Fetal and adult sheep adrenal cortical cells contain glucocorticoid receptors

Specific receptors for glucocorticoids were identified in the fetal and adult sheep adrenal cortex by a whole-cell binding assay using [3H]triamcinolone acetonide ([3H]TA) as the radiolabelled ligand. [3H]TA binding sites were saturable and of high affinity, with dissociation constant (Kd) of 2-3nM. Scatchard analysis revealed a single class of binding sites with a binding capacity (Bmax) of 207 and 5 fmol/10(6) cells for d100 fetuses and adults, respectively. By single point analyses at saturating [3H]TA concentration, we found that glucocorticoid receptors (GR) were present in the fetal adrenal cortex as early as d60. Highest concentrations were found at d100-110. GR decreased to d125, then increased to term (approx. d145) before declining again in newborn and adult animals. This demonstration of glucocorticoid receptors in ovine fetal adrenal cortical cells provides a mechanistic basis for the concept that glucocorticoids may act, perhaps in a paracrine or autocrine fashion, to influence adrenocorticotropin (ACTH)-induced activation of fetal adrenal function and the events leading to parturition.     

A specific, photolabile and irreversible antagonist (L662,025) of the PAF-receptor

PAF (0.2 microM) induced maximal platelet aggregation in human PRP and [3H]-PAF (1-5 nM) binding to platelet membrane preparations had Kd value of 3.8 nM and Bmax of 200 fmoles/mg of protein. Without UV irradiation, a synthetic azido tetrahydrofuran derivative L662,025 was a reversible and competitive PAF-receptor antagonist with IC50 values of 5.6 +/- 0.3 microM (platelet aggregation) and 1.0 +/- 0.25 microM (receptor binding). Photolysis of L662,025 in the presence of PRP produced an irreversible inhibition of platelet aggregation and specific binding of [3H]-PAF (1 nM). L662,025 did not affect collagen- or ADP-induced human platelet aggregation before or after photolysis. It is a new probe that can be used to identify and characterize the PAF-receptor.     

Binding of pyrimidin-2-one ribonucleoside by cytidine deaminase as the transition-state analogue 3,4-dihydrouridine and the contribution of the 4-hydroxyl group to its binding affinity

Cytidine deaminase, purified to homogeneity from constitutive mutants of Escherichia coli, was found to bind the competitive inhibitors pyrimidin-2-one ribonucleoside (apparent Ki = 3.6 x 10(-7) M) and 5-fluoropyrimidin-2-one ribonucleoside (apparent Ki = 3.5 x 10(-8) M). Enzyme binding resulted in a change of the lambda max of pyrimidin-2-one ribonucleoside from 303 nm for the free species to 239 nm for the bound species. The value for the bound species was identical with that of an oxygen adduct formed by combination of hydroxide ion with 1,3-dimethyl-2-oxopyrimidinium (239 nm), but lower than that of a sulfur adduct formed by combination of the thiolate anion of N-acetylcysteamine with 1,3-dimethyl-2-oxopyrimidinium (259 nm). The results suggest that pyrimidin-2-one ribonucleoside is bound by cytidine deaminase as an oxygen adduct, probably the covalent hydrate 3,4-dihydrouridine, rather than intact or as an adduct involving a thiol group of the enzyme. In dilute solution at 25 degrees C, the equilibrium constant for formation of a single diastereomer of 3,4-dihydrouridine from pyrimidin-2-one ribonucleoside was estimated as approximately 4.7 x 10(-6), from equilibria of dissociation of water, protonation of 1-methylpyrimidin-2-one, and combination of the 1,3-dimethylpyrimidinium cation with the hydroxide ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

The primary structure of human apolipoprotein A-IV

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Opioid-binding protein (OBCAM) is rich in β-sheets

Based on circular dichroism (CD) and the sequence-predictive method, the opioid-binding cell adhesion molecule (OBCAM) consisted of one half -sheets and one fourth -helices. This is consistent with significant sequence homology of the protein to several members of the immunoglobulin (Ig) superfamily, particularly cell adhesion molecules, which are rich in -sheets. Hydropathy analysis suggests that hydrophobic and hydrophilic regions were evenly distributed along the sequence, but the NH₂- and COOH-termini were hydrophobic. Hydrophobic moments and Fourier-transform amphipathic analyses further suggest that residues 23–30 and 83–93 were amphiphathie -sheets. The overall conformation of OBCAM was unaltered by adding linoleic acid, which is required for opioid ligand binding.     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

Albumin — vitamin D-binding protein haplotypes in Asian-Pacific populations

Summary We have determined the various haplotypic combinations between alleles as well as restriction fragment length polymorphisms of two linked genetic markers, albumin and vitamin D-binding protein or group-specific component, in a number of Asian-Pacific populations. Using the partial maximum likelihood method, we constructed a phylogenetic network from the haplotype frequencies to assess relationships among the populations sampled. No systematic linkage disequilibrium was detected between most of the combinations, suggesting a lack of operation of any selection pressure at the two loci. The phylogenetic analysis confirmed the known interrelationships among various populations in the Asian-Pacific region. The Australian aborigines clustered closely with the non-Austronesian-speaking highlanders from Papua New Guinea, as expected. Similarly, the Austronesian-speaking Polynesians, Micronesians, and the Southeast Asians branched off together as a separate group. The position of the Austronesian-speaking Tolais from New Britain with respect to other populations from the Southwest Pacific was anomalous. The Tolais revealed a strong affinity with the Australian aborigines, which is inexplicable. The populations from China formed a tight cluster with other populations from the Asian-Pacific region. Genetic interrelationships of these populations with the white Australians were remote, which is in accordance with the known affinities of various human racial groups.     

Postural dynamics of walking in humans

The dynamics of postural control in human biped locomotion were studied using(1) a model, and(2) experimentally applied impulsive force disturbances. The model was planar, and contained five rigid segments, articulating at frictionless pin joints. The model was used to identify joint torque combinations which would successfully correct for an impulsive force disturbance applied at different points in the walking cycle. The simulation results suggested that(1) early responses (within 80ms) can be effective in compensating for impulsive disturbances,(2) the same strategies which successfully counteract similar disturbances during quiet standing are also effective in certain phases of the walking cycle,(3) modifications in the response strategies are needed to accomodate differences in the dynamics over the stride cycle, and(4) the swing leg is ineffective in compensating for disturbances in the short term. These model predictions were tested experimentally. Subject responses to an impulsive force disturbance applied during walking were studied. The electromyographic results generally support the model predictions.