The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA

In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys⁹⁷–Lys²⁷⁴), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by ∼25 bp. The solution structure determined by NMR revealed that the globular domain (Met¹⁵³–Thr²³⁷) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.     

Synthesis and biological activities of NB-506 analogues modified at the glucose group

A new indolocarbazole compound, NB-506 (1), modified at the glucose group yielded a beta-D-glucopyranoside, J-107,088 (2), which showed potent anticancer activity. A beta-D-ribofuranoside, J-109,534 (3), was found to be 6 times more potent than J-107,088 at inhibiting topoisomerase I.     

Depression of mitochondrial metabolism by downregulation of cytoplasmic deacetylase, HDAC6

Mitochondria perform multiple functions critical to the maintenance of cellular homeostasis. Here we report that the downregulation of histone deacetylase 6 (HDAC6) causes a reduction in the net activity of mitochondrial enzymes, including respiratory complex II and citrate synthase. HDAC6 deacetylase and ubiquitin-binding activities were both required for recovery of reduced mitochondrial metabolic activity due to the loss of HDAC6. Hsp90, a substrate of HDAC6, localizes to mitochondria and partly mediates the regulation of mitochondrial metabolic activity by HDAC6. Our finding suggests that HDAC6 regulates mitochondrial metabolism and might serve as a cellular homeostasis surveillance factor.     

Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.     

Multiplex PCRs for assignment of Staphylocoagulase types and subtypes of type VI Staphylocoagulase

Staphylocoagulases (SCs) have been classified by the differences in antigenicity using a serological method. We have developed a system to classify them based on the nucleotide differences in SC genes (coa). The system was composed of three multiplex PCRs (M-PCRs): M-PCR:A, identifying types III, IV, VII, and VIII; M-PCR:B, identifying types I, II, V, and VI; M-PCR:C, identifying three subtypes of type VI. In this study, we found that coa genes of the serotype VI were not identical, but classified into three subtypes based on the nucleotide differences, especially in D2 and the central region: VIa, the coa gene carried by stp12 from human; and VIb and VIc, the coa genes carried by strains IFH556 and IFH514 isolated from bovine raw milk. The primer pair used in M-PCR:B was designed to identify all three subtypes of type VI coa. The results showed that coa types of 154 out of 155 Staphylococcus aureus strains from various origins assigned by M-PCR:A and B were identical to those obtained by serological methods, leaving a serotype IV strain unclassifiable. All 73 type VI strains were classified into one of three subtypes by M-PCR:C. Furthermore, we found that type VIa and VIb strains carried characteristic pyrogenic toxin superantigen genes, while no toxin genes were identified in type VIc strains, suggesting the correlation between the subtype of type VI coa gene and the carriage of genomic islands. Our results showed that these M-PCRs are convenient methods for SC typing that might be useful for epidemiological studies.     

Winter lipid depletion of juvenile walleye pollock Theragra chalcogramma in the Doto area, northern Japan

Seasonal variation in body size and nutritional condition of juvenile walleye pollock Theragra chalcogramma was examined to elucidate the mechanism underlying their first-winter survival on the continental shelf of the Doto area, northern Japan, based on monthly samples collected over 2 years. Stored lipid mass was highest during autumn, but 93% (2004) and 80% (2005) of lipids were exhausted by the onset of winter. Lipid levels in the winter of 2004 remained low (7–14% of the autumnal maximum), and there was reduced growth rate until the spring, whereas in 2005 lipid levels were higher and more variable (10–46% of the maximum) and some growth occurred. An analysis of the allometric relationships between body size and stored energy showed that larger individuals accumulated disproportionately more energy in the autumn, but the advantage disappeared prior to the winter. In January 2004, stored lipid energy was low throughout the Doto continental shelf relative to the continental slope area. These results suggest that winter feeding opportunities on the shelf are severely limited but not completely absent. Previous studies have shown that winter temperatures on the shelf are lower than those in the slope area. It is possible that juvenile T. chalcogramma survive winter on the shelf without a high level of pre-winter lipid storage because the occasional feeding in the cold shelf water benefits energy conservation.     

Solution conformation of conotoxin GI determined by 1H nuclear magnetic resonance spectroscopy and distance geometry calculations

Conformational analysis of conotoxin GI, one of the neurotoxic peptides produced by a marine snail, genus Conus, was performed by a combination of nuclear magnetic resonance spectroscopy (NMR) and distance geometry calculations. The resulting conformers on minimization of the target function were classified into two groups. The difference in the structures of the conformers is mainly due to the difference in the orientation of the side chain of the tyrosyl residue. The results show that the solution structure of conotoxin GI satisfies the conformational requirements for the biological activity of an antagonist toward nicotinic cholinergic receptors elucidated in a series of studies on alkaloids. The structure is discussed on the basis of the results of comparison of the atomic arrangements of the active sites of snake venom peptides and molecular models based on the results of secondary structure prediction.     

Cricket mate selection as a spatial discounting phenomenon without learning

Many animals prefer small local benefits to large but temporally or spatially distant benefits, and this preference is termed “temporal discounting” or “spatial discounting.” Although temporal discounting has been studied in many taxa, only a limited number of papers have primarily focused on spatial discounting. Here, a mate-choice experiment was conducted using female house crickets (Acheta domesticus) tasked with selecting between a non-attractive local male and an attractive distant male; training procedures were not performed. The results showed that females that had never heard the calling songs of males exhibited spatial discounting behavior. This finding may be key to determining the fundamental differences between temporal and spatial discounting.