Search

Effective amelioration of type II diabetes requires therapies that increase both glucose uptake activity per cell and skeletal muscle mass. Myristic acid (14:0) increases diacylglycerol kinase (DGK) δ protein levels and enhances glucose uptake in myotubes in a DGKδ-dependent manner. However, it is still unclear whether myristic acid treatment affects skeletal muscle mass. In this study, we found that myristic acid treatment increased the protein level of β-tubulin, which constitutes microtubules and is closely related to muscle mass, in C2C12 myotubes but not in the proliferation stage in C2C12 myoblasts. However, lauric (12:0), palmitic (16:0) and oleic (18:1) acids failed to affect DGKδ and β-tubulin protein levels in C2C12 myotubes. Moreover, knockdown of DGKδ by siRNA significantly inhibited the increased protein level of β-tubulin in the presence of myristic acid, suggesting that the increase in β-tubulin protein by myristic acid depends on DGKδ. These results indicate that myristic acid selectively affects β-tubulin protein levels in C2C12 myotubes via DGKδ, suggesting that this fatty acid improves skeletal muscle mass in addition to increasing glucose uptake activity per cell.

Tenascin-X (TNX) is an extracellular matrix glycoprotein for which a deficiency results in a recessive form of classical-like Ehlers-Danlos syndrome (clEDS), a heritable connective tissue disorder with hyperextensible skin without atrophic scarring, joint hypermobility, and easy bruising. Notably, patients with clEDS also suffer from not only chronic joint pain and chronic myalgia but also neurological abnormalities such as peripheral paresthesia and axonal polyneuropathy with high frequency. By using TNX-deficient (Tnxb−/−) mice, well-known as a model animal of clEDS, we recently showed that Tnxb−/− mice exhibit hypersensitivity to chemical stimuli and the development of mechanical allodynia due to the hypersensitization of myelinated A-fibers and activation of the spinal dorsal horn. Pain also occurs in other types of EDS. First, we review the underlying molecular mechanisms of pain in EDS, especially that in clEDS. In addition, the roles of TNX as a tumor suppressor protein in cancer progression have been reported. Recent in silico large-scale database analyses have shown that TNX is downregulated in various tumor tissues and that high expression of TNX in tumor cells has a good prognosis. We describe what is so far known about TNX as a tumor suppressor protein. Furthermore, some patients with clEDS show delayed wound healing. Tnxb−/− mice also exhibit impairment of epithelial wound healing in corneas. TNX is also involved in liver fibrosis. We address the molecular mechanism for the induction of COL1A1 by the expression of both a peptide derived from the fibrinogen-related domain of TNX and integrin α11.

Hydrogen-rich water (HW) has been suggested to possess antioxidant properties of value in treatments of lifestyle diseases and for prevention of latent pathologies. To date, the potential benefits of HW against the deleterious effects of excessive salt intake and hypertension have not been investigated. Here, we first examined the effects of HW or HW supplemented with 0.1% ascorbic acid (HWA) on spontaneously hypertensive rats (SHR) that had been fed a normal diet. In comparison to control rats given distilled water (DW), we found that HW did not significantly influence systolic blood pressure (SBP) or diastolic blood pressure (DBP) in SHR; however, the increase in SBP and DBP were inhibited in the HWA group. Next, four groups of SHR were given DW, 0.1% ascorbic acid-added DW (DWA), HW, or HWA in combination with a 4% NaCl-added diet. SHR fed the 4% NaCl-added diet showed increased hypertension; HWA treatment resulted in a significant reduction in blood pressure. The HWA group tended to have lower plasma angiotensin II levels than the DW group. In addition, urinary volumes and urinary sodium levels were significantly lower in the HWA group than the DW group. Urinary isoprostane, an oxidative stress marker, was also significantly lower in the HWA group, suggesting that the inhibitory effect of HWA on blood pressure elevation was caused by a reduction in oxidative stress. These findings suggest a synergistic interaction between HW and ascorbic acid, and also suggest that HWA ingestion has potential for prevention of hypertension.

Extracellular matrix tenascin‑X (TNX) is the largest member of the tenascin family. Our previous study demonstrated that TNX was involved in hepatic dysfunction, including fibrosis, in mice that were administered a high‑fat and high‑cholesterol diet with high levels of phosphorus and calcium. The present study investigated whether overexpression of both the fibrinogen domain of TNX (TNX‑FG) and integrin α11, one of the TNX cell surface receptors, induces

Introduction: Whether hypothermic cardiopulmonary bypass could attenuate both blood coagulation and platelet activation compared to normothermic cardiopulmonary bypass remains elusive. Methods: Biocompatibility of a polymer-coated cardiopulmonary bypass circuit was comparatively assessed by plasma proteomics between juvenile pigs undergoing hypothermic (23°C) cardiopulmonary bypass and those undergoing normothermic (37°C) cardiopulmonary bypass (n = 6, respectively). Plasma samples were taken three times: 5 minutes after initiation of cardiopulmonary bypass (T5, before cooling), just before declamping and rewarming (Tc), and just before termination of cardiopulmonary bypass (Trw, 120 minutes). Proteomic analysis was quantitively performed by isobaric tags for relative and absolute quantification labeling. Thrombin–antithrombin complexes (TAT III) were measured by enzyme immunoassay, and vitamin K–dependent protein C (PROC), β-thromboglobulin (TG), and P-selectin were measured by enzyme-linked immunosorbent assay. Blood gas analyses evaluated oxygenator performance. Results: Hypothermic cardiopulmonary bypass had a significantly higher PaO2 at Tc and lower PaCO2 at Trw than normothermic cardiopulmonary bypass. Two hundred twenty-four proteins were identified with statistical criteria of both protein confidence (>95%) and false discovery rate (Conclusion: Hypothermic cardiopulmonary bypass attenuated platelet degranulation/blood coagulation and maintained better oxygenator performance compared to normothermic cardiopulmonary bypass in juvenile pigs.

Housing conditions can affect the well-being of laboratory animals and thereby affect the outcomes of experiments. The appropriate environment is essential for the expression of natural behavior in animals. Here, we compared survival rates in four inbred mouse strains maintained under three different environmental conditions. Three mouse strains (C57BL/6J, C3H/HeN, and DBA/2J) housed under environmental enrichment (EE) conditions showed improved survival; however, EE did not alter the survival rate of the fourth strain, BALB/c. None of the strains showed significant differences in body weights or plasma corticosterone levels in the three environmental conditions. For BALB/c mice, the rates of debility were higher in the EE group. Interestingly, for C57BL/6J and C3H/HeN mice, the incidence of animals with alopecia was significantly lower in the EE groups than in the control group. It is possible that the enriched environment provided greater opportunities for sheltering in a secure location in which to avoid interactions with other mice. The cloth mat flooring used for the EE group was bitten and chewed by the mice. Our findings suggest that depending on the mouse strains different responses to EE are caused with regard to health and survival rates. The results of this study provide basic data for further studies on EE.

Alzheimer’s disease (AD) is a common dementia disease in the elderly. To get a better understanding of the pathophysiology, we performed a proteomic analysis of the urine exosomes (U-exo) in AD model mice (J20). The polymer precipitation method was used to isolate U-exo from the urine of 3-month-old J20 and wild-type (WT) mice. Neuron-derived exosome (N-exo) was isolated from U-exo by immunoprecipitation. iTRAQ-based MALDI TOF MS/MS was used for proteomic analysis. The results showed that compared to WT, the levels of 61 and 92 proteins were increased in the J20 U-exo and N-exo, respectively. Gene ontology enrichment analysis demonstrated that the sphingolipid catabolic process, ceramide catabolic process, membrane lipid catabolic process, Aβ clearance, and Aβ metabolic process were highly enriched in U-exo and N-exo. Among these, Asah1 was shown to be the key protein in lipid metabolism, and clusterin, ApoE, neprilysin, and ACE were related to Aβ metabolism and clearance. Furthermore, protein–protein interaction analysis identified four protein complexes where clusterin and ApoE participated as partner proteins. Thus, J20 U-exo and N-exo contain proteins related to lipid- and Aβ-metabolism in the early stages of AD, providing a new insight into the underlying pathological mechanism of early AD.

Joint hypermobility syndrome (JHS) (also termed hypermobility type Ehlers-Danlos syndrome, hEDS) is a heritable connective tissue disorder that is characterized by generalized joint hypermobility, chronic pain, fatigue, and minor skin changes. Initially, it was reported that there is a small subset of patients with JHS/hEDS who have haploinsufficiency of tenascin-X (TNX). However, the relationship between TNXB and JHS/hEDS has not been reported at all afterwards. EDS was reclassified into thirteen types in 2017, and the causative gene of JHS/hEDS remained to be identified. Therefore, in this study in order to determine whether JHS/hEDS can be diagnosed by the concentrations of serum form of TNX (sTNX), we measured the concentrations of sTNX in 17 JHS/hEDS patients. The sTNX concentrations in half of the JHS/hEDS patients were significantly lower than those in healthy individuals. No mutations, insertions or deletions were detected in the TNX exon sequence of the JHS/hEDS patients except for one in patient. That patient has a heterozygous mutation. A correlation between sTNX concentration and mutation of the TNXB genomic sequence was not found in the JHS/hEDS patients. These results indicate that the decrease in sTNX concentration could be used as a risk factor for JHS/hEDS.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PA). The η isozyme of DGK is abundantly expressed in C2C12 myoblasts. However, the role of DGKη in skeletal muscle cells remains unknown. In the present study, we showed that DGKη was downregulated at an early stage of myogenic differentiation. The knockdown of DGKη by siRNAs significantly inhibited C2C12 myoblast proliferation but did not inhibit differentiation. Moreover, the suppression of DGKη expression decreased the expression levels of mammalian target of rapamycin (mTOR), which is a key regulator of cell proliferation, and fatty acid synthase (FASN), which catalyzes the de novo synthesis of fatty acids for cell proliferation and is transcriptionally regulated via mTOR signaling. Furthermore, the knockdown of mTOR or raptor, which is a component of mTOR complex 1 (mTORC1), decreased the amount of FASN. These results indicate that DGKη regulates myoblast proliferation through the mTOR (mTORC1)-FASN pathway. Interestingly, the knockdown of mTOR reduced the expression levels of DGKη, implying mutual regulation between DGKη and mTOR. In DGKη-knockdown myoblasts, C30-C36-PA species, mTOR activators, were decreased, suggesting that the modulation of mTOR activity through these PA species also plays an important role in myoblast proliferation.

Background Cystatin C (CST3), a ubiquitously expressed cysteine protease inhibitor, is implicated in several neurological diseases. Here, we have developed an accurate CST3 measurement system based on liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods LC-MS/MS based measurement for CSF CST3 was validated by determination of assay precision, accuracy and recovery. The values were compared with those measured by immunoassay. Glycosylation of CST3 in CSF was analyzed by Western blotting and lectin blotting. Results Measuring standard CST3 by LC-MS/MS produced a linear standard curve that correlated with assigned values (r2 = 0.99). Both intra- and inter-assay variation was Conclusions Our results suggest that LC-MS/MS-based CST3 measurement is a robust method with higher detection ability. Such method could be useful for the diagnosis and monitoring of neurological diseases.

Tenascin-X (TNX), an extracellular matrix protein, is abundantly expressed in peripheral nerves. However, the physiological role of TNX in peripheral nerves remains unknown. In this study, we found that actin levels in sciatic nerves of TNX-deficient mice were markedly decreased. Since actin was highly expressed in endothelial cells in wild-type sciatic nerves, we assessed morphological alterations of blood vessels in TNX-null sciatic nerves. The density of blood vessels was significantly decreased and the size of blood vessels was larger than those in wild-type sciatic nerves. Immunofluorescence showed that TNX was expressed by Schwann cells and fibroblasts in sciatic nerves. The results suggest that TNX secreted from Schwann cells and/or fibroblasts is involved in blood vessel formation in peripheral nerves.

Extracellular matrix glycoprotein tenascin‑X (TNX) is the largest member of the tenascin family. In the present study, the contribution of TNX to liver dysfunction was investigated by administration of high‑fat and high‑cholesterol diet with high levels of phosphorus and calcium (HFCD) to wild‑type (WT) and TNX‑knockout (KO) mice. After 16 weeks of HFCD administration, the ratio of liver weight to body weight was approximately 22% higher in the HFCD‑fed WT mice compared with the HFCD‑fed TNX‑KO mice, indicating hepatomegaly in HFCD‑fed WT mice. Histological analyses with hematoxylin and eosin staining at 21 weeks revealed that hepatocyte hypertrophy in HFCD‑fed TNX‑KO mice was suppressed to 85% of that in HFCD‑fed WT mice. By contrast, there was a 1.2‑fold increase in lipid deposition in hepatocytes from HFCD‑fed TNX‑KO mice compared with HFCD‑fed WT mice at 18 weeks, as demonstrated by Oil Red O staining. In addition, TNX‑KO mice at 21 weeks and 27 weeks post‑HFCD administration exhibited significant suppression of inflammatory cell infiltrate to 51 and 24% of that in WT mice, respectively. Immunofluorescence analysis for type I collagen and Elastica van Gieson staining demonstrated a clear hepatic fibrosis progression in HFCD‑fed WT mice at 27 weeks, whereas hepatic fibrosis was undetected in HFCD‑fed TNX‑KO mice. The present findings indicated that TNX deficiency suppressed hepatic dysfunction induced by HFCD administration.

Polyamines have beneficial properties to prevent aging-associated diseases. Raw soybean has relatively high polyamine contents; and the fermented soybean natto is a good source of polyamines. However, detailed information of diversity of polyamine content in raw soybean is lacking. The objectives of this study were to evaluate differences of polyamines among raw soybeans and select the high polyamine-containing cultivar for natto production. Polyamine contents were measured chromatographically in 16 samples of soybean, which showed high variation among soybeans as follows: 93–861 nmol/g putrescine, 1055–2306 nmol/g spermidine, and 177–578 nmol/g spermine. We then confirmed the high correlations of polyamine contents between raw soybean and natto (r = 0.96, 0.95, and 0.94 for putrescine, spermidine, and spermine, respectively). Furthermore, comparison of the polyamine contents among 9 Japanese cultivars showed that ‘Nakasen-nari’ has the highest polyamine contents, suggesting its suitability for enhancement of polyamine contents of natto.

Tenascin-X (TNX) is a member of the extracellular matrix glycoprotein tenascin family, and TNX deficiency leads to Ehlers-Danlos syndrome, a heritable human disorder characterized mostly by skin hyperextensibility, joint hypermobility, and easy bruising. TNX-deficient patients complain of chronic joint pain, myalgia, paresthesia, and axonal polyneuropathy. However, the molecular mechanisms by which TNX deficiency complicates pain are unknown. Here, we examined the nociceptive behavioral responses of TNX-deficient mice. Compared with wild-type mice, TNX-deficient mice exhibited mechanical allodynia but not thermal hyperalgesia. TNX deficiency also increased pain sensitivity to chemical stimuli and aggravated early inflammatory pain elicited by formalin. TNX-deficient mice were significantly hypersensitive to transcutaneous sine wave stimuli at frequencies of 250 Hz (Aδ fiber responses) and 2000 Hz (Aβ fiber responses), but not to stimuli at frequency of 5 Hz (C fiber responses). In addition, the phosphorylation levels of extracellular signal-related kinase, an active neuronal marker, and the activity of NADPH-diaphorase, a neuronal nitric oxide activation marker, were enhanced in the spinal dorsal horns of TNX-deficient mice. These results suggest that TNX deficiency contributes to the development of mechanical allodynia and hypersensitivity to chemical stimuli, and it induces hypersensitization of myelinated A fibers and activation of the spinal dorsal horn.

Transplantation with mesenchymal stem cells (MSCs) has been reported to promote functional recovery in animal models of ischemic stroke. However, the molecular mechanisms underlying the therapeutic effects of MSC transplantation have been only partially elucidated. The purpose of this study was to comprehensively identify changes in brain proteins in rats treated with MSCs for ischemic stroke, and to explore the multi-target mechanisms of MSCs using a proteomics-based strategy. Twenty-eight proteins were found to be differentially expressed following B10 MSC transplantation in adult male Wistar rats, as assessed using isobaric tagging for relative and absolute protein quantification (iTRAQ). Subsequent bioinformatic analysis revealed that these proteins were mainly associated with energy metabolism, glutamate excitotoxicity, oxidative stress, and brain structural and functional plasticity. Immunohistochemical staining revealed decreased expression of EAAT1 in the phosphate-buffered saline group as opposed to normal levels in the B10 transplantation group. Furthermore, ATP levels were also significantly higher in the B10 transplantation group, thus supporting the iTRAQ results. Our results suggest that the therapeutic effects of B10 transplantation might arise from the modulation of the acute ischemic cascade via multiple molecular pathways. Thus, our findings provide valuable clues to elucidate the mechanisms underlying the therapeutic effects of MSC transplantation in ischemic stroke.

Introduction: To consider alternative mechanisms that give rise to a refluxing uretero-vesical junction (UVJ), we hypothesized that children with a common heritable urinary tract defect, vesico-ureteric reflux (VUR), may have a defect in the extracellular matrix composition of the UVJ and other tissues that would be revealed by assessment of the peripheral joints. Hypermobile joints can arise from defects in the extracellular matrix within the joint capsule that affect proteins, including Tenascin XB. Methods: We performed an observational study of children with familial and non-familial VUR to determine the prevalence of joint hypermobility, renal scarring, and DNA sequence variants in Tenascin XB. Results: The majority of children, 27/44, exhibited joint hypermobility using the Beighton scoring system. This included 15/26 girls (57.6%) and 12/18 boys (66.6%), which is a significantly higher prevalence for both sexes when compared to population controls (p

Hypothermia is used for several h during cardiac and aortic surgery to protect ischemic organs. Therapeutic hypothermia (TH) is used for ≤24 h as a treatment for comatose patients after the return of spontaneous circulation (ROSC) following cardiac arrest. The proteomic approach may provide unbiased data on alterations in the abundance of proteins during TH. The objective of this study was to assess the effects of cooling/rewarming on the plasma proteome during TH after ROSC and to identify the mechanism underlying its therapeutic effects. A total of nine comatose adult patients, resuscitated shortly after cardiac arrest, were cooled to 34°C for 24 h and slowly rewarmed to 36°C. A quantitative gel-free proteomic analysis was performed using the isobaric tag for relative and absolute quantification labeling tandem mass spectrometry. Plasma samples were obtained prior to cooling and rewarming, and immediately after rewarming, from all patients during TH after ROSC. A total of 92 high-confidence proteins were identified. Statistically significant alterations were observed (>1.2-fold increase or

Tenascin-X (TNX), a glycoprotein of the extracellular matrix (ECM), is expressed in various tissues and plays an important role in ECM architecture. The TNXB gene encoding TNX is known as the gene responsible for classic-like Ehlers-Danlos syndrome (clEDS). To date, the role of TNX in dermal, muscular and obstetric features has been reported, but its role in bone homeostasis remains to be clarified. In this study, we found significant bone loss and upregulation of osteoclast marker gene expression in TNX-deficient mice. Further, TNX deficiency in the bone marrow promoted multinucleation of osteoclasts and resulted in increased bone resorption activity. These results indicate that multinucleated osteoclasts are the cause of bone loss in a TNX-deficient environment. Our findings provide new insight into the mechanism of osteoclast differentiation mediated by TNX and the pathology of clEDS.

The morphogenesis of mammalian embryonic external genitalia (eExG) shows dynamic differences between males and females. In genotypic males, eExG are masculinized in response to androgen signaling. Disruption of this process can give rise to multiple male reproductive organ defects. Currently, mechanisms of androgen-driven sexually dimorphic organogenesis are still unclear. We show here that mesenchymal-derived actomyosin contractility, by MYH10, is essential for the masculinization of mouse eExG. MYH10 is expressed prominently in the bilateral mesenchyme of male eExG. Androgen induces MYH10 protein expression and actomyosin contractility in the bilateral mesenchyme. Inhibition of actomyosin contractility through blebbistatin treatment and mesenchymal genetic deletion induced defective urethral masculinization with reduced mesenchymal condensation. We also suggest that actomyosin contractility regulates androgen-dependent mesenchymal directional cell migration to form the condensation in the bilateral mesenchyme leading to changes in urethral plate shape to accomplish urethral masculinization. Thus, mesenchymal-derived actomyosin contractility is indispensable for androgen-driven urethral masculinization., Alvin Acebedo, Kentaro Suzuki et al. show that mesenchymal-derived actomyosin contractility is required for androgen-dependent urethral masculinization in mouse embryos. They also show that actomyosin contractility regulates mesenchymal cell migration during the developmental process.

Background Complete deficiency of an extracellular matrix tenascin-X (TNX) leads to a classical type of Ehlers-Danlos syndrome (EDS). TNX haploinsufficiency is a cause of hypermobility type of EDS. Human TNX is also present in a serum form (sTNX) with a molecular size of 140 kDa. In this study, we established a method for quantification of sTNX using nano-liquid chromatography tandem mass spectrometry (LC/MS/MS) with selected/multiple reaction monitoring. Methods Twelve abundant protein-depleted sera were reduced, alkylated, and digested with Lys-C and trypsin. Subsequently, the digests were fractionated by strong cation exchange chromatography. Optimal and validated transitions of precursor and product ions of the peptides from sTNX were developed on a triple quadrupole mass spectrometer. Results Serum concentrations of sTNX of healthy individuals were quantified as an average of 144 ng/ml. However, sTNX was not detected by this method in serum from a patient with a classical type of EDS in whom sTNX was not found by Western blot analysis. The limit of quantification (LOQ) of sTNX by nano-LC/MS/MS method was 2.8 pg whereas the detection sensitivity of sTNX by Western blot analysis was 19 pg. The nano-LC/MS/MS method is more sensitive than Western blot analysis. Conclusions The quantification method will be useful for diagnosis and risk stratification of EDS caused by TNX deficiency and haploinsufficiency.

Among numerous possible choices of effort estimation methods, analogy-based software effort estimation based on Case-based reasoning is one of the most adopted methods in both the industry and research communities. Solution adaptation is the final step of analogy-based estimation, employed to aggregate and adapt to solutions derived during the case-based reasoning process. Variants of solution adaptation techniques have been proposed in previous studies; however, the ranking of these techniques is not conclusive and shows conflicting results, since different studies rank these techniques in different ways. This paper aims to find a stable ranking of solution adaptation techniques for analogy-based estimation. Compared with the existing studies, we evaluate 8 commonly adopted solution techniques with more datasets (12), more feature selection techniques included (4), and more stable error measures (5) to a robust statistical test method based on the Brunner test. This comprehensive experimental procedure allows us to discover a stable ranking of the techniques applied, and to observe similar behaviors from techniques with similar adaptation mechanisms. In general, the linear adaptation techniques based on the functions of size and productivity (e.g., regression towards the mean technique) outperform the other techniques in a more robust experimental setting adopted in this study. Our empirical results show that project features with strong correlation to effort, such as software size or productivity, should be utilized in the solution adaptation step to achieve desirable performance. Designing a solution adaptation strategy in analogy-based software effort estimation requires careful consideration of those influential features to ensure its prediction is of relevant and accurate.

Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). DGKδ is highly expressed in the skeletal muscle, and a decrease in DGKδ expression increases the severity of type 2 diabetes. However, the role of DGKδ in myogenic differentiation is still unknown. The present study demonstrated that DGKδ expression was down-regulated in the early stage of C2C12 myogenic differentiation almost concurrently with a decrease in cyclin D1 expression. The knockdown of DGKδ by DGKδ-specific siRNAs significantly increased the levels of cyclin D1 expression at 48 h after C2C12 myogenic differentiation. In contrast, at the same time, the knockdown of DGKδ decreased the levels of myogenin expression and the number of myosin heavy chain (MHC)-positive cells. These results indicate that DGKδ regulates the early differentiation of C2C12 myoblasts via controlling the down-regulation of cyclin D1 expression. Moreover, the suppression of DGKδ expression increased the phosphorylation levels of conventional and novel protein kinase Cs (cnPKCs). Furthermore, DGKδ suppression increased the levels of cyclin D1 and phospho-cnPKCs even at the first 24 h of myogenic differentiation. These results suggest that DGKδ controls the down-regulation of cyclin D1 expression by attenuating the PKC signaling pathway for C2C12 myogenic differentiation.

Joint hypermobility syndrome (JHS) (also termed Ehlers-Danlos syndrome, hypermobility type) is a heritable connective tissue disorder which is characterized by generalized joint hypermobility, chronic pain, dizziness, fatigue, and minor skin changes. However, it has yet to be determined in patients with JHS whether specific genetic factors are involved in the risk of developing the disorder. Therefore, interventions have been limited to symptomatic treatments, and biomarkers for diagnosis and therapy have not yet been identified. In the present study, to identify potential serum biomarkers for JHS, we examined proteins with differential levels in sera from patients with JHS and in sera from control individuals using isobaric tags for relative and absolute quantitation (iTRAQ) labeling in combination with nano LC-MALDI-TOF/TOF-MS/MS followed by ProteinPilot analysis. In the sera of patients with JHS, a total of 106 proteins with differential levels were identified, and they were further narrowed down to 6 proteins (p

Background. Comprehensive analysis of proteome differentially expressed in response to surgery or drug treatment is useful to understand biological responses to dispensed interventions. Here we investigated expression changes in sera of patients who suffered from calcific aortic stenosis (CAS), before and after surgery for aortic valve replacement.Materials and Methods. Sera obtained before and after surgery with depletion of highly abundant proteins were analyzed with iTRAQ labeling followed by nanoLC-MALDI-TOF/TOF-MS/MS.Results. Fifty-one proteins shared in five patients were identified with differential levels in postsurgical and presurgical sera. Finally, 16 proteins that show statistically significant levels in patients’ sera compared with those in control sera (P<0.05) were identified. Most of the identified proteins were positive acute-phase proteins. Among three proteins other than acute-phase proteins, we confirmed increased levels of antithrombin-III and zinc-α-2-glycoprotein in postsurgical sera by Western blot analysis using other CAS patients’ sera. Furthermore, antithrombin-III and zinc-α-2-glycoprotein were not found among proteins with differential levels in postsurgical and presurgical sera of patients with aortic aneurysms that we identified in a previous study.Conclusions. The results indicated that antithrombin-III and zinc-α-2-glycoprotein would become unique monitoring proteins for evaluating pathophysiological and biochemical processes occurring before and after surgery for CAS.

Introduction: Interaction of blood with a cardiopulmonary bypass (CPB) circuit activates the coagulation-fibrinolysis, complement and kinin-kallikrein systems that are mainly supported by proteases and their inhibitors. Methods: Biocompatibility of a new polymer-coated (SEC-coated) CPB circuit was globally evaluated and compared with that of a non-coated CPB circuit by quantitative proteomics, using isobaric tags for relative and absolute quantification labeling tandem mass spectrometry. Plasma samples were taken three times (5 min after initiation of CPB, just before declamping and just before termination of CPB) in 12 pigs undergoing 120 min of CPB with the SEC-coated CPB circuit or a non-coated CPB circuit (n = 6, respectively). Results: Identified were 224 proteins having high protein confidence (>99%) and false discovery rate (FDR) Conclusion: The new polymer (SEC)-coated CPB circuit effectively attenuated upregulation of proteins compared to the non-coated CPB circuit. These proteins were associated with both proteases/protease inhibitors and platelet degranulation.

Patients with tenascin-X (TNX)-deficient type Ehlers-Danlos syndrome (EDS) do not exhibit delayed wound healing, unlike classic type EDS patients, who exhibit mutations in collagen genes. Similarly, in TNX-knockout (KO) mice, wound closure of the skin is normal even though these mice exhibit a reduced breaking strength. Therefore, we speculated that the wound healing process may be affected in the absence of TNX. In this study, to investigate the effects of TNX absence on wound healing-related properties, we performed collagen gel contraction assays with wild-type (WT) and TNX-KO mouse embryonic fibroblasts (MEFs). Collagen gels with embedded TNX-KO MEFs showed significantly greater contraction than those containing WT MEFs. Subsequently, we assessed collagen gel contraction-related properties, such as the activities of matrix metalloproteinase (MMP)-2 and MMP-9 and the protein and mRNA expression levels of transforming growth factor β1 (TGF-β1) in the collagen gels. The activities of MMP-2 and MMP-9 and the expression level of TGF-β1 were elevated in the absence of TNX. Furthermore, filopodia-like protrusion formation, cell proliferation, migration, and collagen expression in MEFs were promoted in the absence of TNX. These results indicate that these wound healing-related properties are affected in a TNX-deficient extracellular environment.

Oxygen reduction reaction (ORR) activities of Pt/Au(111) and Pt/Au(100) bimetallic surfaces prepared using molecular beam epitaxy were evaluated in this study. Surface structures of the bimetallic surfaces were verified using a scanning tunneling microscope under ultra-high vacuum conditions. The deposition of a 0.3-nm-thick Pt on a clean Au(111) at 300 K (300K-Pt0.3nm/Au(111)) generated a corrugated Pt(111) epitaxial layer on which monoatomic-height Pt islands are present. The ORR activity estimated from the kinetically controlled current density at 0.9 V vs. RHE was ca. two times greater than that of the clean Pt(111). In contrast, the 473K-Pt0.3nm/Au(111) as well as 300K-Pt0.3nm/Au(100) exhibited small hydrogen-related charges, thereby were less ORR active than the corresponding clean Pt substrate surfaces. These results suggest that atomic arrangements of surface Pt atoms, particularly the morphologies of the most dense plane of Pt(111), correlate with the enhancement in the ORR activity of Pt–Au bimetallic nanoparticles.

Oxygen reduction reaction (ORR) activity and stability of a molecular beam epitaxially (MBE) prepared Pt-enriched Ni0.3nm/Pt(111) surface was investigated. Reflection high-energy electron diffraction (RHEED) patterns, and a scanning tunneling microscopic (STM) image indicated that the Pt-enriched surface has long-range-ordered six-fold symmetry with atomic-scale corrugations. ORR activity was estimated by kinetically-controlled current density at 0.9 V vs. a reversible hydrogen electrode, and the as-prepared Pt-enriched surface showed 8-times-higher ORR activity than clean Pt(111). The activity steeply reduced during potential cycling between 0.6 V and 1.0 V. After 1000 potential cycles, the enhancement factor was 2.5 and a cyclic voltammetry (CV) curve exhibited an increase in (EC) electron charge for the Had&des region accompanied by the emergence of a 0.13 V redox feature caused by (110) surface defects. These results suggest that the electrochemical stability of the underlying Ni atoms determines the durability of Pt-Ni alloy catalysts.

Pt-deposited Au(1 1 1) surfaces were prepared by molecular beam epitaxy (MBE). Surface structures of the samples were verified with reflection high-energy electron diffraction (RHEED), scanning tunneling microscopy (STM) and infrared reflection absorption spectroscopy (IRRAS) for adsorbed carbon monoxide in an ultra-high vacuum (UHV) condition. The UHV-results show that epitaxial growth of Pt(1 1 1) on clean Au(1 1 1). A 0.3-nm-thick Pt deposition found to almost cover the Au surface. In addition, monoatomic-height Pt islands were observed on the topmost surface. Electrochemical properties were evaluated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV) using a rotating disk electrode apparatus set in an N2-purged glove box. A CV curve of the Pt0.3nm/Au(1 1 1) recorded in the potential region of 0.05–1.0 V vs. reversible hydrogen electrode (RHE) was similar to that of clean Pt(1 1 1), except for the lack of ‘butterfly’ peaks at 0.8 V. Oxygen reduction reaction (ORR) activities of the samples were evaluated by kinetic controlled current densities at 0.9 V; the activity of the Pt0.3nm/Au(1 1 1) was ca. 1.8 times higher than that of the clean Pt(1 1 1). During CV curve measurements of the Pt0.3nm/Au(1 1 1) in the region of 0.05–1.7 V, a redox feature at 0.13 V became apparent; the ORR activity evaluated after the CV measurements was higher than the as-prepared sample. An UHV-STM image collected after re-transfer from the glove box showed two to three monoatomic-height Pt mounds whose slopes were 10–20°. The results clearly showed that surface defects at the topmost Pt(1 1 1) layer such as steps contribute to the ORR enhancement of MBE-prepared Pt/Au(1 1 1).

Nano-polycrystalline diamond has been used to obtain a glitch-free X-ray absorption spectrum under high pressure. The advantage and capability of nano-polycrystalline diamond anvils is discussed by a comparison of the glitch map with that of single-crystal diamond anvils., Nano-polycrystalline diamond (NPD) [Irifune et al. (2003 ▶), Nature (London), 421, 599] has been used to obtain a glitch-free X-ray absorption spectrum under high pressure. In the case of conventional single-crystal diamond (SCD) anvils, glitches owing to Bragg diffraction from the anvils are superimposed on X-ray absorption spectra. The glitch has long been a serious problem for high-pressure research activities using X-ray spectroscopy because of the difficulties of its complete removal. It is demonstrated that NPD is one of the best candidate materials to overcome this problem. Here a glitch-free absorption spectrum using the NPD anvils over a wide energy range is shown. The advantage and capability of NPD anvils is discussed by a comparison of the glitch map with that of SCD anvils.

Aortic aneurysm is a complex multifactorial disease with genetic and environmental risk factors. It is often accompanied by aortic calcification. Here, to uncover proteins that are significantly changed in calcified abdominal aortic aneurysms (CAAs) and calcified thoracic aortic aneurysms (CTAs) compared with those in adjacent normal aorta tissues, comprehensive analysis of differentially expressed proteins in their tissues was performed by a quantitative proteomic approach with iTRAQ labeling in combination with nanoLC-MALDI-TOF/TOF-MS/MS followed by ProteinPilot analysis. The proteomic analysis revealed 138 and 134 proteins differentially expressed in CAAs and CTAs in contrast to neighboring normal aorta tissues with high confidence, respectively. Significantly increased expression (≥1.3-fold) was found in 41 and 28 proteins, whereas decreased expression (

Reversible protein phosphorylation represents a major mechanism of signal transduction in a variety of cellular functions. An understanding of proteome-wide phosphorylation dynamics is important to obtain an overview of the whole signal transduction network. However, a systematic analysis for differentially expressed phosphoproteins under serum-stimulated response is lacking. Here, an easy and fast approach for the identification of differentially expressed phosphoproteins was used. After enrichment of phosphoproteins from serum-stimulated cell lysates by immobilized metal affinity chromatography, a quantitative proteomic approach with isobaric tag for absolute and relative quantitation labeling in combination with nanoLC-MALDI-TOF/TOF-MS/MS followed by ProteinPilot analysis was used. Consequently, 506 differentially expressed phosphoproteins were identified. Among them, 22 proteins that had a reproducible phosphorylation site at Ser or Thr were identified. Out of these 22 phosphoproteins, 7 are mainly involved in splicing. Among the 22 proteins, it was found that extracellular matrix tenascin-X is phosphorylated, although there is little quantitative change by the serum stimulation. MS/MS analysis revealed a novel phosphorylation site of tenascin-X, Thr1841, located in the loop region between the 10th and 11th fibronectin type III repeats. The phosphorylation of tenascin-X would be considered in clarifying its function in the future.

The geology and petrology of the Itoigawa–Omi area of the Hida Gaien belt have been reexamined with pioneering metamorphic zonal mapping of glaucophane schist facies done by Banno (1958). A geologic study shows that crystalline schists of ca. 262-381 Ma, 450 Ma garnet amphibolite, amphibolite, 520 Ma jadeitite, albitite, and rodingite, reaching 10 km long and 2-5 km wide, occur as tectonic blocks of serpentinite forming the Omi serpentinite melange (OSM). Along the Omi–River of the eastern side of the OSM, chlorite, garnet, and biotite zones of pelitic schists are again recognized across several tectonic blocks of schist, indicating the slightly broken nature of a large metamorphic unit that resulted probably from exhumation of the serpentinite melange belt. Kfs and chlorite are common throughout pelitic schists along the Omi–River. Thus, biotite- and oligclase-forming reactions may have been K-feldspar + chlorite → muscovite + biotite + quartz + H2O, and muscovite + clinozoisite + quartz → K-feldspar + anorthite + H2O, respectively. Glaucophane described by Banno (1958) in the chlorite zone of the Omi River has been recognized in composite grains consisting of hornblende core, gluacophane mantle, and actinolite rim. In contrast, glaucophane appears in a few samples along with eclogitic assemblages as plurifacial minerals before, during, and after eclogite facies in the pelitic schists of the western side of OSM (EC unit). Thus, the regional metamorphism described along the Omi–river of OSM (EA unit) is not a retrogressive metamorphism of the EC unit, but a regional hydrothermal recrystallization just before or during exhumation in the serpentinite melange belt.

Interstitial extracellular matrix tenascin-X (iTNX) with about 450 kDa is prominently present in various tissues. Previously, we identified the serum form of TNX (sTNX) with 200 kDa in the mouse. In the present study, in order to investigate distinctive features and functions of sTNX, a plasmid encoding the recombinant mouse sTNX was constructed. As a control, we also constructed a plasmid encoding mouse 450-kDa iTNX and a plasmid encoding 250-kDa iTNX, which lacks the region of 200-kDa sTNX from 450-kDa iTNX. In cells stably expressing each recombinant TNX, a more than 7-fold larger amount of 200-kDa sTNX was released into conditioned medium than the amounts of 250-kDa iTNX and 450-kDa iTNX released into the medium. We previously reported that a splice isoform of iTNX (340-kDa iTNX) binds to vascular endothelial growth factor B (VEGF-B) as well as to VEGF-A. Therefore, the ability of VEGF-A and VEGF-B to bind to 200-kDa sTNX was examined by a co-immunoprecipitation assay in comparison with the binding abilities to 250-kDa iTNX and 450-kDa iTNX. It was found that sTNX strongly bound to VEGF-A and VEGF-B, compared with the binding abilities of other iTNX proteins. Based on the results of assays of incorporation of 5-ethynyl-2'-deoxyuridine (EdU), we found that purified recombinant 200-kDa sTNX both alone and in combination with VEGF-A or basic fibroblast growth factor (bFGF) can weakly promote DNA synthesis in proliferating vascular endothelial cells (UVfemale symbol2 cells). These results suggest that sTNX possesses weak activity for proliferation of endothelial cells.

Extracellular matrix glycoprotein tenascin-X (TNX) is the largest member of the tenascin family. In this study, we investigated the adhesive properties of TNX and the signaling pathway to be induced to mouse fibroblast L cells on TNX substrate. Approximately 45% of evaluable cells used in the cell adhesion assay were attached to purified TNX but did not spread and were rounded on TNX. The remaining 55% of cells were detached from the TNX substrate and were floating in the conditioned medium. In rounded cells on TNX, phosphorylation of focal adhesion kinase (FAK) was diminished compared with that in cells on control phosphate buffered saline (PBS). To better understand the pathways that lead to the detachment of cells on the TNX substrate, we examined phosphorylation of p38 mitogen-activated protein (MAP) kinase. Phosphorylation of p38 MAP kinase was observed in the rounded cells on TNX in a dose-dependent manner, and the maximum effect was observed at 30 min on TNX. Inhibition of p38 MAP kinase alpha expression by RNA interference partially suppressed the TNX-induced cell detachment. These results suggest that the p38 MAP kinase is a major mediator of TNX-induced cell detachment.

XB-S is a protein with an amino-terminal-truncated form of tenascin-X (TNXB). However, the precise roles of XB-S in vivo are unknown. In this study, to determine the role of XB-S in vivo, we screened XB-S-binding proteins. FLAG-tagged XB-S was transiently introduced into 293T cells. Then its associated proteins were purified by immunoprecipitation using an anti-FLAG antibody and its components were identified by mass spectrometric analyses. Mitotic motor kinesin Eg5 was identified in the immunoprecipitates. XB-S and Eg5 proteins were co-localized in the cytoplasm in interphase and mitosis, but XB-S did not localize on mitotic spindle microtubules, on which Eg5 prominently localized in mitosis. As for Eg5 binding to XB-S, glutathione S-transferase-fused XB-S expressed in vitro directly bound to full-length Eg5 translated in reticulocyte lysate, and the XB-S-binding region was located in the motor domain of Eg5. Furthermore, during cell cycle progression XB-S showed a similar expression profile to that of Eg5. These results suggest possible involvement of XB-S in the function of Eg5.

XB-S is an amino-terminal truncated protein of tenascin-X (TNX) in humans. The levels of the XB-S transcript, but not those of TNX transcripts, were increased upon hypoxia. We identified a critical hypoxia-responsive element (HRE) localized to a GT-rich element positioned from − 1410 to − 1368 in the XB-S promoter. Using an electrophoretic mobility shift assay (EMSA), we found that the HRE forms a DNA–protein complex with Sp1 and that GG positioned in − 1379 and − 1378 is essential for the binding of the nuclear complex. Transfection experiments in SL2 cells, an Sp1-deficient model system, with an Sp1 expression vector demonstrated that the region from − 1380 to − 1371, an HRE, is sufficient for efficient activation of the XB-S promoter upon hypoxia. The EMSA and a chromatin immunoprecipitation (ChIP) assay showed that Sp1 together with the transcriptional repressor histone deacetylase 1 (HDAC1) binds to the HRE of the XB-S promoter under normoxia and that hypoxia causes dissociation of HDAC1 from the Sp1/HDAC1 complex. The HRE promoter activity was induced in the presence of a histone deacetylase inhibitor, trichostatin A, even under normoxia. Our results indicate that the hypoxia-induced activation of the XB-S promoter is regulated through dissociation of HDAC1 from an Sp1-binding HRE site.

We developed an easy and fast method to isolate extracellular matrix tenascin-X (TNX) from various tissues in mice based on TNX antibody affinity purification. We purified approximately 350-kDa cellular interstitial TNX (iTNX) from the spleen, liver and kidney as well as 200-kDa serum TNX (sTNX). Since the nature and significance of glycosylation in TNX remains to be elucidated, glycobiochemical properties of purified TNX were characterized by lectin blot analysis. Lectin blots by Con A, LCA, PHA-E4, RCA120 or WGA revealed the presence of N-glycan in the cellular TNX and especially complex-type N-glycan in the serum TNX. In addition, the iTNX from liver and kidney also possessed O-glycan based on the reaction to PNA. The binding to AAL indicated that iTNX from the three tissues possesses fucose linked alpha1,6 to a pentasaccharide core, whereas sTNX does not. The reaction to SSA but not to MAM suggested the presence of sialic acid linked alpha2,6 to galactose in both cellular and serum TNX. Lectin blots of trypsin-treated iTNX from the spleen also demonstrated that WGA alone reacts to the t300 product derived from the amino-terminal 300-kDa portion.

BACKGROUND Hypothermia is utilized in cardiac and aortic surgery to protect organs from ischemic reperfusion injury. Although the cooled body is invariably rewarmed after the procedure, it is still unknown whether the rewarmed body regains its former biological state. This study determined the modulatory effects of hypothermia on the human myocardial proteome and whether subsequent rewarming restores the proteome to the state prior to cooling. METHODS AND RESULTS A quantitative proteomic analysis was performed using isobaric tags for relative and absolute quantification labeling tandem mass spectrometry. Right atrial samples were taken 3 times (pre, during and post cooling) during deep hypothermic cardiopulmonary bypass (CPB) from 8 patients with aortic arch aneurysms and 3 corresponding time points during normothermic CPB from 8 patients with ascending aortic or valsalva aneurysms. In total, 697 proteins were identified, with 222 proteins having high protein confidence. Bioinformatic analyses revealed significant downregulation of 19 proteins associated with energy production at hypothermic cardioplegic arrest. On rewarmed beating, 10 proteins remained downregulated, including those regulating cardiac contraction and adaptor proteins, although levels of the aforementioned 19 downregulated proteins returned to their initial values. Additional echocardiographic evaluation demonstrated that hypothermia preserved the variables of diastolic function to a greater extent than normothermic surgery. CONCLUSIONS Rewarming restores the human myocardial proteome to the pre-cooled state, except for proteins regulating cardiac contraction and adaptor proteins.

We previously disclosed a novel extracellular matrix tenascin-X (TNX) , the largest member of the tenascin family. So far, we have made efforts to elucidate the roles of TNX. TNX is involved in collagen deposition, collagen fibrillogenesis, and modulation of collagen stiffness. Homozygous mutations in TNXB, the gene encoding TNX, cause a classic-type Ehlers-Danlos syndrome (EDS) , a heritable connective tissue disorder, whereas haploinsufficiency of TNXB and heterozygous mutations in TNXB are associated with hypermobility-type EDS. Recently, we performed proteomic analyses of calcific aortic valves (CAVs) compared with relatively adjacent normal tissues to understand the underlying molecular mechanisms of dystrophic valvular calcification. Interestingly, we found that TNX was the protein with the greatest decrease in expression among the differentially expressed proteins and that expression levels of proteins modulating collagen structure and function, such as type I collagen and decorin, were also decreased in CAVs. In this review, I will discuss about the decreased level of collagen due to the reduction of expression levels of proteins that play regulatory roles in collagen functions such as fibril organization and fibrillogenesis in CAVs.

The interstitial extracellular matrix tenascin-X (iTNX), which has a molecular mass of roughly 450 kDa, is expressed at high levels in muscular tissues and skin. In this study, we identified the serum form of TNX (sTNX) with a molecular mass of 200 kDa in the mouse. Western blot analysis with specific antibodies against fibronectin type III-like (FNIII) repeats of TNX and N-terminal sequence analysis of 200-kDa sTNX revealed that the N-terminus of sTNX is located in the juncture between the 16th FNIII (M16) and 17th FNIII (M17) repeats of iTNX. The 200-kDa sTNX contains 15 FNIII repeats and a fibrinogen domain identical to the Cterminal portion of the iTNX. TNX-deficient mice lacked not only iTNX but also sTNX. Furthermore, 200-kDa sTNX was generated by cleavage of the spleen iTNX by spleen homogenate, and its generation was inhibited by protease inhibitors. These results suggest that sTNX is generated by proteolytic cleavage of iTNX.

Ascidians are hermaphrodites releasing sperm and eggs simultaneously, but many species are self-sterile because of a self/nonself-recognition system in spermegg interaction. Here, we show that a 70-kDa vitelline coat protein, HrVC70, consisting of 12 epidermal growth factor-like repeats, plays a key role in self/nonself recognition during ascidian fertilization. We discovered that the amount of HrVC70 of the self-sterile mature oocytes is markedly higher than that of the self-fertile immature oocytes and that the selfsterile mature oocytes become self-fertile by acid treatment, which is able to release the HrVC70 from isolated vitelline coats. In addition, fertilization is strongly inhibited by the pretreatment of sperm with HrVC70 from a different individual, but not from the same individual, and the number of nonself sperm bound to HrVC70-agarose was significantly higher than that of self-sperm. A sequence analysis of HrVC70 disclosed that several amino acid residues in a restricted region are substituted at an individual level, with no identical sequences among the 10 individuals tested. Furthermore, genomic DNA analysis revealed that the epidermal growth factor-like domains correspond to the exons, and each intron is highly conserved among even- and odd-numbered introns, suggesting that multiple gene duplications or amplification of this region might have taken place during evolution. It was also found that diversity in cDNA sequences is derived from genomic DNA polymorphism probably elicited by crossing over and specific nucleotide substitutions. These results indicate that HrVC70 is a candidate allogeneic recognition molecule in the gamete interaction of the ascidian Halocynthia roretzi .

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro.

Tenascin-X (TNX) is an extracellular matrix protein that is highly expressed in the peripheral nervous system as well as muscular tissues, especially the heart and skeletal muscle. However, the expression manner and the physiological role of TNX in the peripheral nervous system have not been fully investigated. In this study, we elucidated its distribution in adult mouse sciatic nerves by immunohistochemical staining. TNX was found to be localized in the perineurium and the endoneurium of sciatic nerve fibers. To examine the physiological role of TNX, we investigated sciatic nerves of TNX-deficient mice that are viable and fertile and have no obvious deficits in general performance. The thickness of myelin sheaths and the size of the individual axons in these mice appeared normal. The ultrastructure of the sciatic nerves of TNX-deficient mice were similar to those of wild-type mice. Thus, the lack of a discernible phenotype in the sciatic nerves of TNX-deficient mice suggests that TNX has either a redundant or a very subtle function in the macromolecular organization in the peripheral nerve.

The roles of extracellular matrix glycoproteins belonging to the tenascin family in the regulation of tumor cell proliferation, invasion, and metastasis are not known. To address this issue, we generated tenascin-X (TNX) and tenascin-C (TNC) double knockout mice and compared findings in these mice with those in single knockout (TNX + / + TNC - / - and TNX - / - TNC + / +) mice. We investigated the proliferation and invasion of B16-BL6 melanoma cells after these cells had been injected into the footpads of mice in each group. The primary tumor size and invasion to the ankle adjacent to the primary tumor site were examined at 35 days after injection of the melanoma cells. The primary tumor size in TNX - / - TNC + / + mice was significantly larger than that in wild-type mice, but those of TNX + / + TNC - / - and double knockout mice were comparable to that in the wild-type mice. On the other hand, invasion to the ankle was obviously promoted in TNX - / - TNC + / + and double knockout mice compared with that in the wild-type mice, but invasion to the ankle in TNX + / + TNC - / - mice was only slightly promoted. Gelatin zymography confirmed increased matrix metalloproteinase (MMP)-9 activity in the dorsal skin of TNX - / - TNC + / +, TNX + / + TNC - / - and double knockout mice. However, the amounts of MMP-9 mRNA in the dorsal skins of all mice were almost the same, indicating that the increased activity of MMP-9 in the single and double knockout mice is regulated at the MMP-9 processing level. These results indicate that MMP-9 is activated in all TN-deficient mice, but that TNX exerts a greater effect on tumor invasion than does TNC.

原料の約50%に都市型廃棄物を利用して製造されたセメントは, 普通ポルトランドセメントと比較して, C3Aと塩素が概して多く含まれる. 塩素が多いことから, 鉄筋コンクリートに適用した場合には, 鉄筋腐食を引き起こすことが懸念されている. 本研究では, セメント硬化体に含まれる細孔溶液に着目し, 細孔溶液の組成ならびにその経時変化の観点から鉄筋腐食の可能性について検討を行った. その結果都市型廃棄物を原料として製造されるセメントでは, 細孔溶液中の塩化物イオン濃度が普通ポルトランドセメントの場合と比較して高くなるものの塩化物イオン濃度と水酸化物イオン濃度の比は低く, 鉄筋が腐食する指標とされる値は大きく下回ることが明らかになった. これらより, 鉄筋腐食の可能性に関しては, 普通ポルトランドセメントと同様に考えてもよいと考えられる.

Ehlers-Danlos syndrome (EDS) is a clinically and genetically heterogeneous disorder. Using a customized targeted exome-sequencing system we identified nonsense mutations in TNXB in a patient who had recurrent gastrointestinal perforation due to tissue fragility. This case highlights the utility of targeted exome sequencing for the diagnosis of congenital diseases showing genetic heterogeneity, and the importance of attention to gastrointestinal perforation in patients with tenascin-X deficient type EDS.