Biomedical subjects

P Sehl

Publications and source records attributed to P Sehl.

5 recordsLinked to original sources

PubMed1999

Gene expression analysis by transcript profiling coupled to a gene database query.

We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

Animals↗

PubMed1998

Receptor-specific ligands distinguish natriuretic peptide receptors-A and -C in primate tissues.

Systemic clearance of atrial natriuretic peptide (ANP) is in part due to neutral endopeptidase (NEP) proteolysis and natriuretic peptide receptor-C (NPR-C) mediated endocytosis. Biological responses to ANP are primarily mediated by the membrane guanylyl cyclase-A/natriuretic peptide receptor-A (NPR-A). Analogs of ANP selective for NPR-A and/or resistant to NEP may have increased activity in those tissues where NPR-C and NEP are coexpressed with NPR-A. The analog of ANP termed vANP; [(R3D, G9T, R11S, M12L, G16R)ANP] is selective for human NPR-A with at least 10,000 fold reduction in affinity for human NPR-C. We report that rat NPR-A is insensitive to 10 nM vANP, demonstrating the limitations of this species in evaluating human therapeutic candidates. As an alternative approach we tested the binding and potency of receptor-selective and NEP-resistant ANP analogs in rhesus monkey tissues. Competition binding studies with a simplified version of vANP, sANP [(G9T, R11S, G16R)rANP], in rhesus monkey kidney and lung membrane preparations shows displacement of 125I-ANP from only a fraction of the total ANP receptor population, 30 and 85%, respectively. The remaining ANP binding sites can be occupied with the NPR-C selective ligand cANP(4-23). These data strongly suggest that only two classes of ANP receptor are present in these membrane preparations, NPR-A and NPR-C. The NEP resistant sANP derivative called sANP(TAPR) was 8 fold more potent (ED50 = 0.6 nM) than rANP (ED50 = 5 nM) in stimulating cGMP production in the lung membrane preparation. Our results demonstrate that the rhesus monkey natriuretic peptide receptors reflect the pharmacology of the human receptors, and that this species may be suitable to determine the role of NPR-C and NEP in peptide clearance and attenuating functional responses.

Amino Acid Sequence↗

PubMed1997

A soluble tissue factor mutant is a selective anticoagulant and antithrombotic agent.

One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant ("rTFAA") was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF x FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.

Animals↗

PubMed1996

Novel analog of atrial natriuretic peptide selective for receptor-A produces increased diuresis and natriuresis in rats.

Atrial natriuretic peptide (ANP) binds to natriuretic peptide receptor-A (NPR-A), a membrane guanylyl cyclase, and to natriuretic peptide receptor-C (NPR-C), which plays a role in peptide clearance. Rat ANP (rANP) mutants that bind rat NPR-A selectively over rat NPR-C were isolated from randomized libraries of rANP-display phage by differential panning. One variant was identified with reduced NPR-C binding; rANP (G16R, A17E, Q18A) [rANP(REA18)]. Synthetic rANP(REA18) was equipotent with rANP in stimulating cGMP production from cloned rat NPR-A (ED50 = 1.8 nM) and was reduced in NPR-C binding by approximately 200-fold. When infused into conscious rats at 0.325 microg/min for 30 min rANP elicited an identical decrease in blood pressure compared with 0.25 microg/min of rANP(REA18), however the natriuretic (P < 0.05) and diuretic (P = 0.07) responses to rANP(REA18) were greater. These data are consistent with a role for NPR-C as a local decoy receptor attenuating NPR-A effects in the kidney, where these receptors are coexpressed. Improved NPR-A specificity could provide more effective natriuretic peptides for treatment of acute renal failure or heart failure.

Amino Acid Sequence↗

PubMed1995

Agonist selectivity for three species of natriuretic peptide receptor-A.

We determined the nucleotide sequence of mouse natriuretic peptide receptor-A (NPR-A) cDNA and compared the revised deduced amino acid sequence with those of rat and human NPR-A. The ligand selectivity of these three receptor/guanylyl cyclases was examined by whole-cell stimulation of cGMP production. The 28-amino acid atrial natriuretic peptide (ANP) has only one difference among these three species, i.e., human Met-12 versus rat and mouse Ile-12. However, despite the nearly invariant ANP sequence among these species, ANP analogs have marked differences in ED50 values and maximal cGMP responses among the three receptors. With the natriuretic peptide analogs we tested, human NPR-A is less sensitive than rat or mouse NPR-A to changes in the 17-amino acid, disulfide-bonded ring of ANP and to the species differences in brain natriuretic peptide (BNP) but is more sensitive to deletions in the carboxyl tail of ANP. The ANP determinants of agonist potency have therefore changed for different species of NPR-A. This is reflected in the amino acid sequence divergence in the receptor extracellular domains and in the divergence and specificity of BNP among species. Our results suggest that the coevolution of NPR-A and BNP has thus been constrained within the context of the conserved ANP sequence.

Amino Acid Sequence↗