Immunocytochemical localization of the functional fraction of lipoprotein lipase in the perfused heart

The functional (heparin-releasable) fraction of myocardial lipoprotein lipase (LPL) has been located at the lumen surface of capillary endothelium by means of an indirect immunocytochemical perfusion method for electron microscopy. The primary step immunoreactant was an IgG fraction of goat antiserum directed against LPL from rat heart. The second step antibody, conjugated with horseradish peroxidase, was rabbit IgG directed against goat IgG. Peroxidase reaction product, when present, appeared at the surface an in invaginations of the lumenal plasma membrane of capillary endothelium and also on chylomicrons adherent to that membrane. The highest coverage by such product occurred when the highest heparin-releasable heart LPL activity was attained after fat-feeding of rats. Coverage was low when a low level of heparin-releasable heart LPL activity was induced by carbohydrate-feeding. Coverage was very low in the perfused hearts after heparin-release of functional LPL activity. The positive association between these immunocytochemical results and actual levels of functional LPL activities indicates that functional LPL in the isolated rat heart is at the lumen surface of capillary endothelium.     

Biosynthesis of penicillin N and cephalosporin C: Antibiotic production and other features of the metabolism of a Cephalosporium sp

1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.     

Production of monoclonal antibodies against avian LHRH-I

Summary Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm² tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse. This work was funded by the Maryland Agricultural Experiment Station artical no. A4975, contribution no. 8019.     

Evidence that the effect of physical exercise on NK cell activity is mediated by epinephrine

The present study was designed to test the hypothesis that the changes in natural killer (NK) cell activity in response to physical exercise were mediated by increased epinephrine concentrations. Eight healthy volunteers 1) exercised on a bicycle ergometer (60 min, 75% of maximal O2 uptake) and 2) on a later day were given epinephrine as an intravenous infusion to obtain plasma epinephrine concentrations comparable with those seen during exercise. Blood samples were collected in the basal state, during the last minutes of exercise or epinephrine infusion, and 2 h later. The NK cell activity (lysis/fixed number of mononuclear cells) increased during exercise and epinephrine infusion and dropped below basal levels 2 h afterward. The increased NK cell activity during exercise and the epinephrine infusion resulted from an increased concentration of NK (CD16+) cells in the peripheral blood. On the other hand, the decreased NK cell activity demonstrated 2 h after exercise and epinephrine infusion did not simply reflect preferential removal of NK cells from the blood, because the proportion of CD16+ cells was normalized. On the basis of the finding that indomethacin abolished the suppressed NK cell activity in vitro and the demonstration of a twofold increase in the proportion of monocytes (CD14+ cells) 2 h after exercise and epinephrine infusion, we suggest that, after stress, prostaglandins released by monocytes are responsible for downregulation of NK cell function. Our findings support the hypothesis that increased plasma epinephrine during physical stress causes a redistribution of mononuclear subpopulations that results in altered function of NK cells.     

Palatine torus in the Greenlandic Norse.

Physical anthropologists have long been intrigued by the distinctive oral tori expressed by the medieval Norse populations of Iceland and Greenland. To assess the temporal and spatial variation of one form of oral tori, palatine torus, observations were made on all available Greenlandic Norse skeletons, as well as on samples of medieval Icelanders and Norwegians. In terms of temporal variation, 12th to 14th century (medieval) Greenlanders from the Eastern and Western settlements exhibited higher frequencies and more pronounced expressions of palatine torus compared with early 11th century Greenlanders. The early Greenlandic sample closely approximated the medieval Icelandic and Norwegian samples for total torus frequency, although the Norwegians exhibited the trait to a less pronounced degree. As degree of expression is the most distinctive aspect of torus variation among the Norse, some combination of environmental factors, including increased masticatory stress and chronic undernutrition, probably accounts for most of the difference between settlement period and medieval Greenlanders. Although palatine torus may be hereditary in part, environmental factors play a significant role in the expression of this trait.     

Free radical-mediated platelet activation by hemoglobin released from red blood cells.

It is known that the rate of thrombus formation depends on interaction between platelets and erythrocytes, but the mechanism of this process has remained obscure. We here show that nanomolar levels of hemoglobin released from damaged red blood cells can induce platelet aggregation. The molecular mechanism is not receptor-based, but involves oxidation of oxyhemoglobin by platelet-derived hydrogen peroxide, with subsequent generation of a small unknown free radical species, detected by ESR spectroscopy. Methemoglobin and carbon monoxide-treated hemoglobin are unable to cause platelet activation or radical formation. The aggregation of platelets induced by hemoglobin is completely blocked by catalase or radical scavengers. These findings indicate a role for a novel extracellular free radical second messenger in the activation of platelets.     

Two-dimensional NMR, circular dichroism, and fluorescence studies of PP-50, a synthetic ATP-binding peptide from the beta-subunit of mitochondrial ATP synthase.

PP-50, a peptide based on residues 141-190 of the beta-subunit of mitochondrial F1-ATPase, contains the GX4GKT consensus region for nucleoside triphosphate binding and has been shown to bind ATP [Garboczi, D.N., Shenbagamurthi, W.K., Hullihen, J., & Pedersen, P.L. (1988) J. Biol. Chem. 263, 812-816]. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (KD = 17.5 microM) with a 1:1 stoichiometry as shown by titrations measuring the partial quenching of ATP fluorescence by PP-50. CD spectra of PP-50 at pH 4.0 and at low ionic strength show 5.8% helix, 30.2% beta-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% beta-structure, and 48% coil. Increasing the ionic strength to 50 mM KCl also increases the structure, changing the CD to 7.4% helix, 64.4% beta-structure, and 28.2% coil. The 600-MHz proton NMR spectrum of PP-50, at pH 4.0 and low ionic strength, has been assigned by 2D methods (TOCSY, DQF-COSY, and NOESY with jump-return water suppression). Based on strong d alpha N NOEs, J alpha N values, and NH chemical shifts differing from random coil values, regions of extended structure are detected from residues 1-7 and 43-48. Based on dNN, dNN(i,i+2), and d alpha N(i,i+2) NOEs and 3J alpha N values, possible type I' and type I turns are found from residues 11-14 and 31-34, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)