Proteomic method for the quantification of methionine sulfoxide

Glycoxidation and lipoxidation reactions contribute to the chemical modification of proteins during the Maillard reaction. Reactive oxygen species, produced during the oxidation of sugars and lipids in these processes, irreversibly oxidize proteins. Methionine is particularly susceptible to oxidation, yielding the oxidation product methionine sulfoxide (MetSO). Here we describe a method for the analysis of MetSO using proteomic techniques. Using these techniques, we measured MetSO formation on the model protein RNase during aerobic incubations with glucose and arachidonate. We also evaluated the susceptibility of MetSO to reduction by NaBH4, a commonly used reductant in the analysis of Maillard reaction products.

Mass spectrometric analysis of glucose-modified ribonuclease

RNase A (1 mM) was incubated with glucose (0.4 M) at 37degreesC for up to 14 days in phosphate buffer (0.2 M, pH 7.4), digested with trypsin and analysed by LC-MS. The major sites of fructoselysine formation were Lys(1), Lys(7), Lys(37) and Lys(41). Three of these sites (Lys(7), Lys(37) and Lys(41)) were also the major sites of N-epsilon-(carboxymethyl)lysine formation.

Enzyme-catalysed synthesis and reactions of benzene oxide/oxepine derivatives of methyl benzoates

A series of twelve benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl benzoate, allied to evidence of the migration and retention of a carbomethoxy group ( the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-( trifluoromethyl) benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.

Transport Properties of Self-Consolidating Concrete

The permeability of concrete is influenced by the porosity and the interconnectivity of the pores in the cement paste and the microcracks in concrete, especially in the interface of paste-aggregate. The movements of gases, liquids, and ions through concrete is important because of their interactions with concrete constituents, including pore water, which can alter the integrity of concrete directly and indirectly, leading to the deterioration of structures. This study reports the findings from an investigation carried out to study the effect of the mixture variations on the durability of medium- and high-strength self-consolidating concrete (SCC). The mixture variations studied include the type of mineral admixtures, such as limestone powder (LSP) and pulverized fuel ash (PFA), and viscositymodifying admixtures (VMA) for both medium- and high-strength SCC. Air permeability, water permeability, capillary absorption, and chloride diffusivity were used to assess the durability of SCC mixtures in comparison with normal, vibrated concretes. The results showed that SCC mixtures, for medium- and high-strength grades using PFA followed by LSP, have lower permeability properties compared with normal concretes. SCC made with VMA had a higher sorptivity, air permeability, and water permeability compared with other SCC mixtures, which can be attributed to higher watercement ratio (w/c) and lack of pore filling effect. An in-place migration coefficient was obtained using the in-place ion migration test. This was used to compare the potential diffusivity of different concretes. The results indicated that SCC, for both grades of strength, made with PFA showed much lower diffusivity values in comparison with other mixtures, whereas the SCC mixtures with VMA showed higher diffusivity.

Protein expression profiling during chick retinal maturation: a proteomics-based approach

Background: The underlying pathways that drive retinal neurogenesis and synaptogenesis are still relatively poorly understood. Protein expression analysis can provide direct insight into these complex developmental processes. The aim of this study was therefore to employ proteomic analysis to study the developing chick retina throughout embryonic (E) development commencing at day 12 through 13, 17, 19 and post-hatch (P) 1 and 33 days.

Results: 2D proteomic and mass spectrometric analysis detected an average of 1514 spots per gel with 15 spots demonstrating either modulation or constitutive expression identified via MS. Proteins identified included alpha and beta-tubulin, alpha enolase, B-creatine kinase, gamma-actin, platelet-activating factor (PAF), PREDICTED: similar to TGF-beta interacting protein 1, capping protein (actin filament muscle Z line), nucleophosmin 1 (NPM1), dimethylarginine dimethylaminohydrolase, triosphoaphate isomerase, DJ1, stathmin, fatty acid binding protein 7 (FABP7/B-FABP), beta-synuclein and enhancer of rudimentary homologue.

Conclusion: This study builds upon previous proteomic investigations of retinal development and represents the addition of a unique data set to those previously reported. Based on reported bioactivity some of the identified proteins are most likely to be important to normal retinal development in the chick. Continued analysis of the dynamic protein populations present at the early stages and throughout retinal development will increase our understanding of the molecular events underpinning retinogenesis.

Novel Venom Proteins Produced by Differential Domain-Expression Strategies in Beaded Lizards and Gila Monsters (genus Heloderma)

The origin and evolution of venom proteins in helodermatid lizards were investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: 1) The first full-length sequences of lethal toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral monodomain, monoproduct gene (beta-defensin) that underwent three tandem domain duplications to encode a tetradomain, monoproduct with a possible novel protein fold; 2) an ancestral monodomain gene (encoding a natriuretic peptide) was medially extended to become a pentadomain, pentaproduct through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by posttranslational proteolysis; and 3) an ancestral multidomain, multiproduct gene belonging to the vasoactive intestinal peptide (VIP)/glucagon family being mutated to encode for a monodomain, monoproduct (exendins) followed by duplication and diversification into two variant classes (exendins 1 and 2 and exendins 3 and 4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isoforms within each class. These results highlight the importance of utilizing evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.

Variation in RTN3 and PPIL2 Genes Does not Influence Platelet Membrane β-Secretase Activity or Susceptibility to Alzheimer’s Disease in the Northern Irish Population

beta-site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of beta-amyloid peptides (A beta), which are proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (beta-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulates BACE1 expression. The present study investigated whether there was any association between genetic variation in RTN3 and PPIL2, and either risk for AD, or levels of platelet beta-secretase activity, in a large Northern Irish case-control sample. Four hundred and sixty-nine patients with a diagnosis of probable AD (NINCDS-ADRDA criteria) and 347 control individuals (MMSE > 28/30) were genotyped. SNPs in both genes were selected by downloading genotype data from the International HapMap Project (Phase II) and tags selected using multimarker approach in Haploview, where r (2) > 0.8 and LOD > 3.0. Non-synonymous SNPs of interest were also included. Genotyping was performed by Sequenom iPLEX and TaqMan technologies. Alleles, genotypes and multi-marker haplotypes were tested for association with AD, and platelet beta-secretase activities were measured for a subset of individuals (n = 231). Eight SNPs in RTN3 and 7 in PPIL2 were genotyped. We found no significant associations between allele, genotype or haplotype frequencies and risk of AD. Further, there was no effect of genotype on platelet membrane beta-secretase activity. We conclude that common or potentially functional genetic variation in these BACE1 interacting proteins does not affect platelet membrane beta-secretase activity or contribute to risk of AD in this population.

Effect of Aspirin or Resistant Starch on Colorectal Neoplasia in the Lynch Syndrome

Background: Observational and epidemiologic data indicate that the use of aspirin reduces the risk of colorectal neoplasia; however, the effects of aspirin in the Lynch syndrome (hereditary nonpolyposis colon cancer) are not known. Resistant starch has been associated with an antineoplastic effect on the colon.

Detection of aberrant behaviour in home environments from video sequence

We introduce an application for the detection of aberrant behaviour within home based environments, with a focus on repetitive actions, which may be present in instance of persons suffering from dementia. Video based analysis has been used to detect the motion of a person within a given scene in addition to tracking them over the time. Detection of repetitive actions has been based on the analysis of a person's trajectory using the principles of signal correlation. Along with the ability to detect repetitive motion the developed approach also has the ability to measure the amount of activity/inactivity within the scene during a given period of time. Our results showed that the developed approach had the ability to detect all patterns in the data set examined with an average accuracy of 96.67%. This work has therefore validated the proposed concept of video based analysis for the detection of repetitive activities.

Adjuvant chemotherapy in oestrogen-receptor-poor breast cancer: patient-level meta-analysis of randomised trials

Background: The long-term effects of adjuvant polychemotherapy regimens in oestrogen-receptor-poor (ER-poor) breast cancer, and the extent to which these effects are modified by age or tamoxifen use, can be assessed by an updated meta-analysis of individual patient data from randomised trials. Methods: Collaborative meta-analyses of individual patient data for about 6000 women with ER-poor breast cancer in 46 trials of polychemotherapy versus not (non-taxane-based polychemotherapy, typically about six cycles; trial start dates 1975-96, median 1984) and about 14 000 women with ER-poor breast cancer in 50 trials of tamoxifen versus not (some trials in the presence and some in the absence of polychemotherapy; trial start dates 1972-93, median 1982). Findings: In women with ER-poor breast cancer, polychemotherapy significantly reduced recurrence, breast cancer mortality, and death from any cause, in those younger than 50 years and those aged 50-69 years at entry into trials of polychemotherapy versus not. In those aged younger than 50 years (1907 women, 15% node-positive), the 10-year risks were: recurrence 33% versus 45% (ratio of 10-year risks 0·73, 2p<0·00001), breast cancer mortality 24% versus 32% (ratio 0·73, 2p=0·0002), and death from any cause 25% versus 33% (ratio 0·75, 2p=0·0003). In women aged 50-69 years (3965 women, 58% node-positive), the 10-year risks were: recurrence 42% versus 52% (ratio 0·82, 2p<0·00001), breast cancer mortality 36% versus 42% (ratio 0·86, 2p=0·0004), and death from any cause 39% versus 45% (ratio 0·87, 2p=0·0009). Few were aged 70 years or older. Tamoxifen had little effect on recurrence or death in women who were classified in these trials as having ER-poor disease, and did not significantly modify the effects of polychemotherapy. Interpretation: In women who had ER-poor breast cancer, and were either younger than 50 years or between 50 and 69 years, these older adjuvant polychemotherapy regimens were safe (ie, had little effect on mortality from causes other than breast cancer) and produced substantial and definite reductions in the 10-year risks of recurrence and death. Current and future chemotherapy regimens could well yield larger proportional reductions in breast cancer mortality.