Molecular and pharmacological characterization of the murine tachykinin NK(3) receptor.

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.

Identification of a small-molecule, nonpeptide macrophage scavenger receptor antagonist.

Class A scavenger receptor (SR-A) antagonists may prevent the initiation of atherosclerosis, because a recent report found that SR-A/apolipoprotein E (apoE) double-knockout mice had 60% smaller lesions than apoE single-knockout littermates. We transfected human embryonic kidney (HEK) 293 cells with SR-A type I or II receptors to find small-molecule antagonists. Uptake of 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL) showed that among common polyanionic ligands, polyinosine was the most potent, with an IC50 of 0.74 microgram/ml, whereas the novel compound (E)-methyl 4-chloro-alpha-[4-(4-chlorophenyl)-1, 5-dihydro-3-hydroxy-5-oxo-1-(2-thiazolyl)-2H-pyrrol-2-ylidene]benzene acetate gave an IC50 of 6.1 microgram/ml (13 microM). The novel antagonist also inhibited DiI-AcLDL uptake in cultured human peripheral and rat peritoneal macrophages with IC50 values of 21 microM and 17 microM, respectively. With [125I]AcLDL as ligand for transfected HEK 293 cells, binding/uptake and degradation at 37 degrees C for 5 h was saturable and selective. In a comparison of both types of receptor, we found no difference between the capacity of SR-AI or SR-AII for either binding or degradation. Polyinosine competed both [125I]AcLDL binding and degradation with a Ki of 1 microgram/ml, whereas the novel antagonist competed with a Ki of 19 microgram/ml (40 microM) and 8.6 microgram/ml (18 microM), respectively, for binding and degradation. Saturation binding in the presence of the ionophore monensin indicated that the novel compound behaved as a noncompetitive antagonist and perhaps as an allosteric effector. This is the first report to describe a small-molecule macrophage scavenger receptor antagonist. Utilization of this permanently transfected HEK 293 cell line will allow the identification of more potent macrophage scavenger receptor antagonists, so that their utility as therapeutics for atherosclerosis can be determined.

Characterization of the rpoC gene of Streptomyces coelicolor A3(2) and its use to develop a simple and rapid method for the purification of RNA polymerase.

The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparison of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoCHIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoCHIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoCHIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoCHIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.

Characterization of functional chemokine receptors (CCR1 and CCR2) on EoL-3 cells: a model system to examine the role of chemokines in cell function.

A growing family of proteins, known as the chemokines, play an important role in the recruitment and activation of inflammatory cells. The purpose of these studies was to characterize the chemokine receptors present on human sodium butyrate differentiated EoL-3 cells (dEoL-3 cells). Using a combination of 3' rapid amplification of cDNA ends and nested polymerase chain reaction, we detected mRNA for CC chemokine receptor (CCR)1, CCR2, CCR3 and low level of CCR5. Radioligand binding studies demonstrated high-affinity saturable binding for both 125I-macrophage inflammatory protein (MIP)-1alpha and 125I-regulated upon activation normal T cell expressed and secreted (RANTES) with Kd values of 1.4 and 7 nM, respectively. Competition binding with chemokines demonstrated exactly the same rank order of potency for displacement of both ligands: MIP-1alpha approximately monocyte chemoattractant protein (MCP)-3 approximately RANTES > MIP-1beta >> MCP-1 >>> IL-8. RANTES, MCP-3 and MIP-1alpha all produced concentration-dependent transient increases in intracellular calcium concentrations in dEoL-3 cells. Desensitization studies indicated that RANTES, MIP-1alpha and MCP-3 interacted at the same receptor, which is identical in characterization to the cloned CCR1. 125I-MCP-1 also demonstrated high-affinity satuable binding to dEoL-3 cells with a Kd value of 0.4 nM. Competition studies showed that MCP-3 was slightly more potent than MCP-1 and MCP-2. MIP-1alpha, MIP-1beta and RANTES were unable to displace 125I-MCP-1. Addition of either MCP-1 or MCP-3 produced a concentration-dependent elevation of intracellular calcium with a maximun response 2-fold higher than that seen with RANTES or MIP-1alpha. Desensitization studies indicated that MCP-1 and MCP-3 function through CCR2 on these cells. Thus binding and functional studies indicate that dEoL-3 cells express functional CCR1 and CCR2 and that these cells may serve as an important system with which to study the regulation and role of these receptors.

Nonpeptide tachykinin receptor antagonists: I. Pharmacological and pharmacokinetic characterization of SB 223412, a novel, potent and selective neurokinin-3 receptor antagonist.

The in vitro and in vivo pharmacological profile of SB 223412 [(S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carbo xamide], a novel human NK-3 (hNK-3) receptor antagonist, is described. SB 223412 demonstrated enantioselective affinity for inhibition of [125I][MePhe7]neurokinin B (NKB) binding to membranes of CHO cells expressing the hNK-3 receptor (CHO hNK-3). SB 223412, the (S)-isomer, (Ki = 1.0 nM), has similar affinity as the natural ligand, NKB (Ki = 0.8 nM) and another nonpeptide NK-3 receptor antagonist, SR 142801 (Ki = 1.2 nM). SB 223412 was selective for hNK-3 receptors compared with hNK-1 (>10,000-fold selective) and hNK-2 receptors (>140-fold selective), and selectivity was further demonstrated by its lack of effect, in concentrations up to 1 or 10 microM, in >60 receptor, enzyme and ion channel assays. SB 223412 enantioselectively inhibited the NKB-induced Ca++ mobilization in HEK 293 cells stably expressing the hNK-3 receptor. SB 223412 (10-1,000 nM) produced concentration-dependent rightward shifts in NKB-induced Ca++ mobilization concentration-response curves with a Kb value of 3 nM. In addition, SB 223412 antagonized senktide-induced contraction in the isolated rabbit iris sphincter muscle (Kb = 1.6 nM). In mice, oral administration of SB 223412 produced dose-dependent inhibition of behavioral responses induced by the NK-3 receptor-selective agonist, senktide (ED50 = 12.2 mg/kg). Pharmacokinetic evaluation of SB 223412 in rat and dog indicated low plasma clearance, oral bioavailability and high and sustained plasma concentrations after 4 to 8 mg/kg oral dosages. The preclinical profile of SB 223412 (high affinity, selectivity, reversibility and oral activity) suggests that it will be a useful tool compound to define the physiological and pathophysiological roles of NK-3 receptors.

The Streptomyces galP1 promoter has a novel RNA polymerase recognition sequence and is transcribed by a new form of RNA polymerase in vitro.

We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions -34, -36, and -11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The -35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.

Advances in heterologous gene expression by Streptomyces.

During the past year, noteworthy advances have been made in our ability to express foreign genes in Streptomyces. These advances were due, in part, to a detailed examination of the critical parameters that limit expression by Streptomyces of soluble forms of the human T-cell receptor CD4. Significant progress has also been made in our understanding of transcriptional regulation and protease gene expression. Application of this knowledge to expression vector design and the construction of alternative expression hosts should improve our ability to easily and routinely express foreign genes in Streptomyces.

Soluble forms of the human T cell receptor CD4 are efficiently expressed by Streptomyces lividans.

We have developed a new gene expression and secretion system for Streptomyces lividans and used it to produce soluble forms of a human T-cell receptor CD4 at levels greater than 300 mg/l. The system uses the transcription, translation and secretion signals of the serine protease inhibitor gene STI-II which is naturally produced by S. longisporus. Using these signals, soluble derivatives of CD4 were secreted directly into the culture supernatant as correctly processed soluble, biologically active proteins. High level expression of the CD4 proteins depended on the transcription initiation signal, the amino acid sequence surrounding the signal peptide cleavage site and temporally controlled protease activities. We discuss these results in the context of the potential of this system for producing other eukaryotic proteins in Streptomyces.

Identification of a complex operator for galP1, the glucose-sensitive, galactose-dependent promoter of the Streptomyces galactose operon.

The galP1 promoter is responsible for galactose-dependent, glucose-sensitive transcription of the galactose utilization operon of Streptomyces coelicolor and Streptomyces lividans. We describe the characterization of mutations that were positioned directly upstream of the apparent transcription start site of galP1 and that resulted in deregulated expression. Certain combinations of base changes within a series of hexamers that lie within two pairs of direct repeat sequences resulted in significant expression from galP1 in the absence of inducer. These motifs are further implicated in regulation by the observation that DNA fragments containing the hexamers and direct repeat sequences resulted in increased transcription from the chromosomal copy of galP1 on multicopy plasmids in the absence of galactose. We suggest that these hexamers and direct repeat sequences constitute an operator for the negative regulation of the Streptomyces gal operon.

Secretion of interleukin-1 beta and Escherichia coli galactokinase by Streptomyces lividans.

The functionality of the Streptomyces lividans beta-galactosidase signal peptide to direct heterologous protein export was examined. The signal peptide plus eight amino acids of mature protein were sufficient to export not only a naturally exported protein, interleukin-1 beta, but also a naturally occurring cytoplasmic protein, Escherichia coli galactokinase. Interestingly, cells which expressed yet exported galactokinase were phenotypically Gal-. The potential use of the exported galactokinase system for the isolation and characterization of mutations within signal peptides and the export machinery of the host is discussed.

Gene organization and structure of the Streptomyces lividans gal operon.

We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes. Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK. Comparison of the inferred protein sequences for the S. lividans gal gene products to the corresponding E. coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes. Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism.

Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon.

Galactose utilization in Streptomyces lividans was shown to be controlled by an operon that is induced in the presence of galactose and repressed by glucose. Two promoters, galP1 and galP2, which direct transcription of two distinct polycistronic transcripts, have been identified. galP1 is located immediately upstream of the operon and is induced in the presence of galactose. This promoter directs transcription of the galT, galE, and galK genes. The second promoter, galP2, is located within the operon just upstream of the galE gene. This promoter is responsible for constitutive transcription of the galE and galK genes. Comparison of the S. lividans gal operon to the Escherichia coli gal operon indicates the presence of a constitutive promoter positioned upstream of galE in both operons. We suggest that coupling the operon's constitutive promoter to the galE gene fulfills a physiological requirement for constitutive UDPgalactose 4-epimerase expression in Streptomyces.

Interferon action: transcriptional control of a gene specifying a 56,000-Da protein in Ehrlich ascites tumor cells.

The exposure of cells to interferons enhances the accumulation of particular mRNAs and of the corresponding proteins. A cDNA clone (clone 202) complementary to an mRNA (202 mRNA) whose level is enhanced over 12-fold in mouse Ehrlich ascites tumor cells upon exposure to beta-interferon for 10 hr has previously been isolated. The level of this mRNA was also increased in other beta-interferon-responsive mouse cell lines (i.e., L929, L1210S) but not in a line (L1210R) which is not responsive to beta-interferon. The extent of induction in Ehrlich ascites tumor cells depended on the beta-interferon concentration and reached its maximal level between 300 and 1000 units of interferon/ml. Nuclei isolated from Ehrlich ascites tumor cells which had been exposed to beta-interferon produced in vitro more 202 specific RNA than nuclei from control Ehrlich ascites tumor cells: an increase in this production was detectable 2 hr after beginning the exposure of the cells to 1000 units/ml of beta-interferon and the increase reached its maximal level, around 18-fold, after 18 hr exposure. Much, if not all of this increase, appeared to be due to an increase in the rate of synthesis of the RNA and not to a decrease in its rate of turnover. The 202 mRNA was translated in a reticulocyte lysate into a 56,000-Da protein.

Characterization of Streptomyces promoter sequences using the Escherichia coli galactokinase gene.

A gene fusion system that uses the Escherichia coli galK gene has been developed to characterize Streptomyces transcriptional regulatory sequences. The system consists of galK-deficient Streptomyces lividans mutants and plasmids containing the E. coli galK gene with its natural ribosome-binding site and sites upstream of galK for insertion of transcription signals. Expression of the E. coli galK gene in S. lividans can be quantitated by either an enzymatic or immunoblot assay or detected by genetic complementation of an S. lividans galK- mutant. The utility of the plasmid to select, detect and assess promoter function was examined using the S. lividans XP55 and S. fradiae aph gene promoters. The potential use of the galK fusion system to isolate and characterize Streptomyces transcription signals is discussed.