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PubMed1992

Suprasensitivity to calcitonin gene-related peptide but not vasoactive intestinal peptide in women with chronic pelvic pain.

Chronic pelvic pain in women is associated with radiological evidence of pelvic venous dilatation and reduced flow, termed 'pelvic congestion'. The aim of this study was to elucidate a possible role in this condition for vasoactive intestinal peptide and calcitonin gene-related peptide, both localized in perivascular nerves in the ovaries and uterus. Healthy volunteers and women with chronic pelvic pain and venous congestion received intravenous infusions of vasoactive intestinal peptide (n = 15), calcitonin gene-related peptide (n = 15) or a bland infusate (n = 7). Changes in the uterovaginal and skin blood flow were assessed by continuous measurement of vaginal, axillary, cheek and hand temperature. During calcitonin gene-related peptide infusion median hand temperature changes were +0.97 degrees C in women with pelvic pain and -0.03 degrees C in healthy volunteers (p < 0.05). There were no differences between groups in hand and cheek temperature responses to vasoactive intestinal peptide infusion. Vasoactive intestinal peptide and calcitonin gene-related peptide appeared to dilate the uterovaginal vasculature in healthy subjects but not in those with pelvic pain. Vasoactive intestinal peptide and calcitonin gene-related peptide did not provoke pain in healthy subjects but in those with pelvic pain, symptoms were significantly exacerbated during calcitonin gene-related peptide infusion but not by vasoactive intestinal peptide. Changes in plasma follicle stimulating hormone, luteinizing hormone and oestradiol during either infusion were not significant. These findings indicate greater sensitivity to calcitonin gene-related peptide in women with pelvic pain and suggest a possible underlying neurovascular disorder.

Adult↗
PubMedDate not supplied

Improvement of the T-cell response to a non-immunogenic peptide by its tandem association with a highly efficient T-helper peptide.

The 45-69 peptide, an helper T-cell epitope derived from HIV nef protein, is strongly immunogenic. A T-cell proliferative response was observed following immunization of Lou/M rats with 45-69 peptide administered in low dose and without any adjuvant. It is already known that the T-cell response to the 115-131 peptide of Sm28GST antigen, a protein of the parasite Schistosoma mansoni, requires the presence of a carrier or the use of peptidic constructs. We demonstrate here that a T-cell response against the 115-131 peptide can be obtained in the absence of adjuvant using peptidic constructs (115-45 and 45-115 peptides) resulting from tandem synthesis of 115-131 and 45-69 peptides. A covalent association of both peptides is necessary, since the co-injection of 45-69 and 115-131 peptides is not sufficient to induce a detectable anti-115-131 T-cell response. The mutual orientation between the respective tandem peptides (45-115 and 115-45) is critical for the T-cell response. These peptidic constructs possess distinct properties of antigenicity and immunogenicity but both allowed to reveal the existence of a 115-131 specific T-cell response normally undetectable using 115-131 peptide alone. This immunopharmacological approach should be useful in the rational design and construction of vaccines.

Adjuvants, Immunologic↗
PubMed1991

Evidence that some preganglionic sympathetic neurons in the rat contain vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunoreactivities.

Physiological studies have established that preganglionic sympathetic nerve fibers innervating the rat superior cervical ganglion release a second transmitter, in addition to acetylcholine. Based on pharmacological and histochemical investigations, possible candidates for this non-cholinergic neurotransmitter include vasoactive intestinal peptide and peptide histidine isoleucine amide. For example, previous immunohistochemical studies have demonstrated that antisera raised against both of these peptides stain neural processes in the rat preganglionic cervical sympathetic trunk and in the superior cervical ganglion. In the present study, it was found that, when the cervical sympathetic trunk was ligated, vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities built up on both sides of the ligature. In addition, examination of the thoracic spinal cord in colchicine-treated animals revealed vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivies in neuronal cell bodies in the intermediolateral cell column and in the region of the lateral funiculus adjacent to it. In a second group of animals in which retrograde tracing techniques were used, these two regions of the spinal cord were shown to contain most of the cell bodies of the preganglionic neurons that project to the superior cervical ganglion. Smaller numbers of retrogradely labeled neurons were found dorsal to the central canal and in the nucleus intercalatus. When either vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunostaining and retrograde labeling were examined in the same animals, double-labeled neurons were found in the intermediolateral cell column and in the lateral funiculus. These data demonstrate that vasoactive intestinal peptide- and peptide histidine amide-like immunoreactivities are present in certain of the preganglionic neurons that project to the superior cervical ganglion, supporting the hypothesis that vasoactive intestinal peptide and peptide histidine isoleucine amide are released in the ganglion when these preganglionic neurons are activated.

Animals↗
PubMed1994

[Pro34]peptide YY is a Y1-selective agonist at peptide YY/neuropeptide Y receptors.

We have investigated binding and functional effects of a new peptide YY analogue, [Pro34]peptide YY, at Y1 and Y2-like subtypes of receptors for peptide YY and neuropeptide Y. In binding studies [Pro34]peptide YY had a similarly high affinity as peptide YY to human Y1-like receptors in SK-N-MC cells, a human neuroblastoma cell line of presumed neurogenic origin, and HEL cells, a human cell line derived from a patient with Hodgkin's disease. In functional studies [Pro34]peptide YY stimulated Ca2+ elevations in both Y1-like receptor cell lines with similar potency and efficacy as peptide YY. In contrast to peptide YY [Pro34]peptide YY was 1000-fold less potent in binding to Y2-like receptors in porcine splenic membranes and lacked agonistic effects in another Y2-like receptor-mediated model system, i.e. inhibition of [3H]serotonin release from rat cerebral cortical slices. Thus, [Pro34]peptide YY is a highly Y1-selective full agonist of peptide YY/neuropeptide Y receptors. [Pro34]peptide YY could be useful for studying the importance of Y receptor subtypes in mediating peptide YY physiological actions.

Amino Acid Sequence↗
PubMed2005

Rational design of complementary peptides to the betaAmyloid 29-42 fusion peptide: an application of PepDesign.

Peptides in solution currently exist under several conformations; an equilibrium which varies with solvent polarity. Despite or because of this structure versatility, peptides can be selective biological tools: they can adapt to a target, vary conformation with solvents and so on. These capacities are crucial for cargo carriers. One promising way of using peptides in biotechnologies is to decipher their medium-sequence-structure-function relationships and one approach is molecular modelling. Only few "in silico" methods of peptide design are described in the literature. Most are used in support of experimental screening of peptide libraries. However, the way they are made does not teach us much for future researches. In this paper, we describe an "in silico" method (PepDesign) which starts by analysing the native interaction of a peptide with a target molecule in order to define which points are important. From there, a modelling protocol for the design of 'better' peptides is set. The PepDesign procedure calculates new peptides fulfilling the hypothesis, tests the conformational space of these peptides in interaction with the target by angular dynamics and goes up to the selection of the best peptide based on the analysis of complex structure properties. Experimental biological assays are finally used to test the selected peptides, hence to validate the approach. Applications of PepDesign are wide because the procedure will remain similar irrespective of the target which can be a protein, a drug or a nucleic acid. In this paper, we describe the design of peptides which binds to the fusogenic helical form of the C-terminal domain of the Abeta peptide (Abeta29-42).

Amino Acid Sequence↗
PubMed2004

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides.

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.

Animals↗
PubMed1994

Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor.

The islets of Langerhans contain four distinct endocrine cell types producing the hormones glucagon, insulin, somatostatin and pancreatic polypeptide. These cell lineages are thought to arise from a common, multipotential progenitor cell whose identity has not been well established. The pancreatic and intestinal hormone, peptide YY, has been previously identified in glucagon-producing cells in islets; however, transgenic mice expressing Simian Virus 40 large T antigen under the control of the peptide YY gene expressed the oncoprotein in beta, delta and pancreatic polypeptide cells, and occasionally developed insulinomas, suggesting relationships between peptide YY-producing cells and several islet cell lineages. The four established pancreatic islet cell types were examined for coexpression of peptide YY in islets of normal and transgenic mice throughout development. Peptide YY immunoreactivity was identified in the earliest endocrine cells in the fetal pancreas and was coexpressed in each islet cell type during development. Peptide YY showed a high degree of co-localization with glucagon- and insulin-producing cells in early pancreatic development, but by adulthood, peptide YY was expressed in less than half of the alpha cells and was no longer expressed in beta cells. Peptide YY was also coexpressed with somatostatin and pancreatic polypeptide when these cell types first appeared, but most delta and pancreatic polypeptide cells continued to express peptide YY throughout development. The use of conditions that distinguish peptide YY from the related peptides, pancreatic polypeptide and neuropeptide Y, as well as the ability of the peptide YY gene to direct expression of a reporter gene in islets of transgenic mice, establishes expression of peptide YY in the earliest pancreatic endocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Extensive alanine substitutions increase binding affinity of an influenza nucleoprotein peptide to HLA-Aw68 and do not abrogate peptide-specific CTL recognition.

Class I MHC molecules bind peptides in the endoplasmic reticulum and present them at the cell surface to circulating CD8+ T cells for analysis. We have examined binding of peptides and stabilization of HLA-Aw68 class I molecules using synthetic peptide variants of an influenza virus nucleoprotein peptide, NP91-99 (KTGGPIYKR). We have demonstrated that insertion of increasing numbers of alanines in the center of the peptide (between P and I), to increase a natural bulging out of the peptide-binding cleft, results in a large decrease in thermal stability. Although there is a great decrease in the t 1/2 of the MHC/peptide complex for NP-1A compared with NP91-99, a T cell line, stimulated by NP91-99, recognizes NP-1A efficiently. Peptide variants with three or more alanines do not show saturable binding to HLA-Aw68 and also are not recognized by the T cell line. Thermal studies show that polyalanine peptides with minimal anchors and nearly all TCR contact residues exchanged stabilized HLA-Aw68 to high temperatures. Additionally, some of these polyalanine peptides are recognized by T cell lines generated against NP91-99. Analysis of the peptide sequences show that the stabilization effects are not due to the hydrophobicity of the peptide. These data suggest that the strength of binding of peptides to HLA-Aw68 is not only dictated by the presence of anchor residues but also by the lack of unfavorable contacts between the peptide ligand and class I MHC-binding cleft.

Alanine↗
PubMed2000

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217.

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.

Amino Acid Motifs↗
PubMed2000

Identification of a ras oncogene peptide that contains both CD4(+) and CD8(+) T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization.

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2(d)) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. This peptide reflected ras sequence 4-16, and contained the substitution of Gly to Val at position 12 ¿i.e., 4-16(Val12)¿. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide ¿i.e., 4-12(Val12)¿, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Valpha- and Vbeta-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Valpha1 and Vbeta9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4(+) T cell helper peptide ¿i.e., 5-17(Val12)¿ lacking the class I N-terminus anchor site, enhanced the production of the CD8(+) T cell response. Finally, immunization with plasmid DNA encoding the ras 4-16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4(+) and CD8(+) T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8(+) CTL response, likely due to the intimate coexistence of CD4(+) help, which may have implications in peptide- or DNA-based immunotherapies.

Animals↗
PubMed1994

Systematic identification of H-2 Kd binding peptides and induction of peptide specific CTL.

Most peptides with putative MHC I restricted sequence motifs do not bind to the corresponding MHC I nor induce cytolytic T cells. There exist additional constraints which limit peptide binding and immunogenicity. To identify immunogenic peptides in novel protein sequences, it will be necessary to first evaluate peptide binding to MHC I. In this study, a soluble single chain fusion protein SC-Kd was used to evaluate potential Kd binding peptides from the sequences of mouse mammary tumor virus gag and env proteins. A total of 27 peptides were identified which displayed the reported Kd restricted motif. Of the 27 peptides, six demonstrated strong to moderate binding to SC-Kd. The strongest binding peptides expressed tyrosine or phenylalanine at position 2 and leucine at the C-terminus. The capability of MMTV peptides to induce CTL corresponds to their SC-Kd binding activity. Of the six peptides that demonstrated moderate to strong binding, five induced CTL in BALB/c mice. These peptides induced CTL after 1-3 in vivo immunizations followed by 5 day in vitro stimulation. Furthermore, a single in vitro stimulation of naive lymphocytes with strong-binding G425 was sufficient to induce significant CTL activity. Weak or non-binding peptides did not induce CTL. Therefore, peptide binding to SC-Kd is a predictive indicator of CTL inducing activity.

Amino Acid Sequence↗
PubMedDate not supplied

Improvement of the T-cell response to a non immunogenic peptide by its tandem association with a highly efficient T-helper peptide.

The 45-69 peptide, an helper T-cell epitope derived from the HIV nef protein is strongly immunogenic. A T-cell proliferative response was observed following immunization of Lou/M rats with 45-69 peptide administered in low dose and without any adjuvant. It is already known that the T-cell response to the 115-131 peptide of Sm28GST antigen, a protein of the parasite Schistosoma mansoni, requires the presence of a carrier of the use of peptidic constructs. We demonstrate here that a T-cell response against the 115-131 peptide can be obtained in the absence of adjuvant using peptidic constructs (115-45 and 45-115 peptides) resulting from tandem synthesis of 115-131 and 45-69 peptides. A covalent association of both peptides is necessary, since the coinjection of 45-69 and 115-131 peptides is not sufficient to induce a detectable anti-115-131 T-cell response. The mutual orientation between the respective tandem peptides (45-115 and 115-45) is critical for the T-cell response. These peptidic constructs possess distinct properties of antigenicity and immunogenicity but both allowed to reveal the existence of a specific T-cell response normally undetectable using 115-131 peptide alone. This immunopharmacological approach should be useful in the rational design and construction of vaccines.

Adjuvants, Immunologic↗
PubMed1995

Passive adsorption of immunologically active and inactive synthetic peptides to polystyrene is influenced by the proportion of non-polar residues in the peptide.

A well-known drawback in the use of synthetic peptides as solid-phase antigens in immunoassays is that positive controls confirming the presence of the peptide on the solid phase are not always present. We therefore evaluated the applicability of a recently described enzyme immunoassay (EIA) method by which the presence of peptides is detected by biotinylation (BioEIA) of alpha- and/or epsilon-amino groups after passive adsorption. This approach allows the rapid screening of a large number of proteins and peptides in respect to passive adsorption to plastic surfaces. When using irradiated polystyrene microplates we found that 240 (94%) of 256 synthetic peptides, covering 85% of the complete hepatitis C virus (HCV) sequence, passively adsorbed to polystyrene. When comparing the results from the BioEIA to the peptide reactivity of human sera it was obvious that the absence of serum reactivities was not due to lack of peptide adsorption to the plates. Using 192 peptides the relation between the signal-to-cutoff ratio (S/CO) in the BioEIA and the amino acid content of the individual peptides was further analyzed. The S/CO ratio was related to the number of epsilon NH2 groups (Lys residues) present in the peptide (P < 0.001, Kruskal-Wallis). We separately related the amino acid content of 68 peptides with Lys and 124 peptides lacking Lys to the S/CO ratio in the BioEIA. In both cases it was found that an increasing amount of nonpolar residues such as Ala, Phe, Ile, Met, and Val (P < 0.05, respectively) in the peptides was related to a lower S/CO ratio in the BioEIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed2001

Molecular mimicry in type 1 diabetes mellitus revisited: T-cell clones to GAD65 peptides with sequence homology to Coxsackie or proinsulin peptides do not crossreact with homologous counterpart.

Type 1 diabetes mellitus is a T-cell mediated autoimmune disease in which the insulin-producing pancreatic beta cells are selectively destroyed. Molecular mimicry and T-cell crossreactivity to beta-cell autoantigens and environmental agents with sequence similarities have been a proposed mechanism underlying the pathogenesis of type 1 diabetes, but actual crossreactivity has not yet been demonstrated. We isolated and investigated T cells reactive to GAD65 peptides and homologous peptides of the Coxsackie virus protein P2C and proinsulin from recent onset type 1 diabetes patients, and tested their fine specificity and cytokine production profile. Six T-cell lines specific for GAD65 peptides (amino acids 491-530) with homology to proinsulin (B20-C14) were isolated from six newly diagnosed patients with type 1 diabetes, but none of the stable T-cell lines crossreacted to the homologous proinsulin peptides. Similarly, none of four T-cell lines reactive to GAD65 peptides (amino acids 247-280) with sequence homology to Coxsackie P2C (amino acids 30-50) crossreacted to the homologous viral peptide. Two T-cell lines corecognized a GAD65 peptide and a Coxsackie P2C peptide. However, the antigen-specific T-cell clones from these T-cell lines were reacting either with the GAD65 peptide or the Coxsackie P2C peptide using different restriction elements without crossreacting to the homologous peptide. Our data demonstrate that homologous peptides previously proposed to serve as targets for crossreactivity indeed are immunogenic. Yet, T-cell clones did not crossreact with linear sequence homologies, despite strong T-cell responses to individual peptides.

Amino Acid Sequence↗
PubMed1999

Composition of the peptide fraction in human blood plasma: database of circulating human peptides.

A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, beta2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aalpha-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.

Amino Acid Sequence↗
PubMedDate not supplied

Multivalent thioether-peptide conjugates: B cell tolerance of an anti-peptide immune response.

Antibodies which bind beta2-glycoprotein I (beta2GPI) are associated with antiphospholipid syndrome. Synthetic peptide mimotopes have been discovered which compete with beta2GPI for binding to selected anti-beta2GPI. A thiol-containing linker was attached to the N-terminus of two cyclic thioether peptide mimotopes, peptides 1a and 1b. The resulting peptides, with linker attached, were reacted with two different haloacetylated platforms to prepare four tetravalent peptide-platform conjugates to be tested as B cell toleragens. The linker-containing peptides were reacted with maleimide-derivatized keyhole limpet hemocyanin (KLH) to provide peptide-KLH conjugates. Peptides 1a and 1b were also modified by acylation with 3-(4'-hydroxyphenyl)propionic acid N-hydroxysuccinimidyl ester. The resulting hydroxyphenyl peptides were radioiodinated and used to measure anti-peptide antibody levels. The KLH conjugates were used to immunize mice to generate an anti-peptide immune response. The immunized mice were treated with the conjugates or saline solution and boosted with the appropriate peptide-KLH conjugate. Three of the four conjugates suppressed the formation of anti-peptide antibody. The stabilities of the conjugates in mouse serum were measured, and the relative stabilities did not correlate with ability to suppress antibody formation.

Animals↗
PubMed1991

Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes.

The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Structure-transport relationship for the intestinal small-peptide carrier: is the carbonyl group of the peptide bond relevant for transport?

PURPOSE: The objective of this study was to determine the influence of the peptide bond with emphasis on the carbonyl group on the interaction with and transport by the intestinal small-peptide carrier. Therefore enalapril, a known substrate for the small-peptide carrier, has been modified to an analogue with a reduced peptide bond, enamipril. The transport characteristics of both compounds have been determined. METHODS: The in vitro transport studies were performed using rat ileum in Ussing chambers. The transport of enalapril and enamipril were measured in a concentration range from 0.5-8 mM in both directions across the ileum. in the presence and absence of inhibitors. The interaction with the small-peptide carrier was studied by evaluating the ability of enalapril and its analogue enamipril to inhibit the transport rate of amoxycillin. RESULTS: Enalapril shows, besides passive diffusion (P(m)3.06+/-0.14 . 10(-6)cm/s), saturable transport kinetics (Jmax = 16+/-5 nmol/h.cm2, Km = 1.86+/-0.64 mM) which can be inhibited with 10 mM cephalexin. The analogue with a reduced peptide bond does not show saturable transport from the mucosal to the serosal side, and cephalexin does not inhibit the flux of enamipril. However, the transport of enamipril from the serosal to mucosal side of the intestinal membrane is saturable and can be inhibited by 100 microM verapamil. Although enamipril is not a substrate for the small-peptide carrier in contrast to enalapril, both enalapril and enamipril are able to inhibit the active transport of amoxycillin with a K(i) of 0.41+/-0.24 mM and 0.24+/-0.12 mM respectively. CONCLUSIONS: The reduction of the peptide bond of enalapril results in a compound, enamipril, which does not show polarized and saturable transport from the mucosal to the serosal side of the intestinal tissue. Also because the transport of enamipril cannot be inhibited by cephalexin, the analogue with the reduced peptide bond is no longer a substrate for the intestinal small-peptide carrier. Therefore, it can be concluded that the carbonyl group is an essential structural requirement for transport by the small-peptide carrier. In contrast, the interaction with the small-peptide carrier is still present, shown by the inhibition of the fluxes of amoxycillin. Reduction of the peptide bond of enalapril resulted in a new substrate for the P-glycoprotein efflux pump.

Amoxicillin↗