Design, Recombinant Fusion Expression and Biological Evaluation of Vasoactive Intestinal Peptide Analogue as Novel Antimicrobial Agent.

Antimicrobial peptides represent an emerging category of therapeutic agents with remarkable structural and functional diversity. Modified vasoactive intestinal peptide (VIP) (VIP analogue 8 with amino acid sequence "FTANYTRLRRQLAVRRYLAAILGRR") without haemolytic activity and cytotoxicity displayed enhanced antimicrobial activities against Staphylococcus aureus (S. aureus) ATCC 25923 and Escherichia coli (E. coli) ATCC 25922 than parent VIP even in the presence of 180 mM NaCl or 50 mM MgCl2, or in the range of pH 4-10. VIP analogue 8 was expressed as fusion protein thioredoxin (Trx)-VIP8 in E. coli BL21(DE) at a yield of 45.67 mg/L. The minimum inhibitory concentration (MIC) of the recombinant VIP analogue 8 against S. aureus ATCC 25923 and E. coli ATCC 25922 were 2 µM. These findings suggest that VIP analogue 8 is a promising candidate for application as a new and safe antimicrobial agent.

Determination of binding constants of vasoactive intestinal peptide to poly(amidoamine) dendrimers designed for drug delivery using ACE.

The purpose of the present paper was to study at physiological pH the affinity between vasoactive intestinal peptide (VIP) and four poly(amidoamine) dendrimers (PAMAMs) designed for drug delivery. Therefore, a fast and reproducible CE method was first developed to analyze the strongly basic peptide. To allow an accurate determination of binding constant (K) values, the ability to suppress peptide adsorption onto the silica capillary of nonpermanent coatings (poly(ethylene oxide) (PEO), low and medium relative molecular masses poly(diallyldimethylammonium chloride) (PDDA)) or poly(acrylamide) permanent coating (PAA) was evaluated. Very good intraday repeatability of VIP migration times and peak areas (0.1-0.6 and 2.9-4.9% RSD, respectively) was obtained using two of the investigated coatings (PEO and PDDA with medium molecular mass). ACE combined with these dynamic coatings was then employed to evaluate K between VIP and two amine-terminated PAMAM dendrimers of generation 2 and 5 (G2.NH2, G5.NH2) and two carboxyl-terminated PAMAM derivatives of generation 2 and 5 (G2.COOH, G5.COOH). Binding constant of (6.7 +/- 1.1) x 10(4)/M could be determined for the couple VIP/G5.NH2, while no affinity was evidenced between VIP and all other dendrimers investigated. These results suggest that G5.NH2 might be an interesting carrier for the delivery of VIP.

Isolation and characterization of four VIP-related peptides from red-bellied newt, Cynops pyrrhogaster.

Four novel bioactive peptides were isolated from the red-bellied newt, Cynops pyrrhogaster, using a bioassay system monitoring the rectum contraction of the Japanese quail, Coturnix japonica. As these peptides are structurally related to vasoactive intestinal polypeptide (VIP), we termed these peptides newt VIP-related peptides 1, 2, 3, and 4 (NVRP-1, -2, -3, and -4). The primary sequences of these peptides were determined to be HSDAVFTDNYSRLLGKTALKNYLDGALKKE (NVRP-1), HSDAVFTDNYSRLLAKTALKNYLDGALKKE (NVRP-2), HSDAVFT-DNYSRLLGKIALKNYLDEALKKE (NVRP-3), and HSDAVFTDNYSRLLGKT-ALKNYLDSALKKE (NVRP-4). The N-terminal regions of these NVRPs possessed homology at the amino-acid level to various VIP, while the NVRP C-termini differed from VIPs significantly. All of the VIP consist of 28 amino-acid residues with amidated forms at the C-termini, whereas NVRPs possess 30 amino-acid residues and have free forms at the C-termini. NVRPs exert relaxant activities on isolated quail rectums in a dose-dependent manner, with threshold concentrations between 1 x 10(-8) and 3 x 10(-8) M. NVRPs also exhibited potent relaxant activities acting on the newt duodenum at 3 x 10(-8) M. As yet, this is the first isolation of biologically active VIP-related peptides from urodele.

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors.

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.

Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue.

The measurement of neuropeptides in complex biological tissue samples requires efficient and appropriate extraction methods so that immunoreactivity is retained for subsequent radioimmunoassay detection. Since neuropeptides differ in their molecular mass, charge and hydrophobicity, no single method will suffice for the optimal extraction of various neuropeptides. In this study, dental pulp tissue was obtained from 30 human non-carious teeth. Of the three different neuropeptide extraction methods employed, boiling in acetic acid in the presence of protease inhibitors yielded the highest levels of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). High pressure liquid chromatography (HPLC) analysis of dental pulp tissue verified the authenticity of the neuropeptides extracted.

The augmentation of pituitary adenylate cyclase-activating polypeptide (PACAP) in streptozotocin-induced diabetic rats.

Pituitary adenylate cyclase activating polypeptide (PACAP), which was isolated from ovine hypothalamic extract, has been shown to have a physiological role in the regulation of insulin or islet functions. In streptozotocin (STZ)-induced diabetic rats, we examined the content of PACAP immunoreactivity and gene expression of three specific receptors. Four weeks after administration of STZ (50 mg/kg), plasma glucose levels increased 3.3-fold, and plasma insulin levels decreased to one-tenth as compared with the control. The content of PACAP immunoreactivity in the pancreas potently increased by 30%, but the content of vasoactive intestinal polypeptide (VIP) immunoreactivity was not changed. In the other tissues, the content of PACAP immunoreactivity did not significantly change except in the hypothalamus, which showed a 10% increment. In the expression level of PACAP/VIP receptors, semi-quantitative RT-PCR analysis revealed that VIP1/PACAP receptor mRNA significantly increased as compared with the other two types of receptors in the pancreas of STZ-induced diabetic rats. These findings suggest that PACAP and VIP1/PACAP receptor might be involved in the pathophysiology of diabetes mellitus.

An improved method to obtain a single recombinant vasoactive intestinal peptide (VIP) analog.

The vasoactive intestinal peptide (VIP) is an ubiquitous peptide of great potential for applications. Development of new bioactive VIP analogs using production in recombinant E coli has been carried out in our laboratory. This work presents a new multimeric fusion protein expressing several VIP units separated by factor Xa cleavage site linkers. The steps leading from the affinity purification of the fusion protein and its processing by the factor Xa to the full characterization of the new bioactive improved VIP analog are also described.

Production, analysis and bioactivity of recombinant vasoactive intestinal peptide analogs.

Recombinant vasoactive intestinal polypeptide (VIP) analogs were expressed in Escherichia coli as a fusion protein containing tandemly repeated multiple copies of a synthetic VIP gene joined to glutathione S-transferase. The encoded protein contains VIP units separated by a linker peptide, potentially excisable by a double cleavage with endoprotease factor Xa and hydroxylamine. Expression of different polyVIP genes, from 1 to 32 units, was detected and the production of a 16 VIP polymer was performed. MonoVIP analogs appended by 5 or 10 amino acids at their C terminus were released by factor Xa from this polymerized product. They were then submitted to hydroxylamine cleavage to remove the linker sequence to finally obtain a recombinant VIP analog devoid of any amino acid extension. The biological activity of the recombinant polyVIP and VIP analogs was tested. Although less efficient than the natural neuropeptide, some of these components bound to VIP receptor, activated adenylate cyclase in human colonic adenocarcinoma cells and displayed a relaxation activity on guinea pig tracheal rings.

Somatostatin-, vasoactive intestinal peptide-, and granulin-like peptides isolated from intestinal extracts of goldfish, Carassius auratus.

Three new peptides were originally isolated from intestinal extracts of goldfish. They were structurally related with somatostatin, vasoactive intestinal peptide (VIP), and granulin (GRN) and thus termed goldfish somatostatin (gSS-28), gVIP, and gGRN, respectively. The primary structures of these peptides were determined as: SVESSNHLPA 10RERKAGCKNF20YWKGFTSC for the gSS-28; HSDAVFTDNY10SRYRKQMAAK20KYLNSVLA-NH2 for the gVIP, and VIHCDSSTIC10 PDGTTCCLSP20YGVWYCCPFS30MGQCCRDGIH40CCRHGYHCDS50TSTHCLR for the gGRN. The amino acid sequence of the gSS-28 was more similar (79-86% similarity) to somatostatins obtained in anglerfish, flounder, and sculpin, but far (21% similarity) from the catfish somatostatin, whereas goldfish and catfish belong to the same superorder. The structure of the gVIP was closely related to that of the cod VIP; only one residue (Tyr13) being substituted for Phe13 in the cod VIP. Comparing amino acid sequences of VIPs obtained in various vertebrates, the primary structure of this peptide was revealed to be relatively well conserved among vertebrates. In addition, the dose-response curve for the effect of gVIP on the short-circuit current (Isc) across the eel intestine was similar to that of human VIP, suggesting that VIPs in vertebrates have similar effect. The amino acid sequence of gGRN was 96% identical to that of carp GRN-1; only two residues (Ser6-Ser7) being substituted for Ala6-Ala7 in the carp GRN-1. The physiological significance of these peptides is discussed.

Frog vasoactive intestinal polypeptide and galanin: primary structures and effects on pituitary adenylate cyclase.

Vasoactive intestinal polypeptide (VIP) and galanin were isolated in pure form from the stomach of the European green frog, Rana ridibunda. Frog VIP is identical to the previously characterized VIP from chicken and alligator. The primary structure of frog galanin contains only two amino acid substitutions (asparagine for histidine at position 23 and histidine for tyrosine at position 26) compared with porcine galanin. The data indicate that evolutionary pressure to conserve the amino acid sequence of both peptides during the evolution of amphibia to mammals has been strong. Synthetic frog VIP produced a dose-dependent increase in cAMP concentration in frog anterior pituitary fragments. The potency of the peptide (ED50 = 1.2 x 10(-6) M; mean +/- SE; n = 8) was comparable to that of porcine VIP (EC50 = 1.3 x 10(-6) M), but was approximately 10-fold less than that of frog pituitary adenylate cyclase-activating polypeptide [PACAP-(1-38); ED50 = 1.1 x 10(-7) M] in the same system. The increases in cAMP concentrations produced by maximal doses of PACAP (10(-5) M) and VIP (10(-5) M) were not additive. The data suggest that the effects of both peptides are mediated through a common PACAP-preferring receptor that is pharmacologically different from the mammalian PACAP type I receptor. Synthetic frog galanin also produced a dose-dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 9.3 x 10(-8) M) consistent with a possible role for the peptide as a hypophysiotropic factor in amphibians.

Purification and structural characterization of vasoactive intestinal polypeptide from the trout and bowfin.

Vasoactive intestinal polypeptide (VIP) was purified from extracts of the stomachs of the rainbow trout, Oncorhynchus mykiss, and the bowfin, Amia calva. The primary structure of VIP from both species was the same: His-Ser-Asp-Ala-Ile-Phe-Thr-Asp-Asn-Tyr10- Ser-Arg-Phe-Arg-Lys-Gln-Met-Ala-Val-Lys20-Lys-Tyr-Leu-Asn-Ser-Val- Leu-Thr. This amino acid sequence shows only one amino acid substitution (Val5-->Ile) compared with the common sequence of VIP from the chicken, alligator, and European green frog. The structural identity of VIP from the trout and bowfin is consistent with the close phylogenetic relationship between the Salmoniformes and the Amiiformes and the data indicate that pressure to conserve the complete primary structure of VIP during vertebrate evolution has been very strong.

All prepro-VIP-derived peptides, except PHI/PHV, are expressed in the female rat anterior pituitary and increased by estrogen.

The expression of VIP precursor products: prepro-VIP(22-79), peptide histidine isoleucine (PHI), peptide histidine valine (PHV), prepro-VIP(111-122), VIP, prepro-VIP(156-170), and prepro-VIP mRNA in the anterior pituitary of estrogen-treated, ovariectomized rats, of ovariectomized controls, and of sham-operated controls was examined. Using radioimmunoassays based on antisera against each of the prepro-VIP sequences, we found that all sequences were expressed and markedly induced by estrogen, except PHI and PHV, which both were undetectable. By immunohistochemistry, it appeared that the number of cells immunoreactive for each of these sequences was increased in the estrogen-treated animals. However, PHI/PHV-immunoreactive cells could not be detected, despite the use of four different PHI antisera with different specificities. Estrogen treatment increased the prepro-VIP mRNA as judged by Northern blotting. In situ hybridization signals for both VIP mRNA and PHI mRNA were observed in few pituitary cells from control animals whereas strong positive signals were observed in a larger number of cells after estrogen treatment. The findings show that estrogen causes activation of the VIP gene expression in anterior pituitary cells, and that the absence of PHI and PHV probably is due to translational or posttranslational events.

Extraction and quantitation of neuropeptides in bone by radioimmunoassay.

The feasibility of extracting and quantifying neuropeptides in bone by radioimmunoassay was investigated in a study including 60 diaphyseal rat femora. Substance P, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide, previously identified in bone by immunohistochemistry, were extracted from separate homogenates of bone, periosteum and bone marrow in a solution of 4% EDTA and 2 M acetic acid. Measurable amounts of all four neuropeptides in bone, periosteum and bone marrow were obtained by radioimmunoassay in a reproducible manner. The neuropeptide immunoreactivities were characterized by reverse-phase high performance liquid chromatography. Among the four neuropeptides analyzed, neuropeptide Y consistently exhibited the highest concentrations in the different tissues. Overall, cortical bone showed the lowest neuropeptide concentrations. The concentration of vasoactive intestinal polypeptide was higher in periosteum than in bone marrow, whereas that of calcitonin gene-related peptide was uniform in these tissues. The distributional differences observed in bone tissue may be explained by a variety of physiological roles attributed to neuropeptides such as regulation of nociception, vasoactivity, immune function and local bone metabolism. The described methodology offers a new means of investigating a neuropeptidergic involvement in various disorders of the skeleton.

Extraction of neuropeptides from joint tissue for quantitation by radioimmunoassay. A study in the rat.

The feasibility of extracting neuropeptides from rat knee joints for quantitation by radioimmunoassay was tested. The investigation, based on 25 adult Lewis rats, focused on substance P, calcitonin gene-related peptide, neuropeptide Y, and vasoactive intestinal polypeptide. The relative recovery of the peptides in different extraction media was assessed Both knee joints including the articulating epiphysis were dissected and cut into small pieces. The series was divided into five subgroups, 10 joints in each, for extraction in five different media: 1) 1 M acetic acid in 4% EDTA, 2) 2 M acetic acid in 4% EDTA, 3) neutral water in 4% EDTA, 4) 2 M acetic acid in 4% EDTA and 95% alcohol, and 5) 2 M acetic acid without EDTA. Measureable concentrations of the four neuropeptides were reproducibly assessed by RIA. Although all extraction media provided measurable concentrations, 2 M acetic acid in 4% EDTA was found to give the highest overall yield of the four neuropeptides analyzed. Reverse-phase HPLC confirmed that the immunoreactivities assessed by RIA corresponded to the four neuropeptides of interest. Experimental and clinical evidence suggest a neurogenic involvement in the pathophysiology of inflammatory joint disease, e.g., rheumatoid arthritis. The extraction procedure described offers a means of determining neuropeptide concentrations in joint tissue under normal and pathologic conditions by RIA.

The presence of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and preproVIP(111-122) in the mouse pineal gland.

A low to moderate number of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI)-immunoreactive nerve fibers with prominent boutons-en-passage were demonstrated in the pineal gland of the mouse. The two peptides, which are parts of the same precursor molecule, were distributed identically in the gland. Positive fibers were present in the connective tissue septae in the gland, in the pineal capsule, and in the pineal parenchyma. No VIP-PHI-immunoreactive elements were found in the deep pineal gland, in the pineal stalk, or in habenular and posterior commissures. This morphological distribution of immunoreactive nerve fibers, which is similar to the distribution in other mammals, indicates that the VIP/PHI fibers of the mouse pineal gland originate exclusively from perikarya in a peripheral ganglion, presumably one of the cholinergic ganglia of the head. No evidence for a VIPergic central innervation was found. VIP and PHI are connected via a bridging peptide equivalent to amino acids 111-122 of the precursor (preproVIP(111-122)). In order to demonstrate the possible existence of this peptide in intra-pineal nerve fibers, antisera directed against a synthetic sequence identical to preproVIP(111-122) and immunohistochemistry were applied. PreproVIP(111-122)-immunoreactive nerve fibers were observed in the mouse pineal gland, with the same distribution pattern and morphology as those immunoreactive for VIP and PHI. To quantify the peptide-immunoreactivities, 50 mice pineals were pooled, extracted, and the concentrations were measured radioimmunologically. The concentrations of the VIP and preproVIP(111-122) immunoreactivities were 1.7 and 2.0 pmol/g, respectively, whereas the concentration of PHI was 0.9 pmol/g.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of neuropeptides extracted from canine intestine.

Quantitative determination of neuropeptides in biologic tissues by radioimmunoassay requires both an efficient extraction of neuropeptides as well as maintenance of immunochemical reactivity. Vasoactive intestinal peptide, substance P, and met5-enkephalin were chosen for this study because they are neuropeptides which appear to be involved in multiple physiologic systems. Since all three neuropeptides have a methionine residue within their amino acid sequence, oxidation of methionine to methionine-sulfoxide during the extraction process could diminish their immunochemical reactivity. Multiple factors that might be important in extracting these neuropeptides from canine intestine, including pH of the solvent, tissue homogenization, heating, and addition of enzyme inhibitors, were examined. Concentrations of vasoactive intestinal peptide-like immunoreactivity and substance P-like immunoreactivity were significantly higher in acidic solvents, and tissue homogenization appeared to increase the concentrations of these two neuropeptides. Substance P-like immunoreactivity was increased by heating after tissue homogenization, suggesting heat-induced denaturation of tissue enzymes liberated by homogenization. Separation of acidic tissue extracts by high performance liquid chromatography followed by radioimmunoassay for all three neuropeptides revealed minor acid-induced oxidation of substance P. These results should be useful for planning the extraction of these three neuropeptides from other tissues.

Neuroendocrine peptides (NPY, GRP, VIP, somatostatin) from the brain and stomach of the alligator.

Despite the important position of the reptiles in phylogeny, relatively few regulatory peptides from reptilian species have been characterized structurally. Neuropeptide Y was isolated from the brain of the alligator, Alligator mississippiensis, and vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), its COOH-terminal decapeptide (GRP-10), and somatostatin-14 were isolated from the alligator stomach. The primary structures of NPY and somatostatin-14 are the same as the corresponding peptides from the human, whereas alligator VIP is identical to chicken VIP. The amino acid sequence of GRP (Ala-Pro-Ala-Pro-Ser-Gly-Gly-Gly-Ser-Ala10-Pro-Leu-Ala-Lys-Ile-Tyr -Pro-Arg-Gly-Ser20-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2) contains an additional residue and six substitutions compared with chicken GRP, but alligator GRP-10 is the same as chicken GRP-10. Bombesin was not detected in the stomach extract. The data confirm that evolutionary pressure to conserve the amino acid sequence of NPY and somatostatin-14 has been very strong but demonstrate that pressure to conserve the complete primary structure of GRP has been less than that for other neuroendocrine peptides. The identity of chicken and alligator VIP is consistent with the known close phylogenetic relationship between crocodilians and birds.

Preparative peptide purification by cation-exchange and reversed-phase perfusion chromatography.

Cation exchange was compared to reversed-phase chromatography for the preparative purification of a 28-residue peptide (vasoactive intestinal polypeptide) on the 100-mg scale. Optimized high-speed, high-resolution methods were developed for both chromatographic modes on POROS Perfusion Chromatography flow-through particle chromatography columns. While both methods appeared to provide similar purity, the cation exchange column had approximately ten times the loading capacity per unit column volume as the reversed-phase column. Five-minute methods for desalting the cation exchange-purified peptide and analysis of fractions were developed using small reversed-phase columns. The cation-exchange method was scaled up to process 95 mg of crude peptide in a 12-min run.