Prostaglandin I2 is less relaxant than prostaglandin E2 on the lamb ductus arteriosus.

Prostaglandin (PG) I2 and its stable metabolite, 6-keto-PGF1alpha, were tested on the isolated ductus arteriosus from mature fetal lambs. PGI2 relaxed the ductus in high doses (threshold 10(-6)M) and its activity disappeared on standing at room temperature for 30 minutes. 6-keto-PGF1alpha was inactive at all doses. By contrast, PGE2 produced a dose-dependent relaxation over a range between 10(-10) and 10(-6)M. These findings confirm that PGE2 is the most potent ductal relaxant among the known derivatives of arachidonic acid. PGE2 probably maintains ductus patency in the fetus and, together with PGE1, remains the compound of choice in the management of newborns requiring a viable ductus for survival.     

Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B.

We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin. These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells. Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with alpha-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR promoter in an effort to overexpress these proteins. The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the pR promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the pR promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells. It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.     

Evidence for a role of prostaglandin I2 and thromboxane A2 in the ductus venosus of the lamb

The prostaglandin (PG) endoperoxide, PGH2, and the thromboxane (TX) A2 analog, 9,11-epithio-11,12-methano-TXA2, were tested in vitro on the ductus venosus sphincter from fetal (premature and mature) and neonatal (1-day-old) lambs. PGH2 relaxed the indomethacin-contracted fetal ductus in a dose-dependent manner and its action was reduced after treatment with 15-hydroperoxyarachidonic acid. In contrast, reduced glutathione did not affect the PGH2 relaxation in the indomethacin-treated ductus, nor did it modify the response of the untreated ductus to constrictor stimuli. Unlike PGH2, the stable 9 alpha,11 alpha-epoxymethano-PGH2 analog contracted the vessel. Similarly, the TXA2 analog was a contractile agent, its action exceeding that of the PGH2 analog in potency and efficacy. The TXA2 analog was active on preparations from both premature (minimum 117 days gestation) and mature lambs, but a maximal effect was attained during the perinatal period. These results confirm the existence of a PG-mediated relaxing mechanism in the ductus venosus and suggest that the active compound is PGI2. This mechanism is likely responsible for keeping the ductus patent in the fetus. TXA2, formed within the liver parenchyma, is well suited for playing a role in postnatal closure of the vessel.     

A peroxide/ascorbate-inducible catalase from Haemophilus influenzae is homologous to the Escherichia coli katE gene product.

Bacterial catalases are induced by exposure to peroxide (e.g., Escherichia coli katG) or entry into stationary phase (e.g., E. coli katE). To study regulatory systems in Haemophilus influenzae, we complemented an E. coli rpoS mutant, which is unable to induce katE in stationary phase, with a plasmid library of H. influenzae Rd- chromosomal DNA. Nineteen complementing clones with a catalase-positive phenotype were obtained and characterized after screening about 10(5) transformants. All carried the same structural gene for an H. influenzae catalase. The DNA sequence of this gene, called hktE, encodes a 508-amino-acid polypeptide with strong homology to eukaryotic catalases and E. coli katE. However, hktE is regulated like E. coli katG, with catalase activity increasing 10-fold and hktE mRNA levels increasing 4-fold upon exposure to ascorbic acid, which serves to generate hydrogen peroxide. Mutations in the known global regulatory genes of H. influenzae--crp, cya, and sxy--do not affect the inducibility of hktE. The hktE gene maps to a 225-kb segment of the H. influenzae chromosome in a region encoding resistance to spectinomycin.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.