Enzyme immunoassay of gastrin releasing peptide (GRP)-like immunoreactivity in milk

A sensitive and specific enzyme immunoassay (EIA) for gastrin releasing peptide (GRP)-like immunoreactivity was developed using enzyme-labeled antigen. The synthetic carboxy-terminal fragment of human GRP(12-27) was conjugated with beta-D-galactosidase for EIA. The minimum amount of GRP-like immunoreactivity detectable by this method was 0.24 femtomol/well (6 picomol/liter). The level of GRP-like immunoreactive substance in bovine foremilk was about 150 nanomol/liter, the level of which was more than hundredfold higher than that in normal milk or calf serum.     

Effects of porcine gastrin releasing peptide (GRP) on canine antral motility and gastrin release in vivo

The effect of GRP on the vivo canine antrum was investigated. GRP caused a dose-dependent increase in antral gastrin output which was not significantly altered by administration of tetrodotoxin. The higher doses of GRP administered also caused excitation of antral motility which was abolished by tetrodotoxin, a finding in contrast to previous in vitro results demonstrating bombesin-induced antral smooth muscle contraction to be tetrodotoxin-resistant. These data suggest that in the vivo canine model GRP causes antral gastrin release via non-reurally mediated mechanisms (probably by acting directly on the G-cell) and excites antral motility via neurally-mediated mechanisms.     

Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization

Hybrid antibacterial peptide CA-MA (cecropinA(1-8)-magainin2(1-12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA-MA in methylotrophic yeast, Pichia pastoris, the gene of CA-MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZalpha-A. The SalI-linearized plasmid pPICZalpha-CA-MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96h with 1.0% methanol at 28 degrees C, pH 5.0. Recombinant CA-MA was purified by reversed-phase HPLC and 22 mg pure active CA-MA was obtained from 1L fermentation culture. Tricine-SDS-PAGE indicated that recombinant CA-MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA-MA demonstrated a single large signal for the molecular ion [M+2H+](2+) at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA-MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA-MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.     

Aging and gender affect the response of thyrotropin (TSH) to gastrin releasing peptide (GRP) in rats

We had previously shown that GRP acts directly at the pituitary gland inhibiting basal and TRH-stimulated TSH secretion in adult male rats. In this study we showed a gender dimorphism in this response of old animals pituitaries to GRP. In both female and male young adult animals, GRP-incubated pituitaries showed approximately 50% less basal and TRH-stimulated TSH secretion to the medium, without affecting the pituitary content of TSH. However, GRP did not have any significant effect upon TSH secretion in old male rats, but the old female showed the same degree of response to GRP as the young adult female rat, regarding basal and TRH-stimulated TSH secretion, while the TSH pituitary content after GRP incubation was higher than that of the young female group. Our data suggest a loss of thyrotrope responsiveness to GRP in aged male rats that could contribute to the decrease in TSH pituitary stores leading to lower basal and TRH-stimulated TSH secretion. Meanwhile, the preservation of GRP responsiveness could help in the relative maintenance of these parameters in the old female rat.     

A two dimensional 1H NMR study of the solution conformation of gastrin releasing peptide

An almost complete assignment of the 1H NMR spectrum of gastrin releasing peptide in dimethyl sulphoxide solution and aqueous solution has been carried out using two dimensional NMR techniques. The chemical shifts in both solvents have been compared with the corresponding values in random coil polypeptides and it is concluded that gastrin releasing peptide adopts little short or long range order under either solvation conditions.     

Bombesin/oligoarginine fusion peptides for gastrin releasing peptide receptor (GRPR) targeted gene delivery

The development of non-viral gene delivery systems, with the capacity to overcome most of the biological barriers facing gene delivery, is challenging. We have developed peptide-based, multicomponent, non-viral delivery systems, incorporating: a bombesin peptide ligand (BBN(6–14)), to selectively target the gastrin releasing peptide receptor (GRPR); oligoarginine peptides (hexa- (R₆) and nona-arginine (R₉)), for plasmid DNA (pDNA) condensation; and GALA, to facilitate endosome escape. The uptake and endosome escape efficiency of bombesin/oligoarginine and bombesin/oligoarginine/GALA fusion peptides for oligonucleotide delivery was evaluated in terms of their complex size, cellular uptake, endosome escape, and cellular toxicity. Complex size and cell uptake studies demonstrated that the nona-arginine/bombesin delivery system was more efficient at condensing and delivering pDNA into PC-3 prostate cancer cells compared to the hexa-arginine/bombesin delivery system. Further, competition with free bombesin peptide, and comparative uptake studies in Caco-2 cells, which express GRPR at a lower level, suggested that GRPR contributes to the targeted uptake of this system. The addition of GALA into the nona-arginine/bombesin-based system further increased the pDNA cellular uptake at all tested N/P ratios; facilitated endosomal pDNA release; and had limited effects on cell viability. In conclusion, the delivery system combining BBN(6–14) with nona-arginine and GALA had optimal characteristics for the delivery of pDNA into the GRPR overexpressing cell line PC-3.     

Motor effects of gastrin releasing peptide (GRP) and bombesin in the canine stomach and small intestine

Close intraarterial injections of synthetic porcine gastrin releasing peptide (GRP) or bombesin stimulated contractions in the stomach and inhibited ongoing contractile activity in the small intestine of anaesthetized dogs. Contractile activity of the circular muscle was recorded by serosal strain gauges and phasic activity when desired was elicited by local field stimulation or intraarterial motilin injections. In the stomach (corpus and antrum) following tetrodotoxin blockade of field-stimulated contractions, the contractile response to either peptide was not present, suggesting that stimulation of receptors on nerves initiated contractions in the stomach. Similarly, in the small intestine, the inhibitory response was eliminated by tetrodotoxin suggesting a neural receptor. Pre-treatment with reserpine did not alter the inhibitory response, either in the presence or absence of atropine, therefore, adrenergic inhibitory mechanisms did not appear to be involved. The concentration of bombesin producing 50% inhibition of field stimulation (ED50) was increased following treatment with the putative M1 muscarinic antagonist, pirenzipine suggesting activation of M1 cholinergic inhibitory receptors by bombesin. After blockade by atropine of field-stimulated contractions and the contractile response to intraarterial acetylcholine, the ED50 for bombesin inhibition of motilin contractions was increased. After muscarinic blockade, the residual inhibitory response of GRP/bombesin may involve activation of a neural non-cholinergic non-adrenergic inhibitory mechanism. These results suggest that GRP and bombesin act to alter motility in the dog in vivo by affecting neural activity.     

Monoclonal antibodies against human abnormal (des-gamma-carboxy)prothrombin specific for the calcium-free conformer of prothrombin

A monoclonal antibody JO1 X 1 was prepared against human abnormal prothrombin using the hybridoma technique. The clone secreting this antibody was selected on the basis of the ability of this antibody to bind to abnormal prothrombin, but not to prothrombin, in the presence of calcium ions. The antibodies were purified by affinity chromatography in EDTA on columns of prothrombin-Sepharose. Bound antibodies were eluted with 15 mM CaCl2. The kinetics of dissociation of antibody from the antibody-prothrombin complex with the addition of calcium ions fit a first-order kinetic model. Increasing CaCl2 concentration increased the rate of antibody-prothrombin dissociation. Ca(II) and Mn(II) inhibited antibody-prothrombin interaction; half-maximal binding was observed at 0.9 and 4 mM, respectively. Mg(II) had little effect on antibody-antigen interaction. The JO1 X 1 antibody bound fragment 1, fragment (1-39), abnormal prothrombin, and prothrombin equivalently in the presence of EDTA, but did not bind to des(1-44)prothrombin in the presence of EDTA or prothrombin in the presence of CaCl2. These results indicate that the monoclonal antibody JO1 X 1 is conformation specific for the calcium-free conformer of prothrombin and directed against an antigenic determinant near the NH2 terminus of prothrombin expressed in the 1-39 region of the protein. This analysis provides confirmation of the presence of a metal-free conformer of prothrombin.     

Detection of RNA and specific antibodies against influenza virus A (H5N1) in human samples

Results of inspection 451 persons in the age of from 19 till 58 years and 2 children in the age of till 2 years on presence of specific antibodies and RNA of the infuenza virus A (H5N1) are submitted. RNA of infuenza virus A (H5N1) in researched samples is not revealed. Specific antibodies to a infuenza virus A (H5N1) in a blood serum of 211 patients and donors it is not revealed.     

Secondary structure of the human growth hormone releasing factor (GRF 1-29) by two-dimensional 1H-nmr spectroscopy

The secondary structure of the human growth hormone releasing factor (GRF 1-29) in a solution mixture of 60% aqueous phosphate buffer:40% 2,2,2-trifluoroethanol-d₃ has been investigated by two-dimensional ¹H-nmr spectroscopy. Sequential resonance assignments and elements of secondary structure were obtained from phase-sensitive correlation spectroscopy, relayed coherence spectroscopy, and nuclear Overhauser spectroscopy experiments. The observation of a large number of α-amide and amide-amide interresidual nuclear Overhauser effect connectivities as well as the existence of 11 slowly exchanging amide protons indicates that the peptide adopts a well-defined secondary structure most likely constituted of a single long helix. This conclusion is consistent with the CD measurements.     

Quantification of the bombesin/gastrin releasing peptide antagonist RC-3095 by liquid chromatography-tandem mass spectrometry

Bombesin (BN) and its mammalian equivalent, gastrin-releasing peptide (GRP), stimulate cell proliferation and are involved in the pathogenesis of several types of human cancer. BN/GRP and their receptors were shown to be critical for the growth of various human malignancies, such as small-cell lung, prostate, ovary, stomach and breast cancers in the human tumor xenograft model. In the present study, a fast, sensitive, robust method was developed for the determination and quantification of a BN/GRP receptor antagonist RC-3095 (D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leupsi(CH2NH)Leu-NH2), in human plasma by liquid chromatography coupled with tandem mass spectrometry. RC-3095 was extracted from 0.2 ml human plasma by protein precipitation using cold acetonitrile (0.4 ml). The method has a chromatographic run of 10 min using a C(8) analytical column (150 mm x 4.6 mm i.d.) and the linear calibration curve over the range was linear from 20 to 10000 ng ml(-1) (r(2)>0.994). The between-run precision, based on the relative standard deviation replicate quality controls, was 5.7% (60 ng ml(-1)), 7.1% (600 ng ml(-1)) and 6.8% (8000 ng ml(-1)). The between-run accuracy was +/-0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively. The developed procedure allows the quantitative determination of peptide RC-3095 for pharmacokinetics studies in human plasma.     

The [Lys(-2)-Arg(-1)-des(17-21)]-endothelin-1 peptide retains the specific Arg(-1)-Asp8 salt bridge but reveals discrepancies between NMR data and molecular dynamics simulations

The [des(17-21)]-endothelin-1 (CSH-ET) and [Lys(-)(2)-Arg(-)(1)-des(17-21)]-endothelin-1 (KR-CSH-ET) peptides, designed by removing the five-residue hydrophobic tail from the endothelin-1 (ET-1) and [Lys(-)(2)-Arg(-)(1)]-endothelin-1 (KR-ET-1) peptides, respectively, were synthesized. Previous studies on KR-ET-1 showed that, in contrast to ET-1, this engineered compound displays a pH-dependent conformational change related to the formation of a stabilizing salt bridge between the Arg(-)(1) and Asp(8) side chains. CD and NMR spectra indicate that CSH-ET and KR-CSH-ET display conformational behavior similar to those of ET-1 and KR-ET-1, respectively. The short salt bridge-stabilized KR-CSH-ET peptide therefore appears to be an attractive elementary scaffold for drug design. The solution structure of the salt-bridged form of KR-CSH-ET was determined by NMR at pH 4.5 and is very similar to the corresponding form of the parent KR-ET-1 peptide. Molecular dynamics simulations of the salt-bridged form of KR-CSH-ET were performed using both the GB/SA implicit solvation scheme or an explicit solvation and the particle-mesh Ewald method for long-range electrostatic calculation. Unexpectedly, the Arg(-)(1)-Asp(8) salt bridge does not display in the simulation the stability that could be expected from the experimental data. The cooperative involvement of a cation-pi interaction in formation of the salt bridge has been hypothesized. Difficulties in accurately simulating cation-pi interactions might be responsible for the lack of stability in the simulation. At this time, however, no definitive explanation for the observed discrepancy between experiments and simulations is available, and further experimental studies appear to be necessary to fully understand in atomic detail the pH-dependent conformational change observed in the KR-ET-1 series.     

Induction of human immunodeficiency virus (HIV-1) envelope specific cell-mediated immunity by a non-homologous synthetic peptide

Background

Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide.

Methodology/Principal Findings

The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants.

Conclusions/Significance

For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.     

Vasopressin(1-8) (des-glycinamide9-[Arg8]vasopressin) is an endogenous peptide present in rat plasma

Using a radioimmunoassay for [Arg8]vasopressin(1-8) (des-glycinamide9-[Arg8]vasopressin; DGAVP) endogenous immunoreactive DGAVP (IR-DGAVP) was detected in extracts of plasma prepared from trunk blood of male Wistar rats. The IR-DGAVP was further characterized by reversed-phase high pressure liquid chromatography (HPLC). One of the two immunoreactive peaks obtained by HPLC coeluted with synthetic DGAVP and did not cross-react in a radioimmunoassay specific for [Arg8]vasopressin(1-9) (AVP). The other showed the chromatographical and radioimmunological characteristics of AVP. Analysis by HPLC of plasma prepared from fresh blood spiked with 3H-AVP indicated that under the experimental conditions employed no DGAVP was formed during extraction. The results indicate that DGAVP is present in rat plasma, possibly as an endogenous metabolite of AVP.     

Gram-scale syntheses of the (1-->3)-linked and (1-->4)-linked hyaluronan disaccharides

The first gram-scale syntheses of two hyaluronan disaccharides are described. Construction of the (1-->4)-linked disaccharide 12 was achieved in 12% overall yield using 2,3-bis-dimethyl acetal protection in combination with chlorosilane-induced carbamate cleavage methodologies. The uronic acid functionality was installed using TEMPO oxidation with NaOCl as the hypochlorite source. The (1-->3)-linked disaccharide 18 was achieved in 7% overall yield utilizing acetonide protection in addition to the chlorosilane-induced carbamate cleavage methodology and the TEMPO oxidation.     

The human lactoferrin-derived peptide hLF1-11 exerts immunomodulatory effects by specific inhibition of myeloperoxidase activity

Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are considered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte-macrophage differentiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may exert its modulatory effects on human monocytes by specific inhibition of MPO activity.     

Rapid immunoassay to detect antibodies against human T-cell lymphotropic virus type 1 (HTLV-1) by the recombinant immunodominant antigens

A rapid immunochromatographic assay, using the recombinant immunodominant antigens of HTLV-1, has been developed to detect circulating antibodies to HTLV-1. The method was compared with an enzyme-linked immunosorbent assay by evaluating 1,631 serum or plasma samples. This HTLV-1 rapid assay was easy to perform and required no special equipment which provided visual result within 5 min with an excellent sensitivity and specificity in detecting HTLV-1 infection.