Peptides and the development of double- and triple-resonance solid-state NMR of aligned samples.

Peptides have been instrumental in the development of solid-state nuclear magnetic resonance (NMR) spectroscopy, and their roles in the development of solid-state NMR of aligned samples is reviewed. In particular, the roles of synthetic peptides in the development of triple-resonance methods are described. Recent developments of pulse sequences and NMR probes for triple-resonance NMR of aligned samples are presented.     

Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures

Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 α-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06–0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as −7°C.     

The renal urate transporter SLC17A1 locus: confirmation of association with gout

Introduction

Two major gout-causing genes have been identified, the urate transport genes SLC2A9 and ABCG2. Variation within the SLC17A1 locus, which encodes sodium-dependent phosphate transporter 1, a renal transporter of uric acid, has also been associated with serum urate concentration. However, evidence for association with gout is equivocal. We investigated the association of the SLC17A1 locus with gout in New Zealand sample sets.

Methods

Five variants (rs1165196, rs1183201, rs9358890, rs3799344, rs12664474) were genotyped across a New Zealand sample set totaling 971 cases and 1,742 controls. Cases were ascertained according to American Rheumatism Association criteria. Two population groups were studied: Caucasian and Polynesian.

Results

At rs1183201 (SLC17A1), evidence for association with gout was observed in both the Caucasian (odds ratio (OR) = 0.67, P = 3.0 × 10^-6) and Polynesian (OR = 0.74, P = 3.0 × 10^-3) groups. Meta-analysis confirmed association of rs1183201 with gout at a genome-wide level of significance (OR = 0.70, P = 3.0 × 10^-8). Haplotype analysis suggested the presence of a common protective haplotype.

Conclusion

We confirm the SLC17A1 locus as the third associated with gout at a genome-wide level of significance.     

Myxoma virus M141R expresses a viral CD200 (vOX-2) that is responsible for down-regulation of macrophage and T-cell activation in vivo

M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25(+) infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R(+) dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly.     

Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair

Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair. The relationship of this repair defect to disease traits remains unclear, given that crosslink sensitivity is recapitulated in FA mouse models without most of the other disease-related features. Mice deficient in Mus81 are also defective in crosslink repair, yet MUS81 mutations have not been linked to FA. Using mice deficient in both Mus81 and the FA pathway protein FancC, we show both proteins cooperate in parallel pathways, as concomitant loss of FancC and Mus81 triggered cell-type-specific proliferation arrest, apoptosis and DNA damage accumulation in utero. Mice deficient in both FancC and Mus81 that survived to birth exhibited growth defects and an increased incidence of congenital abnormalities. This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity. Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.     

Androgen dynamics in vitro in the normal and hyperplastic human prostate gland

The dynamics of uptake and metabolism in vitro of androgens by normal and hyperplastic human prostate glands was studied by means of a new experimental design proposed by Gurpide & Welch (1969). Prostate slices were perfused with a medium containing [(3)H]testosterone and [(14)C]androstenedione, or 5alpha-dihydro-[(3)H]testosterone and [(14)C]testosterone. The entry into the slices, the irreversible metabolism, the conversion between the compounds and the tissue retention or ;uptake' of the steroids were measured at the steady state. A similar portion of the three androgens entered the tissue and was irreversibly metabolized. Conversion of testosterone into 5alpha-dihydrotestosterone was much greater than the interconversion of testosterone and androstenedione. The prostate slices retained 5alpha-dihydrotestosterone at a concentration three times that in the medium, whereas testosterone and androstenedione were retained to a smaller extent. At a steroid concentration of 0.11mumol/l in the medium, the various parameters did not differ significantly in experiments performed with slices from normal and hyperplastic glands. When the steroid concentration in the medium was increased tenfold, however, a difference between normal and hyperplastic glands was evident. The normal glands increased the uptake and metabolism proportionally to the elevation of the steroid concentration in the medium. In the hyperplastic glands the entry and metabolism lagged behind the increase in steroid supply, whereas the tissue uptake became disproportionately high. The possible causes of this finding are discussed.     

The Oxidative Cost of Acoustic Signals: Examining Steroid Versus Aerobic Activity Hypotheses in a Wild Bird

Vertebrate vocalizations are widespread secondary sexual signals used for mate attraction and territory defence, and variation in signal quality is often condition dependent and impacts reproductive outcomes. Although vocal signal performance is known to reflect various aspects of male quality, few studies have examined the underlying mechanisms mediating its costs and hence its honesty. Using a population of Arctic‐breeding snow buntings (Plectrophenax nivalis), we compared the ‘Oxidation Handicap Hypothesis’, which predicts that testosterone‐induced increases in oxidative stress provide a direct mechanistic basis for ensuring the honesty of many secondary sexual signals, to the ‘Aerobic Activity Hypothesis, which predicts that it is the aerobic activity involved with signal production (i.e. vocal performance or defending a large territory) and not testosterone directly that links signal quality and oxidative stress. Males singing at faster rates had higher levels of both reactive oxygen metabolites and non‐enzymatic antioxidant capacity in the plasma (i.e. without an increase in overall oxidative stress), enabling certain males to produce high‐quality signals while also mitigating the costs of an associated increase in oxidative stress. However, these results were completely independent of plasma testosterone levels, supporting the role of aerobic performance in directly affecting oxidative stress. Although song performance was not linked to reproductive parameters in our data set, our research is the first to test these competing hypotheses in a behavioural trait and results suggest that oxidative stress may be an underlying physiological cost preventing low‐quality individuals from producing high‐quality signals.