Binding affinity of adenosine receptor agonists and antagonists at human cloned A3 adenosine receptors

The A3 adenosine receptor is one of the four adenosine receptors which have thus far been identified. Cloning of the A3 receptor from animal species such as rat, sheep and human has shown that there are interspecies differences in its peripheral distribution, and binding affinity for various adenosine receptor ligands. The adenosine derivative, 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA), is a potent A3 receptor agonist which is used as a reference drug. In this report we have characterized the binding of selected adenosine receptor agonists and antagonists to HEK 293 cells transfected with the human A3 adenosine receptor using [125I]AB-MECA as radioligand. HE-NECA and NECA were the most potent compounds showing Ki values in the low nanomolar range, while the recently discovered non-xanthine A2A receptor antagonists ZM 241385, SCH 58261 and SCH 63390 showed affinity values in the micromolar range. These data further indicate the need to examine the affinity of new adenosine receptor ligands directly in human A3 receptors.     

Antibody catalysis of peptidyl-prolyl cis-trans isomerization in the folding of RNase T1

An antibody generated to an alpha-keto amide containing hapten 1 catalyzes the cis-trans isomerization of peptidyl-prolyl amide bonds in peptides and in the protein RNase T1. The antibody-catalyzed peptide isomerization reaction showed saturation kinetics for the cis-substrate, Suc-Ala-Ala-Pro-Phe-pNA, with a kcat/Km value of 883 s-1.M-1; the reaction was inhibited by the hapten analog 13 (Ki = 3. 0 +/- 0.4 microM). Refolding of denatured RNase T1 to its native conformation also was catalyzed by the antibody, with the antibody-catalyzed folding reaction inhibitable both by the hapten 1 and hapten analog 13. These results demonstrate that antibodies can catalyze conformational changes in protein structure, a transformation involved in many cellular processes.     

Effects of exogenous recombinant ovine interferon tau on circulating concentrations of progesterone, cortisol, luteinizing hormone, and antiviral activity; interestrous interval; rectal temperature; and uterine response to oxytocin in cyclic ewes

Interferon tau (IFN tau) is the conceptus-produced antiluteolytic signal in ruminants. Three experiments examined the effects of s.c. administration of recombinant ovine (ro)IFN tau on interestrous interval (IEI), oxytocin (OT)-induced uterine prostaglandin F2alpha metabolite (PGFM) production, rectal temperature (RT), respiration rate (RR), and plasma concentrations of progesterone, cortisol, LH, and antiviral activity (AVA) in plasma and uterine flushings. In experiment I, 20 ewes were treated s.c. with either 0, 1, 2, or 4 mg/day roIFN tau (0.7 x 10(8) U/mg; 5 ewes/dosage) from Days 11 to 15 of the estrous cycle (estrus = Day 0) and were challenged with OT (30 IU) on Day 15. Jugular blood samples were collected at -10, 0, 10, 20, 30, 40, 50, and 60 min relative to the OT challenge and assayed for PGFM. Recombinant oIFN tau increased IEI (16.7, 18.7, and 22.6 +/- 0.6 days for 0, 2, and 4 mg roIFN tau, respectively, p < 0.01). Recombinant oIFN tau did not affect peak PGFM response to OT (2309 +/- 172 pg/ml; p > 0.1). However, the 4 mg/day dosage delayed the time to peak PGFM (32.4 vs. 47.5 +/- 3.4 min; p < 0.01, 0 vs. 4 mg) and resulted in approximately 200% higher concentrations of PGFM at 60 min post-OT (0 vs. 4 mg/day, p < 0.07). Experiment II was similar to experiment I, except that only the 0- and 4-mg/day dosages of roIFN tau were administered. Ewes were hysterectomized on Day 16, and assay of uterine flushes detected no AVA from ewes treated with either 0 or 4 mg/day roIFN tau. In experiment III, 20 ewes were treated s.c. with either 0, 2, 4, or 6 mg roIFN tau on Day 12. Blood samples, RT, and RR were obtained at frequent intervals for 24 h, and plasma was assayed for progesterone, cortisol, LH, and AVA. Plasma AVA, which increased in a dose-dependent manner, was detectable within 60 min and remained elevated at 24 h compared to control values. RT (elevated 0.5-1.0 degrees C), RR, and cortisol increased in response to all dosages of roIFN tau, with peak values occurring 150-180 min postinjection. For all dosages of roIFN tau, plasma progesterone declined from 120 to 360 min posttreatment and then returned to pretreatment values by 24 h (p < 0.01) as compared to controls. Overall, exogenous roIFN tau altered uterine PGFM response to OT from a pulse to a gradual and sustained elevation and extended IEI with only a transient decline in progesterone and mild hyperthermia, effects that are not expected to compromise pregnancy.     

Interaction of the von Willebrand factor with platelets and thrombosis

The human von Willebrand factor (vWf) is a multimeric glycoprotein present in plasma, platelets, endothelial cells and subendothelium and synthesized in endothelial cells and megakaryocytes. vWf plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; quantitative and qualitative abnormalities of vWf cause the most common congenital bleeding disorder in man, the von Willebrand disease. vWf stabilizes factor VIII and interacts with subendothelial components and with platelet membrane receptors. The multimeric structure of vWf provides an array of binding sites which allows multivalent interactions with its ligands, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. In the last years, remarkable progress has been made toward understanding the structure of vWf protein and gene, and the elucidation of many structure-function relationships, which may result in improved therapeutic intervention for vWD patients, and in the development of effective strategies for antithrombotic therapy.