Pascual Ferrara

SR141716A, a potent and selective antagonist of the brain cannabinoid receptor

SR141716A is the first selective and orally active antagonist of the brain cannabinoid receptor. This compound displays nanomolar affinity for the central cannabinoid receptor but is not active on the peripheral cannabinoid receptor. In vitro, SR141716A antagonises the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and adenylyl cyclase activity in rat brain membranes. After intraperitoneal or oral administration SR141716A antagonises classical pharmacological and behavioural effects of cannabinoid receptor agonists. This compound should prove to be a powerful tool for investigating the in vivo functions of the anandamide/cannabinoid system.

Monoallelically Expressed Gene Related to p53 at 1p36, a Region Frequently Deleted in Neuroblastoma and Other Human Cancers

We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.

The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor

In this work, the 100-kDa neurotensin (NT) receptor previously purified from human brain by affinity chromatography (Zsürger, N., Mazella, J., and Vincent, J. P. (1994) Brain Res. 639, 245–252) was cloned from a human brain cDNA library. This cDNA encodes a 833-amino acid protein 100% identical to the recently cloned gp95/sortilin and was then designated NT3 receptor-gp95/sortilin. The N terminus of the purified protein is identical to the sequence of the purified gp95/sortilin located immediately after the furin cleavage site. The binding of iodinated NT to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized extracts of COS-7 cells transfected with the cloned cDNA was saturable and reversible with an affinity of 10–15 nm. The localization of the NT3 receptor-gp95/sortilin into intracellular vesicles was in agreement with previous results obtained with the purified receptor and with gp95/sortilin. Affinity labeling and binding experiments showed that the 110-kDa NT3 receptor can be partly transformed into a higher affinity (K d = 0.3 nm) 100-kDa protein receptor by cotransfection with furin. This 100-kDa NT receptor corresponded to the mature form of the receptor. The NT3/gp95/sortilin protein is the first transmembrane neuropeptide receptor that does not belong to the superfamily of G-protein-coupled receptors.

Intramolecular Masking of Nuclear Import Signal on NF-AT4 by Casein Kinase I and MEKK1

T cell activation requires the import of NF-AT transcription factors to the nucleus, a process promoted by calcineurin-dependent dephosphorylation and inhibited by poorly understood protein kinases. Here, we report the identification of two protein kinases that oppose NF-AT4 nuclear import. Casein kinase Ialpha directly binds and phosphorylates NF-AT4, resulting in the inhibiton of NF-AT4 nuclear translocation. MEKK1 indirectly suppresses NF-AT4 nuclear import by stabilizing the interaction between NF-AT4 and CKIalpha. CKIalpha thus acts to establish an intramolecular masking of the nuclear location signal on NF-AT4, while MEKK1 augments this mechanism, and may further provide a link to signal transduction pathways regulating NF-AT4.

Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor

A human neurotensin receptor (hNTR) cDNA was cloned from the colonic adenocarcinoma cell line HT29. The cloned cDNA encodes a putative peptide of 418 amino acids with 7 transmembrane domains. The amino acid sequence of the hNTR is 84% identical to the rat NTR [Neuron, 4 (1990) 847‐854]. Transfection of this cDNA into COS cells results in the expression of receptors with pharmacological properties similar to those found with HT29 cells. Northern blot analysis using the hNTR cDNA probe indicated a single transcript of 4 kb in the brain, the small intestine and blood mononuclear cells.

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. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

Molecular cloning of a human β3-adrenergic receptor cDNA

We report the molecular cloning of a β3‐adrenergic receptor β3‐AR cDNA from human brown adipose tissue. The cDNA‐encoded protein is identical to the previously cloned β3‐AR but with 6 additional amino acids at the C‐terminus. The C‐terminus is shared by the β3 receptors expressed in human neuroblastoma cells [SK‐N‐MC] [Mol. Pharmacol. 42 (1992) 964‐970]. Furthermore, using a polymerase chain reaction strategy we have cloned and sequenced the β3‐AR introns. Sequence analysis demonstrates that the human β3‐AR gene comprises at least 3 exons and 2 introns and that the most abundant β3‐AR transcripts encode a protein with an exon 3‐derived C‐terminus. Interestingly, although a similar organization has been found in rodent genes, the rat β3‐AR transcripts encode a receptor with an exon 2‐derived C‐terminus.

Interleukin-13 stimulates interleukin-6 production by human keratinocytes. Similarity with interleukin-4.

Interleukin‐13 (IL‐13) is a recently described human lymphokine which is produced by activated T‐cells. Its effect on the production of IL‐6 by normal keratinocytes and keratinocyte cell lines of human origin was studied and compared to that of IL‐4. IL‐13, similarly to IL‐4, stimulated IL‐6 expression by these cells in a dose‐ and time‐dependent manner. Contamination with endotoxin was excluded by the use of polymyxin B and heat‐inactivated cytokines. Further, we showed that IL‐13, like IL‐4, not only stimulated IL‐6 production but also was able to induce overexpression of this cytokine in response to an inflammatory signal such as lipopolysaccharide (LPS). In a previous study, we demonstrated that IL‐13, by inhibiting IL‐6 and other cytokines produced by monocytes, exhibited an ‘anti‐inflammatory profile’ comparable to that displayed by IL‐4. In contrast, we show here that IL‐13, by stimulating IL‐6 production by keratinocytes, may favour the installation of an inflammatory process at a local level and, here again, it acted like IL‐4. Therefore, according to the type of target cell, these two ‘TH2 type’ cytokines induce similar opposing effects on IL‐6 production and are likely to be important cytokines in the regulation of inflammation at both systemic and local levels.

Characterization of a peripheral-type benzodiazepine-binding site in the mitochondria of Chinese hamster ovary cells

The isoquinoline carboxamide derivative [3H]PK11195, a ligand for the peripheral‐type benzodiazepine (BZD) receptor, binds to Chinese hamster ovary (CHO) cell mitochondria in a specific and saturable manner. Scatchard analysis showed the presence of a single‐binding site with an apparent dissociation constant (K d) of 12.0 ± 1.0 nM and a maximal binding capacity of 23.0 ± 2.0 pmol/mg protein. The pharmacological characterization of this CHO BZD‐binding site, based on the displacement of [3H]PK11195 by several drugs of known binding specificity, indicated that it is of the peripheral‐type. The photoaffinity probe [3H]PK14105, a nitrophenyl derivative of [3H]PK11195, specifically labeled a 17 kDa CHO mitochondrial protein. This 17 kDa protein was purified from digitonin‐solubilized mitochondria by gel‐filtration chromatography and two reverse‐phase HPLC steps. The purified material migrated as a single band on silver stained or autoradiographed SDS‐polyacrylamide gels, and had an amino acid composition corresponding to a 17 kDa protein rich in Leu, Val, Ala, Gly, and Pro. Analysis of the amino‐terminal sequence of the purified 17 kDa protein revealed a blocked amino‐terminus.

Protein depletion and refeeding change the proportion of mouse liver glutathione S-transferase subunits

The effect of protein depletion followed by refeeding with a normal diet on the content of mouse liver cytosolic proteins was studied. By peptide-mass fingerprinting and N-terminal sequencing, three polypeptides whose contents changed with dietary protein level were identified as glutathione S-transferases (GST) Yb1, Yc and Yf subunits. Five days of depletion caused the increase of Yb1 and Yf (21.6% and 78.5%, respectively) and the decrease of Yc (31.2%). After two days of refeeding, Yb1 and Yc were practically restored, while the neoplastic marker Yf remained higher (63.4%). None of the nutritional conditions tested induced new GSTs. While protein depletion-refeeding altered the ratios between the constitutive GST subunits, total liver GST content and activity were unaffected by depletion and slightly increased by refeeding. The increased amounts of Yb1 and Yf, and the maintenance of total GST content, indicate that during protein depletion, the GST subunits levels are controlled by mechanisms different from the majority of cytosolic proteins.