Membrane-bound glycoconjugates of fetal mouse erythropoietic cells with special reference to phagocytosis by hepatic macrophages

Using lectin and colloidal iron (CI) stainings in combination with neuraminidase digestion, glycoconjugates on the surface of erythropoietic cells of the yolk sac and liver in fetal mice were examined. Fetal hepatic macrophages were capable of distinguishing between phagocytozed and non-phagocytozed erythroid elements as described in our previous study. Marked differences between these two elements could be ultrahistochemically detected on their cell surface. The phagocytozed elements, such as nuclei expelled from erythroblasts and degenerating primitive erythroblasts, faintly bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a weak peanut agglutinin (PNA) binding. In contrast, erythroblasts at various maturation stages, erythrocytes and normal primitive erythroblasts heavily bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a moderate PNA binding. No differences in binding of either concanavalin agglutinin,Ricinus communis agglutinin-I or PNA were noted between phagocytozed and non-phagocytozed erythroid elements. Desialylation appears to be one of the most important signs for the recognition mechanism of fetal macrophage phagocytosis. During maturation of hepatic erythroblasts, sialic acid changes its affinity forLimax flavus agglutinin from strong to weak, and soybean agglutinin binding sites disappear at the basophilic erythroblast stage. Glycoconjugates on polychromatophilic erythroblasts acquire similar compositions to those of erythrocytes.     

An improved method for cell and spore counts during aerobic cultivation of crystal toxin producingBacillus sphaericus

Summary A simple improved method including sonication treatment is proposed to determine accurate cell and spore counts forBacillus sphaericus, a microorganism which can form cell aggregates. Sonication (10 watt power output) for 40 seconds after dilution of culture broth was effective in dispersing clumps of cells and spores without disruption. This improved method gave approximately 2 times higher cell and spore counts compared with the conventional counting methods (without sonication).     

The effects of naloxone on canine splanchnic arterial smooth muscle

The pharmacological properties of naloxone on vascular smooth muscle in vitro were examined using canine mesenteric arterial segments. Naloxone exerted two different effects on the artery: (A) naloxone at a high concentration (3 X 10(-4) M) produced a nonspecific vasodilation; and (B) naloxone at lower concentrations (3 X 10(-7), 3 X 10(-6), and 3 X 10(-5) M) augmented the vasoconstrictor effects of epinephrine and norepinephrine without altering KCl- or serotonin-induced constriction. Naloxone's augmenting effect on epinephrine-induced constriction was dose dependent. Even when the arterial strips were incubated in low calcium (0.8 mM) or calcium free Kreb's solution, naloxone (3 X 10(-5) M) still augmented epinephrine-induced constriction. With respect to naloxone's effect on another alpha-adrenoreceptor agonist, naloxone (3 X 10(-5) M) failed to alter phenylephrine-induced constriction. Naloxone's augmenting effect on norepinephrine-induced constriction was abolished when the specimens were incubated with 10(-5) M normetanephrine, while naloxone (3 X 10(-5) M) still augmented the constriction even when the specimens were incubated with 10(-5) M cocaine. These results suggest that naloxone at lower concentrations may augment the constrictor responses to catecholamines, at least in part, by inhibiting the extraneuronal uptake of those catecholamines.     

Identification of cells by backscattered electron imaging of silver stained bulk tissues in scanning electron microscopy

Anuran tadpole tail muscle was stained en bloc by a modified light microscope silver stain for light microscopy and freeze-fractured in liquid nitrogen after partial dehydration with ethanol. The fractured specimens were observed in both secondary electron and backscattered electron modes in a scanning electron microscope. Since the cell nuclei specifically stained with silver provided high contrast against the unstained background due to atomic number contrast of backscattered electron image, various cells were easily identified by a comparison of secondary electron images and compositional images of backscattered electron signals.     

A fluorimetric determination of the activity of glycolipid transfer protein and some properties of the protein purified from pig brain

The fluorimetric method of Correa-Freire et al. (Correa-Freire, M.C., Barenholz, Y. and Thompson, T.E. (1982) Biochemistry 21, 1244-1248) to measure glucosylceramide transfer between phospholipid bilayers has been applied to the determination of the activity of glycolipid transfer protein purified from pig brain. The transfer of pyrene-labeled galactosylceramide (PyrGalCer) from donor to acceptor vesicles was measured by a decrease in the intensity ratio of eximer (E) to excited monomer (M). A sensitive determination of the glycolipid transfer activity is possible by the fluorimetric method without separation of the donor and acceptor vesicles. The newly developed fluorimetric assay of glycolipid transfer protein was used to study the effects of N-ethylmaleimide, HgCl2 and sugars on the transfer activity. The treatment with N-ethylmaleimide inactivated the activity to about 40%. The activity was almost completely inactivated by the treatment with HgCl2. Monosaccharides and methyl-alpha-D-glucoside had no inhibitory effect on the transfer activity. A marked and immediate drop of the E/M ratio was observed by the addition of glycolipid transfer protein to vesicles containing PyrGalCer at a protein-to-PyrGalCer molar ratio of 1.56:1. The result suggests a complex formation of glycolipid transfer protein with PyrGalCer.     

Stimulation of phospholipase A2 activity by high osmotic pressure on cholesterol-containing phospholipid vesicles

Bovine serum albumin conjugates of guanosine prepared by the periodate method was used as immunogen to elicit guanosine antibodies in rabbits. The specificities of the antibodies were studied by the inhibition of their binding to [3H]Gox-red, [32P]DNA and [3H]RNA by related non-radioactive compounds. A population of antibodies is specific to Gox-red with an average association constant of around 10(7) M-1 at 4 degrees C. There are a population of antibodies which bind to [32P]ssDNA and [3H]RNA specifically at guanosine residues. RNA binding antibodies were separated into two populations by affinity chromatography.     

Inhibition of growth of mammalian cell cultures by extracts of arginine-utilizing mycoplasmas

Mechanisms of the inhibition of growth of mammalian cell cultures caused by mycoplasmal infection were investigated by using cell-free extracts of 14 species of mycoplasmas. In four mammalian cell lines tested, the growth of two cell lines, FM3A and MDCK, was inhibited by the extracts of arginine-utilizing mycoplasmas, whereas that of the other two cell lines, Vero and LLC-MK2, was not inhibited by extracts of either arginine- or glucose-utilizing mycoplasmas. These results suggest that there are two types of cell cultures, one susceptible and the other insusceptible to arginine-utilizing mycoplasmas. In a series of experiments using FM3A cells, it was found that the growth inhibition caused by the extracts of arginine-utilizing mycoplasmas was due to removal of arginine from the medium by the action of arginine deiminase present in the extracts and that none of the metabolic products of arginine had any effect on the growth. A highly positive correlation (r = 0.96, P less than 0.01) was observed between the activity of arginine deiminase and the growth-inhibiting activity of extracts of arginine-utilizing mycoplasmas.     

Interaction between histamine and vagal stimulation on tracheal smooth muscle in dogs

We examined the interaction between histamine and vagal efferent activity on airway smooth muscle reactivity in 11 anesthetized vagotomized dogs using an isolated closed segment of the intrathoracic trachea filled with Tyrode solution under an isovolumetric condition. Intratracheal pressure change was measured as an index of tracheal smooth muscle tone. The administration into the tracheal segment of histamine (0.1 or 1.0 mg/ml) in six dogs and methacholine chloride (0.001 or 0.01 mg/ml) in the other five dogs elevated intratracheal pressure by about 5 cmH2O. The electrical stimulation of the peripheral ends of both of the cut cervical vagus nerves in the presence of histamine produced significantly greater responses than the additive responses of these two stimuli applied individually (two-way analysis of variance, P less than 0.025). However, the combined effects of vagal stimulation and methacholine were not significantly different from the additive responses of these two stimuli applied individually. The average values of intratracheal pressure elevated by the combined effects of vagal stimulation and histamine were significantly higher than those obtained by the combination of vagal stimulation and methacholine (two-way analysis of variance, P less than 0.01). This suggests that histamine potentiates tracheal smooth muscle reactivity to electrical vagal stimulation, which may contribute to the hyperreactivity observed in patients with asthma.     

Fluctuations in pulmonary fibrin decomposing activities (plasmin and non-plasmin activities) in an endotoxin DIC model in rats

Non-plasmin fibrinolysis enzyme was extracted from the lung and spleen of conventional rats (Thrombos. Haemostas., 1979), although the enzyme was not found in germfree rats, suggesting the possibility that the enzyme may participate in the defence mechanism of the body. The present study was made in an attempt to determine the behavior of non-plasmin fibrinolysis enzyme of the lung tissue in the DIC model of conventional rats induced by a single injection of bacterial endotoxin. The plasminogen-activator activity of the lung tissue, and the fibrinogen level, platelet count, urea nitrogen and plasminogen-activator activity in the blood were also measured. Examination of the lung tissue in the DIC rats indicated a remarkable increase in non-plasmin fibrinolysis activity and a disappearance of plasminogen-activator activity. Inhibitor studies using t-AMCHA and DFP demonstrated that the increased non-plasmin fibrinolysis activity was not derived from activated plasmin, but from serine protease. The disappearance of plasminogen-activator activity in the lung and increase of plasminogen-activator activity in the blood suggested a release of the activator from the lung into the blood due to the endotoxin injection.