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Mohammed Abdullahel Amin, Sachihiro Matsunaga, Susumu Uchiyama, Kiichi Fukui; Depletion of nucleophosmin leads to distortion of nucleolar and nuclear structures in HeLa cells. Biochem J 1 November 2008; 415 (3): 345–351. doi: https://doi.org/10.1042/BJ20081411., NPM (nucleophosmin; also known as B23) is an abundantly and ubiquitously expressed multifunctional nucleolar phosphoprotein, which is involved in numerous cellular processes, including ribosome biogenesis, protein chaperoning and centrosome duplication; however, the role of NPM in the cell cycle still remains unknown. In the present study, we show dynamic localization of NPM throughout the cell cycle of HeLa cells. Using a combination of RNAi (RNA interference) and three-dimensional microscopy we show that NPM is localized at the chromosome periphery during mitosis. We also demonstrate that depletion of NPM causes distortion of nucleolar structure as expected and leads to unexpected dramatic changes in nuclear morphology with multiple micronuclei formation. The defect in nuclear shape of NPM-depleted cells, which is clearly observed by live-cell imaging, is due to the distortion of cytoskeletal (α-tubulin and β-actin) structure, resulting from the defects in centrosomal microtubule nucleation. These results indicate that NPM is an essential protein not only for the formation of normal nucleolar structure, but also for the maintenance of regular nuclear shape in HeLa cells.

Cytochrome c(552) (PH c(552)) from moderately thermophilic Hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome c(552) (HT c(552)) from thermophilic Hydrogenobacter thermophilus and cytochrome c(551) (PA c(551)) from mesophilic Pseudomonas aeruginosa. To understand the mechanism of stabilization of PH c(552), we introduced mutations into PH c(552) at five sites, which, in HT c(552), are occupied by the amino acids responsible for stability higher than the less stable PA c(551). When PH c(552) Val-78 was mutated to Ile, as found in HT c(552), the resulting variant showed increased stability. Mutation of Ala-7, Met-13, and Tyr-34 to the corresponding residues in PA c(551) (Phe, Val, and Phe, respectively) resulted in destabilization. We also found that PH c(552) Lys-43 contributed to stability through the formation of an attractive electrostatic interaction with Asp-39. These results suggest that the intermediate stability of PH c(552) is due to the amino acids at these five sites.

Chromosomes are supermolecules that contain most of the DNA within a cell and are visible under optical and electron microscopes. Although they were observed at the earliest stage of genetics, their fundamental structure is not yet understood. The reasons for this are debated among researchers; however, it is clear that the accumulation of metaphase chromosomes for their biochemical analysis has been a significant challenge. In this chapter, a method is described for accumulating and preparing human metaphase chromosomes in sufficient amount to perform their proteome analysis. Preparation and separation methods of chromosome proteins are described, followed by a protocol for their identification by mass spectrometry.

Chromosomes are supermolecules that contain most of the DNA within a cell and are visible under optical and electron microscopes. Although they were observed at the earliest stage of genetics, their fundamental structure is not yet understood. The reasons for this are debated among researchers; however, it is clear that the accumulation of metaphase chromosomes for their biochemical analysis has been a significant challenge. In this chapter, a method is described for accumulating and preparing human metaphase chromosomes in sufficient amount to perform their proteome analysis. Preparation and separation methods of chromosome proteins are described, followed by a protocol for their identification by mass spectrometry.

The frequency dependence of permeability has been calculated for soft magnetic thin films which have a uniaxial anisotropy with both amplitude and angular dispersion. These calculations have been performed for amorphous Co-Nb films, whose anisotropy dispersion has been determined by low-frequency FMR measurements. Comparison of the measured permeability with calculations suggests that the effective uniaxial anisotropy is dispersed more widely and with a smaller amplitude than is indicated by FMR measurements. The magnetization ripple is inferred to be one of the causes of the low value for the effective field.

Magnetic and magneto-optical properties have been investigated for Pd/Co multilayered films prepared by rf sputtering method. The perpendicular magnetic anisotropy of the films increases with decreasing bilayer period λ and with decreasing Co composition. The maximum value of the anisotropy K⊥ obtained is 3.8×106 erg/cc, where λ and Co composition are 11A and 28 at%, respectively. The saturation magnetization increases as the λ decreases and approaches the same value as alloys. The Kerr spectra of the multilayered films and those of alloy films are alike in having a peak around the wavelength of 400 nm, and they become almost the same for λ

Measurements of the saturation magnetostriction constant ? 3 of amorphous wires have been difficult using conventional techniques, due to their very small diameters (10~125 ?m). This paper describes the stable measurement of the magnetostriction of amorphous wires, using a magnetization rotation model and applying a compressive stress to positive-magnetostriction wires and a tensile stress to negative-magnetostriction wires. A value for ? S of 35 ppm was obtained for an as-prepared Fe 77.5 Si 7.5 B 15 wire at room temperature.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N--I and I--U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.

The barnacle relies for its attachment to underwater foreign substrata on the formation of a multiprotein complex called cement. The 20 kDa cement protein is a component of Megabalanus rosa cement, although its specific function in underwater attachment has not, until now, been known. The recombinant form of the protein expressed in bacteria was purified in soluble form under physiological conditions, and confirmed to retain almost the same structure as that of the native protein. Both the protein from the adhesive layer of the barnacle and the recombinant protein were characterized. This revealed that abundant Cys residues, which accounted for 17% of the total residues, were in the intramolecular disulfide form, and were essential for the proper folding of the monomeric protein structure. The recombinant protein was adsorbed to calcite and metal oxides in seawater, but not to glass and synthetic polymers. The adsorption isotherm for adsorption to calcite fitted the Langmuir model well, indicating that the protein is a calcite-specific adsorbent. An evaluation of the distribution of the molecular size in solution by analytical ultracentrifugation indicated that the recombinant protein exists as a monomer in 100 mm to 1 m NaCl solution; thus, the protein acts as a monomer when interacting with the calcite surface. cDNA encoding a homologous protein was isolated from Balanus albicostatus, and its derived amino acid sequence was compared with that from M. rosa. Calcite is the major constituent in both the shell of barnacle base and the periphery, which is also a possible target for the cement, due to the gregarious nature of the organisms. The specificity of the protein for calcite may be related to the fact that calcite is the most frequent material attached by the cement.

Standard immunogold-labeling methods in transmission electron microscopy (TEM) are unable to locate immunogold particles in the depth direction. This inability does not only concern bulky whole mounts, but also sections. A partial solution to the problem is stereo inspection. However, three-dimensional reconstruction of immunogold-labeled structures, that is, immuno-electron tomography (IET), is a correct solution for this inconsistency. Striking improvement in resolution is achieved: the 1.4-nm immunogold particles are shown in IET that are not detected in the original tilt series. IET is not restricted to laboratories with advanced medium- or high-voltage TEM and super-computing facilities; the methods we have developed for whole-mounted chromosomes and also for whole-mounted cytoskeleton of fibroblasts work remarkably well with ordinary 80-kV TEMs equipped with a goniometer to collect tilt series for IET on film. In addition, free programs are available to produce three-dimensional reconstructions even without high-performance computers. These improvements make it possible to many laboratories without modern facilities to perform IET reconstruction with standard TEM apparatus.

Ma N., Matsunaga S., Takata H., et al. Nucleolin functions in nucleolus formation and chromosome congression. Journal of Cell Science, 120, 12, 2091. https://doi.org/10.1242/jcs.008771., A complex structure, designated the chromosome periphery, surrounds each chromosome during mitosis. Although several proteins have been shown to localize to the chromosome periphery, their functions during mitosis remain unclear. Here, we used a combination of high-resolution microscopy and RNA-interference-mediated depletion to study the functions of nucleolin, a nucleolar protein localized at the chromosome periphery, in interphase and mitosis. During mitosis, nucleolin was localized in the peripheral region including the vicinity of the outer kinetochore of chromosomes. Staining with an antibody specific for nucleolin phosphorylated by CDC2 revealed that nucleolin was also associated with the spindle poles from prometaphase to anaphase. Nucleolin depletion resulted in disorganization of the nucleoli at interphase. Furthermore, nucleolin-depleted cells showed a prolonged cell cycle with misaligned chromosomes and defects in spindle organization. The misaligned chromosomes showed syntelic kinetochore-microtubule attachments with reduced centromere stretching. Taken together, our results indicate that nucleolin is required for nucleolus formation, and is also involved in chromosome congression and spindle formation.

A comparative proteome analysis of human metaphase chromosomes between a typical epithelial-like cell, HeLa S3, and a lymphoma-type cell, BALL-1, was performed. One-dimensional (1-D) SDS-PAGE and radical-free and highly reducing two-dimensional electrophoresis (RFHR 2-DE) detected more than 200 proteins from chromosomes isolated from HeLa S3 cells, among which 189 proteins were identified by mass spectrometry (MS). Consistent with our recent four-layer structural model of a metaphase chromosome, all the identified proteins were grouped into four distinct levels of abundance. Both HeLa S3 and BALL-1 chromosomes contained specific sets of abundant chromosome structural and peripheral proteins in addition to less abundant chromosome coating proteins (CCPs). Furthermore, titin array analysis and a proteome analysis of the ultra-high molecular mass region indicated an absence of titin with their molecular weight (MW) more than 1000 kDa. Consequently, the present proteome analyses together with previous information on chromosome proteins provide the comprehensive list of proteins essential for the metaphase chromosome architecture.

Background: Cell-based assays utilizing digital image cytometry yield multivariate sets of information measuring the efficacy of medicines/chemicals. The use of a HeLa cell line that expresses a GFP-Histone-H1 fusion protein further enhances the performance of these systems, avoiding the use of dyes that may have detrimental influence on cells. Aside from the mitotic index, the distribution of the cell-cycle phases during mitosis can be used as measures of drug/treatment efficacy. Quantification of these parameters, however, requires skill and is time consuming. The purpose of this research was therefore to create a classifier to be incorporated into a system that can automatically identify the cell-cycle phases in a given image. Methods: Features based on the shape and texture of the chromosomal regions in images of live HeLa cells were measured and analyzed. Linear discriminant functions were calculated for the eight cell-cycle phases: interphase, prophase, prometaphase, metaphase, early anaphase, anaphase, telophase and cytokinesis. Results: The multistage linear discriminant classifier developed had an average classification efficiency of 87.30%. Conclusion: We demonstrated the possibility of creating a classifier to discriminate between cell-cycle phases using shape and texture features of chromosomal regions. The classifier can be fused to an algorithm for image segmentation, forming a system to automatically and rapidly measure the aforementioned parameters. The results can then be collated to constitute an assay assessing the effects of a drug or treatment on mammalian cells. © 2007 International Society for Analytical Cytology

Standard immunogold-labeling methods in transmission electron microscopy (TEM) are unable to locate immunogold particles in the depth direction. This inability does not only concern bulky whole mounts, but also sections. A partial solution to the problem is stereo inspection. However, three-dimensional reconstruction of immunogold-labeled structures, that is, immuno-electron tomography (IET), is a correct solution for this inconsistency. Striking improvement in resolution is achieved: the 1.4-nm immunogold particles are shown in IET that are not detected in the original tilt series. IET is not restricted to laboratories with advanced medium- or high-voltage TEM and super-computing facilities; the methods we have developed for whole-mounted chromosomes and also for whole-mounted cytoskeleton of fibroblasts work remarkably well with ordinary 80-kV TEMs equipped with a goniometer to collect tilt series for IET on film. In addition, free programs are available to produce three-dimensional reconstructions even without high-performance computers. These improvements make it possible to many laboratories without modern facilities to perform IET reconstruction with standard TEM apparatus.

Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition.

Bacterial 3alpha-hydroxysteroid dehydrogenase, which belongs to a short-chain dehydrogenase/reductase family and forms a dimer composed of two 26-kDa subunits, catalyzes the oxidoreduction of hydroxysteroids in a coenzyme-dependent manner. This enzyme also catalyzes the oxidoreduction of nonsteroid compounds that play an important role in xenobiotic metabolism of bacteria. We performed an x-ray analysis on the crystal of Ps3alphaHSD, the enzyme from Pseudomonas sp. B-0831 complexed with NADH. The resulting crystal structure at 1.8A resolution showed that Ps3alphaHSD exists as a structural heterodimer composed of apo- and holo-subunits. A distinct structural difference between them was found in the 185-207-amino acid region, where the structure in the apo-subunit is disordered whereas that in the holo-subunit consists of two alpha-helices. This fact proved that the NADH binding allows the helical structures to form the substrate binding pocket even in the absence of the substrate, although the region corresponds to the so-called "substrate-binding loop." The induction of alpha-helices in solution by the coenzyme binding was also confirmed by the CD experiment. In addition, the CD experiment revealed that the helix-inducing ability of NADH is stronger than that of NAD. We discuss the negative cooperativity for the coenzyme binding, which is caused by the effect of the structural change transferred between the subunits of the heterodimer.

Susumu Uchiyama, Tetsuo Tomiyama, Tsutomu Kai, Kazuhiro Hukada, Koichi Kajiyama, and Yuji Kobayashi 1 Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 5650871, Japan; 2 Graduate School of Science, Nagoya University, Chikusa, Nagoya, 460814, Japan; Faculty of Science, Tokyo Metropolitan University, Hachioji, Tokyo, 1920364, Japan; and Admon Science Inc., Fujieda, Shizuoka, 4260007, Japan.

The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range.

We have studied the structure-thermostability relationship using cytochromes c from mesophilic and thermophilic bacteria; Pseudomonas aeruginosa (PAc(551)) growing at 37 degrees C and Hydrogenobacter thermophilus (HTc(552)) at 72 degrees C and showed that only five residues primarily differentiate their stabilities. For a more comprehensive study, we found Hydrogenophilus thermoluteolus (Pseudomonas hydrogenothermophila) growing at 52 degrees C and showed the moderate stability of the cytochrome c from this bacterium (PHc(552)). To explore the stabilization mechanisms, the crystal structure of PHc(552) was determined by X-ray analysis. The solution structure of HTc(552) elucidated previously by NMR was refined using distributed computational implementation. Furthermore, the recently reported crystal structure of HTc(552) has become available [Travaglini-Allocatelli, C. et al. (2005) J. Biol. Chem. 280, 25729-25734]. When the structures of these three cytochromes c were combined, this revealed that the five residues, corresponding to those mentioned above, determine the difference of stabilities among them as well. These facts suggested the stabilization mechanisms as follows: (1) improved van der Waals interactions by packing optimization at the N-terminal helix, (2) attractive electrostatic interactions with the heme propionate group, and (3) favorable van der Waals interaction with the heme. This comparative study, by supplementing the structural information of PHc(552) with its complementary feature, demonstrates that just a small number of amino acid residues determine the overall molecular stability by means of additivity of the effects of their substitutions. It is interesting that, in naturally occurring proteins, these adaptation strategies are accommodated by these bacteria to survive in the wide range of thermal conditions.

The NAD(P)(+)-dependent enzyme 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3alpha-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 A. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 A, and contained two molecules, reflecting dimer formation of 3alpha-HSD, in the asymmetric unit.

Background: Single-chain fragment variable region (scFv) in a chimeric antigen receptor (CAR) is a key component that directly binds the target antigen and transmits an activating signal into the CAR-T cells, subsequently triggering its effector function against the target cell. Thus, the affinity of scFv is considered to be critically important for CAR-T-cell function. However, optimal scFv affinity to induce maximal CAR function remains unclear. Methods: In this study, we constructed anti-CD20 scFv based on the reported sequence of humanized anti-CD20 monoclonal antibody with five different affinities: 20-vv (Kd value=7.07 nM), -fv (7.21), -fa (10.09), -sv (12.56), and -sa (14.66). Each scFv was then fused to an IgG4 hinge, a CD3-zeta chain, a CD28 costimulatory domain, and a truncated version of the epidermal growth factor receptor (tEGFR) as a transduction and selection marker. CD8-positive T cells from a healthy donor were activated with anti-CD3/28 beads, transduced with the CARs on day 3, and enriched by selection with anti-EGFR mAb. CAR-T cells were expanded using anti-CD3/28 beads and used in the subsequent analysis. Results: To determine the lytic activity according to the scFv affinity, we tested cytotoxicity against CD20-transduced K562 cells (K562-CD20) by 51Cr releasing assay. CAR-T cells with the three highest-affinity scFv (20CAR-vv, -fv, -fa) effectively and equally lysed K562-CD20, while CAR-T cells with the two lowest-affinity scFv (20CAR-sv, -sa) failed to exhibit significant cytotoxicity (20CAR-vv, -fv, -fa, -sv, and -sa: 60.2±2.85%, 57.3±2.03%, 57.0±3.82%, 2.8±0.1%, and 2.0±0.64% at an E:T ratio = 10:1, respectively). To examine CAR-T-cell activation, cytokine secretion in the supernatant after 16 hours of culture with K562-CD20 was analyzed. Similarly to the cytotoxicity, 20CAR-vv, -fv, and -fa produced cytokine, but 20CAR-sv and -sa did not. The highest-affinity CAR-T (20CAR-vv) produced significantly higher IFN-g and IL-2 than the other CAR-T cells [IFN-g (pg/ml): 7644±326, 3380±179, 4718±315, 4.9±0.4, and 5.4±1.2; IL-2 (pg/ml): 4179±177, 1071±30, 1379±118, 0, and 0, respectively]. We next analyzed cell proliferation upon stimulation with K562-CD20 over 72 hours. 20CAR-vv resulted in rather poor absolute cell proliferation, while CAR-T cells with the second and third highest-affinity scFv (20CAR-fv, -fa) resulted in preferable proliferation (1.22±0.20-, 2.52±0.12-, 2.01±0.42-, 0.65±0.11-, and 1.15±0.13-fold expansion, respectively). To explore the possible reason for the difference of CAR-T proliferation, we evaluated activation-induced cell death (AICD) at 24 hours after the K562-CD20 stimulation (Annexin-V/PI staining). 20CAR-vv underwent significantly higher AICD than the other CAR-T cells (68.4±1.1, 39.6±0.7, 37.9±5.3, 25.9±11.2, and 16.1±1.2% cell death). Finally, we tested cell proliferation and AICD of the three highest-affinity scFv CAR-T cells (-vv, -fv, and -fa) upon CD20 high and low stimulation. In this experiment, we used CD20-transduced CEM cells, which are controlled to express high and low CD20: CEM-CD20 high (CD20 = 143,000 molecules/cell, the same level as other B-cell lines) and CEM-CD20 low (5,320 molecules/cell, the same level as the rituximab refractory cell line). The replacement of the stimulator from CD20-CEM high to low resulted in partial reduction of AICD to a level comparable to those of other 20CARs (-vv, 62.7%→33.0%, -fv, 56.0%→37.4%, –fa, 55.3%→27.7%), but the cell proliferation upon stimulation did not improve (-vv, 1.2-, -fv, 2.6-, -fa, 2.7-fold expansion after CD20-CEM low stim). These results suggest that the higher-affinity scFv was not necessarily associated with better CAR-T proliferation after ligation and may inhibit cell proliferation, possibly by multiple factors besides AICD. Conclusions: We observed that scFv with affinity above a certain level is needed to induce CAR-T function. Higher-affinity scFv induced more robust cytokine production; however, excessively high-affinity scFv in CAR can counteract T-cell proliferation, which may be partially associated with increased AICD. Optimization of scFv affinity is desirable for effective CAR-T production. Disclosures No relevant conflicts of interest to declare.

X-ray analysis has been carried out on a crystal of the collagen model peptide (Hyp(R)-Hyp(R)-Gly)10 [where Hyp(R) is 4(R)-hydroxyproline] with 1.5 A resolution. The triple-helical structure of (Hyp(R)-Hyp(R)-Gly)10 has the same helical parameters and Rich and Crick II hydrogen bond patterns as those of other collagen model peptides. However, our full-length crystal structure revealed that almost all consecutive Hyp(R) residues take the up-up pucker in contrast to putative down-up puckering propensities of other collagen model peptides. The unique feature of thermodynamic parameters associated with the conformational transition of this peptide from triple helix to single coil is that both enthalpy and entropy changes of the transition are much smaller than those of other model peptides such as (Pro-Pro-Gly)10 and (Pro-Hyp(R)-Gly)10. To corroborate the precise structural information including main- and side-chain dihedral angles and intra- and interwater bridge networks, we estimated the degrees of hydration by comparing molecular volumes observed experimentally in solution to those calculated ones from the crystal structure. The results showed that the degree of hydration of (Hyp(R)-Hyp(R)-Gly)10 is comparable to that of (Pro-Hyp(R)-Gly)10 in the triple-helical state, but the former was more highly hydrated than (Pro-Hyp(R)-Gly)10 in the single-coil state. Because hydration reduces the enthalpy due to the formation of a hydrogen bond with a water molecule and diminishes the entropy due to the restriction of water molecules surrounding a peptide molecule, we concluded that the high thermal stability of (Hyp(R)-Hyp(R)-Gly)10 is able to be described by its high hydration in the single-coil state.

Calreticulin (CRT) is a multifunctional Ca2+-binding protein that mainly functions in the endoplasmic reticulum as a molecular chaperone for newly synthesized proteins. Recently we reported the protein composition of human metaphase chromosomes (Uchiyama et al., 2004), which included CRT. Here we describe new characteristics of CRT in vitro as well as its localization on the surface of metaphase chromosomes in vivo. CRT was detected in the chromosomal fraction by Western blotting and its binding partners were identified as core and linker histones by ligand overlay assay. Surface plasmon resonance sensor analyses revealed that CRT is bound to chromatin fibers. Moreover, we found that CRT has both supercoiling activity, which assists core histone assembly into chromatin fibers, and binding ability to histone H2A/H2B dimers and histone H3/H4 tetramers. Unlike the chromosome scaffold proteins, indirect immunofluorescent staining revealed that CRT is located on the surface of metaphase chromosomes. These results suggest that CRT plays a role which involves chromatin dynamics on the surface of mitotic chromosomes.

For the rational design of a stable collagen triple helix according to the conventional rule that the pyrrolidine puckerings of Pro, 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) should be down at the X-position and up at the Y-position in the X-Y-Gly repeated sequence for enhancing the triple helix propensities of collagen model peptides, a series of peptides were prepared in which X- and Y-positions were altogether occupied by HypR, HypS, fProR or fProS. Contrary to our presumption that inducing the X-Y residues to adopt a down-up conformation would result in an increase in the thermal stability of peptides, the triple helices of (HypS-HypR-Gly)10 and (fProS-fProR-Gly)10 were less stable than those of (Pro-HypR-Gly)10 and (Pro-fProR-Gly)10, respectively. As reported by Bachinger's and Zagari's groups, (HypR-HypR-Gly)10 which could have an up-up conformation unfavorable for the triple helix, formed a triple helix that has a high thermal stability close to that of (Pro-HypR-Gly)10. These results clearly show that the empirical rule based on the conformational preference of pyrrolidine ring at each of X and Y residues should not be regarded as still valid, at least for predicting the stability of collagen models in which both X and Y residues have electronegative groups at the 4-position. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

By optimizing the concentration and time of treatment with hydroxyurea (HU), a DNA synthesis inhibitor, and trifluralin, a microtubule inhibitor, a highly effective (over 60%) cell cycle synchronization method for rye and barley meristem cells was developed. Chromosome suspensions containing highly purified and morphologically intact rye and barley chromosomes were prepared from the meristems of their root tips by homogenization. Digoxigenin-labeled 5S rDNA was used as a probe in FISH for the rye chromosomes in the suspension, and biotin-labeled 17S rDNA and centromeric DNA were used in FISH for the rye and barley chromosome suspensions, respectively. Bright signals were detected at the specific regions of interest on the chromosomes. The results indicate that the method developed in this study is useful for selection and sorting of chromosomes that are not distinguishable by other means, using specific fluorescent labeling by FISH of the chromosomes in suspension.

The amino-acid sequence of cytochrome c 552 (PH c 552) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c 552), and from a mesophile, Pseudomonas aeruginosa (PA c 551). The PH c 552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c 552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C2221, with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 A. The crystals diffract X-rays to around 2.1 A resolution.

Five amino acid residues responsible for extreme stability have been identified in cytochrome c552 (HT c552) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c552, were replaced with the corresponding residues in the homologous but less stable cytochrome c551 (PA c551) from Pseudomonas aeruginosa. The quintuple HT c552 variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c551, suggesting that the five residues in HT c552 necessarily and sufficiently contribute to the overall stability. In the three HT c552 variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by –13.3 kJ mol–1, followed by A7F/M13V (–3.3 kJ mol–1) and then I78V (–1.5 kJ mol–1). The order of destabilization in HT c552 was the same as that of stabilization in PA c551 with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol–1, respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability., This work was supported in part by grants from Hiroshima University, the Noda Institute for Scientific Research, and the Japanese Ministry of Education, Science and Culture (grants-in-aid for Scientific Research on Priority Areas).

Susumu Uchiyama, Shouhei Kobayashi, Hideaki Takata, Takeshi Ishihara, Naoto Hori, Tsunehito Higashi, Kayoko Hayashihara, Takefumi Sone, Daisuke Higo, Takashi Nirasawa, Toshifumi Takao, Sachihiro Matsunaga, Kiichi Fukui. Proteome Analysis of Human Metaphase Chromosomes. Journal of Biological Chemistry, Volume 280, Issue 17, 2005, Pages 16994-17004. https://doi.org/10.1074/jbc.M412774200., DNA is packaged as chromatin in the interphase nucleus. Dining mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite > 1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into sis groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein frame-work for future research on this topic.

The Aurora kinase family is a well-characterized serine/threonine protein kinase family that regulates different processes of mitotic events. Although functions of animal and yeast Aurora kinases have been analyzed, plant aurora kinases were not identified and characterized. We identified three Aurora kinase orthologs in Arabidopsis thaliana and designated these as AtAUR1, AtAUR2, and AtAUR3. These AtAURs could phosphorylate serine 10 in histone H3, in vitro. Dynamic analyses of GFP-fused AtAUR proteins revealed that AtAUR1 and AtAUR2 localized at the nuclear membrane in interphase and located in mitotic spindles during cell division. AtAUR1 also localized in the cell plates. AtAUR3 showed dot-like distribution on condensed chromosomes at prophase and then localized at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes. The localization of AtAUR3 during mitosis is very similar to that of phosphorylated histone H3. Interestingly, an overexpression of AtAUR3 induces disassembly of spindle microtubules and alteration of orientation of cell division. Our results indicate that plant Aurora kinases have different characters from that of Aurora kinases of other eukaryotes.

Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (HT c(552)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552) variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c(551), suggesting that the five residues in HT c(552) necessarily and sufficiently contribute to the overall stability. In the three HT c(552) variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 kJ mol(-1)) and then I78V (-1.5 kJ mol(-1)). The order of destabilization in HT c(552) was the same as that of stabilization in PA c(551) with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.

The complete thermal-unfolding profiles of both oxidized and reduced forms of cytochrome c551 (PA) from mesophilic Pseudomonas aeruginosa and cytochrome c552 (HT) from thermophilic Hydrogenobacter thermophilus were obtained by the newly developed pressure-proof cell compartment installed in a circular dichroic spectrometer, which facilitates protein thermal-unfolding experiments up to 180 degrees C. The thermodynamic cycle, which relates protein stability and redox function, indicated that the redox potentials of PA and HT in the native state are regulated by the stability of the oxidized proteins rather than by that of the reduced ones.

Human endothelin-1 (ET-1) is a potent cardiovascular bioactive peptide. Its activity is based on the C-terminal residues, e.g., Trp 21 in particular. Recently, we reported an NMR solution structure of ET-1, which has a C-terminal hydrophobic core around Tyr 13. This C-terminal conformation does not agree with a previously reported X-ray crystal structure. To clarify the discrepancy, we performed photo- CIDNP NMR in combination with MALDI-TOF MS. The photo-CIDNP results revealed that the Tyr 13 aromatic ring is concealed in a hydrophobic interaction. MALDI-TOF MS experiments showed this is an intramolecular interaction in monomeric form, which is also supported by sedimentation analysis and two-dimensional NMR cross-peak line shapes. Thus, we confirmed the intramolecular hydrophobic core around Tyr 13 in aqueous solution, which agrees with the solution structure. The C-terminal conformational discrepancy between the solution and crystal was caused by the intermolecular hydrogen bond between Tyr 13 of one molecule and Asp 8 of the other in a dimer-like formation of crystalline ET-1. On the other hand, we indicated that endothelin-3, another isoform of the endothelin, has an apparent self-association equilibrium under the same condition in which three tyrosines participate. Human endothelin-1 (ET-1) 1 is a potent cardiovascular bioactive peptide (1). Because of its complex bioactivities related to heart failure, hypertension, angiogenesis, cancer, etc. (2-5), ET-1 has been an important target for drug discovery at many pharmaceutical companies. The three- dimensional structures of ET-1 have been determined by X-ray crystallography and NMR spectroscopy (6-10); however, there was a large conformational discrepancy in the C-terminal part, which is conserved in the endothelin family and is unambiguously critical for the bioactivities ( 11). The discrepancy mainly resulted from structural dispersion of the part in the solution structures ( 7-10). Therefore, for years, only an ET-1 crystal structure (6) has been utilized in the drug design process for the ET-1 receptor antagonists. The most important pharmacophore of ET-1 is a residue

The collagen model peptide (Pro-Pro-Gly)10 is known to fold into a triple helix in solution. So far, the triple helix has been considered to exist as a single state. However, our previous study of (Pro-Pro-Gly)10 in solution has indicated the presence of two different states of the triple helix, a lower (HL) and a higher temperature state (HH). In the present study, these triple-helical states were investigated in more detail by NMR. Complete stereospecific assignments of the methylene protons of the proline residues were accomplished by the use of NOESY and TOCSY spectra. The temperature dependence of the 1H chemical shifts showed that the HL-to-HH thermal transition can be attributed to a conformational change of the first proline (Pro1) residues of the (Pro-Pro-Gly) triplets. Since TOCSY spectra with a 10 ms mixing-time confirmed a down puckering of these Pro residues in the HL state, but interconverting down and up puckerings in the HH state, the HL-to-HH thermal transition corresponds to conformational changes of the pyrrolidine rings of the Pro1 residues from an uniform down puckering to a more flexible state. The results confirm that thermal unfolding of the triple helix proceeds through the intermediate HH state.

The minimum size of a closed nano-space in which cells can survive was determined using 4-nl nanowells. One or two cells could divide in the nanowell. Our results suggest that the cell division activity in the nano-space is determined by the conflict between intercellular effects and consumption of substrates.

The flow cytometry and chromosome imaging method were jointly used for analyzing genome content and chromosomal DNA content of hexaploid wheat (AABBDD), hexaploid triticale (AABBRR), tetraploid wheat (AABB), and AA, BB, DD genome donors and RR genome rye. Their genome sizes were 34.4 pg, 40.9 pg, 26.2 pg, 12.1 pg, 13.7 pg, 10.5 pg, and 16.9 pg, respectively. The 2C nuclear DNA content of BB genome donor with 13.7 pg was the highest value among the other genome donors, AA or DD. The genome content of tetraploid wheat, unlike hexaploid wheat or hexaploid triticale, was larger than the sum of the genomes of AA and BB genome donors. The DNA content of each chromosome ranged from 1.22 pg in DD genome donor to 2.61 pg in rye. Each chromosome peak was divided into three to four groups. Only one chromosome was included in the highest chromosomal DNA peak in hexaploid wheat, tetraploid wheat, DD genome donor and rye but two chromosomes in AA, BB genome donors, and hexaploid triticale. Correlation between 2C nuclear DNA content and chromosome density volume was the highest value compared with the other chromosomal parameters of chromosome area, or chromosome length.

The backbone dynamics of ribosome recycling factor (RRF) from Escherichia coli in water were characterized by (15)N NMR relaxation analysis and molecular dynamics (MD) simulation. RRF is composed of two domains connected by a joint region that consists of two peptide chains, such that the overall structure seems to mimic that of tRNA. MD trajectories indicated that the relative orientation of domains varies on the nanosecond time scale. We analyzed the observed (15)N T(1), T(2), and NOE using an extended model-free spectral density function in which the domain motions with a nanosecond time scale were considered. At 30 degrees C, the order parameters of slow motion () were determined to be approximately 0.9 for domain I and 0.7 for domain II, respectively. These values indicate that domain I is nearly fixed on the molecular diffusion frame, and domain II is wobbling in a cone for which the semi-angle is about 30 degrees.

The human cell cycle was synchronized and the chromosomes were isolated by a centrifugation method using two representative solutions for chromosome isolation (a polyamine buffer, PAB and citric acid solution, CAS) and fixatives. The centrifugation method yielded sufficient amounts of human metaphase chromosomes. Observation of the isolated chromosomes by scanning electron microscopy (SEM) revealed two types of surface structure which have been repeatedly reported to date: the human chromosomes in the PAB were relatively smooth but covered irregularly with scaly structures, while the surface of the chromosomes in the CAS exhibited a dense fibrous structure with a uniform diameter of 50-70 nm. Comparison of proteins extracted from chromosomes isolated with the PAB and CAS clearly indicated the removal of linker histones, H1, from chromosomes isolated with the CAS. These findings imply that the two different images of human chromosomes frequently observed by SEM are due to the removal of peripheral chromosomal materials including linker histones and/or the depletion of linker histones which prevent the surface chromatin fibers from scattering.

Thermal stability has been investigated for the structure and the perpendicular anisotropy Ku of Nd44Gd56/Fe89Co11 multilayer films with the bilayer period of 1 nm, where Ku becomes the maximum. From the results of isothermal annealing at temperature Ta, it has been found that the saturation magnetization Ms scarcely changes up to Ta=400 °C, while Ku is almost constant up to Ta=200 °C but decreases rather quickly for Ta above 200 °C. The decrease in Ku for Ta≳260 °C is accompanied by the decrease in the peak intensity of low angle x‐ray diffraction, namely by the deterioration of the layered structure.

A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.

Paramagnetic NMR and optical studies of the oxidized forms of mesophile Pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (F7A/V13M/F34Y/E43Y/V78I), and thermophile Hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins. The strength of heme axial coordinations was found to correlate with the overall protein thermostability.

Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.

Recording properties have been investigated for a magnetooptical disk made of Pt/Co multilayers. The disk exhibits good recording properties for both light intensity modulation and field modulation methods. Reasonable recording properties, namely a carrier-to-noise ratio (CNR) of 46 dB and a block error rate of 1.9%, have been obtained for the field modulation method which seems to satisfy usual standards. However, in the case of low modulation fields, the Pt/Co disk exhibits lower CNR than conventional disks made of rare earth-transition metal amorphous films because of lower recording carrier. >

The effect of buffer layers on giant magnetoresistance has been investigated for Ni66Fe16Co18/Cu multilayers with Fe, FeCo, NiFeCo and CoZr buffer layers. The structure and the antiferromagnetic coupling strength of the multilayers were found to depend on the crystal structure of buffer layers. At a Cu layer thickness of 2.2 nm, which correponds to the 2nd peak of MR ratio, the multilayers with NiFeCo and CoZr buffer layers exhibited much smaller saturation fields than those with Fe and FeCo buffer layers.

The structural and magnetic properties of FeCo/Zr multilayers prepared by the rf sputtering method have been investigated. The multilayers with bilayers of Fe 70 Co 30 (2.5 nm)/Zr(1–2 nm) exhibited a coercive force as low as 0.1 Oe and a uniaxial anisotropy as small as 0.2 Oe after annealing at 500°C in a field parallel to the easy axis.