Airway constrictor effects of leukotriene D4 in dogs with hyperreactive airways

Leukotriene D₄ (5 μg/ml) aerosol constricts airways of dogs with nonspecific airway hyperreactivity but not of mongrel dogs which lack nonspecific airway hyperreactivity. R_L increased 200 + 25% and C_dyn decreased to 77 ± 5% of the pre-challenge value. LTD₄ (10 μg/ml) produced no further increase. Atropine (0.2 mg/kg) prevented the increase in R_L and decrease in C_dyn, suggesting that part of the effect of LTD₄ on airways is neurally mediated.     

Precursor frequency of antigen-specific T cells: effects of sensitization in vivo and in vitro

Limiting dilution analysis (LDA) of primary lymphocyte cultures was used to determine the frequency of keyhole limpet hemocyanin (KLH)-specific precursors in the peripheral blood of unimmunized individuals. The KLH-specific precursor frequencies ranged from 1:150,000 to 1:340,000. In contrast, frequencies of KLH-specific cells in the blood from immune donors ranged from 1:25,000 to 1:42,000. LDA of KLH-stimulated primary cultures indicated that the frequency of KLH-specific cells increased with time in culture reaching a four- to fivefold expansion relative to the frequency obtained prior to culture. The data presented suggest that the enhanced kinetics of secondary T-cell responses observed after in vitro sensitization are due to a decrease in the proportion of lymphocytes which exhibit a suppressor phenotype.     

The role of divalent cations in interactions between lymphokines and macrophages

The divalent cation requirements of lymphokine-mediated alterations in macrophage function (activation and inhibition of migration) were examined. Normal rabbit alveolar macrophages exposed to incubation supernatants of antigen-stimulated sensitized lymphocytes (lymphokine) were activated, manifested by increased adherence and enhanced bactericidal activity, as compared with control cells. This lymphokine-mediated activation was dependent upon the presence of extracellular Mg²⁺ (but not Ca²⁺). Our data from both current and previous studies suggest that Mg²⁺ influx is necessary for initiation or support of the macrophage activation process. The divalent cation requirements for lymphokine (MIF)-induced inhibition of macrophage migration differed from that of the activation phenomenon. Specifically, both Ca²⁺ and Mg²⁺ were required for expression of MIF activity. Adsorption experiments indicate that these cations are needed for binding of MIF to the macrophage surface.     

Crenation and cupping of the red cell: A new theoretical approach. Part I. Crenation

Crenation can be thought of as a surface instability caused by intrinsic precurvature of the membrane. Mathematical modeling, on the presupposition that the red blood cell is a thin shell consisting of a connected (coupled) bilayer having uniformly distributed elastic properties shows that crenation can be initiated by negative precurvature, that is, intrinsic curvature having its concavity directed towards the outside of the cell. This is contrary to the currently accepted view which attributes the effect to positive precurvature of an unconnected bilayer. Crenation and the biconcave shape can coexist in the red cell. This suggests that the bilayer must be connected even when the cell is crenated because the biconcave shape could not otherwise be maintained. The progressive development of crenation to more advanced stages, such as the echinocyte type III and the spheroechinocyte can be accounted for if the outer layer of the membrane is stressed beyond the range where strain is proportional to stress. This is consistent with the extremely small radius of curvature at the tips of the crenations.Certain small variations in the uncrenated biconcave shape of the red cell can be interpreted mathematically as due either to negative intrinsic curvature or to shear resistance. Since, however, a small amount of negative precurvature has been shown to be capable of inducing crenation, it is unlikely to be the cause of the variations in the biconcave shape. These must therefore be due to shear resistance.In the light of this new approach, membrane molecular models based on the assumption that crenation is due to positive precurvature need reconsideration.     

Reduced muscarinic receptor plasticity in frontal cortex of aged rats after chronic administration of cholinergic drugs

Age-related differences in muscarinic receptor plasticity were observed in young, adult and senescent Fischer 344 rats (3, 9 and 27 months old, respectively) following the chronic, intracerebroventricular (ivt) administration of a cholinergic agonist, oxotremorine, or antagonist, methylatropine. After three weeks treatment of young rats with ivt oxotremorine, the maximum number (Bmax) of 3H-QNB binding sites in frontal cortex, determined by saturation experiments, was reduced by 27%, with no apparent change in the affinity (Kd) of 3H-QNB for the muscarinic receptor. Conversely, chronic ivt methylatropine administered to 3 month old animals caused a 29% increase in Bmax with no significant change in Kd. Adult animals showed a somewhat lesser degree of muscarinic receptor plasticity (16% down-regulation after oxotremorine, 22% up-regulation after methylatropine). However, 3H-QNB binding parameters in frontal cortex from senescent rats were not significantly altered following identical treatments with oxotremorine or methylatropine. Thus, muscarinic receptor adaptation to chronic, cholinergic drug administration was impaired in aged animals. This reduced receptor plasticity with aging could have important implications for the long-term drug treatment of elderly patients and for the therapeutic efficacy of cholinergic drugs in age-related neurological disorders, such as Alzheimer's disease.     

Mitochondrial regulation in sea urchins. I. Mitochondrial ultrastructure transformations and changes in the ADP:ATP ratio at fertilization.

Mitochondrial ultrastructural transformations have been examined in intact eggs and embryos from three sea urchins, Arbacia punctulata, Strongylocentrotus purpuratus, and Lytechinus pictus. Following fertilization, naturally occurring ultrastructural transformations (designated as condensed to orthodox) were observed to occur in mitochondria of all three families. The ratios of the soluble ADP and ATP pools were examined in eggs of S. purpuratus before and after fertilization in order to test whether the ultrastructural transformations reflect a decrease in relative size of the ADP pool following fertilization. Our data indicate that there is a decrease in the ADP:ATP ratio at fertilization. These findings and their implications are discussed with respect to presently accepted theories on mitochondrial regulation.     

Crenation and cupping of the red cell: A new theoretical approach. Part II. Cupping

Typical, axisymmetrical cup shaped cells have been carefully measured and the shapes analyzed mathematically. The results show that the strain energy of a cup shaped cell is always higher than that of a biconcave cell except when the two layers of the membrane involved in resistance to bending are free to slide over one another. This is true whether intrinsic curvature of the membrane is positive, negative or zero. If the two layers can slide over one another, the cup shape becomes the lower energy form. Shear resistance, if appreciable, must cause the cup cell to buckle. Photomicrographs of cup shaped cells show buckled configurations characteristic of those of a partly deflated thin-walled rubber ball, which is a similar object having a low ratio of bending/shear strength.In light of these findings, the cup shape of the red cell can no longer be considered as evidence of intrinsic membrane curvature of opposite sign to that of the crenated cell, but appears to indicate a phase change either in the hydrophobic interior of the bimolecular membrane or in some equivalent interface.     

Human B-cell mitogenic responsiveness to lectins: the requirement for T cells.

The mitogenic potential of normal human peripheral blood lymphocyte subpopulations in response to the plant lectins phytohemagglutin, erythroaggluting phytohemagglutinin, leukoagglutinating phytohemagglutinin, and pokeweed mitogen has been examined. Doubly purified B-, Null, and T-cell fractions were used by themselves and in conjunction with small percentages (1 to 10%) of other subpopulations. Purified human B and Null cells were found to be nonreactive. However, when incubated with as little as 1% T cells, significant mitogenic responsiveness by B and Null cells was observed. This helper effect was apparent with both normal and irradiated T cells. It would thus appear that mitogenic responsiveness of unseparated mononuclear cells is predominantly a function of T cells either as responding cells or as necessary helper cells.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.     

Metals in biochemistry : P.M. Harrison and R.J. Hoare,Chapman & Hall,New York, 1980, 80 pp. $5.95 (paper)

An ultrafiltration technique was used to study stripping by glycine of the first copper and zinc ion equivalents bound by bovine, dog, and rat serum albumins at pH 7.5. Affinity of dog serum albumin for copper was poorer than for the other albumins, consistent with the absence in the former albumin of the copper binding site present at the amino terminus of the latter albumins. Affinities of all three proteins for zinc were similar, suggesting that the albumin amino terminus is not the primary zinc ion binding site.     

Characterization of cross-reacting antigens on the Epstein-Barr virus envelope and plasma membranes of producer cells.

A rabbit antiserum has been prepared against the B95-8 transforming strain of EBV. The antiserum has a high virus neutralizing titer (approximately 1:1000) against both the marmoset B95-8 EBV and the human P3HR-1 EBV. The neutralizing antibodies may be absorbed completely with EBV producer cell lines, but not with nonproducer cell lines or producer cell lines treated with phosphonoacetic acid (PAA) so as to be nonproducer. After repeated absorption with PAA-treated B95-8, the serum remains reactive with the membranes of producer cell lines as judged by immunofluorescence or the 125I--Staphylococcal protein A radioimmunoassay. Thus the neutralizing antigens are expressed on the membranes of producer cell lines and may be purified from this source using the serum and 125I--Staph A binding as an assay. The ability of the serum to differentiate between producer and nonproducer cells by means of cell surface determinants has been exploited to achieve a separation of these two populations from the same culture. Immunoprecipitation by the protein A technique shows that the serum recognizes two polypeptides from producer cells of approximate molecular weights 150,000 and 75,000.     

Early degranulation of human neutrophils: immunocytochemical studies of surface and intracellular phagocytic events.

Degranulation of azurophil and specific granules after phagocytic challenge with E. coli for 5 sec to 10 min was investigated in the human polymorphonuclear neutrophil (PMN). PMN were stained simultaneously with fluorescein and rhodamine-labeled monospecific antisera to myeloperoxidase (MPO) and lactoferrin (LF) to identify azurophil and specific granules, respectively, within single cells. Fixation was designed to preserve or disrupt differential permeability of cell membrane to fluorescent conjugates in order to study granule translocation. Within 5 sec after phagocytic challenge, MPO and LF appeared on the cell surface coating the bacteria as granule contents leaked from the incompletely formed phagolysosomes. The phagocytic cup, shown by scanning electron microscopy as large and circular, appeared by immunofluorescent markers to be outlined by curvilinear staining for both granule markers, and was always coincident with bacterial localization. MPO and LF appeared singly or simultaneously on the cell surface, suggesting that degranulation to the surface was random. Sequential phagocytic events were demonstrated by comparing staining intensities for each granule marker on the surface and intracellularly within single cells. LF sometimes appeared on the cell surface independent of the nascent phagosome, suggesting that perturbation of the cell membrane by bacteria may cause some specific granule extrusion not limited to the phagosome. These results imply that bacteria make contact with granule-associated anti-microbial substances within 5 sec after phagocytosis is initiated and that free communication of granule constituents occurs between the newly forming phagolysosome and the extracellular space.     

Renal cortical cells in primary monolayer culture. Enzymatic changes and morphological observations.

Stress fibers and bands of intermediate filaments (100 Å) were studied in cultured non-muscle cells using laser microbeam techniques. Wavelengths of 532, 537 and 280 nm were used, and no artificial chromophores were employed. Lesions were assayed using a combination of phase contrast, polarizing and transmission electron microscopy (TEM). (1) Stress fibers 1–2 μm in diameter were narrowed or completely servered by irradiation at 532, 537 and 280 nm. Stress fibers could be grouped into two classes: (a) those whose severed ends separated during the first few seconds following laser irradiation (46% of fibers irradiated); (b) those fibers which showed no movements (54%). Microtubules which paralleled stress fibers persisted in the presence of colcemid for up to 5 h, and alignment of the severed stress fiber ends was maintained even in their absence. Injured stress fibers appear to be repaired within 1 h of irradiation. (2) Bands of 100 Å filaments were induced in non-muscle cells in secondary cultures of neonatal rat heart by exposure to colcemid. Lesions which appeared as phase dense spots were induced in these bands by irradiation at 532, 537 and 280 nm. The positions of the lesions in the band relative to one another did not change over several hours despite movements of the entire band. These studies demonstrate that (a) stress fibers may be an excellent system in which to study subcellular repair; (b) induced bands of 100 Å filaments probably move passively in the cells containing them; (c) laser irradiation of cytoplasmic filaments in non-muscle cells does not require the introduction of an artificial chromophore.     

Effects of antioxidants on cyclooxygenase and lipoxygenase activities in intact human platelets: Comparison with indomethacin and etya

Five antioxidative agents (BW755C, 1-naphtol, NDGA, propylgallate and quercetin) were compared with indomethacin and ETYA for their effects on (¹⁴C) arachidonic acid metabolism by cyclooxygenase (CO) and lipoxygenase (LPO) enzymes in intact human platelets. All tested compounds inhibited CO activity in a concentration-dependent manner. LPO activity was suppressed by NDGA, propylgallate, quercetin and ETYA but strongly enhanced by BW755C, 1-napthol and indomethacin. Whereas NDGA and ETYA showed almost equipotent inhibitory effects towards both fatty acid oxygenases, propylgallate and quercetin were found to be respectively 6.5 and 4 times better inhibitors of LPO than of CO activities.These data indicate that antioxidants affect arachidonic acid metabolism in intact human platelets in different ways: BW755C and 1-naphtol exerted the same activity as indomethacin, a selective CO blocker, whereas NDGA, propylgallate and quercetin behaved as ETYA, a dual CO-LPO inhibitor. Considering their inhibition selectivity, propylgallate and quercetin may serve as prototypes for more specific blockers of LPO activity.     

Reactivity of amino acids in the azo coupling reaction: I. Dependence of their reactivity on pH

Azo coupling reactions of N-α-acetylhistidine, N-α-acetyltyrosine, and N-α-acetyllysine with p-methylbenzenediazonium ion were investigated as model reactions to obtain information on the relative reactivity of the histidine, tyrosine, and lysine moieties of protein, separated from structural effects. The azo coupling yields of the amino acids increased as the pH of the reaction medium was increased, indicating that the ractive species are the imidazole anion of histidine, the phenolate anion of tyrosine, and the neutral ε-amino group of lysine. It was calculated, based on percentage yields of the azo products, that the imidazole anion is more reactive than the phenolate anion and the ε-amino group, respectively.     

A modified microassay for human macrophage activation: I. Activation by antigen-stimulated lymphocytes and correlation with delayed hypersensitivity

The degree of activation of glass-adherent human blood monocyte-macrophages cultured with autologous lymphocytes was assessed by measurement of [¹⁴C]glucosamine uptake. In the absence of streptokinase-streptodornase (SK-SD) or purified protein derivative of tuberculin (PPD) minimal incorporation of the labeled compound occurred. Enhanced glucosamine uptake in the presence of antigen was positively correlated with cell donor-delayed skin hypersensitivity (PPD) and in vitro lymphoproliferative response (PPD, SK-SD). Increasing antigen or mononuclear cell concentrations resulted in increasing macrophage glucosamine uptake. Lymphoblasts, cell clumping, and macrophages with prominent pseudopodia were seen on stained monolayers of stimulated cells. Radioautography of such monolayers showed that the radiolabel was present only in mononuclear phagocytes. Adherent cell protein also generally increased in stimulated monolayers but did not account for the enhanced glucosamine uptake. Measurement of radioactive glucosamine incorporation into human macrophages is a useful tool to assess their degree of activation by lymphocytes stimulated by specific antigen.     

Collagen synthesis by cultures of stromal cells from normal human and keratoconus corneas.

Keratoconus is a disease which thins and scars the central cornea. Confluent cultures of corneal stromal cells were derived from patients with keratoconus. The collagen synthesized by these cultures was compared to the collagen synthesized by age-matched normal human corneal stromal cultures. Although the amount of collagen and the types of collagen synthesized were similar, relative proportion of type I collagen and A, B chains produced was significantly altered in keratoconus cultures. The DEAE-cellulose chromatograms of procollagen in the medium fraction were different, not only between normal control and keratoconus cultures but also among keratoconus patients.     

Immunoadjuvant activity of pyran copolymer: I. Evidence for direct stimulation of T-lymphocytes and macrophages

A single injection of pyran copolymer has been shown to greatly increase the number of hemolytic plaque forming cells to sheep erythrocytes (sRBC). Pyran given from 1 day before to 2 days after sRBC inoculation increased both specific activity and plaques/spleen, suggesting that macrophage activation was probably not responsible for the enhancement seen. In addition, pyran given 1 day prior to the primary injection of sRBC was found to increase the secondary response to SRBC given alone. As similar experiments using thymectomized irradiated bone marrow reconstituted mice showed no increase in specific activity following pyran administration, it was unlikely that pyran was acting directly on B cells. Furthermore, experiments measuring the antibody response to Escherichia coli lipopolysaccharide, a thymic independent antigen, pyran did not increase the response to this antigen. In contrast to the above, pyran delayed and depressed cell mediated cytotoxicity to the allogeneic DBA/2 P815 mastocytoma. However, no difference in the titers of cytotoxic antibody against mastocytoma cells was seen between pyran-treated and normal animals. Pyran was mitogenic for spleen cells in vitro. However, following the administration of pyran in vivo, mitogen induced blastogenesis in vitro to PHA and LPS was inhibited and this inhibition was determined to be macrophage-dependent. These results are consistent with a model in which the immunoregulatory effects of pyran act through macrophages and T-lymphocytes.