380 results on '"Mizuno, Kazunori"'
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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]
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- 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]
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- 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
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- 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]
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- 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. Increased mitochondrial fission induces NLRP3/cGAS-STING mediated pro-inflammatory pathways and apoptosis in UVB-irradiated immortalized human keratinocyte HaCaT cells.
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Li, Can, Zhu, Yuying, Liu, Weiwei, Hayashi, Toshihiko, Xiang, Wendie, He, Sijun, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, and Ikejima, Takashi
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MITOCHONDRIA , *KERATINOCYTES , *PYROPTOSIS , *APOPTOSIS , *REACTIVE oxygen species ,KERATINOCYTE differentiation - Abstract
Ultraviolet B (UVB) irradiation causes skin inflammation and apoptosis. Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in skin damages, little is known about the roles of mitochondrial dynamics in these processes. UVB irradiation increases abnormal mitochondrial content but decreases mitochondrial volume in immortalized human keratinocyte HaCaT cells. UVB irradiation resulted in marked upregulation of mitochondrial fission protein dynamin-related protein 1 (DRP1) and downregulation of mitochondrial outer membrane fusion proteins 1 and 2 (MFN1 and MFN2) in HaCaT cells. Mitochondrial dynamics was discovered to be crucial for NLRP3 inflammasome and cGAS-STING pathway activation, as well as the induction of apoptosis. Inhibition of mitochondrial fission by treatments with a DRP1 inhibitor, mdivi-1, or with DRP1-targeted siRNA, efficiently prevented UVB-induced NLRP3/cGAS-STING mediated pro-inflammatory pathways or apoptosis in the HaCaT cells, whereas inhibition of mitochondrial fusion with MFN1and 2 siRNA increased these pro-inflammatory pathways or apoptosis. The enhanced mitochondrial fission and reduced fusion caused the up-regulation of reactive oxygen species (ROS). Application of an antioxidant, N -acetyl- l -cysteine (NAC), which scavenges excessive ROS, attenuated inflammatory responses through suppressing NLRP3 inflammasome and cGAS-STING pathway activation, and rescued cells from apoptosis caused by UVB-irradiation. Together, our findings revealed the regulation of NLRP3/cGAS-STING inflammatory pathways and apoptosis by mitochondrial fission/fusion dynamics in UVB-irradiated HaCaT cells, providing a new strategy for the therapy of UVB skin injury. [Display omitted] • UVB perturbs mitochondrial fission/fusion dynamics in HaCaT cells. • Disturbed mitochondrial dynamics control the amount of mitochondria. • Disturbed mitochondrial dynamics regulate NLRP3/cGAS-STING mediated pro-inflammatory pathways and apoptosis. • ROS are responsible for pro-inflammatory pathways and apoptosis initiated by the dysregulated mitochondrial dynamics. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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171. Impaired mitophagy causes mitochondrial DNA leakage and STING activation in ultraviolet B-irradiated human keratinocytes HaCaT.
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Li, Can, Zhu, Yuying, Liu, Weiwei, Xiang, Wendie, He, Sijun, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, and Ikejima, Takashi
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MITOCHONDRIAL DNA , *MEMBRANE permeability (Biology) , *KERATINOCYTES , *MITOCHONDRIAL membranes , *LEAKAGE , *MITOCHONDRIAL pathology , *WRINKLES (Skin) - Abstract
Ultraviolet B (UVB) irradiation causes skin damages. In this study, we focus on the involvement of mitochondrial disorders in UVB injury. Surprisingly, UVB irradiation increases the amounts of mitochondria in human immortalized keratinocytes HaCaT. However, further analysis shows that ATP levels decreased by UVB treatment in accordance with the collapse of mitochondrial membrane potential (MMP), suggesting an accumulation of dysfunctional mitochondria in UVB-irradiated HaCaT cells. Mitophagy, mainly mediated by PINK1 and parkin, is critical for the elimination of damaged mitochondria. Western blot results show that the levels of both PINK1 and parkin are decreased in UVB-irradiated cells, indicating the impairment of mitophagy. Silencing the expression of PINK1 or parkin by transfection of siRNA shows essentially the same damage to the cells as UVB irradiation does, including increased mitochondrial amount, decreased MMP and ATP production, and enhanced apoptosis, evidencing that repression of PINK1/parkin-mediated mitophagy plays a primary cause of UVB-caused cells damages. We previously found that HaCaT cells exposed to UVB showed activation of the cGAS-STING pathway and apoptosis. Here, silencing PINK1 or parkin also increases the protein levels of cGAS and STING, facilitates nuclear accumulation of NF-κB, and promotes the transcription of IFNβ, suggesting for the activation of STING pathway. Mitophagy impairment either by UVB-irradiation or by PINK1/parkin silencing initiates caspase-3-mediated apoptosis, as shown by the activation of caspase-3 and cleavage of PARP, as well as the increase of Hoechst-positive stained cells and Annexin V-positive cells. Further studies find that Bax-mediated permeabilization of mitochondrial membrane is critical for cell apoptosis, as well as the cytosolic leakage of mtDNA in UVB-treated cells, which results in cGAS-STING activation, and these processes are negatively-regulated by PINK1/parkin-mediated mitophagy. This study reveals the involvement of dysfunctional mitochondria due to impaired mitophagy in the damaging effect of UVB irradiation on HaCaT cells. Restoring the mitophagy has the potential to be developed as a new strategy to protect skin from UVB damages. [Display omitted] • UVB decreases mitophagy and accumulates dysfunctional mitochondria in HaCaT cells. • UVB reduces PINK1/parkin expression that is involved in mitophagy of damaged mitochondria. • Mitophagy arrest caused by UVB leads to the activation of cGAS-STING/apoptosis pathway. • Mitophagy arrest caused by UVB increases mitochondrial membrane permeability. • Mitophagy arrest caused by UVB results in the mtDNA leakage into cytosol. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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172. Combined use of dasatinib and quercetin alleviates overtraining-induced deficits in learning and memory through eliminating senescent cells and reducing apoptotic cells in rat hippocampus.
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Wang, Chenkang, Kang, Yu, Liu, Panwen, Liu, Weiwei, Chen, Wenhui, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, and Ikejima, Takashi
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DASATINIB , *MEMORY disorders , *QUERCETIN , *HIPPOCAMPUS (Brain) , *ORAL drug administration , *MEMORY testing - Abstract
Excessive physical exercise (overtraining, OT) charactered by long-term and excessive training results in the damage of multiple vital tissues including hippocampus which plays a critical role in learning and memory. A combination of dasatinib (D) plus quercetin (Q) (D+Q) belongs to senolytic drugs which selectively kill senescent cells in vitro and vivo. In this study, the rats that suffered a five-week excessive swimming training were subjected to the oral administration of D+Q. D+Q alleviated the decline in exercise performance of OT rats during the swimming training, and prevented learning and memory deficits in Morris water maze, Y-maze and novel object recognition tests after excessive swimming training. Analytical results by SA-β-gal staining and western blotting showed that D+Q significantly reduced senescent cells with repressed expression of senescence-related proteins, p53 and p21, in hippocampus. Nissl and immunohistochemical staining showed that D+Q significantly attenuated neuronal loss caused by apoptosis. Interestingly, we observed elevated level of cleaved caspase 3, an apoptosis executor protein, in p21 positive hippocampus cells by D+Q treatment in immunofluorescent staining, suggesting that senescent cells were induced to apoptosis in D+Q-treated rats. The positive control drug, silibinin, showed similar protective effect against OT, but did not induce the apoptosis of senescent cells, suggesting a difference in the protective mechanisms. These results indicated that D+Q alleviates overtraining-induced deficits in learning and memory through elimination of senescent cells and reduction of apoptotic cell number. [Display omitted] • Dasatinib + quercetin treatment prevents OT-induced learning and memory deficits. • Dasatinib + quercetin induces apoptosis of senescent cells in hippocampus. • Dasatinib + quercetin alleviates apoptotic neuronal loss in hippocampus. [ABSTRACT FROM AUTHOR]
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- 2023
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173. Type I collagen reduces lipid accumulation during adipogenesis of preadipocytes 3T3-L1 via the YAP-mTOR-autophagy axis.
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Gao, Yanfang, Ma, Kai, Kang, Yu, Liu, Weiwei, Liu, Xiaoling, Long, Xinyu, Hayashi, Toshihiko, Hattori, Shunji, Mizuno, Kazunori, Fujisaki, Hitomi, and Ikejima, Takashi
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ADIPOGENESIS , *FREE fatty acids , *LIPIDS , *WEIGHT loss , *COLLAGEN , *EXTRACELLULAR matrix , *CELL survival , *GLYCOLIPIDS - Abstract
The extracellular matrix (ECM) regulates cell behavior through signal transduction and provides a suitable place for cell survival. As one of the major components of the extracellular matrix, type I collagen is involved in regulating cell migration, proliferation and differentiation. We present a system in which 3T3-L1 preadipocyte cells are induced for adipogenic differentiation on type I collagen coated dishes. Our previous study has found that type I collagen inhibits adipogenic differentiation via YAP activation. Here we further reveal that type I collagen inactivates autophagy by up-regulating mTOR activity via the YAP pathway. Under collagen-coating conditions, co-localization of lysosomes with mTOR was increased and the level of downstream protein p-S6K was elevated, accompanied by a decrease in the level of autophagy. Autophagy is negatively correlated with adipogenesis under type I collagen coating. Through the YAP-autophagy axis, type I collagen improves glycolipid metabolism accompanied by increased mitochondrial content, enhanced glucose uptake, reduced release of free fatty acids (FFAs) and decreased intracellular lipid accumulation. Our findings provide insight into the strategy for dealing with obesity: Type I collagen or the drugs with inhibitory effects on autophagy or YAP, have a potential to accelerate the energy metabolism of adipose tissue, so as to better maintain the homeostasis of glucose and lipids in the body, which can be used for achieving weight loss. [Display omitted] • Type I collagen inhibits autophagy by up-regulating YAP-mTOR pathway. • Autophagy is negatively correlated with adipogenesis under type I collagen coating. • Type I collagen improves glucose and lipid metabolism through the YAP-autophagy axis. [ABSTRACT FROM AUTHOR]
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- 2022
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174. Silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in liver cell lines.
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Song, Xiao-Yu, Liu, Peng-Cheng, Liu, Wei-Wei, Zhou, Jia, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, and Ikejima, Takashi
- Abstract
Alcoholic liver disease has become one of the main causes of liver injury, and its prevention and cure are important medical tasks. Silibinin, a natural flavonoid glycoside, is a conventional hepatic protectant. This study elucidates the modulation of ferroptosis in silibinin's protective effects on ethanol- or acetaldehyde-induced liver cell damage by using human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells. Our results show that ferroptosis is induced in the cells treated with ethanol or acetaldehyde, as evidenced by the increased ROS stress and iron level. Silibinin resolves the oxidative stress and reduces iron level. Ferroptosis induced by ethanol- or acetaldehyde involving nuclear receptor co-activator 4 (NCOA4)-dependent autophagic degradation of ferritin, a protein for storing iron is rescued by silibinin. PINK1 and Parkin-mediated mitophagy is arrested in ethanol- or acetaldehyde-treated cells but reversed by silibinin. Ferritin degradation and ROS level are further increased when PINK1 or Parkin is silenced in the cells treated with ethanol or acetaldehyde. Collectively, our study reveals that silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in two liver cell lines, HepG2 and HL7702 cells, providing new therapeutic strategies for alcoholic liver injury. [Display omitted] • Ethanol or acetaldehyde triggers ferroptosis in hepatocytes. • Autophagy is involved in ferroptosis treated with ethanol or acetaldehyde. • Ethanol or acetaldehyde reduces PINK1/Parkin-mediated mitophagy in ferroptosis. • Silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in two liver cell lines. • Silibinin enhances mitophagy in ethanol- or acetaldehyde-induced liver injury. [ABSTRACT FROM AUTHOR]
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- 2022
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175. Protective effects of silibinin against ethanol- or acetaldehyde-caused damage in liver cell lines involve the repression of mitochondrial fission.
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Song, Xiao-Yu, Liu, Peng-Cheng, Liu, Wei-Wei, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, and Ikejima, Takashi
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LIVER cells , *ACETALDEHYDE , *SILIBININ , *CELL lines , *MITOCHONDRIAL proteins , *MITOCHONDRIA - Abstract
Silibinin is a natural polyphenolic flavonoid, isolated from the seeds of the milk thistle of Silybum marianum (L.) Gaertn. Silibinin has been widely used clinically as a traditional medicine for liver diseases. This study investigated the protective role of silibinin in ethanol- or acetaldehyde-induced apoptosis in human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells, focusing on elucidation of the underlying mechanism in vitro. The toxicity of ethanol or acetaldehyde was evaluated by MTT assay. Apoptosis-related proteins, mitochondrial fission-associated proteins and mitochondrial fusion-associated proteins were analyzed by western blotting and immunofluorescence microscopy. Present experimental results demonstrated that silibinin improved cell viability, reduced the enzyme activities of AST/ALT and ALDH/ADH, inhibited apoptosis and recovered mitochondrial function in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. Silibinin reduced the expression of mitochondrial fission-associated proteins, dynamin-related protein 1 (DRP1), but increased mitochondrial fusion-associated proteins, optic atrophy 1 (OPA1) and mitofusin 1 (MFN1). Accordingly, inhibition of DRP1 activity with its pharmacological inhibitor or siDRP1 efficiently attenuated ethanol- or acetaldehyde-induced apoptosis, whereas activation of DRP1 by using staurosporine (STS) further increased apoptosis in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. The results show that silibinin protects cells against ethanol- or acetaldehyde-induced mitochondrial fission that results in apoptosis. • Ethanol or acetaldehyde induces apoptosis in HepG2 and HL7702 cells. • Silibinin reduces ethanol- or acetaldehyde-induced apoptosis. • Ethanol or acetaldehyde aggravates mitochondrial fission resulting in apoptosis. • Silibinin restores ethanol- or acetaldehyde-caused mitochondrial disorders. [ABSTRACT FROM AUTHOR]
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- 2022
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176. Diffusion-weighted MRI of exercise-induced acute renal failure (ALPE)
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Ohta, Kazuhide, Yokoyama, Tadafumi, Shimizu, Masaki, Mizuno, Kazunori, Sakazume, Shinobu, Fujiki, Takuma, Saikawa, Yutaka, and Yachie, Akihiro
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- 2011
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177. Silibinin ameliorates depression/anxiety-like behaviors of Parkinson's disease mouse model and is associated with attenuated STING-IRF3-IFN-β pathway activation and neuroinflammation.
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Liu, Xiumin, Chen, Wenhui, Wang, Chenkang, Liu, Weiwei, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, and Ikejima, Takashi
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LABORATORY mice , *PARKINSON'S disease , *NEUROINFLAMMATION , *SILIBININ , *APATHY , *ANIMAL disease models , *TEST anxiety - Abstract
• MPTP induces depression/anxiety and hippocampal damage in PD model mice. • STING-IRF3-IFN- β inflammation pathway is involved in hippocampal damage. • Silibinin improves the depressive and anxious-like behaviors of mice with PD. • Silibinin inhibits STING-IRF3-IFN- β pathway. • Silibinin protects hippocampal neurons and attenuates neuroinflammation. Depression and anxiety are common neuropsychiatric symptom of Parkinson's disease (PD), reflecting reduced quality of life in patients with PD. Silibinin (silybin), a flavonoid extracted and isolated from the fruit of Silybum marianum (L.) Gaertn, is widely used for the treatment of hepatic diseases. We report here that silibinin shows anti-depressant and anti-anxiety effects on 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced model mice with PD. All the results of open field test, elevated plus maze test, tail suspension test and forced swimming test demonstrated that silibinin administration significantly attenuated MPTP-induced depression/anxiety. Hematoxylin-eosin (HE) staining and Nissl staining results showed that MPTP injection caused the damage of hippocampal neurons, but this was ameliorated by oral administration of silibinin. Silibinin significantly restored hippocampal levels of 5-hydroxyptramine (5-HT) and noradrenaline (NA), two important neurotransmitters for regulating mood, which decreased in MPTP-injected mice. Neuroinflammation, as reflected by the increased expressions of IL-1 β , TNF α and IFN- β , was marked in the hippocampus of MPTP-treated mice, accompanying increased stimulator of interferon genes (STING) and interferon regulatory factor-3 (IRF3). Silibinin administration, however, down-regulated the levels of IL-1 β , TNF α and IFN- β , as well as STING and IRF3, protecting MPTP-induced PD model mice. These findings indicate that silibinin has a potential of being further developed as a therapeutic for depression and anxiety in PD. [ABSTRACT FROM AUTHOR]
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- 2021
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178. 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]
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- 2008
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179. Silibinin attenuates motor dysfunction in a mouse model of Parkinson's disease by suppression of oxidative stress and neuroinflammation along with promotion of mitophagy.
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Liu, Xiumin, Liu, Weiwei, Wang, Chenkang, Chen, Yinzhe, Liu, Panwen, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, and Ikejima, Takashi
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LABORATORY mice , *PARKINSON'S disease , *SILIBININ , *NEUROINFLAMMATION , *OXIDATIVE stress , *ELLAGIC acid - Abstract
• Silibinin reverses motor function deficits in behavioral tests in mice with PD. • Silibinin protects dopaminergic neurons from MPTP-induced neurodegeneration. • Silibinin ameliorates oxidative stress and α -synuclein aggregation in PD mice. • Silibinin mitigates NLRP3 activation and pro-inflammatory cytokine production. • Silibinin promotes mitophagy in the MPTP-treated mice. Silybum marianum (L.) Gaertn has been widely used to obtain a drug for the treatment of hepatic diseases. Silibinin (silybin), a flavonoid extracted and isolated from the fruit of S. marianum is investigated in our study to explore its motor protective potential on Parkinson's disease (PD) model mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). PD is a neurodegenerative disease that causes a debilitating movement disorder, characterized by a progressive loss of nigrostriatal (substantia nigra and striatum) dopaminergic neurons. Several studies have proven that neurodegeneration is aggravated by neuroinflammation, oxidative stress and/or the presence of α -synuclein (α -syn) aggregation. Essentially no causal therapy for PD exists at present. Our results demonstrate that silibinin significantly attenuates MPTP-induced movement disorder in behavioral tests. Immunohistochemical analysis shows that MPTP injection results in the loss of dopaminergic neurons in the substantia nigra, and the decrease of the striatal tyrosine hydroxylase. However, MPTP-injected mice were protected against dopaminergic neuronal loss by oral administration of silibinin (280 mg/kg) that increased expressions of PTEN-induced putative kinase 1 (PINK1) and Parkin, suggesting mitophagy activation. The neuroprotective mechanism of silibinin involves not only reduction of mitochondrial damage by repressing proinflammatory response and α -syn aggregation, but also enhancement of oxidative defense system. Namely, protection of dopaminergic nerves is due to promotion of mitophagy, leading to clearance of the toxic effects of damaged mitochondria. These findings suggest that silibinin has a potential to be further developed as a therapeutic candidate for PD. [ABSTRACT FROM AUTHOR]
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- 2021
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180. Reconstituted type V collagen fibrils as cementing materials in the formation of cell clumps in culture.
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Kihara, Takanori, Takemura, Yukitoshi, Imamura, Yasutada, Mizuno, Kazunori, and Hayashi, Toshihiko
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COLLAGEN , *FIBROBLASTS , *PEPSIN , *CONNECTIVE tissues , *CELL culture , *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. [ABSTRACT FROM AUTHOR]
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- 2004
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181. Silibinin treatment protects human skin cells from UVB injury through upregulation of estrogen receptors.
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Liu, Weiwei, Wang, Fang, Li, Can, Otkur, Wuxiyar, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, Onodera, Satoshi, and Ikejima, Takashi
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ESTROGEN receptors , *SILIBININ , *SMALL interfering RNA , *CELL death , *SKIN injuries - Abstract
Ultraviolet B (UVB) from the sunlight is a major environmental cause for human skin damages, inducing cell death, inflammation, senescence and even carcinogenesis. The natural flavonoid silibinin, clinically used as liver protectant, has protective effects against UVB-caused skin injury in vivo and in vitro. Silibinin is often classified as a phytoestrogen, because it modulates the activation of estrogen receptors (ERs). However, whether silibinin's estrogenic effect contributes to the skin protection against UVB injury remains to be elucidated. The issue was explored in this study by using the human foreskin dermal fibroblasts (HFF) and human non-malignant immortalized keratinocytes (HaCaT). In HFF, pre-treatment with silibinin rescued UVB-irradiated cells from apoptosis. Interestingly, silibinin increased the whole cellular and nuclear levels of ERα and ERβ in UVB-irradiated cells. Activation of ERs by treatment with estradiol elevated the cell survival and reduced apoptosis in UVB-treated cells. ERα agonist increased cell survival, while its antagonist decreased it. ERβ agonist also increased cell survival, but the antagonist had no effect on cell survival. Transfection of the cells with the small interfering RNAs (si-RNAs) to ERα or ERβ diminished the protective effect of silibinin on UVB-irradiated cells. In UVB-treated HaCaT cells, both ERα and ERβ were increased by silibinin treatment. Inhibition of activation and expression of ERα or ERβ by specific antagonists and si-RNAs, respectively, reduced cell survival in UVB-treated HaCaT cells regardless of silibinin treatment. Taken together, it is summarized that silibinin up-regulates both ERα and ERβ pathways in UVB-treated dermal HFF cells and epidermal HaCaT cells, leading to protection of skin from UVB-damage. Unlabelled Image • Silibinin protects human foreskin fibroblasts from UVB-induced cell death. • Silibinin increases nuclear estrogen receptor α and β in UVB-treated fibroblasts. • Upregulation of estrogen receptor α and β contributes to silibinin's cytoprotection. • Increase in estrogen receptors by silibinin protects UVB-treated HaCaT cells. [ABSTRACT FROM AUTHOR]
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- 2021
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182. Effect of silibinin on ethanol- or acetaldehyde-induced damge of mouse primary hepatocytes in vitro.
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Song, Xiao-Yu, Li, Rong-Hua, Liu, Wei-Wei, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, and Ikejima, Takashi
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ACETALDEHYDE , *SILIBININ , *LIVER cells , *AUTOPHAGY , *CELL survival , *APOPTOSIS - 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. • Acetaldehyde (ACH) induced apoptosis in liver cells; • AcH induced autophagy in liver cells; • Silibinin protects AcH-induced death, but not ethanol-induced death of hepatocytes. [ABSTRACT FROM AUTHOR]
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- 2021
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183. Silibinin-induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells.
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Si, Lingling, Fu, Jianing, Liu, Weiwei, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, Onodera, Satoshi, and Ikejima, Takashi
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SMALL interfering RNA , *TRIPLE-negative breast cancer , *APOPTOSIS , *CANCER cells , *CELLS , *CYTOPROTECTION , *PLANT mitochondria - 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. [ABSTRACT FROM AUTHOR]
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- 2020
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184. Silibinin ameliorates STZ-induced impairment of memory and learning by up- regulating insulin signaling pathway and attenuating apoptosis.
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Liu, Panwen, Cui, Lingyu, Liu, Bo, Liu, Weiwei, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Ushiki-Kaku, Yuko, Onodera, Satoshi, and Ikejima, Takashi
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PSYCHIATRIC treatment , *SILIBININ , *TYPE 2 diabetes , *TAU proteins , *INSULIN - Abstract
• Silibinin significantly ameliorates STZ-caused cognitive and memory deficit. • Silibinin attenuates the neuronal loss and degeneration induced by STZ treatment. • Silibinin exerts a neuroprotective effect through inhibiting STZ-induced hyper-phosphorylation of tau proteins. • Silibinin can recover the function of insulin/IGF1 signaling pathway. 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. [ABSTRACT FROM AUTHOR]
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- 2020
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185. Type I collagen or gelatin stimulates mouse peritoneal macrophages to aggregate and produce pro-inflammatory molecules through upregulated ROS levels.
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Zhang, Xuan, Chen, Yi-Ran, Zhao, Ye-Li, Liu, Wei-Wei, Hayashi, Toshihiko, Mizuno, Kazunori, Hattori, Shunji, Fujisaki, Hitomi, Ogura, Takayuki, Onodera, Satoshi, and Ikejima, Takashi
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PERITONEAL macrophages , *COLLAGEN , *CELL aggregation , *MOLECULES , *EXTRACELLULAR matrix , *GELATIN - Abstract
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. 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). MTT assay, flow cytometric analysis of ROS, biochemical detection of antioxidant levels, ELISA assay, and western blot were used. 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 (PGE 2) 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. 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. • Mouse Peritoneal macrophages form multicellular aggregates on collagen or gelatin-coated surface. • Col I and/or gelatin stimulates MPMs to form cell aggregates and induce pro-inflammatory cytokine production. • ROS contribute to the cell aggregation and the production of pro-inflammatory molecules in MPMs on collagen I- or gelatin-coated dishes. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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186. Generation of bone-specific lysyl hydroxylase 2 knockout mice and their phenotypes.
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Tsuneizumi K, Kasamatsu A, Saito T, Fukushima R, Taga Y, Mizuno K, Sunohara M, Uzawa K, and Yamauchi M
- Abstract
Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of type I collagen. This modification is critical for the formation of stable hydroxylysine-aldehyde derived collagen cross-links, thus, for the stability of collagen fibrils. Though dysfunction of LH2 causes Bruck syndrome, recessive osteogenesis imperfecta with joint contracture, the molecular mechanisms by which LH2 affects bone formation are still not well understood. Since the Plod2 knockout mice are embryonically lethal, we generated bone-specific LH2 conditional knockout mice (bsLH2-cKO) using the osteocalcin-Cre/loxP system, and evaluated phenotypes of femurs. LH2 mRNA and protein levels assessed by qPCR, immunohistochemistry and Data Independent Acquisition proteomics were all markedly low in bsLH2-cKO femurs when compared to controls. Lysine hydroxylation of both carboxy- and amino-terminal telopeptides of an α1(I) chain were significantly diminished resulting in reduction of the hydroxylysine-aldehyde derived cross-links. The collagen fibrils in bsLH2-cKO appeared to be thicker, often fused and irregular when compared to controls. In addition, bone mineral density and mechanical properties of bsLH2-cKO femurs were significantly impaired. Taken together, these data demonstrate that LH2-catalyzed modification and consequent cross-linking of collagen are critical for proper bone formation and mechanical strength., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)
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- 2024
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187. Inhibition of YAP/TAZ pathway contributes to the cytotoxicity of silibinin in MCF-7 and MDA-MB-231 human breast cancer cells.
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Fu J, Liu W, Liu S, Zhao R, Hayashi T, Zhao H, Xiang Y, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
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- Female, Humans, Adaptor Proteins, Signal Transducing metabolism, Cell Line, Tumor, Cell Proliferation drug effects, Cell Survival drug effects, Gene Expression Regulation, Neoplastic drug effects, Intracellular Signaling Peptides and Proteins metabolism, MCF-7 Cells, Molecular Docking Simulation, Phosphoproteins metabolism, Signal Transduction drug effects, Trans-Activators metabolism, Transcription Factors metabolism, Verteporfin pharmacology, Breast Neoplasms metabolism, Breast Neoplasms drug therapy, Breast Neoplasms pathology, Silybin pharmacology, Transcriptional Coactivator with PDZ-Binding Motif Proteins antagonists & inhibitors, Transcriptional Coactivator with PDZ-Binding Motif Proteins drug effects, Transcriptional Coactivator with PDZ-Binding Motif Proteins metabolism, YAP-Signaling Proteins antagonists & inhibitors, YAP-Signaling Proteins drug effects, YAP-Signaling Proteins metabolism
- Abstract
Breast cancer is one of the most common cancers threatening women's health. Our previous study found that silibinin induced the death of MCF-7 and MDA-MB-231 human breast cancer cells. We noticed that silibinin-induced cell damage was accompanied by morphological changes, including the increased cell aspect ratio (cell length/width) and decreased cell area. Besides, the cytoskeleton is also destroyed in cells treated with silibinin. YAP/TAZ, a mechanical signal sensor interacted with extracellular pressure, cell adhesion area and cytoskeleton, is also closely associated with cell survival, proliferation and migration. Thus, the involvement of YAP/TAZ in the cytotoxicity of silibinin in breast cancer cells has attracted our interests. Excitingly, we find that silibinin inhibits the nuclear translocation of YAP/TAZ in MCF-7 and MDA-MB-231 cells, and reduces the mRNA expressions of YAP/TAZ target genes, ACVR1, MnSOD and ANKRD. More importantly, expression of YAP1 gene is negatively correlated with the survival of the patients with breast cancers. Molecular docking analysis reveals high probabilities for binding of silibinin to the proteins in the YAP pathways. DARTS and CETSA results confirm the binding abilities of silibinin to YAP and LATS. Inhibiting YAP pathway either by addition of verteporfin, an inhibitor of YAP/TAZ-TEAD, or by transfection of si-RNAs targeting YAP or TAZ further enhances silibinin-induced cell damage. While enhancing YAP activity by silencing LATS1/2 or overexpressing YAP
S127/397A , an active form of YAP, attenuates silibinin-induced cell damage. These findings demonstrate that inhibition of the YAP/TAZ pathway contributes to cytotoxicity of silibinin in breast cancers, shedding lights on YAP/TAZ-targeted cancer therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)- Published
- 2024
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188. Collagen I protects human keratinocytes HaCaT against UVB injury via restoring PINK1/parkin-mediated mitophagy.
- Author
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Zhu Y, Xiang W, He S, San Z, Liu W, Wu J, Hayashi T, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
- Subjects
- Humans, Keratinocytes metabolism, Ubiquitin-Protein Ligases metabolism, Protein Kinases metabolism, Mitophagy, Apoptosis
- Abstract
Collagen I is a major component of extracellular matrix in human skin, and is also widely used in a variety of skin-care products. In this study, we investigated the modulatory roles of collagen I on human immortalized keratinocytes HaCaT, especially when cells were irradiated with UVB. Interestingly, the cells grown on plates coated by molecular collagen I, but not fibrillar collagen I, acquired certain resistance against UVB damages, as shown by increased survival and reduced apoptosis. The accumulation of dysfunctional mitochondria in UVB-treated cells was attenuated by molecular collagen I-coating. Interestingly, molecular collagen I rescued the loss of mitochondrial biogenesis in cells treated with UVB. Loss of PINK1/parkin-mediated mitophagy was dominant for the accumulation of dysfunctional mitochondria after UVB irradiation. Of note, cells cultured on molecular collagen I-precoated plates exhibited reserved mitophagy after UVB irradiation, as reflected by the enhanced protein level of PINK1/parkin, increased mitochondrial ubiquitin and the co-localization of lysosomes and mitochondria. Moreover, in UVB-treated cells, inhibiting mitophagy by Cyclosporin A, or by silencing PINK1 or parkin, disturbed the resolution of mitochondrial stress and reduced the protective effect of molecular collagen I, indicating that mitophagy is pivotal for the protection of collagen I against UVB damage in keratinocytes HaCaT. Collectively, this study reveals an unexpected protective role of collagen I, which facilitates mitophagy to rescue cells under UVB irradiation, providing a new direction for clinical application of collagen products., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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189. Isolation of type I collagen homotrimer from human placenta with LC-MS monitoring of the α1(I)/α2(I) chain ratio.
- Author
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Taga Y, Kiriyama-Tanaka T, and Mizuno K
- Subjects
- Female, Pregnancy, Humans, Chromatography, Liquid, Liquid Chromatography-Mass Spectrometry, Placenta, Tandem Mass Spectrometry, Collagen Type I chemistry, Collagen chemistry
- Abstract
The general molecular form of type I collagen is heterotrimer consisting of two α1(I) chains and one α2(I) chain. However, α111(I) homotrimer is rarely observed in vivo, especially in pathological tissues such as cancer. Here we utilized a previously developed LC-MS method that can accurately and sensitively quantitate α1(I) and α2(I) chains to distinguish type I collagen homotrimer from human placenta. By monitoring with the LC-MS method, the α1(I)/α2(I) chain ratio was found to be high in the supernatant of salt precipitation with >2.8 M NaCl at neutral pH. Type I collagen homotrimer was successfully isolated using optimized sequential salt fractionation and confirmed to show previously reported features of the homotrimer, including high thermal stability and overmodification. These data clearly indicate that placental tissue contains α111(I) homotrimer. Our LC-MS method can sensitively detect the rare form of type I collagen and can help understand its physiological and pathological significance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuki Taga reports a relationship with Nippi Inc. that includes: employment. Tomomi Kiriyama-Tanaka reports a relationship with Nippi Inc. that includes: employment. Kazunori Mizuno reports a relationship with Nippi Inc. that includes: employment., (Copyright © 2023 Elsevier B.V. All rights reserved.)
- Published
- 2024
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190. Corrigendum to "DNA damage-triggered activation of cGAS-STING pathway induces apoptosis in human keratinocyte HaCaT cells" [Mol. Immunol. 131 (2021) 6222].
- Author
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Li C, Liu W, Wang F, Hayashi T, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
- Published
- 2023
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191. Dietary collagen peptides alleviate exercise-induced muscle soreness in healthy middle-aged males: a randomized double-blinded crossover clinical trial.
- Author
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Kuwaba K, Kusubata M, Taga Y, Igarashi H, Nakazato K, and Mizuno K
- Subjects
- Adult, Male, Middle Aged, Humans, Cross-Over Studies, Diet, Fatigue, Muscle, Skeletal, Dietary Supplements, Myalgia prevention & control, Myalgia drug therapy, Exercise physiology
- Abstract
Background: Post-exercise muscle soreness and fatigue can negatively affect exercise performance. Thus, it is desirable to attenuate muscle soreness and fatigue and promote recovery even for daily exercise habits aimed at maintaining or improving health., Methods: This study investigated the effects of dietary collagen peptides (CPs) on post-exercise physical condition and fitness in healthy middle-aged adults unfamiliar with exercise. Middle-aged males ( n = 20, 52.6 ± 5.8 years) received the active food (10 g of CPs per day) or the placebo food for 33 days in each period of the randomized crossover trial (registered at the University Hospital Medical Information Network Clinical Trials Registry with UMIN-CTR ID of UMIN000041441). On the 29th day, participants performed a maximum of five sets of 40 bodyweight squats. Muscle soreness as the primary outcome, fatigue, the maximum knee extension force during isometric muscle contraction of both legs, the range of motion (ROM), and the blood level of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) were assessed before and after the exercise load., Results: The analysis set was the per-protocol set ( n = 18, 52.6 ± 6.0 years) for efficacy and the full analysis set ( n = 19, 52.8 ± 5.9 years) for safety. The visual analog scale (VAS) of muscle soreness immediately after the exercise load was significantly lower in the active group than in the placebo group (32.0 ± 25.0 mm versus 45.8 ± 27.6 mm, p < 0.001). The VAS of fatigue immediately after the exercise load was also significantly lower in the active group than in the placebo group (47.3 ± 25.0 mm versus 59.0 ± 22.3 mm, p < 0.001). Two days (48 hours) afterthe exercise load, muscle strength was significantly higher in the active group than in the placebo group (85.2 ± 27.8 kg versus 80.5 ± 25.3 kg, p = 0.035). The level of CPK did not change over time. The level of LDH increased slightly but was not different between the groups. No safety-related issues were observed., Conclusions: These results showed that dietary CPs alleviated muscle soreness and fatigue and affected muscle strength after exercise load in healthy middle-aged males.
- Published
- 2023
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192. Silibinin ameliorates STING-mediated neuroinflammation via downregulation of ferroptotic damage in a sporadic Alzheimer's disease model.
- Author
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Liu P, Chen W, Kang Y, Wang C, Wang X, Liu W, Hayashi T, Qiu Z, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
- Subjects
- Rats, Mice, Animals, Silybin pharmacology, Silybin therapeutic use, Down-Regulation, Neuroinflammatory Diseases, Streptozocin adverse effects, Disease Models, Animal, Alzheimer Disease chemically induced, Alzheimer Disease drug therapy, Alzheimer Disease metabolism, Neuroprotective Agents pharmacology, Neuroprotective Agents therapeutic use
- Abstract
Ferroptosis, an iron-dependent cell death, is caused by lipid peroxidation. Noteworthily, accumulation of iron and lipid peroxidation are found in the proximity of the neuritic plaque, a hallmark of Alzheimer's disease (AD), but the relationship between ferroptosis and neuroinflammation in AD is unclear. Silibinin, extracted from the Silybum marianum, is possibly developed as an agent for AD treatment from its neuroprotective effect, but the effect of silibinin on sporadic AD that accounts for more than 95% of AD remains unclear. To determine whether silibinin alleviates the pathogenesis of sporadic AD and investigate the underlying mechanisms, STZ-treated HT22 murine hippocampal neurons and intracerebroventricular injection of streptozotocin (ICV-STZ) rats, a sporadic AD model, were used in this study. Results show that silibinin not only promotes survival of STZ-treated HT22 cells, but also ameliorates the cognitive impairment and anxiety/depression-like behavior of ICV-STZ rats. We here demonstrate that silibinin evidently inhibits the protein level of p53 as well as upregulates the protein level of cystine/glutamate antiporter SLC7A11 and ferroptosis inhibitor GPX4, but not p21, leading to the protection against STZ-induced ferroptotic damage. Immunofluorescent staining also shows that accumulation of lipid peroxidation induced by ferroptotic damage leads to increased fluorescence of 8-oxo-deoxyguanosine (8-OHDG), a maker of oxidized DNA. The oxidized DNA then leaks to the cytoplasm and upregulates the expression of the stimulator of interferon gene (STING), which triggers the production of IFN-β and other inflammatory cascades including NF-κB/TNFα and NLRP3/caspase 1/IL-1β. However, the treatment with silibinin blocks the above pathological changes. Moreover, in HT22 cells with/without STZ treatment, GPX4-knockdown increases the protein level of STING, indicating that the ferroptotic damage leads to the activation of STING signaling pathway. These results imply that silibinin exerts neuroprotective effect on an STZ-induced sporadic AD model by downregulating ferroptotic damage and thus the downstream STING-mediated neuroinflammation., (Copyright © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2023
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193. Silibinin alleviates ferroptosis of rat islet β cell INS-1 induced by the treatment with palmitic acid and high glucose through enhancing PINK1/parkin-mediated mitophagy.
- Author
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Du Q, Wu X, Ma K, Liu W, Liu P, Hayashi T, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
- Subjects
- Animals, Rats, Glucose pharmacology, Mitophagy, Palmitic Acid pharmacology, Protein Kinases genetics, Reactive Oxygen Species metabolism, Silybin pharmacology, Ubiquitin-Protein Ligases metabolism, Diabetes Mellitus, Experimental, Diabetes Mellitus, Type 2, Ferroptosis
- Abstract
Type 2 diabetes (T2DM) is induced by the abundance of glucose and lipids, which causes glucolipotoxicity to the pancreatic β-cells. Silibinin is a natural flavonoid possessing the regulatory activity on insulin production and therapeutic activity in diabetic mice; however, its effect on glucolipotoxicity is not fully explained. This in vitro study investigates the effects of silibinin on palmitic acid (PA) and high glucose (HG)-induced cell loss and ferroptosis of rat insulinoma INS-1 cells. In the cells treated with PA and HG, expressions of glucose transporter 4 (Glut4) and carnitine acyltransferase I (CPT1) for β-oxidation of fatty acids are reduced. Mitochondria are the metabolic organelles for glucose and fatty acids. The mitochondrial membrane potential (MMP) and ATP production were decreased, while the ROS level was elevated in the cells treated with PA and HG, indicating an induction of mitochondrial disorder. Cell loss was partially rescued by ferroptosis inhibition, suggesting an involvement of ferroptosis in the cells treated with PA and HG. More importantly, the increases in total iron, lipid ROS, MDA and COX-2, and the decrease in ferroptosis inhibitory molecules GSH, GPX4 and FSP1 appeared in the cells treated with PA and HG, confirming the occurrence of ferroptosis. Moreover, PINK1/parkin-mediated mitophagy, a vital process for selective elimination of damaged mitochondria, was blocked. Interestingly, silibinin rescued the mitochondria, restricted the ferroptosis and restored the mitophagy. By using the pharmacological stimulator and inhibitor of mitophagy, and si-RNA transfection to silence PINK1 expression, silibinin's protective effect against ferroptosis caused by PA and HG treatment was found to depend on mitophagy. Collectively, our current study reveals the new mechanisms for the protection of silibinin against the injury of INS-1 cells treated with PA and HG, elucidates the participation of ferroptosis in glucolipotoxicity, highlighting the involvement of mitophagy in defense against ferroptotic cell death., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Inc.)
- Published
- 2023
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194. A recombinant technique for mapping functional sites of heterotrimeric collagen helices: Collagen IV CB3 fragment as a prototype for integrin binding.
- Author
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Boudko SP, Konopka EH, Kim W, Taga Y, Mizuno K, Springer TA, Hudson BG, Moy TI, and Lin FY
- Subjects
- Animals, Humans, Binding Sites, Protein Binding, Protein Structure, Secondary, Mutation, Protein Domains, Collagen Type IV chemistry, Collagen Type IV genetics, Collagen Type IV metabolism, Integrins chemistry, Integrins metabolism, Protein Multimerization
- Abstract
Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α
1 β1 and α2 β1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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195. Inhibition of GluN2B pathway is involved in the neuroprotective effect of silibinin on streptozotocin-induced Alzheimer's disease models.
- Author
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Liu P, Wang C, Chen W, Kang Y, Liu W, Qiu Z, Hayashi T, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
- Subjects
- Rats, Mice, Animals, Receptors, N-Methyl-D-Aspartate metabolism, Amyloid beta-Peptides metabolism, Silybin pharmacology, Streptozocin, Brain-Derived Neurotrophic Factor metabolism, Molecular Docking Simulation, Disease Models, Animal, Alzheimer Disease chemically induced, Alzheimer Disease drug therapy, Neuroprotective Agents pharmacology, Neuroprotective Agents therapeutic use
- Abstract
Background: Over-activation of N-methyl-D-aspartate receptors (NMDARs) is involved in sporadic Alzheimer's disease. Silibinin, a natural flavonoid gained from the seeds of Silybum marianum, exerts neuroprotective effects on sporadic AD models, but its impacts on NMDARs remain unknown., Purpose: To study silibinin's regulatory effects on NMDARs pathway in sporadic AD models., Methods: MTT assay, western blotting, confocal microscopy, flow cytometry, RT-PCR, and siRNA transfection etc. were used for cellular and molecular studies. The direct interactions between silibinin and NMDAR subunits were evaluated by computational molecular docking, drug affinity responsive target stability (DARTS) assay and cellular thermal shift assay (CETSA). Y maze test, novel objects recognition test and Morris water maze test were conducted to examine the learning and memory ability of rats., Results: An in vitro AD model was established by treating HT22 murine hippocampal neurons with streptozotocin (STZ), as evidenced by the amyloid β (Aβ) deposition and hyperphosphorylation of tau proteins. Silibinin shows protection of neurons against STZ-induced cell damage. It is noteworthy that STZ-induced cellular calcium influx is inhibited by silibinin-treatment, indicating the possible modulation of calcium channels. Studies on NMDARs, the most widely distributed calcium channel, by using molecular docking, DARTS and CESTA, reveal that the GluN2B subunit, but not GluN2A, is the potential target of silibinin. Further studies using the pharmacological agonist (NMDA) and the GluN2B-specific inhibitor (Ifenprodil) or siRNA, indicate that the protection by silibinin treatment from STZ-induced cytotoxicity is medicated through interference with GluN2B-containing NMDARs, followed by the upregulation of CaMKIIα/ BDNF/ TrkB signaling pathway and improved levels of synaptic proteins (SYP and PSD-95). The results in vivo using rats intracerebroventricularly injected with STZ (ICV-STZ), a well-established sporadic AD model, confirm that silibinin improves learning and memory ability in association with modulation of the GluN2B/CaMKIIα/ BDNF/TrkB signaling pathway., Conclusion: Inhibiting over-activation of GluN2B-containing NMDARs is involved in the neuroprotective effect of silibinin on STZ-induced sporadic AD models., Competing Interests: Declaration of Competing Interest We wish to confirm that there are no potential conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2022 Elsevier GmbH. All rights reserved.)
- Published
- 2023
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196. Lysyl hydroxylase 3-mediated post-translational modifications are required for proper biosynthesis of collagen α1α1α2(IV).
- Author
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Ishikawa Y, Taga Y, Coste T, Tufa SF, Keene DR, Mizuno K, Tournier-Lasserve E, and Gould DB
- Subjects
- Humans, Glycosylation, Protein Processing, Post-Translational, Collagen metabolism, Connective Tissue Diseases, Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase genetics, Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase metabolism
- Abstract
Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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197. IGF-1R/YAP signaling pathway is involved in collagen V-induced insulin biosynthesis and secretion in rat islet INS-1 cells.
- Author
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Zhu Y, Chen S, Liu W, Xu F, Lu J, Hayashi T, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
- Subjects
- Animals, Collagen Type V pharmacology, Phosphorylation, RNA, Small Interfering metabolism, Rats, Transcription Factors metabolism, Insulin biosynthesis, Islets of Langerhans metabolism, Receptor, IGF Type 1 metabolism, Signal Transduction, YAP-Signaling Proteins metabolism
- Abstract
Purpose: Type V collagen (collagen V) is one of the important components of extracellular matrix (ECM) in pancreas. We previously reported that pre-coating collagen V on the culture dishes enhanced insulin production in INS-1 rat pancreatic β cells. In this study, we investigate the underlying mechanism., Results: Insulin biosynthesis and secretion are both increased in INS-1 cells cultured on collagen V-coated dishes, accompanied by the reduced nuclear translocation of Yes-associated protein (YAP), a transcriptional co-activator. YAP, the downstream effector of Hippo signaling pathway, plays an important role in the development and function of pancreas. Inhibition of YAP activation by verteporfin further up-regulates insulin biosynthesis and secretion. Silencing large tumor suppressor (LATS), a core component of Hippo pathway which inhibits activity of YAP by phosphorylation, by siRNA transfection inhibits both insulin biosynthesis and secretion. In the present study, the protein level of insulin-like growth factor 1 receptor (IGF-1 R), detected as the upstream molecule of YAP, is reduced in the INS-1 cells cultured on the dishes coated with collagen V. The silencing of IGF-1 R by siRNA transfection further enhances insulin biosynthesis and secretion. IGF-1 treatment reduces collagen V-induced up-regulation of insulin biosynthesis and secretion, accompanying the increased nuclear YAP., Conclusion: Inhibition of IGF-1 R/YAP signal pathway is involved in collagen V-induced insulin biosynthesis and secretion in INS-1 cells.
- Published
- 2022
- Full Text
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198. Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes.
- Author
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Terajima M, Taga Y, Nakamura T, Guo HF, Kayashima Y, Maeda-Smithies N, Parag-Sharma K, Kim JS, Amelio AL, Mizuno K, Kurie JM, and Yamauchi M
- Subjects
- Collagen metabolism, Hydroxylation, Lysine metabolism, Protein Processing, Post-Translational, Collagen Type I metabolism, Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase genetics, Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase metabolism
- Abstract
Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hyl
ald )-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald -derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells., (© 2022. The Author(s).)- Published
- 2022
- Full Text
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199. Silibinin relieves UVB-induced apoptosis of human skin cells by inhibiting the YAP-p73 pathway.
- Author
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Liu WW, Wang F, Li C, Song XY, Otkur W, Zhu YY, Hayashi T, Mizuno K, Hattori S, Fujisaki H, and Ikejima T
- Subjects
- Apoptosis, Humans, Infant, Newborn, Molecular Docking Simulation, RNA, Small Interfering genetics, RNA, Small Interfering pharmacology, Silybin pharmacology, Silymarin pharmacology
- Abstract
Excessive exposure to UVB induces skin diseases. Silibinin, a flavonolignan used for treating liver diseases, is found to be effective against UVB-caused skin epidermal and dermal cell damage. In this study we investigated the molecular mechanisms underlying. Human nonmalignant immortalized keratinocyte HaCaT cells and neonatal human foreskin fibroblasts HFFs were exposed to UVB irradiation. We showed that pre-treatment with silibinin dose-dependently decreased UVB-induced apoptosis of HaCaT cells. Furthermore, we showed that silibinin treatment inhibited nuclear translocation of YAP after UVB irradiation. Molecular docking analysis and DARTS assay confirmed the direct interaction of silibinin with YAP. Silencing YAP by siRNA had no influence on the survival of HaCaT cells, whereas inhibiting classical YAP-TEAD signaling pathway by siRNA targeting TEAD1 or its pharmaceutical inhibitor verteporfin further augmented UVB-induced apoptosis, suggesting that YAP-TEAD pathway was prosurvival, which did not participate in the protective effect of silibinin. We then explored the pro-apoptotic YAP-p73 pathway. p73 was upregulated in UVB-irradiated cells, but reduced by silibinin cotreatment. The mRNA and protein levels of p73 target genes (PML, p21 and Bax) were all increased by UVB but decreased by silibinin co-treatment. Inhibiting p73 by using siRNA reduced UVB-induced apoptosis, suggesting that downregulation of p73 was responsible for the cytoprotective effect of silibinin. In HFFs, the upregulated YAP-p73 pathway by UVB irradiation was also suppressed by silibinin. Collectively, YAP-p73 pathway is a major cause of the death of UVB-exposed epidermal HaCaT cells and dermal HFFs. Silibinin directly inhibits YAP-p73 pathway, exerting the protective action on UVB-irradiated skin cells., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)
- Published
- 2022
- Full Text
- View/download PDF
200. Identification of a highly stable bioactive 3-hydroxyproline-containing tripeptide in human blood after collagen hydrolysate ingestion.
- Author
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Taga Y, Iwasaki Y, Tometsuka C, Funato N, Shigemura Y, Kusubata M, and Mizuno K
- Abstract
There are increasing reports demonstrating high bioavailability of 4-hydroxyproline (4Hyp)-containing oligopeptides after oral ingestion of collagen hydrolysate and their bioactivity. In contrast, no study investigates the fate of another collagen-specific but minor amino acid, 3Hyp. Here, we identified Gly-3Hyp-4Hyp tripeptide in human blood at high concentrations, comparable to other 4Hyp-containing oligopeptides, after ingesting porcine skin collagen hydrolysate. Additionally, Gly-3Hyp-4Hyp uniquely maintained the maximum concentration until 4 h after the ingestion due to its exceptionally high resistance to peptidase/protease demonstrated by incubation with mouse plasma. In mice, oral administration of collagen hydrolysate prepared from bovine tendon, which contains a higher amount of 3Hyp, further increased blood Gly-3Hyp-4Hyp levels compared to that from bovine skin. Furthermore, Gly-3Hyp-4Hyp showed chemotactic activity on skin fibroblasts and promoted osteoblast differentiation. These results highlight the specific nature of the Gly-3Hyp-4Hyp tripeptide and its potential for health promotion and disease treatment., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
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