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Post-translational modifications (PTMs) of histone N-terminal tails in nucleosome core particle (NCP), such as acetylation, play crucial roles in regulating gene expression. To unveil the regulation mechanism, atomic-level structural analysis of in-vitro modified NCP is effective with verifying the PTMs of histones. So far, identification of PTMs of NCP originating from living cells has mainly been performed using mass spectrometry (MS) techniques, such as bottom-up approach. The bottom-up approach is the most established method for protein characterization, but it does not always provide sufficient information on the acetylated sites of lysine residues in the histone tails if trypsin digestion is carried out. For histone proteins, which have many basic amino acids, trypsin generates too many short fragments that cannot be perfectly analyzed by tandem MS. In this study, we investigated the in vitro acetylation sites in the histone H3 tail using a top-down sequence analysis, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiment, in combination with aminopeptidase digestion. Aminopeptidase can cleave peptide bonds one-by-one from the N-terminus of peptides or proteins, generating N-terminally truncated peptides and/or proteins. As a result, it was identified that this method enables sequence characterization of the entire region of the H3 tail. Also, application of this method to H3 in in-vitro acetylated NCP enabled assigning acetylation sites of H3. Thus, this method was found to be effective for obtaining information on in-vitro acetylation of NCP for structural biology study.

Microarrays containing 45 different lectins were analyzed to identify global changes in the glycosylation of serum glycoproteins from mice exposed to whole-body γ-radiation. The results showed that radiation exposure increased and decreased the relative amounts of α-2,3- and α-2,6-sialic acids, respectively. The expression of α-2,3- and α-2,6-sialyltransferase genes in the liver was analyzed to determine whether changes in their expression were responsible for the sialic acid changes. The increase in α-2,3-sialic acid correlated with St3gal5 upregulation after radiation exposure; however, a decrease in St6gal1 expression was not observed. Analysis of a PCR array of genes expressed in irradiated mouse livers revealed that irradiation did not alter the expression of most of the included genes. These results suggest that glycomic screening of serum glycoproteins using lectin microarrays can be a powerful tool for identifying radiation-induced changes in the post-translational addition of sugar moieties to proteins. In addition, the results indicate that altered sialylation of glycoproteins may be an initial response to acute radiation exposure.

We used high-performance liquid chromatography to separate urine obtained from whole-body gamma-irradiated mice (4 Gy) before analyzing each fraction with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry to identify radiation-responsive molecules. We identified two candidates: hepcidin antimicrobial peptide 2 (hepcidin-2) and peptide fragments of kidney androgen-regulated protein (KAP). We observed that peak increases of hepcidin-2 in urine were delayed in a dose-dependent manner (1 Gy and above); however, the amount of KAP peptide fragments showed no correlation with radiation dose. In addition, an increase in hepcidin-2 after exposure to relatively low radiation doses (0.25 and 0.5 Gy, respectively) was biphasic (at 8–48 h and 120–168 h, respectively, after irradiation). The increase in hepcidin-2 paralleled an increase in hepcidin-2 gene (Hamp2) mRNA levels in the liver. These results suggest that radiation exposure directly or indirectly induces urinary excretion of hepcidin-2 at least in part by the upregulation of Hamp2 mRNA in the liver.

The interaction between camphor and sodium dodecyl sulfate (SDS) was experimentally investigated to understand restoration of Marangoni flow around a camphor disk on water under the addition of SDS. With an increase in the concentration of SDS, the flow velocity decreased to almost zero up to the critical micelle concentration (cmc), but increased above the cmc. Speed of Marangoni flow around the camphor disk increased with an increase in the scale of the aqueous channel. 13C NMR, NOE experiments, and mass spectrometry were used to evaluate the interaction between camphor and SDS molecules, which increased the dissolution rate of camphor into the SDS aqueous solution. The experimental results suggest that an increase in the dissolution of camphor into SDS micelles induces the restoration of Marangoni flow.

Spinal cord ischemic injury is the most devastating sequela of descending and thoracoabdominal aortic surgery. Motor-evoked potentials (MEPs) have been used to intraoperatively assess motor tract function, but it remains unclear whether MEP monitoring can decrease the incidence of postoperative motor deficits. Therefore, we reviewed multicenter medical records of patients who had undergone descending and thoracoabdominal aortic repair (both open surgery and endovascular repair) to assess the association of MEP monitoring with postoperative motor deficits.Patients included in the study underwent descending or thoracoabdominal aortic repair at 12 hospitals belonging to the Japanese Association of Spinal Cord Protection in Aortic Surgery between 2000 and 2013. Using multivariable mixed-effects logistic regression analysis, we investigated whether intraoperative MEP monitoring was associated with postoperative motor deficits at discharge after open and endovascular aortic repair.We reviewed data from 1214 patients (open surgery, 601 [49.5%]; endovascular repair, 613 [50.5%]). MEP monitoring was performed in 631 patients and not performed in the remaining 583 patients. Postoperative motor deficits were observed in 75 (6.2%) patients at discharge. Multivariable logistic regression analysis revealed that postoperative motor deficits at discharge did not have a significant association with MEP monitoring (adjusted odds ratio [OR], 1.13; 95% confidence interval [CI], 0.69-1.88; P = .624), but with other factors: history of neural deficits (adjusted OR, 6.08; 95% CI, 3.10-11.91; P.001), spinal drainage (adjusted OR, 2.14; 95% CI, 1.32-3.47; P = .002), and endovascular procedure (adjusted OR, 0.45; 95% CI, 0.27-0.76; P = .003). The sensitivity and specificity of MEP25% of control value for motor deficits at discharge were 37.8% (95% CI, 26.5%-49.5%) and 95.5% (95% CI, 94.7%-96.4%), respectively.MEP monitoring was not significantly associated with motor deficits at discharge.

Nitric oxide (NO) exerts a ubiquitous influence on cellular signaling, largely through S-nitrosylation/denitrosylation of cysteines in proteins. The ability to analyze S-nitrosylation directly by mass spectrometry (MS) would be highly useful in understanding the action of NO in various biological processes. However, S-nitrosylation has never been detected by ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) MS because the NO group completely dissociates during the ionization process. In this study, tetraphenylporphyrin (TPP) was used as matrix additive with 2,5-dihydroxybenzoic acid (DHB) to evaluate its suppressive effect on the NO group dissociation of S-nitrosylated peptides. Samples were subjected to MS and MS/MS analysis by MALDI-TOF MS and MALDI-QIT-TOF MS, respectively. In MS analysis using TPP as a matrix additive, the presumably intact molecular ion [M+H]+ peak was also observed, although the NO-dissociated ions [M−NO+H]+ were mainly detected. In contrast, in the absence of TPP, this [M+H]+ peak was not observed. MS/MS analysis of this ion indicated that the peptides contained S-nitrosylated cysteines. Furthermore, other reagents with radical scavenging activity (retinoic acid, α-tocopherol, ascorbic acid, naringenin, and 3,5-di-tert-butyl-4-hydroxytoluene) were tested to examine the possibility of their use as matrix additives for S-nitrosylation detection. As with TPP, these additives showed a suppressive effect on NO group dissociation. In conclusion, radical scavenging reagents are effective additive candidates for detecting S-nitrosylation directly with UV-MALDI MS.

The demand for establishment of high-throughput biodosimetric methods is increasing. Our aim in this study was to identify low-molecular-weight urinary radiation-responsive molecules using electrospray ionization Fourier transform mass spectrometry (ESI-FT MS), and our final goal was to develop a sensitive biodosimetry technique that can be applied in the early triage of a radiation emergency medical system. We identified nine metabolites by statistical comparison of mouse urine before and 8 h after irradiation. Time-course analysis showed that, of these metabolites, thymidine and either thymine or imidazoleacetic acid were significantly increased dose-dependently 8 h after radiation exposure; these molecules have already been reported as potential radiation biomarkers. Phenyl glucuronide was significantly decreased 8 h after radiation exposure, irrespective of the dose. Histamine and 1-methylhistamine were newly identified by MS/MS and showed significant, dose-dependent increases 72 h after irradiation. Quantification of 1-methylhistamine by enzyme-linked immunosorbent assay (ELISA) analysis also showed a significant increase 72 h after 4 Gy irradiation. These results suggest that urinary metabolomics screening using ESI-FT MS can be a powerful tool for identifying promising radiation-responsive molecules, and that urinary 1-methylhistamine is a potential radiation-responsive molecule for acute, high-dose exposure.

Amphiphilic fluoroalkyl end-capped N-(1,1-dimethyl-3-oxobutyl)acrylamide–acryloylmorpholine cooligomer/fluorescein nanocomposites afforded brilliant yellow-colored solutions in not only protic solvents such as methanol and ethanol but also protic-like solvents such as dichloromethane and 1,2-dichloroethane, respectively. However, the corresponding non-fluorinated cooligomer/fluorescein composites and parent fluorescein gave the colorless solutions under similar conditions. On the other hand, unexpectedly, such brilliant yellow-colored solutions provided by these fluorinated nanocomposites completely disappeared in aprotic solvents such as N,N-dimethylformamide, dimethyl sulfoxide, and tetrahydrofuran. Thus, these fluorinated fluorescein nanocomposites can exhibit a coloring–decoloring behavior through solvatochromic response.

Cell surface heparan sulfate proteoglycans (HSPGs) play significant roles in the regulation of developmental signaling, including vascular endothelial growth factor (VEGF), fibroblast growth factor, Wnt and bone morphogenetic protein signaling, through modification of their sulfation patterns. Recent studies have revealed that one of the functions of heparan sulfate 6-O-endosulfatase (Sulf) is to remove the sulfate from the 6-O position of HSPGs at the cell surface, thereby regulating the binding activities of heparan sulfate (HS) chains to numerous ligands and receptors in animal species. In this study, we focused on the sea urchin Hemicentrotus pulcherrimus homolog of Sulf (HpSulf), and analyzed its expression pattern and functions during development. HpSulf protein was present throughout development and localized at cell surface of all blastomeres. In addition, the HS-specific epitope 10E4 was detected at the cell surface and partially colocalized with HpSulf. Knockdown of HpSulf using morpholino antisense oligonucleotides (MO) caused abnormal morphogenesis, and the development of MO-injected embryos was arrested before the hatched blastula stage, indicating that HpSulf is necessary for the early developmental process of sea urchin embryos. Furthermore, we found that injection of HpSulf mRNA suppressed the abnormal skeleton induced by overexpression of HpVEGF mRNA, whereas injection of an inactive form of HpSulf mRNA, containing mutated cysteines in the sulfatase domain, did not have this effect. Taken together, these results suggest that HpSulf is involved in the regulation of various signal transductions, including VEGF signaling, during sea urchin development.

We have previously demonstrated that RNA interference-mediated suppression of xanthine dehydrogenase (XDH), the rate-limiting enzyme in purine degradation, causes defects in the normal growth and development of Arabidopsis thaliana. Here, we investigated a possible role for XDH in drought tolerance, since this enzyme is also implicated in plant stress responses and acclimatization. When XDH-suppressed lines were subjected to drought stress, plant growth was markedly reduced in conjunction with significantly enhanced cell death and H2O2 accumulation. This drought-hypersensitive phenotype was reversed by pretreatment with exogenous uric acid, the catalytic product of XDH. These results suggest that fully functional purine metabolism plays a role in the Arabidopsis drought acclimatization.

We investigated the topology and dynamics of melittin within the liposomes using mass spectrometry combined with acetylation. According to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry/mass spectrometry (MALDI-QIT-TOF MS/MS) analyses, melittin within the liposomes was mono acetylated; with the acetylated position being the N-terminal of melittin. This acetylation followed first-order kinetics. The rate constant was less than that of the acetylation of melittin in aqueous solution. Thus, it is suggested that the observed rate constant is that for the release of the N-terminal region of melittin to a water phase from the hydrophobic core of the liposomes. This approach has opened a new method in the study of dynamics of transmembrane helices within the membranes using mass spectrometry.

The La-Si binary phase diagram under a high pressure of 13.5 GPa was experimentally constructed. New superconducting silicides LaSi{sub 5} and LaSi{sub 10} were found, which have peritectic decomposition temperatures at 1000 and 750 deg. C, respectively. The single crystal X-ray structural analysis revealed that there are two polymorphs in LaSi{sub 5}. The alpha-form obtained by heating a molar mixture of LaSi{sub 2} and 3 Si at about 700 deg. C or by a rapid cooling from 1000 deg. C under pressure crystallizes with the space group C2/m and the lattice parameters a=15.11(3), b=4.032(6), c=8.26(1) A, and beta=109.11(1){sup o}. The beta-form obtained by a slow cooling from 800-950 deg. C to 600 deg. C under pressure has the same space group but with slightly different lattice parameters, a=14.922(7), b=3.906(2), c=8.807(4) A, and beta=107.19(1){sup o}. The beta-form is formed during the incomplete transformation of the alpha-form on cooling, and has always been obtained as a mixture with the alpha-form. The compound can be characterized as a Zintl phase with a polyanionic framework {sub i}nfinity{sup 3}[Si{sub 5}]{sup 3-} with large tunnels running along the b axis hosting lanthanum ions. In the beta-form, three of the five Si sites are disordered. The twomore » polymorphs contain one dimensional sila-polyacene ribbons, Si ladder polymer, running along the b axis. The alpha-form showed superconductivity with the transition temperature T{sub c} of 11.5 K. LaSi{sub 10} crystallizes with the space group 6{sub 3}/mmc and the lattice parameters a=9.623(4), c=4.723(3) A. It is composed of La containing Si{sub 18} polyhedra (La-Si{sub 18}) of hexagonal beer-barrel shape, which form straight columns by stacking along the c-axis via face sharing. One-dimensional columns of La-Si{sub 18} barrels are edge-shared, and bundled with infinite Si trigonal bipyramid chains via corner sharing. The Si atoms in the straight chains have a five-fold coordination. LaSi{sub 10} became a superconductor with T{sub c}=6.7 K. The ab initio calculation of the electric band structures showed that alpha-LaSi{sub 5} and LaSi{sub 10} are metallic, and the conduction electrons mainly come from Si-3p orbitals. - Graphical abstract: In the study of the phase diagram of the La-Si binary system under high pressure and high temperature conditions, new Si-rich superconducting LaSi{sub 5} (T{sub c}=11.5 K) and LaSi{sub 10} (T{sub c}=6.7 K) have been found. LaSi{sub 5} contain unique one-dimensional sila-polyacene ribbons, and LaSi{sub 10} has a hexagonal columnar structure formed by face shared La-Si{sub 18} polyhedra.« less

Glycogen synthase kinase-3beta (GSK-3beta) is known to play a role in the regulation of the dynamics of microtubule networks in cells. Here we show the role of GSK-3beta in the proper formation of the mitotic spindles through an interaction with GCP5, a component of the gamma-tubulin ring complex (gammaTuRC). GCP5 bound directly to GSK-3beta in vitro, and their interaction was also observed in intact cells at endogenous levels. Depletion of GCP5 dramatically reduced the GCP2 and gamma-tubulin in the gammaTuRC fraction of sucrose density gradients and disrupted gamma-tubulin localization to the spindle poles in mitotic cells. GCP5 appears to be required for the formation or stability of gammaTuRC and the recruitment of gamma-tubulin to the spindle poles. A GSK-3 inhibitor not only led to the accumulation of gamma-tubulin and GCP5 at the spindle poles but also enhanced microtubule nucleation activity at the spindle poles. Depletion of GCP5 rescued this disrupted organization of spindle poles observed in cells treated with the GSK-3 inhibitor. Furthermore, the inhibition of GSK-3 enhanced the binding of gammaTuRC to the centrosome isolated from mitotic cells in vitro. Our findings suggest that GSK-3beta regulates the localization of gammaTuRC, including GCP5, to the spindle poles, thereby controlling the formation of proper mitotic spindles.

We have quantified the photomovement behavior of a suspension of Euglena gracilis representing a behavioral response to a light gradient. Despite recent measurements of phototaxis and photophobicity, the details of macroscopic behavior of cell photomovements under conditions of light intensity gradients, which are critical to understand recent experiments on spatially localized bioconvection patterns, have not been fully understood. In this paper, the flux of cell number density under a light intensity gradient was measured by the following two experiments. In the first experiment, a capillary containing the cell suspension was illuminated with different light intensities in two regions. In the steady state, the differences of the cell numbers in the two regions normalized by the total number were proportional to the light difference, where the light intensity difference ranged from 0.5–2.0 μmol m−2 s−1. The proportional coefficient was positive (i.e., the bright region contained many microorganisms) when the mean light intensity was weak (1.25 μmol m−2 s−1), whereas it was negative when the mean intensity was strong (13.75 μmol m−2 s−1). In the second experiment, a shallow rectangular container of the suspension was illuminated with stepwise light intensities. The cell number density distribution exhibited a single peak at the position where the light intensity was about Ic ≃ 3.8 μmol m−2 s−1. These results suggest that the suspension of E. gracilis responded to the light gradient and that the favorable light intensity was Ic.

Protein tyrosine nitration is an important post-translational modification. A variety of nitrated proteins are reported in Arabidopsis leaves and seedlings, sunflower hypocotyls, and pea roots. The identities of nitrated proteins are species-/organ-specific, and chloroplast proteins are most nitratable in leaves. However, precise mechanism is unclear. Here, we investigated nitroproteome in tobacco leaves following exposure to nitrogen dioxide. Proteins were extracted, electrophoresed and immunoblotted using an anti-3-nitrotyrosine antibody. Mass spectrometry and FASTA search identified for the first time an exclusive nitration of pathogenesis-related proteins, PR-1, PR-3 and PR-5, which are reportedly located in the apoplast or the vacuole. Furthermore, Tyr36 of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.

Site-specific hydrogen/deuterium (H/D) exchange was investigated using a model protein, Escherichia coli dihydrofolate reductase, by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) coupled with two-step protease digestion by pepsin and carboxypeptidase P. Carboxypeptidase, which is generally inactive under quenching conditions (0°C and pH 2.9), produced some overlapping peptide sequences in the presence of 3.9 mM NaCl as a stabilizer. From differences in the masses of these peptides, we successfully determined the deuterium incorporation of backbone amide hydrogens of six residues—Met16 (M20 loop), Glu17 (M20 loop), Met92 (βE), Ala117 (βF-βG loop), Cys152 (βH), and Phe153 (βH)—at a higher resolution using the program Isotopica, which is capable of deconvoluting a complex isotope envelope associated with the isotope distribution of deuterated peptides. The H/D exchange kinetics data obtained were highly consistent with the local structure and fluctuation of these sites as revealed by nuclear magnetic resonance (NMR) and X-ray crystallography. These results demonstrate that MALDI MS coupled with two-step digestion is a useful tool for studying the site-specific H/D exchange of proteins without the deuterium scrambling that occurs in the gas-phase collision-induced dissociation method.

Many static protein structures have been determined by X-ray crystallography and nuclear magnetic resonance (NMR). Protein dynamics has also been studied. The structure and dynamics of proteins provide critical information about their functional mechanisms. Recently, mass spectrometry has become an essential method to analyze protein topological structures and protein dynamics. We performed topological analysis of membrane proteins using mass spectrometry coupled with chemical cross-linking. The combination of chemical cross-linking and mass spectrometry is a powerful method for analyzing the topological organization of protein structures, protein complexes, and the interface between interacting subunits. In this paper, we introduce our strategies for studying the membrane-protein and protein-protein interaction sites of P450 17α with its redox partners, NADPH-cytochrome P450 reductase and cytochrome b5.

vitamin D is 25-hydroxylated in the liver, before being activated by 1α-hydroxylation in the kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vitamin D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem 2004;279(22):22848-56]. In this study, we examine whether human CYP2J2 that exhibits 73 0x1.39bf8p-890mino acid homology to rat CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found significant 25-hydroxylation activity toward vitamin D3 (turnover number, 0.087 min-1), vitamin D2 (0.16 min-1), and 1α-hydroxyvitamin D3 (2.2 min-1). Interestingly, human CYP2J2 hydroxylated vitamin D2, an exogenous vitamin D, at a higher rate than it did vitamin D3, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D3 (1.4 min-1) more efficiently than vitamin D2 (0.86 min-1). Our study demonstrated that human CYP2J2 exhibits 25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D3 and D2. c 2006 Elsevier Inc. All rights reserved.

Morphological changes of DMPC, DLPC, and DPPC bilayers containing melittin (lecithin/melittin molar ratio of 10:1) around the gel-to-liquid crystalline phase transition temperatures (Tc) were examined by a variety of biophysical methods. First, giant vesicles with the diameters of approximately 20 microm were observed by optical microscopy for melittin-DMPC bilayers at 27.9 degrees C. When the temperature was lowered to 24.9 degrees C (Tc = 23 degrees C for the neat DMPC bilayers), the surface of vesicles became blurred and dynamic pore formation was visible in the microscopic picture taken at different exposure times. Phase separation and association of melittin molecules in the bilayers were further detected by fluorescent microscopy and mass spectrometry, respectively. These vesicles disappeared completely at 22.9 degrees C. It was thus found that the melittin-lecithin bilayers reversibly undergo their fusion and disruption near the respective Tcs. The fluctuation of lipids is, therefore, responsible for the membrane fusion above the Tc, and the association of melittin molecules causes membrane fragmentation below the Tc. Subsequent magnetic alignments were observed by solid-state (31)P NMR spectra for the melittin-lecithin vesicles at a temperature above the respective Tcs. On the other hand, additional large amplitude motion induced by melittin at a temperature near the Tc breaks down the magnetic alignment.

Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.

Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.

Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock-out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF)alpha in epithelial cells, and induced IL-1beta and TNFalpha production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.

Summary During prometaphase, dense microtubule nucleation sites at centrosomes form robust spindles that align chromosomes promptly. Failure of centrosome maturation leaves chromosomes scattered, as seen routinely in cancer cells, including myelodysplastic syndrome (MDS). We previously reported that the Miki ( LOC253012 ) gene is frequently deleted in MDS patients, and that low levels of Miki are associated with abnormal mitosis. Here we demonstrate that Miki localizes to the Golgi apparatus and is poly(ADP-ribosyl)ated by tankyrase-1 during late G2 and prophase. PARsylated Miki then translocates to mitotic centrosomes and anchors CG-NAP, a large scaffold protein of the γ-tubulin ring complex. Due to impairment of microtubule aster formation, cells in which tankyrase-1, Miki, or CG-NAP expression is downregulated all show prometaphase disturbances, including scattered and lagging chromosomes. Our data suggest that PARsylation of Miki by tankyrase-1 is a key initial event promoting prometaphase.

The large GTPase dynamin is strongly accumulated in the constricted area including midzonal microtubules of dividing cells. The proline-rich domain (PRD) of dynamin has been considered as a microtubule-binding domain. However, it remains unclear how PRD controls dynamin-microtubule interaction in mitotic cells. Here, we found that the microtubule-binding activity of PRD is low in dynamin-2. One of the mitosis-specific kinase activities to PRD in HeLa cells was identified as cyclin B-Cdc2 kinase. The kinase phosphorylated PRD at Ser(764) and/or Thr(766) and reduced the microtubule-binding activity of PRD. These results suggest that phosphorylation of PRD by cyclin B-Cdc2 kinase plays an important role to control dynamin-2-microtubule interaction in mitotic HeLa cells.

Localized pattern of bioconvection was newly observed in a suspension of Euglena gracilis , which was a photosensitive micro-organism. The suspension was exposed bright illumination from the bottom, in which the cells swam away from the light source. Then high-density spots, i.e., settling the cells, were formed at a part of a sealed chamber. This localized pattern was contrast with a general bioconvection where pattern was generated whole of a chamber. The experimental observations were reproduced by a mathematical model that was based on the phototaxis of individual cells in both vertical and lateral directions. Our results indicate that convection is maintained by upward swimming, as with general bioconvection, and the localization originates from lateral phototaxis.

We examined the biotransformation of enantiomeric pairs of enones such as pulegone and carvone in recombinant Escherichia coli expressing Nicotiana tabacum pulegone reductase. It was found that recombinant E. coli cells acquired the ability for stereospecific hydrogenation of the exocyclic Cdouble bond; length as m-dashC double bond of pulegone. However, stereospecificity in hydrogenation with the recombinant E. coli cells was opposite to that in hydrogenation with N. tabacum cells. On the other hand, the isolated recombinant pulegone reductase (rPRase) from the recombinant E. coli cells catalyzed hydrogenation of the exocyclic Cdouble bond; length as m-dashC double bond of pulegone; the hydrogen atoms participating in the reduction at C-8 and C-4 of pulegone originate from the pro-4R hydrogen of NADPH and the medium (H2O), respectively. Stereospecificity was lost in the hydrogenation of pulegone with the isolated rPRase, but was recovered when bovine serum albumin was added to the enzymatic reaction as an auxiliary factor.

Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.

Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long-term depression (LTD) and spinogenesis, were investigated, in response to 17beta-estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi-electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nm estradiol. This enhancement of spinogenesis was completely suppressed by mitogen-activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl-pyrazole-trinyl)tris-phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4-hydroxyphenyl)-propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the ERalpha agonist, a search was made for synaptic ERalpha using purified RC-19 antibody qualified using ERalpha knockout (KO) mice. Localization of ERalpha in spines of principal glutamatergic neurons was demonstrated using immunogold electron microscopy and immunohistochemistry. ERalpha was also located in nuclei, cytoplasm and presynapses.

Wnt-5a is a representative ligand that activates a β-catenin-independent pathway in Wnt signalling. In the present paper, the roles of the post-translational modifications in the actions of Wnt-5a were investigated. We found that Wnt-5a is modified with palmitate at Cys104 and glycans at Asn114, Asn120, Asn311 and Asn325. The palmitoylation was not essential for the secretion of Wnt-5a, but was necessary for its ability to suppress Wnt-3a-dependent T-cell factor transcriptional activity and to stimulate cell migration. Wnt-5a activated focal adhesion kinase and this activation also required palmitoylation. Wild-type Wnt-5a induced the internalization of Fz (Frizzled) 5, but a Wnt-5a mutant that lacks the palmitoylation site did not. Furthermore, the binding of Wnt-5a to the extracellular domain of Fz5 required palmitoylation of Wnt-5a. These results indicate that palmitoylation of Wnt-5a is important for the triggering of signalling at the cell surface level and, therefore, that the lipid-unmodified form of Wnt-5a cannot activate intracellular signal cascades. In contrast, glycosylation was necessary for the secretion of Wnt-5a, but not essential for the actions of Wnt-5a. Thus the post-translational palmitoylation and glycosylation of Wnt-5a are important for the actions and secretion of Wnt-5a.

The 70S ribosome from Escherichia coli is a supermacro complex (MW: 2.7MDa) comprising three RNA molecules and more than 50 proteins. We have for the first time successfully analyzed the flexibility of 70S ribosomal proteins in solution by detecting the hydrogen/deuterium exchange with mass spectrometry. Based on the deuterium incorporation map of the X-ray structure obtained at the time of each exchange, we demonstrate the structure–flexibility–function relationship of ribosome focusing on the deuterium incorporation of the proteins binding ligands (tRNA, mRNA, and elongation factor) and the relation with structural assembly processes.

We have studied amino acid formation by UV (193 nm) irradiation to organic molecules (amines, alcohols and amides) in aqueous solution. Among several types of detected amino acids, small aliphatic amino acids (Gly and alpha-, beta-Ala and alpha-, beta-, gamma-ABA) were quantitatively identified. Among these small aliphatic amino acids, certain amino acids were formed in its free form, even before hydrolysis, contrary to the results of UV irradiation to a gas mixture of CO, NH3, and H2O, where amino acids were hardly detected before hydrolysis. The species distribution of identified amino acids showed a dependence on the starting organic molecules, and also on the presence of ammonia. The formation processes of the identified small aliphatic amino acids were investigated with the aid of electrospray ionization (ESI) MS and MS/MS measurements of photoproducts. Possible formation processes of these amino acid precursors from each starting molecules are proposed. By identifying the amino acid precursor, which has a chiral carbon atom, a new possibility is suggested for asymmetric photosynthesis of amino acid from achiral organic molecules.

Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock-out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF)alpha in epithelial cells, and induced IL-1beta and TNFalpha production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.

Fertilization is an indispensable step for formation of a zygote in sexual reproduction, leading to species survival. When mating occurs, sperm is transported to the female reproductive tracts via the seminal plasma (SP). SP is derived from male accessory sex glands and it plays pivotal roles for fertilization in animals. However, molecular mechanisms of SP or a fluid derived from male accessory sex glands for successful fertilization remain unclear. Here, we report that in male quail the cloacal gland (CG) produces prostaglandin F2α (PGF2α) that contributes to successful fertilization. PGF2α, as well as the secretion of CG (CGS), induced vaginal contractions and caused the opening of the entrance of the sperm storage tubules, the structures responsible for the long-term sperm storage and fertilization. The removal of CGS from the male before mating reduced the fertility, but the supplementation of CGS or PGF2α rescued the subfertility. We further showed that male CG contains glucose that is utilized as energy source for the intrinsic sperm mobility after transportation to female vagina. This mechanism, in concert with the excitatory effects of PGF2α enables successful fertilization in the domestic bird.

A 20-kDa protein (p20) with a GTP binding activity was purified from the cultured cells of Glycine max (soybean). The amino acid sequence of p20 showed 65% identity in a 23 amino acid overlap against the Kunitz-type trypsin inhibitor of soybean reported. Furthermore, it was found that a Kunitz-type soybean trypsin inhibitor of commercial origin also binds GTP.

Background The objective of this study was to identify substantially increased proteins in bovine cystic follicular fluid (FF) in order to clarify the pathology and etiology of bovine ovarian follicular cysts (BOFC). Methods Proteins in normal and cystic FF samples were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were compared using silver stained gel images with PDQuest image analysis software. Peptides from these increased spots were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and were identified based on the NCBI database by a peptide mass fingerprinting method. Results Comparative proteomic analysis showed 8 increased protein spots present in cystic FF. MS analysis and database searching revealed that the increased proteins in cystic FF were bovine mitochondrial f1-atpase (BMFA), erythroid associated factor (EAF), methionine synthase (MeS), VEGF-receptor, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), heat shock protein 70 (HSP70), β-lactoglobulin (BLG) and succinate dehydrogenase Ip subunit (SD). Conclusion Our results suggest that these proteins are overexpressed in BOFC, and that they may play important roles in the pathogenesis of BOFC. Furthermore, these proteins in the FF could be useful biomarkers for BOFC.