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Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a [99mTc]chelator peptide on the N-terminus, could measure levels of MYC mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in nude mice. We prepared the chelator-MYC PNA-IGF1 peptide, as well as a 4-nt mismatch PNA control, by solid-phase synthesis. We imaged MCF7 xenografts scintigraphically and measured the distribution of [99mTc]probes by scintillation counting of dissected tissues. MCF7 xenografts in nude mice were visualized at 4 and 24 h after tail vein administration of the [99mTc]PNA probe specific for MYC mRNA, but not with the mismatch control. The [99mTc]probes distributed normally to the kidneys, livers, tumors, and other tissues. Molecular imaging of oncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might provide additional genetic characterization of preinvasive and invasive breast cancers.
A strong beta-sheet forming peptide was conjugated to PEO and utilized to guide the structure formation process toward well-defined, tape-like structures with millimeters in length, about 2 mum width, and approximately 50 nm height. The aggregation tendency of the peptide was temporarily suppressed for ease of synthesis by the integration of multiple switch-peptide defect segments into the peptide backbone. A subsequent rearrangement in the defects re-establishes the native peptide backbone and triggers the assembly by switching the aggregation properties on.
A brief survey of the history of peptide chemistry from Theodore Curtius to Emil Fischer to Bruce Merrifield is first presented. The discovery and development of peptide ligation, i.e. of actual chemical synthesis of proteins are described. In the main chapter, 'Synthesis of Proteins by Chemical Ligation' a detailed discussion of the principles, reactivities and mechanisms involved in the various coupling strategies now applied (ligation, chemical ligation, native chemical ligation) is given. These include coupling sites with cysteine and methionine (as well as the seleno analogs), histidine, glycine and pseudo-prolines, 'unrestricted' amino-acid residues (using the Staudinger reaction), as well as solid-phase segment coupling by thioligation of unprotected peptides. In another section, 'Synthesis of beta-peptides by Thioligation', couplings involving beta2- and beta3-peptides are described (with experimental details).
We have been investigating the role of proteolytic enzymes in the inactivation of peptide neurotransmitters in the marine snail Aplysia. Previous studies (Squire, C. R., Talebian, M., Menon, J. G., Dekruyff, S. D., Lee, T. D., Shively, J. E., and Rothman, B. S. (1991) J. Biol. Chem. 266, 22355-22363) showed that neuroactive fragments of the neurotransmitter alpha-bag cell peptide (alpha-BCP) were rapidly degraded (t1/2 = 0.5-2.7 min) in plasma, hemolymph that had been cleared by centrifugation. Degradation was caused by one or more enzymes resembling mammalian leucine amino-peptidase (LAP, EC 3.4.11.1). In this report we show that three other Aplysia peptide neurotransmitters, beta-BCP(1-5) (Arg-Leu-Arg-Phe-His), FMRFa (Phe-Met-Arg-Phe-amide), and SCPB(1-9) (Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met-amide) are rapidly degraded (t1/2 = 0.3-2.4 min) in plasma by apparently the same LAP-like enzyme(s). Our findings strongly suggest that the LAP-like enzyme(s), by means of its broad substrate specificity and access to the extracellular spaces of the nervous system in vivo, plays a significant role in the inactivation of many Aplysia peptide neurotransmitters, and they raise the possibility that proteolytic enzymes in the extracellular fluid contribute significantly to the inactivation of peptide neurotransmitters in other animal species.
To assess the role of growth factors in proliferative disorders of the breast, we assayed breast cyst fluid from 70 patients for calcitonin-related peptides. Cyst fluids (5.4 +/- 6.6 ml) (mean +/- SD) (n = 70) contained 10,499 +/- 8272 pg/ml of gastrin-releasing peptide (GRP)-like immunoreactivity in 66 of 70 samples. Calcitonin gene-related peptide (CGRP)-like immunoreactivity was found in 64 of 64 samples tested (3842 +/- 2048 pg/ml). Calcitonin-like immunoreactivity was detected in 47 of 69 samples (185 +/- 106 pg/ml). Significant correlations were found for GRP versus volume, CGRP, and calcitonin, for calcitonin versus volume and CGRP, and for CGRP versus volume. Extracts of two human breast carcinoma cell lines (MCF-7 and BT-20) contained measurable GRP-like immunoreactivity. We conclude that GRP-, CGRP-, and calcitonin-like immunoreactivities are present in human breast cyst fluid and that GRP-like immunoreactivity is present in two established human breast carcinoma cell lines. High concentrations of GRP-like immunoreactivity in both breast cyst fluid and breast carcinoma tissue, taken together with the known mitogenic and trophic activities of this peptide, support the hypothesis that GRP may be an important factor in human breast disease.
Natriuretic peptide receptor A (NPR-A/GC-A) and B (NPR-B/GC-B) are members of the transmembrane guanylyl cyclase family that mediate the effects of natriuretic peptides via the second messenger, cGMP. Despite numerous reports of these receptors being down-regulated in response to various pathological conditions, no studies have actually measured desensitization and receptor internalization in the same cell line. Furthermore, the ligand-dependent trafficking properties of NPR-A remain controversial, whereas nothing is known about the trafficking of NPR-B. In this report, we tested whether down-regulation explains the ligand-dependent desensitization of NPR-A and NPR-B and characterized their trafficking properties using a combination of hormone-binding and antibody-based assays. Quantitative partition analysis indicated that (125)I-atrial natriuretic peptide (ANP) was rapidly released into the medium after 293T cells stably expressing NPR-A were warmed from 4 degrees to 37 degrees C. High-performance liquid chromatography fractionation of medium supplemented with the protease inhibitor phosphoramidon indicated that the (125)I-ANP was mostly intact. In contrast, (125)I-ANP purified from medium bathing cells expressing NPR-C, a receptor known to internalize natriuretic peptides, was degraded. Cleavable biotinylation and noncleavable biotinylation assays indicated that neither NPR-A nor NPR-B was internalized or degraded in response to natriuretic peptide binding. In contrast, agonist-dependent internalization of a G protein-coupled receptor was clearly apparent in the same cell line. Finally, we show that NPR-A and NPR-B are desensitized in cells in which they are not internalized. We suggest that mechanisms other than receptor down-regulation account for the desensitization of NPR-A and NPR-B that occurs in response to various physiological and pathological stimuli.
After over a decade of studies on aspartic protease inhibitors and water-soluble prodrugs, we have been developing a novel method, since 2003, called 'O-acyl isopeptide method', for the synthesis of peptides containing difficult sequences. With our recent discoveries of 'O-acyl isodipeptide unit' and the 'racemization-free segment condensation method', this method has further evolved as a general synthetic method for peptides. Moreover, 'Click Peptide', which could be a powerful tool for identifying the pathological functions of amyloid beta peptides in Alzheimer's disease, represents a valuable use of the isopeptide method in Chemical Biology-oriented research.
OBJECTIVES: The objectives of the current study were to define for the first time the roles of the natriuretic peptide (NP) receptors and neutral endopeptidase (NEP) in mediating and modulating the renal actions of Dendroaspis natriuretic peptide (DNP), a new therapeutic synthetic NP. BACKGROUND: Recent reports have advanced the therapeutic potential of a newly described synthetic NP called DNP. Dendroaspis natriuretic peptide is a 38-amino acid peptide recently isolated from the venom of Dendroaspis augusticeps (the green mamba snake). METHODS: Synthetic DNP was administered intra-renally at 5 ng/kg/min to 11 normal anesthetized dogs, 5 of which received the NP receptor antagonist HS-142-1 (3 mg/kg intravenous bolus) while the remaining 6 dogs received an infusion of the NEP inhibitor, candoxatrilat (8 and 80 microg/kg/min) (Pfizer, Sandwich United Kingdom). RESULTS: Intra-renal DNP resulted in marked natriuresis associated with increased urinary cyclic guanosine monophosphate excretion (UcGMPV), glomerular filtration rate (GFR), and renal blood flow (RBF) and decreased distal fractional sodium reabsorption (FNaR) compared with baseline. HS-142-1 attenuated the natriuretic response to DNP, resulting in decreased UcGMPV, GFR, and RBF and increased distal FNaR. In contrast, low and high doses of NEP inhibitor did not potentiate the renal actions of DNP. CONCLUSIONS: We report that the NP receptor blockade attenuated the renal actions of synthetic DNP and that the NEP inhibitor did not alter the renal response to DNP. This latter finding is a unique property of synthetic DNP, as distinguished from other known NPs, supporting its potential as a therapeutic agent.
Mammalian antimicrobial proteins, such as defensins and cathelicidin, have stimulating effects on host leukocytes. Cathelin-related antimicrobial peptide (CRAMP), the orthologue of human cathelicidin/LL-37, is the sole identified murine cathelicidin. CRAMP has been shown to have both antimicrobial and angiogenic activities. However, whether CRAMP, like human cathelicidin/LL-37, also exhibits a direct effect on the migration and function of leukocytes is not known. We have observed that CRAMP, like LL-37, was chemotactic for human monocytes, neutrophils, macrophages, and mouse peripheral blood leukocytes. CRAMP also induced calcium mobilization and the activation of MAPK in monocytes. CRAMP-induced calcium flux in monocytes was desensitized by MMK-1, an agonistic ligand specific for formyl peptide receptor-like-1 (FPRL1), and vice versa, suggesting the use of FPRL1 by CRAMP as a receptor. Furthermore, CRAMP induced the chemotaxis of human embryonic kidney 293 cells transfected with either FPRL1 or mouse formyl peptide receptor-2, the mouse homologue of FPRL1, but not by untransfected parental human embryonic kidney 293 cells, confirming the use of FPRL1/mouse formyl peptide receptor-2 by CRAMP. Injection of CRAMP into mouse air pouches resulted in the recruitment predominantly of neutrophils and monocytes, indicating that CRAMP acts as a chemotactic factor in vivo. Finally, simultaneous administration of OVA with CRAMP to mice promoted both humoral and cellular Ag-specific immune responses. Thus, CRAMP functions as both a chemoattractant for phagocytic leukocytes and an enhancer of adaptive immune response.
We have directly compared atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and N-terminal pro-ANP (N-ANP) as markers of patients with left ventricular ejection fraction (LVEF) < or = 35%, as measured by radionuclide ventriculography. Venous blood samples were obtained from an unselected group of 87 patients who had been referred for assessment of ventricular function. ANP, BNP, and N-ANP were measured by radioimmunoassay using commercial kits. Receiver-operating characteristic analysis was used for the objective assessment of the diagnostic performance of each assay. There was a weak negative correlation between LVEF and plasma levels of ANP-li (r = -0.50,), BNP-li (r = -0.57), and N-ANP-li (r = -0.49) (p <0.01 for each peptide). Areas under the receiver-operating characteristic curves for BNP (0.880) and N-ANP (0.832) were not significantly different from each other, but were both significantly greater than the value for ANP (0.761): BNP versus ANP, p <0.01; and N-ANP versus ANP, p <0.05. The optimal sensitivity and specificity of each assay for the detection of patients with LVEF < or = 35% were: BNP > 4 pmol/L-sensitivity 1.0, specificity 0.58; N-ANP >200 pmol/L-sensitivity 0.95, specificity 0.35; and ANP >10 pmol/L-sensitivity 0.90, specificity 0.30. Plasma concentrations of BNP and N-ANP provide sensitive indicators of moderate to severe LV dysfunction; both peptides, are objectively superior to ANP for identifying patients with LVEF < or = 35%. These simple tests could be used to screen patients with suspected ventricular dysfunction to reduce the demand for further cardiac investigations.
We have investigated the presentation and CTL recognition of an HLA A*1101-restricted CTL peptide epitope AVFDRKSDAK (AVF)(3), derived from the EBV nuclear antigen (EBNA) 4, in the context of alleles belonging to the A3-supertype, A*0101, 0301, 1101, 3101, 3301, and 6801. The peptide binds to a A*6801 molecule as efficiently as to A*1101. The A*6801:AVF complex is recognized by some A*1101-restricted AVF- specific CTL clones. However, A*6801-positive (A*6801+) EBV-transformed lymphoblastoid cell lines (LCLs) are not killed by the same effectors. Furthermore, two A*6801+ donors did not mount an AVF-specific CTL response in vitro and lacked detectable AVF-specific effectors. Thus, this epitope is either subdominant, or non-immunogenic in the context of A*6801. These characteristics correlate with low stability of this MHC:peptide complex in living cells. We also demonstrate that a highly conserved AVF-specific TCR that dominates the AVF-specific CTL response in the majority of A*1101+ individuals recognizes the A*6801 molecule as a crossreactive alloantigen. Therefore, deletion of AVF-specific T cells may contribute to the non-immunogenicity or subdominance of the peptide in A*6801+ individuals.
A simple method is described for the purification of peptide-M and MCD-peptide from bee venom. The steps are Sephadex G-50 gel filtration of pH 4.5 (Steps 1 and 2) and chromatography on silica gel (Steps 3 and 4). The peptides are homo geneous with respect to thin-layer chromatography, electrophoresis at pH 2.0, and amino acid analysis. Peptide-M contains 24 amino acid residues and has a calculated molecular weight of 2750.
To determine the role of vasoactive peptides such as endothelin (ET), calcitonin gene-related peptide (CGRP) and human atrial natriuretic peptide (hANP) in the regulation of blood pressure in uremic patients, and to determine the effect of various types of dialyzer membranes on hemodialysis (HD)-induced changes in plasma levels of such peptides, plasma ET, CGRP and hANP were measured in HD patients and patients on continuous ambulatory peritoneal dialysis (CAPD). Plasma levels of ET, CGRP, and hANP were significantly higher in HD and CAPD patients than in healthy subjects. There were no significant differences in plasma levels of ET, CGRP, and hANP between hypertensive and normotensive HD patients, and no significant correlation was observed between HD-induced changes in plasma levels and changes in blood pressure. Plasma levels of ET decreased when HD was performed using high-flux membranes, such as polyacrylonitrile (PAN), polymethyl methacrylate (PMMA) and cellulose triacetate (CTA), but did not decrease using a saponified cellulose (SC) membrane. Plasma levels of CGRP decreased in the case of PAN, but increased significantly with PMMA and showed no change with SC and CTA. Plasma levels of hANP decreased in all types of dialyzer membranes due to decreased secretion. These results indicate that the effect of HD on plasma levels of ET and CGRP, but not hANP, depends on the type of dialyzer membrane used.
Urinary C-peptide (UCP) is a noninvasive measure of integrated insulin production, and the usefulness of 24-h collections has been previously reported. Only small numbers of subjects have been studied using shorter urine collections. To see how well 4-h urine collections for C-peptide (UCP) correlate with serum immunoreactive insulin (SI) and plasma C-peptide (PCP), we studied 41 healthy subjects (19 men, 22 women) using as a stimulus a 600-kcal mixed meal and the same mixed meal after oral prednisone. UCP values correlated best with the area under the curves for SI (r = 0.457, P less than 0.001) and PCP (r = 0.557, P less than 0.001). UCP was also significantly correlated with peak SI (r = 0.382, P less than 0.001), peak PCP (r = 0.496, P less than 0.001), fasting SI (r = 0.297, P = 0.007), and fasting PCP (r = 0.341, P = 0.007) values. Urinary C-peptide was significantly correlated with SI and PCP concentrations in a broad range of physiologic values for SI and PCP supporting the usefulness of UCP as a simple, noninvasive measure of beta-cell function. Four-hour collections for UCP may be useful in further studies of beta-cell function.