Preparation and antigenic properties of 5alpha-dihydrotestosterone-11-(O-carboxymethyl) oxime-BSA conjugate.

The C-11 (O-carboxymethyl) oxime derivative of 5-alphadihydrotestosterone (5alphaDHT) has been prepared. Due to steric hindrance at C-11, a novel two step procedure was used to introduce the (O-carboxymethyl) oxime at this position. Condensation of this oxime to bovine serum albumin afforded a conjugate which produced anti-5alphaDHT sera inoculated rabbits. Apart from a 30% cross reaction with testosterone, the antisera was reasonably specific for 5alphaDHT.     

Influence of Plant Hormones on Ethylene Production in Apple, Tomato, and Avocado Slices during Maturation and Senescence

Ethylene production by tissue slices from preclimacteric, climacteric, and postclimacteric apples was significantly reduced by isopentenyl adenosine (IPA), and by mixtures of IPA and indoleacetic acid, and of IPA, indoleacetic acid, and gibberellic acid after 4 hours of incubation. Ethylene production by apple (Pyrus malus L.) slices in abscisic acid was increased in preclimacteric tissues, decreased in climacteric peak tissues, and little affected in postclimacteric tissues. Indoleacetic acid suppressed ethylene production in tissues from preclimacteric apples but stimulated ethylene production in late climacteric rise, climacteric, and postclimacteric tissue slices. Gibberellic acid had less influence in suppressing ethylene production in preclimacteric peak tissue, and little influenced the production in late climacteric rise, climacteric peak, and postclimacteric tissues. IPA also suppressed ethylene production in pre- and postclimacteric tissue of tomatoes (Lycopersicon esculentum) and avocados (Persea gratissima). If ethylene production in tissue slices of ripening fruits is an index of aging, then IPA would appear to retard aging in ripening fruit, just as other cytokinins appear to retard aging in senescent leaf tissue.     

Effect of splenectomy on the expression of regulatory T cell activity.

The effect of adult splenectomy on the expression of suppressor and amplifier T cell activity was examined with respect to the serum antibody response to Type III pneumococcal polysaccharide (SSS-III) by using a sensitive radioimmunoassay. Suppressor T cell activity, as measured by the degree of low-dose paralysis induced, was not impaired in the least by splenectomy; however, amplifier T cell activity was almost completely eliminated within 7 days after splenectomy. These findings indicate that suppressor T cell activity is not confined solely to the spleen, the major site of antibody synthesis after immunization with SSS-III, and that the spleen may be an important site for the generation and/or maintenance of amplifier T cell activity.     

Steroid estrogen conjugates in hens' urine II. identifications of some minor conversion products of intramuscularly injected [4-14c]estrone

[4-¹⁴C]Estrone was injected intramuscularly into two mature laying Rhode Island Red hens. Radioactive steroids and steroid conjugates recovered from the urine on Amberlite XAD-2 columns were fractionated on columns (100 cm) of DEAE-Sephadex A-25 by NaCl gradients. The presences of the following were confirmed, the figures in brackets indicating average proportions as per cent of total radioactivity recovered after Sephadex column chromatography: -the 3-β-glucuronides of estrone (10. 9) and of estradiol-17α plus estradiol-17β(9.8); the 17-β-glucuronides of estradiol-17α plus estradiol-17β (2.1); the 3-sulfates of estrone (14. 5) and of estradiol-17α plus estradiol-17β (27. 4); and the disulfates of estradiol-17α plus estradiol-17β (2. 3). The following additional conjugates were identified:-a β-glucuronide of 16-epiestriol (0.2) and a β-glucuronide of 16-ketoestradiol-17β (0. 2); the 3-sulfates of 16-epiestriol (1. 4), of 17-epiestriol (0. 9), of 16, 17-epiestriol (0. 7), of 16-keto-estradiol-17β (1. 1), and of 2-methoxyestrone (0. 7). Some evidence was obtained for the presence of 16, 17-epoxy-estratrienol-3-sulfate (1.9).     

Immune response of Tilapia aurea exposed to Salmonella typhimurium

Tilapia aurea showed a specific immune response to Salmonella typhimurium. S. typhimurium was introduced into the gut of T. aurea by force-feeding. S. typhimurium was isolated from the fish viscera after 15 days, but at 30 days viable cells were not detected. T. aurea had an antibody titer to S. typhimurium after 30 days which was fivefold greater than the natural background antibody titer. An elevated antibody titer was not indicative of active bacterial infection.     

Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding

In order to identify residues required for the binding of interleukin-8 (IL-8) to its receptor, mutants were constructed in which clusters of charged amino acids were systematically replaced with alanine along the entire IL-8 sequence. The mutants were tested for their ability to induce a receptor-mediated rise in cytosolic free Ca2+, a property of wild-type IL-8 which can readily be detected by flow cytometry using neutrophils loaded with the calcium probe Indo-1. Eleven of the 12 mutants caused neutrophil calcium mobilization at 5 nM; the exception being a triple alanine mutant at positions K3, E4, and R6, which was inactive at all concentrations tested (150 nM maximum). A second set of mutants was generated in which residues 1-15 were individually mutated to alanine. Mutants E4A, L5A, or R6A were all inactive in the Ca2+ assay at 5 nM and competed poorly with 125I-IL-8 for neutrophil receptor binding; I10A, E4A, L5A, and R6A had approximately 30-, 100-, 100-, and 1000-fold reduced affinity, as compared with control IL-8, respectively. The nuclear magnetic resonance structure of IL-8 indicates that, in solution, the side chains of E4, L5, R6, and I10 point away from the core of the protein and do not participate in any intramolecular hydrogen bonds or salt bridges (Clore, G. M., and Gronenborn, A. M. (1991) J. Mol. Biol. 217, 611-620).     

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.