Melatonin Binding Sites

The distribution and characterization of specific melatonin binding sites were studied using 125I-melatonin. Autoradiography revealed only three sites of specific melatonin binding in brain: the suprachiasmatic nuclei, the median eminence, and the small part of choroid plexus at the caudal end of the fourth ventricle. Two other sites were detected outside the CNS: the anterior pituitary and the retina. The specific binding of 125I-melatonin was saturable and reversible. The dissociation constant (KD) of the binding sites was 60 pM. The concentration of the binding sites (Bmax) in the median eminence was 26 fmol/mg protein, and in the pituitary 3 fmol/mg protein. Specificity of the binding sites was tested by displacement of 125I-melatonin. The order of potency--melatonin much less than N-acetyl-5-hydroxytryptamine less than 5-methoxytryptamine much less than 5-hydroxytryptamine = 3,4-dihydroxyphenylethylamine = noradrenaline--shows high specificity of the binding sites for melatonin.     

Interaction of l-Prolyl-l-Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a delayed rectifier K+ channel in NG108-15 neuroblastomaX glioma cells: Gating kinetics and the effects of enrichment of membrane phospholipids with arachidonic acid

Summary The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca²⁺ levels much in excess of 50 m. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca²⁺ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.Deceased     

桑蚕促前胸腺激素的作用与前胸腺分泌活动的某些特点

本工作以前胸腺的体外器官培养技术和蜕皮激素的放射免疫分析法(MH-RIA)相结合,研究了桑蚕(Bombyx mori)促前胸腺激素(PTTH)的作用与前胸腺分泌的某些特点。结果表明,被PTTH激活后的前胸腺,在一定的时相过程内合成并分泌脱皮甾类激素;前胸腺本体不积累蜕皮甾类激素;PTTH对前胸腺的作用是积累性的;五龄不同天数的前胸腺合成分泌脱皮甾类激素的能力不同,并有不同的剂量反应。     

Somatotropin antagonism of insulin-stimulated glucose utilization in 3T3-L1 adipocytes

It is well established that somatotropin (GH) antagonizes insulin action in vivo and that supraphysiologic concentrations of GH frequently result in insulin resistance and glucose intolerance. However, the demonstration of an anti-insulin activity by GH in vitro has been difficult. This study, therefore, set out to determine whether cultures of 3T3-L1 adipocytes could be used to examine the anti-insulin activity of GH. The ability of insulin to stimulate glucose utilization by 3T3-L1 adipocytes increases approximately five-fold during the first 4 days following treatment of the cells with a differentiation medium. It was found that glucose utilization in 3T3-L1 adipocytes is regulated in a reciprocal fashion by insulin and GH. Bovine or human GH directly inhibit up to 50% of insulin-stimulated [14C]-glucose incorporation into lipids in a concentration-dependent manner. The 3T3-L1 sensitivity to GH appears to be at the maximum (50% inhibition of an insulin response) immediately following removal of the cells from the differentiation medium and remains essentially constant during the subsequent 4 days. The GH inhibition of insulin action does not appear to be due GH enhancement of cellular degradation of insulin, competitive binding of GH to the insulin receptor, or GH-induced decrease in cell number. The 3T3-L1 adipocyte system appears to be a sensitive and reliable in vitro model with which to study the molecular mechanisms involved in both GH antagonism of insulin action and development of hormone responsiveness during cellular differentiation into adipocytes.     

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.     

Endogenous cholesterol synthesis is sufficient for ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture, fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented medium. This work was supported by Finnish Culture Foundation.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Immunohistochemical localization of Na+-dependent glucose transporter in rat jejunum

Summary Glucose is actively absorbed via a Na⁺-dependent active glucose transporter (Na-GT) in the small intestine. We raised a polyclonal antibody against the peptide corresponding to amino acids 564–575 of rabbit intestinal Na-GT, and localized it immunohistochemically in the rat jejunum. By means of immunofluorescence staining, Na-GT was located at the brush border of the absorptive epithelial cells of the intestinal villi. Electron-microscopic examination showed that Na-GT was localized at the plasma membrane of the apical microvilli of these cells. Little Na-GT was found at the basolateral plasma membrane. Along the crypt-villus axis, all of the absorptive epithelial cells in the villus were positive for Na-GT. In addition to the brush border staining, the supranuclear positive staining, which was shown to be the Golgi apparatus by use of electron microscopy, was seen in cells located between the base to the middle of the villus. Cells in crypts exhibited little or no staining for Na-GT. Goblet cells scattered in the intestinal epithelium were negative for Na-GT staining. These observations show that Na-GT is specific to the apical plasma membrane of the absorptive epithelial cells, and that the onset of Na-GT synthesis may occur near the crypt-villus junction.     

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.