Nucleosome assembly and disassembly pathways in vitro.

Structural fluctuations of nucleosomes modulate the access to internal DNA in eukaryotic cells; clearly characterisation of this fundamental process is crucial to understanding gene regulation. Here we apply PhAST (Photochemical Analysis of Structural Transitions) to monitor at a base pair level, structural alterations induced all along the DNA upon histone binding or release. By offering the first reliable, detailed comparison of nucleosome assembly and disassembly in vitro, we reveal similarities and differences between the two processes. We identify multiple, sequential intermediate states characterised by specific PhAST signals whose localisation and amplitude reflect asymmetries of DNA/histone interactions with respect to the nucleosome pseudo dyad. These asymmetries involve not only the DNA extremities but also regions close to the pseudo dyad. Localisations of asymmetries develop in a consistent manner during both assembly and disassembly processes; they primarily reflect the DNA sequence effect on the efficiency of DNA-histone binding. More unexpectedly, the amplitude component of PhAST signals not only evolves as a function of intermediate states but does so differently between assembly and disassembly pathways. Our observation of differences between assembly and disassembly opens up new avenues to define the role of the DNA sequence in processes underlying the regulation of gene expression. Overall, we provide new insights into how the intrinsic properties of DNA are integrated into a holistic mechanism that controls chromatin structure.

Photochemical analysis of structural transitions in DNA liquid crystals reveals differences in spatial structure of DNA molecules organized in liquid crystalline form.

The anisotropic shape of DNA molecules allows them to form lyotropic liquid crystals (LCs) at high concentrations. This liquid crystalline arrangement is also found in vivo (e.g., in bacteriophage capsids, bacteria or human sperm nuclei). However, the role of DNA liquid crystalline organization in living organisms still remains an open question. Here we show that in vitro, the DNA spatial structure is significantly changed in mesophases compared to non-organized DNA molecules. DNA LCs were prepared from pBluescript SK (pBSK) plasmid DNA and investigated by photochemical analysis of structural transitions (PhAST). We reveal significant differences in the probability of UV-induced pyrimidine dimer photoproduct formation at multiple loci on the DNA indicative of changes in major groove architecture.

High-resolution biophysical analysis of the dynamics of nucleosome formation.

We describe a biophysical approach that enables changes in the structure of DNA to be followed during nucleosome formation in in vitro reconstitution with either the canonical "Widom" sequence or a judiciously mutated sequence. The rapid non-perturbing photochemical analysis presented here provides 'snapshots' of the DNA configuration at any given moment in time during nucleosome formation under a very broad range of reaction conditions. Changes in DNA photochemical reactivity upon protein binding are interpreted as being mainly induced by alterations in individual base pair roll angles. The results strengthen the importance of the role of an initial (H3/H4)2 histone tetramer-DNA interaction and highlight the modulation of this early event by the DNA sequence. (H3/H4)2 binding precedes and dictates subsequent H2A/H2B-DNA interactions, which are less affected by the DNA sequence, leading to the final octameric nucleosome. Overall, our results provide a novel, exciting way to investigate those biophysical properties of DNA that constitute a crucial component in nucleosome formation and stabilization.

Development and psychometric properties of the Japanese heart failure knowledge scale.

Knowledge about their own condition is important for patients with heart failure (HF). No valid, reliable, and easily administered instrument is available to measure this knowledge in clinical practice. In this study, a HF knowledge scale was developed, and its psychometric properties were tested. Items related to knowledge about HF were extracted from relevant guidelines. Content validity of the items was confirmed by an expert panel including a cardiologist and nurses specialized in treatment and care of patients with HF. A self-administered questionnaire was then distributed to 187 patients with HF (64.0 ± 12.1 years, males 69%). In 62% patients, a left ventricular ejection fraction of < 50% was identified. Exploratory factor analysis demonstrated the one-dimensionality of the 15-item HF knowledge scale. Mean score was 10.7 ± 3.0 (range, 0-15). Known-group validity testing revealed a significant difference in HF knowledge score between patients newly diagnosed with HF and patients experienced with HF (9.4 ± 3.2 versus 10.8 ± 2.9, P = 0.043). In addition, HF knowledge scale scores were correlated with HF self-care scores assessed by the European Heart Failure Self-Care Behavior Scale for evaluation of criterion validity (ρ = -0.304, P < 0.001). Cronbach's alpha was 0.79, and item-total correlation was 0.22-0.51, thereby suggesting that the reliability of the scale was acceptable. Acceptable validity and reliability were demonstrated for the HF knowledge scale developed in this study. This instrument could be useful in evaluation of patient knowledge about HF.

Influence of membrane curvature on the structure of the membrane-associated pleckstrin homology domain of phospholipase C-delta1.

The effects of geometric properties of membranes on the structure of the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain were investigated using solid state (13)C NMR spectroscopy. Conformations of the PLC-delta1 PH domain at the surfaces of multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and micelles were examined to evaluate the effects of membrane curvature on the PH domain. An increase in curvature of the water-hydrophobic layer interface hinders membrane-penetration of the amphipathic alpha2-helix of the PH domain that assists the membrane-association of the PH domain dominated by the phosphatidylinositol 4,5-bisphosphate (PIP(2)) specific lipid binding site. The solid state (13)C NMR signal of Ala88 located at the alpha2-helix indicates that the conformation of the alpha2-helix at the micelle surface is similar to the solution conformation and significantly different from those at the MLV and SUV surfaces which were characterized by membrane-penetration and re-orientation. The signal of Ala112 which flanks the C-terminus of the beta5/beta6 loop that includes the alpha2-helix, showed downfield displacement with decrease in the interface curvature of the micelles, SUV and MLV. This reveals that the conformation of the C-terminus of the beta5/beta6 loop connecting the beta-sandwich core containing the PIP(2) binding site and the amphipathic alpha2-helix is sensitive to alterations of the curvature of lipid bilayer surface. It is likely that these alterations in the conformation of the PLC-delta1 PH domain contribute to the regulatory mechanisms of the intracellular localization of PLC-delta1 in a manner dependent upon the structure of the molecular complex containing PIP(2).

Aminosilane multilayer formed on a single-crystalline diamond surface with controlled nanoscopic hardness and bioactivity by a wet process.

Diamond could be an excellent support for nanodevices utilizing biomolecules if it is covered with a polymer layer immobilizing a variety of biomolecules. We report a wet method to form a 3-aminopropyltriethoxysilane (APTES) multilayer with a controlled hardness, roughness, and capacity for immobilizing protein. The method is feasible in typical biochemical laboratories where biomolecules are prepared. Atomic force microscopy (AFM) revealed that the surface geometries and nanoscopic hardness of the multilayers on an oxygen-terminated single-crystalline diamond surface depended on the dielectric constant of the solvent; the smaller the constant, the harder the layer. The hard multilayers had holes and APTES aggregates on the surfaces, while less hard ones had homogeneous surfaces with rare holes and little aggregates. The secondary deposition of APTES in a solvent with a large dielectric constant on a hard multilayer removed the holes, and further treatment of the multilayer in acidic ethanol solution diminished the aggregates. Such a surface can immobilize streptavidin with enough specificity against nonspecific adsorption using a combination of polyethylene glycol reagents. The results of a scratching test and nanoindentation test with AFM provided consistent results, suggesting some universality of the scratching test independent of the tip structure of the cantilever. The mechanism of formation of multilayers on the diamond surface and their binding to it is discussed.

[Symptoms of sick house syndrome and contributory factors; study of general dwellings in Hokkaido].

OBJECTIVE: The aim of this study was to clarify the "Sick House Syndrome" which has recently received increasing attention, and to investigate relationships between symptoms and the state of general dwellings in Hokkaido. METHODS: Questionnaires were sent to residents in 1775 dwellings, mainly solitary houses built or remodeled within the past few years by 24 construction companies in Sapporo and its environs, and answers was received from 564. The questionnaires included queries about building structure and characteristics, the residents' habits in the home, and subjective symptoms. We requested one resident who had the most severe symptoms in the dwelling to answer a questionnaire about symptoms. We classified the symptoms into 11 categories, and selected those that developed or were aggravated after the building or remodeling. We defined dwellings in which inhabitants complained of one or more categories of symptoms as the group with sick-house-related disease (developed or aggravated group: DA group), and those in which the inhabitants complained of two or more symptoms as the group with sick house syndrome (more than one organic symptom group: MO group)". Associations between symptoms and dwellings were then studied. RESULTS: There were 201 dwellings for which residents complained of symptoms (37.2%). Of these, 94 were in the DA group (16.7%), and 57 (10.1%) in the MO group. The symptoms that developed or were aggravated after building or remodeling of the dwellings were throat, 7.1%, dermal, 6.9%, psychoneural, 5.3%, eye, 5.1%, and nasal problems, 4.1%. Unpleasant odors form furniture were significant in both groups (DA: crude odds ratio (OR) 2.66, MO: OR 3.24). Use of aromatics was significant in group DA (OR 1.78). Condensation on windows and mold growth in the dwellings were significant in both groups (condensation on windows; DA: OR 2.98, MO: OR 3.32, mold growth; DA: OR 3.11, MO: OR 3.24). In addition, the percentage of dwellings for which residents complained of symptoms increased with signs of dampness (condensation on windows and mold growth). On logistic regression analysis, condensation on windows and mold growth were significant in both groups, and unpleasant odors from furniture in the MO group. CONCLUSION: It is suggested that symptoms of sick house syndrome are associated with high humidity such as condensation on windows and mold growth, odors from furniture and use of aromatics.