Functional diversification of the toll-like receptor gene family

Phylogenetic analyses supported the hypothesis that the vertebrate toll-like receptors (TLRs) include two very ancient groups that arose by gene duplication prior to the divergence of protostomes and deuterostomes: (1) the TLR1 family (including mammalian TLR1, TLR2, TLR6, and TLR10); and (2) a clade including the remainder of mammalian TLRs. Correlating data on ligand type, subcellular localization, and gene expression in leukocytes and other tissues with the phylogeny provided evidence that certain major functional specializations within the TLRs occurred after ancient gene duplication events and that these traits have been retained through further events of gene duplication. For example, the recognition of bacterial lipoproteins appears to have arisen in the ancestor of the TLR1 family and continues to characterize members of that family whose ligands are known. Likewise, expression on the endosomal membrane and the recognition of nucleic acids appears to have been arisen in the ancestor of the TLR7 family and some related TLRs. On the other hand, gene expression patterns across tissues appear to have been much more volatile over the evolution of the vertebrate TLRs, since genes may show expression profiles similar to those of distantly related genes but dissimilar to those of closely related genes. Thus, the vertebrate TLRs provide an example of a multi-gene family in which gene duplication has been followed by extensive changes in certain aspects of gene function, while others have been conserved throughout vertebrate history. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.     

Structure and function of SirC from Bacillus megaterium: a metal-binding precorrin-2 dehydrogenase

In Bacillus megaterium, the synthesis of vitamin B(12) (cobalamin) and sirohaem diverges at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD(+) as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 A (1 A = 0.1 nm) resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional sirohaem synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give sirohaem. Guided by the structure, in the present study a number of active-site residues within SirC were investigated by site-directed mutagenesis. No active-site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion with that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with an independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of sirohaem and cobalamin.     

Evaluation of the repeatability of ultra-performance liquid chromatography-TOF-MS for global metabolic profiling of human urine samples

The application of reversed-phase ultra-performance liquid chromatography, based on the use of sub 2 microm particles, combined with time-of-flight mass spectrometry has been investigated for the production of global metabolite profiles from human urine. The stability and repeatability of the methodology, which employed gradient elution, was determined by the repeat analysis of a pooled quality control (QC) sample. As seen in previous studies conducted with conventional LC-MS an element of system conditioning was required to obtain reproducible data, as the initial injections were unrepresentative. However, once the system had equilibrated excellent repeatability in terms of retention time, signal intensity and mass accuracy was seen providing confidence that for this matrix, the within-day repeatability of UPLC-TOF-MS was sufficient to assure data quality in global metabolic profiling applications.     

Long-term mutagenic effects of ionising radiation on mice which vary in their p53 status

The tumor suppressor gene p53 plays a major role in the maintenance of genomic integrity. The impact that variations in cellular turnover rates and sensitivity to DNA damage will have on the effectiveness of p53 in this role was examined by following the induction and persistence of mutations in the brain and small intestine of mice after exposure to ionising radiation (IR). The examination of mutagenesis was carried out using the pUR288 LacZ plasmid-based mouse model-consisting of mice containing a target gene for mutation analysis integrated into every cell. In addition the mice varied in their p53 status. The tissues were compared at post-irradiation time-points from 24h to 3 months. The mutation frequencies (MFs) in the p53 wildtype and heterozygous brains peaked at 24h post-irradiation, and then returned to background or close to background levels, respectively. The p53 nullizygous brain showed a more fluctuating MF pattern, but returned to background levels by 3 months, indicating that the effect of the loss of p53 did not result in lasting differences in the response to mutation induction in the brain. In the intestine, there was a different pattern; in the wildtype and heterozygous animals, the MFs increased from 24h to a peak at 4 weeks post-irradiation, before decreasing towards background levels at 3 months. The MFs in the intestine from the nullizygous animals did not decrease significantly between 4 weeks and 3 months, illustrating that the loss of p53 had a greater impact in this tissue than the brain. The variation in mutation frequencies and the type of mutations generated after DNA damage suggests that while p53 plays a significant role in the maintenance of genomic integrity, other mechanisms, such as the drive to replicate in progenitor cells, can reduce its effectiveness as the "guardian of the genome".     

Chemical and principal-component analyses of the essential oils of Apioideae taxa (Apiaceae) from central Balkan

The volatile constituents of the essential oils of 23 taxa belonging to the Apioideae subfamily were studied in detail. The investigated taxa were Pimpinella serbica (Vis.) Bentham & Hooker, Libanotis montana Cr., Cnidium silaifolium (Jacq.) Simk. ssp. orientale (Boiss.) Tutin, Bupleurum praealtum L., B. sibthorpianum S. S. var. diversifolium (Roch.) Hay, Aegopodium podagraria L., Torilis anthriscus (L.) Gmel., Orlaya grandiflora (L.) Hoffm., Laserpitium siler L., Laser trilobum (L.) Brokh., Chaerophyllum aureum L., C. hirsutum L., C. temulum L., Pastinaca sativa L., P. hirsuta Pancic., Tordylium maximum L., Physospermum cornubiense (L.) DC., Peucedanum alsaticum L., P. oreoselinum (L.) Moench, P. cervaria (L.) Cuss., P. austriacum (Jacq.) Koch, P. longifolium W. et K., and P. officinale L. All of these species grow wild in the central part of the Balkan Peninsula. The essential oils were found to be complex mixtures of various compounds, more than 100 constituents being in each taxon, with contributions of main products never exceeding 25% of the total content. Sesquiterpene hydrocarbons were found to be the main group of constituents of all taxa, except for Peucedanum species, where monoterpene hydrocarbons were identified as the main components. The chemotaxonomic value of the essential-oil composition is discussed according to results of principal-component analysis (PCA). The essential-oil composition mainly reflects current taxonomic relationships between the investigated taxa.     

Acid cleavable PEG-lipids for applications in a ternary gene delivery vector

A novel class of pH-sensitive PEG lipids bearing acid-cleavable acetal linkages and short PEG chains have been synthesised and used in ternary vector formulations. The cleavage pH was influenced by structural components including the terminal PEG moiety and spacer length.     

General practitioner attitudes towards referral of eating-disordered patients: a vignette study based on the theory of planned behaviour

Objective The study examined individual differences between general practitioners (GPs) to determine their impact on variations in intention to refer a hypothetical patient with disordered eating to specialist eating disorder services. The study also examined the impact of patient weight on intention to refer.Method GPs within three primary care trusts (PCTs) were posted a vignette depicting a patient with disordered eating, described as either normal weight or underweight. A questionnaire was developed from the theory of planned behaviour to assess the GPs' attitudes, perception of subjective norms, perceived behavioural control, and intention to refer the patient. Demographic details were also collected.Results Responses were received from 88 GPs (33%). Intention to refer the patient was significantly related to subjective norms and cognitive attitudes. Together these predictors explained 86% of the variance in the intention to refer. GP or practice characteristics did not have a significant effect on the GPs' intention to refer, and nor did the patient's weight.Conclusion Despite National Institute for Health and Clinical Excellence current guidance, patient weight did not influence GPs' decisions to refer. Much of the variance in actual referral behaviour may be explained by cognitive attitudes and subjective norms. Interventions to reduce this variation should be focused on informing GPs about actual norms, and best practice guidelines.