The ubiquitous neuropeptide galanin controls numerous functions such as endocrine secretions, intestinal motility, and behavioral activities. These regulatory effects of galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive guanine nucleotide binding proteins Gj/G0 as transducing elements. We report here the isolation of a cDNA coding for a human galanin receptor from a Bowes melanoma cell line cDNA expression library, by using a radioligand binding strategy. The nucleotide sequence ofthe cloned receptor reveals an open reading frame encoding a 349-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the guanine nucleotide binding protein-coupled neuropeptide receptor family. The cloned receptor expressed in COS cells specifically binds human, porcine, and rat galanin with high affinity (Kd in the nanomolar range) and mediates the galanin inhibition of adenylate cyclase. A 2.8-kb galanin receptor transcript was identified in several human tissues. Cloning of this galanin receptor should enhance our knowledge of its distribution, structure, and function in human physiology and pathophysiology.Galanin, a 29-amino acid neuropeptide (30 amino acids in humans), was originally isolated from pig intestine (1) and later reported to be widely distributed in the central and peripheral nervous systems of numerous species (2). Galanin is unrelated to the other known families of regulatory peptides and, to date, remains the only known member of its own family. It exerts multiple regulatory functions such as (i) control of endocrine and exocrine pancreatic secretions, (ii) regulation of intestinal motility, and (iii) modulation of behavioral, cognitive, and sensory functions such as feeding, learning, memory, and nociception (for reviews, see refs. 2 and 3). Galanin exerts its actions via binding to specific membrane receptors (4). Biochemical and molecular studies, performed in brain and pancreas, indicate that the galanin receptor is a glycoprotein of 54 kDa (5-7) coupled to the inhibitory guanine nucleotide binding (G) protein Gi, identified as Gil, Gi2, and Gi3 in pancreatic 83 cells (8,9). Depending on the target tissue, different pathways for intracellular signaling by galanin are involved: inhibition of adenylate cyclase (10), blockage of voltage-dependent Ca2+ channels (11), and activation of ATP-sensitive K+ channels (12). Structure-activity studies, with galanin fragments (13-15) and chimeric peptides (16,17), generally emphasize the importance of the N-terminal fragment of galanin for the interaction with the peptide receptor. These studies also raise the possibility of the existence of galanin receptor subtypes, an issue that may be properly addressed with the molecular cloning of the galanin receptor.In this context, we describe here the expression cloning of a cDNA encoding a galanin receptor from the human Bowes melanoma cell line (18) (vol/vol) fetal calf serum, 6 mM glutami...
A series of triterpene compounds characterized by a stringent structure-activity relationship were identified as potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication. Currently studied betulinic derivatives have 50% inhibitory concentrations (ICss) agaeHIV-1 strain III/LAI in the 10 nM range in several cellular infection assays but are inactive against HIV-2. These compounds did not significantly inhibit the in vitro activities of several purified HIV-l enzymes. Rather, they ap ed to block virus infection at a postbinding, envelope-dependent step involved in the fusion of the virus to the cell membrane. CCID5o) and cultured for 5 days. Cell viability was assessed by the spectrophotometric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-based assay (9). Mock-infected cultures were carried out in parallel to determine the degree of cytotoxicity of the compounds. p24 core antigen in culture supernatants was determined by ELISA (NEK060B kit from New England Nuclear). RT activity was measured by a poly(rA) 3H SPA scintillation proximity assay (NK9020 from Amersham). The antiviral assay using MT4 cells was as described (10). The effect of compounds on the kinetics of viral production in human peripheral blood mononuclear cells (PBMCs) was also assayed. PBMCs from seronegative donors were isolated by Ficoll/Hypaque (Pharmacia) and stimulated for 3 days with phytohemagglutinin (2.5 pg/ml; Difco). Cells were then washed three times with medium, pelleted, and incubated with 50-100 CCID50 of virus stock per ml. After 1 hr at room temperature, cells were resuspended in RPMI 1640 medium containing 10%6 fetal bovine serum, 10%6 T-cell growth factors (Lymphocult, Biotest Diagnostics, Danville, NJ), and anti-interferon a neutralizing antibody (Bayer, Wuppertal, F.R.G.) at 80 units/ml. Cells were plated at 106 per ml in six-well culture plates (Costar) in the presence of the compound to be tested. At 3, 7, 10, and 14 days after infection, cultures were harvested and viable cells were examined by trypan blue exclusion. Aliquots of supernatants were frozen at -700C until tested for p24 antigen, and cells were resuspended at the same density in complete fresh medium with the same concentration of the tested drug.Determination of Cytoplasmic Proviral DNA. Shortly after infection, the proviral DNA obtained from a selective extraction was quantified by PCR amplification. H9 cells were preincubated in the presence or absence of the drug to be tested for 1 hr, infected with HIV-1 (LAI) at 500 CCIDso/ml for 1 hr, and grown in culture medium for another hour. Cells were then washed intensively, and cytoplasmic proviral DNA was extracted by a digitonin/proteinase K/RNase Abbreviations: AZT, 3'-azido-3'-deoxythymidine; HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cell; sCD4, soluble CD4; RT, reverse transcriptase. tTo whom reprint requests should be addressed. 3564The publication costs of this article were defrayed in part by page charge payment. This article mus...
Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine͞plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine͞plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine͞plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid͞DNA ratio. The most active lipopolyamine͞DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.
An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 imol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30TC under the control of an E. coli promoter and ribosome-binding site.
A novel series of omega-aminoalkanoic acid derivatives of betulinic acid were synthesized and evaluated for their activity against human immunodeficiency virus (HIV). The anti-HIV-1 activity of several members of this new series was found to be in the nanomolar range in CEM 4 and MT-4 cell cultures. The optimization of the omega-aminoalkanoic acid side chain is described. The presence of an amide function within the side chain was found important for optimal activity. RPR 103611 (14g), a statine derivative, was found to be inactive against HIV-1 protease, reverse transcriptase, and integrase as well as on gp120/CD4 binding. "Time of addition" experiments suggested interaction with an early step of HIV-1 replication. As syncytium formation, but not virus-cell binding, seems to be affected, betulinic acid derivatives are assumed to interact with the postbinding virus-cell fusion process.
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