A quantitative characterisation of meals and their contexts in a sample of 25 to 49-year-old Spanish people.

To date there have been few large-scale studies empirically examining context factors and the relationships between food choice and meal occasion. This study was conducted online and used 24-h dietary recall to capture the foods eaten by a sample of 25 to 49-year-old Spanish people (n=831), as well as some of the situational and social contexts of those meal occasions. Canonical correspondence analysis of the 3086 recorded eating occasions was used to map food and contexts and reveal associations between food categories and the meal occasions in which they were consumed.

Impact and outcomes of routine microstaging of sentinel lymph nodes in breast cancer: significance of the pN0(i+) and pN1mi categories.

Background: In 2003, the American Joint Committee on Cancer (AJCC) initiated the 6th edition staging criteria, including pN0(i+) and pN1mi categories for breast cancer. However, the clinical significance of these categories is debated in the literature.

Methods: A prospective registry was used to identify patients staged with sentinel lymph node (SLN) biopsy.

Replacement of the axial histidine heme ligand with cysteine in nitrophorin 1: spectroscopic and crystallographic characterization.

To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant.

The real-time monitoring of the thermal unfolding of tetramethylrhodamine-labeled actin.

Modification of actin at Cys (374) with tetramethylrhodamine maleimide (TMR-actin) has been used for visualization of actin filaments and to produce high-resolution crystal structures of actin. We show that TMR-actin exhibits a 21% decrease in absorbance at 557 nm upon thermal unfolding, likely due to the movement of TMR to a more hydrophobic environment upon rapid unfolding and protein aggregation. We took advantage of this property to test models of actin protein unfolding.

Community treatment orders are not a good thing.

Does politicians' enthusiasm for community treatment orders lie primarily in the area of public protection? If so, can such orders reduce homicide rates? Is there adequate evidence of their value, given their adverse effects on individual liberty? This well-researched and provocative debate will enlighten readers on these and many more of the complicated questions surrounding this issue.

Mental capacity and psychiatric admission.

Many patients lack capacity to consent to treatment on admission, but not all qualify for treatment under the Mental Capacity Act

Pyrazolopyridines as a novel structural class of potent and selective PDE4 inhibitors.

Optimisation of a high-throughput screening hit resulted in the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective inhibitors of Phosphodiesterase 4 (PDE4). Herein, we describe early SAR studies around this novel template highlighting preferred substituents and rationalization of SAR through X-ray crystal structures of analogues bound to the PDE4 active site. Pyrazolopyridine 20a was found to be a potent and selective PDE4 inhibitor which also inhibits LPS induced TNF-alpha production from isolated human peripheral blood mononuclear cells and has an encouraging rat PK profile suitable for oral dosing.

EPR and ENDOR studies of cryoreduced compounds II of peroxidases and myoglobin. Proton-coupled electron transfer and protonation status of ferryl hemes.

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, [Fe(IV)=O]2+, but can be the protonated ferryl, [Fe(IV)-OH]3+. We show here that cryoreduction of the EPR-silent Compound II by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O]2+ or [Fe(IV)-OH]3+, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and 1H and 14N ENDOR spectroscopies. To give the broadest view of the properties of Compounds II we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES.

Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence spectroscopy.

Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths, with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket.

Replacement of tyrosine residues by phenylalanine in cytochrome P450cam alters the formation of Cpd II-like species in reactions with artificial oxidants.

Our previous rapid-scanning stopped-flow studies of the reaction of substrate-free cytochrome P450cam with peracids [Spolitak et al. (2005) J Biol Chem 280:20300-20309; (2006) J Inorg Biochem 100:2034-2044] spectrally characterized compound I [ferryl iron plus a porphyrin pi-cation radical (Fe(IV) = O/Por(+))], as well as Cpd ES (Fe(IV) = O/Tyr.).

Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems.

Caldariomyces fumago chloroperoxidase catalyzes the oxidative dehalogenation of chlorophenols by a mechanism involving two one-electron steps.

We have employed rapid scan stopped-flow spectroscopy to examine whether the mechanism of oxidative dehalogenation catalyzed by C. fumago chloroperoxidase (CCPO) involves two consecutive one-electron steps or a single two-electron oxidation. First, we optimized the formation of CCPO compound I (CCPO-I) [Fe(IV)=O/porphyrin radical] and CCPO compound II (CCPO-II) [Fe(IV)=O] for use in double mixing rapid scan stopped-flow experiments.

The ferric-hydroperoxo complex of chloroperoxidase.

The hydroperoxo-ferric complex, or Compound 0 (Cpd 0), is an unstable transient intermediate common for oxygen activating heme enzymes such as the cytochromes P450, nitric oxide synthases, and heme oxygenases, as well as the peroxidases and catalases which utilize hydrogen peroxide as a source of oxygen and reducing equivalents. Detailed understanding of the mechanism of oxygen activation and formation of the higher valent catalytically active intermediates in heme enzyme catalysis requires the structural and spectroscopic characterization of this immediate precursor, Cpd 0. Using the method of cryoradiolytic reduction of the oxy-ferrous heme complex, we have prepared and characterized hydroperoxo-ferric complex in chloroperoxidase (CPO) and compared this to the same intermediate generated in cytochrome P450 CYP101.

Overexpression of cardiac actin with baculovirus is promoter dependent.

The influence of the promoter and an N-terminal hexahistidine tag on human cardiac actin (ACTC) expression and function was investigated using four baculovirus constructs. It was found that both non-tagged ACTC and hisACTC expression from the p10 promoter was higher than from the polh promoter. Characterization showed that an N-terminal hexahistidine tag has a negative effect on ACTC.

Stimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling.

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion.

Horse heart myoglobin catalyzes the H2O2-dependent oxidative dehalogenation of chlorophenols to DNA-binding radicals and quinones.

The heme-containing respiratory protein, myoglobin (Mb), best known for oxygen storage, can exhibit peroxidase-like activity under conditions of oxidative stress. Under such circumstances, the initially formed ferric state can react with H2O2 (or other peroxides) to generate a long-lived ferryl [Fe(IV)=O] Compound II (Cpd II) heme intermediate that is capable of oxidizing a variety of biomolecules. In this study, the ability of Mb Cpd II to catalyze the oxidation of carcinogenic halophenols is demonstrated.

Yeast actin with a subdomain 4 mutation (A204C) exhibits increased pointed-end critical concentration.

Characterizing mutants of actin that do not polymerize will advance our understanding of the mechanism of actin polymerization and will be invaluable for the production of short F-actin structures for structural studies. To circumvent the problem of expressing dominant lethal nonpolymerizing actin in yeast, we adopted a cysteine engineering strategy. Here we report the characterization of a mutant of yeast actin, AC-actin, possessing a single pointed-end mutation, A204C.

A high-throughput assay shows that DNase-I binds actin monomers and polymers with similar affinity.

Previous conflicting reports suggest that DNase-I binds F-actin with either equal or drastically different K(D) values compared to G-actin. We developed a high-throughput DNase-I inhibition assay to determine the K(D) of DNase-I for F-actin. We confirmed that phalloidin-stabilized F-actin is protected from depolymerization by DNase-I and that the critical concentration at the pointed end of phalloidin-F-actin is 45.

Family perspective on community treatment orders: a New Zealand study.

Background: People with serious mental disorders typically live with family members. Despite increasing interest in compulsory community treatment for such patients, the experience and views of their family members have been little studied.

Material: Qualitative interviews with 27 family members, whose relatives have been subject to compulsory community treatment.

N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts.

The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1(+/+) and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles.

Purification and characterization of a nonpolymerizing long-pitch actin dimer.

Atomic resolution structures of filamentous actin have not been obtained owing to the self-association of actin under crystallization conditions. Obtaining short filamentous actin complexes of defined lengths is therefore a highly desirable goal. Here we report the production and isolation of a long-pitch actin dimer employing chemical crosslinking between wild-type actin and Q41C/C374A mutant actin.

Rapid kinetics investigations of peracid oxidation of ferric cytochrome P450cam: nature and possible function of compound ES.

Previously, we reported spectroscopic properties of cytochrome P450cam compound I, (ferryl iron plus a porphyrin pi-cation radical (Fe(IV)=O/Por(+))), as well as compound ES (Fe(IV)=O/Tyr()) in reactions of substrate-free ferric enzyme with m-chloroperbenzoic acid [T. Spolitak, J.H.

Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors.

Rapid mixing of substrate-free ferric cytochrome P450(BM3)-F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450(CAM) and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme.