Establishment of vascular endothelial cell lines in a serum-free culture and the discovery of endothelin and a Vasoactive Intestinal Contractor (VIC)

Immortal vascular endothelial cell lines were established and utilized for the production of an endothelium-derived contraction factor (EDCF) in a serum-free medium. After the discovery of Endothelin (21 amino acid peptide, ET) as an EDCF, a prepro ET cDNA isolated from human tissue was used to examine the expression of ET and its regulation in human endothelial cells. A gene family of ET was shown in mouse by using prepro ET cDNA as a probe. Thus, a novel peptide, Vasoactive Intestinal Contractor (VIC) homologous to ET was deduced from the sequence of one of these genes. VIC was confirmed to induce vasocontraction as well as intestinal contraction. Northern blot analysis indicated that this gene was expressed in the intestine but not in endothelial cells. A cloning and sequencing of prepro VIC cDNA from mouse intestine suggest that a VIC-like peptide, as well as VIC, are co-synthesized by cleavage from prepro VIC with 160 amino acids.     

The prepro vasoactive intestinal contractor (VIC)/endothelin-2 gene (EDN2): structure, evolution, production, and embryonic expression

Murine vasoactive intestinal contractor (VIC) and its human analog endothelin-2 (ET2) are potent vasoactive hormones composed of 21 amino acids. To study the structural characteristics of the VIC/ET2 gene (HGMW-approved symbol EDN2), we isolated the full length of the mouse VIC gene. Sequence analysis indicates that a biologically active mature VIC peptide is produced from a 175-residue precursor protein; preproVIC (PPVIC). Several remarkable similarities of the PPVIC gene to the human preproendothelin-1 gene strongly suggest that the two genes have arisen from a common progenitor by gene duplication. Transfection of ACHN adenocarcinoma cells with the cDNA resulted in the production of VIC peptide. VIC production was increased by the deletion of the 3'-untranslated region, which contains an AU-rich mRNA destabilizing sequence. Increased PPVIC gene expression during the late embryonic stage suggests an important function in development. This study provides the basis for disruption and regulation analysis of the gene, which may lead to a better understanding of VIC/ET2's physiological significance.     

cDNA cloning and chromosomal assignment of the endothelin 2 gene: vasoactive intestinal contractor peptide is rat endothelin 2.

Four members of the endothelin family of vasoactive and mitogenic peptides have been identified: human endothelins 1, 2, and 3 (ET1, ET2, and ET3, respectively) and mouse vasoactive intestinal contractor (VIC). To characterize the mRNA encoding ET2, a 192-bp fragment of the ET2 gene, amplified by the polymerase chain reaction from human genomic DNA, was used to screen cell lines and tissues for ET2 gene expression. ET2 mRNA was detected in a cell line (HTB119) derived from a human lung small cell carcinoma, and an ET2 cDNA was cloned from a cDNA library prepared from HTB119 mRNA. DNA prepared from human-mouse somatic hybrid cell lines was used to assign the gene encoding ET2 (EDN2) to the 1p21----1pter region of chromosome 1, demonstrating that EDN2 is not linked to genes encoding ET1 (EDN1; chromosome 6) and ET3 (EDN3; chromosome 20). Southern blot hybridization revealed a single gene in human and rat genomes that hybridized with the ET2 gene fragment, and the rat gene was cloned. The endothelin peptide encoded by the rat gene differed from ET2 at 1 of 21 residues and was identical to mouse VIC. We conclude that VIC is the mouse and rat analogue of the human ET2 gene.     

A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity

A new peptide family (endothelin (ET] consisting of three members in mammals appears to be present in mice according to genomic Southern blot analysis. Two ET-related genes were identified by cloning and sequence analysis of a mouse genome. One encoded a peptide identical to porcine and human vasoconstrictor peptide ET, and the other encoded a novel peptide differing from ET in 3 amino acid residues, with 4 cysteines in the same positions as in ET. This novel peptide was synthesized and confirmed to have in vivo pressor activity similar to that of ET. Northern blot analysis, however, indicated the gene of this novel peptide to be expressed only in the intestine, and not in other tissues or cell lines, or endothelial cells. Furthermore, the peptide evoked a strong contractile response in the guinea pig ileum. This peptide may thus be reasonably classified as a gastrointestinal peptide, vasoactive intestinal contractor.     

cDNA cloning, sequence analysis and tissue distribution of a precursor for vasoactive intestinal contractor (VIC)

A full-length cDNA encoding preprovasoactive intestinal contractor (PPVIC) has been cloned. From the deduced 160 amino acid PPVIC, the mature VIC is predicted to be produced via a 37 residue intermediate, big VIC. The PPVIC also contains a VIC-like peptide of 16 amino acids structurally related to to the amino-terminal residues of VIC and flanked by pairs of dibasic amino acids, putative processing sites. RNA blot hybridization with PPVIC cDNA confirmed the PPVIC gene to be expressed in the small and large intestinal tract in a tissue specific manner.     

Vasoactive intestinal contractor, a novel peptide, shares a common receptor with endothelin-1 and stimulates Ca2+ mobilization and DNA synthesis in Swiss 3T3 cells

Vasoactive intestinal contractor peptide (VIC), a novel member of the endothelin family, stimulated a rapid increase in the intracellular Ca2+ concentration in fura-2-loaded Swiss 3T3 cells. Sequential addition of VIC and endothelin-1 (ET1) demonstrated the induction of both homologous and heterologous desensitization. VIC was as potent as ET1 in displacing the binding of 125I-ET1 and in stimulating mitogenesis in Swiss 3T3 cells. These findings suggest that VIC and ET1 share a common receptor in Swiss 3T3 cells.     

Molecular cloning and characterization of a novel rat CXC chemokine, rPBP, the homologue of human and mouse PBP

We have previously cloned the mouse platelet basic protein (mPBP), a homologue of human PBP, from mouse thymic stromal cells. Using EST alignment and RT-PCR, the rat homologue of human and mouse PBP was cloned from lung and named as rPBP. The complete open reading frame and part of the 3'- and 5'-non-coding regions were obtained through rapid amplification of cDNA ends. The rPBP cDNA encodes a protein of 111 amino acids containing a signal peptide of 37 amino acids at the N-terminus, with the mature protein of 74 amino acids. The rPBP is a new member of ELR+CXC chemokines. The mature protein of rPBP shares 69% and 45% homology with mouse and human PBP, respectively. In situ hybridization assay revealed rPBP to be predominantly localized in the pulmonary vascular endothelial cells. The eukaryotic expression vector pCDNA3-rPBP was constructed and transiently transfected into COS-7 cells. In the in vitro chemotaxis assay, the polymorphonuclear leukocytes (PMNs) were chemoattracted to the supernatants from transfected COS-7 cells in a dose-dependent manner. The implication of rPBP found in rat lung is that this chemokine may have the function to recruit PMNs to fight against pulmonary infection.     

Structure of a full-length cDNA clone for the prepro alpha 2(I) chain of human type I procollagen. Comparison with the chicken gene confirms unusual patterns of gene conservation.

A cDNA clone from a human placental library was found to consist of an essentially full-length cDNA of 4.6 kb for the prepro alpha 2(I) chain of type I procollagen. Nucleotide sequencing of the 5'-end of the cDNA provided a sequence of 1617 nucleotide residues and codons for 539 amino acid residues not previously defined. Comparison of the complete structure of the prepro alpha 2(I) cDNA with previously reported sequences for the chicken pro alpha 2(I) gene indicated that 83% of 1366 total amino acid residues were conserved. In the alpha-chain domain 84% of 1014 amino acid residues were conserved. Also, there was conservation of the previously noted preference for U and C in the third position of codons for glycine, proline and alanine. One major difference between the human and the chicken prepro alpha 2(I) chain was that the human chain contained 21 fewer proline residues, an observation that probably explains why the triple helix of human type I procollagen unfolds at temperatures that are 1-2 degrees C lower. In parallel experiments, sequencing of intron-exon boundaries for nine exons of genomic subclones confirmed and extended previous observations that the pro alpha 2(I) gene, like other genes from fibrillar collagens, has an unusual 54-base pattern of exon sizes that is highly conserved through evolution.     

Isolation of a cDNA that encodes the peptide core of the secretory granule proteoglycan of rat basophilic leukemia-1 cells and assessment of its homology to the human analogue

It has been previously shown that a single gene is used to encode the peptide core of the extracellular proteoglycan of rat L2 yolk sac tumor cells and the intracellular proteoglycan of rat basophilic leukemia (RBL)-1 cells. In order to determine if the predicted amino acid sequences of these proteoglycans are identical as well as to isolate a full length cDNA encoding a rat secretory granule proteoglycan, a cDNA library was prepared from RBL-1 cells and screened with the 165-base pair 5'----XmnI fragment of pPG-1, a partial cDNA which encodes the rat L2 cell proteoglycan peptide core. Based on the consensus nucleotide sequence of two full length RBL-1 cell-derived cDNAs, the 5' untranslated region of the mRNA that is expressed in RBL-1 cells is shorter than that expressed in the rat L2 cells although the coding regions of the mRNAs from the two cell types are identical. These findings indicate that the targeting of proteoglycans to an intracellular or extracellular compartment is a cell-specific event which is independent of the translated peptide core. Since the RBL-1 cell and the rat L2 cell proteoglycans have different types of glycosaminoglycans bound to them, it can also be concluded that the selection of the type of glycosaminoglycan that will be synthesized onto a peptide core is a cell-specific event which is not exclusively dependent on the translated peptide core. When the predicted amino acid sequence of the RBL-1 cell proteoglycan peptide core was compared to the predicted sequence of the homologous human molecule from HL-60 cells, 48% of the amino acids were identical. The N terminus was the most highly conserved area of the molecule. This region of the peptide core, which precedes the serine-glycine repeat region, is likely to be of critical importance for the biosynthesis and/or function of these proteoglycans. Analysis of 10 different mouse/hamster somatic cell hybrid lines with a SspI----3' fragment of the rat L2 cell cDNA revealed that, as in the human, the gene that encodes the mouse analogue of this peptide core resides on chromosome 10.     

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.     

The sequence of human serum albumin cDNA and its expression in E. coli

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.     

Molecular cloning of nucleobindin, a novel DNA-binding protein that contains both a signal peptide and a leucine zipper structure.

We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mouse produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier. We purified this factor, a 55 kD protein that we termed nucleobindin (Nuc), and obtained its cDNA clone. Although the gene for Nuc encodes a signal peptide and, in fact, Nuc was identified as a secreted protein, Nuc had a DNA-binding property. The putative polypeptide predicted from the cDNA sequence featured a signal peptide, a leucine zipper structure and a basic amino acid-rich region. The DNA-binding property of Nuc was destroyed by deletion of either the leucine zipper structure or the basic amino acid-rich region. The amino acid sequences of Nuc are highly conserved between mouse and human. We discuss the possible role of Nuc in autoimmunity.     

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.