Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr virus (EBV) is documented as the important etiologic agent of nasopharyngeal carcinoma (NPC) but the mechanism of development and pathogenesis induced by EBV is presently unclear. Hypermethylation of epithelial-cadherin (E-cadherin) promoter has been shown to be induced in NPC cell line by EBV LMP1 via DNA methyltransferase activation. EBV genomes and hypermethylation of E-cadherin promoter were investigated in NPC tissues to evaluate the role of EBV in the hypermethylation and pathogenesis of NPC. METHODS: Methylation-specific polymerase chain reaction (MSP) was performed to detect E-cadherin promoter hypermethylation in paraffin embedded tissues from patients with NPC and normal nasopharyngeal tissues. EBV genomes were detected by PCR in the tissue samples. RESULTS: Hypermethylation of E-cadherin promoter and EBV were predominantly detected in undifferentiated and non-keratinizing NPC compared to those in squamous cell NPC. Hypermethylation of E-cadherin was found in 28 of 38 (73.7%) patient samples. EBV was detected in 22 of the 28 (78.6%) NPC samples demonstrating E-cadherin hypermethylation. EBV genomes and hypermethylation were not detected in normal nasopharyngeal tissues. Significant association was found between E-cadherin hypermethylation and EBV genomes (p<0.001; Fisher's exact test). Hypermethylation of E-cadherin was more frequently detected in advanced stages compared to those in early stages of NPC (p=0.036; Fisher's exact test). CONCLUSIONS: The high incidence of EBV with the consistency of E-cadherin hypermethylation, particularly in undifferentiated and non-keratinizing NPC suggests the role of EBV in the hypermethylation. EBV exists at early stage of NPC that induces the hypermethylation and contributes to progression of the disease to the advanced stage of NPC.

Osteoblast differentiation and bone formation gene expression in strontium-inducing bone marrow mesenchymal stem cell.

Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is an important step of bone formation. We studied the in vitro induction of hMSCs by using strontium ranelate, a natural trace amount in water, food and human skeleton. The mRNA synthesis of various osteoblast specific genes was assessed by means of reverse transcription polymerase chain reaction (RT-PCR). In the hMSCs culture, strontium ranelate could enhance the induction of hMSCs to differentiate into osteoblasts. Cbfa1 gene was earlier expressed on day 4 of cell culture (the control group, on day 14) and osteonectin on day 11 (control, on day 21). The early Cbfa1 expression indicates that strontium could enhance osteoblastic differentiation. The detection of osteonectin using strontium induction indicates the role of strontium in enhancing bone remodeling, bone structure stabilization of hydroxyapatite molecule and collagen fibril organization. The cultured hMSCs in the presence of strontium expressed genes of bone extracellular matrix: collagen type I, bone sialoprotein and osteocalcin on the same days as control (same medium with no strontium). Concentration of strontium ranelate has been recommended to be optimized in between 0.2107 - 21.07 microg/ml whereas the high concentration up to 210.7 microg/ml have delayed effect on osteoblastic differentiation with delayed expression on Cbfa1 and osteonectin, and inhibitory effect on bone sialoprotein expression. In addition, strontium could help cell expansion by maintaining cell proliferation rate of hMSCs and osteoblast lineage. We recommend that the strontium is an important factor for inducing mesenchymal stem cells to differentiate into osteoblasts with further enhancement on bone formation. This model might provide a useful cell source for tissue engineering and bone repair.

Pancreatic arteriovenous malformation combined with portal thrombosis.

We encountered a case of portal vein thrombosis (PVT) after treatment for portal hypertension due to pancreatic arteriovenous malformation (PAVM). A 75-year-old man was admitted for the treatment of esophageal varices. Diffuse PAVM and aneurysm in the celiac and superior mesenteric arteries were detected via abdominal computed tomography and angiography. Although endoscopical sclerotherapy was performed, PVT was identified after the treatment and variceal bleeding continued. Autopsy was performed and the thrombus and malformation were pathologically confirmed. This case indicates that PVT can be associated with PAVM.

MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity.

Adipocytes secrete a variety of bioactive molecules that affect the insulin sensitivity of other tissues. We now show that the abundance of monocyte chemoattractant protein-1 (MCP-1) mRNA in adipose tissue and the plasma concentration of MCP-1 were increased both in genetically obese diabetic (db/db) mice and in WT mice with obesity induced by a high-fat diet. Mice engineered to express an MCP-1 transgene in adipose tissue under the control of the aP2 gene promoter exhibited insulin resistance, macrophage infiltration into adipose tissue, and increased hepatic triglyceride content. Furthermore, insulin resistance, hepatic steatosis, and macrophage accumulation in adipose tissue induced by a high-fat diet were reduced extensively in MCP-1 homozygous KO mice compared with WT animals. Finally, acute expression of a dominant-negative mutant of MCP-1 ameliorated insulin resistance in db/db mice and in WT mice fed a high-fat diet. These findings suggest that an increase in MCP-1 expression in adipose tissue contributes to the macrophage infiltration into this tissue, insulin resistance, and hepatic steatosis associated with obesity in mice.

Histological and cytogenetic characterization of bone marrow in relation to prognosis and diagnosis of myelodysplastic syndromes.

Bone marrow (BM) histology of 102 myelodysplastic syndromes (MDS) patients was analyzed retrospectively. All the cases were reclassified according to the World Health Organization (WHO) classification. Karyotype study was conducted for all except one. Fifteen of the MDS cases were hypoplastic. The cellularity in bone marrow histology is sometimes ineffective in the differential diagnosis of MDS and aplastic anemia (AA). Nonetheless, a marked decrease in the number of megakaryocytes (average, 0.3/mm(2); range, 0-2/mm(2)) even in the hyperplastic foci of the marrow of AA was the most important histological feature differentiating AA from MDS, whereas the number of megakaryocytes increased in most MDS cases (44/mm(2); range, 1-240/mm(2)) and also in hypoplastic MDS (14/mm(2); range, 8-26/mm(2)). Hyperplastic marrow had a significantly high frequency of progress to acute myeloid leukemia (AML) and hypoplastic MDS had a lower rate of progress to AML. Severe myelofibrosis had a significantly poor prognosis. An increase in CD34-positive cells in MDS indicated a high rate of progress to AML. As for the patients with refractory cytopenia with multilineage dysplasia (RCMD; the new category under the WHO classification), the increased number of megakaryocytes was correlated with poor prognosis.

Modulation of mouse RANKL gene expression by Runx2 and PKA pathway.

Runx2 regulates the target genes characteristic of osteoblastic phenotypes, while exerting diverse and sometimes controversial effects on osteoblastic cells depending on their differentiation stage. Receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) is a membrane bound cytokine essential for osteo(chondro)clastogenesis. During endochondral ossification, while Runx2-positive hypertrophic chondrocytes express RANKL, the steady-state expression of the RANKL gene in osteoblastic cells is, at later stages, kept at a relatively low level to sustain the established bone. The aim of this study was to elucidate the mechanism whereby Runx2 and the protein kinase A (PKA) pathway modulate RANKL expression, especially from the viewpoint of their functions in RANKL basic promoter activity and in chromatin structural changes in osteoblastic/stromal cells. Osteoblastic/stromal cell lines derived from normal and Runx2-deficient mice were used to analyze endogenous RANKL gene expression by real-time reverse transcription (RT)-PCR, the acetylation status of the H3 and H4 histone proteins associated with the 5'-flanking region of the RANKL gene by chromatin immunoprecipitation, and the exogenously transfected RANKL gene promoter activity both in the steady-state and under PKA-activated conditions. Here, we demonstrate that Runx2 suppresses steady-state RANKL gene expression by condensing chromatin, while showing a slightly positive effect on RANKL basic promoter activity. Besides acting through the CRE-like region (-0.96 kb) of the RANKL gene promoter, forskolin (FK) treatment transactivates the RANKL gene by antagonizing the function of Runx2, by reducing Runx2 mRNA expression and by opening the chromatin conformation far upstream (more than 40 kb) of the RANKL gene.

Expression profile of genes related to osteoclastogenesis in mouse growth plate and articular cartilage.

Based on developmental fate and function, cartilage tissue is broadly classified into transient cartilage (e.g. growth plate, GP) and permanent cartilage (e.g. articular cartilage, AC). The former eventually disappears and is replaced by bone during the endochondral ossification process, whereas the latter retains its permanency. Osteo(chondro)clasts, multinucleated giant cells of the monocyte/macrophage lineage, are selectively induced in the GP during endochondral ossification and play central roles in the resorption of cartilagenous matrices. The aim of this study was to investigate the factors determining the GP-specific recruitment of osteo(chondro)clasts. We especially focused on the expression pattern of the receptor activator of NF-kappaB ligand (RANKL), an essential factor for osteo(chondro)clast differentiation, and on that of epigenetic and transcriptional factors affecting RANKL gene expression. Knee joints of male BALB/c mice aged 8 weeks were dissected and subjected to immunohistochemical analysis using anti-RANKL, Runx2, Dlx5 and Msx2 antibodies. The methylation status of the mouse RANKL gene promoter in both the GP and the AC was analyzed by sodium bisulfite mapping using microdissected mouse tissue. The expression of BMP-2, -3, -4, -6 and type X collagen mRNA was examined by in situ hybridization (ISH). At the boundary between the calcifying cartilage and the hypertrophic chondrocytes of the GP, RANKL-expressing chondrocytes overlapped those expressing Runx2, Dlx5 and Msx2, near numerous osteo(chondro)clasts. Although similar BMP-2 and -4 expression was observed in chondrocytes in both the GP and the AC as well as in maturing osteoblasts, a rather restricted BMP-6 expression pattern was observed in resting and proliferating chondrocytes in the GP. On the other hand, sodium bisulfite mapping showed that mostly non-CpG methylation was similarly scattered in a non-specific manner in chondrocytes in the GP and the AC. Taken together with the fact that putative Runx2 binding elements are located in the RANKL promoter, our data suggest that Runx2, an essential transcription factor for skeletal development, is also a key regulator of RANKL expression in chondrocytes in the GP. Furthermore, a selective and sequential expression of a subset of BMP and of transcription factors may define the expression pattern of RANKL through Runx2.

PKC pathway and ERK/MAPK pathway are required for induction of cyclin D1 and p21Waf1 during 12-o-tetradecanoylphorbol 13-acetate-induced differentiation of myeloleukemia cells.

Treatment of human promyelocytic leukemia cell HL60 with 12-o-tetradecanoylphorbol 13-acetate (TPA) induces growth arrest, differentiation towards the monocyte/macrophage lineage, and expression of cell cycle-regulating genes cyclin D1 and p21Waf1. First, we demonstrated that p21Waf1 expression was increased by TPA in other leukemia cell lines also, including THP-1, U937, and KG-1, which differentiate into monocytes/macrophages by TPA. Secondly, we demonstrated the signal transduction pathways of cyclin D1 and p21Waf1 expressions in TPA-treated HL60 cells. Induction of cyclin D1 expression in TPA-treated HL60 cells was inhibited with protein kinase C (PKC) inhibitor bisindolylmaleimide I and mitogen activated protein kinase kinase (MEK) inhibitor PD98059. Induction of p21Waf1 expression in TPA-treated HL60 cells was inhibited with PKC inhibitor bisindolylmaleimide I and Gö6976, MEK inhibitor PD98059, and p38 mitogen-actibated protein kinase (MAPK) inhibitor SB202190. Thus, cyclin D1 and p21Waf1 expressions are considered to be induced via PKC and extracellular signal-regulated kinase/mitogen-activated protein kinase (MAPK/ERK) pathways in TPA-treated HL60 cells. The upregulation of p21Waf1 seems to play a critical role in TPA-induced cell differentiation by suppressing cyclin dependent kinase activity , while the upregulation of cyclin D1 seems to be compensated by p21Waf1.

Ray tracing analysis of overlapping objects in refraction contrast imaging.

We simulated refraction contrast imaging in overlapping objects using the ray tracing method. The easiest case, in which two columnar objects (blood vessels) with a density of 1.0 [g/cm3], run at right angles in air, was calculated. For absorption, we performed simulation using the Snell law adapted to the object's boundary. A pair of bright and dark spot results from the interference of refracted X-rays where the blood vessels crossed. This has the possibility of increasing the visibility of the image.

Enhanced expression of programmed death-1 (PD-1)/PD-L1 in salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: Programmed death-1 (PD-1) mediates a negative signal and introduces tolerance for lymphocytes. Dysfunction of the PD-1 pathway is thought to result in autoimmune diseases such as rheumatoid arthritis (RA). To investigate the role of the PD-1/PD-L system in the pathology of Sjögren's syndrome (SS), we examined the expression of PD-1 and its ligand PD-L1 in salivary lymphocytes and salivary glands from patients with SS. METHODS: Flow cytometry analysis was used to determine expression of PD-1 in SS salivary lymphocytes. Intracellular staining of interleukin 10 (IL-10) was performed after stimulation with PMA and ionomycin. Indirect immunohistochemistry was used to investigate the expression of PD-1 and PD-L1. RESULTS: The mean fluorescence intensity of PD-1 expression in SS salivary lymphocytes was significantly higher than that from healthy controls and patients with RA or systemic lupus erythematosus. PD-1-positive SS salivary lymphocytes expressed IL-10 intracellularly upon PMA/ionomycin stimulation. Immunohistochemical analysis showed that PD-1 was expressed on infiltrating lymphocytes in salivary gland from 52% of SS patients, and PD-L1 was expressed on ductal and acinar epithelial cells from 68% of SS patients. In vitro analysis using HSG cells revealed that PD-L1 was induced by interferon-gamma but not by tumor necrosis factor-alpha and IL-1beta. CONCLUSION: PD-1 is expressed on T lymphocytes and PD-L1 on epithelial cells from inflamed salivary glands of patients with SS, which suggests that dysfunction of the PD-1/PD-L1 pathway may be related to tolerance for lymphocytes, which causes SS.

How to help Acehnese helping themselves?--a note after a visit with Kobe University medical team.

On December 26, Aceh Province in Indonesia was hit by the worst earthquake and tsunami. A medical team from Kobe University Graduate School of Medicine visited two of the affected areas on the eastern coast of Aceh: Sigli and Lhokseumawe. This article provides a simple description of experience and assessment derived from the visit. The disaster has left Indonesia with complex problems, which will take a long time to overcome. A continuity of the aid and assistance from various resources is crucial to help Indonesia rebuilt the affected areas. These continuous efforts will provide great contribution for Acehnese people to recover and rebuild their life after tsunami.

Methylation adjacent to negatively regulating AP-1 site reactivates TrkA gene expression during cancer progression.

Nerve growth factor and its high-affinity receptor TrkA are thought to be involved in the progression of various cancers. This study investigated the mechanism that regulates aberrant or increased TrkA expression in various cancer cell lines and in the course of pancreatic cancer progression. We found that the negative cis-acting AP-1-like sequence TGAGCGA was located in the 5'-untranslated region of the TrkA gene. Sodium bisulfite mapping revealed that steady-state TrkA expression correlated positively with the accumulation of methylated CpG around the AP-1-like site. Electrophoretic mobility shift assay showed that the AP-1-like site was bound mainly by c-Jun homodimers; the binding was directly blocked by Sss I methylase-induced methylation or by an excess of oligonucleotides containing consensus AP-1 sequences. Consequently, activation of TrkA gene expression by methylation was considered to be caused by the direct interference of c-Jun binding to the negatively regulating AP-1-like site. Furthermore, the accumulation of methylated CpG around the AP-1-like site was also observed with increased TrkA immunohistochemical staining in cases of advanced pancreatic adenocarcinoma with extensive perineural invasion. Unlike global methylation at CpG islands that leads to gene silencing, specific methylation at non-CpG islands would play a crucial epigenetic role in the versatility and plasticity of TrkA expression during cancer progression.

Estimation of contrast of refraction contrast imaging compared with absorption imaging-basic approach.

PURPOSE: We discuss the usefulness of the refraction contrast method using highly parallel X-rays as a new approach to minute lung cancer detection. The advantages of refraction contrast images are discussed in terms of contrast, and a comparison is made with absorption images. MATERIALS AND METHODS: We simulated refraction contrast imaging using globules with the density of water in air as models for minute lung cancer detection. The contrast intensified by bright and dark lines was compared on a globule with the contrast of absorption images. We adopted the Monte Carlo simulation to determine the strength of the profile curve of the photon counts at the detector. RESULTS: The obtained contrasts were more intense by two to three digits than those obtainable with the absorption contrast imaging method. CONCLUSION: The contrast in refraction contrast imaging was more intense than that obtainable with absorption contrast imaging. A two to three digit improvement in contrast means that it is possible to greatly reduce the exposure dose necessary for imaging. Therefore, it is expected to become possible to detect the interfaces of soft tissues, which are difficult to capture with conventional absorption imaging, at low dosages and high resolution.

Congenital semilunar valvulogenesis defect in mice deficient in phospholipase C epsilon.

Phospholipase Cepsilon is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cepsilon is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cepsilon. These results suggest a crucial role of phospholipase Cepsilon downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.

PKClambda regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic beta cells.

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.

Primary pulmonary myxoma.

We report a case of a primary pulmonary myxoma. The patient was a 69-year-old previously healthy human. Computed tomographic scan demonstrated a well-circumscribed tumor with a diameter of approximately 13 mm in the right upper lobe. We performed a right upper lobectomy under video-assisted thoracoscopy. Microscopic examination of the tumor disclosed elongated and stellate cells in a myxoid stroma, typical of myxoma.

Crucial role of phospholipase Cepsilon in chemical carcinogen-induced skin tumor development.

Mutational activation of the ras proto-oncogenes is frequently found in skin cancers. However, the nature of downstream signaling pathways from Ras involved in skin carcinogenesis remains poorly understood. Recently, we and others identified phospholipase C (PLC) epsilon as an effector of Ras. Here we have examined the role of PLCepsilon in de novo skin chemical carcinogenesis by using mice whose PLCepsilon is genetically inactivated. PLCepsilon(-/-) mice exhibit delayed onset and markedly reduced incidence of skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the papillomas formed in PLCepsilon(-/-) mice fail to undergo malignant progression into carcinomas, in contrast to a malignant conversion rate of approximately 20% observed with papillomas in PLCepsilon(+/+) mice. In all of the tumors analyzed, the Ha-ras gene is mutationally activated irrespective of the PLCepsilon background. The skin of PLCepsilon(-/-) mice fails to exhibit basal layer cell proliferation and epidermal hyperplasia in response to TPA treatment. These results indicate a crucial role of PLCepsilon in ras oncogene-induced de novo carcinogenesis and downstream signaling from TPA, introducing PLCepsilon as a candidate molecular target for the development of anticancer drugs.

1 alpha,25 dihydroxyvitamin D3 rapidly regulates the mouse osteoprotegerin gene through dual pathways.

UNLABELLED: 1 alpha,25(OH)(2)D(3) rapidly and transiently suppressed OPG gene expression both by accelerating the degradation of mRNA and by suppressing promoter activity. The latter process was mediated through the AP-1 binding site by a reduction in the proportion of phospho-c-Jun in a JNK-independent manner. INTRODUCTION: Osteoclastogenesis is regulated by an integrated network of numerous bone metabolic factors, among which 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)] promotes osteoclastogenesis by reciprocally upregulating the expression of RANKL and downregulating that of osteoprotegerin (OPG). MATERIALS AND METHODS: To analyze the mechanism by which 1 alpha,25(OH)(2)D(3) suppresses OPG, we characterized cis-acting elements of the mouse OPG gene and assessed the post-transcriptional modifications by actinomycin D assays. RESULTS: 1 alpha,25(OH)(2)D(3) rapidly and transiently suppressed OPG expression and shortened the half-life of OPG mRNA; additionally, the c-Jun homodimer bound to the AP-1 binding site (TGACTGA, -293/-287) and maintained steady-state transcription of the OPG gene. Furthermore, mutation of the AP-1 site negated 1 alpha,25(OH)(2)D(3)-driven OPG suppression. Moreover, 1 alpha,25(OH)(2)D(3) treatment of ST2 cells decreased the amount of phosphorylated c-Jun protein (phospho-c-Jun), while the total amount of c-Jun remained constant; however, the amount of phosphorylated Jun N-terminal kinase (JNK) was nearly unchanged by 1 alpha,25(OH)(2)D(3) treatment. CONCLUSION: Taken together with the observation that the OPG promoter has no consensus negative vitamin D-responsive elements, these data suggest that 1 alpha,25(OH)(2)D(3) transrepresses mouse OPG by reducing the proportion of phospho-c-Jun in a JNK-independent manner. Our data indicated that short-term treatment with 1 alpha,25(OH)(2)D(3) effectively downregulated OPG expression both by accelerating the degradation of OPG mRNA and by transrepressing the OPG gene through its AP-1 binding site in the catabolic phase. The OPG gene became insensitive to 1 alpha,25(OH)(2)D(3) treatment, however, and reverted to its steady-state expression level over time, leading to the anabolic phase of the effect of 1 alpha,25(OH)(2)D(3) on bone.

Imaging of fine structure of bone sample with high coherent X-ray beam and high spatial resolution detector.

In this study, we observed bone specimens of the mouse using a very high coherence beam and high spatial resolution detector (zooming tube: approximately 0.7 micron resolution) and successfully obtained images of the Haversian canal, osteocytes, and osteoclasts.