Receptor binding of guinea pig and pig vasoactive intestinal peptides by rat lung.
Ontology highlight
ABSTRACT:
SUBMITTER: Paul S
PROVIDER: S-EPMC1135123 | biostudies-other | 1988 Sep
REPOSITORIES: biostudies-other
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Project description:The broad spectrum of vasoactive intestinal polypeptide (VIP) cellular functions are mediated by high-affinity binding sites. To determine regulation of the VIP receptor gene expression, we have isolated and characterized two genomic clones that contain the first three exons and the 5' flanking region of the VIP receptor gene. Using RNase protection assays, receptor gene expression was detected in adult rat lung, liver and intestine, but not in fetal lung, indicating that VIP receptor is expressed in diverse tissues, and its expression is differentially regulated during lung development. The transcription start site of the gene was mapped to a cytosine residue, 76 bp upstream from the ATG initiation codon. Transfection into rat lung cell lines shows that although 126 bp of the VIP receptor 5' DNA sequences are capable of activating VIP receptor gene basal transcription 30-fold over a promoterless control, 488 bp of the 5' sequences further induce this activation to 97-fold over control. However, inclusion of up to 859 bp 5' sequences results in a decrease in basal promoter activity (31-fold over control), indicating that while sequences between -126 and -488 bp contain potential enhancer sequences, sequences between -488 and -859 bp may include a transcriptional repressor sequence. Deletion analysis shows that transcription factor Sp1 plays an important role in activating basal promoter of the VIP receptor gene. DNase I footprinting and gel-mobility-shift assays show that Sp1 binds to its consensus binding sites in the VIP receptor promoter, suggesting that interaction of Sp1 with VIP receptor promoter transactivates this gene.
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Project description:Guinea pigs represent an important model for a number of infectious and non-infectious pulmonary diseases. The guinea pig genome has recently been sequenced to full coverage, opening up new research avenues using genomics, transcriptomics and proteomics techniques in this species. In order to further annotate the guinea pig genome and to facilitate future pulmonary proteomics in this species we constructed a 2-D guinea pig proteome map including 486 protein identifications and post translational modifications (PTMs). The map has been up-loaded to the UCD 2D-PAGE open access database (http://proteomics-portal.ucd.ie/). Transit peptides, N-terminal acetylations and other PTMs are available via Peptideatlas (ftp://PASS00619:NM455hi@ftp.peptideatlas.org/). This dataset is associated with a research article published in the Journal of Proteomics [1].
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Project description:Phosphodiesterase II from extracts of intestinal mucosa of rat and guinea pig was purified by chromatography on DEAE-cellulose, CM-cellulose and agarose. The rat enzyme was purified 350-550-fold, with recoveries ranging up to 46%. The best purification of the guinea-pig enzyme was 15-fold, and the recovery was only 2.6%, the large loss occurring during chromatography on DEAE-cellulose and agarose. The poor results with the guinea-pig enzyme reflect the difficulty in obtaining a truly soluble material. Repeated sonication of the crude guinea-pig preparations yielded material that was initially soluble but tended to re-aggregate quickly. Purification of the rat phosphodiesterase II increased its thermostability, the temperature of half-inactivation being increased from 54degrees to 60degreesC. Both enzymes had a Km value of 4 X 10(-5) M with thymidine 3'-(2,4-dinitrophenyl) phosphate as substrate and showed similar pH optima for activity. Both enzymes were inhibited slightly in 0.1 M-MgC12 or 2M-urea and much more strongly in 2M-(NH4)2SO4 or 6M-NaC1. The guinea-pig enzyme was usually inhibited more than the rat enzyme. The Arrhenius plots of the two enzymes differed slightly in slope, but both were biphasic, showing breaks between 30degrees and 40degreesC. It was concluded that the two enzymes were markedly similar in behaviour and that the differences found were related to the different degrees of purification attained by the procedures described.
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Project description:Glyco-engineered recombinant antibodies are currently being developed as the next generation therapeutics to treat human diseases, including cancer, autoimmunity and infection. Antibodies lacking core fucosylation show great increase in affinity for FcγRIIIA, leading to an improved receptor-mediated effector function. While afucosyl human IgG1 exhibits 50-100-fold increase in antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism underlying the anti-cancer effect of some approved therapeutic antibodies, it is not clear whether such glyco-engineered antibodies would find similar use for infectious disease. Due to the species difference, human antibodies may have different binding properties towards corresponding IgG receptors from animals used for modeling infection and intoxication. During the course of studying a recombinant human IgG1 in neutralizing diphtheria toxin (DT) in Guinea pigs (Cavia porcellus), we identified a previously uncharacterized Guinea pig protein H0VDZ8 from UNIPROT database that shows high sequence homologies to human FcγRIIIA and mouse FcγRIV. This Fcγ receptor, which we named as gpFcγRIV, also demonstrates functional similarity although not to the same extent as the human and mouse counterparts, in that it binds to afucosyl human and mouse IgG much stronger than to the wild type antibodies. Thus, Guinea pigs can be used to compare the efficacies of wild type vs. afucosyl anti-DT human IgG1 in toxin removal and animal protection. Molecular and functional characterization of human FcγRIIIA and mouse FcγRIV equivalents in other species could expand the list of preclinical animal models for testing afucosyl human antibodies in treating various human diseases.
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Project description:Pituitary adenylate-cyclase-activating polypeptide (PACAP) stimulates release of several anterior pituitary hormones by interacting with PACAP receptors on pituitary cells. To learn more about the distribution and possible regulatory roles of PACAP and its receptors in human pituitary adenomas, we investigated the expression of vasoactive intestinal polypeptide (VIP) and PACAP binding sites using receptor autoradiography, PACAP and PACAP/VIP receptor (PVR) mRNAs by reverse transcription polymerase chain reaction (RT-PCR), conventional in situ hybridization, and catalyzed reporter deposition in situ hybridization (CARD-ISH) analyses. PACAP mRNA was expressed in normal human hypothalamus, which was used as a positive control, but not in pituitary adenomas. Receptor autoradiography showed PACAP types I and II binding sites in all groups of pituitary adenomas, except prolactinomas. The highest levels were present in gonadotroph and null cell adenomas. PVR-2 mRNA was expressed in normal pituitaries and in all groups of pituitary adenomas by RT-PCR, whereas PVR-1 and -3 mRNAs were expressed in all groups of pituitary adenomas, except for most prolactinomas. Conventional in situ hybridization studies with digoxigenin-labeled probes demonstrated weak staining for PVR-1, -2, and -3 mRNAs in most tissues. The CARD-ISH technique, which increased the sensitivity of the in situ hybridization method, also revealed PVR-2 mRNA expression in all adenomas, whereas PVR-1 and -3 mRNAs were detected in nearly all adenomas except for prolactinomas. The presence of PACAP mRNA in the hypothalamus, but not in normal anterior pituitary or in pituitary adenomas, and the differential expression of PVRs in adenomas indicate a selective regulatory endocrine and paracrine role of PACAP in normal and neoplastic anterior pituitary cells.
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