Decreased superoxide anion radical production by rat alveolar macrophages following inhalation of ozone or nitrogen dioxide

$\text{In vivo}$ exposure of rats to ozone or nitrogen dioxide results in a dose-dependent decrease in superoxide anion radical production (O₂^?·) by alveolar macrophages isolated from the exposed animals. When alveolar macrophages from ozone-exposed animals were stimulated with phorbol myristate acetate (PMA, a non-phagocytic stimulus of O₂^?· production) the decrease in O₂^?· production ranged from 85.9% of control at 3.2 ppm-hrs ozone to 7% of control at 10.5 ppm-hrs. In a similar fashion, O₂^?· production by PMA-stimulated macrophages from NO₂-exposed rates ranged from 78% of control at 18.3 ppm-hrs NO₂ down to 14.5% of control at 51 ppm-hrs. Since the viability of the alveolar macrophages obtained from ozone or nitrogen dioxide-exposed animals was 88% or better in all cases as judged by both Trypan blue exclusion and lactate dehydrogenase release, the decreased ability of these cells to produce superoxide anion radical cannot be attributed to a pollutant effect on cell viability. This diminution in superoxide anion radical production by alveolar macrophages from the pollutant-exposed animals might account, in part, for the ability of these 2 air pollutants to potentiate bacterial infections in laboratory animals.     

Polyadenylation of nascent RNA during the embryogenesis of Ilyanassa obsoleta.

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated $\text{32}\text{64}$ cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

Collagenase from rabbit pulmonary alveolar macrophages.

Rabbit pulmonary alveolar macrophages produce a collagenase which lyses labeled collagen gels, specifically cleaves collagen types I, II and III, is inhibited by ethylenediaminetetraacetate, cysteine, dithiothreitol and serum but is not inhibited by a serine protease inhibitor. Alveolar macrophage collagenase activity can be enhanced by $\text{in vivo}$ BCG activation, $\text{in vitro}$ latex, silica or mycobacterium activation and by $\text{in vitro}$ uncovering of latent enzymatic activity with trypsin treatment. The production of collagenase by unactivated alveolar macrophages and the presence of “latent” collagenase in culture media of alveolar macrophages are examples of significant differences between alveolar and peritoneal macrophages.     

Rat alpha-fetoprotein: in vitro production of four molecular variants by clonal cell lines.

Mass and six clonal cultures derived from Morris hepatoma 7777 by standard tissue culture techniques synthesize and secrete alpha-fetoprotein $\text{in vitro}$. During serial passage, the alpha-fetoprotein which accumulates in the media of these cultures contains two concanavalin A-affinity molecular variants. Each of the concanavalin A-affinity molecular variants shows two electrophoretic variants. Mass and clonal cell populations of hepatoma cells continue to secrete $\text{in vitro}$four molecular variants of rat alpha-fetoprotein known to occur $\text{in vivo}$. These results demonstrate for the first time that individual hepatoma cells have the potential to synthesize four molecular variants of alpha-fetoprotein and that this phenotypic property is maintained during serial subculture $\text{in vitro}$.     

Isolation and purification of the sexual agglutination substance of mating type a cells in Saccharomyces cerevisiae

The substance responsible for the sexual agglutinability was successfully solubilized by a newly established autoclaving method from the surface of mating type $\text{a}$ cells of $\text{Saccharomyces cerevisiae}$ and purified by DEAE cellulose chromatography, gel filtration, affinity chromatography and electrophoresis. The substance was found to consist of at least two different glycoprotein subunits. The molecular weight of the substance was estimated to be about 23,000 daltons by gel filtration. The substance was univalent in its biological activity and specifically masked the sexual agglutinability of the mating type α cells. The substance formed a complementary complex with the agglutination substance from α cells in vitro.     

Monovalent concanavalin A

The preparation of monovalent concanavalin A was achieved by incubating metal-free concanavalin A with trypsin at room temperature for 50 hrs. The digest was subjected to affinity chromatography on a column of ovomucoid-agarose to remove the trypsin and subsequent chromatography on Bio-Gel P-150 to resolve the monovalent fragment. Monovalent concanavalin A bound Mn²⁺, Ca²⁺ and p-nitrophenyl-α-D-mannopyranoside as determined by ultraviolet difference spectroscopy. The modified protein would not precipitate glycogen or agglutinate $\text{Bacillus subtilis}$ cells. The fragment did, however, prevent the agglutination of $\text{B. subtilis}$ by native concanavalin A. Preparation of monovalent concanavalin A could not be achieved unless metals were first removed from the native protein.     

Rodent macrophages metabolize 25-hydroxyvitamin D3in,vitro

Rodent macrophages metabolized 25-hydroxyvitamin D₃ to an unidentified metabolite during $\text{in}\text{,}\text{vitro}$ incubations. The production of this macrophage-derived metabolite of 25-hydroxyvitamin D₃ increased as the substrate concentration was raised. A two step high pressure liquid chromatography system revealed a unique elution position of this macrophage-derived metabolite that did not match the elution positions of any of the vitamin D₃ metabolites available in this laboratory. This unique metabolite was formed $\text{in}\text{,}\text{vitro}$ within one minute by incubated macrophages although its formation increased gradually up to 60 minutes of incubation.     

Dynamics of changes in the surface of normal cultivated rat cells after a single exposure to the carcinogen 7,12-dimethylbenz(a)anthracene]

Changes in the cell surface after a single treatment with 7,12-dimethylbenz(a)anthracene (DMBA) of newborn rat carcass in cell culture have been studied by means of the agglutination reaction with concanavalin A. DMBA was shown to cause alterations in the cell surface. At 0.5 mkg/ml of DMBA, the difference in agglutinability of treated and untreated cells persists for 30 days. At 0.1 mkg/ml of DMBA, the agglutinability of drug-treated and control cells was similar on the 4th day after removal of carcinogen. A prolonged culturing of control cells results in an increased agglutinability of cells with concanavalin A, and in 2.5 months it becomes indistinguishable from the agglutinability level of tumor cells with concanavalin A. In 5 months, drastic karyotypic changes are registered in control cultures.     

Effect of metabolic inhibitors on the agglutination of tumor cells by concanavalin A and Ricinus communis agglutinin

The agglutinations of rat ascites tumor cells by concanavalin A and by $\text{Ricinus communis}$ agglutinin were inhibited by low temperature, 2,4-dinitrophenol and cytochalasin B but not by cycloheximide. These metabolic inhibitors, however, did not inhibit the binding of the agglutinins to the cells. These results suggest that the agglutination was dependent on an active process; probably on a microfilament system responsible for cell surface movement which requires ATP, but not on protein synthesis.     

Polyamines and platelet aggregation

High blood concentrations of the naturally occurring polyamines have been reported in leukemia, psoriasis, cystic fibrosis and polycythemia rubra vera. Spermidine and spermine inhibit $\text{in}$$\text{vitro}$ plate-let aggregation of platelet rich plasma preparations in which ADP and Ristocetin are the agglutinating agents. The proposal is made that these organic cations may modulate $\text{in}$$\text{vivo}$ platelet agglutinability.     

Concanavalin A-mediated agglutinability of Balbc3T3 cells grown in media supplemented with different phosphatidylcholines

$\text{Balb}\text{c3T3}$ cells were grown near confluency in media supplemented with 10% fetal calf serum and than exposed for 24 hours to media containing different phosphatidylcholines bound to delipidated fetal calf serum. Compared to cells grown in regular media, 3T3 cells exposed to media containing dioleoyl-phosphatidylcholine dramatically increased their agglutinability by Concanavalin A. Exposure to several other phosphatidylcholines had no effect.     

Regulation of concanavalin A agglutination by the extracellular matrix

The agglutinability of rat C₆ glioma cells by concanavalin A (Con A) depends upon cell density. From sparse density to near confluency agglutinability increases as cell density rises. Both the half-maximal concentration and the maximum amplitude of agglutination by Con A are functions of cell density, but are separate cell parameters differing in the extent to which they are affected by density and the point at which they become insensitive to further density increases. Both trypsin and EDTA reduce cell agglutinability. The similarity in recovery kinetics between low density cells and cells dissociated with EDTA or trypsin suggests that low density cells may lose the same surface agglutination component(s) removed by trypsin and EDTA. Density-dependent regulation of Con A agglutinability is anchorage dependent; cells grown in suspension display no such phenomenon. The cooperative cell regulation of agglutinability is mediated by the extracellular matrix, or micro-exudate. The matrix contains two activities: low density cultures produce a matrix inhibitor of Con A agglutinability, while high density cultures produce a matrix promotor.     

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37°C to 22°C with an activation energy of $\text{11.8 ± 0.2}\text{kcal/mol}$ in this region. There is a sharp decrease in agglutination rate below 22°C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 $\text{(2,2-}\text{dimethyl-5-dodecyl-5-methyloxazolidine}\text{-N-}\text{oxide}$) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.     

Glutaraldehyde fixation and the mechanism of erythrocyte agglutination by concanavalin a and soybean agglutinin

To evaluate the importance of lectin receptor mobility and clustering for enhanced cell agglutinability, the effect of glutaraldehyde fixation on the agglutinability of human erythrocytes by concanavalin A and soybean agglutinin was investigated. Agglutinability was evaluated in unperturbed Microtiter^® plates. Fixation increased slightly the agglutinability of the erythrocytes by both lectins. Fixation did not alter trypsin-enhanced agglutinability. Furthermore, when fixed erythrocytes were trypsinized, their agglutinability increased to the level of unfixed, trypsinized erythrocytes.The kinetics of agglutination of fixed and unfixed erythrocytes were monitored in an electronic particle counter. The shear forces associated with the kinetic experiments diminished fixed-cell to fixed-cell agglutination, i.e., both lectins gave slower kinetics of agglutination with fixed erythrocytes than with unfixed erythrocytes. In contrast, the kinetics of concanavalin A-mediated agglutination of trypsinized-fixed erythrocytes mixed with equal numbers of trypsinized-unfixed erythrocytes were indistinguishable from the rapid kinetics of agglutination of trypsinizedunfixed erythrocytes alone. Light microscopy revealed aggregates composed of fixed and unfixed erythrocytes.We conclude that glutaraldehyde fixation does not diminish the agglutinability of human erythrocytes under low-shear conditions. Our results indicate that the enhanced agglutination of trypsinized erythrocytes is not dependent on clustering of lectin receptors. The disruption of agglutination of fixed erythrocytes by shear forces that do not disrupt agglutination of fixed erythrocytes with unfixed erythrocytes suggests that the rigidity of the fixed erythrocyte may prevent stable aggregate formation by fixed erythrocytes alone.     

A quantitative assay for concanavalin A- and Ricinus communis agglutinin-mediated agglutinations of rat ascites hepatoma cells: Relationship between concanavalin a binding and cell agglutination

A simple quantitative assay method was developed for the agglutination of rat ascites hepatoma cells mediated by Concanavalin A or Ricinus communis agglutinin. This method was based on the principle that the turbidity of a cell suspension is proportional to the sum of the cross-sectional area of cells and aggregates. As predicted by the theoretical consideration, the turbidity decreased when cells were aggregated and the decrease was a function of the average number of the cells in aggregates.The agglutinability of the cells, judged by this method, showed a maximum value at a certain concentration of the agglutinin. By further addition of the agglutinin, the agglutinability slightly decreased from the maximum. These phenomena were observed both for Concanavalin A and Ricinus communis agglutinin. The binding and the agglutination experiments using [³H] concanavalin A revealed that the binding to approx. 20% of the total receptors caused a maximal agglutination. This suggested that the receptors responsible for the agglutination constitute only a small part of the total receptors on the surface.     

Postinhibitory overshoot of insulin release by gastric inhibitory polypeptide

Following intravenous infusion of somatostatin $\text{in vivo}$ occasionally there is a large rebound overshoot of insulin release. An $\text{in vitro}$ model to simulate this phenomenon was made by perfusing rat pancreas with gastric inhibitory polypeptide (GIP) during simultaneous perfusion with somatostatin. Adding GIP (100 ng/ml) to the perfusate for 2 minutes beginning either 3 or 9 minutes before terminating the somatostatin perfusion produced a large overshoot in insulin release. The magnitude of overshoot was greater when medium contained 300 mg/dl glucose that when it contained 150 mg/dl glucose. Perfusion with GIP for 2 minutes beginning 9 minutes before increasing the glucose concentration of the medium from 30 to 300 mg/dl elicited a large increase in both the acute and second-phase release of insulin. These suggest that post-inhibitory overshoot of insulin release after somatostatin may be produces $\text{in vitro}$ by the suppressed action of stimulatory hormones such as GIP. Prior infusion with GIP can also potentiate glucose-stimulated insulin increase.     

Effect of metabolic state on agglutination of human erythrocytes by concanavalin

Intact freshly drawn or stored human erythrocytes, which show little agglutination by concanavalin A, become agglutinable by this lectin in the presence of adenosine. α-Methylglucose (10 mM) completely inhibits this agglutination. The concanavalin A agglutination shows no sensitivity to vinblastine or cytochalasin B.Resealed membranes prepared with ATP in lysing and resealing medium give modest agglutinability, while the presence of adenosine in both the lysing and the resealing medium results in a substantial agglutinability of the resealed membranes.Mild trypsin treatment of the erythrocytes causes an enhanced sensitivity to adenosine activation of the concanavalin A agglutination, while extensive trypsin treatment produced highly agglutinable erythrocytes that show no response to the presence of adenosine in the lectin solution. The extensively treated erythrocytes also show concanavalin A agglutination at temperatures below 37°C, under conditions in which intact or moderately treated erythrocytes do not agglutinate, with or without adenosine present.Results suggest that the adenosine activation of concanavalin A agglutination of intact human erythrocytes is mediated through a metabolic conversion of adenosine to a rapidly turned over metabolite which participates directly in the activation of agglutination. The agglutinability does not appear to depend on whole cell ATP levels, but may involve a particular pool of ATP.The effect of variation of cellular metabolic state and the response of particular systems involved in lectin-mediated agglutinability to cellular metabolism seem to be worth consideration in explaining the frequently large differences in agglutinability of und in cells indifferent biological states, such as those encountered in normal and transformed cells.     

The release of phospholipase A2 from aggregated platelets and stimulated macrophages of sheep

Phospholipase A₂ release from platelets and macrophages was assayed by means of a modified, rapid bioassay based on the hydrolysis of $\text{E. coli}$ membranes. Enzyme activity was found in the supernatants of both washed, aggregated platelets and concanavalin A stimulated macrophages from afferent lymph of sheep. Phospholipase A₂ may be related to the vasoactive moiety secreted by antigen or mitogen stimulated macrophages.     

A lysosomal factor that interconverts multiple forms of rat liver tyrosine aminotransferase.

A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase $\text{in}$$\text{vitro}$ with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated $\text{in}$$\text{vitro}$ by 2.5 mM MgCl₂ and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to $\text{in}$$\text{vitro}$ interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of $\text{in}$$\text{vitro}$ manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.     

Effect of metabolic state on agglutination of human erythrocytes by concanavalin A.

Intact freshly drawn or stored human erythrocytes, which show little agglutination by concanavalin A, become agglutinable by this lectin in the presence of adenosine. alpha-Methylglucose (10 mM) completely inhibits this agglutination. The concanavalin A agglutination shows no sensitivity to vinblastine or cytochalasin B. Resealed membranes preparaed with ATP in lysing and resealing medium give modest agglutinability, while the presence of adenosine in both the lysing and the resealing medium results in a substantial agglutinability of the resealed membranes. Mild trypsin treatment of the erythrocytes causes an enhanced sensitivity to adenosine activation of the concanavalin A agglutination, while extensive trypsin treatment produced highly agglutinable erythrocytes that shown no response to the presence of adenosine in the lectin solution. The extensively treated erythrocytes also show concanavalin A agglutination at temperatures below 37 degrees C, under conditions in which intact or moderately treated erythrocytes do not agglutinate, with or without adenosine present. Results suggest that the adenosine activation of concanavalin A agglutination of intact human erythrocytes is mediated through a metabolic conversion of adenosine to a rapidly turned over metabolite which participates directly in the activation of agglutination. The agglutinability does not appear to depend on whole cell ATP levels, but may involve a particular pool of ATP. The effect of variation of cellular metabolic state and the response of particular systems involved in lectin-mediated agglutinability to cellular metabolism seem to be worth consideration in explaining the frequently large differences in agglutinability of und in cells in different biological states, such as those encountered in normal and transformed cells.