Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor.

The mouse C5a receptor gene was isolated using the human C5a receptor cDNA probe recently described (Gerard, N. P., and C. Gerard. 1991. Nature 349:614). By analogy with the human gene, the mouse homolog contains two exons with the 5' untranslated region and initiating methionine codon present in exon 1 and the remainder of the molecule in exon 2. Generation of an expressible cDNA for the mouse C5a receptor was accomplished using the polymerase chain reaction and a sense oligodeoxynucleotide primer which included an initiation codon just 5' to the sequence encoding the N-linked glycosylation site. When transfected into human 293 kidney epithelial cells the cloned cDNA directs expression of a binding site for human C5a anaphylatoxin with a binding constant of 2.5 +/- 0.3 nM; the human C5a receptor expressed under identical conditions has a Kd of 1.7 +/- 0.2 nM. Overall, the deduced amino acid sequences of the receptors are 65% identical given the analogous gene structures. Alignment of the sequences as seven transmembrane segment receptors reveals that the greatest structural diversity (approximately 70%) exists in the putative extracellular domains. In contrast, species differences among other members of this family of seven membrane-spanning receptors is generally only 10 to 20%, even for receptors whose ligands are relatively small and not expected to interact with sites on the extracellular surfaces. A high degree of structural identify is observed for the C5a receptors in the transmembrane segments and in all but one of the loops predicted to exist in the cytoplasm. Inasmuch as critical structures responsible for high affinity binding of the 74 amino acid polypeptide to both C5a receptors involve features conserved between species, these data provide the starting point for mutagenesis studies to determine the nature of the binding and activation sites for the chemotactic receptors. Additionally, these data provide a reagent for immunologic and molecular genetic studies on the role of C5a receptors in inflammatory models.     

Bradykinin and ATP stimulate L-arginine uptake and nitric oxide release in vascular endothelial cells.

The effects of bradykinin and ATP on L-arginine transport and nitric oxide (NO) production were studied in porcine aortic endothelial cells cultured and perfused on microcarriers and deprived of L-arginine for 24 h. Stimulation of cells with bradykinin (100 nM) or ATP (100 microM) resulted in a rapid increase in L-arginine uptake and NO release. In the presence of nitro-L-arginine (100 microM), an inhibitor of NO synthase, the stimulatory effect of bradykinin on L-arginine uptake was partially inhibited while NO release was completely abolished. Nitro-L-arginine alone was not an inhibitor of basal L-arginine transport, suggesting that its inhibitory action was not directly on the L-arginine transporter but a result of the inhibition of NO generation. These data indicate that during agonist-stimulated NO production there is a concomitant increase in the transport of L-arginine into endothelial cells providing a mechanism for the continual generation of NO.     

Retinol and α-tocopherol concentrations in whole fish commonly fed in zoos and aquariums

Retinol (n = 17 spp.) and α-tocopherol (n = 9 spp.) concentrations in whole fish utilized for captive animal feeding programs were determined by high-performance liquid chromatography (HPLC) following routine storage and preparation after commercial purchase by two zoological institutions. Vitamin A activity was calculated from retinol values and ranged from 55 IU/100 g (immature herring) to >2,000 IU/100 g (salmon) on an as-fed basis. α-Tocopherol values, a measure of vitamin E activity, ranged from 0.9 IU/100 g (butterfish) to 12.3 IU/100 g (tilapia) on a wet basis. Vitamin levels in whole fish were intermediate to values previously quantified for muscle or liver tissues alone. Vitamin concentrations in fish livers were quantified separately in seven of these species; liver contributed 35–63% of total retinol measured and 8–34% of total α-tocopherol. Based on these analyses, whole fish commonly fed in zoos, aquariums, and marine zoological parks would appear to meet vitamin A requirements established for most species without additional supplementation, whereas levels of vitamin E quantified indicate a need for supplementation of diets for piscivores.     

Expression of aChlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein

A single-chain antibody fragment has been constructed for an antibody that binds to theChlamydia specific carbohydrate structure of the lipopolysaccharide. Single-chain protein was expressed and secreted into the periplasmic space ofE. coli as a fusion protein with the maltose binding protein. The fusion protein was purified in one step by virtue of its ability to bind to maltose. In a sandwich ELISA, the eluted protein boundChlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein. The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation.     

Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro

The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro. One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant. The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media. These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.     

P-element-mediated enhancer detection allows rapid identification of developmentally regulated genes and cell specific markers in Drosophila

We have employed a new technique in Drosophila that allows in vivo detection of genomic regulatory elements using a beta-galactosidase reporter gene. A translational fusion of the reporter gene to the P-transposase gene, which is encoded by the P-transposon of Drosophila, places the expression of beta-galactosidase under the control of the weak P-transposase promoter. Flies carrying single insertions of this P-element construct at different locations in the Drosophila genome frequently stain for beta-galactosidase activity in a temporally and spatially restricted fashion in embryos, larvae and adult ovaries, reflecting the influence of nearby genomic regulatory elements on the P-transposase promoter. This technique is a powerful tool as it can be used to produce very many different cell markers and to isolate developmentally regulated genes in Drosophila. We discuss the implications of our results and the applications of the technique to further the study of Drosophila development.     

S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate

S-(N-methylcarbamoyl)glutathione, a chemically-reactive glutathione conjugate, has been isolated from the bile of rats administered methyl isocyanate and characterized, as its N-benzyloxycarbonyl dimethylester derivative, by tandem mass spectrometry. The ability of this glutathione adduct to donate an N-methylcarbamoyl moiety to the free -SH group of cysteine was evaluated in vitro with the aid of a highly specific thermospray LC/MS assay procedure. The glutathione adduct reacted readily with cysteine in buffered aqueous media (pH 7.4, 37 degrees C) and after 2 hr, 42.5% of the substrate existed in the form of S-(N-methylcarbamoyl)cysteine. The reverse reaction, i.e. between the cysteine adduct and free glutathione, also took place readily under these conditions. It is concluded that conjugation of methyl isocyanate with glutathione in vivo affords a reactive S-linked product which displays the potential to carbamoylate nucleophilic amino acids. The various systemic toxicities associated with exposure of animals or humans to methyl isocyanate could therefore be due to release of the isocyanate from its glutathione conjugate, which thus may serve as a vehicle for the transport of methyl isocyanate in vivo.     

The development of gamma-glutamyltransferase in a pig renal-epithelial-cell line in vitro. Relationship to amino acid transport.

The pig kidney-cell line, LLC-PK1, possesses gamma-glutamyltransferase with properties similar to those of the purified renal enzyme. gamma-Glutamyltransferase activity increases after cells are seeded at low density to reach values comparable with those found in kidney cortex when the cells are fully confluent. This increase is paralleled by an increase in alpha-methyl D-glucoside uptake. In contrast, the uptakes of L-leucine and L-alanine decrease during this time. These results suggest that gamma-glutamyltransferase is not important as a mediator of the renal transport of neutral amino acids.     

NITROGEN FIXATION RATE AND CHLOROPHYLL CONTENT OF THE LICHEN PELTIGERA CANINA EXPOSED TO SULFUR DIOXIDE

In general, the rate of nitrogen fixation decreased when the lichen Peltigera canina (L.) Willd. was exposed to sulfur dioxide gas at levels from 0.1 to 500 ppm; at 30 ppm, however, nitrogen fixation was stimulated. Chlorophyll content decreased as level of sulfur dioxide increased.     

Myosin light chain kinase binding to plastic

Methionine-81 and/or -8 of the transmembrane sialoglycoprotein, glycophorin A, have been specifically alkylated with ¹³CH₃I to produce the sulfonium ion derivatives [S-[¹³C]methylmethionine-8]glycophorin A and [S-[¹³C]methylmethionine-8 and -81]glycophorin A. ¹³C NMR spectra of these species show that the resonances of the methyl groups of the modified glycophorins occur at 26.1 ppm downfield from Me₄Si. A spin-lattice relaxation time of 0.4 was observed for the ¹³C-enriched methyl resonances of the sulfonium ion derivatives of Met-8 and -81, which corresponds to an effective correlation time of < 2× 10^?10 s. Demethylation of the 2 glycophorin A sulfonium ion species with 2-mercaptoethanol produces native glycophorin A which now has the ε-carbon of the methionine residue(s) 45% isotopically enriched. The ε-carbon of Met-8 was found to occur at 15.7 ppm downfield from Me₄Si whereas the ε-carbon of Met-81 exhibited an unusual chemical shift of 2.0 ppm downfield from Me₄Si. The spin-lattice relaxation time of both resonances was found to be ～0.3 s.