Crystal Structures of the Catalytic Domain of a Novel Glycohydrolase Family 23 Chitinase from Ralstonia sp. A-471 Reveals a Unique Arrangement of the Catalytic Residues for Inverting Chitin Hydrolysis

Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) has a catalytic domain sequence similar to goose-type (G-type) lysozymes and, unlike other chitinases, belongs to glycohydrolase (GH) family 23. Using NMR spectroscopy, however, Ra-ChiC was found to interact only with the chitin dimer but not with the peptidoglycan fragment. Here we report the crystal structures of wild-type, E141Q, and E162Q of the catalytic domain of Ra-ChiC with or without chitin oligosaccharides. Ra-ChiC has a substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. Mutation analyses based on this structural information indicated that a highly conserved Glu-141 acts as a catalytic acid, and that Asp-226 located at the roof of the tunnel activates a water molecule as a catalytic base. The unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases.     

Pericentric Heterochromatin Generated by HP1 Protein Interaction-defective Histone Methyltransferase Suv39h1

Pericentric regions form epigenetically organized silent heterochromatin structures that accumulate histone H3 lysine 9 trimethylation (H3K9me3) and HP1. At pericentric regions, Suv39h is the major enzyme that generates H3K9me3. Suv39h also interacts directly with HP1, a methylated H3K9-binding protein. However, it is not well characterized how HP1 interaction is important for Suv39h accumulation and Suv39h-mediated H3K9me3 formation at the pericentromere. To address this, we introduced the HP1 binding-defective N-terminally truncated mouse Suv39h1 (ΔN) into Suv39h-deficient embryonic stem cells. Interestingly, pericentric accumulation of ΔN and ΔN-mediated H3K9me3 was observed to recover, but HP1 accumulation was only marginally restored. ΔN also rescued DNA methyltransferase Dnmt3a and -3b accumulation and DNA methylation of the pericentromere. In contrast, other pericentric heterochromatin features, such as ATRX protein association and H4K20me3, were not recovered. Finally, derepressed major satellite repeats were partially silenced by ΔN expression. These findings clearly showed that the Suv39h-HP1 binding is dispensable for pericentric H3K9me3 and DNA methylation, but this interaction and HP1 recruitment/accumulation seem to be crucial for complete formation of heterochromatin.     

Which wavelength does the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae), prefer? Electrophysiological and behavioral studies using light-emitting diodes (LEDs)

The cigarette beetle, Lasioderma serricorne (Fabricius), is an important pest insect that consumes a variety of dry foods. It is known that UV light traps attract this species. However, less attention has been paid to its preferred wavelength. First, we investigated the spectral sensitivity of the compound eye. Next, we compared the attraction efficiency of LEDs of different colors (wavelengths). Our results showed that ultraviolet (UV, 375 nm) and blue (470 nm) LEDs attracted the most cigarette beetles of both sexes, irrespective of mating or oviposition status, although the UV LED consistently tended to attract the most beetles. Although the primary sensitivity peak of the compound eye was 520 nm, the green LED (520 nm) scarcely attracted beetles. Although the reason for the difference between the peaks in spectral sensitivity and attraction of beetles awaits further studies, whether UV and/or blue LEDs is more effective as a practical light trap for controlling L. serricorne beetle is discussed in this study.     

Photosensitized protein damage by dimethoxyphosphorus(V) tetraphenylporphyrin

For the purpose of the basic study of photodynamic therapy, the activity of the water-soluble P(V)porphyrin, dimethoxyP(V)tetraphenylporphyrin chloride (DMP(V)TPP), on photosensitized protein damage was examined. The quantum yield of singlet oxygen generation by DMP(V)TPP (0.64) was comparable with that of typical porphyrin photosensitizers. Absorption spectrum measurement demonstrated the binding interaction between DMP(V)TPP and human serum albumin, a water-soluble protein. Photo-irradiated DMP(V)TPP damaged the amino acid residue of human serum albumin, resulting in the decrease of the fluorescence intensity from the tryptophan residue of human serum albumin. A singlet oxygen quencher, sodium azide, could not completely inhibit the damage of human serum albumin, suggesting that the electron transfer mechanism contributes to protein damage as does singlet oxygen generation. The decrease of the fluorescence lifetime of DMP(V)TPP by human serum albumin supported the electron transfer mechanism. The estimated contribution of the electron transfer mechanism is 0.64. These results suggest that the activity of DMP(V)TPP can be preserved under lower oxygen concentration condition such as tumor.     

Purification and Characterization of Amidase which Participates in Nitrile Degradation

Amidase was purified from the cell-free extract of acetonitrile-grown Arthrobacter sp. J-1 by a procedure involving protamine sulfate precipitation, ammonium sulfate fractionation, and column chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200. The overall purification was 47-fold. The purified enzyme was homogeneous as judged by ultracentrifugal analysis and disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 300,000 and 320,000 by disc gel electrophoresis and gel filtration, respectively. The enzyme was possibly composed of eight identical subunits of a molecular weight of 39,000. The isoelectric point was 3.8. The enzyme catalyzed the stoichiometric hydrolysis of acetamide to form acetic acid and ammonia. The enzyme was active toward acetamide, acrylamide and propionamide and the Km values were 0.97, 23.3 and 8.05 mm, respectively. The enzyme showed acyltransferase activity.     

Optical effects on the surrounding structure during quantitative analysis using indocyanine green videoangiography: A phantom vessel study

Various reports have been published regarding quantitative evaluations of intraoperative fluorescent intensity studies using indocyanine green (ICG) with videoangiography (VAG). The effects of scattering and point‐spread functions (PSF) on quantitative ICG‐VAG evaluations have not been investigated. Clinically, when ICG is administered through the peripheral vein, it reaches the tissue intra‐arterially. To achieve more reliable intraoperative quantitative intensity evaluations, we examined the impact of high‐intensity structures on close areas. The study was conducted using a phantom model and surgical fluorescent microscope. A region of interest (ROI) was created for the vessel model and another ROI was created within 3 cm of that. With an ROI of 6.8 mm in the vessel phantom model, 10% intensity was confirmed, even though there was no fluorescent structure. Intensity decreased gradually as the ROI moved further from the vessel model. Our study results suggest that the presence of a high‐intensity structure and the size of the ROI may affect quantitative intensity evaluations using ICG‐VAG. Results of linear regression analysis indicate that the relationship of intensity (Y) and distance (X) is as follows: Y(real/A) = 29 Exp(?0.062X) + 164.3 Exp(?1.81X). The optical effect should be considered when performing an intraoperative intensity study with a surgical microscope.      

Identifying critical interactions in complex competition dynamics between bean beetles

Identifying behavioural basis of competitive relationship is essential to understand outcome of interspecific competition. However, it remains difficult to investigate demographic effect of competitive behaviour, because various kinds of behaviours may co‐occur in the competition and make the dynamics far complicated in nonlinear ways. We report that the behavioural basis of interspecific interaction can be identified, by focusing on the timescale difference from the occurrence of each behaviour to the appearance of its demographic effect. Between two bean beetles, Callosobruchus chinensis and C. maculatus, major interspecific interactions are resource competition (RC) at the larval stage and reproductive interference (RI) at the adult stage. RC has longer time lag than RI, because effect of RC appears in the adult number of the next generation through larval competition while effect of RI appears instantaneously in the adult number through early death of females. If we detect two effects with different time lags from the competition dynamics, an effect with intergenerational time lag and with no time lag would be considered as RC and RI, respectively. We applied empirical dynamic modelling approach, which is a nonlinear time series analysis for detecting causal interactions and the strength, to two published datasets of experimental competition between those beetles. Results showed the significant causality from the winner species to the loser one in both experiments, but the causality time lag differed between experiments: the causality had no time lag in the C. chinensis‐win data, while intergenerational time lag in C. maculatus‐win data. Furthermore, detection of the causality with intergenerational time lag from C. maculatus to C. chinensis in both experiments suggests interplay of constant RC and variable RI which can reverse the outcome. This study is the first successful case study that links behavioural‐level interactions to demographic‐level effects in interspecific competition.     

Inhibition of amyloid fibril formation in the variable domain of λ6 light chain mutant Wil caused by the interaction between its unfolded state and epigallocatechin-3-O-gallate

Background

Light chains are abnormally overexpressed from disordered monoclonal B-cells and form amyloid fibrils, which are then deposited on the affected organ, leading to a form of systemic amyloidosis known as AL (Amyloid Light chain) amyloidosis. A green tea catechin, epigallocatechin-3-O-gallate (EGCG), which is thought to inhibit various amyloidoses, is a potent inhibitor of amyloid fibril formation in AL amyloidosis.