186 results on '"Mizuno, Kazunori"'
Search Results
152. Peculiar Effect of Urea on the Interaction of Type I Collagen with Heparin on Chromatography
- Author
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Mizuno, Kazunori, primary and Hayashi, Toshihiko, additional
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- 1994
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153. Crystal structure of the collagen model peptide (Pro-Pro-Gly)4-Hyp-Asp-Gly-(Pro-Pro-Gly)4 at 1.0 Å resolution.
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Okuyama, Kenji, Kawaguchi, Tatsuya, Shimura, Masaki, Noguchi, Keiichi, Mizuno, Kazunori, and Bächinger, Hans Peter
- Abstract
The single-crystal structure of the collagen-like peptide (Pro-Pro-Gly)
4 -Hyp-Asp-Gly-(Pro-Pro-Gly)4 , was analyzed at 1.02 Å resolution. The overall average helical twist (θ = 49.6°) suggests that this peptide adopts a 7/2 triple-helical structure and that its conformation is very similar to that of (Gly-Pro-Hyp)9 , which has the typical repeating sequence in collagen. High-resolution studies on other collagen-like peptides have shown that imino acid-rich sequences preferentially adopt a 7/2 triple-helical structure (θ = 51.4°), whereas imino acid-lean sequences adopt relaxed conformations (θ < 51.4°). The guest Gly-Hyp-Asp sequence in the present peptide, however, has a large helical twist (θ = 61.1°), whereas that of the host Pro-Pro-Gly sequence is small (θ = 46.7°), indicating that the relationship between the helical conformation and the amino acid sequence of such peptides is complex. In the present structure, a strong intermolecular hydrogen bond between two Asp residues on the A and B strands might induce the large helical twist of the guest sequence; this is compensated by a reduced helical twist in the host, so that an overall 7/2-helical symmetry is maintained. The Asp residue in the C strand might interact electrostatically with the N-terminus of an adjacent molecule, causing axial displacement, reminiscent of the D-staggered structure in fibrous collagens. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 436-447, 2013. [ABSTRACT FROM AUTHOR]- Published
- 2013
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154. Fragility of Reconstituted Type V Collagen Fibrils with the Chain Composition of α1(V)α2(V)α3(V) Respective of the D-Periodic Banding Pattern.
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Mizuno, Kazunori, Bächinger, Hans Peter, Imamura, Yasutada, Hayashi, Toshihiko, and Adachi, Eijiro
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The triple-helical domains of two subtypes of type V collagen were prepared from human placenta, one with the chain composition of [α1(V)]2α2(V) (Vp112) and the other with the chain composition of α1(V)α2(V)α3(V) (Vp123) with limited pepsin treatment. In order to characterize the triple-helical domain of the type Vp123 collagen molecule, the reconstituted aggregate structure formed from the pepsin-treated collagen was compared by using transmission electron microscopy. The diameter of the fibrils reconstituted from types pepsin-treated type Vp123 collagen and type Vp112 collagen was highly uniform and less than the D-periodicity at all the temperatures examined, suggesting that the major triple-helical domain of both subtypes has a potency to limit their lateral growth. Both fibrils were approximately 45 nm in width and showed the D-periodic banding pattern along their axes at 34°C. In contrast to type Vp112, the reconstituted type Vp123 fibrils showed no banding pattern along their axes when they were reconstituted at 37°C. The banded fibrils once reconstituted from type Vp123 at 34°C tend to lose their characteristic pattern within 60 min when they were incubated at 37°C. One explanation is that a slightly higher content of hydrophobic residues of type Vp123 collagen than those of type V112p collagen augmented the intermolecular interaction that disturbs the D-periodicity governed essentially by electrostatic interactions. Taken together with recent data in Col5a3 gene-targeted mice, the results suggest that type V123 collagen exists not only as a periodic banded fibril but also as nonfibrillar meshwork structures. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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155. Crystal structure of (Gly-Pro-Hyp)9: Implications for the collagen molecular model.
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Okuyama, Kenji, Miyama, Keita, Mizuno, Kazunori, and Bächinger, Hans Peter
- Abstract
Collagens have long been believed to adopt a triple-stranded molecular structure with a 10/3 symmetry (ten triplet units in three turns) and an axial repeat of 29 Å. This belief even persisted after an alternative structure with a 7/2 symmetry (seven triplet units in two turns) with an axial repeat of 20 Å had been proposed. The uncertainty regarding the helical symmetry of collagens is attributed to inadequate X-ray fiber diffraction data. Therefore, for better understanding of the collagen helix, single-crystal analyses of peptides with simplified characteristic amino acid sequences and similar compositions to collagens have long been awaited. Here we report the crystal structure of (Gly-Pro-Hyp)
9 peptide at a resolution of 1.45 Å. The repeating unit of this peptide, Gly-Pro-Hyp, is the most typical sequence present in collagens, and it has been used as a basic repeating unit in fiber diffraction analyses of collagen. The (Gly-Pro-Hyp)9 peptide adopts a triple-stranded structure with an average helical symmetry close to the ideal 7/2 helical model for collagen. This observation strongly suggests that the average molecular structure of collagen is not the accepted Rich and Crick 10/3 helical model but is a 7/2 helical conformation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 607-616, 2012. [ABSTRACT FROM AUTHOR]- Published
- 2012
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156. The effect of deuterium oxide on the stability of the collagen model peptides H-(Pro-Pro-Gly)10-OH, H-(Gly-Pro-4(R)Hyp)9-OH, and Type I collagen.
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Mizuno, Kazunori and Bächinger, Hans Peter
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- 2010
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157. Two crystal modifications of (Pro-Pro-Gly)4-Hyp- Hyp-Gly-(Pro-Pro-Gly)4 reveal the puckering preference of Hyp(X) in the Hyp(X):Hyp(Y) and Hyp(X):Pro(Y) stacking pairs in collagen helices.
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Okuyama, Kenji, Morimoto, Tatsuya, Narita, Hirotaka, Kawaguchi, Tatsuya, Mizuno, Kazunori, Bächinger, Hans Peter, Guanghan Wu, and Noguchi, Keiichi
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MOLECULAR structure ,CRYSTALLOIDS (Botany) ,PEPTIDES ,PROLINE hydroxylase ,TWINNING (Crystallography) ,CRYSTAL growth ,MOLECULAR association ,HELICES (Algebraic topology) - Abstract
Two crystal modifications of a collagen model peptide, (Pro-Pro-Gly)
4 -Hyp-Hyp-Gly-(Pro-Pro-Gly)4 [where Hyp is (4R,2S)-L-hydroxyproline], showed very similar unit-cell parameters and belonged to the same space group P21 . Both crystals exhibited pseudo-merohedral twinning. The main difference was in their molecular-packing arrangements. One modification showed pseudo-hexagonal packing, while the other showed pseudo-tetragonal packing. Despite their different packing arrangements, no significant differences were observed in the hydration states of these modifications. The peptide in the pseudo-tetragonal crystal showed a cyclic fluctuation of helical twists with a period of 20 Å, while that in the pseudo-hexagonal crystal did not. In these modifications, the puckering conformations of four of the 12 Hyp residues at the X position of the Hyp(X)-Hyp(Y)-Gly sequence were in the opposite conformations to the previous hypothesis that Hyp(X) residues involved in Hyp(X):Hyp(Y) and Hyp(X): Pro(Y) stacking pairs prefer up-puckering and down-puckering conformations, respectively. Detailed investigation of the molecular interactions between Hyp(X) and adjacent molecules revealed that these opposite conformations appeared because the puckering conformation, which follows the hypothesis, is subject to steric hindrance from the adjacent molecule. [ABSTRACT FROM AUTHOR]- Published
- 2010
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158. High-resolution structures of collagen-like peptides [(Pro-Pro-Gly)4-Xaa-Yaa-Gly-(Pro-Pro-Gly)4]: Implications for triple-helix hydration and Hyp(X) puckering.
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Okuyama, Kenji, Hongo, Chizuru, Wu, Guanghan, Mizuno, Kazunori, Noguchi, Keiichi, Ebisuzaki, Shutoku, Tanaka, Yuji, Nishino, Norikazu, and Bächinger, Hans Peter
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- 2009
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159. Effect of the -Gly-3( S)-hydroxyprolyl-4( R)-hydroxyprolyl- tripeptide unit on the stability of collagen model peptides.
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Mizuno, Kazunori, Peyton, David H., Hayashi, Toshihiko, Engel, Jürgen, and Bächinger, Hans Peter
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PEPTIDES , *HYDROXYLATION , *COLLAGEN , *THERMAL analysis , *DYNAMICS , *TEMPERATURE measurements - Abstract
In order to evaluate the role of 3( S)-hydroxyproline [3( S)-Hyp] in the triple-helical structure, we produced a series of model peptides with nine tripeptide units including 0–9 3( S)-hydroxyproline residues. The sequences are H-(Gly-Pro-4( R)Hyp) l-(Gly-3( S)Hyp-4( R)Hyp) m-(Gly-Pro-4( R)Hyp) n-OH, where ( l, m, n) = (9, 0, 0), (4, 1, 4), (3, 2, 4), (3, 3, 3), (1, 7, 1) and (0, 9, 0). All peptides showed triple-helical CD spectra at room temperature and thermal transition curves. Sedimentation equilibrium analysis showed that peptide H-(Gly-3( S)Hyp-4( R)Hyp)9-OH is a trimer. Differential scanning calorimetry showed that replacement of Pro residues with 3( S)Hyp residues decreased the transition enthalpy, and the transition temperature increases by 4.5 °C from 52.0 °C for the peptide with no 3( S)Hyp residues to 56.5 °C for the peptide with nine 3( S)Hyp residues. The refolding kinetics of peptides H-(Gly-3( S)Hyp-4( R)Hyp)9-OH, H-(Gly-Pro-4( R)Hyp)9-OH and H-(Gly-4( R)Hyp-4( R)Hyp)9-OH were compared, and the apparent reaction orders of refolding at 10 °C were n = 1.5, 1.3 and 1.2, respectively. Replacement of Pro with 3( S)Hyp or 4( R)Hyp has little effect on the refolding kinetics. This result suggests that the refolding kinetics of collagen model peptides are influenced mainly by the residue in the Yaa position of the -Gly-Xaa-Yaa- repeated sequence. The experiments indicate that replacement of a Pro residue by a 3( S)Hyp residue in the Xaa position of the -Gly-Xaa-4( R)Hyp- repeat of collagen model peptides increases the stability, mainly due to entropic factors. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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160. Chapter 3 - Regulation of Phenotypes of Human Aorta Endothelial Cells and Smooth Muscle Cells in Culture by Type IV Collagen Aggregates
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Hayashi, Toshihiko, Hirose, Motohiro, Yamano, Hiroko, Takeda, Yasushi, Kosugi, Hiroaki, Kihara, Takanori, Imamura, Yasutada, Mizuno, Kazunori, Nakazato, Koichi, Yoshikawa, Kiwamu, Kajimura, Daisuke, Takahashi, Seiichiro, and Adachi, Eijiro
- Published
- 2003
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161. Threonine in Collagen Triple-helical Structure.
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Jiravanichanun, Nattha, Mizuno, Kazunori, Bächinger, Hans Peter, and Okuyama, Kenji
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COLLAGEN ,AMINO acids ,PEPTIDES ,HYDROGEN ,NONMETALS - Abstract
The cuticle collagen of some invertebrates form the stable triple-helix despite the low content of imnio acid residues and the unusual high content of Thr residues in the Y position of the repeated -Xaa-Yaa-Gly- sequences, The crystal structure of HqPro-Pro-Gly)
4 (R)Hyp-Thr-Gly-(Pro-Pro-Gly)4 -OH peptide was analyzed by X-ray diffraction method. The side-chain conformation of Thr and the water-mediated hydrogen bond patterns are shown. The OH group of Thr acts like that of 4(R)Hyp and makes the similar water-mediated hydrogen bond patterns as 4(R)Hyp in a triple-helix [ABSTRACT FROM AUTHOR]- Published
- 2006
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162. The Crystal Structure of the Collagen-like Polypeptide (Glycyl-4(R)-hydroxyprolyl-4(R)-hydroxyprolyl)9 at 1.55 Å Resolution Shows Up-puckering of the Proline Ring in the Xaa Position.
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Schumacher, Maria, Mizuno, Kazunori, and Bächinger, Hans Peter
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PEPTIDES , *COLLAGEN , *PROLINE , *EXTRACELLULAR matrix proteins , *THERMODYNAMICS , *CONNECTIVE tissues - Abstract
The collagen triple helix is characterized by the repeating sequence motif Gly-Xaa-Yaa, where Xaa and Yaa are typically proline and (2S,4R)-4-hydroxyproline (4(R)Hyp), respectively. Previous analyses have revealed that H-(Pro-4(R)Hyp-Gly)10-OH forms a stable triple helix, whereas H-(4(R)Hyp-Pro-Gly)10-OH does not. Several theories have been put forth to explain the importance of proline puckering and conformation in triple helix formation; however, the details of how they affect triple helix stability are unknown. Underscoring this, we recently demonstrated that the polypeptide Ac(Gly-4(R)Hyp-4(R)Hyp)10-NH2 forms a triple helix that is more stable than Ac-(Gly-Pro-4(R)Hyp)10-NH2. Here we report the crystal structure of the H-(Gly-4(R)Hyp4(R)Hyp)9-OH peptide at 1.55 ρ resolution. The puckering of the Yaa position 4(R)Hyp in this structure is up (Cγ exo), as has been found in other collagen peptide structures. Notably, however, the 4(R)Hyp in the Xaa position also takes the up pucker, which is distinct from all other collagen structures. Regardless of the notable difference in the Xaa proline puckering, our structure still adopts a 7/2 superhelical symmetry similar to that observed in other collagen structures. Thus, the basis for the observed differences in the thermodynamic data of the triple helix↔ coil transition between our peptide and other triple helical peptides likely results from contributions from the unfolded state. Indeed, the unfolded state of the H-(Gly-4(R)Hyp-4(R)Hyp)9-OH peptide seems to be stabilized by a preformed polyproline II helix in each strand, which could be explained by the presence of a unique repeating intra-strand water-mediated bridge observed in the H-(Gly-4(R)Hyp-4(R)Hyp)9-OH structure, as well as a higher amount of trans peptide bonds. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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163. The Peptides Acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 and Acetyl-(Gly-Pro-3(S)Hyp)10-NH2 Do Not Form a Collagen Triple Helix.
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Mizuno, Kazunori, Hayashi, Toshihiko, Peyton, David H., and Bächinger, Hans Peter
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COLLAGEN , *PEPTIDES , *EXTRACELLULAR matrix proteins , *PROTEINS , *CIRCULAR dichroism , *MAGNETIC resonance imaging - Abstract
Hydroxylation of proline residues in the Yaa position of the Gly-Xaa-Yaa repeated sequence to 4(R)-hydroxyproline is essential for the formation of the collagen triple helix. A small number of 3(S)-hydroyxyproline residues are present in most collagens in the Xaa position. Neither the structural nor a biological role is known for 3(S)hydroxyproline. To characterize the structural role of 3(S)-hydroxyproline, the peptide Ac-(Gly-3(S)Hyp4(R)Hyp)10-NH2 was synthesized and analyzed by circular dichroism spectroscopy, analytical ultracentrifugation, and ¹H nuclear magnetic resonance spectroscopy. At 4 °C in water the circular dichroism spectrum indicates that this peptide was in a polyproline-II-like secondary structure with a positive peak at 225 nm similar to Ac-(Gly-Pro4(R)Hyp)10-NH2. The positive peak at 225 nm almost linearly decreases with increasing temperature to 95 °C without an obvious transition. Although the peptide Ac(Gly-Pro-4(R)Hyp)10-NH2 forms a trimer at 10 °C, sedimentation equilibrium experiments indicate that Ac-(Gly3(S)Hyp-4(R)Hyp)10-NH2 is a monomer in water at 7 °C. To study the role of 3(S)-hydroxyproline in the Yaa position, we synthesized Ac-(Gly-Pro-3(S)Hyp)10-NH2. This peptide also does not form a triple helix in water. ¹H Nuclear magnetic resonance spectroscopy data (including line widths and nuclear Overhauser effects) are entirely consistent, with neither Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 nor Ac-(Gly-Pro-3(S)Hyp)10-NH2 forming a triple helix in water. Therefore 3(S)-hydroxyproline destabilizes the collagen triple helix in either position. In contrast, when 3(S)-hydroxyproline is inserted as a guest in the highly stable-Gly-Pro-4(R)Hyp- repeated host sequence, Ac-(GlyPro-4(R)Hyp)3-Gly-3(S)Hyp-4(R)Hyp-(Gly-Pro-4(R)HyP)4Gly-Gly-NH2 forms as stable a trimer (Tm = 49.6 °C) as Ac-(Gly-Pro-4(R)Hyp)8-Gly-Gly-NH2 (Tm = 48.9 °C). Given that Ac-(Gly-Pro-4(R)Hyp)3-Gly-4(R)Hyp-Pro-(Gly-Pro4(R)Hyp)4-Gly-Gly-NH2 forms a triple helix nearly as stable as the above two peptides (Tm = 45.0 °C) and the knowledge that Ac-(Gly-4(R)Hyp-Pro)10-NH2 does not form a triple helix, we conclude that the host environment dominates the structure of host-guest peptides and that these peptides are not necessarily accurate predicters of triple helical stability. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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164. Separation of the Subtypes of Type V Collagen Molecules, [αl(V)]2α2(V) and α1(V)α2(V)α3(V), by Chain Composition-Dependent Affinity for Heparin: Single α1(V) Chain Shows Intermediate Heparin Affinity between Those of the Type V Collagen Subtypes ...
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Mizuno, Kazunori and Hayashi, Toshihiko
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- 1996
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165. CD8αα memory effector T cells descend directly from clonally expanded CD8α+βhighTCRαβ T cells in vivo
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Konno, Akihiro, Okada, Kanae, Mizuno, Kazunori, Nishida, Mika, Nagaoki, Shuya, Toma, Tomoko, Uehara, Takahiro, Ohta, Kazuhide, Kasahara, Yoshihito, Seki, Hidetoshi, Yachie, Akihiro, and Koizumi, Shoichi
- Abstract
Whereas most peripheral CD8+αβ T cells highly express CD8αβ heterodimer in healthy individuals, there is an increase of CD8α+βlowor CD8αα αβ T cells in HIV infection or Wiskott-Aldrich syndrome and after bone marrow transplantation. The significance of these uncommon cell populations is not well understood. There has been some question as to whether these subsets and CD8α+βhighcells belong to different ontogenic lineages or whether a fraction of CD8α+βhighcells have down-regulated CD8β chain. Here we assessed clonality of CD8αα and CD8α+βlowαβ T cells as well as their phenotypic and functional characteristics. Deduced from surface antigens, cytotoxic granule constituents, and cytokine production, CD8α+βlowcells are exclusively composed of effector memory cells. CD8αα cells comprise effector memory cells and terminally differentiated CD45RO−CCR7−memory cells. T-cell receptor (TCR) Vβ complementarity-determining region 3 (CDR3) spectratyping analysis and subsequent sequencing of CDR3 cDNA clones revealed polyclonality of CD8α+βhighcells and oligoclonality of CD8α+βlowand CD8αα cells. Importantly, some expanded clones within CD8αα cells were also identified within CD8α+βhighand CD8α+βlowsubpopulations. Furthermore, signal-joint TCR rearrangement excision circles concentration was reduced with the loss of CD8β expression. These results indicated that some specific CD8α+βhighαβ T cells expand clonally, differentiate, and simultaneously down-regulate CD8β chain possibly by an antigen-driven mechanism. Provided that antigenic stimulation directly influences the emergence of CD8αα αβ T cells, these cells, which have been previously regarded as of extrathymic origin, may present new insights into the mechanisms of autoimmune diseases and immunodeficiencies, and also serve as a useful biomarker to evaluate the disease activities.
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- 2002
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166. Gravimetric determination of cadmium with ο-phenanthroline and iodide
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YOSHIDA, Hitoshi, primary, MIZUNO, Kazunori, additional, TAGA, Mitsuhiko, additional, and HIKIME, Seiichiro, additional
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- 1976
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167. Comment on Microfibrillar structure of type I collagen in situ by Orgel et al. (2006), Proc. Natl Acad. Sci. USA, 103, 9001-9005.
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Okuyama, Kenji, Bächinger, Hans Peter, Mizuno, Kazunori, Boudko, Sergei, Engel, Jürgen, Berisio, Rita, and Vitagliano, Luigi
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LETTERS to the editor ,COLLAGEN - Abstract
A letter to the editor is presented in response to the article "Microfibrillar Structure of Type 1 Collagen in Situ," published in the "Proceedings of the National Academy of Sciences."
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- 2009
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168. Analysis of Phase Transitions in Graph-Coloring Problems Based on Constraint Structures.
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Goos, G., Hartmanis, J., van Leeuwen, J., Mizoguchi, Riichiro, Slaney, John, Carbonell, Jaime G., Siekmann, Jörg, Mizuno, Kazunori, Hayashimoto, Atsuki, and Nishihara, Seiichi
- Abstract
Situations very similar to phase transitions (PTs) have been observed in constraint satisfaction problems. In our analysis, applying the backtracking-based tree search algorithm with Brélaz heuristic to the graph-coloring problems with three colors (3GCPs), we first reconfirmed PT phenomena. We then traced the backtracking history for variables for which thrashing appears to occur in the hardest problems. As a result, we found a local key structure of a graph, or a rigid pair, which is a pair of nodes to each of which the same color must be assigned, and is included in a subgraph such as Fig. l(a) in 3GCPs. We found many rigid pairs in extraordinarily hard problems, in which very heavy trial-and-error repetitions of coloring are performed until the same color is assigned to rigid pairs. [ABSTRACT FROM AUTHOR]
- Published
- 2000
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169. Reconstitution of banding fibrils from the subtypes of type V collagen with the chain compositions of [α1(V)I 2 α2(V) and α1(V)α2(V)α3(V)
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Mizuno, Kazunori, Adachi, Eijiro, and Hayashi, Toshihiko
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- 1997
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170. Crystal Structure of Human Type III Collagen Gly991-Gly1032 Cystine Knot-containing Peptide Shows Both 7/2 and 10/3 Triple Helical Symmetries.
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Boudko, Sergei P., Engel, Jürgen, Okuyama, Kenji, Mizuno, Kazunori, Bächinger, Hans Peter, and Schumacher, Maria A.
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COLLAGEN , *PEPTIDES , *EHLERS-Danlos syndrome , *EXTRACELLULAR matrix proteins , *AMINO acids - Abstract
Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal cystine knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)n or (Gly-Pro-Hyp)n. To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal cystine knot region, to 2.3Å resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly991-Gly1032 structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the cystine knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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171. Effect of silibinin on ethanol- or acetaldehyde-induced damge of mouse primary hepatocytes in vitro.
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Song XY, Li RH, Liu WW, Hayashi T, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
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- Animals, Apoptosis drug effects, Autophagy drug effects, Cell Survival drug effects, Cells, Cultured, Mice, Acetaldehyde toxicity, Ethanol toxicity, Hepatocytes drug effects, Protective Agents pharmacology, Silybin pharmacology
- Abstract
Silibinin, one of the flavonoids isolated from milk thistle seeds of Silybum marianum, has hepatoprotective properties against toxins in clinical. However, the detailed mechanisms have remained unclear. This study investigates the underlying mechanism of silibinin in the protection against ethanol- or acetaldehyde-induced damage of neonatal mouse primary hepatocytes in vitro. The results show that ethanol inhibited proliferation of hepatocytes in a time (12, 24, 36 h) and dose-dependent (0-800 mM) manner. However, silibinin did not show protective effect on ethanol (500 mM)-induced suppression of hepatocyte proliferation. Acetaldehyde, the toxic metabolite of ethanol, appearing immediately in individuals after drink also inhibited the proliferation of hepatocytes in a dose-dependent (0-12 mM) manner. Surprisingly, silibinin significantly increased the cell viability and reduced the leakage of alanine amino transferase (ALT) and aspartate amino transferase (AST) in acetaldehyde-treated hepatocytes, suggesting that silibinin protected cell injury caused by acetaldehyde treatment. The apoptosis-inducing effect of acetaldehyde was demonstrated by the increased number of cells in sub-G1 phase as well as caspase-3 activation. Further study shows that acetaldehyde induced autophagy in the hepatocytes. The autophagy inhibitors, 3-Methyladenine (3-MA) and chloroquine (CQ), further decreased the viability of cells treated with acetaldehyde, suggesting that autophagy plays a protective role against apoptosis. Consistently, silibinin (20 μM) significantly reduced the activation of caspase 3 or apoptosis and increased the conversion of LC3-I to LC3-II or autophagy. Taken together, it is concluded that silibinin does not repress the ethanol- induced hepatocyte injury, whereas silibinin reduces acetaldehyde-caused hepatocyte injury through down-regulation of apoptosis and up-regulation of autophagy., (Copyright © 2020. Published by Elsevier Ltd.)
- Published
- 2021
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172. 4-Phenylbutyric acid enhances the mineralization of osteogenesis imperfecta iPSC-derived osteoblasts.
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Takeyari S, Kubota T, Ohata Y, Fujiwara M, Kitaoka T, Taga Y, Mizuno K, and Ozono K
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- Cell Differentiation, Child, Preschool, Collagen Type I biosynthesis, Collagen Type I genetics, Collagen Type I metabolism, Endoplasmic Reticulum metabolism, Fibroblasts metabolism, Humans, Mutation, Osteoblasts cytology, Osteogenesis Imperfecta metabolism, Protein Folding, Calcification, Physiologic drug effects, Induced Pluripotent Stem Cells drug effects, Osteoblasts drug effects, Osteogenesis Imperfecta pathology, Phenylbutyrates pharmacology
- Abstract
Osteogenesis imperfecta (OI) is a heritable brittle bone disease mainly caused by mutations in the two type I collagen genes. Collagen synthesis is a complex process including trimer formation, glycosylation, secretion, extracellular matrix (ECM) formation, and mineralization. Using OI patient-derived fibroblasts and induced pluripotent stem cells (iPSCs), we investigated the effect of 4-phenylbutyric acid (4-PBA) on collagen synthesis to test its potential as a new treatment for OI. Endoplasmic reticulum (ER) retention of type I collagen was observed by immunofluorescence staining in OI patient-derived fibroblasts with glycine substitution and exon skipping mutations. Liquid chromatography-mass spectrometry analysis revealed excessive glycosylation of secreted type I collagen at the specific sites in OI cells. The misfolding of the type I collagen triple helix in the ECM was demonstrated by the incorporation of heat-dissociated collagen hybridizing peptide in OI cells. Type I collagen was produced excessively by OI fibroblasts with a glycine mutation, but this excessive production was normalized when OI fibroblasts were cultured on control fibroblast-derived ECM. We also found that mineralization was impaired in osteoblasts differentiated from OI iPSCs. In summary, treatment with 4-PBA normalizes the excessive production of type I collagen, reduces ER retention, partially improves misfolding of the type I collagen helix in ECM, and improves osteoblast mineralization. Thus, 4-PBA may improve not only ER retention, but also type I collagen synthesis and mineralization in human cells from OI patients., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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173. Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.
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Ishikawa Y, Taga Y, Zientek K, Mizuno N, Salo AM, Semenova O, Tufa SF, Keene DR, Holden P, Mizuno K, Gould DB, Myllyharju J, and Bächinger HP
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- Animals, Collagen genetics, Collagen metabolism, Collagen Type I genetics, Collagen Type V genetics, Endoplasmic Reticulum, Rough metabolism, Hydroxylation, Hydroxylysine metabolism, Lysine metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Procollagen-Proline Dioxygenase genetics, Protein Conformation, Protein Processing, Post-Translational genetics, Collagen Type I metabolism, Collagen Type V metabolism, Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase metabolism, Procollagen-Proline Dioxygenase metabolism
- Abstract
Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen., Competing Interests: Conflict of interest The authors declare that they have no competing interests related to this work., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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174. Natural flavonoid silibinin promotes the migration and myogenic differentiation of murine C2C12 myoblasts via modulation of ROS generation and down-regulation of estrogen receptor α expression.
- Author
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Long X, Gao Y, Liu W, Liu X, Hayashi T, Mizuno K, Hattori S, and Ikejima T
- Subjects
- Animals, Antineoplastic Agents, Phytogenic pharmacology, Cell Proliferation, Cells, Cultured, Estrogen Receptor alpha genetics, Estrogen Receptor alpha metabolism, Mice, Myoblasts cytology, Myoblasts drug effects, Cell Differentiation, Cell Movement, Estrogen Receptor alpha antagonists & inhibitors, Gene Expression Regulation drug effects, Muscle Development, Myoblasts physiology, Reactive Oxygen Species metabolism, Silybin pharmacology
- Abstract
Skeletal muscle regeneration is a complex process, involving the proliferation, migration, and differentiation of myoblasts. Recent studies suggest that some natural flavanones stimulate myogenesis. However, the effect of plant estrogen, silibinin, on the regulation of myoblast behaviors is unclarified. In this study, we investigated the effects of silibinin on immortalized murine myoblast C2C12 in the aspects of proliferation, migration, differentiation along with underlying mechanisms. The results show that silibinin at concentrations below 50 μM enhanced the migration and differentiation of C2C12 cells, but had no effect on cell proliferation. Silibinin significantly promoted the production of ROS, which appeared to play important roles in the migration and differentiation of the myoblasts. Interestingly, among ROS, the superoxide anion and hydroxyl radical were associated with the migration, whereas hydrogen peroxide contributed to the myogenic differentiation. We used ER agonist and antagonist to explore whether estrogen receptors (ERs), which are affected by silibinin treatment in the silibinin-enhanced C2C12 migration and differentiation. Migration was independent of ERs, whereas the differentiation was associated with decreased ERα activity. In summary, silibinin treatment increases ROS levels, leading to the promotion of migration and myogenic differentiation. Negative regulation ERα of differentiation but not of migration may suggest that ERα represses hydrogen peroxide generation. The effect of silibinin on myoblast migration and differentiation suggests that silibinin may have therapeutic benefits for muscle regeneration.
- Published
- 2020
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175. Quantitative Analysis of the Positional Distribution of Hydroxyproline in Collagenous Gly-Xaa-Yaa Sequences by LC-MS with Partial Acid Hydrolysis and Precolumn Derivatization.
- Author
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Taga Y, Kusubata M, and Mizuno K
- Subjects
- Amino Acid Sequence, Chromatography, Liquid, Hydrolysis, Mass Spectrometry, Collagen chemistry, Hydrochloric Acid chemistry, Hydroxyproline analysis, Trifluoroacetic Acid chemistry
- Abstract
Collagen is extensively modified by various enzymes, including prolyl hydroxylases. Pro residues at the Yaa position of repeating Gly-Xaa-Yaa amino acid sequences are mostly hydroxylated to 4-hydroxyproline (4Hyp), which is essential for the thermal stability of collagen triple helix. In contrast, Pro residues at the Xaa position are rarely modified to 3Hyp and 4Hyp, the biological function of which is poorly understood. Overall estimation of prolyl hydroxylation with discrimination of the position (Xaa or Yaa) and hydroxylation type (4Hyp or 3Hyp) has been difficult to perform using traditional methods. In the present study, we developed a novel position-specific analytical method featuring LC-MS detection of collagenous Gly-containing dipeptides, including Gly-Pro, Pro-Gly, Gly-4Hyp, Gly-3Hyp, and 4Hyp-Gly, after partial acid hydrolysis and precolumn derivatization using 3-aminopyridyl- N -hydroxysuccinimidyl carbamate (APDS). We performed acid hydrolysis at 55 °C with HCl/trifluoroacetic acid/water (2:1:1, v/v) to avoid peptide inversion and imbalanced peptide generation observed for collagenous model peptides. The positional distribution of Pro, 4Hyp, and 3Hyp can be calculated from the relative concentrations of the APDS-derivatized dipeptides, and in combination with amino acid analysis, we can determine their absolute contents at the Xaa and Yaa positions. Bovine type I, III, and V collagens were analyzed by the established method, and the amount of 4Hyp was higher than that of 3Hyp at the Xaa position in type I and III collagens. In addition, we clearly showed that collagen extracted from earthworm cuticles has an extremely high content of Xaa position 4Hyp, reaching over 10% of the total amino acids.
- Published
- 2020
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176. Silibinin-induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells.
- Author
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Si L, Fu J, Liu W, Hayashi T, Mizuno K, Hattori S, Fujisaki H, Onodera S, and Ikejima T
- Subjects
- Autophagy drug effects, Cell Line, Tumor, Dynamins pharmacology, Gene Knockdown Techniques, Humans, Organelle Biogenesis, Protein Kinases genetics, Quinazolinones pharmacology, Ubiquitin-Protein Ligases genetics, Apoptosis drug effects, Mitochondria metabolism, Mitochondrial Dynamics drug effects, Mitophagy drug effects, Silybin pharmacology
- Abstract
We reported previously that higher doses (150-250 μM) of silibinin enhanced fission and inhibited fusion of mitochondria, accompanying apoptosis of double-positive breast cancer cell line MCF-7 cells and triple-negative breast cancer cell line MDA-MB-231 cells. We report here three important questions yet unclarified in the previous study; 1) Whether enhanced fission of mitochondria by the treatment of silibinin leads to mitophagy, 2) Whether mitophagy positively contributes to apoptosis and 3) Whether estrogen receptor-positive (ER
+ ) MCF-7 cells and estrogen receptor-negative (ER- ) MDA-MB-231 cells are affected in a different way by silibinin treatment, since silibinin often works through ERs signaling pathway. Mitophagy driven by Pink1/Parkin signaling, plays an important role in eliminating damaged mitochondria. Indeed, increased expression of Pink1 and the recruitment of Parkin and LC3-II to mitochondria by the treatment with silibinin account for silibinin induction of mitophagy. In this study, the effects of mitochondrial division inhibitor 1 (mdivi-1) and small interfering RNA targeting dynamin-related protein 1 (DRP1) were examined to reveal the effect of mitochondrial fission on mitophagy. As expected, mdivi-1 or siRNA targeting DRP1 reversed silibinin-induced mitochondrial fission due to down-regulation in the expression of DRP1. Inhibition of mitochondrial fission by mdivi-1 prevented induction of mitophagy as well as autophagy in both MCF-7 and MDA-MB-231 cells, indicating that silibinin-induced mitochondrial fission leads to mitophagy. Inhibition of mitochondrial fission efficiently prevented silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells in our previous work, and the second point of the present study, inhibition of mitophagy by Pink1 or Parkin knockdown increased silibinin-induced apoptosis of these cells, respectively, suggesting that the mitophagy induced by silibinin treatment serves as a cytoprotective effect, resulting in reduction of apoptosis of cancer cells in both cells. In the third point, we studied whether estrogen receptors (ERs) played a role in silibinin-induced mitophagy and apoptosis in MCF-7 and MDA-MB-231 cells. ERα and ERβ are not involved in silibinin-induced mitophagic process in MCF-7 and MDA-MB-231 cells. These findings demonstrated that silibinin induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis not through ERs-Pink1 or -Parkin pathway in MCF-7 and MDA-MB-231., (Copyright © 2020. Published by Elsevier Inc.)- Published
- 2020
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177. Silibinin inhibits migration and invasion of breast cancer MDA-MB-231 cells through induction of mitochondrial fusion.
- Author
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Si L, Fu J, Liu W, Hayashi T, Nie Y, Mizuno K, Hattori S, Fujisaki H, Onodera S, and Ikejima T
- Subjects
- Cell Line, Tumor, Cell Movement, Epithelial-Mesenchymal Transition genetics, Female, Humans, Mitochondrial Dynamics genetics, Mitochondrial Membrane Transport Proteins genetics, Neoplasm Proteins genetics, Triple Negative Breast Neoplasms drug therapy, Triple Negative Breast Neoplasms genetics, Triple Negative Breast Neoplasms pathology, Epithelial-Mesenchymal Transition drug effects, Gene Expression Regulation, Neoplastic drug effects, Mitochondrial Dynamics drug effects, Mitochondrial Membrane Transport Proteins biosynthesis, Neoplasm Proteins biosynthesis, Silybin pharmacology, Triple Negative Breast Neoplasms metabolism
- Abstract
Human triple negative breast cancer cells, MDA-MB-231, show typical epithelial to mesenchymal transition associated with cancer progression. Mitochondria play a major role in cancer progression, including metastasis. Changes in mitochondrial architecture affect cellular migration, autophagy and apoptosis. Silibinin is reported to have anti-breast cancer effect. We here report that silibinin at lower concentrations (30-90 μM) inhibits epithelial to mesenchymal transition (EMT) of MDA-MB-231, by increasing the expression of epithelial marker, E-cadherin, and decreasing the expression of mesenchymal markers, N-cadherin and vimentin. Besides, silibinin inhibition of cell migration is associated with reduction in the protein expression of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) and paxillin. In addition, silibinin treatment increases mitochondrial fusion through down-regulating the expression of mitochondrial fission-associated protein dynamin-related protein 1 (DRP1) and up-regulating the expression of mitochondrial fusion-associated proteins, optic atrophy 1, mitofusin 1 and mitofusin 2. Silibinin perturbed mitochondrial biogenesis via down-regulating the levels of mitochondrial biogenesis regulators including mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator (PGC1) and nuclear respiratory factor (NRF2). Moreover, DRP1 knockdown or silibinin inhibited cell migration, and MFN1&2 knockdown restored it. Mitochondrial fusion contributes to silibinin's negative effect on cell migration. Silibinin decreased reactive oxygen species (ROS) generation, leading to inhibition of the NLRP3 inflammasome activation. In addition, knockdown of mitofusin 1&2 (MFN 1&2) relieved silibinin-induced inhibition of NLRP3 inflammasome activation. Repression of ROS contributes to the inhibition of the expression of NLRP3, caspase-1 and IL-β proteins as well as of cell migration. Taken together, our study provides evidence that silibinin impairs mitochondrial dynamics and biogenesis, resulting in reduced migration and invasion of the MDA-MB-231 breast cancer cells.
- Published
- 2020
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178. Silibinin ameliorates STZ-induced impairment of memory and learning by up- regulating insulin signaling pathway and attenuating apoptosis.
- Author
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Liu P, Cui L, Liu B, Liu W, Hayashi T, Mizuno K, Hattori S, Ushiki-Kaku Y, Onodera S, and Ikejima T
- Subjects
- Animals, Cerebral Cortex metabolism, Cognitive Dysfunction chemically induced, Diabetes Mellitus, Experimental chemically induced, Diabetes Mellitus, Experimental metabolism, Diabetes Mellitus, Experimental pathology, Diabetes Mellitus, Experimental prevention & control, Hippocampus pathology, Insulin-Like Growth Factor I metabolism, Male, Memory Disorders chemically induced, Nerve Degeneration chemically induced, Nerve Degeneration pathology, Nerve Degeneration prevention & control, Neuroprotective Agents pharmacology, Phosphorylation drug effects, Rats, tau Proteins metabolism, Apoptosis drug effects, Cognitive Dysfunction prevention & control, Insulin metabolism, Memory Disorders prevention & control, Signal Transduction drug effects, Silybin pharmacology, Streptozocin antagonists & inhibitors
- Abstract
Alzheimer's disease (AD) is a neurodegenerative disease, mainly characterized by cognitive dysfunction and memory impairment. Due to its pathological similarities to type 2 diabetes mellitus (T2DM), such as β-amyloid deposition, oxidative stress, inflammation, disordered glucose metabolism, impaired signaling pathways of insulin and insulin-like growth factor-1 (IGF-1), we speculate that AD is another form of brain diabetes. Clarifying the relationship between T2DM and AD is important for us to better understand the exact pathological mechanisms of AD. Silibinin, a polyphenolic flavonoid extracted from the seeds of Silybum marianum, exerts hepatoprotective, anti- diabetic and neuroprotective effects. Streptozotocin (STZ), which is used to disrupt the insulin signal transduction pathway, could well mimic the sporadic AD models by intracerebroventricular (ICV) injection. Therefore, we selected ICV injection of STZ (ICV-STZ) to investigate the neuroprotective effects of silibinin in rats and to make a foundation for further exploring the relationship between AD and T2DM. ICV-STZ obviously caused memory damage, sharply reduced the number of nissl bodies and destroyed morphological structure of hippocampal neuronal cells, while silibinin attenuated the damages. Moreover, silibinin significantly decreased STZ-induced tau hyperphosphorylation (ser404) in hippocampus and cerebral cortex, markedly inhibited apoptosis of neurons induced by STZ, and up-regulated insulin signal transduction pathway. Silibinin exerts neuroprotective effect in STZ-treated rats, indicating the potential of silibinin for the treatment of AD patients with T2DM in future., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2020
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179. Type I collagen or gelatin stimulates mouse peritoneal macrophages to aggregate and produce pro-inflammatory molecules through upregulated ROS levels.
- Author
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Zhang X, Chen YR, Zhao YL, Liu WW, Hayashi T, Mizuno K, Hattori S, Fujisaki H, Ogura T, Onodera S, and Ikejima T
- Subjects
- Animals, Cell Aggregation, Dinoprostone metabolism, Female, Interleukin-1beta metabolism, Macrophages, Peritoneal metabolism, Mice, NF-kappa B metabolism, Tumor Necrosis Factor-alpha metabolism, Collagen Type I, Gelatin, Macrophages, Peritoneal physiology, Reactive Oxygen Species metabolism
- Abstract
Background: Extracellular matrix (ECM) comprising the environments of multicellular society has a dynamic network structure. Collagen is one of the ubiquitous components of ECM. Collagen affects the inflammatory response by regulating the release of pro-inflammatory cytokines from cells. Gelatin, denatured collagen found temporally in tissues, is supposed to be pathophysiologically involved in tissue remodeling, inflammation caused by tissue damage. Previous reports indicate that, phorbol myristate (PMA)-stimulated human U937 (lymphoma cell line) cells that are often used as macrophage-like cells, show cell aggregations when cultured on type I collagen (col I) or gelatin-coated dishes, accompanying the changes of production and release of proinflammatory factors. However, it still remains to be examined whether collagen and gelatin affects normal macrophages as well., Aim: This study aims to investigate the effect of col. I, the main component of collagenous protein and its denatured product, gelatin, on mouse peritoneal macrophages (MPMs)., Methods: MTT assay, flow cytometric analysis of ROS, biochemical detection of antioxidant levels, ELISA assay, and western blot were used., Results: MPMs formed multicellular aggregates on col. I - and gelatin-coated dishes with a concentration- and time-dependent manner. Further studies showed that the culture on col. I and gelatin up-regulated the protein expression and secretion of pro-inflammatory molecules such as IL-1β, TNFα and prostaglandin E
2 (PGE2 ) in MPMs. The levels were higher in the cells on gelatin than those on col. I. ROS levels are significantly increased in the cells cultured on both col. I- and gelatin-coated dishes, accompanying decreased levels of antioxidant enzyme catalase (CAT) and anti-oxidant glutathione (GSH), and enhanced nuclear translocation of NF-κB., Conclusion: Col I - or gelatin-coated culture induced the formation of multicellular aggregates and increased production of NF-κB-associated pro-inflammatory molecules in MPMs through up-regulation of ROS levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)- Published
- 2019
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180. Posttranslational modifications in type I collagen from different tissues extracted from wild type and prolyl 3-hydroxylase 1 null mice.
- Author
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Pokidysheva E, Zientek KD, Ishikawa Y, Mizuno K, Vranka JA, Montgomery NT, Keene DR, Kawaguchi T, Okuyama K, and Bächinger HP
- Subjects
- Animals, Collagen Type I genetics, Hydroxylation physiology, Mice, Mice, Mutant Strains, Organ Specificity physiology, Procollagen-Proline Dioxygenase genetics, Proline genetics, Proline metabolism, Collagen Type I metabolism, Procollagen-Proline Dioxygenase metabolism, Protein Processing, Post-Translational physiology
- Abstract
Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.
- Published
- 2013
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181. Crystal structure of the collagen model peptide (Pro-Pro-Gly)4-Hyp-Asp-Gly-(Pro-Pro-Gly)4 at 1.0 Å resolution.
- Author
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Okuyama K, Kawaguchi T, Shimura M, Noguchi K, Mizuno K, and Bächinger HP
- Subjects
- Amino Acid Sequence, Collagen chemistry, Peptides chemistry, Protein Conformation, Protein Structure, Secondary
- Abstract
The single-crystal structure of the collagen-like peptide (Pro-Pro-Gly)4 -Hyp-Asp-Gly-(Pro-Pro-Gly)4, was analyzed at 1.02 Å resolution. The overall average helical twist (θ = 49.6°) suggests that this peptide adopts a 7/2 triple-helical structure and that its conformation is very similar to that of (Gly-Pro-Hyp)9, which has the typical repeating sequence in collagen. High-resolution studies on other collagen-like peptides have shown that imino acid-rich sequences preferentially adopt a 7/2 triple-helical structure (θ = 51.4°), whereas imino acid-lean sequences adopt relaxed conformations (θ < 51.4°). The guest Gly-Hyp-Asp sequence in the present peptide, however, has a large helical twist (θ = 61.1°), whereas that of the host Pro-Pro-Gly sequence is small (θ = 46.7°), indicating that the relationship between the helical conformation and the amino acid sequence of such peptides is complex. In the present structure, a strong intermolecular hydrogen bond between two Asp residues on the A and B strands might induce the large helical twist of the guest sequence; this is compensated by a reduced helical twist in the host, so that an overall 7/2-helical symmetry is maintained. The Asp residue in the C strand might interact electrostatically with the N-terminus of an adjacent molecule, causing axial displacement, reminiscent of the D-staggered structure in fibrous collagens., (Copyright © 2013 Wiley Periodicals, Inc.)
- Published
- 2013
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182. Diffusion-weighted MRI of exercise-induced acute renal failure (ALPE).
- Author
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Ohta K, Yokoyama T, Shimizu M, Mizuno K, Sakazume S, Fujiki T, Saikawa Y, and Yachie A
- Subjects
- Adult, Diffusion Magnetic Resonance Imaging, Female, Humans, Male, Tomography, X-Ray Computed, Acute Kidney Injury diagnosis, Acute Kidney Injury etiology, Exercise physiology
- Abstract
Acute renal failure with severe loin pain induced by anaerobic exercise (ALPE) is a rare condition that is accompanied by wedge-shaped contrast enhancement on computed tomography (CT) without evidence of rhabdomyolysis. In two pediatric cases with ALPE, we tried to determine the relationship between findings from CT and magnetic resonance imaging (MRI). Case 1 involved a 13-year-old Japanese girl with a diagnosis of ALPE with normo-uricemia. Contrast-enhanced CT after 24 and 48 h showed a wedge-shaped excretion delay for the contrast media. A clear wedge-shaped signal hyperintensity matching the CT images was obtained by diffusion-weighted MRI. Case 2 involved a 16-year-old boy who presented with a second attack of ALPE after diagnosis of ALPE with hypouricemia 1 year earlier. Only diffusion-weighted imaging was performed. Clear wedge-shaped signal hyperintensity was apparent, similar to Case 1. MRI is safer than contrast-enhanced CT for patients with ALPE. Diffusion-weighted MRI is a very useful examination for diagnosing ALPE, providing noninvasive detection of lesions peculiar to ALPE.
- Published
- 2011
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183. The effect of deuterium oxide on the stability of the collagen model peptides H-(Pro-Pro-Gly)(10)-OH, H-(Gly-Pro-4(R)Hyp)(9)-OH, and Type I collagen.
- Author
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Mizuno K and Bächinger HP
- Subjects
- Antineoplastic Agents pharmacology, Calorimetry, Differential Scanning, Circular Dichroism, Collagen metabolism, Magnetic Resonance Spectroscopy, Oligopeptides metabolism, Protein Stability drug effects, Water chemistry, Collagen chemistry, Deuterium Oxide pharmacology, Oligopeptides chemistry
- Abstract
The collagen triple helix has a larger accessible surface area per molecular mass than globular proteins, and therefore potentially more water interaction sites. The effect of deuterium oxide on the stability of collagen model peptides and Type I collagen molecules was analyzed by circular dichroism and differential scanning calorimetry. The transition temperatures (T(m)) of the protonated peptide (Pro-Pro-Gly)(10) were 25.4 and 28.7 degrees C in H(2)O and D(2)O, respectively. The increase of the T(m) of (Pro-Pro-Gly)(10) measured calorimetrically at 1.0 degrees C min(-1) in a low pH solution from the protonated to the deuterated solvent was 5.1 degrees C. The increases of the T(m) for (Gly-Pro-4(R)Hyp)(9) and pepsin-extracted Type I collagen were measured as 4.2 and 2.2 degrees C, respectively. These results indicated that the increase in the T(m) in the presence of D(2)O is comparable to that of globular proteins, and much less than reported previously for collagen model peptides [Gough and Bhatnagar, J Biomol Struct Dyn 1999, 17, 481-491]. These experimental results suggest that the interaction of water molecules with collagen is similar to the interaction of water with globular proteins, when the ratio of collagen to water is very small and collagen is monomerically dispersed in the solvent.
- Published
- 2010
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184. Two crystal modifications of (Pro-Pro-Gly)4-Hyp-Hyp-Gly-(Pro-Pro-Gly)4 reveal the puckering preference of Hyp(X) in the Hyp(X):Hyp(Y) and Hyp(X):Pro(Y) stacking pairs in collagen helices.
- Author
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Okuyama K, Morimoto T, Narita H, Kawaguchi T, Mizuno K, Bächinger HP, Wu G, and Noguchi K
- Subjects
- Collagen metabolism, Crystallization, Crystallography, X-Ray, Hydroxyproline metabolism, Multiprotein Complexes metabolism, Peptide Fragments metabolism, Protein Binding, Protein Multimerization, Protein Structure, Secondary, Stereoisomerism, Collagen chemistry, Hydroxyproline chemistry, Multiprotein Complexes chemistry, Peptide Fragments chemistry
- Abstract
Two crystal modifications of a collagen model peptide, (Pro-Pro-Gly)(4)-Hyp-Hyp-Gly-(Pro-Pro-Gly)(4) [where Hyp is (4R,2S)-L-hydroxyproline], showed very similar unit-cell parameters and belonged to the same space group P2(1). Both crystals exhibited pseudo-merohedral twinning. The main difference was in their molecular-packing arrangements. One modification showed pseudo-hexagonal packing, while the other showed pseudo-tetragonal packing. Despite their different packing arrangements, no significant differences were observed in the hydration states of these modifications. The peptide in the pseudo-tetragonal crystal showed a cyclic fluctuation of helical twists with a period of 20 A, while that in the pseudo-hexagonal crystal did not. In these modifications, the puckering conformations of four of the 12 Hyp residues at the X position of the Hyp(X)-Hyp(Y)-Gly sequence were in the opposite conformations to the previous hypothesis that Hyp(X) residues involved in Hyp(X):Hyp(Y) and Hyp(X):Pro(Y) stacking pairs prefer up-puckering and down-puckering conformations, respectively. Detailed investigation of the molecular interactions between Hyp(X) and adjacent molecules revealed that these opposite conformations appeared because the puckering conformation, which follows the hypothesis, is subject to steric hindrance from the adjacent molecule.
- Published
- 2010
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185. Re: Microfibrillar structure of type I collagen in situ.
- Author
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Okuyama K, Bächinger HP, Mizuno K, Boudko S, Engel J, Berisio R, and Vitagliano L
- Subjects
- Animals, Collagen Type I metabolism, Collagen Type I ultrastructure, Crystallization methods, Microfibrils ultrastructure, Multiprotein Complexes metabolism, Multiprotein Complexes ultrastructure, Muscles metabolism, Muscles ultrastructure, Protein Conformation, Rats, Research Design, Tendons pathology, Collagen Type I chemistry, Microfibrils chemistry, Multiprotein Complexes chemistry
- Published
- 2009
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186. Reconstituted type V collagen fibrils as cementing materials in the formation of cell clumps in culture.
- Author
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Kihara T, Takemura Y, Imamura Y, Mizuno K, and Hayashi T
- Subjects
- Cell Movement, Cell Proliferation, Collagen Type V chemistry, Electrophoresis, Polyacrylamide Gel, Fibroblasts physiology, Fluorescein-5-isothiocyanate chemistry, Fluorescence, Humans, Lung cytology, Lung embryology, Microscopy, Fluorescence, Staining and Labeling, Cell Adhesion physiology, Cell Aggregation physiology, Cell Line physiology, Collagen Type V metabolism, Fibroblasts cytology
- Abstract
Previous studies have reported that type V collagen is an anti-adhesive substrate for cultured cells in that the cells detach from culture dishes coated with type V collagen molecules or polypeptides derived from them. We have noticed that human fetal lung fibroblasts (TIG-1) initially show no reduction in adherence to and spreading on a dish coated with reconstituted type V collagen fibrils but eventually detach from the dish and form cell clumps. To determine the way in which reconstituted type V collagen fibrils are involved in cell clump formation, we have followed the fate of the fluorescence of type V collagen fibrils pre-labeled with fluorescein isothiocyanate. Essentially, all the fluorescence disappeared from the dish surface as the cells detached and was condensed in the cell clumps. The cells that were recovered from clumps and dissociated into separate cells by trypsin treatment proliferated normally after they were seeded on a bare culture dish. This result and those from gel electrophoresis, fluorescence microscopy, and a cell proliferation assay indicate that the cell detachment from the dish is not caused by cell necrosis or apoptosis but by cellular motility together with the unique features of type V collagen fibrils. Not only the adherence of type V collagen fibrils to TIG-1 cells is much stronger than that to the culture dish, but the fibrils are retained on the cellular surface. The strong adherence of type V collagen fibrils to cells plays a role in cementing TIG-1 cells together.
- Published
- 2004
- Full Text
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