A Strategy Combining Differential Low-Throughput Screening and Virtual Screening (DLS-VS) Accelerating the Discovery of new Modulators for the Orphan GPR34 Receptor.

The DLS-VS strategy was developed as an integrated method for identifying chemical modulators for orphan GPCRs. It combines differential low-throughput screening (DLS) and virtual screening (VS). The two cascaded techniques offer complementary advantages and allow the experimental testing of a minimal number of compounds.

P2y(12), a new platelet ADP receptor, target of clopidogrel.

The binding characteristics of (33)P-2MeS-ADP, a stable analogue of ADP, were determined on CHO cells transfected with the human P2Y(12) receptor, a novel purinergic receptor. These transfected CHO cells displayed a strong affinity for (33)P-2MeS-ADP, the binding characteristics of which corresponded in all points to those observed on platelets. In particular, this receptor recognised purines with the following order of potency: 2MeS-ADP = 2MeS-ATP > ADP = ATPgammaS = ATP >> UTP, a binding profile which is similar to that obtained in platelets.

High-level synthesis of human prolactin in Chinese-Hamster ovary cells.

Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr(-) Chinese- hamster ovary (CHO) cells. One vector, p658-hPRL, contains the hepatitis-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation. The other, pEDdc-hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high-level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA.

An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1.

The neuropeptide neurotensin (NT) interacts with two types of human receptors (hNTR) termed hNTR-1 and hNTR-2. This study describes a monoclonal antibody (MAb) specific for hNTR-1, B-N6. This MAb binds specifically to hNTR-1, but not to hNTR-2 transfected CHO cells.

Antiproliferative effects of SR31747A in animal cell lines are mediated by inhibition of cholesterol biosynthesis at the sterol isomerase step.

SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative activity on lymphocyte cells. Only two subtypes of sigma receptor, namely the sigma1 receptor and emopamil-binding protein, have been characterised molecularly. Only the sigma1 receptor has been shown to bind (Z)N-cyclohexyl-N-ethyl-3-(3-chloro4-cyclohexylphenyl)pro pen-2-ylamine hydrochloride (SR31747A) with high affinity.

Residual host cell protein from continuous cell lines. Effect on the safety of protein pharmaceuticals.

The presence of residual host cell proteins in protein pharmaceuticals obtained from continuous mammalian cell lines has been a point of concern. Clinical experience with different biopharmaceuticals shows that residual protein in these highly purified products does not represent a danger to the health of the patients.

A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions.

In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists.

Cloning of the human IL-13R alpha1 chain and reconstitution with the IL4R alpha of a functional IL-4/IL-13 receptor complex.

The human homologue of the recently cloned murine IL-13 binding protein (IL-13R alpha1) was cloned from a cDNA library derived from the carcinoma cell line CAKI-1. The cloned cDNA encodes a 427 amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The human protein is 74% identical to the murine IL-13R alpha1, and 27% identical to the human IL-13R alpha2.

Emopamil-binding protein, a mammalian protein that binds a series of structurally diverse neuroprotective agents, exhibits delta8-delta7 sterol isomerase activity in yeast.

Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae.

Isolation of an IL-13-dependent subclone of the B9 cell line useful for the estimation of human IL-13 bioactivity.

A novel sub-clone of the B9 hybridoma cell line (B9-1-3) has been selected by cloning following continuous culture in rhIL-13. This cell line shows an increased sensitivity to both hIL-13 and mIL-4 compared to the parental B9 cell line. The proliferative response to IL-13 can be blocked with an anti-IL-4 receptor monoclonal antibody but not with the soluble IL-4 receptor, suggesting that IL-13- and IL-4-binding receptor subunits are distinct but form part of a common receptor complex.

Rapid isolation of highly productive recombinant Chinese hamster ovary cell lines.

An expression system combining a unit for the expression of the gene of interest reinforced by the hepatitis B virus X transactivator and a selectable gene weakened by the insertion of an A+T-rich sequence derived from the 3'-untranslated region of the granulocyte-macrophage colony-stimulating factor mRNA is described. This vector allows rapid one-step isolation of highly productive Chinese hamster ovary clones.

The two soluble forms of the lipopolysaccharide receptor, CD14: characterization and release by normal human monocytes.

CD14, a glycolipid-anchored membrane glycoprotein, acts as a high affinity lipopolysaccharide receptor on leukocytes. We previously reported that the Mono-Mac-6 cell line releases two different soluble forms of CD14 (sCD14) (Labeta et al., Eur.

Purified protein products of rDNA technology expressed in animal cell culture.

During the past 7 years, 14 versions of 7 rDNA proteins have been licensed which are derived from animal cell culture expression systems. These medically useful products have included hormones, coagulation factors, enzymes and a vaccine. Aspects of the molecular complexity, manufacture, control and utilization of these products are discussed.

Human interleukin 1 beta fused to the human growth hormone signal peptide is N-glycosylated and secreted by Chinese hamster ovary cells.

A hybrid gene consisting of the sequences coding for the signal peptide of human growth hormone and the mature form of interleukin-1 beta (IL-1 beta) was chemically synthesized. This sequence was inserted into a eukaryotic expression vector and introduced into Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of recombinant IL-1 beta, which was secreted into the culture medium mainly as a 22-kDa form.

Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2.

We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins.

Characterization of recombinant glycosylated human interleukin 2 produced by a recombinant plasmid transformed CHO cell line.

A recombinant plasmid containing expression units for human pre-interleukin 2 (pre-IL-2) and the selectable marker mouse DHFR, was constructed and used to transform DHFR- CHO cells to the DHFR+ phenotype. Selected colonies were isolated and tested for IL-2 production. Twelve highly IL-2-producing clones were amplified in stepwise increasing concentrations of methotrexate.

Abundant excretion of human growth hormone by recombinant-plasmid-transformed monkey kidney cells.

A recombinant plasmid was constructed permitting the efficient synthesis of human growth hormone (hGH) in monkey cells. The plasmid contains the cDNA sequence of the hGH precursor and controlled by the SV40 early promoter, an intron, and the poly(A)-addition site of the mouse alpha-globin gene. To permit selection of transformed cells, a selectable marker (xanthine-guanine phosphoribosyl transferase; XGPRT) was also introduced into this plasmid.

The effect of cell irradiation on mutation in ultraviolet-irradiated and intact simian virus 40.

The induction of phenotypic wild-type revertants in the progeny of an unirradiated or UV-irradiated temperature-sensitive late mutant of simian virus 40 was studied after low multiplicity passages in normal or UV-irradiated confluent monkey kidney cells. The production of wild-type revertants in the progeny of undamaged tsBC245 was followed by infecting the cells at distinct times after irradiation of the cells. Mutation frequencies reached a maximum when infection was delayed for 3--4 days after irradiation of the host cells, and declined gradually thereafter.

In vitro synthesis of adenovirus type 5 T antigens. II. Translation of virus-specific RNA from cells transformed by fragments of adenovirus type 5 DNA.

Virus-specific cytoplasmic RNA was isolated from rat cell lines transformed by fragments of adenovirus type 5 DNA, and the RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes. RNA was isolated from cell lines transformed by the following fragments: XhoI-C (leftmost 15.5%), HindIII-G (leftmost 8%), and HpaI-E (leftmost 4.

In vitro synthesis of adenovirus type 5 T antigens. I. Translation of early region 1-specific rna from lytically infected cells.

Translation of early RNA specific for the leftmost early region 1 of adenovirus type 5 DNA in a rabbit reticulocyte cell-free system resulted in the synthesis of proteins with the following molecular weights: 65,000 (65K), 54K, 42 to 34K, 29K, 25K, 19K, and 18K. All of these proteins could be immunoprecipitated with hamster antitumor serum. The 42 to 34K proteins mapped in early region 1a, and those with molecular weights of 65K, 19K, and 18K mapped in early region 1b.

UV-reactivation, virus production and mutagenesis of SV40 in VU-irradiated monkey kidney cells.

The survival of UV-irradiated simian virus 40 (SV40) is higher in VU-irradiated than in non-irradiated monolayers of BSC-1 monkey cells. A similar reactivation is found when cells are infected with SV40-DNA, suggesting that reactivation acts on viral DNA. The enhanced reactivation of VU-irradiated SV40 and SV40-DNA is optimal when infection is delayed for 2--3 days after irradiation of the cells.

An Escherichia coli mutant with an altered elongation factor Tu.

A thermosensitive mutant of Escherichia coli has been isolated that is unable to replicate the bacteriophage MS2 at 42 degrees but permits phage production at 37 degrees . Thermal inactivation studies of the supernatant enzymes show that this mutant contains a factor essential for the polymerization of phenylalanine from phenylalanyl-tRNA that at 50 degrees is more rapidly inactivated than the corresponding wild-type factor. The elongation factor Tu (EF-Tu) was isolated and purified to apparent homogeneity as the EF-Tu.