Sequential 1H-NMR assignments of mouse
Cd7-metallothionein-1 (MT1) have
been carried out by standard homonuclear NMR methods and
the use of an accordion–heteronuclear multiple quantum
correlation ...(HMQC) experiment for establishing the metal,
113Cd2+, to cysteine connectivities.
The three-dimensional structure was then calculated using
the distance constraints from two-dimensional nuclear Overhauser
effect (NOE) spectroscopy spectra and the Cys–Cd
connectivities as input for a distance geometry-dynamical
simulated annealing protocol in X-PLOR 3.851. Similar to
the mammalian MT2 isoforms, the homologous primary structure
of MT1 suggested two separate domains, each containing
one metal cluster. Because there were no interdomain constraints,
the structure calculation for the N-terminal β- and
the C-terminal α-domain were carried out separately.
The structures are based on 409 NMR constraints, consisting
of 381 NOEs and 28 cysteine–metal connectivities.
The only elements of regular secondary structure found
were two short stretches of 310 helices along
with some half-turns in the α-domain. Structural comparison
with rat liver MT2 showed high similarity, with the β-domain
structure in mouse MT1 showing evidence of increased flexibility
compared to the same domain in MT2. The latter was reflected
by the presence of fewer interresidue NOEs, no slowly exchanging
backbone amide protons, and enhanced cadmium–cadmium
exchange rates found in the β-domain of MT1.
Object
We sought to measure brain metabolite levels in healthy older people.
Materials and methods
Spectroscopic imaging at the level of the basal ganglia was applied in 40 participants aged ...73–74 years. Levels of the metabolites
N
-acetyl aspartate (NAA), choline, and creatine were determined in "institutional units" (IU) corrected for T1 and T2 relaxation effects. Structural imaging enabled determination of grey matter (GM), white matter (WM), and cerebrospinal fluid content. ANOVA analysis was carried out for voxels satisfying quality criteria.
Results
Creatine levels were greater in GM than WM (57 vs. 44 IU,
p
< 0.001), whereas choline and NAA levels were greater in WM than GM 13 vs. 10 IU (
p
< 0.001) and 76 versus 70 IU (
p
= 0.03), respectively. The ratio of NAA/cre was greater in WM than GM (2.1 vs. 1.4,
p
= 0.001) as was that of cho/cre (0.32 vs. 0.16,
p
< 0.001). A low voxel yield was due to brain atrophy and the difficulties of shimming over an extended region of brain.
Conclusion
This study addresses the current lack of information on brain metabolite levels in older adults. The normal features of ageing result in a substantial loss of reliable voxels and should be taken into account when planning studies. Improvements in shimming are also required before the methods can be applied more widely.
Summary Background Some countries fortify flour with folic acid to prevent neural tube defects but others do not, partly because of concerns about possible cancer risks. We aimed to assess any ...effects on site-specific cancer rates in the randomised trials of folic acid supplementation, at doses higher than those from fortification. Methods In these meta-analyses, we sought all trials completed before 2011 that compared folic acid versus placebo, had scheduled treatment duration at least 1 year, included at least 500 participants, and recorded data on cancer incidence. We obtained individual participant datasets that included 49 621 participants in all 13 such trials (ten trials of folic acid for prevention of cardiovascular disease n=46 969 and three trials in patients with colorectal adenoma n=2652). All these trials were evenly randomised. The main outcome was incident cancer (ignoring non-melanoma skin cancer) during the scheduled treatment period (among participants who were still free of cancer). We compared those allocated folic acid with those allocated placebo, and used log-rank analyses to calculate the cancer incidence rate ratio (RR). Findings During a weighted average scheduled treatment duration of 5·2 years, allocation to folic acid quadrupled plasma concentrations of folic acid (57·3 nmol/L for the folic acid groups vs 13·5 nmol/L for the placebo groups), but had no significant effect on overall cancer incidence (1904 cancers in the folic acid groups vs 1809 cancers in the placebo groups, RR 1·06, 95% CI 0·99–1·13, p=0·10). There was no trend towards greater effect with longer treatment. There was no significant heterogeneity between the results of the 13 individual trials (p=0·23), or between the two overall results in the cadiovascular prevention trials and the adenoma trials (p=0·13). Moreover, there was no significant effect of folic acid supplementation on the incidence of cancer of the large intestine, prostate, lung, breast, or any other specific site. Interpretation Folic acid supplementation does not substantially increase or decrease incidence of site-specific cancer during the first 5 years of treatment. Fortification of flour and other cereal products involves doses of folic acid that are, on average, an order of magnitude smaller than the doses used in these trials. Funding British Heart Foundation, Medical Research Council, Cancer Research UK, Food Standards Agency.
Upon storage under aerobic conditions metallothioneins (MTs) form a new species, which is characterized by a molecular mass approximately twice the size of monomeric MT and shifted (113/111)Cd- and ...(1)H-NMR resonances. The investigation of this oxidative dimerization process by NMR spectroscopy allowed us to structurally characterize this MT species that has been described to occur in vivo and might be synthesized under conditions of oxidative stress. The oxidative dimer was characterized by the formation of an intermolecular cysteine disulphide bond involving the alpha-domain, and a detailed analysis of chemical shift changes and intermolecular nuclear Overhauser effects points towards a disulphide bond involving Cys(36). In contrast to the metal-bridged (non-oxidative) dimerization, the metal-cysteine cluster structures in both MT domains remain intact and no conformational exchange or metal-metal exchange was observed. Also in contrast to the many recently reported oxidative processes which involve the beta-domain cysteine groups and result in the increased dynamics of the bound metal ions in this N-terminal domain, we found no evidence for any increased dynamics in the alpha-domain metals following this oxidation. Therefore these findings provide additional corroboration that metal binding in the C-terminal alpha-domain is rather tight, even under conditions of a changing cellular oxidation potential, compared with the more labile/dynamic nature of the metals in the N-terminal beta-domain cluster under similar conditions.
CD40, a member of the tumour necrosis factor receptor family, is expressed on the surface of B lymphocytes where its ligation provides a potent survival signal. CD40 is also expressed in basal ...epithelial cells and in a number of different carcinomas where its function remains unknown. We observed that contrary to the studies in normal B cells, CD40 ligation in carcinoma cell lines and in normal primary epithelial cells resulted in growth inhibition and enhanced susceptibility to apoptosis induced by anti-neoplastic drugs, TNF-alpha, Fas and ceramide. This effect was also observed in CD40-transfected Rat-1 fibroblasts. The expression of Bcl-2 did not affect growth inhibition induced by CD40 ligation in epithelial cells but the Epstein - Barr Virus-encoded latent membrane protein 1 (LMP1) blocked the effect. Whilst transient expression of LMP-1 resulted in the inhibition of epithelial cell growth, this effect was not observed with a LMP1 mutant lacking the binding domain for TRAF3, a protein which may mediate signal transduction by interacting with the cytoplasmic domains of both CD40 and LMP1. Transient expression of TRAF3 also inhibited epithelial cell growth, whilst expression of a dominant-negative TRAF3 partially blocked the inhibitory effect of CD40 ligation and of transient LMP1 expression. These results suggest that CD40 regulates epithelial cell growth in a manner mimicked by LMP1 and implicate TRAF3 as a common mediator in the transduction of the growth inhibitory signals generated via the CD40 and LMP1 pathways.
The objective of this project was to analyse the current access to in-patient stroke services and MDT rehabilitation in an acute stroke centre and to compare these services to the recommended ..."National Clinical Guidelines and Recommendations for the Care of People with Stroke and TIA" (IHF 2010). A retrospective chart review was carried out, recording activity statistics of all patients admitted with acute stroke over a three-month period. 73 patients (male = 40, 54.8%) were included. Patients were discharged from the stroke service after a mean stay of 20.2 days (SD = 19.3). 76.7% (N = 56) of patients were admitted to the acute stroke unit (ASU). The mean length of time from admission to first assessment 3.4 days (SD. = 2.68), with an average of 138 minutes of treatment received per day across all disciplines. This is compared to the IHF's recommendation of patients being assessed within 24-48 hours of admission and receiving 180 minutes of treatment across all disciplines. As demands for stroke MDT services increase, it is important to recognise the benefits of increasing staff and resources to maintain and continue to improve standards of care.
Monkeys are becoming important translational models of neurodegenerative disease. To facilitate model development, we measured cerebrospinal fluid (CSF) concentrations of key biomarkers in healthy ...male and female cynomolgus and rhesus macaques. Amyloid beta (Aβ40, Aβ42), tau (total tau t‐tau, phosphorylated tau pThr181), and neurofilament light (NfL) concentrations were measured in CSF of 82 laboratory‐housed, experimentally naïve cynomolgus (n = 33) and rhesus (n = 49) macaques. Aβ40 and Aβ42 were significantly higher in rhesus, and female rhesus were higher than males. NfL and t‐tau were higher in males, and NfL was higher in rhesus macaques. p‐tau was not affected by species or sex. We also examined whether sample location (lumbar or cisterna puncture) affected concentrations. Sample acquisition site only affected NfL, which was higher in CSF from lumbar puncture compared to cisterna magna puncture. Establishing normative biomarker values for laboratory‐housed macaque monkeys provides an important resource by which to compare to monkey models of neurodegenerative diseases.
The interleukin-2 (IL-2) receptor gamma-chain is a common component of several members of the cytokine receptor superfamily including those for IL-2, IL-4, IL-7, IL-9, IL-15, and possibly IL-13, and ...has recently been renamed the common gamma-chain (gamma c-chain). Transfection experiments have shown that the gamma c-chain participates in signal transduction by IL-2, IL-4 and IL-7, but a functional role for the gamma c-chain in biological responses by normal T cells and B cells to these cytokines has not been established. In this study, we have used X-linked severe combined immunodeficiency (X-SCID) as a naturally occurring gamma c-chain gene disruption model to examine the role of the gamma c-chain in human B-cell responses to IL-2, IL-4, IL-13, and IL-15. Our experiments show that B cells from two X-SCID patients with characterized gamma c-chain gene mutations do not respond to IL-2 or IL-15, but respond as well or better than normal B cells to both IL-4 and IL-13 in assays for B-cell activation, proliferation, and IgE secretion. This finding raises important questions about the function of the gamma c-chain in receptors for IL-4 and IL-13, and the nature of the immune defect in X-SCID.
A cattle genetic linkage map was constructed which marks about 90% of the expected length of the cattle genome. Over 200 DNA polymorphisms were genotyped in cattle families which comprise 295 ...individuals in full sibling pedigrees. One hundred and seventy-one loci were found linked to one other locus. Twenty nine of the 30 chromosome pairs are represented by at least one of the 36 linkage groups. Less than a 50 cM difference was found in the male and female genetic maps. The conserved loci on this map show as many differences in gene order compared to humans as is found between humans and mice. The conservation is consistent with the patterns of karyotypic evolution found in the rodents, primates and artiodactyls. This map will be important for localizing quantitative trait loci and provides a basis for further mapping.
It is hypothesised that endothelial cell reactive antibodies (ECRA) play a role in the progression of PAD through activation of endothelial cells and the release of inflammatory cytokines. We aimed ...to test this hypothesis by assessing levels of ECRA, E-selectin and IL-6 in patients with PAD of varying severity in a case controlled study.
Patients were assessed clinically and with ankle–brachial pressure indices. Patients with critical ischaemia (CI, n=30), stable claudicants (SC, n=30), and age-matched controls (AMC, n=20) were studied. Antibody, E-selectin and IL-6 levels were measured using ELISA.
ECRA levels were significantly raised in the CI group over AMC.
IL-6 levels were significantly elevated in both SC and CI over the control group and in CI over SC.
There were no significant differences in E-selectin levels between the AMC, SC and CI.
Our findings support the hypothesis that autoantibodies play a role in promoting PAD by elevating IL-6. The absence of an elevation in E-selectin in this study may be due to its short half-life, and casts doubt on its value as a marker of inflammation in atherosclerosis.
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