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Metabolites of arachidonic acid (AA) released into bronchoalveolar lavage fluid of animals exposed to hyperoxia have previously been implicated as mediators of pulmonary oxygen toxicity. The alveolar macrophage (AM) represents an important potential source of these eicosanoids. We have therefore investigated the effects of in vitro hyperoxia (95% O2/5% CO2) versus normoxia (95% air/5% CO2) on the metabolism of AA in the AM of the rat. Exposure to 95% O2 for up to 72 h did not impair the viability or affect the protein content of cultured AMs. Hyperoxia for 24 to 72 h increased the accumulation of free AA liberated from endogenous stores in cultures of resting AMs. Despite this increase in free AA, no changes in synthesis of thromboxane B2, prostaglandin (PG) E2, PGF2 alpha, leukotriene (LT) B4, or LTC4 were observed in resting AMs exposed to hyperoxia for up to 72 h. This was not due to degradation of eicosanoids in hyperoxia. However, formation of cyclooxygenase metabolites from exogenously supplied AA was reduced in hyperoxia-incubated AMs, suggesting that hyperoxia inhibited the cyclooxygenase enzyme. In AMs stimulated with calcium ionophore A23187, both AA release and synthesis of cyclooxygenase and lipoxygenase eicosanoids were augmented after incubation in hyperoxia for 24 to 72 h. The increase in A23187-stimulated LTB4 synthesis caused by hyperoxia was inhibited by the antioxidants catalase, superoxide dismutase, and the intracellular cysteine loading agent L-2-oxothiazolidine-4-carboxylic acid, suggesting that the augmentation by hyperoxia of A23187-induced AA metabolism was mediated by reactive oxygen metabolites. Thus, hyperoxia has complex effects on AA metabolism in the AM, which include the ability to augment the release of AA and formation of bioactive eicosanoids. These findings support a possible role for eicosanoid synthesis by the AM in the pathogenesis of oxygen toxicity of the lung.
Am J Respir Cell Mol Biol 1990 Jan
PMID:Complex effects of in vitro hyperoxia on alveolar macrophage arachidonic acid metabolism. 215 14

Carbon tetrachloride and bromotrichloromethane are both metabolized by cytochrome P-450 in the presence of phenyl-N-t-butyl nitrone PBN) to the PBN/trichloromethyl (PBN/.CCl3) and the PBN carbon dioxide anion (PBN/.CO2-) radical adducts in the liver. The formation of the latter but not the former species in perfused liver was reduced markedly by prior depletion of hepatic glutathione with either diethyl maleate or buthionine sulfoximine treatments. In microsomal incubations, the PBN/.CO2- radical adduct was detected only upon the addition of cytosol. In microsomal incubations containing PBN, CCl4, and GSH, but no added cytosol, a novel radical adduct with distinctive coupling constants was detected. This radical adduct's ESR spectrum exhibited 13C isotope effects when it was formed in an incubation containing 13CCl4 or Br13CCl3. The presence of GSH in the radical adduct is postulated based on the radical adduct's hydrophilicity and slow rate of rotation in solution. The detection of this new radical adduct, PBN/[GSH-.CCl3], establishes the reaction of GSH with a CCl4-derived free radical as a significant event in the metabolism of CBrCl3 and CCl4. The cytosolic conversion of PBN/[GSH-.CCl3] into PBN/.CO2- has been demonstrated and characterizes the PBN/.CO2- radical adduct as the product of metabolism of PBN/[GSH-.CCl3], a primary radical adduct. Thus, it is concluded that GSH rather than oxygen is obligatory for the formation of PBN/.CO2- from .CCl3 in intact cells.
Mol Pharmacol 1990 Mar
PMID:Reaction of glutathione with a free radical metabolite of carbon tetrachloride. 215 56

Migration of epithelial cells to cover areas of injury is thought to be important in the repair process following airway insult. Insulin is reported to be a growth factor for bronchial epithelial cells, and growth factors have been known to be chemotactic for many types of cells. Thus, we hypothesized that insulin may be a chemoattractant for bronchial epithelial cells. To evaluate this, we prepared bronchial epithelial cells and measured their chemotactic activity toward insulin. Bronchial epithelial cells were isolated by overnight digestion with bacterial protease, filtered through 100-microns nitex mesh, and then cultured at 1 x 10(6) cells/ml in tissue culture dishes in medium 199 supplemented with transferrin, insulin, epidermal growth factor, hydrocortisone, antibiotics, and 10% FCS for 3 d. The cultured cells were rinsed twice to remove supplements, trypsinized and resuspended at 1 x 10(6) cells/ml in medium 199 without supplements, and used as the cell source for chemotaxis. Chemotactic activity of bronchial epithelial cells was measured by the blindwell chamber technique using 8-microns Nuclepore filter membranes coated with 0.1% gelatin. The cells were added to the top wells in a 48-multiwell chamber with insulin in the bottom wells and incubated for 6 h at 37 degrees C, 5% CO2. Bronchial epithelial cells migrated in response to insulin in a dose-dependent manner up to an optimal dose of insulin, 100 micrograms/ml, and decreased at higher concentrations. The number of migrated cells per 10 high power fields was 33.7 +/- 1.9 at the optimum and 3.7 +/- 0.7 without insulin (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1990 Jun
PMID:Bronchial epithelial cells respond to insulin and insulin-like growth factor-I as a chemoattractant. 218 58

Androgen action is largely determined by the formation of dihydrotestosterone in target tissues. In women, androstenedione is the major precursor of dihydrotestosterone production in female genital skin. The present study was initiated to determine whether androstenedione is converted to dihydrotestosterone primarily via testosterone or 5 alpha-androstane-3,17-dione (5 alpha-androstanedione), and to examine the pathway of androstenedione metabolism in genital skin. Genital skin was obtained from 9 normal premenopausal women and 2 normal men. Each tissue was incubated with [3H]androstenedione in RPMI-1640 medium for 1 h at 37 degrees C in 95% O2/5% CO2. The metabolites were separated and purified by paper partition and thin-layer chromatography. The conversions of androstenedione to 5 alpha-androstanedione and to androsterone were similar (10.45 +/- 1.46 and 11.04 +/- 2.04%/200 mg tissue), and were approx. 12, 8 and 23 times higher than the conversion of androstenedione to testosterone, dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol, respectively. The male samples showed a similar pattern of metabolism. These data indicate that 5 alpha-androstanedione is the most important intermediate in the conversion of androstenedione to dihydrotestosterone. The data also confirm the importance of 5 alpha-reductase activity over that of 17 beta-hydroxysteroid oxidoreductase activity in the expression of androgen action in women.
J Steroid Biochem Mol Biol 1990 Sep
PMID:Androstenedione is an important precursor of dihydrotestosterone in the genital skin of women and is metabolized via 5 alpha-androstanedione. 224 46

The first six normal stages of cleavage and blastocyst formation in vitro of the brown antechinus and the stripe-faced dunnart are described based on 429 embryos from 37 dunnarts and 143 embryos from 32 antechinus and an information from previous studies. Embryos were cultured in Dulbecco's modified Eagle's medium with high glucose and 10% foetal calf serum at 35 degrees C and 5% CO2 in air. Seven antechinus and 18 dunnart embryos were cultured in Rose chambers and filmed by time-lapse cinematography, with an exposure of 0.2 sec and at a rate of 1 frame/30 sec. A rim of zona was formed around the yolk mass during early cleavage. Blastomeres attached to this rim, ensuring that the zona in the hemisphere containing the yolk mass was the first to be lined by blastomeres during blastocyst formation, which is completed at about the 32-cell stage. Two foci were established during cleavage. The descendants of the first cell to divide at the four-cell stage included, at both the fourth and fifth division, the first cell to divide and the cell with the shortest cell cycle time and had the shortest average cell cycle time at each division. The descendants of the cell opposite the first cell to divide at the four-cell stage included, at both the fourth and fifth divisions, the last cell to divide and the cell with the longest cell cycle time and had the longest average cell cycle time at each division.
Mol Reprod Dev 1990 May
PMID:Time-lapse analysis and normal stages of development of cleavage and blastocyst formation in the marsupials the brown antechinus and the stripe-faced dunnart. 234 46

To investigate the role of cellular fatty acid content on the susceptibility of airway epithelial cells to hyperoxic injury, monolayer cultures of rabbit tracheal epithelial (TE) cells were grown to confluence in serum-free media with or without a commercial mixture of cholesterol esters and phospholipid-rich lipoproteins (Excyte III, Miles-Pentex, Kankakee, IL) in conjunction with arachidonic acid complexed to BSA. Monolayer cultures were then exposed to control (5% CO2/air) or hyperoxic atmospheres (95% oxygen/5% CO2) for 2 h using an in vitro system in which cells were maintained at a gas-liquid interface analogous to in vivo conditions. Hyperoxic injury was assessed by cell viability (trypan blue exclusion) and by the generation of lipid peroxides measured as thiobarbituric acid (TBA) reactive substances. Changes in TE cell and cell culture effluent fatty acid content induced by exposure to control or hyperoxic atmospheres were analyzed by gas chromatography. TE cells grown in lipid-unsupplemented media had fatty acid profiles characteristic of essential fatty acid deficiency, whereas the fatty acid content of lipid-supplemented TE cells more closely resembled those of acutely recovered TE cells. Lipid-unsupplemented cells were more susceptible to hyperoxic injury as demonstrated by decreased viability and increased production of TBA-reactive substances compared to cells maintained in lipid-supplemented media. In both lipid-supplemented and unsupplemented cells, hyperoxic exposure was associated with a decreased relative cellular content of the monounsaturated and polyunsaturated fatty acids (PUFA) and an increased content of saturated fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1990 Aug
PMID:Effect of fatty acid profiles on the susceptibility of cultured rabbit tracheal epithelial cells to hyperoxic injury. 237 48

We have used tow previously characterized models of hyperpnea in vivo and different modes of ventilation in the isolated perfused rat lung to investigate changes in the phospholipid content of tubular myelin-rich (PLalv-1) and -poor (PLalv-2) fractions isolated from lavaged material and of two lamellar body subfractions. A vesicular lamellar body subfraction (lbB) was preferentially released during 30-min swimming. However, during the subsequent 3-h recovery period there was a preferential supplementation of the classic-appearing lamellar body fraction (lbA). Hyperpnea induced by exposure to 5% CO2/13% O2/82% N2 led to an increase in lbA after 12 h and in lbB after 48 h. Whereas PLalv-2 was elevated above control values after 8 h, PLalv-1 remained unchanged until 24 h. In the perfused lung isolated from rats infused with [methyl-3H]choline chloride 3 h previously, salbutamol, a deep breath, and increased tidal volume (VT) all increased total alveolar phospholipids; however, the pattern of change was very different. Salbutamol markedly elevated PLalv-1 and increased the specific activity of both alveolar fractions. In contrast, a single deep breath increased PLalv-2 while slightly increasing the specific activity of PLalv-1. Finally, an increased VT decreased PLalv-1 while inducing a large increase in PLalv-2; it increased specific activity in both alveolar fractions. Both salbutamol and an increased VT decreased phospholipids in lbA. We conclude that lbA and lbB vary in their response to different stimuli. In vivo, PLalv-1, the tubular myelin-rich fraction, remains very constant, a fact consistent with its being the controlled variable in surfactant homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1990 Sep
PMID:Effect of pattern of breathing on subfractions of surfactant in tissue and alveolar compartments of the adult rat lung. 239 Feb 66

A culture vessel consisting of two independent chambers separated only by the growth substrate is described. Cells may be cultured on both sides of the growth substrate. Culture medium and gas exposure can independently be controlled in both compartments. Human hair follicles have been used as source of keratinocytes and the bovine eye lens capsule has been explored as growth substrate. The presence of 5% CO2 in air in the lower compartment appears to have a significant effect on the morphology of the cultures. When the cultures are being exposed to air with 5% CO2, the culture medium being applied in the lower compartment, formation of corneocytes characteristic for adult stratum corneum is induced, as evidenced by light and electron microscopy. To the knowledge of the authors, this stage of differentiation in vitro has not been obtained with previously described systems. Differentiation of the lower cell layers has been characterised with specific antibodies. The possible use of the system for applied and pure scientific research is discussed.
Mol Biol Rep 1985 Oct
PMID:Differentiation of keratinocytes in vitro: a new culture vessel mimicking the in vivo situation. 241 9

The capsule of Bacillus anthracis is an important virulence factor consisting of poly-D-glutamic acid. The genetic region required for the encapsulation was cloned in Escherichia coli from the capsule plasmid pTE702, using a selection procedure based on an immunodiffusion assay. The cloned region directed synthesis of the capsule both in E. coli and B. anthracis. Capsule synthesis from these clones, as in the wild type, was dependent upon the presence of CO2. However, encapsulation directed by the cloned fragment was less marked than from pTE702. Another region enhancing capsulation was shown to exist on pTE702. The minimum size of the encapsulation region was defined to within 2.7 kb DNA and shown to be essential for the encapsulation in B. anthracis.
Mol Microbiol 1988 May
PMID:Cloning and CO2-dependent expression of the genetic region for encapsulation from Bacillus anthracis. 245 47

The products released by Leishmania major promastigotes incubated with [1-13C]glucose as sole exogenous carbon source were identified using nuclear magnetic resonance (NMR). Under aerobic (95% O2/5% CO2) conditions, acetate, succinate, and small amounts of pyruvate, D-lactate, and glycerol were released in addition to CO2. Under anaerobic (95% N2/5% CO2) conditions, the relative amounts of products formed changed and alanine was also released. The changes in the rates of glucose consumption and product formation during the aerobic to anaerobic transition were measured. Under hypoxic conditions (O2 less than 0.2%), glucose consumption was decreased by about 50%. Under completely anaerobic conditions (100% N2), glucose consumption almost ceased (a total reverse Pasteur effect). The inclusion of 5% CO2 in the gas phase restored hypoxic and anaerobic glucose consumption to the aerobic rate, and increased production of succinate, pyruvate, and D-lactate. Thus, CO2 and very low concentrations of O2 have strong regulatory effects on L. major glucose metabolism. A quantitative carbon balance showed that the NMR-identified products accounted for only about 25% of the glucose carbons consumed under aerobic conditions. CO2, measured as the release of 14CO2 from [U-14C]glucose, accounted for an additional 25% of the glucose consumed. About 11% of the glucose carbon was incorporated into trichloroacetic acid-insoluble products, mostly lipid. Large amounts of label from [U-14C]glucose were incorporated into the intracellular pools of alanine, glutamate, glutamine, and aspartate, indicating that CO2 from unlabeled amino acids contributed to the carbon balance. Under anaerobic conditions, all the glucose carbons consumed could be accounted for solely by the NMR-identified products.
Mol Biochem Parasitol 1989 Mar 01
PMID:Carbon dioxide abolishes the reverse Pasteur effect in Leishmania major promastigotes. 249 56


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