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R Seyer

Publications and source records attributed to R Seyer.

36 records · Page 2Linked to original sources
PubMed1989

Synthesis of a biotinylated, iodinatable, and photoactivatable probe for angiotensin receptors.

We propose here a biotinyl-aminohexanoyl-[Ala1, Phe(4N3)8]angiotensin II analog as a radioiodinatable and photoactivatable probe for covalent labeling, detection and isolation of angiotensin receptors. A combination of solid phase and minimum-protection segment-coupling strategy using hexafluorophosphate of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium (BOP) as a coupling reagent is proposed for the synthesis of this probe. Optimized yields were obtained by HPLC monitoring of all reactions. A complete n.m.r. study suggests an extended conformation of this molecule, allowing a simultaneous recognition of receptor and avidin. The probe binds with high affinity (Kd = 2 nM) to angiotensin II receptors from rat liver membranes.

Amino Acid Sequence↗
PubMed1988

Peptides mimicking the flap of human renin: synthesis, conformation, and antibody recognition.

Four peptides related to human renin flap region have been synthesized. Two of them are ring closed through appropriately designed disulfide bridges. Structure analysis involving IR and NMR techniques and recognition by polyclonal human renin antibodies provides support for a beta-hairpin secondary structure of the cyclized peptides identical with that presented by the flap section in the speculative human renin model [Blundell, T., Sibanda, B. L., & Pearl, L. (1983) Nature (London) 304, 273-275; Sibanda, B. L., Blundell, T., Hobart, P. M., Fogliano, M., Bindra, J. S., Dominy, B. W., & Chirgwin, J. M. (1984) FEBS Lett. 174, 102-111].

Antibodies↗
PubMed1988

New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives.

The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed.

2,4-Dinitrophenol↗
PubMed1988

Design of angiotensin II derivatives suitable for indirect affinity techniques: potential applications to receptor studies.

The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-AII (Bio-Ahx-AII) and dinitrophenyl- aminohexanoyl-AII (Dnp-Ahx-AII). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (Kd values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala1,Phe(4N3)8)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido AII-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.

Affinity Labels↗
PubMed1988

[Production and characterization of human antirenin antibodies prepared with synthetic immunogens].

Specific blockade of renin angiotensin system can be obtained both by enzymatic inhibitors and by passive and active immunization against renin. Recent studies have shown that synthetic peptides mimicking a protein segment can be used as immunogens to elicit antibodies which react with the parent molecule. In order to develop synthetic antirenin antigens we have selected peptidic sequences from human active renin and synthesized the corresponding peptides to produce antibodies able to recognize the entire human molecule and to inhibit its enzymatic activity. Three dimensional models of human renin were used to predict 17 putative epitopes. Then 18 peptides related to 13 potential antigenic determinants were synthesized by solid or liquid phase technic and 11 were shown to be antigenic when tested by their binding to several polyclonal and monoclonal human renin antibodies. The peptides were injected into rabbits and antisera tested by radio-immunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) showed that nine peptides were immunogenic. Mainly, antiserum raised against the peptide mimicking the beta-hairpin 81-50 of active human renin which lies across the catalytic cleft, produced a 25 p. 100 inhibition of plasma renin activity at a 1: 50 dilution. Immunoglobulins, purified from antibodies raised against this epitopes, bound labelled renin and inhibited enzymatic activity of pure human renin on its synthetic tetradecapeptide substrate, in a dose dependent manner.

Antibodies↗
PubMed1987

Production and characterization of human renin antibodies with region-oriented synthetic peptides.

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.

Amino Acid Sequence↗
PubMed1987

Production and characterization of human renin antibodies prepared with synthetic immunogens.

Renin is an aspartyl protease that plays a key role in regulating blood pressure. The aim of this study was to use synthetic peptides to produce antibodies able to recognize the entire human renin molecule and to inhibit its enzymatic activity. Two three-dimensional renin models were used to predict 17 putative epitopes. Then 18 peptides related to 13 potential antigenic determinants were synthesized by solid- or liquid-phase technique, and 11 were shown to be antigenic when tested by their binding to several polyclonal human renin antibodies. The peptides were injected into rabbits, and antisera tested by radio-immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) showed that nine peptides were immunogenic. The peptide related to the flap region which lies across the renin catalytic cleft showed potentially useful characteristics. Immunoglobulin G, purified from antibodies raised against this epitope, bound labelled renin and inhibited its enzymatic activity in a dose-dependent manner. This approach constitutes the basis for the development of a synthetic antirenin vaccine able to inhibit the renin-angiotensin system specifically.

Amino Acid Sequence↗
PubMed1987

Iodination of vasopressin analogues with agonistic and antagonistic properties: effects on biological properties and affinity for vascular and renal vasopressin receptors.

Twelve L- and D-tyrosine-containing vasopressin analogues were prepared in their mono- and diiodinated forms. These include six arginine vasopressin (AVP) vascular (V1) type antagonists/antidiuretic (V2) agonists, four V1/V2 antagonists, and two V1/V2 agonists, one of which is AVP itself. Ten peptides were iodinated on the tyrosyl residue in position 2; two were iodinated on a tyrosyl amide residue replacing the glycyl amide residue at position 9. All peptides were tested both for their biological activities in vivo (rat vasopressor and antidiuretic tests) and for their ability to bind to vasopressin receptors of the V1 (vascular) and V2 (renal) types from rat liver and rat kidney membranes, respectively. It is shown that monoiodination of the tyrosyl residue in the vasopressin analogues that were tested either preserves or reduces to a highly variable extent the in vivo and in vitro biological activities of these analogues. In most cases diiodonitation resulted in a marked decrease in biological activity. The effects of iodination on the affinity of vasopressin analogues for hepatic V1 receptors and renal V2 receptors were more related to the affinity of the noniodinated peptide for these receptors than to the biological properties (antagonist versus agonist) of the tested analogues, the nature (L versus D) of the iodinated tyrosyl residue, or the position (2 versus 9) at which this residue was introduced. The loss of affinity due to iodination was usually more pronounced for peptides exhibiting high affinity for vasopressin receptors. However, we show that among the monoiodinated peptides some (especially monoiodinated [2-D-Tyrosine]-AVP) retained enough affinity for vasopressin binding sites to suggest that their radioiodinated conterparts would be promising labeled ligands for use in studies in vasopressin receptors.

Animals↗
PubMed1986

Analysis of inactive renin by renin profragment monoclonal antibodies.

Two peptides were synthesized, corresponding to the sequences (-19 to -7) and (-26 to -17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (-19 to -7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.

Antibodies, Monoclonal↗
PubMed1986

Solution synthesis of antigenic and inhibiting peptides of the human renin prosegment-1. [Tyr47, Nle53] preprorenin-(47-60) peptide methyl ester.

The [Tyr47, Nle53] preprorenin (47-60) peptide methyl ester, a tetradecapeptide related to the human renin prosegment, has been synthesized using a three-segment coupling strategy. Selective deprotection of the segments before coupling allowed an easy removal of the final tetradecapeptide side chain-protecting groups by acidolysis and an easy purification. Antibodies raised against this peptide bound the plasmatic inactive renin.

Amino Acid Sequence↗
PubMed1986

Solution synthesis of antigenic and inhibiting peptides of the human renin prosegment-2. [Tyr40] preprorenin-(40-50) peptide methyl ester.

The [Tyr40] preprorenin (40-50) peptide methyl ester, an undecapeptide related to the human renin prosegment, has been synthesized using a stepwise strategy with hydrogenolisable protections on the side chains. The final deprotection was very difficult as observed by 1H NMR and reversed phase HPLC. 2D 1H NMR spectroscopy of the purified peptide allowed the assignment of all protons.

Amino Acid Sequence↗
PubMed1985

Inhibition of human renin by synthetic peptides derived from its prosegment.

The primary structure of human preprorenin has recently been determined from its cDNA sequence. It includes a 46-amino acid NH2-terminal prosegment. Six peptides corresponding to the entire prosegment (9-40), except for the NH2-terminal (1-8) and COOH-terminal (41-46) ends have been synthesized. These peptides were tested for their inhibitory effect on human plasma renin activity. Boc-Tyr-Thr-Thr-Phe-Lys-Arg-Ile-Phe-Leu-Lys-Arg-Met-Pro-OMe (where Boc represents t-butoxycarbonyl and OMe represents methoxy) (h Y(9-20) and its fragment Boc-Leu-Lys-Arg-Met-Pro-OMe h (16-20) were the most potent inhibitors with IC50 values of 2 X 10(-4) and 3 X 10(-4)M, respectively. Peptides located near the COOH-terminus were less inhibitory. The inhibitory capacity of h (16-20) was studied further on highly purified human renin acting on either pure human angiotensinogen or a synthetic human tetradecapeptide substrate. In both of these assays its inhibitory potency was about 10-fold greater than that found on plasma renin activity. Peptide h (16-20) was 3-6 times less potent in inhibiting human renin than its mouse counterpart m (15-19) was in inhibiting mouse renin. Kinetic studies carried out with h (16-20) showed a mixed type of inhibition. When human angiotensinogen was used as substrate, Ki and K'i values were 17.7 +/- 3.9 and 2.9 +/- 0.9 microM, respectively. These studies showed that human renin, like mouse renin and pepsin, can be inhibited by peptides derived from its prosegment. In addition, as in the case of pepsin, they suggest that the NH2-terminal part of the prosegment interacts more strongly with the active enzyme.

Amino Acid Sequence↗
PubMed1985

Immunologic identification of both plasma and human renal inactive renin as prorenin.

Antibodies were raised against a synthetic tetradecapeptide which is a component of the non-renin portion (prosegment) of human renin precursor. Inactive renin from human kidney and plasma strongly adsorbed to a gel coupled to immunoglobulins purified from such an antiserum. These results suggest that renal and circulating inactive human renins contain in their structure the prosegment of prorenin.

Antibody Specificity↗
PubMed1984

Renin inhibition by synthetic peptides related to mouse and human renin prosegments.

Recently the nucleotide sequences of the genes coding for mouse and human renins have been elucidated and the primary structures of the corresponding renin precursors deduced. The existence of a prosegment suggests that renin is biosynthesized as an inactive zymogen prorenin. Three peptides related to the region 11-19 of the mouse submaxillary prosegment have been synthesized and tested as inhibitors of pure mouse renin acting on a synthetic porcine substrate. Inhibition of renin activity was effective, with IC50s in the 10(-6) M range. Butyloxycarbonyl-leucyl-lysyl-lysyl-methionyl-proline methyl ester (15-19) was the most potent inhibitor with a K value of 2.3 X 10(-6) M at 37 degrees C in 0.5 M citrate/phosphate buffer (pH 6.0). Peptides 16-20 and 9-20 of the human renin prosegment and peptides 11-19 and 15-19 of the mouse prosegment were tested on human plasma renin activity and found to be inhibitory, with IC50s of 3 to 5 X 10(-4) M. These results demonstrate that the renin prosegment is a renin inhibitor and support the hypothesis that prorenin is an inactive zymogen.

Animals↗
PubMed1981

[Preparation of antisera against some sequences of somatostatin synthetized on resin. Application to the immunological detection of somatostatin systems: preliminary results (author's transl)].

Specific SRIF(1-14) fragments were synthetized on resin using conventional procedures. Rabbits received subcutaneously peptidyl resins in complete Freund adjuvant emulsion. The presence of antibodies was assessed by immunocytochemical and radioimmunological assays. 1. Peptidyl resins lead to antibodies production; their specificity depends on sequence and molecular configuration of the peptide on the resin. Anti-resin antibodies were not detected. 2. In the brain, SRIF(1-4) (in rat) and SRIF(10-13) (in garden-dormouse) can be demonstrated in neurophysins--positive cells of both paraventricular and supraoptic nucleus, but never in hypothalamic or extrahypothalamic SRIF(1-14)--positive neurophysin negative cells. 3. Endocrine cells of pancreatic islets contain SRIF(6-9) (in man) or SRIF(10-13) (in rat, mouse, garden-dormouse); generally, these cells are not detected by SRIF(1-14) anti-serum. Moreover, SRIF(10-13) positive cells are also detected by specific glucagon antibodies. However, it cannot be concluded that SRIF(10-13) antibodies reveal the common Thr-Phe-Thr-Ser fragment in the entire glucagon molecule. It is postulated that antibodies to several SRIF tetrapeptides reveal molecular fragments provided by the functional cleavage of an hypothetical prohormone or by the inactivation of SRIF(1-14) molecule in target cells.

Animals↗
PubMed1971

[The social worker].

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Allied Health Personnel↗