Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14.

Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions.

We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity ( approximately 60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA ( approximately 1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a approximately 26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of 125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.

Production of monoclonal antibodies in COS and CHO cells.

Recent advances in the generation of genetically engineered monoclonal antibodies have enhanced the importance of COS cells as expression systems for rapidly producing sufficient quantities of these proteins for preliminary biochemical and biophysical analysis. In order to meet the demand for clinical supplies, a gradual increase has occurred in the usage of dihydrofolate reductase negative (DHFR-) Chinese hamster ovary (CHO) cells for large-scale antibody production. Using a variety of mammalian expression vectors and selection/amplification protocols, CHO cell lines capable of producing monoclonal antibodies at levels exceeding 1 gl-1 can now be obtained in an almost routine fashion. For the applications of monoclonal antibodies to expand into additional therapeutic areas, however, a 5-10-fold increase over current highest expression levels may still need to be achieved.

Cloning, expression and functional characterization of type 1 and type 2 steroid 5 alpha-reductases from Cynomolgus monkey: comparisons with human and rat isoenzymes.

The Cynomolgus monkey may provide an alternative pharmacological model in which to evaluate the efficacy of novel inhibitors of the two known human steroid 5 alpha-reductase (SR) isoenzymes. To evaluate the suitability of this species at the level of the molecular targets, a Cynomolgus monkey prostate cDNA library was prepared and screened using human SR type 1 and 2 cDNAs as hybridization probes. Two distinct cDNA sequences were isolated encoding the monkey type 1 and 2 SR isoenzymes. These sequences share 93 and 95% amino acid sequence identity with their human enzyme counterparts, respectively. Difference in monkey type 1 SR, however, was found within the contiguous four amino acids corresponding to the regions in the human and rat sequences that have been proposed previously to influence steroid and inhibitor affinities. Subsequently, both monkey cDNAs were individually expressed in a mammalian cell (CHO) line. Enzyme activities of both monkey SRs were localized to the membrane fractions of CHO cell extracts. Like the human and rat enzymes, the monkey type 1 and type 2 SRs were most active at neutral and low pH, respectively. The results of inhibition studies with over 30 known SR inhibitors, including epristeride, 4MA, and finasteride, indicate that the monkey SR isoenzymes are functionally more similar to the human than the rat homologues. The results from initial velocity and inhibition studies as functions of pH with the human and monkey type 2 SRs also compare favorably. These results, together, suggest that the monkey SR isoenzymes are structurally and functionally comparable on a molecular level to their respective human counterparts, supporting the relevance and use of the Cynomolgus monkey as a pharmacological model for in vivo evaluation of SR inhibitors.

The soluble form of E-selectin is an asymmetric monomer. Expression, purification, and characterization of the recombinant protein.

The gene coding for a soluble form of human E-selectin (sE-selectin) has been expressed in Chinese hamster ovary (CHO) cells. Cells seeded into a hollow fiber reactor secreted protein at a level of 160 mg/liter. The protein was purified to > 95% pure and low endotoxin (< 2 ng/mg), using physiological pH and buffers. The amino acid composition and N-terminal sequence were as predicted from the cDNA sequence. HL-60 cells bound to sE-selectin-coated plates in a dose-dependent manner, and this binding could be blocked up to 100% by pretreatment of HL60 cells with sE-selectin. The concentration of sE-selectin required for 50% inhibition was 1 microM. This value puts an upper limit for the affinity of E-selectin for its natural receptor. sE-selectin also inhibited inflammatory migration of neutrophils in a selective fashion. Purified sE-selectin exhibited a broad band of M(r) approximately 75,000 on nonreducing SDS-PAGE. sE-selectin eluted with M(r) approximately 310,000 from size exclusion chromatography at physiological pH and buffers, suggesting an oligomeric state. Matrix-assisted laser-desorption MS gave a molecular weight of 80,000, while the minimum monomer molecular weight from the gene sequence should be 58,571, demonstrating that the monomeric molecule thus expressed had 27% carbohydrate. Equilibrium analytical ultracentrifugation gave an average solution molecular weight of 81,600 (+/- 4,500). Velocity ultracentrifugation gave a sedimentation coefficient of 4.3 S and, from this, an apparent axial ratio of 10.5:1, assuming a prolate ellipsoid of revolution. An analysis of the NMR NOESY spectra of sE-selectin, sialyl-Lewis X, and sE-selectin with sialyl-Lewis X demonstrates that the recombinant protein binds sialyl-Lewis X productively. Hence, in solution, sE-selectin is a functional elongated monomer.

Human AT1 receptor is a single copy gene: characterization in a stable cell line.

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT1) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT1 receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT1 receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT1 receptor in a pure cell system, the recombinant human AT1 receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT1-D6, was found to express abundant levels of recombinant receptor [430 +/- 15 fmol/mg] exhibiting high affinity [KD = 0.15 +/- 0.02 nM] for [125I][SAR1, Ile8] angiotensin II (SIA). The pharmacological profile of ligands competing for [125I] SIA binding to the expressed receptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5'-(gamma-thio) triphosphate [GTP gamma S]. Angiotensin II evoked a rapid efflux of 45Ca2+ from LhAT1-D6 cells that was blocked by AT1 receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT1-D6 cell line provides a powerful tool to explore the human AT1 receptor regulation.

DHFR coamplification of t-PA in DHFR+ bovine endothelial cells: in vitro characterization of the purified serine protease.

High-level expression of human tissue-type plasminogen activator was accomplished in endothelial cells by a novel approach to dihydrofolate reductase (DHFR) coamplification in DHFR+ cells. A tripartite mammalian expression vector coding for DHFR, neomycin phosphotransferase, and the t-PA gene was introduced into bovine endothelial cells by transfection and selection for G418 resistance. Upon methotrexate selection of these transformants, we obtained endothelial cells that had amplified the plasmid-encoded DHFR and t-PA genes. As a result, cell lines were isolated that efficiently produced t-PA (greater than 4 pg/cell.day). This t-PA was purified and compared with recombinant t-PA produced in Chinese hamster ovary cells. These two t-PA samples differed in carbohydrate composition, and amounts of 530 and 527 amino acid forms but had similar in vitro activity.

Use of two different deoxyribonucleic acid probes to detect Y chromosome deoxyribonucleic acid in subjects with normal and altered Y chromosomes.

Four experiments evaluated the sensitivity and specificity of molecular techniques to detect human Y chromosome deoxyribonucleic acid. In experiment I, electrophoretic separation of normal male deoxyribonucleic acid fragments after digestion with endonuclease Hae III revealed two male-specific bands of 3.4 and 2.1 kilobase (kb). These bands were not visible if the fraction of male deoxyribonucleic acid in mixed samples was less than 0.3. In experiment II, by means of a repetitive copy Y deoxyribonucleic acid probe (pS4) mapped to Yq12, a male-specific 2.3 kb band was detectable in mixtures of 2.5 ng of male deoxyribonucleic acid and 997.5 ng of 45,X female deoxyribonucleic acid. In experiment III, hybridization with the pS4 probe was performed on the deoxyribonucleic acid of 20 subjects with a normal or a variant Y chromosome. In experiment IV, deoxyribonucleic acid from the same subjects was hybridized to a single copy probe (4B-2) mapped to the Yq11 region. Deoxyribonucleic acid from category A subjects (n = 8) with cytologically normal Y chromosomes hybridized to both deoxyribonucleic acid probes. Deoxyribonucleic acid from category B subjects (n = 2), including a variant Y chromosome that was negative for Q-banding but positive for C-bands, hybridized with the distal pS4 and proximal 4B-2 probes. Deoxyribonucleic acid from category C subjects (n = 10) with variant Y chromosomes uniformly negative for Q- and C-bands, did not hybridize with the pS4 probe. Deoxyribonucleic acid from three of the 10 category C subjects did hybridize to the more proximal sequence-detecting 4B-2 probe. Deoxyribonucleic acid from the remaining seven subjects in category C did not hybridize with either of the deoxyribonucleic acid probes.

The gene for the human immune interferon receptor is located on chromosome 6.

When 32P-labeled human recombinant immune interferon gamma (Hu-[32P]IFN-gamma) is crosslinked to human cells with disuccinimidyl suberate, a complex with a molecular size of approximately equal to 117,000 Da was identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The formation of this complex is inhibited when the binding is performed in the presence of excess unlabeled Hu-IFN-gamma. The specific formation of the 117,000-Da complex is not observed in mouse L cells or Chinese hamster ovary cells. This complex shows all of the criteria that identify it as the Hu-IFN-gamma receptor or its binding subunit. The same complex can be formed following binding and covalent crosslinking of Hu-[32P]IFN-gamma to some hamster-human or mouse-human somatic cell hybrids. The presence of human chromosome 6 in the hybrids is necessary and sufficient for the formation of this complex. More specifically, the long arm of chromosome 6 seems sufficient. Therefore, we have localized the gene for the Hu-IFN-gamma receptor (or its binding subunit) to the long arm of human chromosome 6. The presence of this chromosome in the somatic cell hybrids is not adequate, however, to confer antiviral resistance to the hybrids in the presence of Hu-IFN-gamma.

Differentiation alters the unstable expression of adenine phosphoribosyltransferase in mouse teratocarcinoma cells.

Three multipotent mouse teratocarcinoma stem lines, all exhibiting unstable expression for the purine salvage enzyme adenine phosphoribosyltransferase (APRT) were used for the isolation of differentiated cell lines from neoplasms developed in syngeneic mice. Two of the stem cell lines (DAP1B and DAP1C) exhibited homozygous deficiencies for APRT expression while the third stem cell line (E140) exhibited a heterozygous deficiency (Turker, M.S., Smith, A.C., and Martin, G.M.; Somat. Cell Mol. Genet.; 10:55-69; 1984). A total of 16 morphologically differentiated cell lines were established from these neoplasms; most were no longer tumorigenic. Differentiated cell lines derived from the E140-induced tumors segregated homozygous deficient mutants in a single step, consistent with their retention of the heterozygous deficient state. Differentiated homozygous deficient cell lines gave rise to phenotypic revertants at very high frequencies (10(-1) to 10(-2)). The majority of these putative revertants, however, yielded cell-free extracts with little or no detectable APRT activity. These putative revertants were capable of adenine salvage and were therefore termed APRT pseudorevertants. Since the APRT pseudorevertant phenotype was only observed in the differentiated progeny of the APRT deficient stem cell lines, we conclude that this change in the nature of the revertant phenotype was a consequence of cellular differentiation.

Cloning of a functional human adenine phosphoribosyltransferase (APRT) gene: identification of a restriction fragment length polymorphism and preliminary analysis of DNAs from APRT-deficient families and cell mutants.

A complete human APRT gene has been isolated from a lambda phage genomic library using cloned mouse APRT DNA as a probe. The human gene, contained in a recombinant lambda phage designated lambda Huap15, is functional by virtue of its capacity to transfer human APRT activity to Aprt- mouse recipient cells after phage-mediated transfection. Digestion of lambda Huap15 DNA with BamH1 generated a 2.2-kb fragment that is the only fragment of eight produced to hybridize with the mouse APRT gene. This 2.2-kb BamH1 fragment is a unique, single copy sequence, and has been used to identify a restriction fragment length polymorphism (RFLP) associated with the APRT locus. Taq1 digestion and Southern blot analysis of DNAs from 49 unrelated individuals produced three different patterns. DNAs of 30 individuals produced a restriction pattern of three labeled fragments about 500 bp, 600 bp, and 2.1 kb in size, which is characteristic for individuals homozygous for the more common allele. Two individuals homozygous for the less frequent allele displayed labeled fragments of 500 bp and 2.7 kb. The remaining 17 DNA samples produced all four labeled bands as expected for heterozygous individuals. The frequency of heterozygotes in the population is about 35%, while the frequency of the less common allele is about 0.21. Restriction enzyme analysis of DNAs from two APRT-deficient brothers and from an unrelated heterozygote revealed no gross deletions or rearrangements, nor the Taq1 polymorphism.

Plasmid, phage, and genomic DNA-mediated transfer and expression of prokaryotic and eukaryotic genes in cultured human cells.

Transfection of mammalian cells with genomic DNA and cloned genes is now relatively routine. However, the vast majority of studies have used rodent cells as recipients. Here we describe efficient transfection of two human cell lines, the hypoxanthine guanine phosphoribosyltransferase (HPRT)-deficient HeLa line, D98/AH-2, and the adenine phosphoribosyltransferase (APRT)-deficient HT1080 line, HTD114. D98/AH-2 cells were transfected with the pSV2-gpt plasmid of Mulligan and Berg, which contains the E. coli xanthine-guanine phosphoribosyltransferase (gpt) gene, and Gpt + transfectants were selected in HAT medium. HTD114 cells were transfected with (1) genomic hamster DNA, and ouabain resistant transfectants were selected in 5 X 10(-7)M ouabain; (2) with hamster and mouse genomic DNA, and Aprt + cells were selected in AAA medium; (3) with plasmids containing either the cloned hamster or mouse APRT genes, and Aprt + cells were selected; and (4) with phage particles containing a cloned mouse APRT gene, and Aprt + cells were selected. Transfection efficiencies ranged from 0.25 to 1.5 X 10(3) transfectants per microgram DNA, and in certain cases secondary transfections were done. Foreign DNA in recipients was detected by blot hybridization, and the expression of foreign genes was detected by cell growth in selective media and the expression of enzymes characteristic of the species of the donor DNA. The majority of transfectants showed stable expression of the transgenome.

Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.