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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. 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. Inhibition of IGF-1 R/YAP signal pathway is involved in collagen V–induced insulin biosynthesis and secretion in INS-1 cells. [ABSTRACT FROM AUTHOR]

Acute renal failure with severe loin pain induced by anaerobic exercise (ALPE) is a rare condition that is accompanied by wedge-shaped contrast enhancement on computed tomography (CT) without evidence of rhabdomyolysis. In two pediatric cases with ALPE, we tried to determine the relationship between findings from CT and magnetic resonance imaging (MRI). Case 1 involved a 13-year-old Japanese girl with a diagnosis of ALPE with normo-uricemia. Contrast-enhanced CT after 24 and 48 h showed a wedge-shaped excretion delay for the contrast media. A clear wedge-shaped signal hyperintensity matching the CT images was obtained by diffusion-weighted MRI. Case 2 involved a 16-year-old boy who presented with a second attack of ALPE after diagnosis of ALPE with hypouricemia 1 year earlier. Only diffusion-weighted imaging was performed. Clear wedge-shaped signal hyperintensity was apparent, similar to Case 1. MRI is safer than contrast-enhanced CT for patients with ALPE. Diffusion-weighted MRI is a very useful examination for diagnosing ALPE, providing noninvasive detection of lesions peculiar to ALPE., Author(s): Kazuhide Ohta [sup.1] , Tadafumi Yokoyama [sup.2] , Masaki Shimizu [sup.2] , Kazunori Mizuno [sup.1] , Shinobu Sakazume [sup.1] , Takuma Fujiki [sup.3] , Yutaka Saikawa [sup.3] , Akihiro [...]

Extracellular matrix (ECM) plays an important role in tissue repair, cell proliferation, and differentiation. Our previous study showed that collagen I and collagen V differently regulate the proliferation of rat pancreatic β cells (INS-1 cells) through opposite influences on the nuclear translocation of β-catenin. In this study, we investigated the β-catenin pathway in INS-1 cells on dishes coated with collagen I or V. We found that nuclear translocation of the transcription factor Yes-associated protein (YAP) was enhanced by collagen I and suppressed by collagen V, but had no effect on INS-1 cell proliferation. Morphologically, INS-1 cells on collagen V-coated dishes showed stronger cell-to-cell adhesion, while the cells on collagen I-coated dishes showed weaker cell-to-cell adhesion in comparison with the cells on non-coated dishes. E-cadherin played an inhibitory role in the proliferation of INS-1 cells cultured on collagen I or collagen V coated dishes via regulation of the nuclear translocation of β-catenin. Integrin β1 was enhanced with collagen I, while it was repressed with collagen V. The integrin β1 pathway positively regulated the cell proliferation. Inhibition of integrin β1 pathway restored the protein level of E-cadherin and inhibited the nuclear translocation of β-catenin in the cells on collagen I-coated dishes, but no effect was observed in the cells on collagen V-coated dishes. In conclusion, collagen I enhances the proliferation of INS-1 cells via the integrin β1 and E-cadherin/β-catenin signaling pathway. In INS-1 cells on collagen V-coated dishes, both integrin β1 and E-cadherin/β-catenin signal pathways are involved in the inhibition of proliferation. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) has a marked influence on adipose tissue development. Adipose tissue formation is initiated with proliferation of preadipocytes and migration before undergoing further differentiation into mature adipocytes. Previous studies showed that collagen I (col I) provides a good substratum for 3T3‐L1 preadipocytes to grow and migrate. However, it remains unclear whether and how col I regulates adipogenic differentiation of preadipocytes. This study reports that lipid accumulation, representing in vitro adipogenesis of the 3T3‐L1 preadipocytes or the mouse primary adipocyte precursor cells derived from subcutaneous adipose tissue in the inguinal region is inhibited by the culture on col I, owing to downregulation of adipogenic factors. Previous study shows that col I enhances 3T3‐L1 cell migration via stimulating the nuclear translocation of yes‐associated protein (YAP). In this study, we report that downregulation of YAP is associated with in vitro adipogenesis of preadipocytes as well as with in vivo adipose tissue of high‐fat diet fed mice. Increased expression of YAP in the cells cultured on col I‐coated dishes is correlated with repression of adipogenic differentiation processes. The inactivation of YAP using YAP inhibitor, verteporfin, or YAP small‐interfering RNA enhanced adipogenic differentiation and reversed the inhibitory effect of col I. Activation of YAP either by the transfection of YAP plasmid or the silence of large tumor suppressor 1 (LATS1), an inhibitory kinase of YAP, inhibited adipogenic differentiation. The results indicate that col I inhibits adipogenic differentiation via YAP activation in vitro. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) molecules have multiple functions; prevention of cytotoxicity, provision of mechanical support, cell adhesive substrates and structural integrity in addition to mediation of cellular signaling. In this study, we report that the proliferation of INS-1 cells cultured on collagen I-coated dishes is enhanced, but it is inhibited on collagen V-coated dishes. Inhibitory proliferation on collagen V-coated is not due to apoptosis induction. Silibinin decreases hepatic glucose production and protects pancreatic β-cells, as a potential medicine for type II diabetes. Silibinin up-regulates the proliferation of cells cultured on both collagen I- and V-coated dishes. Collagen-coating regulates gene expression of collagen in a collagen type-related manner. Silibinin increases mRNA expression of collagen I in the cells on collagen I- and V-coated dishes; however, silibinin decreases collagen V mRNA expression on collagen I- and V-coated dishes. Collagen I-coating significantly enhances nuclear translocation of β-catenin, while collagen V-coating reduces it. Differential effects of silibinin on collagen I mRNA and collagen V mRNA can be accounted for by the finding that silibinin enhances nuclear translocation of β-catenin on both collagen I- and V-coated dishes, since phenomenologically nuclear translocation of β-catenin enhances collagen I mRNA but represses collagen V mRNA. These results demonstrate that nuclear translocation of β-catenin up-regulates proliferation and collagen I gene expression, whereas it down-regulates collagen V gene expression of INS-1 cells. Differential gene expressions of collagen I and V by nuclear β-catenin could be important for understanding fibrosis where collagen I and V may have differential effects. [ABSTRACT FROM AUTHOR]

Purpose: Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I-coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface. Material and methods: MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, enzyme-linked immunosorbent assay, and western blot were used. Results: Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, the autophagy and ROS. Autophagy induction is attenuated by N-acetyl-L-cysteine treatment, but the treatment with autophagy inhibitor, 3-methyladenine, does not affect ROS levels, suggesting ROS are upstream of autophagy in the regulation axis of differentiated U937 cells on gelatin-coated surface. Further study confirmed that upregulation of autophagy was responsible for ROS-induced cell aggregation and production of pro-inflammatory mediators. Conclusion: The results suggest that gelatin-coating promotes the aggregation of PMA-treated U937 cells and the production of pro-inflammatory mediators by ROS-autophagy signaling pathway. [ABSTRACT FROM AUTHOR]

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. To study silibinin's regulatory effects on NMDARs pathway in sporadic AD models. 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. 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. Inhibiting over-activation of GluN2B-containing NMDARs is involved in the neuroprotective effect of silibinin on STZ-induced sporadic AD models. [Display omitted] [ABSTRACT FROM AUTHOR]

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]

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]

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]

The single-crystal structures of three collagen-like host-guest peptides, (Pro-Pro-Gly)-Hyp-Yaa-Gly-(Pro-Pro-Gly) [Yaa = Thr, Val, Ser; Hyp = (4 R)-4-hydroxyproline] were analyzed at atomic resolution. These peptides adopted a 7/2-helical structure similar to that of the (Pro-Pro-Gly) peptide. The stability of these triple helices showed a similar tendency to that observed in Ac-(Gly-Hyp-Yaa)-NH (Yaa = Thr, Val, Ser) peptides. On the basis of their detailed structures, the differences in the triple-helical stabilities of the peptides containing a Hyp-Thr-Gly, Hyp-Val-Gly, or Hyp-Ser-Gly sequence were explained in terms of van der Waals interactions and dipole-dipole interaction between the Hyp residue in the X position and the Yaa residue in the Y position involved in the Hyp(X):Yaa(Y) stacking pair. This idea also explains the inability of Ac-(Gly-Hyp-alloThr)-NH and Ac-(Gly-Hyp-Ala)-NH peptides to form triple helices. In the Hyp(X):Thr(Y), Hyp(X):Val(Y), and Hyp(X):Ser(Y) stacking pairs, the proline ring of the Hyp residues adopts an up-puckering conformation, in agreement with the residual preference of Hyp, but in disagreement with the positional preference of X in the Gly-Xaa-Yaa sequence. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 628-640, 2011. [ABSTRACT FROM AUTHOR]

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]

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]

Abstract: Graph colorability (COL), is a typical constraint satisfaction problem to which phase transition phenomena (PTs), are important in the computational complexity of combinatorial search algorithms. PTs are significant and subtle because, in the PT region, extraordinarily hard problem instances are found, which may require exponential-order computational time to solve. To clarify PT mechanism, many studies have been undertaken to produce very hard instances, many of which were based on generate-and-test approaches. We propose a rather systematic or constructive algorithm that repeats the embedding of 4-critical graphs to arbitrarily generate large extraordinarily hard 3-colorability instances. We demonstrated experimentally that the computational cost to solve our generated instances is of an exponential order of the number of vertices by using a few actual coloring algorithms and constraint satisfaction algorithms. [Copyright &y& Elsevier]

In this paper, the authors propose a method of systematically generating 3-colorable (3COL) graph instances for which a great deal of time and effort are required to obtain a solution (or to determine solvability). This method, which differs from the conventional approach of selecting hard instances from among randomly generated instances, aims to reliably generate hard problems by repeatedly embedding 3COL minimal unsolvable structures in the initial graph. The generated instances conform to various conditions of problems for which the solution searches that had been identified conventionally are extremely difficult. Also, the authors use several solution search algorithms to experimentally verify that the required effort is an exponential order of the number of vertices. © 2007 Wiley Periodicals, Inc. Electron Comm Jpn Pt 3, 90(12): 27–35, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecjc.20359 [ABSTRACT FROM AUTHOR]

Collagen has a triple helical structure comprising strands with a repeating Xaa-Yaa-Gly sequence. L-Proline (Pro) and 4(R)-hydroxyl-L-proline (4(R)Hyp) residues are found most frequently in the Xaa and Yaa positions. However, in natural collagen, 3(S)- hydroxyl-L-proline (3(S)Hyp) occurs in the Xaa positions to varying extents and is most common in collagen types IV and V. Although 4(R)Hyp residues in the Yaa positions have been shown to be critical for the formation of a stable triple helix, the role of 3(S)Hyp residues in the Xaa position is not well understood. Indeed, recent studies have demonstrated that the presence of 3(S)Hyp in the Xaa positions of collagen-like peptides actually has a destabilizing effect relative to peptides with Pro in these locations. Whether this destabilization is reflected in a local unfolding or in other structural alterations of the collagen triple helix is unknown. Thus, to determine what effect the presence of 3(S)Hyp residues in the Xaa positions has on the overall conformation of the collagen triple helix, we determined the crystal structure of the polypeptide H-(Gly-Pro-4(R)Hyp)3- (Gly-3(S)Hyp-4(R)Hyp)2-(Gly-Pro-4(R)Hyp)4-OH to 1.80 Å resolution. The structure shows that, despite the presence of the 3(S)Hyp residues, the peptide still adopts a typical 7/2 superhelical symmetry similar to that observed in other collagen structures. The puckering of the Xaa position 3(S)Hyp residues, which are all down (Cγ-endo), and the ϕ/ψ, dihedral angles of the Xaa 3(S)Hyp residues are also similar to those of typical collagen Pro Xaa residues, Thus, the presence of 3(S)Hyp in the Xaa positions does not lead to large structural alterations in the collagen triple helix. [ABSTRACT FROM AUTHOR]

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]