Biomedical subjects

Susumu Uchiyama

Publications and source records attributed to Susumu Uchiyama.

At least 19 recordsLinked to original sources

PubMed2026

Impact of transfection optimization on adeno-associated virus purification performance and vector quality.

Recombinant adeno-associated virus (rAAV) has shown great promise as a viral vector for gene therapy. However, efficient manufacture of high-quality rAAV to meet clinical demands remains challenging. Here, we optimized rAAV production via a design of experiments (DoE) approach to evaluate the effects of five transfection parameters on vector genome titer, full particle ratio (FE ratio), and viral protein (VP) stoichiometry. The DoE model showed that no single set of transfection conditions could maximize all three responses simultaneously. Subsequently, the materials from DoE-optimized transfections were purified by affinity chromatography followed by anion exchange chromatography (AEX). We observed that AEX recovery declined when capsid loading was high, a situation caused by the combination of high titer with low FE ratio, reducing some of the gains in titer achieved by transfection optimization. Furthermore, AEX had limited capacity to enrich full particles from materials with a very low initial FE ratio. Finally, materials from DoE-optimized transfections showed improved transduction efficiency, which was associated with the ratios of VP1 and VP2 to total VP in the capsid. Overall, this study demonstrates that transfection-derived quality attributes affect purification outcomes and overall vector quality. Our findings highlight the necessity of strategically balancing multiple quality attributes during transfection optimization and provide insights for integrated upstream transfection and downstream purification development.

Adeno-associated virus↗

PubMed2007

Characterization of a splicing variant of plant Aurora kinase.

Aurora kinases play a key role in chromosome segregation and cytokinesis. In plants, three Aurora kinases (AtAUR1-AtAUR3) have been identified in Arabidopsis thaliana. Here, we report an AtAUR2 splicing variant (AtAUR2S), which lacks the fourth exon encoding a part of the kinase domain of AtAUR2. AtAUR2S was shown to have lost its kinase activity to phosphorylate histone H3 at Ser10; however, it maintained its ability to bind to histone H3. The localization pattern of AtAUR2S was the same as that of AtAUR2. The findings suggest that AtAUR2S affects cell division by competing with AtAUR2.

Amino Acid Sequence↗

PubMed2006

Apo- and holo-structures of 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831. Loop-helix transition induced by coenzyme binding.

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.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗

PubMed2006

Crystallization and preliminary X-ray analysis of the complex of NADH and 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831.

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.

3-Hydroxysteroid Dehydrogenases↗

PubMed2006

Structure of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus: comparative study on the thermostability of cytochrome c.

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.

Amino Acid Sequence↗

PubMed2006

Aurora kinase is required for chromosome segregation in tobacco BY-2 cells.

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.

Arabidopsis Proteins↗

PubMed2006

Regional and segmental flexibility of antibodies in interaction with antigens of different size.

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.

Animals↗

PubMed2005

Effect of hydration on the stability of the collagen-like triple-helical structure of [4(R)-hydroxyprolyl-4(R)-hydroxyprolylglycine]10.

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.

Circular Dichroism↗

PubMed2005

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse.

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.

Animals↗

PubMed2005

Characterization and dynamic analysis of Arabidopsis condensin subunits, AtCAP-H and AtCAP-H2.

Condensin complexes are thought to play essential roles in mitotic chromosome assembly and segregation in eukaryotes. To date, two condensin complexes (condensin I and II) have been identified. Both complexes contain two structural maintenance of chromosome (SMC) subunits and three non-SMC subunits. In plants, little is known about the localization and function of all the condensin subunits. Here, we report the analyses on the localization of a non-SMC subunit of Arabidopsis condensin I and II, AtCAP-H, and AtCAP-H2, respectively. Our study indicated that localization of AtCAP-H and AtCAP-H2 is dynamically changed through the mitotic cell cycle using GFP-tagged AtCAP-H and AtCAP-H2 in tobacco cultured cells. They are localized at mitotic chromosomes from prometaphase to telophase. However, their localization in interphase is quite different. AtCAP-H was mainly found in the cytoplasm whereas AtCAP-H2 was mainly found in a nucleolus. It is revealed using GFP-tagged deletion mutant s of AtCAP-H that the kleisin-gamma middle domain (GM domain) is a unique domain only in AtCAP-H, responsible for chromosomal localization. We propose that the GM domain of CAP-H is essential for its chromosomal localization at mitosis and thus proper function of CAP-H. Differences in localization of AtCAP-H and AtCAP-H2 at interphase also suggest their functional differentiation.

Adenosine Triphosphatases↗

PubMed2005

Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix.

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.

Amino Acid Sequence↗

PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of a conserved domain in plants and prokaryotes from Pyrococcus horikoshii OT3.

A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 53.69, c = 159.2 A. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 A. A complete data set from crystals of the SeMet-substituted protein was also obtained.

Amino Acid Motifs↗

PubMed2005

Cloning, expression, crystallization and preliminary X-ray characterization of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus.

The amino-acid sequence of cytochrome c552 (PH c552) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c552), and from a mesophile, Pseudomonas aeruginosa (PA c551). The PH c552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C222(1), 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.

Cloning, Molecular↗

PubMed2005

Proteome analysis of human metaphase chromosomes.

DNA is packaged as chromatin in the interphase nucleus. During 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 six 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 framework for future research on this topic.

Chromatin↗

PubMed2005

Collagen-like triple helix formation of synthetic (Pro-Pro-Gly)10 analogues: (4(S)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10, (4(R)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10 and (4(S)-fluoroprolyl-4(R)-fluoroprolyl-Gly)10.

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 Hyp(R), Hyp(S), fPro(R) or fPro(S). 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 (Hyp(S)-Hyp(R)-Gly)(10) and (fPro(S)-fPro(R)-Gly)(10) were less stable than those of (Pro-Hyp(R)-Gly)(10) and (Pro-fPro(R)-Gly)(10), respectively. As reported by Bächinger's and Zagari's groups, (Hyp(R)-Hyp(R)-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-Hyp(R)-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.

Collagen↗

PubMed2005

Characterization of plant Aurora kinases during mitosis.

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.

Amino Acid Sequence↗

PubMed2005

Fluorescent labeling of plant chromosomes in suspension by FISH.

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.

Chromosomes, Plant↗

PubMed2004

Five amino acid residues responsible for the high stability of Hydrogenobacter thermophilus cytochrome c552: reciprocal mutation analysis.

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.

Amino Acids↗