Application of ELISA for assessment of antiserum immunoreactivity in endocrine immunocytochemical studies.
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An indirect method of an enzyme-linked immunosorbent assay (ELISA) is described to assess the reactivity of antisera used for the identification of peptide hormone producing cells by immunocytochemistry. Compared with radioimmunoassay and immunodiffusion, the ELISA method has the advantages of simplicity and sensitivity and represents with the hormone adsorbed to a matrix a situation more or less comparable to that in tissue sections. It is concluded that specificity testing of antisera applied in endocrine immunocytochemical studies can best be achieved by application of the ELISA method in combination with appropriate tissue controls. (+info)
Isolation and characterization of alpha-endorphin and gamma-endorphin from single human pituitary glands.
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alpha-Endorphin and gamma-endorphin, two closely related peptides of the pro-opiomelanocortin family with characteristic biological activities, were purified to homogeneity from single human pituitary glands and chemically identified. Isolation of the peptides was based on size fractionation by Sephadex G-75 chromatography followed by two HPLC steps using reverse-phase and paired-ion reverse-phase systems and was monitored by radioimmunoassay. During the isolation procedure alpha- and gamma-endorphin-sized material behaved chromatographically and immunologically indistinguishably from synthetic alpha- and gamma-endorphin. The amino acid composition and NH2-terminus of isolated peptides demonstrated their identity as authentic alpha-endorphin and gamma-endorphin. Acetylated forms were absent. In addition, evidence is provided that large forms with alpha- and gamma-endorphin immunoreactivity detected during gel filtration are human lipotropin-(1-74) and -(1-75), respectively. The data substantiate that alpha-endorphin and gamma-endorphin exist as endogenous peptides in the human pituitary gland. (+info)
Enhancement of murine bone marrow macrophage differentiation by beta-endorphin.
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The present study was performed to investigate the effect of beta-endorphin on macrophage colony-stimulating factor (M-CSF)-induced differentiation of macrophages from bone marrow cells in a semisolid culture system. beta-endorphin increased the number of macrophage colonies when bone marrow cells were cultured in the presence of M-CSF plus lipopolysaccharide (LPS). This was not the case with LPS-unresponsive C3H/HeJ mouse bone marrow cells. alpha-endorphin and gamma-endorphin were as effective as beta-endorphin in enhancing the colony formation. Exogenous interleukin-1 (IL-1), but neither IL-6 nor tumor necrosis factor (TNF), collaborated with beta-endorphin even in the absence of LPS, suggesting that IL-1 is a primary mediator of the effect of LPS. Indeed, anti-IL-1 antibody abolished the collaborative effect of beta-endorphin with LPS. Moreover, IL-1 was effective even for C3H/HeJ mouse bone marrow cells. Naloxone, an antagonist of endorphins for opioid-receptors, completely abrogated the effect of beta-endorphin. In a single-cell culture system, the collaboration between beta-endorphin and IL-1 was revealed by the increase in number and size of macrophage colonies, but collaboration between beta-endorphin and LPS did not occur. These results indicate that, in mixed cell culture, beta-endorphin acts in concert with paracrinal IL-1 induced by LPS to enhance M-CSF-dependent macrophage differentiation from immature precursor cells. (+info)
Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.
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Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed. (+info)
High-efficiency synthesis of human alpha-endorphin and magainin in the erythrocytes of transgenic mice: a production system for therapeutic peptides.
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Chemical synthesis of peptides, though feasible, is hindered by considerations of cost, purity, and efficiency of synthesizing longer chains. Here we describe a transgenic system for producing peptides of therapeutic interest as fusion proteins at low cost and high purity. Transgenic hemoglobin expression technology using the locus control region was employed to produce fusion hemoglobins in the erythrocytes of mice. The fusion hemoglobin contains the desired peptides as an extension at the C end of human alpha-globin. A protein cleavage site is inserted between the C end of the alpha-globin chain and the N-terminal residue of the desired peptide. The peptide is recovered after cleavage of the fusion protein with enzymes that recognize this cleavage signal as their substrate. Due to the selective compartmentalization of hemoglobin in the erythrocytes, purification of the fusion hemoglobin is easy and efficient. Because of its compact and highly ordered structure, the internal sites of hemoglobin are resistant to protease digestion and the desired peptide is efficiently released and recovered. The applicability of this approach was established by producing a 16-mer alpha-endorphin peptide and a 26-mer magainin peptide in transgenic mice. Transgenic animals and their progeny expressing these fusion proteins remain health, even when the fusion protein is expressed at > 25% of the total hemoglobin in the erythrocytes. Additional applications and potential improvements of this methodology are discussed. (+info)
Specific cleavage of amino side chains of serine and threonine in peptides and proteins with S-ethyltrifluorothioacetate vapor.
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A vapor of S-ethyltrifluorothioacetate was found to specifically cleave the amino side of serine and threonine peptide bonds. The cleavage reactions were carried out at 50 degrees C for 6 h-24 h or at 30 degrees C for 24 h. When vapors were generated in a solution containing several conventional organic solvents, the cleavage reactions were reduced or stopped, or modification took place. When the reagent vapor was made in an aqueous solution, the cleavage reaction at glycine residues was enhanced. This reagent did not oxidize any amino acid residues, such as methionine, cysteine and tryptophan. The cleavage was also effective on proteins on membranes blotted or electroblotted from polyacrylamide gels. This method therefore may be used for the peptide mass fingerprinting [Patterson, S. D. (1995) Electrophoresis 16, 1104-1114] after two-dimensional electrophoresis. (+info)