Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer.

Prostaglandin G/H synthase (PGHS), a key enzyme leading to the formation of prostaglandins, is the target of nonsteroidal antiinflammatory drugs. Two forms of the enzyme have been identified, PGHS-1 and PGHS-2. Epidemiological evidence has suggested that aspirin and other nonsteroidal antiinflammatory drugs may reduce the risk of colorectal cancer. We examined by immunoblot analyses the expression of human PGHS-1 and PGHS-2 protein in 25 matched colon cancer and nontumor tissues, 4 premalignant polyps, 5 control colon tissues from noncancer patients, and 3 matched normal and cancerous breast tissue samples. PGHS-1 was detected in all normal and tumor tissue. In contrast, PGHS-2 was not detected in 23 of 25 normal colon tissues but was detected in 19 of 25 colon tumors. PGHS-2 protein was not observed in four human premalignant polyp samples, control colon from noncancer patients, or matched normal or cancerous breast tissues. These results suggest that the beneficial effects of nonsteroidal antiinflammatory drugs in colon cancer may be mediated by inhibition of PGHS-2.

The nucleotide sequence of a voltage-gated chloride channel from the electric organ of Torpedo californica.

The cDNA encoding the voltage-gated chloride channel from the electric organ of Torpedo californica has been isolated and sequenced. The 2.7 kilobase pair cDNA encodes an 810 amino acid polypeptide which is highly homologous at both the DNA (97%) and amino acid (97%) levels to the voltage-gated chloride channel from the electric organ of T. marmorata. The majority of the 24 amino acid differences between the T. californica and T. marmorata voltage-gated chloride channels are clustered in two putative cytoplasmic domains with six differences located between residues 10-92 and 14 differences occurring between residues 576 to 708. Only one amino acid difference occurs in one of the predicted transmembrane domains. The most dramatic difference is an insertion of Asp-Val-Pro-Gly in a large cytoplasmic domain at amino acid residue 627 of the T. californica channel.

Secretion of Cellulomonas fimi exoglucanase by Escherichia coli.

A leader sequence of 41 amino acids (aa) has been proposed as the signal sequence for the exoglucanase (Exg) from Cellulomonas fimi. The ability of this 41-aa peptide to function as a leader sequence has been shown here by gene fusion experiments in Escherichia coli. A hybrid leader sequence containing C-terminal 37 aa of the leader peptide and N-terminal 6 aa of beta-galactosidase (beta Gal) directed export of the Exg into the periplasm of E. coli. In contrast, hybrid beta Gal-Exg proteins in which the leader sequence is not present are retained in the cytoplasm.

Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues.

The rate-limiting step in the formation of prostanoids is the conversion of arachidonic acid to prostaglandin H2 by cyclooxygenase, also known as prostaglandin G/H synthase/cyclooxygenase. Two forms of cyclooxygenase have been characterized: a ubiquitously expressed form (COX-1) and a recently described second form (COX-2) inducible by various factors including mitogens, hormones, serum and cytokines. Here we quantitate by the reverse transcriptase-polymerase chain reaction (RT-PCR) the expression of COX-1 and COX-2 mRNA in human tissues including lung, uterus, testis, brain, pancreas, kidney, liver, thymus, prostate, mammary gland, stomach and small intestine. All tissues examined contained both COX-1 and COX-2 mRNA and could be grouped according to the level of COX mRNA expression. The highest levels of COX mRNAs were detected in the prostate where approximately equal levels of COX-1 and COX-2 transcripts were present. In the lung high levels of COX-2 were observed whereas COX-1 mRNA levels were about 2-fold lower. An intermediate level of expression of both COX-1 and COX-2 mRNA was observed in the mammary gland, stomach, small intestine, and uterus. The lowest levels of COX-1 and COX-2 mRNA were observed in the testis, pancreas, kidney, liver, thymus, and brain.

Mutation of serine-516 in human prostaglandin G/H synthase-2 to methionine or aspirin acetylation of this residue stimulates 15-R-HETE synthesis.

Prostaglandin G/H synthase (PGHS) is a key enzyme in cellular prostaglandin (PG) synthesis and is the target of non-steroidal anti-inflammatory agents. PGHS occurs in two isoforms, termed PGHS-1 and PGHS-2. These isoforms differ in several respects, including their enzymatic activity following acetylation by aspirin. While PG synthesis by both isoforms is inhibited by aspirin, 15-R-hydroxyeicosatetraenoic acid (15-R-HETE) synthesis by PGHS-2, but not PGHS-1, is stimulated by preincubation with aspirin. We have mutated the putative aspirin acetylation site of hPGHS-2, and expressed the mutants in COS-7 cells using recombinant vaccinia virus. Enzyme activity and inhibitor sensitivity studies provide evidence that Ser516 is the aspirin acetylation site of human PGHS-2 and that substitution of a methionine residue at this position can mimic the effects of aspirin acetylation on enzyme activity.

Discovery of a receptor related to the galanin receptors.

We report the isolation of a cDNA clone named GPR54, which encodes a novel G protein-coupled receptor (GPCR). A PCR search of rat brain cDNA retrieved a clone partially encoding a GPCR. In a library screening this clone was used to isolate a cDNA with an open reading frame (ORF) encoding a receptor of 396 amino acids long which shared significant identities in the transmembrane regions with rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%). Northern blot and in situ hybridization analyses revealed that GPR54 is expressed in brain regions (pons, midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal cortex, and striatum) as well as peripheral regions (liver and intestine). In COS cell expression of GPR54 no specific binding was observed for 125I-galanin. A recent BLAST search with the rat GPR54 ORF nucleotide sequence recovered the human orthologue of GPR54 in a 3.5 Mb contig localized to chromosome 19p13.3.

Characterization of arachidonic-acid-metabolizing enzymes in adult Schistisoma mansoni.

Schistosoma mansoni has previously been reported to synthesize a wide range of eicosanoids including prostaglandins, leukotrienes and hydroxyeicosatetraenoic acids (HETEs). Our analysis of arachidonic acid metabolites synthesized by microsomal and cytosolic extracts from adult S. mansoni using thin-layer chromatography and radioimmunoassay techniques indicate the presence of a soluble, enzymatically active lipoxygenase (Lox) and the absence of any cyclooxygenase (Cox) activity. The S. mansoni Lox activity catalyzed the formation of a 15-hydroxyeicosatetraenoic acid (15-HETE)-like species. This activity was calcium-independent and inhibitable by inhibitors of mammalian and plant Lox. The conversion of linoleic acid to a 13-hydroxyoctadecadienoic acid (13-HODE)-like product by S. mansoni extracts indicates that the parasite Lox-homologue is similar to mammalian 15-Lox. Immunoblot analysis of S. mansoni extracts using antisera to different mammalian lipoxygenases detects two immunoreactive proteins with molecular weights similar to plant and mammalian lipoxygenases. In addition, polymerase chain reaction (PCR) amplification of Lox-like sequences from S. mansoni genomic DNA using degenerate primers based on conserved plant and mammalian Lox sequences, generated two PCR products which hybridized to a human 15-Lox cDNA probe. While the role of eicosanoid production in the physiology of S. mansoni is not known, eicosanoids may be essential for normal physiological processes as is the case in other invertebrates. Interestingly, 15-HETE has previously been shown to have immunosuppressive effects in mammals, and this may be related to the ability of the parasite to overcome host immune responses.

Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells.

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.

delta-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA.

Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of delta-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis. The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.

Characterization of the cloned guinea pig leukotriene B4 receptor: comparison to its human orthologue.

A cDNA clone coding for the guinea pig leukotriene B4 (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein-Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B4 (Kd value of approximately 0.4 nM) and were expressed at high levels (Bmax values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [3H]leukotriene B4 specific binding at the recombinant guinea pig BLT receptor is leukotriene B4 > 20-OH-leukotriene B4 > 12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen -1-oic acid) > 12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen -1-oic acid) > 20-COOH-leukotriene B4 > U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexane diol) >> leukotriene C4 = leukotriene D4 = leukotriene E4. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B4 and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca2+ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of Galpha16 was required to achieve a robust Ca2+ driven response. Leukotriene B4 was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B4 analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B4 receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.

Transfer RNA and the formation of the heme and chlorophyll precursor, 5-aminolevulinic acid.

5-Aminolevulinic acid is the first committed precursor for the synthesis of porphyrins such as hemes and chlorophylls. In many organisms aminolevulinate is synthesized from glutamate in a three-step pathway (C5 pathway). The key step in this conversion is a tRNA-mediated reduction of glutamate to glutamate-1-semialdehyde. tRNA is a specific cofactor for an NADPH-dependent enzyme, Glu-tRNA reductase, which is capable of sequence-specific recognition of Glu-tRNA(Glu). tRNA(Glu) is a dual-function molecule; it participates both in protein and in aminolevulinate biosynthesis. This reduction reaction represents a novel role for tRNA where it participates in a metabolic conversion of its amino acid into a low molecular weight metabolite which is subsequently not used in peptide bond synthesis.

Mathematics anxiety and its effect on drug dose calculation.

Eighty learners were randomly selected from 160 first-year nursing students enrolled in an urban community college nursing program in Ontario. They were subsequently divided into control and treatment groups to investigate the effects of different teaching methods on mathematics anxiety and the students' ability to accurately calculate fractional drug doses. The results obtained in this study indicated that there were no statistically significant differences between the control and treatment groups in either mathematics anxiety levels or in arithmetic test performance. These findings counter many of those found in previous investigations. Reasons for these discrepancies are provided along with recommendations for present practice and future research.

Expression of the Synechocystis sp. strain PCC 6803 tRNA(Glu) gene provides tRNA for protein and chlorophyll biosynthesis.

In the cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis 6803) delta-aminolevulinic acid (ALA), the sole precursor for the synthesis of the porphyrin rings of heme and chlorophyll, is formed from glutamate activated by acylation to tRNA(Glu) (G. P. O'Neill, D. M. Peterson, A. Schön, M. W. Chen, and D. Söll, J. Bacteriol. 170:3810-3816, 1988; S. Rieble and S. I. Beale, J. Biol. Chem. 263:8864-8871, 1988). We report here that Synechocystis 6803 possesses a single tRNA(Glu) gene which was transcribed as monomeric precursor tRNA and matured into the two tRNA(Glu) species. They differed in the extent of modification of the first anticodon base, 5-methylaminomethyl-2-thiouridine (O'Neill et al., 1988). The two tRNA species had equivalent capacities to stimulate the tRNA-dependent formation of ALA in Synechocystis 6803 and to provide glutamate for protein biosynthesis in an Escherichia coli-derived translation system. These results are in support of a dual role of tRNA(Glu). The levels of tRNA(Glu) were examined by Northern (RNA) blot analysis of cellular RNA and by aminoacylation assays in cultures of Synechocystis 6803 in which the amount of chlorophyll synthesized was modulated over a 10-fold range by various illumination regimens or by the addition of inhibitors of chlorophyll and ALA biosynthesis. In these cultures, the level of tRNA(Glu) was always a constant fraction of the total tRNA population, suggesting that tRNA(Glu) and chlorophyll levels are regulated independently. In addition, the tRNA(Glu) was always fully aminoacylated in vivo.

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Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli.

A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli.

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of the chlorophyll precursor delta-aminolevulinic acid in cyanobacteria requires aminoacylation of a tRNAGlu species.

In the chloroplasts of higher plants and algae, the biosynthesis of the chlorophyll precursor delta-aminolevulinic acid (ALA) involves at least three enzymes and a tRNA species. Here we demonstrate that in cell extracts of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 ALA was formed from glutamate in a series of reactions in which activation of glutamate by glutamyl-tRNAGlu formation was the first step. The activated glutamate was reduced by a dehydrogenase which displayed tRNA sequence specificity. Fractionation of strain 6803 tRNA by reverse-phase chromatography and polyacrylamide gel electrophoresis yielded two pure tRNAGlu species which stimulated ALA synthesis in vitro. These tRNAs had identical primary sequences but differed in the nucleotide modification of their anticodon. The 6803 tRNAGlu was similar to the sequences of tRNAGlu species or tRNAGlu genes from Escherichia coli and from chloroplasts of Euglena gracilis and higher plants. Southern blot analysis revealed at least two tRNAGlu gene copies in the 6803 chromosome. A glutamate-1-semialdehyde aminotransferase, the terminal enzyme in the conversion of glutamate to ALA in chloroplasts, was detected in 6803 cell extracts by the conversion of glutamate-1-semialdehyde to ALA and by the inhibition of this reaction by gabaculin.

Molecular cloning and functional expression of a Caenorhabditis elegans aminopeptidase structurally related to mammalian leukotriene A4 hydrolases.

In a search of the Caenorhabditis elegans DNA data base, an expressed sequence tag of 327 base pairs (termed cm01c7) with strong homology to the human leukotriene A4 (LTA4) hydrolase was found. The use of cm01c7 as a probe, together with conventional hybridization screening and anchored polymerase chain reaction techniques resulted in the cloning of the full-length 2.1 kilobase pair C. elegans LTA4 hydrolase-like homologue, termed aminopeptidase-1 (AP-1). The AP-1 cDNA was expressed transiently as an epitope-tagged recombinant protein in COS-7 mammalian cells, purified using an anti-epitope antibody affinity resin, and tested for LTA4 hydrolase and aminopeptidase activities. Despite the strong homology between the human LTA4 hydrolase and C. elegans AP-1(63% similarity and 45% identity at the amino acid level), reverse-phase high pressure liquid chromatography and radioimmunoassay for LTB4 production revealed the inability of the C. elegans AP-1 to use LTA4 as a substrate. In contrast, the C. elegans AP-1 was an efficient aminopeptidase, as demonstrated by its ability to hydrolyze a variety of amino acid p-nitroanilide derivatives. The aminopeptidase activity of C. elegans AP-1 resembled that of the human LTA4 hydrolase/aminopeptidase enzyme with a preference for arginyl-p-nitroanilide as a substrate. Hydrolysis of the amide bond of arginyl-p-nitroanilide was inhibited by bestatin with an IC50 of 2.6 +/- 1.2 microM. The bifunctionality of the mammalian LTA4 hydrolase is still poorly understood, as the physiological substrate for its aminopeptidase activity is yet to be discovered. Our results support the idea that the enzyme originally functioned as an aminopeptidase in lower metazoa and then developed LTA4 hydrolase activity in more evolved organisms.

Purification and characterization of Chlamydomonas reinhardtii chloroplast glutamyl-tRNA synthetase, a natural misacylating enzyme.

Glutamyl-tRNA synthetase from Chlamydomonas reinhardtii was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, Mono Q, and Mono S. The apparent molecular mass of the protein when analyzed under both denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 62,000 Da; this indicates that the active enzyme is a monomer. The purified glutamyl-tRNA synthetase was identified as the chloroplast enzyme by its tRNA charging specificity. Reversed-phase chromatography of unfractionated C. reinhardtii tRNA resolved four peaks of glutamate acceptor RNA when assayed with the purified enzyme. The enzyme can also glutamylate Escherichia coli tRNA(2Glu), but not cytoplasmic tRNA(Glu) from yeast or barley. In addition, the enzyme misacylates chloroplast tRNA(Gln) with glutamate. A similar mischarging phenomenon has been demonstrated for the barley chloroplast enzyme (Schön, A., Kannangara, C.G., Gough, S., and Söll, D. (1988) Nature 331, 187-190) and for Bacillus subtilis glutamyl-tRNA synthetase (Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M., and Lapointe, J. (1983) J. Biol. Chem. 258, 753-759).