Conformational analysis in solution of gastrin releasing peptide

Gastrin releasing peptide (GRP) is the first peptide isolated from porcine gastric and intestinal tissues and is homologous to the carboxyl terminus of bombesin (Bn) isolated from the skin of the frog Bombina bombina. It is a member of the Bn-like peptides, which are important in numerous biological and pathological processes. The Bn-like peptides show high sequence homology in their C-terminal regions, but they have different selectivity for their receptors. In particular, GRP selectively binds to the GRP receptor (GRPR). However, the molecular basis for this selectivity remains largely unknown. Here, we report the three-dimensional structure of GRP. Hopefully, it could be helpful in a better understanding of the binding selectivity between GRP and GRPR.     

Gastrin releasing peptide reduces meal size in rats

The satiety-eliciting effect of gastrin-releasing peptide (GRP), a putative mammalian counterpart of bombesin (BBS), was examined in mildly food-deprived rats. Intraperitoneal injections of GRP resulted in a significant decrease of 30-minute food intake at 2, 4, 8 and 16 μg/kg, while 1 μg/kg had no reliable effect. Intraperitoneal GRP at 4 and 8 μg/kg did not suppress 30-minute water consumption by thirsty rats. When the dose-effect curves of GRP and BBS are compared on a molar scale, GRP is approximately 30% less potent than BBS in suppressing food intake. The two dose-effect curves are similar in shape and their regression lines have parallel slopes. These data lend further support to the hypothesis that GRP is a mammalian counterpart of BBS and strengthen the argument that they may function as endogenous satiety factors.     

Catabolism of gastrin releasing peptide and substance P by gastric membrane-bound peptidases

The catabolism of two gastric neuropeptides, the C-terminal decapeptide of gastrin releasing peptide-27 (GRP10) and substance P (SP), by membrane-bound peptidases of the porcine gastric corpus and by porcine endopeptidase-24.11 ("enkephalinase") has been investigated. GRP10 was catabolized by gastric muscle peptidases (specific activity 1.8 nmol min-1 mg-1 protein) by hydrolysis of the His8-Leu9 bond and catabolism was inhibited by phosphoramidon (I50 approx. 10(-8) M), a specific inhibitor of endopeptidase-24.11. The same bond in GRP10 was cleaved by purified endopeptidase-24.11, and hydrolysis was equally sensitive to inhibition by phosphoramidon. SP was catabolized by gastric muscle peptidases (specific activity 1.7 nmol min-1 mg-1 protein) by hydrolysis of the Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10 bonds, which is identical to the cleavage of SP by purified endopeptidase-24.11. The C-terminal cleavage of GRP10 and SP would inactivate the peptides. It is concluded that a membrane-bound peptidase in the stomach wall catabolizes and inactivates GRP10 and SP and that, in its specificity and sensitivity to phosphoramidon, this peptidase resembles endopeptidase-24.11.     

Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine

Summary Bombesin-like and gastrin-releasing peptide (GRP)-like immunoreactivities were localized in nerves of the guinea-pig small intestine and celiac ganglion with the use of antibodies raised against the synthetic peptides. The anti-bombesin serum (preincubated to avoid cross reactivity with substance P) and the anti-GRP serum revealed the same population of neurons. Preincubation of the antibombesin serum with bombesin abolished the immunoreactivity in nerves while absorption of the anti-GRP serum with either bombesin or the 14–27 C-terminal of GRP only reduced the immunoreactivity. The immunoreactivity was abolished by incubation with GRP 1–27.Immunoreactive nerves were found in the myenteric plexus, circular muscle, submucous plexus and in the celiac ganglion. Faintly reactive nerve cell bodies were found in the myenteric ganglia (3.2% of all neurons) but not in submucous ganglia. After all ascending and descending pathways in the myenteric plexus had been cut, reactive terminals disappeared in the myenteric plexus, circular muscle (including the deep muscular plexus) and the submucous plexus on the anal side. After the mesenteric nerves were cut no changes were observed in the intestinal wall but the reactive fibres in celiac ganglia disappeared. It is deduced that GRP/bombesin-immunoreactive nerve cell bodies in myenteric ganglia project from the myenteric plexus to other myenteric ganglia situated further anally (average length 12 mm), anally to the circular muscle (average length 9 mm), anally to submucous ganglia (average length 13 mm) and external to the intestine to the celiac ganglia.It is concluded that the GRP/bombesin-reactive neurons in the intestinal wall represent a distinct population of enteric neurons likely to be involved in controlling motility and in the coordination of other intestinal functions.     

Reduction/pH dual-sensitive PEGylated hyaluronan nanoparticles for targeted doxorubicin delivery

To minimize the side effect of chemotherapy, a novel reduction/pH dual-sensitive drug nanocarrier, based on PEGylated dithiodipropionate dihydrazide (TPH)-modified hyaluronic acid (PEG-SS-HA copolymer), was developed for targeted delivery of doxorubicin (DOX) to hepatocellular carcinoma. The copolymer was synthesized by reductive amination via Schiff's base formation between TPH-modified HA and galactosamine-conjugated poly(ethylene glycol) aldehyde/methoxy poly(ethylene glycol) aldehyde. Conjugation of DOX to PEG-SS-HA copolymer was accomplished through the hydrazone linkage formed between DOX and PEG-SS-HA, and confirmed by FTIR and ¹H NMR spectra. The polymer–DOX conjugate could self-assemble into spherical nanoparticles (∼150 nm), as indicated by TEM and DLS. In vitro release studies showed that the DOX-loaded nanoparticles could release DOX rapidly under the intracellular levels of pH and glutathiose. Cellular uptake experiments demonstrated that the nanoparticles could be efficiently internalized by HepG2 cells. These results indicate that the PEG-SS-HA copolymer holds great potential for targeted intracellular delivery of DOX.     

Epidermal growth factor (EGF) receptor targeted delivery of PEGylated adenovirus

A biotin-polyethylene glycol (PEG)-epidermal growth factor (EGF) conjugate was immobilized onto the surface of avidin-modified adenovirus (ADV-Avi) via biotin-avidin interaction to deliver ADV specifically to EGF receptor over-expressing cancer cells. ADV-Avi/biotin-PEG-EGF complexes showed greatly enhanced intracellular uptake of ADV particles for an EGF receptor positive cell line (A431 cells), compared to naked or PEG alone immobilized ADV. ADV coding an exogenous GFP gene was used to quantitatively evaluate the level of GFP expression. ADV-Avi/biotin-PEG-EGF complexes also exhibited significantly increased extent of GFP expression for A431 cells, but not for MCF-7 cells (an EGF receptor deficient cell line), suggesting that retargeting of ADV to specific cells occurred by tethering of a cell-specific targeting ligand to the distal end of a PEG chain anchored onto the surface of ADV. This study demonstrates that ADV-Avi/biotin-PEG-EGF construct systems can be applied for cell-specific delivery of ADV with simultaneously reducing innate immune responses.     

Ligands of the asialoglycoprotein receptor for targeted gene delivery, part 1: Synthesis of and binding studies with biotinylated cluster glycosides containing N-acetylgalactosamine

In order to develop the non-viral Bioplex vector system for targeted delivery of genes to hepatocytes, we have evaluated the structure-function relationship for a number of synthetic ligands designed for specific interaction with the hepatic lectin ASGPr. Biotinylated ligand derivatives containing two, three or six beta-linked N-acetylgalactosamine (GalNAc) residues were synthesized, bound to fluorescent-labeled streptavidin and tested for binding and uptake to HepG2 cells using flow cytometry analysis (FACS). Uptake efficiency increased with number of displayed GalNAc units per ligand, in a receptor dependent manner. Thus, a derivative displaying six GalNAc units showed the highest uptake efficacy both in terms of number of internalizing cells and increased amount of material taken up per each cell. However, this higher efficiency was shown to be due not so much to higher number of sugar units, but to higher accessibility of the sugar units for interaction with the receptor (longer spacer). Improving the flexibility and accessibility of a trimeric GalNAc ligand through use of a longer spacer markedly influenced the uptake efficiency, while increasing the number of GalNAc units per ligand above three only provided a minor contribution to the overall affinity. We hereby report the details of the chemical synthesis of the ligands and the structure-function studies in vitro.     

Conformational states of the corticotropin releasing factor 1 (CRF1) receptor: detection, and pharmacological evaluation by peptide ligands

Previous corticotropin releasing factor 1 (CRF₁) receptor characterization has been performed using radiolabeled agonists, which bind predominantly the receptor-G-protein complex. The pharmacological profile of other receptor states, and their abundance, remain poorly characterized. Here we investigated the affinity states of the CRF₁ receptor heterologously expressed in Ltk⁻ cells and endogenously expressed in rat cerebellum. In L-CRF₁ cell membranes, three agonist affinity states were detected: a very-high affinity receptor-G-protein complex state (eliminated by GTPγS) bound by [¹²⁵I]sauvagine (43 pM, RG); a high affinity state insensitive to GTPγS bound by [¹²⁵I]sauvagine (1.4 nM, termed R_O); and a low affinity G-protein-uncoupled state detected by sauvagine displacement of [¹²⁵I]astressin, a labeled antagonist (120 nM, R). The relative abundance of RG:R_O:R was 18%:16%:66%. All three states were demonstrated in rat cerebellum with similar relative abundance (15%:16%:69%). The R state bound CRF with low affinity (270–330 nM), displayed a novel rank order of ligand affinity, and represented the majority of the receptor population in both receptor preparations. This study provides a framework to identify CRF₁ receptor conformational states in various receptor preparations.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Immunocytochemical mapping of gastrin/CCK-like peptides in the neuroendocrine system of the blowfly Calliphora vomitoria (Diptera)

Summary The distribution of gastrin/CCK-like immunoreactive material has been studied in the retrocerebral complex of Calliphora. The material reacts with antisera specific for the common COOH terminus of gastrin and CCK but not with N-terminal antisera. The three thoracic ganglia and the fused abdominal ganglia each contain a specific number of symmetrically arranged immunoreactive cells both dorsally and ventrally in pairs on either side of the midline in a sagittal plane. The neuropil of these ganglia also contains a considerable amount of immunoreactive fibres and droplets. Reconstructed axonal pathways suggest that some of the nerve fibres have their origins within the brain and/or the suboesophageal ganglion. Immunoreactive material may also be seen apparently leaving the thoracic ganglion posteriorly via the abdominal nerves, and there is strong evidence of a neurohaemal organ within the dorsal sheath in the region of the metathoracic and abdominal ganglia. There appears to be a direct correlation between the content of peptidergic material of cells and fibres and the age and diet of the flies. The corpus cardiacum contains COOH-terminal specific gastrin/CCK-like material within the intrinsic cells and in the neuropil. It is present also in the cardiac-recurrent nerve entering the corpus cardiacum anteriorly and in the nerves leaving the gland dorsoposteriorly, the aortic or cardiac nerves. It is not observed, however, in the nerves leaving the corpus cardiacum ventroposteriorly, the so-called oesophageal, gastric or crop-duct nerves. The corpus allatum and the hypocerebral ganglion do not contain immunoreactive material of this type. Gastrin/CCK-like and secretin-like immunoreactive materials appear to co-exist in the cells of the corpus cardiacum and co-existence of gastrin/CCK-like and pancreatic polypeptide like substances occurs within certain cells of the thoracic ganglion.     

Optimization of azepanone calcitonin gene-related peptide (CGRP) receptor antagonists: Development of novel spiropiperidines

Several novel spiropiperidine-based CGRP receptor antagonists have been developed that maintain good potency and have reduced potential for metabolism.     

Targeted cationic poly(D,L-lactic-co-glycolic acid) nanoparticles for gene delivery to cultured cells

We developed a new targeted cationic nanoparticulate system composed of poly(D,L-lactic-co-glycolic acid) (PLGA), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and asialofetuin (AF), and found it to be a highly effective formulation for gene delivery to liver tumor cells. The nanoparticles (NP) were prepared by a modified solvent evaporation process that used two protocols in order to encapsulate (NP1 particles) or adsorb (NP2 particles) plasmid DNA. The final particles are in the nanoscale range. pDNA loaded in PLGA/DOTAP/AF particles with high loading efficiency showed a positive surface charge. Targeted asialofetuin-nanoparticles (AF-NP) carrying genes encoding for luciferase and interleukin-12 (IL-12) resulted in increased transfection efficiencies compared to free DNA and to plain (non-targeted) systems, even in the presence of 60% fetal bovine serum (FBS). The results of transfections performed on HeLa cells, defective in asialoglycoprotein receptors (ASGPr-), confirmed the receptor-mediated endocytosis mechanism. In summary, this is the first time that asialoglycoprotein receptor targeting by PLGA/DOTAP/DNA nanoparticles carrying the therapeutic gene IL-12 has been shown to be efficient in gene delivery to liver cancer cells in the presence of a very high concentration of serum, and this could be a potential system for in vivo application.     

Identification of G-proteins coupling to the vasoactive intestinal peptide receptor VPAC(1) using immunoaffinity chromatography: evidence for precoupling.

VPAC(1) receptor subtype-specific G-protein interactions were identified using a strategy that exploits an essential initial signaling event, namely the functional and physical association of the receptor with G-protein. An immunoaffinity purification column was constructed using a previously characterized antibody that had been raised against the first extracellular loop of the VPAC(1) receptor. VPAC(1)/G-protein complexes were solubilized from membranes and copurified. Receptor and Galpha-proteins were detected in eluates using (125)I-VIP labeling and immunoblotting, respectively. Human VPAC(1) transfected in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq. Rat VPAC(1) in brain membranes is coupled to Gs and Gi3. Rat VPAC(1) in lung membranes couples to Gs, Gi3, and Gq. Pretreatment of membranes with VIP increased the level of all G-proteins copurifying with VPAC(1). Immunoaffinity chromatography also revealed VPAC(1) receptor precoupling to G-protein in the absence of VIP pretreatment. This was confirmed using a cross-linking procedure to capture VIP receptor/G-protein complexes in the native membrane milieu prior to solubilization. Precoupling suggests that there is a significant basal level of VPAC(1) receptor activity especially in cells, such as some human malignant tumor cells, that express high levels of receptor.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

T cell antigen receptor (TCR) transmembrane peptides colocalize with TCR,not lipid rafts,in surface membranes

We have previously shown that a synthetic peptide termed core peptide (CP), which corresponds to a sequence within the transmembrane domain of the alpha chain of the T cell antigen receptor (TCR), can inhibit IL-2 production in antigen-stimulated T cells and can suppress inflammation in several T cell-mediated animal models of disease. As the first step in determining the mechanism of CP action, we examined the association of CP with the plasma membrane of human T cells using confocal microscopy. A homogeneous distribution of CP was observed in the plasma membrane of human T cells. This membrane localization was dependent on the presence of positive charges in the CP sequence. CP analogs, containing either neutral or negatively charged amino acids in place of the positive amino acid charges, did not localize within TCR membranes. Following antibody-induced TCR clustering, there was specific colocalization of CP with surface TCR. No association was observed with other cell surface receptors when similarly clustered. Since TCR activation leads to an increased movement of the receptor complex to cholesterol/glycosphingolipid (GSL) plasma membrane microdomains (rafts) we examined whether the association of CP with TCR was raft-driven. TCR clustering led only to a partial colocalization of TCRs with raft GSL, ganglioside GM1, and a complete colocalization of CP with TCRs. We conclude that CP associates specifically with plasma membrane TCRs and not raft lipids.     

Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery

The physical properties of non-viral vector/DNA nanoparticles in physiological aqueous solution are poorly understood. A Fluid Particle Image Analyser (FPIA), normally used for analysis of industrial and environmental fluids, was used to visualise individual (Lys)(16)-containing peptide/DNA particles. Eight (Lys)(16)-containing synthetic peptides were used to generate peptide/DNA particles at a constant + to - charge ratio of 2.8:1 with 10 microg/ml of plasmid DNA in phosphate buffered saline. Dynamic Light Scattering (DLS) and gene delivery studies were also performed. We present the first images of non-viral vector/DNA nanoparticles in physiological aqueous solution, together with precise measurements of individual particle size and shape in solution and, for the first time, an accurate measure of particle number. Particle size and shape, particle number, and efficiency for gene delivery varied markedly with different peptides. Under standard conditions for in vitro gene delivery, we estimate approximately 60 peptide/DNA nanoparticles per target cell, each containing approximately 70,000 plasmids. This novel capacity to image individual vector/DNA nanoparticles in solution and to count them accurately will enable a more precise assessment of non-viral gene delivery systems, and a more quantitative interpretation of gene delivery experiments.     

Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus)

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus–oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.     

A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells

To facilitate nuclear delivery of biomolecules we describe the synthesis of a modular transporter bearing a cellular membrane transport peptide (pAntp) and, as a cargo, a 16-mer peptide nucleic acid (PNA) covalently linked to a nuclear localisation signal (NLS[SV40-T]). Transport peptide and PNA are connected via N-terminal activated cysteine to form cleavable disulphide bonds. Internalization and subsequent delivery of PNA to the nucleus was verified in living and fixed cells by confocal laser scanning microscopy (CLSM) and fluorescence correlation spectroscopy (FCS). Double-labelling experiments indicate the cytoplasmic cleavage of the two modules and the effective nuclear import of the chromophore-tagged cargo. A non-degradable linker between transport module and cargo as well as a construct without NLS did not enable nuclear PNA import under the described experimental conditions. FCS-measurements revealed that most of the PNAs delivered into the cytoplasm by the modular transporter are anchored or encapsulated, indicating that intracellular transport of these compounds is not governed by molecular diffusion. Our results clearly demonstrate efficient compartment-directed transport using a synthetic, non-toxic modular transporter in living cells.