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RB1-inducible coiled-coil 1 (RB1CC1) plays a significant role in the enhancement of the retinoblastoma tumor suppressor (RB1) pathway and is involved in breast cancer development. However, RB1CC1's role in clinical progression of breast cancer has not yet been evaluated, so, as a first step, it is necessary to establish its usefulness as a tool to evaluate breast cancer patients. In this report, we have analyzed the correlation between abnormalities in the RB1CC1 pathway and long-term prognosis, because disease-specific death in later periods (>5 years) of the disease is a serious problem in breast cancer. Breast cancer tissues from a large cohort in Japan were evaluated by conventional immunohistochemical methods for the presence of the molecules involved in the RB1CC1 pathway, including RB1CC1, RB1, p53, and other well-known prognostic markers for breast cancer, such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The correlation between the immunohistochemical results and clinical outcomes of 323 breast cancer patients was analyzed using a Kaplan-Meier log-rank test and a multivariate Cox proportional hazards regression analysis. Absence of nuclear RB1CC1 expression was associated with the worst prognosis (Log-rank test, Chi-Square value = 17.462, p<0.0001). Dysfunction of either one of RB1CC1, RB1, or p53 was associated with the highest risk for cancer-specific death, especially related to survival lasting more than 5 years (multivariate Cox proportional hazard ratio = 3.951, 95% Confidence Interval =1.566-9.967, p = 0.0036). Our present data demonstrate that the combined evaluation of RB1CC1, RB1 and p53 by conventional immunohistochemical analysis provides an accurate prediction of the long-term prognoses of breast cancer patients, which can be carried out as a routine clinical examination.

This study aims to validate ultrasonic anisotropy in 2-dimensional echocardiograms by one-to-one histological correlation. Thirteen echograms were obtained in vitro from 9 specimens of 2 left ventricles of healthy adult beagle dogs and were correlated with corresponding histology in regard to ultrasound orientation relative to the local fiber direction. Median value of pixel echogenicity (255-rank) was lower in areas with parallel (n = 9, 18 ± 13) than in those with oblique (n = 7, 41 ± 14, P < 0.05) and perpendicular (n = 13, 57 ± 21, P < 0.01) ultrasound orientations. Echogenicity in the proximal part within 0.5 cm from an edge was relatively high (n = 9, 33 ± 15; n = 5, 66 ± 27; n = 8, 65 ± 32, respectively) and was different between parallel and perpendicular orientations (P < 0.05). The ratio of the distal adjacent 0.5-cm part to the proximal part echogenicity was lower for either parallel or oblique orientation (0.35 ± 0.25, 0.51 ± 0.16) than for perpendicular orientation (0.95 ± 0.16, P < 0.01, respectively). Acoustic dropouts appeared beyond myocardial areas with parallel or oblique ultrasound orientation. These results disclosed precise acoustic anisotropy in the 2-dimensional echocardiograms.

Background and Importance: Intracranial clear cell meningioma is very rare. We present 3 cases of intracranial clear cell meningiomas genetically characterized by comparative genomic hybridization with a review of the literature., Clinical Presentation: Patient 1 is a 38-year-old woman with a petroclival tumor. Patient 2 is a 60-year-old man with a tumor at the foramen magnum. Patient 3 is a 60-year-old man with a tumor at the posterior clinoid process. Gross total resection was performed in patients 1 and 2. Patient 1 has been free from recurrence for 10 years. Patient 2 had a tumor recurrence at 14 months after the operation. After partial resection, conventional radiotherapy was given, and there was no tumor regrowth at 2 years after radiotherapy. Subtotal resection was performed in patient 3, and no regrowth was detected for 3 months. Histologically, all tumors were composed of cells with clear cytoplasm reactive for periodic acid-Schiff and diagnosed as clear cell meningioma. The MIB-1 and p53 staining indexes were 1.8, 1.7, and 5.6 and 1.1, 1.0, and 5.5, respectively. Comparative genomic hybridization revealed no chromosomal number aberrations in patient 1, numerous losses and gains including loss of chromosome 1 in patient 2, and loss of only 22q in patient 3. Because staining indexes of MIB-1 and p53 were equivalent in 2 patient (patients 1 and 2) with a long follow-up period, the contrary clinical courses are likely associated with genetic characteristics., Conclusion: To the best of our knowledge, this is the first report that suggests association between tumor behavior and genetic characteristics in clear cell meningiomas.

Objective: To determine the nucleotide sequence of the partial mitochondrial (mt) genome and the order of the mitochondrial protein-coding genes for Schistosoma bovis for analysis of possible phylogenetic position of this species in the genus Schistosoma., Methods: The genomic DNA of adult worms were extracted by the GNT-K method. The target regions were amplified by PCR using a degenerated primer and specific primer. The PCR products were purified before ligating into the pGEM1 T-vector system. Recombinant plasmids were amplified in Escherichia coli, extracted and purified using routine methods. The nucleotide sequences were determined with an ABI PRISM 3100-Avant DNA sequencer using a BigDye Terminators v3.1 Cycle Sequencing Kit (Applied Bio-systems, CA, U.S.A.) with two T-vector specific primers (T7 and SP6). Positive colonies were sequenced with two internal specific primers to obtain the full sequence of each fragment on both strands by means of primer walking. Sequences of related schistosomes were retrieved from GenBank and aligned with our data. Gene trees were constructed using neighbor joining methods., Results: The nucleotide sequence was determined and the gene order of this region in S. bovis was found as follows: NADHdehydrogenase4 (nad4)-trnQ (Gln)-trnK(Lys)-NADH dehydrogenase 3(nad3)-trnD (Asp)-NADH dehydrogenase 1(nad1). The gene order covering such region of S. bovis was similar to that of the African Schistosoma species, but strikingly different from the Asian species. Phylogenetic trees inferred from the alignment including partial nad4, nad3, partial nad1 and partial nad4+nad3+nad1 sequence for other 8 Schistosoma spp., respectively, revealed that S. bovis is placed proximally to S. haematobium in the African sub-group, which is identical with those placed by gene order in the African clade., Conclusion: The mtDNA analysis based on mitochondrial DNA sequence and the gene order strongly support the hypothesis that S. bovis belongs to the African schistosome clade rather than the Asian Schistosoma species.

Background: Most biological functions controlled by the brain and their related disorders are closely associated with activation in specific regions of the brain. Neuroproteomics has been applied to the analysis of whole brain, and the general pattern of protein expression in all regions has been elucidated. However, the comprehensive proteome of each brain region remains unclear., Results: In this study, we carried out comparative proteomics of six regions of the adult rat brain: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala using semi-quantitative analysis by Mascot Score of the identified proteins. In order to identify efficiently the proteins that are present in the brain, the proteins were separated by a combination of SDS-PAGE on a C18 column-equipped nano-liquid chromatograph, and analyzed by quadrupole-time of flight-tandem-mass spectrometry. The proteomic data show 2,909 peptides in the rat brain, with more than 200 identified as region-abundant proteins by semi-quantitative analysis. The regions containing the identified proteins are membrane (20.0%), cytoplasm (19.5%), mitochondrion (17.1%), cytoskeleton (8.2%), nucleus (4.7%), extracellular region (3.3%), and other (18.0%). Of the identified proteins, the expressions of glial fibrillary acidic protein, GABA transporter 3, Septin 5, heat shock protein 90, synaptotagmin, heat shock protein 70, and pyruvate kinase were confirmed by immunoblotting. We examined the distributions in rat brain of GABA transporter 3, glial fibrillary acidic protein, and heat shock protein 70 by immunohistochemistry, and found that the proteins are localized around the regions observed by proteomic analysis and immunoblotting. IPA analysis indicates that pathways closely related to the biological functions of each region may be activated in rat brain., Conclusions: These observations indicate that proteomics in each region of adult rat brain may provide a novel way to elucidate biological actions associated with the activation of regions of the brain.

The aim of the present study was to determine whether symptoms of atrial fibrillation (AF) differ between patients with and without subsequent permanent AF. Sixty-four patients (68 +/- 10 years old, 45 males) were recruited. AF follow-up was started at the age of 61 +/- 10 years and accomplished in a median period of 4.9 years (396 person-years). Permanent AF, defined as lasting > 180 days, developed in 17 patients (14 males) (43 per 1000 person-years). The AF follow-up period was longer in the permanent AF group than in the non-permanent AF group (median, 9.8 versus 4.2 years, P < 0.001). For baseline characteristics, hypertension was less frequent in the permanent AF group than in the nonpermanent AF group (18% versus 45%, P < 0.05). A retrospective questionnaire survey regarding initial AF symptoms was conducted. The severity of AF symptoms by a 4-grade scale was significantly milder in the permanent AF group than in the nonpermanent AF group (P < 0.05). Cox proportional hazards model analysis revealed that the severity of initial AF symptoms was related to the subsequent development of permanent AF (hazard ratio 0.46 per grade, 95% confidence interval 0.23 - 0.93, P < 0.05), but age, gender, hypertension, diabetes mellitus, organic heart disease, and left atrial dimension were not. The permanent AF-free rate was significantly lower in 33 patients with mild symptoms than in 31 patients with severe symptoms (log-rank test, P < 0.05). These results point to an inconspicuous feature in the development of permanent AF.

Skeletal fracture healing involves a variety of cellular and molecular events; however, the mechanisms behind these processes are not fully understood. In the current study, we investigated the potential involvement of the signal transducer and activator of transcription 1 (STAT1), a critical regulator for both osteoclastogenesis and osteoblast differentiation, in skeletal fracture healing. We used a fracture model and a cortical defect model in mice, and found that fracture callus remodeling and membranous ossification are highly accelerated in STAT1-deficient mice. Additionally, we found that STAT1 suppresses Osterix transcript levels and Osterix promoter activity in vitro, indicating the suppression of Osterix transcription as one of the mechanisms behind the inhibitory effect of STAT1 on osteoblast differentiation. Furthermore, we found that fludarabine, a potent STAT1 inhibitor, significantly increases bone formation in a heterotopic ossification model. These results reveal previously unknown functions of STAT1 in skeletal homeostasis and may have important clinical implications for the treatment of skeletal bone fracture., ((c) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Valvular heart disease (VHD) is the term given to any disease process involving one or more of the heart valves. The condition can be congenital or acquired, for example as a result of atherosclerosis or rheumatic fever. Despite its clinical importance, the molecular mechanisms underlying VHD remain unknown. We investigated the pathophysiologic role and molecular mechanism of periostin, a protein that plays critical roles in cardiac valve development, in degenerative VHD. Unexpectedly, we found that periostin levels were drastically increased in infiltrated inflammatory cells and myofibroblasts in areas of angiogenesis in human atherosclerotic and rheumatic VHD, whereas periostin was localized to the subendothelial layer in normal valves. The expression patterns of periostin and chondromodulin I, an angioinhibitory factor that maintains cardiac valvular function, were mutually exclusive. In WT mice, a high-fat diet markedly increased aortic valve thickening, annular fibrosis, and MMP-2 and MMP-13 expression levels, concomitant with increased periostin expression; these changes were attenuated in periostin-knockout mice. In vitro and ex vivo studies revealed that periostin promoted tube formation and mobilization of ECs. Furthermore, periostin prominently increased MMP secretion from cultured valvular interstitial cells, ECs, and macrophages in a cell type-specific manner. These findings indicate that, in contrast to chondromodulin I, periostin plays an essential role in the progression of cardiac valve complex degeneration by inducing angiogenesis and MMP production.

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) plays a role in the enhancement of the RB1 pathway through the direct binding to a GC-rich region 201bp upstream (from the initiation ATG) of the RB1 promoter. Here, we identified hSNF5 and p53 as the binding partners of RB1CC1 by immunoprecipitation and immunofluorescence assays. Interaction between these molecules and the RB1 pathway was analyzed by the assays of chromatin immunoprecipitation, luciferase-reporter, reverse transcription-polymerase chain reaction and immunoblot. The tumor growth suppression by RB1CC1 was evaluated by flow cytometry or by a cell growth assay. The nuclear RB1CC1 complex involving hSNF5 and/or p53 activated transcription of RB1, p16 and p21, and suppressed tumor cell growth. Furthermore, nuclear RB1CC1 expression significantly correlated with those of RB1 and p16 in breast cancer tissue in vivo, and the Ki-67 proliferation index was dependent on p53 as well as RB1CC1. The present study indicates that RB1CC1 together with hSNF5 and/or p53 enhances the RB1 pathway through transcriptional activation of RB1, p16 and p21. Evaluation of RB1CC1 expression combined with RB1 and p53 status is expected to provide useful information in clinical practice and future therapeutic strategies in breast cancer.

Rationale: Compensatory lung growth has been well described as a phenomenon in many animal models, but still little is known about the nature, extent, and modulation of such growth. We hypothesized that compensatory lung growth may at least in part recapitulate developmental lung growth, and factors known to be important during normal lung development, such as thyroid transcription factor 1 (TTF-1), may be reactivated during compensatory lung growth., Objectives: To investigate the role of TTF-1 in correlation with the morphological changes during compensatory lung growth., Methods: Sequential changes in TTF-1 expression and morphology were examined in the residual right lung after left pneumonectomy in 9-week-old mice. The effect of temporary knockdown of TTF-1 on compensatory lung growth was also evaluated., Measurements and Main Results: TTF-1 was transiently but significantly elevated at an early stage in compensatory lung growth. Morphologically, a process resembling septation in lung development may have been initiated during this period in the vicinity of the alveolar duct. furthermore, temporary knockdown of ttf-1 transiently but significantly delayed the early phase of compensatory lung growth., Conclusions: These results indicate the influential role of TTF-1 in modulating, and possibly initiating, the early phase of compensatory lung growth. Morphologically, compensatory lung growth may at least in part resemble developmental growth.

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored matrix metalloproteinase regulator, but its functions in cartilage are not fully understood. The aim of the present study was to examine the expression and functions of RECK in human osteoarthritic (OA) cartilage. Quantitative RT-PCR indicated that the expression level of RECK is significantly higher in OA cartilage than in normal cartilage. By immunohistochemical analysis, RECK was localized to chondrocytes in OA cartilage, and the immunoreactivity directly correlated with the Mankin score and degree of chondrocyte cloning and proliferation. In cultured OA chondrocytes, RECK was expressed on the cell surface by glycosylphosphatidylinositol anchoring. The expression was stimulated by insulin-like growth factor-1 and suppressed by interleukin-1 and tumor necrosis factor-alpha. Down-regulation of RECK by small interfering RNA showed reduced spreading and smaller focal adhesions in the chondrocytes. Chondrocyte migration in a monolayer wounding assay was increased by down-regulation of RECK and inhibited by RECK overexpression in an matrix metalloproteinase activity-dependent manner. On the other hand, chondrocyte proliferation was suppressed by RECK silencing, and this was associated with reduced phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, whereas the proliferation was enhanced by RECK overexpression. These data are the first to demonstrate that RECK is up-regulated in human OA cartilage and suggest that RECK plays a role in chondrocyte cloning probably through suppression and promotion of chondrocyte migration and proliferation, respectively.

Purpose: Testicular germ cell tumors (TGCT) have a unique epigenetic profile distinct from that of other types of cancer. Elucidation of these properties has a potential to identify novel markers for TGCTs., Experimental Design: We conducted comprehensive analysis of DNA methyltransferase (DNMT) gene expression in TGCTs. Based on the expression profiles of DNMT genes in TGCTs, we generated a rabbit polyclonal anti-human DNMT3L antibody. We then studied the role of DNMT3L in TGCTs by the treatment of two embryonal carcinoma (EC) cell lines with a small interfering RNA system. Finally, we evaluated the immunohistochemical detection of DNMT3L in TGCT tissues. We also compared the patterns of DNMT3L immunohistochemistry with those of CD30 and SOX2., Results: Among the DNMT genes, we found that mRNA for DNMT3L was specifically expressed in TGCTs, but neither in normal testicular tissues nor in cancer cells of somatic tissue origin. DNMT3L protein was strongly expressed in two EC cell lines, but not in the cell lines of somatic tissue origin. Transfection of small interfering RNA for DNMT3L significantly reduced DNMT3L expression and resulted in growth suppression and apoptosis in EC cells. Immunohistochemical analysis showed that DNMT3L protein was present only in EC cells, but not in the other types of TGCT components and cancer cells of somatic tissue origin. DNMT3L staining was more prominent and specific than CD30 or SOX2 staining for detecting EC cells., Conclusion: DNMT3L is a novel marker and is essential for the growth of human embryonal carcinoma., (Copyright (c) 2010 AACR.)

A hierarchical hologram works in both optical far-fields and near-fields, the former being associated with conventional holographic images, and the latter being associated with the optical intensity distribution based on a nanometric structure that is accessible only via optical near-fields. We propose embedding a nanophotonic code, which is retrievable via optical near-field interactions involving nanometric structures, within an embossed hologram. Due to the one-dimensional grid structure of the hologram, evident polarization dependence appears in retrieving the code. Here we describe the basic concepts, numerical simulations, and experimental results in fabrication of a prototype hierarchical hologram and describe its optical characterization.

Intrabronchial involvement by metastatic tumors from extrapulmonary primary sites is referred to as "endobronchial metastasis", which is an uncommon mode of metastasis. Although various primary sites (for example, breast, colon, and kidney) have been reported previously, endobronchial metastasis of hepatocellular carcinoma (HCC) is rare. Here, we report a case of a 71-year-old woman with hepatitis C-related HCC, in whom respiratory failure due to bilateral endobronchial metastasis occurred. Notably, long tree-like tumor shadows reminiscent of the structure of the bronchial tree were observed in the bilateral lungs on computed tomography scan, and postmortem analysis clearly revealed metastatic HCC growing and spreading extensively in the lumina of the bronchi. This growth pattern is in sharp contrast to the ordinary endobronchial metastasis, which involves a relatively short segment of the unilateral bronchus. This is the first case report of bilateral and extensive spreading of endobronchial metastatic HCC, and this phenomenon should be considered as a cause of dyspnea in advanced HCC patients., (Copyright 2009 Elsevier GmbH. All rights reserved.)

The composition of fatty acids in abdominal subcutaneous adipose tissue and the correlation of fatty acid values of plasma and erythrocytes had not been reported in Japan. The aim of the present study was to investigate the fatty acid composition and correlation of plasma and erythrocyte phospholipids (PL) and adipose triacylglycerols (TG) in 75 adult patients admitted for non-malignant diseases. We also examined the relationship of n-3 and n-6 polyunsaturated fatty acid (PUFA) with patients' characteristics. The total n-3 PUFA were 11.2, 11.8 and 1.9%, and the ratios of n-6/n-3 were 2.41, 1.87 and 8.20 in plasma and erythrocyte PL and adipose TG, respectively. There were the highest correlations for total n-3 PUFA and the n-6/n-3 ratio between plasma and erythrocyte PL and adipose TG. There was a positive correlation between n-3 PUFAs and age, but a negative correlation was found between n-6 PUFAs and age. There was no significant difference in the values of PUFAs in plasma and erythrocyte PL and adipose TG between men and women. The patients with cholesterol cholecystolithiasis showed a significantly lower proportion of eicosapentaenoic acid in plasma and erythrocyte PL than those of the other patients. Our findings suggest that PUFA in plasma and erythrocyte PL may be good biomarkers and more acceptable for studying participants than adipose TG.

An 82-year-old man underwent thoracoscopic upper lobectomy of the left lung for the treatment of the lung cancer. The major complication was asymptomatic chronic trifascicular block. During the surgery, after the upper lobe had been resected, second degree atrioventricular block (Morbitz type II) occurred unexpectedly, soon evolving in complete AV block, with pulse wave disappearing, indicating pulseless electrical activity. Immediately, we used an epicardial pacing wire, and spontaneous circulation returned. Postoperatively, a permanent pacemaker was implanted. Asymptomatic chronic bifascicular block and trifascicular block rarely progress into complete AV block during operation, which we should be prepared in advance. Accordingly in some cases, preoperative insertion of a temporary pacemaker should be considered as a preventive measure.

Although several cytokines and neurotrophic factors induce sympathetic neurons to transdifferentiate into cholinergic neurons in vitro, the physiological and pathophysiological roles of this remain unknown. During congestive heart failure (CHF), sympathetic neural tone is upregulated, but there is a paradoxical reduction in norepinephrine synthesis and reuptake in the cardiac sympathetic nervous system (SNS). Here we examined whether cholinergic transdifferentiation can occur in the cardiac SNS in rodent models of CHF and investigated the underlying molecular mechanism(s) using genetically modified mice. We used Dahl salt-sensitive rats to model CHF and found that, upon CHF induction, the cardiac SNS clearly acquired cholinergic characteristics. Of the various cholinergic differentiation factors, leukemia inhibitory factor (LIF) and cardiotrophin-1 were strongly upregulated in the ventricles of rats with CHF. Further, LIF and cardiotrophin-1 secreted from cultured failing rat cardiomyocytes induced cholinergic transdifferentiation in cultured sympathetic neurons, and this process was reversed by siRNAs targeting Lif and cardiotrophin-1. Consistent with the data in rats, heart-specific overexpression of LIF in mice caused cholinergic transdifferentiation in the cardiac SNS. Further, SNS-specific targeting of the gene encoding the gp130 subunit of the receptor for LIF and cardiotrophin-1 in mice prevented CHF-induced cholinergic transdifferentiation. Cholinergic transdifferentiation was also observed in the cardiac SNS of autopsied patients with CHF. Thus, CHF causes target-dependent cholinergic transdifferentiation of the cardiac SNS via gp130-signaling cytokines secreted from the failing myocardium.

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG), a decoy receptor of RANKL, maintain bone mass by regulating the differentiation of osteoclasts, which are bone-resorbing cells. Endochondral bone ossification and bone fracture healing involve cartilage resorption, a less well-understood process that is needed for replacement of cartilage by bone. Here we describe the role of OPG produced by chondrocytes in chondroclastogenesis. Fracture healing in OPG(-/-) mice showed faster union of the fractured bone, faster resorption of the cartilaginous callus, and an increased number of chondroclasts at the chondroosseous junctions compared with that in wild-type littermates. When a cultured pellet of OPG(-/-) chondrocytes was transplanted beneath the kidney capsule, the pellet recruited many chondroclasts. The pellet showed the ability to induce tartrate-resistant acid phosphatase-positive multinucleated cells from RAW 264.7 cells in vitro. Finally, OPG(-/-) chondrocytes (but not wild-type chondrocytes) cultured with spleen cells induced many tartrate-resistant acid phosphatase-positive multinucleated cells. The expression of RANKL and OPG in chondrocytes was regulated by several osteotropic factors including 1,25-dihydroxyvitamin D(3), PTHrP, IL-1alpha, and TNF-alpha. Thus, local OPG produced by chondrocytes probably controls cartilage resorption as a negative regulator for chondrocyte-dependent chondroclastogenesis.

Background: The aim of the current study was to determine if the slowed exercise oxygen uptake (VO(2)) kinetics, which is developed by myocardial ischemia, would be accompanied by delayed recovery VO(2) kinetics in patients with coronary artery disease (CAD)., Methods and Results: Thirty-seven patients with significant ST depression during treadmill exercise underwent cardiopulmonary exercise testing with cycle ergometer. Measurements performed are the ratios of change in increase in oxygen (O(2)) uptake relative to increase in work rate (DeltaVO(2)/DeltaWR) across anaerobic threshold (AT) and 1 mm ST depression point (ST-dep), the time constants of VO(2) during recovery (T(1/2) VO(2)), stress radio-isotope scintigraphy and coronary angiography. Patients were divided into CAD positive (CAD+) and CAD negative (CAD-) groups, based on coronary angiography. In CAD+, DeltaVO(2)/DeltaWR decreased above AT and ST-dep, in contrast to CAD- patients. The T(1/2) VO(2) in CAD+ (103.1 +/-13.0 s) was greater than that of CAD- (76.5 +/-8.7 s) and showed negative correlations to the ratios of DeltaVO(2)/DeltaWR across AT and ST-dep. These parameters improved in the patients who underwent coronary bypass surgery., Conclusions: Exercise and recovery VO(2) kinetics were slowed when myocardial ischemia was provoked by exercise. Measurement of exercise and recovery VO(2) kinetics improve the accuracy of the exercise electrocardiogram diagnosis of CAD.

The drs gene is an apoptosis-inducing tumor suppressor. Previously, we showed that drs contributes to the suppression of tumor formation by generating drs-knockout mice. In this study, by using drs KO mouse embryonic fibroblasts, we found that drs is involved in the autophagy regulation under low serum culture conditions. Both electron microscopy and GFP-LC3 analyses demonstrated that drs is involved in the maturation process of autophagy from autophagosomes to autolysosomes. In addition, drs could associate with Rab24 and the association between drs and Rab24 was enhanced during autophagy. Drs was also co-localized with Rab24 on punctuated structures during autophagy.

IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Ralpha (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-kappaB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-kappaB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) is a tumor suppressor implicated in the regulation of RB1 (retinoblastoma 1) expression. However, the molecular mechanism of RB1 regulation by RB1CC1 has not been elucidated. Here, we demonstrate that nuclear RB1CC1 binds to the RB1 promoter using chromatin immunoprecipitation assays with anti-RB1CC1 antibody. Luciferase assays with RB1 promoter reporter plasmids revealed that RB1CC1 activated the RB1 promoter through the 201 bp upstream GC-rich region (from the initiation ATG). Electrophoretic mobility shift assay and Western blot analysis supported RB1CC1 binding to the GC-rich region of the RB1 promoter. In addition, the C-terminus of RB1CC1 was required for nuclear localization and subsequent RB1 promoter activation. Furthermore, the expression levels of RB1CC1 and RB1 significantly correlated with in vivo breast cancer tissues as determined by immunohistochemical analysis. These data indicate that nuclear RB1CC1 directly activates the RB1 promoter to enhance RB1 expression in cancer cells. Evaluation of RB1CC1 in various types of human cancer tissues is expected to provide useful information for clinical practice and future therapeutic strategies.

VATER association represents a cluster of Vertebral, Anal, Tracheo-Esophageal, Radial and Renal malformations, and caudal regression syndrome is an entity consisting of a spectrum of congenital anomalies of lower spine and hips associated with genitourinary and lower limb defects. The concurrence of various malformations may be explained by a common defect in blastogenesis, but direct evidence is yet to be accumulated. Here, by the use of autofluorescence and the teratogenic effect of adriamycin, we demonstrated that adriamycin administered to eggs of White Leghorns distributes to the caudal portion of the embryo and foregut epithelium and induces caudal regression and tracheal and pulmonary agenesis. The induction of caudal regression syndrome-like anomaly was developmental stage and dose dependent. Embryos with caudal regression demonstrated tracheoesophageal anomalies, one of the defects included in VATER association. The stages at which anomalies were produced corresponded to that of human embryos between days 22 and 26 (Carnegie stages 10-11). In view of the antitumor activity of adriamycin by intercalating to double-stranded DNA of undifferentiated cells undergoing rapid cell division, it is possible that adriamycin had preferentially attacked cells in the caudal end where pronounced proliferation takes place during this narrow period of greatest susceptibility.

The patient was a 34-year-old man presenting with the right intra-scrotal painless mass. With a diagnosis of right intrascrotal tumor, the patient underwent right high orchiectomy. The pathological diagnosis of pleomorphic rhabdomyosarcoma arisen from the right spermatic cord was made. Computed tomography revealed a single metastasis in the para-vena cava lymph node. Systemic chemotherapy with vincristine, actinomycin D, plus cyclophosphamide (VAC therapy), and etoposide plus cisplatin (EP therapy) were made according to Intergroup Rhabdomyosarcoma Study (IRS)-IV Regimen 45. But the chemotherapy was ineffective and a retoroperitoneal lymphadenectomy (RPLND) was therefore performed. After 3 months following RPLND, the tumor relapsed in a pelvic lymph node involved in right ureter and ileocaecal valve. Resection of the tumor with ileocaecum was performed and then intraoperative radiotherapy (15 Gy) against the tumor bed was performed to ensure the curative effects. After his recovery, he received a total of 6 courses of systemic chemotherapy consisting of vincristin, ifosphamide, etoposide (IRS-IV Regimen 47). The patient was rigorously followed up for 42 months after the final chemotherapy, with no tumor recurrence.

Background: Despite the unstable structure of urinary albumin in kidney diseases, urinary albumin fragments have been identified by denaturing methods such as two-dimensional electrophoresis. This study examined the relationship between the structural heterogeneity of urinary albumin and protease effects., Methods: Urine samples from patients with glomerulonephritis (GN), cardiovascular diseases (CVD), and healthy subjects were analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), Western blot, diagonal 2-dimensional non-reducing/reducing (d2D) SDS PAGE, and albumin zymography., Results: The major band was monomer albumin in CVD and healthy subjects; however, 13 urinary albumin bands ranging from 55 to 172 kDa were identified by non-reducing SDS PAGE in GN. The results from d2D SDS PAGE showed urinary albumin polymerization between disulfide bridges, interactions with other proteins, and reduction induced degradation in GN patients. The results from albumin zymography showed that low-molecular mass forms of albumin did not necessarily correspond to high protease activity. Furthermore, concentrated healthy urine showed similar protease digestion as in GN without low-molecular mass of albumin., Conclusions: The molecular alterations observed cannot be explained only by urinary proteases. The specific alteration of urinary albumin molecules in GN can be attributed to different mechanisms to CVD.

Cytokine signaling via various transcription factors regulates receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-mediated osteoclast differentiation from monocyte/macrophage lineage cells involved in propagation and resolution of inflammatory bone destruction. Protein inhibitor of activated STAT3 (PIAS3) was initially identified as a molecule that inhibits DNA binding of STAT3 and regulates many transcription factors through distinct mechanisms. To analyze PIAS3 function in osteoclasts in vivo, we have generated transgenic mice in which PIAS3 is specifically expressed in the osteoclast lineage using the tartrate-resistant acid phosphatase (TRAP) gene promoter. PIAS3 transgenic mice showed an osteopetrotic phenotype due to impairment of osteoclast differentiation. Overexpression of PIAS3 in RAW264.7 cells suppressed RANKL-induced osteoclastogenesis by inhibiting the expression of c-Fos and NFATc1. Interestingly, PIAS3 inhibits the transcriptional activity of microphthalmia-associated transcription factor (MITF) independent of sumoylation. Down-regulation of PIAS3 markedly enhances RANKL-mediated osteoclastogenesis in RAW264.7 cells. Furthermore, overexpression of PIAS3 in mouse primary osteoblast (POB), down-regulates RANKL expression induced by interleukin-6 (IL-6) cytokine family, and inhibits osteoclast formation from bone marrow macrophages (BMMs) in vitro coculture system. Down-regulation of PIAS3 leads to the accelerated expression of RANKL in POB stimulated with IL-6 and soluble IL-6 receptor (sIL-6R). Taken together, our results clearly indicate that PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts.

The TNF-alpha converting enzyme (TACE/ADAM17) is involved in the proteolytic release of the ectodomain of diverse cell surface proteins with critical roles in development, immunity, and hematopoiesis. As the perinatal lethality of TACE-deficient mice has prevented an analysis of the roles of TACE in adult animals, we generated mice in which floxed Tace alleles were deleted by Cre recombinase driven by a Sox9 promoter. These mutant mice survived up to 9-10 mo, but exhibited severe growth retardation as well as skin defects and infertility. The analysis of the skeletal system revealed shorter long bones and prominent bone loss, characterized by an increase in osteoclast and osteoblast activity. In addition, these mice exhibited hypercellularity in the bone marrow and extramedullary hematopoiesis in the spleen and liver. Flow cytometric analysis of the bone marrow cells showed a sharp increase in granulopoiesis and in the population of c-Kit-1(+) Sca-1(+) lineage(-) cells, and a decrease in lymphopoiesis. Moreover, we found that serum levels of IL-17 and G-CSF were significantly elevated compared with control littermates. These findings indicate that TACE is associated with a regulation of IL-17 and G-CSF expression in vivo, and that the dysregulation in G-CSF production is causally related to both the osteoporosis-like phenotype and the defects in the hematopoietic system.

Background: Rupture of the chordae tendineae cordis (CTC) is a well-known cause of mitral regurgitation. Despite its importance, the mechanisms by which the CTC is protected and the cause of its rupture remain unknown. CTC is an avascular tissue. We investigated the molecular mechanisms underlying the avascularity of CTC and the correlation between avascularity and CTC rupture., Methods and Results: We found that tenomodulin, which is a recently isolated antiangiogenic factor, was expressed abundantly in the elastin-rich subendothelial outer layer of normal rodent, porcine, canine, and human CTC. Conditioned medium from cultured CTC interstitial cells strongly inhibited tube formation and mobilization of endothelial cells; these effects were partially inhibited by small-interfering RNA against tenomodulin. The immunohistochemical analysis was performed on 12 normal and 16 ruptured CTC obtained from the autopsy or surgical specimen. Interestingly, tenomodulin was locally absent in the ruptured areas of CTC, where abnormal vessel formation, strong expression of vascular endothelial growth factor-A and matrix metalloproteinases, and infiltration of inflammatory cells were observed, but not in the normal or nonruptured area. In anesthetized open-chest dogs, the tenomodulin layer of tricuspid CTC was surgically filed, and immunohistological analysis was performed after several months. This intervention gradually caused angiogenesis and expression of vascular endothelial growth factor-A and matrix metalloproteinases in the core collagen layer in a time-dependent manner., Conclusions: These findings provide evidence that tenomodulin is expressed universally in normal CTC in a concentric pattern and that local absence of tenomodulin, angiogenesis, and matrix metalloproteinase activation are associated with CTC rupture.

Amiodarone is an anti-arrhythmic drug for life-threatening tachycardia, but various adverse effects have been reported. Reported herein is an autopsy case of valvular heart disease, in a patient who developed a lung mass (1.5 cm in diameter) and proteinuria (2.76 g/day) after treatment with amiodarone for a long time. The lung mass was highly suspected to be lung cancer on CT and positron emission tomography, but histologically the lesion was composed of lymphoplasmacytic infiltrates in alveolar walls and intra-alveolar accumulation of foamy macrophages containing characteristic myelinoid bodies, indicating that it was an amiodarone-related lesion. In addition, the lung tissue had unevenly distributed hemosiderin deposition, and abnormally tortuous capillaries were seen in the mass and in heavily hemosiderotic lung portions outside the mass. In the kidneys, glomeruli had membrane spikes, prominent swelling of podocytes and subepithelial deposits, which were sometimes large and hump-like. Autoimmune diseases, viral hepatitis, malignant neoplasms or other diseases with a known relationship to membranous glomerulonephritis were not found. The present case highlights the possibility that differential diagnosis between an amiodarone-related pulmonary lesion and a neoplasm can be very difficult radiologically, and suggests that membranous glomerulonephritis might be another possible complication of amiodarone treatment.

The authors describe a very rare case of intravascular large B-cell lymphoma in a woman whose ruptured distal middle cerebral artery (MCA) aneurysms were filled with lymphoma cells. A 69-year-old woman who had undergone artificial graft replacement for an aortic aneurysm presented with transient left hemiparesis. Magnetic resonance imaging demonstrated a small fresh cerebral infarction in the right frontal lobe, although major cervical and cerebral arteries were shown to be intact on MR angiography. Antiplatelet and anticoagulation treatments commenced. On the 21st day after onset, the patient suffered a subarachnoid hemorrhage, and a digital subtraction angiogram revealed aneurysmal lesions in the distal MCA. Based on the histological examination of the resected aneurysms, proliferation of large B-cell lymphoma was identified in the dilated arterial lumen. On the 71st day after ischemic onset, intracranial hemorrhage recurred, and she died. Postmortem examination revealed similar lymphoma cells only in the intimal layer that had grown on the artificial graft, and it was decided that the patient had had intravascular large B-cell lymphoma. The preceding cerebral infarction was thought to be due to occlusion of the distal MCA by tumor embolus, which may be the initial pathological stage in aneurysm formation. For patients with incomprehensible ischemic cerebral stroke, neoplasm must be taken in consideration.

Between 2004 and 2007, we examined a total of 70 waterfowl. Forty of 51 (78.4%) mute swans (Cygnus olor) had amyloidosis. Amyloid deposits were detected in the spleen of 39 of 49 birds (79.6%), liver of 37 of 47 birds (78.7%), intestine of 38 of 50 birds (76.0%), pancreas of 30 of 42 birds (71.4%), kidney of 32 of 47 birds (68.1%), thyroid gland of 20 of 30 birds (66.7%), heart of 26 of 49 birds (53.1%), and lung of 5 of 45 birds (11.1%). In some birds, there was a globular pattern of amyloid deposition or infiltration of foreign-body giant cells around amyloid nodules in the spleen. Immunostaining with anti-AA antibody and Western blotting revealed that all were cases of AA amyloidosis. In sections treated with potassium permanganate, which removes Congo red stain, the green refringence under polarized light had mostly vanished. However, staining was not completely eliminated in some areas. Electron microscopy confirmed that the star-shaped amyloid fibrils were 10 nm in diameter and lacked branching. We also demonstrated amyloid bundles and star-shaped amyloid fibrils. A high percentage (96.3%) of mute swans had an inflammatory condition known as bumblefoot. Swans are useful model for studies of animals that have high amounts of amyloid. This research may help in the elucidation of the mechanism of amyloidogenesis in humans, and more research regarding amyloidosis in birds that are consumed as food is necessary.

Acceleration of amyloid deposition by administration of amyloid fibrils and transmissibility of disease have been reported for several types of amyloidosis. Reactive amyloidosis (AA) occurs in a wide variety of domestic animal species and is characterized by amyloid deposition mainly in spleen, liver, and kidneys. Because the visceral organs of domestic animals have traditionally been used in Asian cuisines, it is important to examine whether dietary ingestion of the organs themselves (rather than purified amyloid fibrils) accelerates AA amyloid deposition. Herein, we show that murine AA amyloidosis develops rapidly after intraperitoneal or oral administration of purified amyloid fibrils or homogenates of amyloid-laden bovine liver. The amyloidosis development in mice was dependent on the concentration of amyloid fibrils or amyloidotic liver homogenates. We found that experimental murine AA amyloidosis was accelerated by dietary ingestion of both purified amyloid fibrils and tissue homogenates that contain amyloid fibrils. We also investigated livers of beef cattle and food chickens to examine whether they contain amyloid-enhancing factor activity. By microscopic examination of hematoxylin and eosin- and Congo red-stained sections, no amyloid deposition was detected in these livers, and no effective activity for experimental induction of AA amyloidosis in mice was detected in homogenates of these livers.

Objective: ADAMs are a gene family of multifunctional proteins. We undertook this study to determine which ADAM species is up-regulated in osteoarthritic (OA) cartilage and to examine its pathobiologic function., Methods: Expression of the 13 different metalloproteinase-type ADAMs was screened by reverse transcription-polymerase chain reaction (PCR), and expression levels of prototype membrane-anchored ADAM-12 (ADAM-12m) were determined by real-time PCR. ADAM-12m expression in articular cartilage was examined by in situ hybridization, immunohistochemistry, and immunoblotting. Chondrocytes were used for functional analyses of ADAM-12m., Results: ADAM-12m was selectively expressed in 87% of OA cartilage, and the expression level was significantly higher in OA cartilage than in normal cartilage. In situ hybridization showed that OA chondrocytes were responsible for the expression. ADAM-12m was immunolocalized on the membranes of OA chondrocytes, and its immunoreactivity correlated directly with the Mankin score and with degrees of chondrocyte cloning and proliferation. Immunoblotting analysis of OA chondrocytes demonstrated an active form of ADAM-12m. ADAM-12m expression in OA chondrocytes was selectively enhanced by transforming growth factor beta (TGFbeta), which also induced chondrocyte proliferation and degradation of insulin-like growth factor binding protein 5 (IGFBP-5). TGFbeta-induced chondrocyte proliferation was inhibited by suppression of IGF-1 signaling. In addition, TGFbeta-induced chondrocyte proliferation, chondrocyte cloning in agarose gel culture, and digestion of IGFBP-5 were inhibited with ADAM inhibitor, anti-ADAM-12 antibody, and small interfering RNA for ADAM-12., Conclusion: These data suggest a novel function of ADAM-12m in chondrocyte proliferation and cloning in OA cartilage through enhanced bioavailability of IGF-1 from the IGF-1-IGFBP-5 complex by selective IGFBP-5 digestion.

Estrogen receptor-related receptors (ERRs) are orphan members of the nuclear receptor superfamily. A single AGGTCA sequence element preceded by three conserved nucleotides has been identified as a specific recognition motif of ERRs. Here we performed systematic analyses of target sequences on all three ERR subtypes, alpha, beta and gamma. In electrophoretic gel-mobility shift assay and transcriptional reporter assays, they exhibited similar patterns of recognition specificities, showing extremely broad ranges of target sequences. We searched a mouse promoter database for a gene carrying possible ERR-binding sequences. The Rb-1 inducible coiled-coil 1 (Rb1cc1) gene was found to contain two putative ERR binding elements, named response element (RE)-1 and RE-2, in the promoter region. In gene reporter assays, RE-2, but not RE-1, functioned as an effective cis-regulatory element for transactivation by ERRalpha in the presence of a coactivator, peroxisome proliferator-activated receptor gamma coactivator-1alpha. Mutational analyses suggested that RE-2 is recognized by ERRalpha partly as a monovalent element, but also as a direct repeat motif separated by four spacer nucleotides. In vivo binding of ERRalpha to the Rb1cc1 promoter region was confirmed by the chromatin immunoprecipitation assay. Thus, Rb1cc1 is a target gene of ERRalpha, driven by a novel type of recognition sequence.

The role of matrix metalloproteinases in disease has been investigated over the last two decades. A focus on this family of proteases is particularly emphasized in two major arthritides in humans, osteoarthritis and rheumatoid arthritis. Early work described the presence of multiple MMP family members in the joint of the disease state and recent advances in the development of new knockout mice and disease models have allowed investigators to directly test the role of the MMP proteases in arthritis. MMP-13 is expressed by chondrocytes and synovial cells in human OA and RA and is thought to play a critical role in cartilage destruction. The recent development of an MMP-13 knockout mouse has documented the important role for this enzyme in cartilage formation and further studies under disease conditions promise to reveal the function of this enzyme in disease pathology. This review describes a body of research that supports the development of novel selective MMP-13 inhibitors with the hope of developing these compounds in clinical trials for the treatment of arthritis.

We have previously reported that the expression of periostin mRNA is significantly repressed in human bladder cancer tissues, and that periostin plays a role as a suppressive factor for invasion and metastasis in the progression of human bladder cancers. In this study, to clarify the role of alternative splicing of periostin in human bladder carcinogenesis, we examined the expression of wild-type (WT) and spliced variants of periostin mRNA in normal bladder and bladder cancer tissues. Although both WT and spliced periostin mRNA were expressed in all normal bladder tissues examined, no WT periostin mRNA was detected in the examined transitional cell carcinomas (TCCs) of the bladder (0/23) or in bladder cancer cell lines (0/6). Spliced variants of periostin were detected in 48% (11/23) of TCC tissues and 33% (2/6) of bladder cancer cell lines. Two types of spliced periostin (Variants I and II) were successfully isolated from bladder cancer tissues, but Variant I, which is predominantly expressed in bladder cancer tissues, did not show suppressor activity on in vitro invasiveness and in vivo metastasis of cancer cells. Immunohistochemical analysis indicated that strong belt-like expression of periostin protein was observed in the stroma just beneath the normal bladder epithelium, while it was mostly attenuated in bladder cancer tissues. These results indicate that the loss of WT periostin by down-regulation and/or alternative splicing, which produces Variant I, is closely correlated with the development of bladder cancer.

We report an autopsied 20-year-old man case of intestinal necrosis associated with megacolon from hypoganglionosis, a pseudo-Hirschsprung's disease. The patient had suffered from severe constipation since two years of age, and presented abdominal distention from age ten. Autopsy revealed marked dilatation and necrosis of the entire large intestine. Although ganglion cells in the intestinal plexus were found throughout the large intestine, their number was reduced to 12-20% of that in the normal control. In pseudo-Hirschsprung's disease, there are occasional cases where an acute abdomen first presents itself in adulthood after running its course as chronic constipation.

Purpose: We compared the negative chronotropic and inotropic effects of landiolol and esmolol, two clinically available short-acting beta1-blockers with high beta1-selectivity, using whole isolated rabbit heart preparations., Methods: Tachycardia was induced by continuous perfusion of 10(-7) M isoproterenol, and we used concentrations of landiolol or esmolol in ascending steps (1 . 10(-6), 3 . 10(-6), 1 . 10(-5), 3 . 10(-5), and 1 x 10(-4) M). Heart rate (HR), left ventricular developed pressure (LVDP), the maximal rates of left ventricular force development (LVdP/dt(max)), and myocardial oxygen consumption (MVO2) were measured and compared., Results: Both landiolol and esmolol produced dosedependent decreases in HR, LVDP, LVdP/dt(max), and MVO2. The HR lowering effects of the two agents were comparable. At concentrations of 3 . 10(-5) and 1 . 10(-4) M, esmolol produced more profound depression of LVDP (47 +/- 26 and 12 +/- 11 mmHg, respectively; mean +/- SD) and reduction of LVdP/dt(max) (650 +/- 287 and 120 +/- 103 mmHg x s(-1)) than landiolol (68 +/- 20 and 64 +/- 20 mmHg, and 897 +/- 236 and 852 +/- 240 mmHg.s(-1), respectively). At the same concentrations, esmolol caused more profound reduction in MVO(2) (40 +/- 11 and 35 +/- 10 microl x min(-1) x g(-1)) than landiolol (50 +/- 8 and 48 +/- 8 microl x min(-1) x g(-1)), respectively., Conclusion: Our results indicate that in the isolated rabbit heart, landiolol and esmolol had equipotent negative chronotropic effects, however, landiolol had a less potent negative inotropic effect than esmolol.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, 4, 5, 8, 9 and 15, members of the ADAMTS gene family, have the ability to degrade a major cartilage proteoglycan, aggrecan, at the specific sites, and thus are called 'aggrecanases'. The expression of these ADAMTS species was examined in human osteoarthritic articular cartilage on reverse transcription-polymerase chain reaction. The results demonstrated the predominant expression of ADAMTS4 in osteoarthritic cartilage, while ADAMTS5 was constitutively expressed in osteoarthritic and normal cartilage. ADAMTS9 was expressed mainly in normal cartilage, whereas no or negligible expression of ADAMTS1, 8 and 15 was observed in either osteoarthritic or normal cartilage. In situ hybridization for ADAMTS4 indicated that chondrocytes in osteoarthritic cartilage expressed the mRNA. Two monoclonal antibodies to ADAMTS4 were developed, and immunolocalized ADAMTS4 to chondrocytes in the proteoglycan-depleted zones of osteoarthritic cartilage, showing a direct correlation with the Mankin scores. Immunoblotting indicated a major protein band of 58 kDa in the chondrocyte culture media and osteoarthritic cartilage tissue homogenates. These data demonstrate that among the six ADAMTS species, ADAMTS4 is mainly expressed in an active form in osteoarthritic cartilage, and suggest that ADAMTS4 may play an important role in the degradation of aggrecan in human osteoarthritic cartilage.

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.

RB1-inducible Coiled-Coil 1 (RB1CC1) has been shown to be a novel tumor suppressor regulating RB1 expression. Neuronal abundance of RB1CC1 is reported to contribute to the non-proliferating enlarged cell phenotype through the maintenance of RB1 and mTOR. To clarify whether RB1CC1 insufficiency is involved in neuronal atrophy and Alzheimer's pathology, we investigated modifications of RB1CC1 as a possible cause of atrophy or death through the disturbance of mTOR signaling in Neuro-2a neuroblastoma cells. We also evaluated the correlation between RB1CC1 and mTOR signaling in a series of Alzheimer's brain tissues. Though RB1CC1 introduction enhanced neurite growth, RNAi-mediated knockdown of RB1CC1 or rapamycin treatment caused neurite atrophy and apoptosis due to mTOR signaling reduction in the differentiated Neuro-2a cells. Both TSC1 and RB1CC1 were equally functional and maintained mTOR signaling, indicated by phospho-S6 (Ser240/244) expression in 69% of Alzheimer's (9/13 cases) and 100% of normal brains (6/6 cases). However, scanty RB1CC1 expression, less than TSC1, caused phospho-S6 disappearance in 31% of Alzheimer's tissues (4/13 cases). These findings suggest that RB1CC1 insufficiency may result in mTOR signaling repression through unbalanced TSC1 abundance and may induce neuronal atrophy. These observations may have implications for the pathogenesis of Alzheimer's disease.

TNF-alpha, a potent proinflammatory cytokine, is synthesized as a membrane-anchored precursor and proteolytically released from cells. Soluble TNF is the primary mediator of pathologies such as rheumatoid arthritis, Crohn's disease, and endotoxin shock. The TNF-alpha converting enzyme (TACE), a disintegrin and metalloprotease 17 (ADAM17), has emerged as the best candidate TNF sheddase, but other proteinases can also release TNF. Because TACE-deficient mice die shortly after birth, we generated conditional TACE-deficient mice to address whether TACE is the relevant sheddase for TNF in adult mice. In this study, we report that TACE inactivation in myeloid cells or temporal inactivation at 6 wk offers strong protection from endotoxin shock lethality in mice by preventing increased TNF serum levels. These findings corroborate that TACE is the major endotoxin-stimulated TNF sheddase in mouse myeloid cells in vivo, thereby further validating TACE as a principal target for the treatment of TNF-dependent pathologies.

The effect of triptolide, which is isolated from Tripterygium Wilfordii, on induction and development of murine AA amyloidosis was studied. In the first experiment, we examined the ability of triptolide to inhibit initiation of amyloidosis. Oral or intraperitoneal administration of 480 microg/kg/day triptolide inhibited splenic amyloid deposition in both rapid and chronic induction models of mouse AA amyloidosis. Moreover, serum amyloid A (SAA) and interleukin (IL)-6 levels were also suppressed remarkably. Triptolide also immediately decreased SAA levels and reduced the incidence of amyloidosis even under conditions of high SAA levels. In the second experiment, we evaluated the influence of triptolide on development and resorption of amyloid deposition. Amyloid deposition was induced in mice by 28 daily injections of casein. After splenic and hepatic biopsies to confirm the presence of amyloid deposits, the mice immediately started to receive daily injections of 480 microg/kg/day triptolide with or without casein. Treatment with triptolide for 35 days and 105 days prevented deposition of amyloid and promoted resorption of splenic amyloid deposits. In conclusion, we show for the first time that triptolide inhibits induction and development of experimental murine amyloidosis. These results suggest that through suppression of IL-6, triptolide can reduce production of SAA. Amyloid deposition is prevented when levels of the amyloid-forming precursor protein SAA are decreased significantly.

Peripherin is a type III neuronal intermediate filament and its expression is involved in the growth and development of the peripheral nervous systems. Peripherin expression has been reported in cutaneous endocrine carcinomas, but not in rectal well-differentiated endocrine neoplasms (carcinoid tumors). Well-differentiated endocrine neoplasms have been reported to have a relatively high incidence of metastasis, but there is no complete parameter for prediction of their malignant behavior. The aim of the present study was to clarify the expression of peripherin in human fetal and adult rectal mucosae and rectal well-differentiated endocrine neoplasms and the significance of peripherin expression in well-differentiated endocrine neoplasms. Expression of peripherin was studied immunohistochemically in 3 fetal hindgut mucosae, 10 non-neoplastic rectal mucosae, 12 rectal well-differentiated endocrine neoplasms without metastases, and 5 well-differentiated endocrine neoplasms with metastases. Adult rectal mucosal epithelial cells did not have peripherin. However, transient expression of peripherin was detected in the subgroup of epithelial cells of the fetal rectum. Peripherin was demonstrated in all of the rectal well-differentiated endocrine neoplasms without metastases (12/12 cases), but was absent from some of the well-differentiated endocrine neoplasms with metastases (3/5 cases). Constant expression of peripherin in rectal well-differentiated endocrine neoplasms without metastases (12/12) seems to reflect the phenotype of the subpopulation of epithelial cells confined to the fetal rectum. However, loss of its expression was observed in cases with metastases (3/5) and can be regarded as an additional parameter to predict the risk of metastasis in rectal well-differentiated endocrine neoplasms.

We previously identified GADD34 (growth arrest and DNA damage protein 34) by screening for genes involved in oncogenic-transformation and/or cellular senescence in Ras-transformed rat F2408 fibroblasts (7EJ-Ras), which exhibit anchorage-independent growth and do not senesce. In the current study, we found that transduction of 7EJ-Ras cells with a retroviral vector expressing GADD34 suppressed their proliferation. Furthermore, we observed that fibroblasts derived from GADD34-knockout mice (GADD34-KO MEFs) did not undergo senescence. Whereas the expression of p21 was decreased in GADD34 KO MEFs, its expression was rescued in these cells by ectopic expression of GADD34 by retroviral transduction. These findings suggest that GADD34 contributes to the regulation of p21 expression, and that it suppresses cellular proliferation through the induction of cellular senescence.

Background: We have experienced instability of hemodynamic state during off-pump coronary artery bypass (OPCAB), especially, circumflex (Cx) artery anastomosis. Although some reports have implied the efficacy of mirlinone in OPCAB anastomosis due to its characteristic inotropic effect without increasing myocardial oxygen consumption, we examined the effect of smaller doses of mirlinone during Cx anastomosis., Methods: Fourteen patients received milrinone (M group) continuously at a rate of 0.15-0.30 microg x kg(-1) min(-1) after sternotomy until the end of operation. Sixteen patients in the control group (C group) received saline. Norepinephrine was concomitantly administered for maintaining systolic blood pressure above 100 mmHg. We measured hemodynamic parameters during Cx anastomosis and postoperative myocardial isozymes, and examined arrhythmias., Results: In the M group mean pulmonary arterial pressure (MPAP) decreased significantly and cardiac index (CI) and SvO2 increased significantly, compared with C group. M group showed lower incidence of atrial fibrillation for 2 days., Conclusions: We conclude that low-dose milrinone has a good influence on intraoperative and postoperative managements of OPCAB surgery.

Comparative genomics is a promising approach for identifying regulatory elements governing the unique spatio-temporal expression patterns of morphogenetic genes. Conserved noncoding genomic sequences are candidate regulatory elements. Here we performed a survey for conserved noncoding elements (CNE) nested within the SALL1 gene; mutations in this gene result in the Townes-Brocks syndrome. A comparison of the genomic sequence between humans and chicken revealed five CNE. Genomic fragments corresponding to each CNE were inserted into reporter cassettes consisting of eGFP cDNA and a minimal promoter. These constructs were electroporated into chick embryos during gastrula, neurula, and pharyngula stages. Among the five CNE that were examined, one 443 bp CNE exhibited tissue-specific enhancer activity. At the neurula stage, the eGFP signal was visualized in the prosencephalon. At the pharyngula stage, the eGFP signal was confined within the anterior neural ridge, which represents one of the morphogenetic centers regulating the patterning of the anterior neural plate. This report identifies, for the first time, an enhancer element of SALL1.

It is known that amyloid-enhancing factor (AEF) shortens the preamyloid phase in experimentally induced AA amyloidosis in mice. Because it is reported that AEF serves as both a nidus and a template for amyloid formation, AA amyloidosis may have transmissibility by a prion-like mechanism. It has been shown that amyloid fibrils also have AEF activity, and amyloid fibrils with AEF activity were named fibril-amyloid enhancing factor (F-AEF). In this study, we investigated methods to inactivate the AEF activity. AEF was extracted from the thyroid gland obtained at autopsy of a patient with AA amyloidosis. Before injection into mice, AEF was treated with several methods for inactivation. Of all the tested treatments, 1 N NaOH, 0.1 N NaOH, and autoclaving consistently demonstrated complete inactivation of AEF. Heat treatment led to incomplete inactivation, but 0.01 N NaOH, 0.001 N NaOH, pepsin, trypsin, pronase, and proteinase K treatment had no effect on AEF activity. By analysis with transmission electron microscopy, the AEF preparation contains amyloid fibrils, and a change of ultrastructure was shown after 1 N NaOH, 0.1 N NaOH, and autoclaving treatment. Furthermore, immunoblotting of AEF with antihuman AA antibody revealed that the protein band was scarcely found after autoclaving, 1 N NaOH, and 0.1 N NaOH treatment. Our results suggest that, similar to Creutzfeldt-Jakob disease (CJD), amyloidosis may require chemical or autoclaving decontamination.

The purpose of the present paper was to investigate the heterogeneous nature of amyloid deposits in the liver, by immunohistochemical and immunochemical examination of liver samples from cases of immunoglobulin lambda light chain amyloidosis (Alambda amyloidosis) with antibodies generated against the peptides corresponding to the three different regions of the lambda light chain. Amyloid deposits in the hepatic artery tended to react better with anti-lambda(118-134) than with anti-lambda(159-175). Amyloid deposits in the space of Disse tended to react weakly or partially with anti-lambda(118-134) but well with anti-lambda(159-175). Amyloid deposits in the portal vein reacted relatively well with both antibodies. By western blotting of water-extracted amyloid in which amyloid deposits were not stained with anti-lambda(118-134) immunohistochemically, the three antibodies detected 27 kDa bands consistent with the full-length Ig lambda chain and some smaller bands. These findings indicate that amyloid deposits may not be homogeneous in the liver of AL amyloidosis, and that molecular heterogeneity of amyloid fibril protein or a difference in the mode of deposition results in the histopathological heterogeneity of AL amyloid deposits even within a single patient.