Stem-like intestinal Th17 cells give rise to pathogenic effector T cells during autoimmunity

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Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development

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Affinity Purification of Synenkephalin-Containing Peptides Including a Novel 23.3-Kilodalton Species

Abstract: Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1 -70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6- kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met⁵]-enkephalin-Arg⁶-Gly⁷-Leu⁸. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3- kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1–206), containing [Leu⁵]- enkephalin at the C-terminus.     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.     

Specific adhesion between pheochromocytoma (PC12) cells and adrenal medullary endothelial cells in co-culture

Summary Chromaffin cells in the adrenal medulla are found in close proximity to capillary endothelial cells, thereby forming the classical endocrine complex. To examine the possible chemical basis of their interaction in more detail, we have grown bovine adrenal medullary endothelial (BAME) cells in monolayer cultures and added to them pheochromocytoma (PC12) cells, a chromaffin tumor cell line of rats. The PC12 cells were chosen because of the similarities they share with adrenal medullary chromaffin cells. PC12 cells rapidly attached to BAME cells cultures, their rate of adhesion being significantly enhanced over binding of PC12 cells to either uncoated plates or to monolayers of unrelated cell cultures. Consistent with this observation, we noted that the extracellular matrix (ECM) derived from the BAME cells did not enhance PC12 cell adhesion and did not promote neurite sprouting as previously described for ECM derived from corneal endothelial cells. The specific adhesion between PC12 and BAME cells could be abolished by cell surface extracts derived from these two cells but not by extracts derived from unrelated cell types. This activity was heat-labile, sensitive to trypsin and, to a lesser extent, to neuraminidase. We therefore conclude that PC12 cells may interact with BAME cells by specific proteinaceous adhesive factors associated with their plasma membranes. These interactions might represent the formative role of cell-cell contacts in the organization of the developing adrenal gland.Abbreviations BAME bovine adrenal medullary endothelial cells - DMEM Dulbecco's modified essential medium - ECM extracellular matrix - EMEM Eagle's modified essential medium - FCS fetal calf serum - PBS phosphate-buffered saline - PC12 rat pheochromocytoma cells     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

Major and trace elements in the twig axes of Norway spruce and the relationship between twig axis and needles

Summary The concentrations of 23 elements have been determined by instrumental neutron activation analysis in the needles and axes of 1-year-old spruce twigs from 12 different sites. Although the twigs had been washed with toluene and tetrahydrofuran prior to analysis, it was found that the amounts of eight elements (Al, Cr, Eu, Fe, La, Sc, Th and V) present were predominantly due to aerosol particles still being retained on the surface of the axes. The results of the remaining 15 elements (Ba, Br, Ca, Cl, Co, Cs, Hg, K, Mg, Mn, Na, P, Rb, Sr and Zn) were not or only slightly influenced by this effect and are considered to represent their inherent concentrations within the axes. With most of these latter 15 elements there are highly significant and linear correlations between the concentrations in the needles and in the axis, which testify to the great similarity of needles and axis of a twig. The axis/needles ratios are constant for every element (except K), but the individual elements show considerable differentiation, as evidenced by their ratios, which have values between 0.5 and 2.8. Inter-element correlations in the axes are mainly found within two groups of elements. With Na, Cl and Br these correlations are probably due to different pollution levels of the different sites, whereas with Mn, Co, Rb and Cs they are due to the pH of the soil. The activity of ¹³⁷Cs from the Chernobyl fallout represents independent confirmation of the results obtained with the stable elements. With twigs sprouted before the deposition of the fallout, ¹³⁷Cs acts as a tracer for aerosol particles, whereas it forms a tracer for the stable Cs inherent to the plant with twigs sprouted after the deposition.