Search

Acylpeptidyl-oligopeptidase (AOP), which has been recently identified as a novel enzyme in a periodontopathic bacterium, Porphyromonas gingivalis, removes di- and tri-peptides from N-terminally acylated polypeptides, with a preference for hydrophobic P1-position amino acid residues. To validate enzymatic properties of AOP, characteristics of two bacterial orthologues from Bacteroides dorei (BdAOP), a Gramnegative intestinal rod, and Lysinibacillus sphaericus (LsAOP), a Gram-positive soil rod, were determined. Like P. gingivalis AOP (PgAOP), two orthologues more efficiently hydrolyzed N-terminal acylated peptidyl substrates than non-acylated ones. Optimal pH was shifted from 7.0 ? 8.9 for N-acylated to 8.5 ? 9.5 for non-acylated substrates, indicating preference for non-charged hydrophobic N-terminal residues. Hydrophobic P1- and P2-position preferences were common in the three AOPs, although PgAOP preferred Leu and the others preferred Phe at the P1 position. In vitro mutagenesis demonstrated that Phe647 in PgAOP was responsible for the P1 Leu preference. In addition, bacterial AOPs commonly liberated acetyl-Ser1-Tyr2 from amelanocyte- stimulating hormone. Taken together, although these three bacterial AOPs exhibited some variations in biochemical properties, the present study demonstrated the existence of a group of exopeptidases that preferentially liberates mainly dipeptides from N-terminally acylated polypeptides with a preference for hydrophobic P1 and P2- position residues., Biochimie, 163, pp.50-27; 2019

Bacterial dipeptidyl-peptidase (DPP) 7 liberates a dipeptide with a preference for aliphatic and aromatic penultimate residues from the N-terminus. Although synthetic substrates are useful for activity measurements, those currently used are problematic, because they are more efficiently degraded by DPP5. We here aimed to develop a potent and specific substrate and found that the kcat/Km value for Phe-Met-methylcoumaryl-7-amide (MCA) (41.40?±?0.83?μM?1?s?1) was highest compared to Met-Leu-, Leu-Leu-, and Phe-Leu-MCA (1.06?3.77?μM?1?s?1). Its hydrolyzing activity was abrogated in a Porphyromonas gingivalis dpp7-knockout strain. Conclusively, we propose Phe-Met-MCA as an ideal synthetic substrate for DPP7., Analytical Biochemistry, 548, pp.78-81; 2018

Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly673 in DPP7 and Arg673 in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly673 exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with Arg673 was confirmed to encode DPP11. These results demonstrated that classification of S46 peptidase is enforceable by the S1 essential residues. Bacteroides DPP11 showed a decreased level of activity towards the substrates, especially with P1-position Glu. Findings of 3D structural modeling indicated three potential amino acid substitutions responsible for the reduction, one of which, Asn650Thr substitution, actually recovered the hydrolyzing activity of Leu-Glu-MCA. On the other hand, the gene currently annotated as DPP7 carrying Gly673 from B. fragilis (BF9343_0130) and Bacteroides ovatus (Bovatus_03382) did not hydrolyze any of the examined substrates. The existence of a phylogenic branch of these putative Bacteroides DPP7 genes classified by the C-terminal conserved region (Ser571-Leu700) strongly suggests that Bacteroides species expresses a DPP with an unknown property. In conclusion, the genus Bacteroides exceptionally expresses three S46-family members; authentic DPP7, a new subtype of DPP11 with substantially reduced specificity for Glu, and a third group of S46 family members., Biochimie, 147, pp.25-35; 2018

Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is one of the major causative organisms of chronic periodontitis. The bacterium utilizes amino acids as energy and carbon sources, and incorporates them mainly as dipeptides. Therefore, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. A DPP7-like activity still remained in a dpp4-7-11 disrupted P. gingivalis ATCC 33277. PGN_0756, currently annotated as a prolyl oligopeptidase, possessed an activity indistinguishable from that of the triple dpp gene-disrupted strain, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with an apparent molecular weight of 66 kDa, while it preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated kcat/Km values for Gly-Phe-MCA and Lys-Ala-MCA of 13.01 and 11.02 μM-1 s-1, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of dpp- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also distributed in prokaryotes., Journal of Biological Chemistry, 289(9), pp.5436-5448; 2014

Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with kcat/Km of 10.62 ± 2.51 μM−1 s−1, while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2- as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1- and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing kcat and lowering Km values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2- and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria., Highlights • Dipeptidyl peptidases are crucial for energy metabolism in Porphyromonas gingivalis. • P. gingivalis DPP7 prefers a hydrophobic amino acid residue at P2 position. • kcat/Km of DPP7 for Met-Leu-MCA is 10.62 ± 2.51 μM−1 s−1. • A hydrophobic P1 residue enhances kcat and a hydrophobic P2 residue lowers Km. • Phe664 of DPP7 and Phe671 of DPP11 primarily afford hydrophobic P2 preference.

The glutamyl endopeptidase family of enzymes from staphylococci has been shown to be important virulence determinants of pathogenic family members, such as Staphylococcus aureus. Previous studies have identified the N-terminus and residues from positions 185-195 as potentially important regions that determine the activity of three members of the family. Cloning and sequencing of the new family members from Staphylococcus caprae (GluScpr) and Staphylococcus cohnii (GluScoh) revealed that the N-terminal Val residue is maintained in all family members. Mutants of the GluV8 enzyme from S. aureus with altered N-terminal residues, including amino acids with similar properties, were inactive, indicating that the Val residue is specifically required at the N-terminus of this enzyme family in order for them to function correctly. Recombinant GluScpr was found to have peptidase activity intermediate between GluV8 and GluSE from Staphylococcus epidermis and to be somewhat less specific in its substrate requirements than other family members. The 185-195 region was found to contribute to the activity of GluScpr, although other regions of the enzyme must also play a role in defining the activity. Our results strongly indicate the importance of the N-terminal and the 185-195 region in the activity of the glutamyl endopeptidases of staphylococci., Biological chemistry, 391(10), pp.1221-1232; 2010

V8 protease (GluV8), a member of the glutamyl endopeptidase I family isolated from the V8 strain of Staphylococcus aureus, is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the autoproteolysis?the use of the prosequence of its homologue (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of four substitutions in the prosequence of GluV8. In the current study, we refined this technique through five amino acid substitutions within the prosequence of GluV8 for complete suppression of the autodegradation. As a result, the recovery of GluV8 proform was enhanced to 20 fg/cell, which was comparable to the level of a constitutive inactive form of GluV8, indicating complete suppression of the autoproteolysis. This mutated propeptide was also effective for the expression of the mature sequence of the glutamyl endopeptidase from Staphylococcus warneri. The recombinant proteins were successfully converted to their active forms through a common cleavage mechanism mediated by thermolysin in vitro. This strategy may shed light on the way for the expression of the proteases that have been scarcely produced in E. coli to date., Analytical Biochemistry, 381(1), pp.74-80; 2008

V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C-terminal His(6)-tagged proGluSE was successfully expressed from the full-length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C-terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.

The 70-kDa heat shock protein (Hsp70) is predominantly present intracellularly as a monomer, but a small population is converted to dimers and oligomers under certain conditions. In the present study, we investigated the dimeric structure of human inducible Hsp70. As reported earlier, the C-terminal client-binding domain (amino acids 382-641) was required for the dimerization. A 40-amino acid deletion in the client-binding domain from either the N-terminus or C-terminus greatly enhanced the dimerization potential of Hsp70. Limited proteolysis indicated that the dimer formed through truncation from the C-terminus had a conformation similar to that of the non-truncated form. Truncation experiments demonstrated that the client-binding sub-domain (amino acids 382-520) with its adjacent region up to amino acid 541 was not sufficient for the dimerization but that the region up to amino acid 561 was sufficient. Interestingly, the dimer formed through truncation from the C-terminus acquired a homomeric disulfide bridge at Cys574., Journal of biochemistry. 139(4), pp.677-687; 2006

Old hen tendon provides a model suitable for the study of calcification in an extracellular matrix. In the present study, we observed the mineralizing substances of hen tendon by scanning electron microscopy of plasma-osmium-coated specimens and by transmission electron microscopy of those processed by a plasma-polymerization film replica method. The mineralizing front area revealed a number of elliptical particles fused to each other and forming rod-like structures oriented parallel to collagen fibrils. The area of advanced mineralization possessed non-mineralizing cavities, in which tendon cells were likely to exist. At this site, we recognized a second form of mineral structure, one in which the crystals had a scale-like morphology and were deposited onto the major first-form mineral component. This crystal form was similar to hydroxyapatite synthesized under wet reaction conditions. These findings strongly suggest that the second form of mineral formed independent of collagen fibrils existed together with the predominant, collagen-dependent form of mineral. We speculate that cell membranes and an extremely slow mineralization process may contribute to the formation of this form of mineral during the mineralization process in the hen tendon.

Magnetization measurements under hydrostatic pressure were performed on S = 1/2 coupled dimer systems TlCuCl 3 and KCuCl 3 under magnetic field H parallel to the [2,0,1] direction. With increasing applied pressure P, the gap in TlCuCl 3 decreases and closes completely at P c = 0.42 ± 0.05 kbar. TlCuCl 3 undergoes pressure-induced quantum phase transition. The gap and Neel temperature are presented as functions of pressure. KCuCl 3 also undergoes pressure-induced quantum phase transition at P c ∼ 8.8 kbar. Pressure dependence of intra- and interdimer interactions in KCuCl 3 are presented.

The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain. An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins.

We have measured the magnetization of the organic compound BIP-BNO (3,5'-bis(N-tert-butylaminoxyl)-3',5-dibromobiphenyl) up to 76 T where the magnetization is saturated. The S = 1/2 antiferromagnetic Heisenberg two-leg spin-ladder model accounts for the obtained experimental data regarding the magnetization curve, which is clarified using the quantum Monte Carlo method. The exchange constants on the rung and the side rail of the ladder are estimated to be J(rung)/kB = 65.7 K and J(leg)/kB = 14.1 K, respectively, deeply in the strong coupling region: J(rung)/J(leg) > 1., 8 pages, 2 figures

1In the present study, we investigated the domain structure and domain–domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90. Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e. Arg7-Gly8, Arg336-Glu337 and Lys552-Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90α. A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions. Thus, HtpG consists of three domains, i.e. Domain A, Met1–Arg336; domain B, Glu337–Lys552; and domain C, Leu553-Ser624, as does HSP90. The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions. As a result, two kinds of domain–domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer. Domain B could be structurally and functionally divided into two subdomains, the N-terminal two-thirds (subdomain BI) that interacted with domain A and the C-terminal one-third (subdomain BII) that interacted with domain C. The C-terminal two-thirds of domain A, i.e. Asp116–Arg336, were sufficient for the binding to domain B. We finally propose the domain organization of an HtpG dimer.

When a rat tail tendon swollen after incubation in guanidine hydrochloride was soaked in an artificial calcification solution, its calcification was promoted. X-ray small angle scattering of the tendon suggested the formation of cracks in the tendon collagen. The cracks may provide the location and sites for nucleations of calcium phosphate crystals. Decorin, a powerful inhibitor of calcification, is eluted out by the guanidine hydrochloride treatment.

生後1年以上のニワトリの足の腱には, 石灰化物の沈着がみられる。さらに, 電子顕微鏡観察によれば, すでに生後100日以上の個体においてその徴候が認められる。本研究では, ニワトリ足腱の異所性石灰化部位の無機成分と有機マトリックス成分をプラズマ重合膜法によりオスミウムコートし, その走査電子顕微鏡観察を行った。その結果, コラーゲンマトリックスと石灰化物のそれぞれに, 新規の構造物を見いだした。第1に, 有機成分を溶解した後には長径が1.86±0.38μm (平均±標準偏差), 短径が1.05±0.16μm (平均±標準偏差) の楕円形の石灰化顆粒がみられ, それらはさらに融合して多数の細長いロッド状の構造を形成していた。さらに透過電顕観察では, 石灰化顆粒の表面には特徴的な67nmの周期的な縞模様が検出され, 本構造が生体内ではコラーゲン線維と接して存在していることが明らかとなった。一方, 脱灰処理後の腱のコラーゲン線維には, 2.8±0.38μmの周期的な波状構造が観察された。しかも, その一端のみが波状を呈する領域が観察され, その場合, 波状部分は必ず石灰化側に, 非波状部分は非石灰化側に位置していた。これらの結果は, 波状のコラーゲン線維構造が顆粒状の石灰化物生成を誘導している可能性を示唆している。

Elastic neutron scattering experiments were carried out to investigate the magnetic structure and the critical behaviour of the mixed triangular antiferromagnetic system CsMn(Br0.19I0.81)3, for which our previous magnetic measurement revealed the macroscopic anisotropy to be negligible. It is found that in the ordered state below TN = 8.29 K, the spin planes in which the spins lie with the 120° structure are tilted from the basal plane, and that the average of the tilting angle is evaluated as cos2 = 0.362, i.e. = 53°. This result suggests that the present system is almost at the Heisenberg limit for the interaction. The critical exponent for the sublattice magnetization is obtained as = 0.28±0.02, which is in agreement with the value predicted for the chiral Heisenberg universality class.

We identified and characterized a novel rat vitamin D receptor isoform (rVDR1), which retains intron 8 of the canonical VDR (rVDR0) during alternative splicing. In this isoform protein directed by the stop codon in this newly identified exon, a part of the ligand binding domain (86 amino acids) is truncated at the C-terminal end but contains 19 extra amino acids. The rVDR1 transcript was expressed at a level 1/15 to 1/20 of that of rVDR0 in the kidney and intestine in adult rats but not in embryos. The recombinant rVDR1 protein showed no ligand binding activity. Homo- and heterodimers of the recombinant rVDR0 and rVDR1 proteins bound to a consensus vitamin D response element (VDRE) but not to consensus response elements for thyroid hormone and retinoic acid. However, unlike rVDR0, rVDR1 did not form a heterodimeric complex with RXR on the VDRE. A transient expression assay showed that this isoform acted as a dominant negative receptor against rVDR0 transactivation. Interestingly, the dominant negative activities of rVDR1 differed among VDREs. Thus, the present study indicates that this new VDR isoform negatively modulates the vitamin D signaling pathway, through a particular set of target genes.

To investigate if K-ras point mutations could occur at an early stage of urethane-induced lung tumorigenesis in mice, we applied a sensitive mutation specific PCR assay technique (MSPA) to DNA from lung at the early stage of this condition. At 7 or 14 d after urethane injection (750 mg/kg, i. p.), pulmonary DNA was isolated and the existence of activated K-ras gene with 61st codon A→T mutation was checked according to the MSPA. This technique can detect mutant alleles in the presence of a very large excess of normal ras alleles ; however, the K-ras point mutation could not be detected by the present MSPA. These results suggest that the K-ras point mutations did not occur at the stage of lung tumorigenesis in mice or that the number of mutant alleles was below the detectable range of the technique.

Porphyromonas gingivalis, an asaccharolytic gram-negative rod-shaped bacterium, expresses the novel Asp/Glu-specific dipeptidyl-peptidase (DPP) 11 (Ohara-Nemoto, Y. et al. (2011) J. Biol. Chem. 286, 38115–38127), which has been categorized as a member of the S46/DPP7 family that is preferential for hydrophobic residues at the P1 position. From that finding, 129 gene products constituting five clusters from the phylum Bacteroidetes have been newly annotated to either DPP7 or DPP11, whereas the remaining 135 members, mainly from the largest phylum Proteobacteria, have yet to be assigned. In this study, the substrate specificities of the five clusters and an unassigned group were determined with recombinant DPPs from typical species, i.e., P. gingivalis, Capnocytophaga gingivalis, Flavobacterium psychrophilum, Bacteroides fragilis, Bacteroides vulgatus, and Shewanella putrefaciens. Consequently, clusters 1, 3, and 5 were found to be DPP7 with rather broad substrate specificity, and clusters 2 and 4 were DPP11. An unassigned S. putrefaciens DPP carrying Ser673 exhibited Asp/Glu-specificity more preferable to Glu, in contrast to the Asp preference of DPP11 with Arg673 from Bacteroidetes species. Mutagenesis experiments revealed that Arg673/Ser673 were indispensable for the Asp/Glu-specificity of DPP11, and that the broad specificity of DPP7 was mediated by Gly673. Taken together with the distribution of the two genes, all 264 members of the S46 family could be attributed to either DPP7 or DPP11 by an amino acid at position 673. A more compelling phylogenic tree based on the conserved C-terminal region suggested two gene duplication events in the phylum Bacteroidetes, one causing the development of DPP7 and DPP11 with altered substrate specificities, and the other producing an additional DPP7 in the genus Bacteroides., Biochimie, 95(4), pp.824-832; 2013

Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di- and tripeptides from the environment as carbon and energy sources. In the present study, we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090, and was present as a 75-kDa form with an N-terminus of Asp22. A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, which has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu, and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0 and the kcat/Km value was higher for Asp than Glu. Those activities were lost by substitution of Ser652 in P. endodontalis and Ser655 in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg670 is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whilst this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg670 interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these gram-negative anaerobic rods., The Journal of Biological Chemistry, 286(44), pp.38115-38127; 2011

Natural mineral Dioptase (Cu6Si6O18 ⋅ 6H2O) is an S = 1/2 antiferromagnet with a unique spin network which consists of helical chains along the c axis connected by inter chain exchange interactions. In order to study the ground state of dioptase, we have performed the magnetization measurement for B//c (easy axis) at 1.5 K and have found the spin-flop transition at 13 T. Moreover, the pressure effect of the magnetic susceptibility is investigated up to 1.78 GPa using the SQUID magnetometer where TN increased while the broad maximum reflecting the low dimensionality of the system decreased by pressure. The results will be discussed in connection with our previous antiferromagnetic resonance (AFMR) measurement results and the phase diagram obtained by Gros et al.

Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE and GluSW, respectively. The order of their protease activities was GluSE, Biological chemistry, 390(3), pp.277-285; 2009

Single-phase bulk samples of the "exotic" CoO2, the x = 0 phase of the AxCoO2 systems (A = Li, Na), were successfully synthesized through electrochemical de-intercalation of Li from pristine LiCoO2 samples. The samples of pure CoO2 were found to be essentially oxygen stoichiometric and possess a hexagonal structure consisting of stacked triangular-lattice CoO2 layers only. The magnetism of CoO2 is featured with a temperature-independent susceptibility of the magnitude of 10-3 emu/mol Oe, being essentially identical to that of a Li-doped phase, Li0.12CoO2. It is most likely that the CoO2 phase is a Pauli-paramagnetic metal with itinerant electrons., Comment: 12 pages, 3 figures

We report here the synthesis of single-phase bulk samples of CoO2, the x = 0 end member of the AxCoO2 systems (A = Li, Na), from a pristine LiCoO2 sample using an electrochemical technique to completely de-intercalate lithium. Thus, synthesized CoO2 samples were found to be oxygen-stoichiometric and possess a crystal structure consisting of stacked triangular-lattice CoO2 layers only. The magnetic susceptibility of the CoO2 sample was revealed to be relatively large in its initial value and then level off as the temperature increases, suggesting that CoO2 is a Pauli-paramagnetic metal with itinerant electrons.

Magnetization measurements under hydrostatic pressure were performed on the S = 1/2 coupled dimer systems TlCuCl3 and KCuCl3. With increasing applied pressure P, the gap in TlCuCl3 decreases and closes completely at Pc = 0.42 ± 0.05 kbar. TlCuCl3 undergoes a pressure‐induced quantum phase transition. The gap and Neel temperature are presented as function of pressure. KCuCl3 also undergoes a pressure‐induced quantum phase transition at Pc = 8.2 ± 0.2 kbar. The magnetic structure of KCuCl3 under high pressure P ⩾ Pc is determined by neutron scattering experiment.

We here investigated the mechanism of self-oligomerization of the 90-kDa heat shock protein (HSP90) molecular chaperone, because it is known that this oligomerization reflects the client-binding activity. The transition temperatures for the self-oligomerization of the full-length forms of human HSP90alpha and HtpG (bacterial HSP90), i.e., 45 and 60 degrees C, respectively, were identical to those for the dissociation of the recombinant N domain (residues 1-400 of human HSP90alpha and residues 1-336 of HtpG in our definition) from the remainder of the molecule. The N domain of human HSP90alpha expressed in Escherichia coli was oligomeric, and the oligomerization activity was localized within residues 311-350, i.e., C-terminally adjacent to the highly immunogenic site (residues 291-304). Particularly, residues 341-350 were critical on oligomerization. On the other hand, residues 289-389 were indispensable for the interaction with the M domain (residues 401-618) of the molecule. Oligomer formation of the N domain was efficiently suppressed by its extension until Lys546, i.e., residues 401-546, which is required for the interaction with the N domain. Among highly conserved amino acids at residues 289-400, Trp297, Pro379, and Phe384 were essential for the interaction with the M domain. With these observations taken together, we propose as the activation mechanism of HSP90 molecular chaperone that heat stress induces the liberation of the oligomerization/client-binding site of residues 311-350 by disrupting the intramolecular interaction between residues 289-389 and 401-546.

A coordination polymer with a two-dimensional square planar lattice network, bis(glycolato)copper(II), shows a large structural phase transition at around 220 K and ferromagnetic ordering at 1.1 K.

Glutamyl endopeptidase from Enterococcus faecalis, designated SprE, is one of the important virulence factors secreted as zymogen. In the present study we expressed recombinant SprE proenzyme (pro-SprE) in Escherichia coli and investigated the in vitro processing to mature SprE. It was found that trypsin could efficiently produce the active form of SprE with the N-terminus Ser1 through cleavage between Arg-1 and Ser1 bond, which was subsequently auto-degraded into inactive species through the cleavage at the Glu6-Asp7 and Glu11-Val12 bonds. Although thermolysin could produce SprE with the N-terminus Leu2, but possessed no proteolytic activity. In contrast to the absolute requirement of the N-terminal Val1 in staphylococcal glutamyl endopeptidases, the N-terminal Ser1 of mature SprE could be substituted by other amino acids despite that Ser showed the maximal activity. Substitution of penultimate Leu2 of SprE to Val2 also reduced the activity to 40% of the wild type. Taken together, we conclude that pro-SprE was converted to mature form with the N-terminus Ser1 by a protease with specificity of trypsin and the length of the N-terminal region rather than specific residue is absolutely required for enzyme activity.

TlCuCl3 is a strongly coupled spin dimer system with the excitation gap Δ/k B =7.7 K . It was found that the gap decreases rapidly at pressures around P=0.2 GPa and the magnetic ordering arises for P>0.4 GPa . The phase diagram for the magnetic field parallel to the [2 0 1] direction obtained at P=0.74 GPa is presented.

An S = 1/2 organic radical F5PNN forms a uniform chain structure at room temperature and the magnetic properties at low temperature is described by the Heisenberg antiferromagnetic alternating chain model with 2J/kB = −5.6 K and α = 0.4. The structural change occurring below 5 K is studied by magnetic measurements. The structural change occurs at different temperatures depending on the condition of the crystal growth. The behaviour is discussed from the aspect of the homogeneity of crystals. The deuterated sample F5PNN-d12 was also synthesized and the magnetic properties are described. The exchange coupling and alternating ratio is estimated to 2J/kB = −4.9 K and α = 0.65.

We have investigated the antiferromagnetic resonance modes of the kagome antiferromagnet Cs2Cu3SnF12 both theoretically and experimentally. High-field electron spin resonance experiments on single crystals were conducted in the ordered phase at different frequencies and temperatures with the external magnetic field parallel to the c axis. Considering three sublattices, we calculated the resonance modes for the motions of the sublattice magnetizations within the framework of the mean field approximation. It was found that the frequency-field diagram is in good agreement with the experimental results.

We report here the synthesis of single-phase bulk samples of CoO2, the x = 0 end member of the AxCoO2 systems (A = Li, Na), from a pristine LiCoO2 sample using an electrochemical technique to completely de-intercalate lithium. Thus, synthesized CoO2 samples were found to be oxygen-stoichiometric and possess a crystal structure consisting of stacked triangular-lattice CoO2 layers only. The magnetic susceptibility of the CoO2 sample was revealed to be relatively large in its initial value and then level off as the temperature increases, suggesting that CoO2 is a Pauli-paramagnetic metal with itinerant electrons., Comment: 18 pages, 5 figures

The mouse vitamin D receptor (VDR)-encoding cDNA was cloned and the coding regions were sequenced. Comparison of the amino-acid sequence to that of humans and rats revealed high homology in the DNA- and ligand-binding domains. Divergence appeared in the internal region between the two domains.

Veillonellophage N2 adsorbed to polysaccharides (PSs) on Veillonella rodentium ATCC 17743 cell wall, and the bacteriophage receptor contained only glucosamine. D(+)-glucosamine hydrochloride (Sigma) also adsorbed the veillonellophage N2. These results therefore indicate that the receptor to the veillonellophage N2 is cell wall PSs. The PSs of the host cells as receptor have been characterized. Glucosamine accounted for approximately 100% of the weight of the PSs. The PSs which were partially resolved by Sephadex G-75 chromatography comprised approximately four glucosamine units. Their primary structure was determined by 400 MHz n.m.r. spectroscopy. One- and two-dimensional 1H-nmr experiments showed the PS to be a branched polymer. Glucosamine linkage was detected in one of the branches.

A protein fraction strongly adsorbed to hydroxyapatite crystals was isolated from Lingula unguis shell. It is an acidic protein complex with a molecular mass of 140 kDa, and abundant in asparatic acid, glutamic acid and glycine. Amino acid analysis indicated an unidentified ninhydrin positive acidic component, but not γ-carboxyglutamic acid. The amino acid composition is similar to that of osteonectin and bone morphogenetic protein. The protein having an affinity for calcium contained somewhat large fractions of an α-helical structure and a β-structure. The conformation did not change by increasing calcium concentration. The protein fraction was a multi-components complex build up by hydrophobic and disulfied bonds.

Bone powder was decalcified and extracted with 0.5 M EDTA and then with McIlvaine buffer at pH 7.4. From the EDTA extract, purified by gel filtration chromatography, three hydroxyproline-containing fractions were obtained which were considered to be degradation products of collagen because of their collagen-like amino-acid composition. The McIlvaine buffer extract was fractionated by ammonium sulfate. From the 0.3 saturation fraction, after reconstitution with ATP, a fragment 700 A in length was observed with the electron microscope. Comparison with segment-long-spacing collagen suggested that this fragment was a partially-degraded product of collagen and consisted of approximately one quarter of the collagen molecule from the terminal-amino end.

多くの金属イオンは硝酸水溶液よりジエチルアミノエチルセルロース(DEAEと略記)に事実上吸着を示さない.この事情はメタノール-硝酸混合溶媒系でも同様であるが,トリウムのみ強い吸着を示すので,カラム法により本元素を選択的に分離することができる.分配係数はメタノール-酸体積比,トリウム負荷量により変わるが,酸濃度の影響は少ない.同一混合溶媒系よりDowex 1に強く吸着するバリウム,ビスマス(III),鉛(II),希土類元素はDEAEには吸着しない.Dowex 1-硝酸(6N)系もトリウム分離系としてすぐれているが,本系はこれに比べて硝酸濃度の許容範囲が広く,またビスマス(III),銅(II),鉛(II)の分離がいっそう容易である.メタノール-硝酸系(20:1,v/v)でDEAE1～2gを含む小カラムを用い,トリウムを30元素より分離した結果を示した.

The presence of pyridinoline in collagen molecule was evidence fluorometrically. From pepsin-solubilized collagen of bovine femur bone, a native type-and SLS fibers were reconstituted, washed, and resolubilized. The solutions, which contain only collagen molecules, emitted the pyridinoline-specific fluorescence.

Various mechanical properties of four kinds of glass fiber-reinforced plasrics irradiated with electron beams were examined at three temperatures; room temperature, 123 K and 77 K. Dynamic viscoelastic properties were measured, and fractography by means of scanning electron microscopy was observed in order to clarify degradation behaviour. A considerable decrease in interlaminar shear strength (ILSS) at room temperature was observed above 60 MGy. On the other hand, the three-point bending strength at 77 K and the ILSS at 123 K decreased with increasing irradiation. Fractography reveals that the degradation of the interface layer between matrix resin and fiber plays an important role in the strength reduction at 123 K and 77 K. These findings suggest that the interface between matrix resin and fiber loses its bondability at 123 K arid 77 K after electron beam irradiation.