Observation of ballistic upstream modes at fractional quantum Hall edges of graphene.

The presence of "upstream" modes, moving against the direction of charge current flow in the fractional quantum Hall (FQH) phases, is critical for the emergence of renormalized modes with exotic quantum statistics. Detection of excess noise at the edge is a smoking gun for the presence of upstream modes. Here, we report noise measurements at the edges of FQH states realized in dual graphite-gated bilayer graphene devices. A noiseless dc current is injected at one of the edge contacts, and the noise generated at contacts at length, L = 4 µm and 10 µm away along the upstream direction is studied. For integer and particle-like FQH states, no detectable noise is measured. By contrast, for "hole-conjugate" FQH states, we detect a strong noise proportional to the injected current, unambiguously proving the existence of upstream modes. The noise magnitude remains independent of length, which matches our theoretical analysis demonstrating the ballistic nature of upstream energy transport, quite distinct from the diffusive propagation reported earlier in GaAs-based systems.

A potential contribution of trappin-2 to the development of vasculopathy in systemic sclerosis.

BACKGROUND: Trappin-2/pre-elafin is an endogenous inhibitor of human neutrophil elastase involved in inflammation, innate immunity and vascular remodelling, which consist of the complex pathological process of systemic sclerosis (SSc). OBJECTIVES: To clarify the potential role of trappin-2 in SSc. METHODS: Serum trappin-2 levels were determined by enzyme-linked immunosorbent assay in 51 SSc and 18 healthy subjects. Trappin-2 expression was evaluated in SSc lesional skin and cultured endothelial cells treated with FLI1 siRNA by immunohistochemistry, reverse transcription-real-time PCR and/or immunoblotting. Friend leukaemia virus integration 1 (Fli1) binding to the PI3 promoter was assessed by chromatin immunoprecipitation. RESULTS: Since serum trappin-2 levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction, SSc patients with normal renal function were analysed. Although serum trappin-2 levels were comparable between diffuse cutaneous SSc, limited cutaneous SSc and control subjects, the prevalence of digital ulcers or elevated right ventricular systolic pressure (RVSP) was significantly higher in SSc patients with elevated serum trappin-2 levels than in those with normal levels. Furthermore, serum trappin-2 levels were significantly increased in SSc patients with digital ulcers or elevated RVSP compared to those without. Moreover, serum trappin-2 levels positively correlated with RVSP values in SSc patients. Importantly, trappin-2 expression was enhanced in small vessels of SSc lesional skin. In cultured endothelial cells, trappin-2 expression was elevated by gene silencing of FLI1 at mRNA and protein levels and Fli1 occupied the PI3 promoter. CONCLUSIONS: Endothelial trappin-2 up-regulation partially due to Fli1 deficiency can be associated with the development of SSc vasculopathy.

Resveratrol directly targets DDX5 resulting in suppression of the mTORC1 pathway in prostate cancer.

Resveratrol has various attractive bioactivities, such as prevention of cancer, neurodegenerative disorders, and obesity-related diseases. Therefore, identifying its direct binding proteins is expected to discover druggable targets. Sirtuin 1 and phosphodiesterases have so far been found as the direct molecular targets of resveratrol. We herein identified 11 novel resveratrol-binding proteins, including the DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5, also known as p68), using resveratrol-immobilized beads. Treatment with resveratrol induced degradation of DDX5 in prostate cancer cells. Depletion of DDX5 caused apoptosis by inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling. Moreover, knockdown of DDX5 attenuated the inhibitory activities of resveratrol against mTORC1 signaling and cancer cell growth. These data show that resveratrol directly targets DDX5 and induces cancer cell death by inhibiting the mTORC1 pathway.

A potential contribution of antimicrobial peptide LL-37 to tissue fibrosis and vasculopathy in systemic sclerosis.

BACKGROUND: LL-37 is an antimicrobial peptide with pleiotropic effects on the immune system, angiogenesis and tissue remodelling. These are cardinal pathological events in systemic sclerosis (SSc). OBJECTIVES: To elucidate the potential role of LL-37 in SSc. METHODS: The expression of target molecules was evaluated by immunostaining and quantitative reverse-transcription real-time polymerase chain reaction in human and murine skin. The mechanisms regulating LL-37 expression in endothelial cells were examined by gene silencing and chromatin immunoprecipitation. Serum LL-37 levels were determined by enzyme-linked immunosorbent assay. RESULTS: In SSc lesional skin, LL-37 expression was increased in dermal fibroblasts, perivascular inflammatory cells, keratinocytes and, particularly, dermal small vessels. Expression positively correlated with interferon-α expression, possibly reflecting LL-37-dependent induction of interferon-α. In SSc animal models, bleomycin-treated skin exhibited the expression pattern of CRAMP, a murine homologue of LL-37, similar to that of LL-37 in SSc lesional skin. Furthermore, Fli1+/- mice showed upregulated expression of CRAMP in dermal small vessels. Fli1 binding to the CAMP (LL-37 gene) promoter and Fli1 deficiency-dependent induction of LL-37 were also confirmed in human dermal microvascular endothelial cells. In the analysis of sera, patients with SSc had serum LL-37 levels significantly higher than in healthy controls. Furthermore, serum LL-37 levels positively correlated with skin score and the activity of alveolitis and were significantly elevated in patients with digital ulcers compared with those without. CONCLUSIONS: LL-37 upregulation, induced by Fli1 deficiency at least in endothelial cells, potentially contributes to the development of skin sclerosis, interstitial lung disease and digital ulcers in SSc.

Fli1 deficiency contributes to the downregulation of endothelial protein C receptor in systemic sclerosis: a possible role in prothrombotic conditions.

BACKGROUND: Endothelial protein C receptor (EPCR), expressed predominantly on endothelial cells, plays a critical role in the regulation of the coagulation system and also mediates various cytoprotective effects by binding and activating protein C. So far, the role of EPCR has not been studied in systemic sclerosis (SSc). OBJECTIVES: To investigate the potential contribution of EPCR to the development of SSc. METHODS: EPCR expression was examined in skin samples and cultivated dermal microvascular endothelial cells by immunostaining, immunoblotting and/or quantitative reverse-transcription polymerase chain reaction. Fli1, binding to the PROCR promoter, was assessed by chromatin immunoprecipitation. Serum EPCR levels were determined by enzyme-linked immunosorbent assay in 65 patients with SSc and 20 healthy subjects. RESULTS: EPCR expression was decreased in dermal small vessels of SSc lesional skin compared with those of healthy control skin. Transcription factor Fli1, deficiency of which is implicated in SSc vasculopathy, occupied the PROCR promoter, and EPCR expression was suppressed in Fli1 small interfering RNA-treated endothelial cells and dermal small vessels of Fli1(+/-) mice. In patients with SSc, decreased serum EPCR levels were associated with diffuse skin involvement, interstitial lung disease and digital ulcers. Furthermore, serum EPCR levels inversely correlated with plasma levels of plasmin-α2-plasmin inhibitor complex (PIC). Importantly, bosentan significantly reversed circulating EPCR and PIC levels in patients with SSc, and the expression of Fli1 and EPCR in dermal small vessels was elevated in patients treated with bosentan compared with untreated patients. CONCLUSIONS: Endothelial EPCR downregulation due to Fli1 deficiency may contribute to hypercoagulation status leading to tissue fibrosis and impaired peripheral circulation in SSc.

A possible contribution of lipocalin-2 to the development of dermal fibrosis, pulmonary vascular involvement and renal dysfunction in systemic sclerosis.

BACKGROUND: Lipocalin-2 is an adipocytokine implicated in apoptosis, innate immunity, angiogenesis, and the development of chronic kidney disease. OBJECTIVES: To investigate the role of lipocalin-2 in systemic sclerosis (SSc). MATERIALS AND METHODS: Serum lipocalin-2 levels were determined by enzyme-linked immunosorbent assay in 50 patients with SSc and 19 healthy subjects. Lipocalin-2 expression was evaluated in the skin of patients with SSc and bleomycin (BLM)-treated mice and in Fli1-deficient endothelial cells by reverse transcriptase-real time polymerase chain reaction, immunoblotting and/or immunohistochemistry. RESULTS: Although serum lipocalin-2 levels were comparable between patients with SSc and healthy controls, the prevalence of scleroderma renal crisis was significantly higher in patients with SSc with elevated serum lipocalin-2 levels than in those with normal levels. Furthermore, serum lipocalin-2 levels inversely correlated with estimated glomerular filtration rate in patients with SSc with renal dysfunction. Among patients with SSc with normal renal function, serum lipocalin-2 levels positively correlated with skin score in patients with diffuse cutaneous SSc with disease duration of < 3 years and inversely correlated with estimated right ventricular systolic pressure in total patients with SSc. Importantly, in SSc lesional skin, lipocalin-2 expression was increased in dermal fibroblasts and endothelial cells. In BLM-treated mice, lipocalin-2 was highly expressed in dermal fibroblasts, but not in endothelial cells. On the other hand, the deficiency of transcription factor Fli1, which is implicated in SSc vasculopathy, induced lipocalin-2 expression in cultivated endothelial cells. CONCLUSIONS: Lipocalin-2 may be involved in renal dysfunction and dermal fibrosis of SSc. Dysregulated matrix metalloproteinase-9/lipocalin-2-dependent angiogenesis due to Fli1 deficiency may contribute to the development of pulmonary arterial hypertension associated with SSc.

Constitutive activation of c-Abl/protein kinase C-δ/Fli1 pathway in dermal fibroblasts derived from patients with localized scleroderma.

BACKGROUND: A noncanonical pathway of transforming growth factor-ß signalling, the c-Abl/protein kinase C-δ (PKC-δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts. OBJECTIVES: To investigate the significance of the c-Abl/PKC-δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc). METHODS: The activation status of the c-Abl/PKC-δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet-derived growth factor receptor inhibitor AG1296 and gene silencing of c-Abl on the expression levels of type I collagen were evaluated by immunoblotting. RESULTS: The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c-Abl were elevated compared with cultured normal fibroblasts and PKC-δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c-Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts. CONCLUSIONS: Constitutive activation of the c-Abl/PKC-δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc.

Endoscopic mucosal resection and endoscopic submucosal dissection for en bloc resection of superficial pharyngeal carcinomas.

BACKGROUND AND STUDY AIM: Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are being used increasingly to treat superficial oropharyngeal and hypopharyngeal carcinomas. The aim of this study was to clarify whether ESD provided better results than EMR for en bloc and complete resection of superficial pharyngeal carcinomas. PATIENTS AND METHODS: A total of 76 superficial pharyngeal carcinomas in 59 consecutively treated patients were included. Patients underwent either conventional EMR (using a transparent cap or strip biopsy) (n = 45 lesions) or ESD (n = 31 lesions) between October 2006 and January 2011. The rates of en bloc resection, complete resection (defined as en bloc resection with tumor-free margins), major complications, and local recurrence were evaluated retrospectively as the therapeutic outcomes. RESULTS: ESD yielded significantly higher rates of both en bloc and complete resection compared with EMR (en bloc 77.4 % [24/31] vs. 37.8 % [17/45], P = 0.0002; complete 54.8 % [17/31] vs. 28.9 % [13/45], P = 0.0379). ESD was more frequently complicated by severe laryngeal edema (4/21 [19.0 %] vs. 1/31 [3.2 %], P = 0.1446) and was also more time-consuming (124.9 ± 65.1 minutes vs. 57.2 ± 69.6 minutes; P = 0.0014). Local recurrence was observed more often after EMR than after ESD (3/45 [6.7 %] vs. 0/31 [0 %]), although this difference did not reach statistical significance (P = 0.2658). CONCLUSIONS: ESD appears to be a superior method of endoscopic resection of superficial pharyngeal carcinomas for achieving both en bloc and complete resection, although these benefits were also associated with a higher incidence of complications and a significantly longer procedure time. Large prospective studies are needed to compare ESD with conventional EMR for superficial pharyngeal carcinomas.

[Thymolipoma associated with myasthenia gravis].

A 42-year-old woman was admitted to our hospital complaining of left ptosis, diplopia, and muscle weakness. A diagnosis of myasthenia gravis was made. Chest roentgenograms, computed tomography (CT), and magnetic resonance imaging (MRI) showed a large anterior mediastial mass and suggested thymolipoma. Extended thymectomy was performed via a median sternotomy. Histopathological examination revealed that the tumor consisted of mature adipose tissue and weighed 1,500 grams, in which thymic tissues with Hassall' s corpuscles but without a germinal center were contained. The histological appearance was compatible with a typical thymolipoma. This is the 24th reported case of thymolipoma associated with myasthenia gravis.

Preoperative visualization of the artery of Adamkiewicz by intra-arterial CT angiography.

BACKGROUND AND PURPOSE: CT and MR angiographies have been reported to visualize the artery of Adamkiewicz (AKA) noninvasively to prevent spinal cord ischemia in surgery of thoracic descending aortic aneurysms. The purpose of this work was to compare the usefulness of CT angiography (CTA) with intra-arterial contrast injection (IACTA) with that of conventional CTA with intravenous contrast injection (IVCTA). MATERIALS AND METHODS: We enrolled 32 consecutive patients with thoracic or thoracoabdominal aortic aneurysms who were scheduled for surgical repair or endovascular stent-graft treatment. All of the CTA images were obtained using a 16-detector row CT scanner and 100 mL of contrast material (370 mg/mL) injected at a rate of 5 mL/s. Contrast was injected via the antecubital veins of 15 patients and via a pig-tail catheter placed at the proximal portion of the descending aorta in 17 patients who underwent IVCTA and IACTA, respectively. Two datasets were reconstructed from 2 consecutive scans. The AKA was identified as a characteristic hairpin curved vessel in the anterior midsagittal surface of the spine and by the absence of further enhancement in the second rather than in the first phase. Continuity between the AKA and aorta was confirmed when the vessel could be traced continuously by paging the oblique coronal multiplanar reconstruction or original axial images. RESULTS: Intra-arterial contrast injection was significantly more sensitive in identifying the AKA than IVCTA: 16 (94.1%) of 17 versus 9 (60.0%) of 15 (P = .033). Continuity between the AKA and aorta through intercostal or lumbar artery was confirmed in 14 (87.5%) of 16 and 5 (55.6%) of 9 of the IACTA and IVCTA groups, respectively. CONCLUSION: Intra-arterial contrast injection detected the AKA at a high rate and verified continuity from the aorta to the AKA.

Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells.

Sodium-dependent vitamin C transporter (SVCT) 2-mediated L-ascorbic acid (AA) uptake is required in osteoblast-like differentiation of MC3T3-E1 cells, and prostaglandin E2 (PGE2) is among the most important local factors in bone formation, but the detailed mechanism by which PGE2 induces osteoblast differentiation remains obscure. We revealed that PGE2 induced AA uptake and osteoblast-like differential markers including alkaline phosphatase, collagen, osteocalcin expression, and mineralization in MC3T3-E1 cells. Inhibition of AA uptake by SVCT2 short isoform functioning as a dominant-negative mutant not only robustly attenuated PGE2-induced markers expression and mineralization, but also decreased their basal levels. However, upregulation of AA uptake resulted from PGE2-induced plasma membrane translocation of cytoplasm SVCT2, and this effect was abolished by pretreatment with EP4 receptor antagonist, AH-23848B or cAMP-dependent protein kinase A (PKA) inhibitor, H-89. Moreover, we showed SVCT2 physically interacted with PKA in immunoprecipitates, and PKA phosphorylated SVCT2 in vitro and in intact cells at Ser402 and Ser639 sites; however, mutation of Ser402 or/and Ser639 in SVCT2 severely diminished SVCT2 translocation in response to PGE2. Together, these results suggest that PGE2-induced SVCT2 plasma membrane translocation through EP4 receptor and subsequent phosphorylation of SVCT2 at Ser402 and Ser639 sites by PKA results in an increase of AA uptake and consequent promotion of osteoblast-like differentiation in MC3T3-E1 cells.

Transient appearance of hepatic natural killer cells with high cytotoxicity and unique phenotype in very young mice.

There were few natural killer (NK) cells in the liver in very young mice at the age of 1-2 weeks. This was because the cell yield from the liver of young mice was low. The percentage of NK cells in the liver of young mice, however, was almost comparable with that in the liver of adult mice. Lymphocytes were isolated from the liver and spleen of C57BL/6 (B6) mice, and NK cytotoxicity and phenotype were herein examined in this study. NK cytotoxicity was extremely high in the liver of very young mice. This phenomenon was seen in the liver of various normal mouse strains. In contrast, the appearance of high cytotoxicity was not seen in NK cells of the spleen, irrespective of mouse strains. The quality of NK cells in the liver of young mice was different from that in adult mice. NK cells in the liver of young mice were mainly CD69(+)Mac-1(-) Fas ligand(+), whereas those in the liver of adult mice were CD69(-)Mac-1(+) Fas ligand(-). These results revealed that the quality of hepatic NK cells changes in the process of ageing. Namely, liver NK cells in very young mice temporarily show the highest NK cytotoxicity and a unique activated phenotype. Physiological meaning of the present phenomenon was discussed.

[Pulmonary pleomorphic carcinoma; report of a case].

We report a case of a 64-year-old man with pleomorphic carcinoma of the lung and thymic cyst. He was admitted to our hospital because of an abnormal shadow observed on chest X-ray. Computed tomography (CT) showed a mass lesion located in the right upper lobe and a non-invasive anterior mediastinal tumor adjacent to the left brachiocepharic vein. On enhanced CT, the lung mass showed central low-attenuation areas with a substantial enhancement in the periphery. Preoperative transbronchial blushing cytology of the mass revealed adenocarcinoma. With a diagnosis of primary lung cancer (cT3N0M0) and mediastinal tumor, an operation was performed through a median sternotomy. The mediastinal tumor was excised and a right upper lobectomy and were also accomplished, because the lung tumor did not show adhesion or pleural invasion. Histopathologic examination of the resected specimen revealed that the lung tumor composed of a mixture of spindle and giant cell features and contained a component of adenocarcinoma and squamous cell carcinoma. This finding yielded a pathological diagnosis of pleomorphic carcinoma (pT2N0M0). The mediastinal tumor was diagnosed as thymic cyst. The postoperative course was uneventful, and he is currently well 6 months after surgery.

Structure and property of self-assemble valinyl bolaform amides having different chirality.

Bolaform amides were designed from N,N'-bis(carboethoxy-L-valinyl)-diaminoethane (1) by linking t-butyloxycarbonyl-valine through ethylenediamine (EDA) to enable spectroscopic and X-ray diffraction analyses. N,N'-Bis(Boc-L-valinyl)-diaminoethane (2) and N,N'-bis(Boc-D-valinyl)-diaminoethane (3) were composed of L-Val and D-Val, respectively. N-(Boc-L-valinyl)-N'-(Boc-D-valinyl)-diaminoethane (4) was composed of both L-Val and D-Val, and was achiral (meso-peptide). Peptide 5 was a 1:1 mixture of 2 and 3, and was also achiral (racemate). These peptides mediated gelation of corn oil at a concentration of approximately 1%. Within crystals, the peptides formed beta-sheet ribbons, but differences were observed in hydrogen-bonding patterns and side-chain arrangements. These differences were also deduced from temperature dependence of amide protons. Force-field calculations based on the crystal structures indicated that association of beta-sheet ribbons had energy benefits, and it was assumed that molecular aggregation progressed spontaneously. These structural studies indicated the chirality of amino acids affected for the properties of bolaform amides.

Interleukin-1beta converting enzyme subfamily inhibitors prevent induction of CD86 molecules by butyrate through a CREB-dependent mechanism in HL60 cells.

To investigate the underlying mechanism for induction of CD86 molecules, we analysed the ability of the histone deacetylase (HDAC) inhibitor, sodium butyrate (NaB), to induce CD86 at the transcriptional level in HL60 cells. Our studies showed that the expression of CD86 on the cell surface was increased by 24 hr of NaB treatment, and the enhancement of CD86 mRNA expression was observed by real-time polymerase chain reaction. When we measured NF-kappaB binding activity, significant activity was induced upon NaB stimulation, which was suppressed by the addition of pyrrolidine dithiocarbamate. Butyrate also induced phosphorylated cAMP response element-binding protein (CREB), which bound to cAMP-responsive elements. Dibutyryl (db) -cAMP induced active CREB and increased the levels of CD86 by 24 hr. These observations indicated that NF-kappaB and/or CREB are crucial for butyrate-dependent activation of CD86 gene expression. We examined the inhibitory effects of various caspase inhibitors on the expression of CD86 in cells treated with NaB, because NaB also induced apoptosis with slow kinetics. Intriguingly, our results demonstrated that inhibitors of the interleukin-1beta converting enzyme subfamily (caspase-1, -4, -5 and -13) blocked the butyrate-induced increase in level of CD86. These inhibitors interfered with CD86 gene transcription in the presence of activated NF-kappaB, whereas phosphorylated CREB was down-regulated in the reactions where these inhibitors were added to inhibit CD86 gene expression. These results suggested that butyrate not only acetylates histones on the CD86 promoter through the suppression of HDAC activity, but that butyrate also regulates CREB-mediated transcription, possibly through the caspase activities triggered by NaB.

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.