Search PubMed⌕ Search

Biomedical subjects

R J Mason

Publications and source records attributed to R J Mason.

At least 217 records · Page 12Linked to original sources
PubMed1977

Isolation and properties of type II alveolar cells from rat lung.

Type II alveolar cells can be isolated and partially purified from adult rat lung by a series of steps that includes enzymatic digestion of the lung with trypsin and separation of cells on a discontinuous albumin density gradient. The yield of the isolated type II cells depends on the supplier and the housing of the rats used to prepare the cells. With specific pathogen-free rats housed in a laminar flow hood, the yield was 20.3 x 10(6) cells per rat, of which 50 per cent were type II cells. With rats from 2 other suppliers and no special housing, the yields were 8.8 and 8.3 x 10(6) cells per rat, of which 67 and 65 per cent were type II cells. The ultrastructural appearance of the isolated cells was similar to that of cells from intact lung, except for some dilatation of the endoplasmic reticulum and the perinuclear space. Most cells (92 +/- 5 per cent) excluded the vital dye, trypan blue. The cells consumed O2 at the rate of 76 +/- 12 nmole per 10(6) cells per hour and released only 5.7 +/- 2.0 per cent of their lactate dehydrogenase, a cytoplasmic enzyme, into the medium after 1 hour of incubation. The isolated type II cells contained disaturated phosphatidylcholine, a major component of purified surface-active material. The cells, however, had a low glucose utilization compared to their O2 consumption, which may indicate an abnormality in the metabolism of glucose. This population of cells could be further purified to 89 per cent type II cells by unit gravity velocity sedimentation.

Animals↗
PubMed1976

Isolation of disaturated phosphatidylcholine with osmium tetroxide.

A simple, rapid, inexpensive method for isolating disaturated phosphatidylcholine from adult rat lung has been devised. Total lipids are reacted with osmium tetroxide dissolved in carbon tetrachloride, and the disaturated phosphatidylcholine is isolated on a column of neutral alumina. More than 99% of the fatty acids in the phosphatidylcholine fraction are saturated and 94% of this material migrates as phosphatidylcholine on subsequent thin-layer chromatography.

Animals↗
PubMed1974

Lipid peroxidation by human blood phagocytes.

Cell suspensions enriched in human blood monocytes, obtained from normal peripheral blood by sedimentation on sodium diatrizoate-Ficoll gradients or from the blood of patients with neutropenia and monocytosis, accumulated malonyldialdehyde, a labile catabolite of lipid peroxidation, during incubations with polystyrene beads or heat-killed Staphylococcus epidermidis. Mixed blood leukocytes principally composed of granulocytes or granulocytes purified by density gradient sedimentation did not accumulate malonyldialdehyde during incubations with these particles, but did when ingesting particles containing linolenate. The phospholipid fatty acid composition of monocyte-enriched and purified granulocyte preparations from the same donors were compared. The molar fraction of arachidonate (20:4) in phospholipids from monocyte-rich preparations was 62% greater than that of purified granulocytes. The findings indicate that human monocytes, possibly because of a greater content of polyunsaturated fatty acids in their membranes, peroxidize a greater quantity of endogenous lipids than granulocytes during endocytosis. Normal human granulocytes have the capacity to peroxidize ingested lipids. However, mixed leukocytes from two patients with chronic granulomatous disease produced little malonyldialdehyde when engulfing linolenate-containing particles. Therefore the capacity to peroxidize lipid is related to cellular oxygen metabolism, a function in which chronic granulomatous disease granulocytes are dificient. Malonyldialdehyde chemically prepared by hydrolysis of tetramethoxypropane, by extraction from peroxidized linolenic acid, or purified from extracts of phagocytizing rabbit alveolar macrophages had bactericidal activity against Escherichia coli and S. epidermidis. Therefore, toxic catabolites of lipid hydroperoxides may potentiate the bactericidal activity of hydrogen peroxide in mononuclear phagocytes.

Aldehydes↗
PubMed1972

Synthesis of dipalmitoyl lecithin by alveolar macrophages.

A reliable, relatively simple method for isolation and quantification of disaturated lecithins is described. In rabbit lung, 34% of the lecithins were disaturated, in alveolar macrophages, 19%. More than 95% of the fatty acids of the disaturated lecithins from lung and alveolar macrophages was palmitic. Hence, the disaturated lecithins from these sources were essentially all dipalmitoyl lecithin. Both heterophils and alveolar macrophages incorporated (14)C-labeled choline and palmitate into disaturated lecithins. Liver slices in which only about 1% of the lecithins were disaturated incorporated very little of these precursors into this fraction. Of the palmitate incorporated in vitro into disaturated lecithins by alveolar macrophages, heterophils, and lung slices, 37% was in the 1 position. In disaturated lecithins isolated from pulmonary lavage fluid, alveolar macrophages, and lung of rabbit 8-12 hr after a single intravenous injection of palmitic-1-(14)C acid, 45% of the (14)C was in position 1. At earlier times, from 20-240 min after injection, the distribution of (14)C was similar in the samples from lung, but in those from alveolar macrophages and lavage fluid, the percentage in position 1 was slightly lower.Glycerol-U-(14)C was incorporated into disaturated lecithins by alveolar macrophages and by lung slices in vitro. Both tissues incorporated very little label from ethanolamine or from methyl-labeled methionine into this fraction. All of the data are consistent with the view that alveolar macrophages synthesize dipalmitoyl lecithin via the cytidine diphosphate-choline pathway.

Animals↗
PubMed1972

Isolation and properties of phagocytic vesicles. II. Alveolar macrophages.

Phagocytic vesicles were obtained by density gradient centrifugation of homogenized rabbit alveolar macrophages that had ingested emulsified paraffin oil contained Oil Red O. The phagocyte vesicles floated and thereby were separated from the soluble fraction and from other cell components which sedimented. The purity of the isolated vesicles was documented by electron microscopy, chemical and enzyme analysis. The vesicles contained 87% of the cell-associated Oil Red O, and were essentially free of DNA, RNA, succinic dehydrogenase, and glucose-6-phosphatase. Acid phosphatase, beta-glucuronidase, and catalase were transferred from the sedimenting fraction to the phagocytic vesicle fraction during phagocytosis, whereas enzyme activities of the soluble fraction remained unchanged. Half of the catalase of resting macrophages was in the pellet fraction and, compared with acid phosphatase, greater amounts of digitonin were required to release full activity. Such differential latency has been described for enzymes of peroxisomes vs. those of lysosomes. Compared with polymorphonuclear leukocyte vesicles studied previously, phagocytic vesicles of macrophages had more electron-dense material and lower Oil Red O:protein, phospholipid:protein, and enzyme:protein ratios. It is thus probable that secondary lysosomes become part of the macrophage vesicle. When paraffin oil particles, the stimulus for phagocytic vesicle formation, were washed away from the macrophages, acquisition of hydrolases by preformed vesicles ceased, i.e. transfer of these enzymes into phagocytic vesicles occurred only during or shortly after the formation of new vesicles. As noted previously by others, the content of acid hydrolases of stimulated alveolar macrophages was doubled in comparison to normal cells. The difference between stimulated and normal macrophages was even more marked when isolated phagocytic vesicles were analyzed. Vesicles from stimulated macrophages had 3-5 times more enzyme activity (per milligram of vesicle protein or per amount of paraffin oil ingested) than did vesicles from normal cells.

Acid Phosphatase↗
PubMed1972

Quantitative studies of phagocytosis by polymorphonuclear leukocytes: use of emulsions to measure the initial rate of phagocytosis.

Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red O emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by N-ethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested. On the other hand emulsions prepared with agents of strong net positive or negative charge were rapidly taken up. The effect of divalent cations on the rate of phagocytosis varied with the nature of the emulsifier, but was not related in any simple, direct fashion to the net surface charge of the particles. However, it has not been conclusively established that charge was the only variable of the emulsion particles employed.

Animals↗
PubMed1972

The appearance of lecithin- 32 P, synthesized from lysolecithin- 32 P, in phagosomes of polymorphonuclear leukocytes.

Rabbit polymorphonuclear leukocytes ingesting paraffin oil particles stabilized with albumin, converted more lysolecithin-(32)P (added to the medium as an albumin complex) to cellular lecithin than did control cells. Almost all of the increment in leukocyte lecithin-(32)P is found in association with the isolated phagocytic vacuoles. About half of lecithin-(32)P of granulocytes incubated first with lysolecithin-(32)P and then reincubated with paraffin particles in a nonradioactive medium is transferred from a sedimentable (presumably membrane) fraction to the phagosomes. Isolated phagosomes or granules by themselves are capable of acylating lysolecithin. The main source of lysolecithin-(32)P for synthesis of cellular lecithin-(32)P, however, appears to be extracellular rather than lysolecithin-(32)P within the cytoplasm or the phagocytic vacuole. We interpret our findings therefore as indicating that lecithin-(32)P in the phagosomes derives chiefly from the outer membrane.

Animals↗
PubMed1972

Lipids of alveolar macrophages, polymorphonuclear leukocytes, and their phagocytic vesicles.

Phagocytic vesicles were isolated from rabbit alveolar macrophages and guinea pig polymorphonuclear leukocytes that had ingested emulsified paraffin oil. Phospholipids and their fatty acids were determined in whole cells and in the phagocytic vesicle and pellet fractions separated from them. The cholesterol-to-phospholipid ratios in the vesicle fractions were distinctly higher than those of the respective whole cells or pellet fractions. The vesicle fractions also had higher phospholipid-to-protein ratios than did the whole cells. The phospholipids of the phagocytic vesicle fraction from macrophages contained relatively more sphingomyelin, lyso-(bis)phosphatidic acid, and phosphatidylserine and less lecithin, phosphatidylethanolamine, and phosphatidylinositol than did the whole cells or pellet fractions. The phospholipids of phagocytic vesicles from polymorphonuclear leukocytes contained significantly more phosphatidylinositol than did the pellet fractions. Lyso(bis)phosphatidic acid, which constituted 15% of the phospholipid in rabbit alveolar macrophages and 25% of that in their phagocytic vesicles, contained almost 60% oleic acid and 20% linoleic acid. This lipid was not detected in rabbit peritoneal macrophages or in rat alveolar macrophages.The polyunsaturated fatty acids of leukocyte phospholipids were chiefly linoleic, whereas in macrophages arachidonic accounted for almost 20% of the total fatty acids. The macrophages produced malondialdehyde when ingesting polystyrene beads or emulsified paraffin oil, from which it was inferred that peroxidation of endogenous lipid can occur during phagocytosis. Polymorphonuclear leukocytes in which less than 3% of phospholipid fatty acids were arachidonic did not produce malondialdehyde during phagocytosis of these inert particles, but did when ingesting an emulsion containing linolenate, thus providing evidence for peroxidation of ingested lipid. Isolated phagocytic vesicles from alveolar macrophages contained lipid peroxides and generated malondialdehyde when incubated with ADP, FeCl(3), and NADH.

Aldehydes↗
PubMed1971

Isolation and properties of phagocytic vesicles from polymorphonuclear leukocytes.

A method for the isolation of intact phagocytic vesicles from guinea pig peritoneal-exudate granulocytes and human peripheral-blood leukocytes is presented. After leukocytes ingested the particles of a stable emulsion of paraffin oil, the uningested emulsion was washed away and the cells were homogenized. The homogenate was placed in the middle of a three-step discontinuous sucrose gradient and centrifuged for 1 hr at 100,000 g. The phagocytic vesicles, containing the low density paraffin-oil particles, were simultaneously washed and collected by floatation, while the other organelles, chiefly granules, sedimented through the lower wash layer, and the particle-free supernatant remained in the middle of the gradient. Emulsion particles stained with Oil Red O were employed to assay the rate of phagocytosis and to mark the location of the particles in subcellular fractions. The dye was extracted from washed cells or cell fractions with dioxane and colorimetrically quantified. The purity of phagocytic vesicles obtained by this method was assessed by electron microscopy, chemical analysis, and assay of enzyme composition. Granule-associated enzymes, acid phosphatase, alkaline phosphatase, beta-glucuronidase, and peroxidase were present in the phagocytic vesicles and originated from the granules. Cyanide-resistant NADH (reduced form of diphosphopyridine nucleotide) oxidase was also found. Enzymes associated with the vesicles exhibited latency to Triton X-100. Uptake of particles and the transfer of total protein and phospholipid into phagocytic vesicles occurred simultaneously Accumulation of acid and alkaline phosphatase in the vesicles continued until phagocytosis ceased. Peroxidase, NADH oxidase, and beta-glucuronidase activities in the phagocytic vesicles, on the other hand, were maximal by 30 min and increased little thereafter even when phagocytosis was still going on.

Acid Phosphatase↗
PubMed1971

The effect of polystyrene beads on cyclic 3',5'-adenosine monophosphate concentration in leukocytes.

After incubation with polystyrene latex beads for 5 min. the cyclic 3',5'-adenosine monophosphate (cyclic AMP) content of human peripheral blood leukocyte suspensions was increased severalfold. Preparations enriched in mononuclear cells and containing only 0-20% polymorphonuclear leukocytes (PMN) and no visible platelets exhibited a quantitatively similar response. Purified fractions of cells containing 85-90% PMN responded to polystyrene beads with a much smaller increase in cyclic AMP content. Phagocytosis of paraffin oil emulsion in the unfractionated mixed human leukocyte preparation was associated with little or no change in cyclic AMP levels. There was no change in cyclic AMP content of rabbit alveolar macrophages or guinea pig PMN during phagocytosis of polystyrene beads. All of these observations are consistent with the view that particle uptake per se does not increase cyclic AMP levels in phagocytic cells. It seems probable that the increase in cyclic AMP concentration that results when unfractionated human blood leukocytes are incubated with polystyrene beads occurs in cells other than PMN.

Animals↗