A non-enzymic procedure for the quantitative analysis of (3-methoxy-4-sulphoxyphenyl)ethylene glycol (MHPG sulphate) in human urine using stable isotope dilution and gas chromatography—mass spectrometry

A method is described for the quantitative analysis of (3-methoxy-4-sulphoxyphenyl)-ethylene glycol (MHPG sulphate) in human urine, based on selected ion monitoring gas chromatography—mass spectrometry and using a specifically deuterium-labelled analogue of MHPG sulphate as internal standard. The procedure involves extraction of the urine sample on Amberlite XAD-2, followed by isolation of MHPG sulphate by column chromatography on Sephadex LH-20. Cleavage of the sulphate conjugate and formation of the MHPG tris(trifluoroscetate) derivative are carried out in a one-step reaction, without recourse to enzymic hydrolysis.     

Species traits explain variation in detectability of UK birds

Capsule Heterogeneous detectability amongst species may impact multi-species bird surveys and if not accounted for, may bias community level conclusions. Estimates of detectability were produced for 195 UK bird species, and detectability was significantly affected by bird size, diet and habitat specialization.

Aims To estimate detectability and understand which species traits may impact detectability.

Methods We estimated the detectability of 195 species of birds in the UK using distance sampling methods and examined the average detectability of genetically related groups. We tested the significance of species traits in describing variation in detectability, whilst controlling for phylogenetic relationships.

Results Passeriformes had the lowest median detectability of 0.37 and Charadriiformes the highest median detectability of 0.65, of the seven largest orders considered. Species most associated with closed habitats such as woodland and urban areas had the lowest detectability. Smaller species had lower detectability than larger species.

Conclusion Heterogeneity in species detectability could lead to biased conclusions, particularly when calculating multi-species indices such as species richness or diversity. Accounting for detectability will be most important in studies that cover a wide range of habitat types or a diverse spread of taxa.     

Characterisation of the immune response to the UK human anthrax vaccine

The UK human anthrax vaccine consists of the alum-precipitated culture supernatant of Bacillus anthracis Sterne. In addition to protective antigen (PA), the key immunogen, the vaccine also contains a number of other bacteria- and media-derived proteins. These proteins may contribute to the transient side effects experienced by some individuals and could influence the development of the PA-specific immune response. Bacterial cell-wall components have been shown to be potent immunomodulators. B. anthracis expresses two S-layer proteins, EA1 and Sap, which have been demonstrated to be immunogenic in animal studies. These are also immunogenic in man so that convalescent and post-immunisation sera contain specific antibodies to Ea1, and to a lesser extent, to Sap. To determine if these proteins are capable of modifying the protective immune response to PA, A/J mice were immunised with equivalent amounts of recombinant PA and S-layer proteins in the presence of alhydrogel. IgG isotype profiles were determined and the animals were subsequently challenged with spores of B. anthracis STI. The results suggest that there was no significant shift in IgG isotype profile and that the presence of the S-layer proteins did not adversely affect the protective immune response induced by PA.     

Formaldehyde mutagenesis in the nematode Caenorhabditis elegans

We have found that formaldehyde is capable of inducing mutations in the nematode Caenorhabditis elegans. 4 concentrations of formaldehyde were tested. At a concentration of 1%, formaldehyde is lethal to the nematode, and 0.01% formaldehyde did not induce any mutations in approx. 60 000 tested chromosomes. 2 concentrations of formaldehyde, 0.1% and 0.07%, were found to be mutagenic, inducing both point mutations and deficiencies in the unc-22 region of linkage group IV.4 of the point mutations have been demonstrated to be alleles of the unc-22 gene and have been mapped within the locus. 2 of the putative deficiencies have been confirmed. Each spans the unc-22 gene and at least 2 other genes in the region. A rough estimate of the forward mutation frequency using 0.1% formaldehyde in this region is 3 × 10⁻⁵, while for 0.07% the frequency is 2 × 10⁻⁴.     

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.     

p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a PDE4/cAMP/PKA pathway

Clearance of fibrin through proteolytic degradation is a critical step of matrix remodeling that contributes to tissue repair in a variety of pathological conditions, such as stroke, atherosclerosis, and pulmonary disease. However, the molecular mechanisms that regulate fibrin deposition are not known. Here, we report that the p75 neurotrophin receptor (p75^NTR), a TNF receptor superfamily member up-regulated after tissue injury, blocks fibrinolysis by down-regulating the serine protease, tissue plasminogen activator (tPA), and up-regulating plasminogen activator inhibitor-1 (PAI-1). We have discovered a new mechanism in which phosphodiesterase PDE4A4/5 interacts with p75^NTR to enhance cAMP degradation. The p75^NTR-dependent down-regulation of cAMP results in a decrease in extracellular proteolytic activity. This mechanism is supported in vivo in p75^NTR-deficient mice, which show increased proteolysis after sciatic nerve injury and lung fibrosis. Our results reveal a novel pathogenic mechanism by which p75^NTR regulates degradation of cAMP and perpetuates scar formation after injury.     

Group A streptococcal phage T12 carries the structural gene for pyrogenic exotoxin type A

Summary In this paper we describe the meiotic pairing behavior of two free duplications in Caenorhabditis elegans. sDp1 is a duplication of approximately 30 map units of the right portion of linkage group I including unc-74 to unc-54. This duplication pairs, recombines, and apparently segregates from one of the normal homologues. A second duplication, sDp2, is a duplication of approximately 15 map units of the left portion of the linkage group. sDp2 was not observed to recombine with the normal homologue but did suppress exchange between the two normal homologues in a sDp2/ ++ / dpy-5 unc-35 heterozygote. Although a number of free duplications have been described previously in Caenorhabditis elegans, none of these have been shown to pair with normal homologues. The meiotic behavior of the duplications described in this paper can be understood assuming the existence in C. elegans chromosomes of pairing sites of the type described in D. melanogaster chromosomes (I. Sandler 1956; Hawley 1980).