Biomedical subjects

E Wünsch

Publications and source records attributed to E Wünsch.

At least 19 recordsLinked to original sources

PubMed2001

The peptides of alpha-aminosuberic acid II. Synthesis of deamino-dicarba-eel-calcitonin sequence 1-9.

The paper describes the synthesis of Asu6-octapeptide derivatives by condensing two alternative pentapeptide fragments with Asu-containing tripeptides. After partial deprotection these linear peptides compounds are subject to cyclization experiments aimed to give the N-terminal [1-9] sequence of deamino-dicarba-eel calcitonin. This is a key substance for the semi-synthesis of the respective analogues of eel calcitonin.

Amino Acid Sequence↗

PubMed2001

Poor responder-high responder: the importance of soluble vascular endothelial growth factor receptor 1 in ovarian stimulation protocols.

This study was designed to detect vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-1) in follicular fluid specimens and to evaluate the importance of sVEGFR-1 with respect to ovarian response to gonadotrophin stimulation. A total of 69 patients was treated for IVF with recombinant human follicle stimulating hormone (FSH). Concentrations of VEGF and sVEGFR-1 were quantified in follicular fluids from oocyte retrievals. Patients were designated to three groups with respect to the number of harvested oocytes: group A, 1-5 oocytes; group B, 6-10 oocytes; group C, >10 oocytes. In group A, 1133 +/- 870 pg VEGF/ml follicular fluid per oocyte were quantified, in group B 426 +/- 262 pg VEGF/ml per oocyte, and in group C 274 +/- 179 pg VEGF/ml per oocyte. Soluble VEGFR-1 concentrations resulted in 1200 +/- 523 pg/ml follicular fluid per oocyte in group A, 255 +/- 193 pg/ml per oocyte in group B, and 79 +/- 69 pg/ml per oocyte in group C. No free sVEGFR-1 could be detected in any follicular fluid. An index to estimate the biological activity of VEGF by dividing VEGF/sVEGFR-1 revealed an increasing availability of VEGF with higher ovarian response to gonadotrophin therapy. In group A this index was 1.03, in group B 1.71, and in group C 3.21. A delicate balance between VEGF and sVEGFR-1 is necessary to allow an adequate ovarian reaction to gonadotrophin therapy. Excess of bio-active VEGF increases the risk for ovarian hyperstimulation syndrome. Excess of sVEGFR-1 results in poor response and goes in parallel with reduced chances for conception.

Adult↗

PubMed2000

The peptides of alpha-aminosuberic acid I. New intermediates for synthetic studies.

The paper describes the synthesis of alpha-aminosuberic acid derivatives suitable for the synthesis of peptides. These include Z-, Boc- and Fmoc-protection on the alpha-amino group, benzyl ester, Boc-hydrazide and Z-hydrazide as well as the free carboxylic function in the side chain, and methyl ester, benzyl ester or free alpha-carboxylic group. Their use is demonstrated on the synthesis of the respective derivatives of Asu-Val-Leu. The enzyme catalyzed reaction was successfully used both as a route to L-Asu from the D,L-compound as well as for the direct synthesis of the optically active tripeptide derivative from the Z-D,L-Asu-OH.

Amino Acids, Dicarboxylic↗

PubMed1997

Enzymatic semisynthesis of dicarba analogs of calcitonin.

The semisynthesis of eel[L-alpha-aminosuberic acid]calcitonin (elcatonin) was accomplished by alpha-chymotrypsin-catalyzed coupling of two peptide segments in a single reaction without the protection of any functional group. The eel calcitonin-(10-32)-peptide was prepared by a gene manipulation. The esters of cyclic desamino nonapeptide (segment 1-9) were synthesized by the conventional solution method including a thermolysin-mediated resolution of DL-alpha-aminosuberic acid via one-step tripeptide synthesis leading to the 7-9 sequence. The main aim of this work was to determine the conditions for protease-catalyzed segment condensation while avoiding a concurrent cleavage of other proteolytically labile peptide bonds in the hormone. The alpha-chymotrypsin condensation strategy under usual conditions led to a complicated mixture of split products with an insignificant amount of the required peptide. When the coupling reaction was carried out at 0 degrees C, the reaction resulted in a satisfactory yield of elcatonin with the complete conversion of the acyl donor (1-9 segment) accompanied by negligible concurrent peptide bond digestion. The same strategy was employed for the preparation of analogous dicarba salmon calcitonin using a synthetic elcatonin-(10-32)-peptide. Both calcitonin analogs exhibited hypocalcemic activity corresponding to the international standard of elcatonin. We demonstrate in this work a peptide synthesis based on the combination of genetic engineering, chemical synthesis and proteinase-catalyzed segment condensation. This approach enables effective incorporation of an unnatural amino acid into calcitonins without the side-chain protection.

Amino Acid Sequence↗

PubMed1995

Effect of 17-norleucine-VIP on gastroduodenal motility relative to serum VIP concentration and blockade of NOS.

Mechanical and electrical activity in the antrum, pylorus, and duodenum was evaluated in the conscious dog, instrumented with seven strain gauges and five platinum electrodes. 17-Norleucine-vasoactive intestinal peptide (17-N-Leu-VIP) or 17-N-Leu-VIP plus NG-nitro-L-arginine methyl ester (L-NAME) was injected intra-arterially close to the pylorus to identify influences of nitric oxide (NO) on effects of VIP. VIP concentration was measured by radioimmunoassay in serum samples collected from the cubital and portal veins before and up to 2 h after VIP injection. VIP (0.004-0.006 mg.kg-1.10 min-1) abolished phasic contractions in the interdigestive state for 16.8 min and in the digestive state for 14.4 min, whereas whole serum VIP concentration rose above 42.4 +/- 13 pmol/l. Administration of L-NAME did not significantly influence the effects of VIP. Aftereffects of VIP, consisting of a reduced motility index, lasted 33 +/- 10.6 min in the interdigestive state and 44.5 +/- 42 min in the digestive state. This VIP aftereffect in the interdigestive state was shortened in time by the addition of L-NAME. The results overall suggest that NO release is a factor only in the aftereffects of VIP.

Animals↗

PubMed1995

Effect of human gastrin-17 with and without acid suppression on human esophageal motility.

The putative role of gastrin for the regulation of esophageal motility is a matter of debate. Accordingly it was the aim of this study a) to examine if physiological postprandial plasma levels of human gastrin-17 (hG-17) can affect esophageal motility, especially the pressure of the lower esophageal sphincter (LESP), and b) to assess the contribution of augmented acid secretion during gastrin infusion. In a first series of experiments postprandial plasma gastrin levels were determined in 8 healthy volunteers following the ingestion of a mixed meal. Gastrin rose from a baseline of 21 +/- 2 pg/ml to 67 +/- 8 pg/ml and returned almost to basal levels within 120 minutes. In a second experimental series the effect of i.v. synthetic human gastrin-17 (hG-17) was studied in 17 volunteers. At a lower dose of 0.75 ng/kg/min hG-17 increased plasma gastrin to 62 +/- 7 pg/ml while a higher dose of 1.5 ng/kg min elicited a supraphysiological increase to 119 +/- 11 pg/ml. Infusion of hG-17 caused a significant increase of the LESP from 19.0 to 25.8 mmHg (p < 0.05, low dose) and from 18.5 mmHg to 23.3 mmHg (p < 0.05, high dose) when compared to the effect of i.v. saline. To exclude effects of augmented acid secretion during hG-17 infusion the experiments were repeated after complete blockade of acid secretion with famotidine 40 mg i.v. After famotidine pretreatment hG-17 caused a similar increase of LESP from 20.1 to 25.9 mmHg (low dose) and from 19.9 to 24.1 mmHg (high dose).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

In man histamine and muscarinergic mechanisms are essential mediators of acid secretion in response to synthetic human gastrin (1-17).

It is still controversial whether gastrin stimulates acid secretion by interacting with specific gastrin receptors on parietal cells or via endogenous mediators, e.g., histamine. Therefore, it was our aim to determine in healthy human volunteers (n = 14; 3 females, 11 males; age 23-28 years) the degree by which the specific histamine H2-receptor antagonist famotidine or the muscarinergic antagonist atropine block acid secretion in response to synthetic human gastrin (hG) (1-17). Famotidine was deliberately administered at a supramaximal dose (40 mg i.v. bolus) to reliably block any and all effects of endogenous histamine on the parietal cells. After an overnight fast famotidine or saline were injected i.v., and gastric secretions were collected via a nasogastric tube for the ensuing 60 min to assess basal secretion. Thereafter, hG (1-17) was infused for 60 min in randomized order at two different rates: 0.75 ng/kg/min resulting in postprandial plasma gastrin levels (55-66 pg/ml), and 1.5 ng/kg/min yielding supraphysiologic levels (110-136 pg/ml). Both rates increased basal acid secretion (meq/10 min) from 0.5 +/- 0.2 to 3.8 +/- 0.6 and 4.7 +/- 0.5, respectively. Famotidine abolished basal acid secretion and completely blocked acid and volume secretion in response to both hG (1-17) doses. After injection of famotidine both hG (1-17) doses resulted in plasma levels exceeding those in controls by 18-27 pg/ml. A similar increase (14-16 pg/ml) was observed after famotidine injection without simultaneous hG (1-17) infusion indicating that this increase was due to the release of endogenous gastrin when the acid feedback inhibition was blocked by famotidine. To study a potential additional role of cholinergic mechanisms the effect of atropine (7 micrograms/kg i.m.) on hG (1-17)-induced acid secretion was examined. Atropine reduced basal acid secretion from 0.8 +/- 0.1 to 0.1 +/- 0.08 meq/15 min. Similarly, the response to 0.75 ng/kg/min hG (1-17) was reduced by 72.9%. Basal gastrin release was not altered by atropine which, however, tended to increase serum gastrin levels during infusion of hG (1-17) by 16-24 pg/ml. We conclude that in man histamine and muscarinic mechanisms are essential mediators of gastrin-stimulated acid secretion. The present data argue against a significant direct effect of gastrin alone on human parietal cells but rather support potentiating interaction with histamine and cholinergic mechanisms.

Adult↗

PubMed1991

A 1H NMR study of human calcitonin in solution.

Human calcitonin (hCT) has been investigated by NMR at 400 MHz in DMSOd6 and in an 85% DMSOd6-15% 1H2O (v/v) cryoprotective mixture. All backbone and side-chain resonances have been assigned, and the secondary structure has been determined in both solvents. In DMSOd6, the simultaneous presence of d alpha N, dNN, and some specific weak medium-range nuclear Overhauser effects, together with the amide temperature coefficients and the analysis of the NH-alpha CH spin-spin coupling constants, indicates that hCT is highly flexible but with three domains (comprising segments Asn3-Gly10, Gln14-Thr21, and Thr25-Ala31) in extended conformations which dynamically transform into isolated beta turns in the N- and C-terminal regions and into adjacent tight turns, resembling a 3(10) helix structure, in the central part. The DMSO-water mixture rigidifies the polypeptide chain, favoring an ordered, extended conformation. NOESY data indicate the presence of a short double-stranded antiparallel beta sheet in the central region made by residues 16-21 and connected by a two-residue hairpin loop formed by residues 18 and 19. Two tight turns, formed by residues 3-6 and 28-31, were also identified. The central beta sheet does not favor an amphipathic distribution of the residues as found for salmon calcitonin [Motta, A., Castiglione Morelli, M. A., Goud, N., & Temussi, P. A. (1989) Biochemistry 28, 7998-8002]. This is in agreement with the smaller tendency of hCT to form the amphipathic alpha helix, postulated to be responsible for the interaction of hCT with lipids. The possible role of the cis-trans isomerism of Pro is discussed.

Amino Acid Sequence↗

PubMed1991

Fully synthetic immunogens. Part III. Synthesis of hinge-peptide/gastrin conjugates and their immunological properties.

As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C-termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N-termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1-14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225-232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor-like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big-gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.

Amino Acid Sequence↗

PubMed1991

Fully synthetic immunogens. Part II. Studies on parallel dimerization of the human IgG1 hinge-fragment 225-232 with N- or C-terminal gastrin related extensions.

The bis-cysteinyl hinge-fragment 225-232 of human IgG1 has been extended at the N- or C-terminus with Nle15-desamido-human-little-gastrin-[5-17] and Nle15-human-little-gastrin-[5-17]-NH2, respectively. Thermodynamically controlled air oxidation of the resulting bis-cysteinyl-peptides led to the predominant formation of the corresponding dimers in parallel alignment despite the incorporation of the immunoglobulin-unrelated gastrin-sequences. These surprising results confirm the high degree of structural information inherent in the hinge-sequence and its intrinsic tendency to fold into the correct structure in terms of cysteine pairings. This protein subdomain-the hinge-peptide-is therefore well suited as core molecule for the design of fully synthetic immunogens with multiple attachment of antigenic determinants.

Amino Acid Sequence↗

PubMed1990

Cyclic hexapeptides related to somatostatin. Synthesis and biological testing.

As a continuation of our program to study the structure-function relationship of the peptide hormone somatostatin, we report the synthesis and biological potencies of a series of cyclic hexapeptide analogs related to somatostatin. The parent peptide of this series was designed by Veber and coworkers, c[-Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11-], and has been reported to be superactive in the inhibition of the release of growth hormone. (The superscript numbers refer to the positions of residues in native somatostatin). The series of analogs has been designed to examine the role of the so-called bridging region, Phe11-Pro6, which has been postulated to be important in maintaining the proper conformation of the biologically active tetrapeptide, Phe-D-Trp-Lys-Thr. We have incorporated peptidomimetics and the retro-inverso modification into the bridging region of the molecule, with the aim of affecting the conformational preferences found in the parent peptide. Results from the biological assay--in vitro inhibition of growth hormone--and the conformational analysis (adjoining paper) of our analogs will provide insight into the relationship between structure and biological activity of somatostatin.

Amino Acid Sequence↗

PubMed1990

On the hypothetical protein F154 of the TTV1 virus from Thermoproteus tenax. Part III: Immunological identification of the protein with anti-peptide antibodies.

To assess the expression of an hypothetical protein F154 encoded in an open reading frame of the DNA sequence of the virus TTV1 identified in the archaebacterium Thermoproteus tenax, two synthetic peptides related to characteristic repeats of the primary structure and corresponding to the sequence portions [Ser81] (73-84) and (79-101) were selectively grafted at their N-termini to the surface-exposed single cysteine residue of iso-1-cytochrome c via the maleimide thiol principle. Antisera were raised in rabbits against the two conjugates and the antipeptide-(79-101) immunoselected antibodies were found to crossreact with a strong protein band in Western blots of cell lysates of TTV1-infected bacteria, thus confirming expression of the protein F154 in Thermoproteus tenax.

Amino Acid Sequence↗

PubMed1990

Side products in the synthesis of secretin.

Analytical studies on side products in the synthesis of secretin led to the discovery of a further side reaction, involving conversion of the carboxamide function of glutamine to glutamic acid gamma-methyl ester. The chemical structure of the side products was determined by NMR spectroscopy upon their tryptic digestion and isolation of the modified secretin fragments. Possible pathways for the observed side reaction are discussed.

Amino Acids↗

PubMed1989

Conformational and biological properties of the Ala10 analogue of human des-Trp1,Nle12-minigastrin.

Synthesis, conformation, and biological properties of the Ala10 analogue of des-Trp1,Nle12-minigastrin are reported. Replacement of the Gly residue in the original sequence with Ala remarkably changes the conformational preference of the hormone in trifluoroethanol. CD and NMR results indicate that the conformational change is mainly located in the C-terminal portion of the molecule, with probable extension of the N-terminal alpha-helix throughout the entire sequence. The structural modification causes a 10-fold decrease in the biological potency of the hormone, which is about as active as the C-terminal tetrapeptide amide. These findings support our previous hypothesis that the optimal bioactive conformation of the native hormone is U-shaped, with mutual interactions among the two end segments.

Alanine↗

PubMed1989

Synthesis of cholecystokinin-related peptides and their biological properties.

Two tyrosine-O-sulfate containing cholecystokinin-related peptides were synthetized, i.e. the undeca- and the dodecapeptide amide corresponding to the C-terminus of this gut hormone; in the undecapeptide the N-terminal serine was O-substituted with the 2-deoxy-alpha-D-galactosyl group. Within the limits of error of the biological assay systems the two CCK peptides exhibited potencies in stimulating amylase secretion from dispersed rat pancreatic acini comparable to those of the CCK deca-, nona- and octapeptide.

Amino Acid Sequence↗

PubMed1989

Synthesis of thiol-functionalized N-acetylmuramyl peptide congeners suitable for their conjugation to target molecules.

N-Acetylmuramyl-dipeptide and tripeptide derivatives containing at the C-terminus a masked thiol function, i.e. the S-tert-butylthiocysteamine residue, were synthesized via direct condensation of N-acetylmuramic acid with the peptide moiety using the dicyclohexylcarbodiimide/N-hydroxysuccinimide procedure. Reduction with tributylphosphine in aqueous organic media generates the free thiol function for a selective conjugation of these immunomodulants with target molecules via unsymmetrical disulfide bridging with a second thiol group by the sulfenohydrazide procedure or via thio-ether linkage by the addition to maleimido--or aziridine-derivatives.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1989

Muramyl-peptide/gastrin conjugates as potential immunogens.

For a selective covalent linkage of muramyl-peptides with human-little-gastrin the maleimide-thiol reaction principle was adopted. For this purpose thiol-functionalized muramyl-peptide derivatives, i.e. N-acetyl-muramyl-alanyl-D-isoglutaminyl-cysteamine and N-acetyl-muramyl-alanyl-D-isoglutaminyl-N epsilon-palmitoyl-lysyl-cysteamine, were reacted with N alpha-maleoyl-beta-alanyl-[15-methoxinine]-human-little-g ast rin-I-[2-17]. The resulting gastrin conjugates were used in immunization experiments on rabbits and mice. Unexpectedly, these gastrin derivatives proved to be poorly immunogenic despite the built-in immunoadjuvanticity. The titers of the antipeptide antibodies as well as of the unspecific immunoglobulins were in the range of those of the control group.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1988

Receptor occupancy and adenylate cyclase activation in rat liver and heart membranes by 10 glucagon analogs modified in position 2,3, 4, 25, 27 and/or 29.

Rat liver and heart membranes were tested for adenylate cyclase activation by glucagon and 10 glucagon analogs mono- or polysubstituted in positions 2-4, 25, 27 and/or 29. The first membranes were, in addition, examined for the capacity of glucagon analogs to inhibit the binding of [125I]iodoglucagon. The monophasic slope of dose-effect curves suggested interaction with one class of glucagon receptors in both tissues, receptors in liver being more sensitive to the ligands and more efficiently coupled to adenylate cyclase than heart receptors. Structure-activity studies on liver membranes revealed that modifications of the beta-turn potential in the 2-4 region by single residue substitutions could lead to partial agonists (with D-Gln3 or Phe4) or to a superagonist (with D-Phe4). The importance of a proper alpha-helix conformation in the C-terminal part of glucagon for binding affinity was also obvious: replacing Trp25, Met27 and Thr29 in combination by Phe25, Leu27 and Thr29-NH2 increased the affinity while single or combined substitutions with Gly25 and/or Nle27 sharply decreased the affinity. Similar trends were less evident but still obvious on heart membranes.

Adenylyl Cyclases↗