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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]

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 (Hylald)-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]

Besides motor disorder, cognitive dysfunction is also common in Parkinson's disease (PD). Essentially no causal therapy for cognitive dysfunction of PD exists at present. In this study, a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD was used to analyze the neuroprotective potential of orally administered silibinin, a proverbial hepatoprotective flavonoid derived from the herb milk thistle (Silybum marianum). Results demonstrated that silibinin administration significantly attenuated MPTP-induced cognitive impairment in behavioral tests. Nissl staining results showed that MPTP injection significantly increases the loss of neurons in the hippocampus. However, these mice were protected by oral administration of silibinin, accompanying reduction in the cell apoptosis in the hippocampus. The hippocampal aggregates of α-synuclein (α-syn) appeared in MPTP-injected mice, but were significantly decreased by silibinin treatment. MPTP injection induced oxidative stress, as evidenced by increased malondialdehyde (MDA) and decreased superoxide dismutase (SOD). The oxidative stress was alleviated by silibinin treatment. Mitochondrial disorder including the decline of mitochondrial membrane potential (MMP) was another signature in the hippocampus of MPTP-treated mice, accompanying increased mitochondrial fission and decreased fusion. Silibinin administration restored these mitochondrial disorders, as expected for the protection against MPTP injury. These findings suggest that silibinin has a potential to be further developed as a therapeutic candidate for cognitive dysfunction in PD. [ABSTRACT FROM AUTHOR]

Skeletal muscle regeneration is a complex process, involving the proliferation, migration, and differentiation of myoblasts. Recent studies suggest that some natural flavanones stimulate myogenesis. However, the effect of plant estrogen, silibinin, on the regulation of myoblast behaviors is unclarified. In this study, we investigated the effects of silibinin on immortalized murine myoblast C2C12 in the aspects of proliferation, migration, differentiation along with underlying mechanisms. The results show that silibinin at concentrations below 50 μM enhanced the migration and differentiation of C2C12 cells, but had no effect on cell proliferation. Silibinin significantly promoted the production of ROS, which appeared to play important roles in the migration and differentiation of the myoblasts. Interestingly, among ROS, the superoxide anion and hydroxyl radical were associated with the migration, whereas hydrogen peroxide contributed to the myogenic differentiation. We used ER agonist and antagonist to explore whether estrogen receptors (ERs), which are affected by silibinin treatment in the silibinin-enhanced C2C12 migration and differentiation. Migration was independent of ERs, whereas the differentiation was associated with decreased ERα activity. In summary, silibinin treatment increases ROS levels, leading to the promotion of migration and myogenic differentiation. Negative regulation ERα of differentiation but not of migration may suggest that ERα represses hydrogen peroxide generation. The effect of silibinin on myoblast migration and differentiation suggests that silibinin may have therapeutic benefits for muscle regeneration. [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]

Human triple negative breast cancer cells, MDA-MB-231, show typical epithelial to mesenchymal transition associated with cancer progression. Mitochondria play a major role in cancer progression, including metastasis. Changes in mitochondrial architecture affect cellular migration, autophagy and apoptosis. Silibinin is reported to have anti-breast cancer effect. We here report that silibinin at lower concentrations (30–90 μM) inhibits epithelial to mesenchymal transition (EMT) of MDA-MB-231, by increasing the expression of epithelial marker, E-cadherin, and decreasing the expression of mesenchymal markers, N-cadherin and vimentin. Besides, silibinin inhibition of cell migration is associated with reduction in the protein expression of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) and paxillin. In addition, silibinin treatment increases mitochondrial fusion through down-regulating the expression of mitochondrial fission-associated protein dynamin-related protein 1 (DRP1) and up-regulating the expression of mitochondrial fusion-associated proteins, optic atrophy 1, mitofusin 1 and mitofusin 2. Silibinin perturbed mitochondrial biogenesis via down-regulating the levels of mitochondrial biogenesis regulators including mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator (PGC1) and nuclear respiratory factor (NRF2). Moreover, DRP1 knockdown or silibinin inhibited cell migration, and MFN1&2 knockdown restored it. Mitochondrial fusion contributes to silibinin's negative effect on cell migration. Silibinin decreased reactive oxygen species (ROS) generation, leading to inhibition of the NLRP3 inflammasome activation. In addition, knockdown of mitofusin 1&2 (MFN 1&2) relieved silibinin-induced inhibition of NLRP3 inflammasome activation. Repression of ROS contributes to the inhibition of the expression of NLRP3, caspase-1 and IL-β proteins as well as of cell migration. Taken together, our study provides evidence that silibinin impairs mitochondrial dynamics and biogenesis, resulting in reduced migration and invasion of the MDA-MB-231 breast cancer cells. [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]

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]

ute 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. [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]

We reported previously that human fibroblasts form clumps when cultured on a dish coated with reconstituted type V collagen fibrils. Essentially all the type V collagen fibrils, initially coated on the dish, were recovered in the cell clumps that had eventually formed during the culture. We interpreted that type V collagen fibrils adhere to cells more strongly than to the dish and are detached by cell movements. In this study, type V collagen was suspended with fibroblasts to examine the fate of the type V collagen fibrils and to determine whether the fibrils affect the behaviour of the cells directly adherent to the dish. The added type V collagen accumulated in the intercellular space concomitantly with the local aggregation of fibroblasts. scanning electron microscope examination indicated that type V collagen fibrils were found in the vicinity of cells in cultures without ascorbic acid where essentially no collagen secretion takes place. These results indicate that type V collagen forms fibrils and the fibrils are accumulated in the intercellular spaces. The accumulated type V collagen fibrils work as a cementing material for cell clump formation. This phenomenon is discussed in relation to the possible involvement of type V collagen fibrils in tissue organization. [ABSTRACT FROM PUBLISHER]

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]

Background: There is increasing concern in Japan over infants with atopic dermatitis (AD) who present with severe systemic complications, such as hypoproteinemia, electrolyte disturbances, and delayed growth and development. They are often associated with extremely increased numbers of circulating eosinophils. However, the clinical significance of eosinophil expansion has not been thoroughly investigated.Methods: This study attempted to determine the significance of eosinophilia and eosinophil activation in infant cases of AD by comparing multiple clinical parameters, indexes of eosinophil activation, and levels of serum cytokines. CD69 expression was determined by flow cytometry. The clinical severity of AD was graded by the severity SCORing of atopic dermatitis (SCORAD) method. Patients were classified into two groups, with and without CD69 on eosinophils. Nuclear lobes were evaluated under a microscopy. Serum levels of eosinophil-derived neurotoxin (EDN), interleukin (IL)-12, IL-18, IL-4, IL-5 and interferon (IFN)-γ were determined by enzyme-linked immunosorbent assay.Results: Patients with CD69-positive eosinophils had significantly higher numbers of eosinophils and platelets, total IgE, and eosinophil nuclear lobes. They also showed growth failure, developmental delay, low serum albumin, and electrolyte disturbances. EDN and IL-18 levels were significantly increased in this group, but the levels of IL-4, IL-5 and IL-12 were not significantly different between the two groups. IFN-γ was not detectable in all patients with AD. Surface expression of CD69 indicates intense systemic allergic inflammation induced in severe cases of AD.Conclusions: Evaluation of eosinophil activation and early therapeutic intervention is mandatory for the treatment of severe AD during infancy. [ABSTRACT FROM AUTHOR]

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]

We investigated the effect of immobilization of hindlimb in a shortened position on proteins in the mouse soleus muscle and determined the molecular properties of a protein that had changed specifically in the atrophied muscle. A protein of 67.5 kDa was increased significantly (P<0.001) by 1.9-fold in the atrophied muscle compared to the control muscle as shown by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE). The isoelectric point of the protein was pH 6.3 in two-dimensional PAGE. We carried out N-terminal and peptide mapping analyses to identify the primary structure of the protein. A 17-amino acid sequence showed 100% homology with the mouse serum albumin precursor. A peptide map of the 67.5-kDa protein was similar to that of mouse serum albumin. Mass spectrometry also showed the protein to be most similar to mouse serum albumin. Thus, based on these criteria, the 67.5-kDa protein was indistinguishable from mouse serum albumin. An immunohistochemical study showed that the 67.5-kDa protein was located mainly in the endomysium of the control muscle and had accumulated in the widened interstitial spaces of the atrophied muscle. A reverse transcription-PCR assay revealed that the albumin gene was expressed in skeletal muscle tissue, but that its expression was significantly lower in the atrophied muscle than in the control muscle (P<0.001). We conclude that albumin is increased in atrophied muscle and suggest that this may be of clinical importance for analyzing the process of muscular atrophy. [ABSTRACT FROM AUTHOR]

The ultrastructure of animal tissues has a graded distribution that is reminiscent of the reconstituted aggregate structures from isolated type I collagen, type V collagen, and type IV collagen aligned in a gradient manner. We can assume that mesenchymal cells with different functions are also located in a graded way in accord with the graded distribution of collagen supramolecular aggregates. It was already known that proliferation, shape, migration, gene expression, and responses to growth factors of the cultured fibroblasts were greatly affected by the type I collagen fibrils. Other differentiated cells such as endothelial cells and smooth muscle cells are surrounded by type IV collagen and/or type V collagen more closely. The reconstituted type IV collagen aggregates in a gel form were tested as culture substrates for these differentiated cells. Of particular interest was the examination of myofibroblasts that are thought responsible for the pathogenesis of fibrotic or cirrhotic tissues. It was found that the type IV collagen gel appeared to restore the behaviors of smooth muscle-related cells including the myofibroblasts into the contractile stage. The whole cells cultured on the type IV collagen gel were totally quiescent and formed a multicellular network. The findings suggest that interrelationships in a graded way between differentiated cells and the specific collagen aggregates might be involved in the homeostasis of tissue structure and function. [ABSTRACT FROM AUTHOR]

The heparin affinities of heat-treated type V collagen α-chains and the triple-helical molecules were evaluated in terms of the NaCl concentration required for prevention of binding to a heparin-Sepharose column. After heat treatment, α1(V) chain required approximately two-fold higher NaCl concentration to pass through the column than the other two chains, α2(V) and α3(V). Thus, the heparin affinity of α1(V) may be approximately two-fold higher than those of the other α(V)-chains. The type V collagen molecules in triple-helical conformation were separated into two fractions at 170 mM NaCl in 20 mM phosphate buffer, pH 7.2, containing 2 M urea; bound and non-bound. The ratio of the three a-chains, αl(V):α2(V):α3(V) was 2: 1 : 0 and 1: 1: 1 in the bound and flow-through fractions, respectively, on analysis by SDS-PAGE. The differential affinity of the two fractions could be accounted for by the number of α1(V) chains in the triple-helical molecule, if these fractions contained triple-helical subtypes with the chain compositions of [α1(V)]2α2(V) and α1(V)α2(V)α3(V), respectively. From the comparison of the NaCl concentration required for prevention of the binding, [α1(V)]2α2(V) had about two-fold higher affinity than α1(V)α2(V)α3(V), and the separated α1(V) chain showed an intermediate affinity. A possible explanation for difference in heparin affinity among the subtypes of molecules and the separated α-chains is that the heparin affinity of type V collagen molecule is governed by the number of α1(V) chains contained in the molecule and that steric restraint in a triple-helical conformation weakens the binding of αl(V) chain to heparin. [ABSTRACT FROM AUTHOR]

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."