Biomedical subjects

L Moroder

Publications and source records attributed to L Moroder.

At least 109 records · Page 6Linked to original sources

PubMed1991

Functional cholecystokinin receptors are distinguished kinetically by biotinyl-Tyr-Gly-(Thr28,Nle31)CCK(25-33) in rat pancreatic acini.

Biotinyl-tyrosine-glycine(Thr28,Nle31)CCK(25-33) (BTG-TN-CCK-9) promoted amylase secretion and phosphatidylinositol (PI) metabolism with the same potency and efficacy as TN-CCK-9 in dispersed rat pancreatic acini. A 1 min preincubation of the ligand with a 20-fold excess of streptavidin completely suppressed this biological activity. On the other hand, amylase secretion and PI metabolism prestimulated with BTG-TN-CCK-9 were blocked within 1-5 min after streptavidin addition. [125I]BTG-TN-CCK-9 bound to high (Kd 0.17 nM) and low (Kd 13 nM) affinity receptors. Its dissociation, in the presence of either streptavidin or TN-CCK-9, showed a rapid component and a slow component. The proportion of tracer dissociating slowly increased with increasing preincubation time as did the proportion of tracer that could not be washed away quickly by acidic treatment, in parallel experiments. This phenomenon occurred less readily at 4 degrees C or in the presence of 1 mM CCCP. In acini preincubated for 30 min with 0.3 nM [125I]BTG-TN-CCK-9 and various concentrations of unlabelled BTG-TN-CCK-9, then washed at neutral pH (in order to eliminate rapidly dissociating ligand preferentially), the tracer displacement curve was shifted leftward, suggesting that rapidly dissociating receptors corresponded to low affinity receptors. When acini were preincubated for 1 min with BTG-TN-CCK-9, then washed at neutral pH with buffer only, we observed residual stimulated secretion over the next 30 min period, that correlated with the BTG-TN-CCK-9 concentration offered during the short preincubation period. This phenomenon was inhibited by streptavidin suggesting that intracellularly accumulated intact BTG-TN-CCK-9 (as shown, by radio-HPLC) promoted residual secretion when free to bind again to cell surface receptors in the absence of streptavidin. Taken collectively, these data suggest the coexistence of at least 2 types (or states) of CCK receptors.

Amylases↗

PubMed1991

Synthetic immunogens. Part IV: Conformational studies on gastrin conjugates with the human immunoglobulin G1 hinge peptide 225-232/225'-232'.

Hybrids of the double-chain bis-cystinyl fragment 225-232/225'-232' of human immunoglobulin G1 (IgG1) with the human little-gastrin sequence 2-17 were found to induce in animals a strong antigastrin humoral immune response with antibody titers comparable to those raised with conventional gastrin/carrier-protein conjugates. The observed gastrin receptor-like specificity of the polyclonal antibodies led to the assumption that the gastrin component of the hybrids is still capable of folding into its preferred bioactive structure and thus to express a similar conformational epitope in the dynamic process of recognition by the B-cell receptors. CD measurements on these hybrid compounds in aqueous and aqueous organic media confirmed the free conformational space for the antigenic gastrin moiety, which is essentially randomly coiled in aqueous solution but retains its ability to fold into the gastrin-specific ordered structure in aqueous organic media as used to mimic the water-limited environment of peptides while interacting with target cells at receptor level. The absence of reciprocal conformational restriction in such hybrid molecules suggests that a compact, rigid heterodetic cyclic structure as the hinge peptide is well suited for the multiple attachment of antigenic sequences in view of the preparation of fully synthetic immunogens.

Amino Acid Sequence↗

PubMed1991

Conformational analysis of a IgG1 hinge peptide derivative in solution determined by NMR spectroscopy and refined by restrained molecular dynamics simulations.

The hinge region links the antigen binding Fab part to the constant Fc domain in immunoglobulins. For the hinge peptide derivative [AcThr(OtBu)-Cys-Pro-Pro-Cys-Pro-Ala-ProNH2]2 the assignment of the 1H and 13C resonances was achieved by two-dimensional nmr techniques: total correlation spectroscopy (TOCSY), nuclear Overhauser enhancement spectroscopy (NOESY), rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), heteronuclear multiple quantum coherence (HMQC) transfer, and a HSQC (modified Overbodenhausen experiment) with high resolution in F1, which was several times folded in F1 but still phase correctable. Conformational relevant parameters (78 nuclear Overhauser effect distance restraints, 3JHH for prochiral assignments, temperature gradients) were determined by nmr and served as input data for molecular dynamics (MD) structure refinement. A simulated model compound corresponding to the [Cys-Pro-Pro-Cys]2 core elongated by the peptide chains in the Fab and Fc direction served as a starting structure for the final MD run. The conformation calculated in in vacuo does not agree with the C2 symmetry required from nmr data, but the structure obtained by a water simulation fulfills the requirement. Here the core of the hinge peptide derivative adopts a polyproline II double helix as in the x-ray structure of IgG1. Hence, segments responsible for the internal flexibility are located outside the core as confirmed by the flexibility of the solvent exposed C termini.

Amino Acid Sequence↗

PubMed1991

Somatostatin 28 interacts with CCK receptor in brain and pancreas.

The ability of somatostatin analogs to interact with the binding of cholecystokinin has been studied in pancreatic and brain cortical membranes. Only the 28 amino-acid forms of somatostatin (S28), [Nle8]S28 and [Des Lys14,DTrp22]S28 were found to inhibit the binding of cholecystokinin to rat pancreatic plasma membranes and to increase the amylase release from pancreatic acini. This effect was independent of somatostatin receptor and resulted from an interaction between S28 and CCK receptor. This interaction was not observed with [Leu8, DTrp22, Tyr25]S28, indicating that this analog does not possess the biological activity of the native peptide and that the iodinated peptide could not label specific S28 receptors. S28 interacted also with CCK receptors in cortical brain membranes. Our results support the concept that S28, but not S14, may function as a regulatory molecule at CCK receptors and emphasize that S28 and S14 may be distinct neuromodulators.

Amylases↗

PubMed1991

Enzyme immunoassay with captured hapten. A sensitive gastrin assay with biotinyl-gastrin derivatives.

Two N-terminal biotinyl-gastrin derivatives were synthesized to investigate the effect of the length and chemical properties of the biotin-spacer on both the capture of the hapten by streptavidin or avidin adsorbed on polystyrene, and the antigenicity of the captured peptide. The observed full retainment of antibody binding capacity of the biotinyl-gastrins upon their immobilization, allowed to develop a sandwich-type ELISA with a sensitivity of one order of magnitude better than the standard ELISA with polystyrene-adsorbed gastrin. This hapten capture system reduces desorption particularly pronounced for low mass peptides, and avoids possible modifications or suppression of epitopes by the adsorption process with concomitant reduction of antibody binding affinity of the antigen. This new type of assay procedure may also represent a useful tool particularly for epitope mapping with relatively low mass synthetic protein fragments.

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

Cyclic hexapeptides related to somatostatin. Conformational analysis employing 1H-NMR and molecular dynamics.

We report the conformational analysis of a series of cyclic hexapeptides related to the hormone somatostatin utilizing 1H NMR spectroscopy and NOE restrained molecular dynamics. The conformational preferences and results from biological analysis of these analogs (previous paper) allow for refinement of the current understanding of the structure-activity relationship of somatostatin. For most of the molecules examined, a beta II' turn about the D-tryptophan-lysine residues, postulated to be required for biological activity, was present. From the NOE restrained molecular dynamics, it can be seen that the turn structure is important for the maintenance of the proper orientation of the side chains of the adjacent phenylalanine, tryptophan and lysine. The biologically active analogs have the side chains of lysine and D-tryptophan extended away from the 18-membered ring in close proximity to each other for a significant portion of the dynamic simulations. Although other conformations are accessible and monitored during the simulations, we believe this is important for biological recognition. The absence of the beta II' turn at the D-tryptophan-lysine disrupts this side chain array producing inactive molecules. The role of the bridging region, the Phe-Pro dipeptide, is to stabilize the beta II' turn and help maintain the proper orientation of the biologically important side chains.

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↗

PubMed1989

A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure.

Biochemical studies on receptors for peptides are most often carried out on affinity-labelled (peptide-receptor) complexes. Necessarily, the assumption is made that a covalent (peptide-receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity-labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity-labelling probe in order to minimize structural modifications occurring within the affinity-labelled cholecystokinin (CCK) receptor protein. The probe was 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK-25-33, (125I-ASD-[Thr28,Ahx31]CCK-25-33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK-25-33 via its N-terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I-ASD-[Thr28,Ahx31]CCK-25-33 was time- and temperature-dependent and reversible. At 25 degrees C, a steady-state level was reached after 60 min and half-maximal dissociation after 38 min. Binding was inhibited by [Thr28,Ahx31]CCK-25-33 and L-364-718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I-ASD-[Thr28,Ahx31]CCK-25-33 identified a glycoprotein of Mr 85,000-100,000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu-C generated a main fragment of Mr 30,000-34,000. The same glycoprotein was photoaffinity labelled with 125I-DTyr-Gly-[Ahx28,31,pNO2Phe33]CCK-26-33 (Ahx, 2-aminohexanoic acid; pNO2Phe,p-nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I-ASD-[Thr28,Ahx31]CCK-25-33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.

Affinity Labels↗

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↗

PubMed1988

Synthesis of the bis-cystinyl-fragment 225-232/225'-232' of the human IgGl hinge region.

In human IgGl the two heavy chains are crosslinked in the central portion of the molecule by two disulfide bridges forming a double chain bis-cystinyl cyclic peptide in parallel alignment. For our synthetic studies we have chosen the sequence portion 225-232/225'-232', i.e. [H-Thr-C1ys-Pro-Pro-C1ys-Pro-Ala-Pro-OH]2. By the use of a combination of the S-tert.-butylthio and the S-acetamidomethyl groups selective cysteine pairings in two successive steps produced the hinge hexadecapeptide in parallel and antiparallel alignment as homogeneous and well characterized compounds. Thiol disulfide interchange experiments on the antiparallel dimer led to over 90% conversion to the parallel isomer. Similarly random air-oxidation was found to generate again mainly the parallel dimer, thus strongly suggesting that this sequence portion contains sufficient structural information for a correct assembly of the two heavy chains of immunoglobulins without decisive contribution of a protein disulfide isomerase.

Amino Acid Sequence↗

PubMed1988

A new reagent for the preparation of glycoconjugates.

The thioglycoside derivative 2-carbazoylethyl 1-thio-beta-D-galactopyranoside hydrochloride was synthesized. Conversion of the carbazoyl group into the reactive azidocarbonyl function leads to a well suited reagent for the preparation of glycoconjugates via amidation of proteins or synthetic carriers in aqueous media.

Glycoconjugates↗