Differential Virtual Screening (DVS) with Active and Inactive Molecular Models for Finding and Profiling GPCR Modulators: Case of the CCK1 Receptor.

We discovered a constitutively activating mutation (CAM) V308E for the neurotensin NT1 receptor. Molecular dynamics (MD) performed for the CAM NT1-V308E exhibiting a high spontaneous activity, and for the wild-type NT1 without basal activity, show dramatic conformational changes for the CAM. To test if the two MD models could be valuable active and inactive templates for building molecular models for other class-A GPCR, supposed active and inactive models were built by homology for the cholecystokinin CCK1 receptor.

Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor.

Heterologous expression of the human neurotensin receptor type I (hNT1-R) has been achieved in the yeast Saccharomyces cerevisiae. Immunoanalysis of membranes prepared from cells expressing a c-myc-tagged version of hNT1-R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT1-R was glycosylated in yeast. High-affinity binding sites for 125I-labeled-[monoiodo-Tyr3]neurotensin ([125I-Tyr3]NT) were detected on hNT1-R-expressing cells with Kd and Bmax values of 3.

The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance.

Study of the higher eukaryotic gene function CDK2 using fission yeast.

In the fission yeast Schizosaccharomyces pombe, cdc2 function is required both in G1 to enter the cell cycle and in G2 to initiate mitosis. In higher eukaryotes, these functions appeared to be shared between several cdc2-like genes including CDK2. Temperature-sensitive mutations in S.

Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses.

The discovery of new cytokines normally relies on a prior knowledge of at least one of their biological effects, which is used as a criterion either for the purification of the protein or for the isolation of the complementary DNA by expression cloning. However, the redundancy of cytokine activities complicates the discovery of novel cytokines in this way, and the pleiotropic nature of many cytokines means that the principal activities of a new cytokine may bear little relation to that used for its isolation. We have adopted an alternative approach which relies on differential screening of an organized subtracted cDNA library from activated peripheral blood mononuclear cells, using the inducibility of lymphokine messenger RNAs by anti-CD28 as a primary screening criterion.

Colicin A lysis protein promotes extracellular release of active human growth hormone accumulated in Escherichia coli cytoplasm.

The colicin A lysis protein (Cal) was used to direct the extracellular release of recombinant proteins produced in Escherichia coli. The cal gene, under the control of its inducible promoter, was introduced into an expression vector encoding the human growth hormone devoid of its signal sequence (Met-hGH). Cal and Met-hGH were simultaneously expressed at two different levels of Met-hGH induction.

High-level production of a peroxisomal enzyme: Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae.

Strains of Saccharomyces cerevisiae producing Aspergillus flavus uricase (Uox) have been constructed. An artificial promoter which combined the upstream and downstream sequences of the GAL7 and ADH2 promoters, respectively, was found to be efficient in directing the synthesis of uaZ mRNAs encoding Uox. A good proportionality between the copy number of the uaZ expression cassette and the level of Uox production was found in the range of 1-10 copies.

Expression and pharmacological characterization of the human peripheral-type benzodiazepine receptor in yeast.

Recently we cloned the cDNA coding for the putative human peripheral-type benzodiazepine receptor (hPBR). This report describes the expression of this cDNA in Saccharomyces cerevisiae and the characterization of the recombinant protein. The expression was achieved by placing the receptor cDNA under the control of a galactose-regulated artificial promoter.

Human recombinant interleukin-1 beta isolated from Escherichia coli by simple osmotic shock.

A synthetic gene coding for the C-terminal 153 amino acids of the human interleukin-1 beta (IL-1 beta) was used to produce large quantities of recombinant IL-1 beta in Escherichia coli. The expression of the synthetic gene was under the control of an inducible promoter. The recombinant protein was released from the cells by an osmotic shock.

Vectors for high conditional expression of cloned genes.

Several multicopy plasmids carrying the control region of bacteriophage lambda lysogeny, including the gene of a thermosensitive repressor CI857 have been constructed. The phages allow high expression of proteins under the transcription control of lambda promoter PR and translation control of Cro. The method has been assayed by measuring expression of either intact beta-galactosidase, truncated beta-galactosidase or beta-galactosidase fused to a mitochondrial gene product.