium periodate; (b) when MIF-containing culture fluids are subjected to precipitation by perchloric acid (final concentration 1 M), the inhibitory activity is recovered in the supernatant; and (c) MIF binds to Sepharose-linked concanavalin A and can be eluted with methyl-alpha-D-glucopyranoside. When MIF-containing culture supernatants are fractionated by isoelectrofocussing, migration inhibitory activity is recovered in a fraction with an isoelectric point of 4.4--4.6.

Animals↗
PubMed1979

Studies on guinea-pig macrophage migration inhibitory factor (MIF). II. Purification of MIF after treatment with reducing and denaturing agents.

Treatment of guinea-pig macrophage migration inhibitory factor (MIF) containing culture supernatants with the denaturing agents guanidine hydrochloride (Gu HCl) or urea, in the presence or absence of the reducing agent 2-mercaptoethanol (2-ME), or with sodium dodecyl sulphate (SDS), does not destroy biological activity. Alkylation of reduced MIF preparations results in a considerable decrease or total loss of MIF activity. Treatment of supernatants with the combinations, Gu HCl and 2-ME or urea and 2-ME results in the recovery of MIF activity in association with molecules less than 30,000 in molecular weight (mol. wt). After removal of the agents by dialysis, MIF activity is found associated with molecules larger than 30,000. The reduction in mol. wt is dependent on the presence of 2-ME. When MIF-containing supernatants are treated with urea and SDS and fractionated by preparative polyacrylamide gel electrophoresis (PAGE) in the presence of the same agents, MIF activity is recovered in the mol. wt range of 42,000--80,000. When supernatants are treated with 2-me, in addition to urea and SDS, and preparative PAGE is performed in their presence, MIF activity is found associated with material having a mol. wt of 15,000--18,000. Analytical SDS-PAGE of this fraction reveals two or three closely grouped bands corresponding to the above mol. wt range.

Acrylonitrile↗
PubMed1978

Enhancement of macrophage adenylate cyclase by microtubule disrupting drugs.

The accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in guinea-pig macrophages exposed to the adenylate cyclase (AC) stimulators prostaglandin E1 (PGE1) and isoproterenol (IP), was markedly enhanced by pretreatment of the cells with colchicine, vinblastine, and podophyllotoxin--agents which prevent microtubule assembly. The same agents did not augment basal cAMP levels. The facilitating effect of the drugs on the response to PGE1 and IP developed both in the absence and presence of a phosphodiesterase (PDE) inhibitor. The same drugs also enhanced the accumulation of cAMP induced by cholera toxin (CT) but the presence of a PDE inhibitor was required for such enhancement to become evident. Pretreatment of macrophages with cytochalasin B, an agent interfering with microfilament function, had no effect on the responsiveness of the cells to AC stimulators. The microtubule stabilizer, deuterium oxide (D2O) partially reversed the colchicine effect. Microtubule disrupting drugs did not block the release of cAMP from the cells into the surrounding medium. Macrophages incubated as monolayers or in suspension showed the same degree of increased responsiveness to stimulators after preexposure to colchicine. Preincubation with the ionophore A23187, which elevates the intracellular concentration of Ca2+, also enhanced the stimulation of AC by PGE1 and IP. Microtubule disrupting agents did not potentiate AC activity in broken cell preparations, whether added to the intact cells before disruption or directly to the enzyme assay mixture, nor did they affect PDE activity of macrophage sonicates. Moderate enhancement of PGE1-induced cAMP formation was also seen in colchicine- and vinblastine-treated lymphocytes. It was concluded that microtubules control the activity of AC by restricting the mobility of membrane receptors. Disruption of microtubules by drugs results in the removal of such restraints and an augmented chance of productive interactions between receptors and catalytic units of AC.

Adenylyl Cyclases↗