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Cluster of differentiation 81 (CD81) is a tetraspanin membrane protein consisting of 4 transmembrane domains and 2 outer membrane loops. CD81 inhibition is a potential treatment for rheumatoid arthritis (RA). Here, we investigated the therapeutic effects of the cytoplasmic RNA vector expressing anti-CD81 antibodies (the anti-CD81 vector) on the ankle joint synovium in collagen-induced arthritis (CIA) rats.Body weight, paw volume, and clinical scores were measured on days 0, 7, and 10 and daily thereafter. On day 28, the ankle joints of the rats were removed and stained with haematoxylin, eosin, and Safranin O. Arthritic changes such as inflammatory cell infiltration, synovial proliferation, articular cartilage destruction, and bone erosion were evaluated by histological scoring.Symptom onset was delayed in the right lower limbs of the rats administered the cytoplasmic RNA vector (CIA + anti-CD81) compared with that in the control group (CIA + control). The CIA + anti-CD81 rats were heavier than the CIA + control rats. The paw volume and clinical scores were significantly lower in the CIA + anti-CD81 than in the CIA + control. The histological scores indicated significantly milder manifestations of RA in the CIA + anti-CD81 than in the CIA + control.Administration of the cytoplasmic RNA vector expressing anti-CD81 antibodies suppressed arthritis and joint destruction in CIA rats. Our findings suggest that the cytoplasmic RNA vector can be used to treat RA.

Purpose CD81 is a member of the tetraspanin family of membrane proteins. Recently, it has been shown that CD81 may be involved in cancer cell proliferation and metastasis. As yet, however, there have been few reports on the expression and role of CD81 in osteosarcoma. Methods The expression of CD81 was investigated in human osteoblast cell line hFOB1.19 and in human osteosarcoma cell lines Saos2, MG63 and 143B. The expression of CD81 was inhibited in osteosarcoma cells using siRNA after which cell proliferation, migration and invasion were assessed. We also used Western blotting to investigate the phosphorylation status of Akt, Erk, JNK and p38, and measured the expression of MMP-2, MMP-9 and MT1-MMP. In addition, we used a CRISPR/Cas9 system to stably knock out CD81 expression in 143B cells, transplanted the cells into mice, and assessed tumor formation and lung metastasis in these mice compared to those in the control group. Results We found that CD81 was expressed in the human osteoblast cell line and in all osteosarcoma cell lines tested. The osteosarcoma cell line 143B exhibited a particularly high level of expression. In addition, we found that osteosarcoma cell proliferation, migration and invasion were decreased after CD81 inhibition, and that the phosphorylation of Akt and Erk was suppressed. Also, the expression levels of MMP-2, MMP-9 and MT1-MMP were found to be suppressed, with MMP-9 showing the greatest suppression. In vivo, we found that mice transplanted with CD81 knockout 143B cells exhibited significantly less tumor formation and lung metastasis than mice in the control group. Conclusion Based on our findings we conclude that inhibition of CD81 suppresses intracellular signaling and reduces tumorigenesis and lung metastasis in osteosarcoma cells.

In this article, the author introduces one interesting method to attract the attention of students considerably to classical music. That is a method to introduce quotation of music. Quotation of music means to quote a work by incorporating some of past composer's melodies or melodies of others to one's composing music. In some cases, it is done intentionally. Quotation is a kind of symbiosis of past composers and recent composers, and we can understand the intension of composers clearly by finding these quotations in the music.

When students of different cultures come together to learn and make music, a special bond is created, and a sense of community is established. Music is a universal language that unites people of all cultures. From March 9-17, 2018, two choirs from New York University, the Modern Madrigals and the Jazz Choir, joined students from Shujitsu University in Okayama, Japan to exchange ideas and make connections through music. The chapter illustrates how the students of NYU and Shujitsu University achieved kyosei goal by sharing the experience of singing.

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro.

Nitrogen-containing bisphosphonates (BPs) are antiresorptive drugs used for the treatment of metabolic bone diseases. Bone marrow stromal cells such as mesenchymal stem cells (MSCs) and MSC-derived osteoblasts that originate from MSCs are known to regulate osteoclast differentiation and activation via the expression of receptor activator of NF-κB ligand (RANKL). Although the effects of nitrogen-containing BPs on osteoclasts and osteoblasts have been well investigated, their effects in MSCs have not been clarified. In this study, we investigated the effects of risedronate (RIS), a nitrogen-containing BP, on osteoblast differentiation, RANKL expression and apoptosis in human and rat MSCs. RIS suppressed the formation of mineralized nodules and mRNA expression of differentiation marker genes such as bone sialoprotein and osteocalcin in MSC-derived osteoblasts. The RANKL expression induced by 1,25-(OH)(2) vitamin D(3) was not affected by RIS in human MSC-derived osteoblasts. In addition, treatment with high-concentration RIS induced chromatin condensation, an apoptosis feature, in MSCs. RIS-induced chromatin condensation was suppressed by a pan-caspase inhibitor zVAD-FMK and a cell-permeable isoprenoid analogue geranylgeraniol. These results indicate that RIS suppressed osteoblast differentiation and induced caspase- and isoprenoid depletion-dependent apoptosis and suggest that the antiresorptive effect of RIS is not mediated by a decrease in the RANKL expression in MSC-derived osteoblasts.

The multistage stacked VIA technology is one of the key technologies for the achievement of high-density packaging, but it appears that the influence of VIA stacking is not sufficiently well understood. The influence of one- to five-level VIA stacking technologies is studied with respect to the parameters of stress and strain from the viewpoint of reliability, comparing the conditions in which these VIAs are located directly on the RFP [Resin Filled PTH (Pin Through Hole)], and the condition in which they are located to the left and right at some distance from the RFP. The maximum stress occurs at the smallest VIA neck, and it is recommended that VIA stacking be designed at some distance from the RFP. Guidelines for optimized design of substrates that have stacked VIAs are provided. © 2010 Wiley Periodicals, Inc. Electron Comm Jpn, 93(11): 1–7, 2010; Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ecj.10320

We reported on a numerical analysis of the material properties that directly control the quality of the analytic results in our previous paper. In this paper, we focus on parameter optimization for the underfill in high density products such as consumer electronics devices. We report on our methodology to optimize the underfill properties through some parameter studies with numerical analysis.It is difficult to select the best underfill to be used for the high density packaging. In this study, some currently available underfills and a newly developed one are used in wafer level chip-sized package technology to compare the reliability of the packages against the stress and strain caused at both solder and underfill under thermal stress.

It is recognized strongly that the reliance of the material properties is extremely important. The material properties that we use in the numerical analysis, controls the quality of the analysis result directly. The best choice to use the correct properties is that we measure them by ourselves. However, it is sometimes difficult to get them by ourselves because of the problem such as expense, equipment and/or time.The reliance of the material properties used in the numerical analysis for the reliability assessment of high density packaging is studied, in this paper, especially focusing on the encapsulant.The result of a numerical analysis with the material properties, that the material supplier announced, did not support the result of the actual hardware testing. On the research process, the material properties were focused, and then I actually measured the material properties with the piece of the actual hardware parts which were constructing this product. As the result, we found the order difference between the material suppliers announced data and our measured data.With this established numerical model, the optimized parameter of the encapsulant has been studied.

Rheumatoid arthritis (RA) is a complex disease including autoimmune disorder and resulting in inflammation. It also shows progressive proliferation of synoviocytes, and these synoviocytes destroys articular structure. In order to understand the mechanism of this abnormal proliferation of RA-originated synoviocytes in molecular level, we analyzed the gene expression profiles by using DNA chips that contain more than 10,000 genes. Comparing the expression profiles of normal and RA-originated synoviocytes, we found several genes that are highly expressed in RA-originated synoviocytes than normal synoviocytes. Among these genes, we focused on one hypothetical protein, cDNA of which contains one reading frame in its DNA fragment, indicating that this fragment is a part of large mRNA structure. The expression of this gene in RA-originated synoviocytes is about three times higher than that in normal synoviocytes by DNA chip analysis. After cDNA cloning of this mRNA, we found that the gene is identical with lumican, which encodes a collagen binding, extracellular matrix protein. This mRNA was widely distributed in many tissues but its alternatively spliced forms are differently expressed in various tissues.

The reliance of the material properties on the numerical analysis has been studied, focusing the encapsulant of POP (Package on Package). The result of the numerical analysis with the material properties, that the material supplier had announced, did not support the result of the actual hardware testing. Through the research process, I had focused the material properties, and then, the material properties were measured actually with the hardware test piece of parts constructing this product. As the result, the order difference between the material supplier announced data and the measured data was observed. It was recognized strongly that the reliance of the material properties is extremely important, and became possible to study the optimized parameter of the encapsulant with the numerical model in which the measured data was applied.

Human bone marrow mesenchymal stem cells (hMSCs), one of the tissue stem cells, have the ability to differentiate into adipocytes, osteoblasts or chondrocytes, leading to the attempt to apply these stem cells to regeneration of bone and cartilage. To analyze characteristics of these cells and explain pluripotency of hMSCs, we analyzed gene expression profiles of hMSC clones by using DNA chips that contain over 10,000 genes per chip. We also analyzed their proliferation rates and cell surface markers. As a result, these hMSC clones showed various proliferation rates indicating that hMSCs contains multiple types of cells in their population. Furthermore, analysis of surface markers, especially analysis of CD34 and CD44 showed that these stem cells are not hematopoietic stem cells but mesenchymal origin. Profiling of gene expression by using DNA chip showed that several genes were specifically expressed in hMSCs, and the highest expression was observed in clone no.12 showing that this clone possesses unique characters as a MSC applicable to regeneration research and regenerative medicine.

When spinach thylakoids were subjected to moderate heat stress (40 degrees C for 30 min), oxygen evolution was inhibited, and cleavage of the reaction center-binding protein D1 of photosystem II took place, producing 23-kDa N-terminal fragments. The D1 cleavage was greatly facilitated by the addition of 0.15 mM ZnCl2 and 1 mM ATP and was completely inhibited by 1 mM EDTA, indicating the participation of an ATP-dependent metalloprotease(s) in the D1 cleavage. Herbicides 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, bromoxynil, and ioxynil, all of which bind to the Q(B) site, inhibited the D1 cleavage, suggesting that the DE-loop of the D1 protein is the heat-sensitive cleavage site. We solubilized the protease by treating the thylakoids with 2 M KSCN and detected a protease activity in the supernatant by gelatin activity gel electrophoresis in the 70-80-kDa region. The antibodies against tobacco FtsH and Arabidopsis FtsH2 reacted with a 70-80-kDa band of the KSCN-solubilized fraction, which suggests the presence of FtsH in the fraction. In accordance with this finding, we identified the homolog to Arabidopsis FtsH8 in the 70-80-kDa region by matrix-assisted laser desorption ionization time-of-flight mass analysis of the thylakoids. The KSCN-solubilized fraction was successively reconstituted with thylakoids to show heat-induced cleavage of the D1 protein and production of the D1 fragment. These results strongly suggest that an FtsH protease(s) is involved in the primary cleavage of the D1 protein under moderate heat stress.

Connective tissue growth factor (CTGF/CCN2), one of the most recently described growth factors, is produced by chondrocytes, vascular endothelial cells, and transforming growth factor (TGF)-beta-stimulated fibroblasts. CTGF was isolated from a chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and found to be normally expressed in cartilage tissues, especially in hypertrophic chondrocytes, and also to stimulate both the proliferation and the differentiation of chondrocytes in vitro. Therefore, CTGF is thought to be one of the most important regulators of endochondral ossification in vivo. Herein we describe the expression pattern of the ctgf gene in the calcifying tissues of normal developing mouse embryos in comparison with that in core binding factor a1 (Cbfa1)-targeted mutant (cbfa1-null) mouse embryos, in which impaired development and growth were characteristically observed in the skeletal system. After 15 days of development (E15), the expression of ctgf was detected in the zone of hypertrophy and provisional calcification, in which ossification proceeds toward the epiphysis during the skeletal development of the mouse embryo. Furthermore, ctgf was expressed in developing molar and incisal tooth germs around the perinatal stage. However, no expression of the gene was found in the cbfa1-null mouse embryos. These results indicate that CTGF may have certain important roles in the development of the calcifying tissues in the mouse embryo.

Background Connective tissue growth factor (CTGF) is known to play a direct role in fibrosis in various organs as a downstream mediator of TGF-β. Objective To evaluate a role in subepithelial fibrosis in the asthmatic airway, we investigated CTGF mRNA expression and CTGF producing cells in the airways of a murine asthma model with allergic inflammation. Methods After repetitive inhalation challenges with ovalbumin (OVA), cell numbers and TGF-β1 concentrations in bronchoalveolar lavage fluid from immunized mice were measured. Collagen deposition in lung tissue was estimated by measuring hydroxyproline content. CTGF mRNA and GAPDH mRNA levels were determined by quantitative RT-PCR method. Immunohistochemistry for CTGF with anti-CTGF antibody was performed. Results Numbers of eosinophils and TGF-β1 concentration increased markedly in BALF on the 7 th day and 14 th day after inhalation challenge with OVA. Hydroxyproline content in lung tissue increased significantly on the 14 th day after inhalation challenge of OVA compared to control. The ratio of CTGF mRNA /GAPDH mRNA in lung tissue in mice exposed to OVA increased 10-fold compared to those exposed to saline. Immunohistochemistry revealed that the number of CTGF-positive cells increased in bronchial submucosa after inhalation challenge of OVA. Conclusions Our results suggested that CTGF might be one of the potential molecules involved in subepithelial fibrosis in murine airways with allergic inflammation.

Objective The investigation of the expression and localization of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24/CCN family member 2 (CTGF/Hcs24/CCN2) in normal and osteoarthritic (OA) cartilage, and quantification of CTGF/Hcs24-positive cells. Methods Cartilage samples of patients ( n =20) with late stage OA were obtained at total joint replacement surgery. Morphologically normal cartilage was harvested for comparison purposes from the femoral heads of 6 other patients with femoral neck fracture. Paraffin-embedded sections were stained by Safranin O. The severity of the OA lesions was divided into four stages (normal, early, moderate, and severe). The localization of protein and mRNA for CTGF/Hcs24 was investigated by immunohistochemistry and in situ hybridization, respectively. The population of CTGF/Hcs24-positive chondrocytes in OA cartilage and chondro-osteophyte was quantified by counting the number of the cells under light microscopy. Results Signals for CTGF/Hcs24 were detected in a small percentage of chondrocytes throughout the layers of normal cartilage. In early stage OA cartilage, the CTGF/Hcs24-positive chondrocytes were localized mainly in the superficial layer. In moderate to severe OA cartilage, intense staining for CTGF/Hcs24 was observed in proliferating chondrocytes forming cell clusters next to the cartilage surface. In chondro-osteophyte, strong signals were found in the chondrocytes of the proliferative and hypertrophic zones. Conclusion CTGF/Hcs24 expression was detected in both normal and OA chondrocytes of human samples. The results of the current study suggested that expression of CTGF/Hcs24 was concomitant with development of OA lesions and chondrocyte differentiation in chondro-osteophyte.

Efforts to reduce the size and increase the density of electronics devices have been accelerated. As one solution, ACF (Anisotropic Conductive Film) has drawn attention for its capability in advanced high density packaging. Though the manufacturing process is simple, the problem of open and/or short circuits occurs and no detailed analysis on this problem has been reported. In this paper, the deformation of conductive particles at ACF connection is studied in detail with TEM (Transmission Electron Microscopy) . The results are discussed with the numerical analysis. It is pointed out that the optimization of such process parameters as temperature and pressure is important. The advantage of combining physical analysis and numerical analysis is emphasized.

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.

Healthy articular cartilage is thought to be maintained by the modulation of Cbfa1 expression, although little is currently known about Cbfa1 expression in such tissues. Therefore, we examined in vivo Cbfa1 transcript levels in the temporomandibular (TM) and knee joints of 3- and 10-week-old male ICR mice (weighing 50–70 g). A digoxigenin-11-UTP-labeled single-stranded RNA probe (0.6 kbp Pst I– Hin dIII fragment of the 3′ of untranslated region in exon 8 of mouse Cbfa1 cDNA) was prepared and in situ hybridization was performed on paraffin-embedded TM and knee joint sections. The antisense probe detected Cbfa1 transcripts in prehypertrophic chondrocytes, but not in the articular surface layer chondrocytes of 3- and 10-week-old mice TMJs. Despite the intense Cbfa1 expression in prehypertrophic chondrocytes, articular surface layer chondrocytes of the knee joints expressed low and undetectable level of Cbfa1 in the 3- and 10-week-old mice, respectively. These results indicate that Cbfa1 are highly expressed in the prehypertrophic chondrocytes presumably for articular tissue remodeling during the entire lifespan of the mouse, whereas Cbfa1 expression is suppressed in the articular surface chondrocytes in the adult mouse TM and knee joints to obtain the permanent cartilage phenotype.

Localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing in mouse ribs were investigated. In situ hybridization demonstrated that CTGF/Hcs24 mRNA was remarkably expressed, especially in hypertrophic chondrocytes and proliferating chondrocytes, in the regions of regenerating cartilage on days 8 and 14 after fracture. CTGF/Hcs24 mRNA was also expressed in proliferating periosteal cells in the vicinity of the fracture sites on days 2 and 8, and in cells in fibrous tissue around the callus on day 8. Northern blot analysis showed that expression of CTGF/Hcs24 mRNA was 3.9 times higher on day 2 of fracture healing than that on day 0. On day 8, it reached a peak of 8.6 times higher than that on day 0. It then declined to a lower level. Immunostaining showed that CTGF/Hcs24 was localized in hypertrophic chondrocytes and proliferating chondrocytes in the regions of regenerating cartilage, and in active osteoblasts in the regions of intramembranous ossification. Although CTGF/Hcs24 was abundant in the proliferating and differentiating cells (on days 8 and 14), immunostaining decreased as the cells differentiated to form bone (on day 20). CTGF/Hcs24 was also detected in cells in fibrous tissue, vascular endothelial cells in the callus, and periosteal cells around the fracture sites. These results suggest that CTGF/Hcs24 plays some role in fracture healing.

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTGF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage.

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here, we present the evidence that the hypoxic induction of angiogenesis by human breast cancer cells (MDA-231) can be ascribed at least in part to CTGF. Our results indicate that (i) CTGF is abundantly present in MDA-231 cells in vitro and in vivo, (ii) its secretion is up-regulated by hypoxia, and (iii) its gene expression is enhanced in MDA-231 cells cultured under hypoxic conditions. These data suggest CTGF may stimulate angiogenesis by paracrine mechanisms, thereby contributing to the invasion of breast cancer cells. This is the first evidence that human cancer cells differentially express CTGF protein and mRNA under the control of hypoxic conditions.

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24) promotes proliferation and differentiation of chondrocytes in culture. We investigated the roles of two major types of mitogen activated protein kinase (MAPK) in the promotion of proliferation and differentiation by CTGF/Hcs24. Here we report the effects of the MAPKK/MEK 1/2 inhibitor, PD098059, and p38 MAPK inhibitor, SB203580, in a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) and rabbit growth cartilage (RGC) cells treated with CTGF/Hcs24. In the proliferation phase, CTGF/Hcs24 induced a ≈ fivefold increase in the phosphorylation of p44/42 MAPK/ERK and a ≈ twofold increase in that of p38 MAPK in an in vivo kinase assay. These inhibitors of MAPKK and MAPK suppressed phosphorylation of ets-like gene-1 (Elk-1) and nuclear activating transcription factor-2 (Atf-2) induced by CTGF/Hcs24 in a dose-dependent manner, respectively. Western blot analysis showed that phosphorylation of ERK was induced from 30 to 60 min and phosphorylation of p38 MAPK from 10 to 15 min after the addition of CTGF/Hcs24 in confluence HCS-2/8 cells. PD098059 suppressed the DNA synthesis of HCS-2/8 cells and RGC cells, while SB203580 did not. On the other hand, the p38 MAPK inhibitor, SB203580, completely inhibited the CTGF/Hcs24-induced synthesis of proteoglycans in HCS-2/8 cells and RGC cells but the MEK1/2 inhibitor, PD098059, did not. These results suggest that ERK mediates the CTGF/Hcs24-induced proliferation of chondrocytes, and that p38 MAPK mediates the CTGF/Hcs24-induced differentiation of chondrocytes.

To investigate the lymphomagenesis of NK/T lymphoma, we comprehensively and systematically analyzed the expression pattern of the human NK/T cell line (NK-YS) genome by cDNA expression array and tissue microarray. We detected significant changes in the gene expression of NK-YS cell line: an increase in 18 and a decrease in 20 genes compared to normal NK cells or peripheral blood mononuclear cells. Among these genes, we found a strong decrease in hematopoietic cell specific protein-tyrosine-phosphatase SH-PTP1 (SHP1) mRNA by cDNA expression array and reverse transcriptase-polymerase chain reaction. Further analysis with standard immunohistochemistry and tissue microarray, which used 207 paraffin-embedded specimens of various kinds of malignant lymphomas, showed that 100% of NK/T lymphoma specimens and more than 95% of various types of malignant lymphoma were negative for SHP1 protein expression. On the other hand, SHP1 protein was strongly expressed in the mantle zone and interfollicular zone lymphocytes in reactive lymphoid hyperplasia specimens. In addition, various kinds of hematopoietic cell lines, particularly the highly aggressive lymphoma/leukemia lines, lacked SHP1 expression in vitro, suggesting that loss of SHP1 expression may be related to not only malignant transformation, but also tumor cell aggressiveness. SHP1 expression could not be induced in either of two NK/T cell lines by phorbol ester, suggesting that genetic impairment or modification with methylation of SHP1 DNA could be one of the critical events in the pathogenesis of NK/T lymphoma. This evidence strongly suggests that loss of SHP1 gene expression plays an important role in multistep tumorigenesis, possibly as an anti-oncogene in the wide range of lymphomas/leukemias as well as NK/T lymphomas.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are three representative neurotrophins responsible for the differentiation and survival of neurons, and their high-affinity receptors are tropomyosin-receptor-kinase (TRK)A, TRKB, and TRKC, respectively. In this study, we investigated the expression of neurotrophins in a mouse periodontal ligament cell line (MPL), by reverse transcription-polymerase chain-reaction (RT-PCR) and enzyme-linked immunoabsorbent assay (ELISA). We also studied the expression of TRK receptors on MPL by immunostaining and the effects of neurotrophins on the proliferation of MPL, with a hypothesis of autocrine mechanism of neurotrophins. Each neurotrophin and TRK receptor was expressed, and neurotrophins enhanced the proliferation of MPL. These findings suggest that the MPL has functional neurotrophin receptors involved in an autocrine function of neurotrophins. The expression level of neurotrophins and TRKs showed the reverse pattern, and we propose an auto-regulatory mechanism of ligands and receptors in accordance with the level of synthesized neurotrophins.

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for fibroblasts, chondrocytes, and vascular endothelial cells. In the present study, we established transgenic (Tg) mice that overproduce CTGF/Hcs24 under the control of mouse type XI collagen promoter. Tg mice could develop and their embryonic and neonatal growth occurred normally. But they showed dwarfism within a few months of birth. X-ray analysis revealed that their bone density was decreased compared with normal mice. The femurs in the hindlimbs in particular showed an apparent low density. These results indicated that overexpression of CTGF/Hcs24 affects certain steps of endochondral ossification. In addition, the testes were much smaller than normal and fertility was affected in Tg mice, indicating that CTGF/Hcs24 may also regulate the embryonic development of the testis.

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C504 and G505 in the cDNA sequences of both cells and C598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

BACKGROUND Connective tissue growth factor (CTGF) predominantly is expressed in hypertrophic chondrocytes and its specific receptors are demonstrated on chondrocytic cells. Therefore, CTGF may be involved in the proliferation and/or differentiation of cartilage cells. In the current study, CTGF expression was examined both in chondrosarcoma and enchondroma to clarify the relation between the expression of CTGF and the grade of malignancy. METHODS The expression of CTGF and proliferating cell nuclear antigen (PCNA) were analyzed immunohistochemically in 34 cartilaginous tumor specimens. Eighteen tumors were determined to be chondrosarcoma including 8 Grade 1 tumors, 6 Grade 2 tumors, and 4 Grade 3 tumors. The percentage of CTGF positive and PCNA positive cells was quantified using at least 500 cells. RESULTS CTGF was expressed in 70.1% of enchondroma cells, 84.0% of Grade 1 chondrosarcoma cells, 53.7% of Grade 2 tumor cells, and 26.8% of Grade 3 tumor cells (ρ = −0.501; P = 0.0053). In chondrosarcoma cases, CTGF expression was correlated closely with tumor grade (ρ = −0.920; P = 0.0001). There was a strong correlation between PCNA expression and tumor grade (ρ = 0.907; P < 0.0001) and a strong negative correlation between CTGF and PCNA expression (ρ = −0.493; P = 0.0061). In chondrosarcoma cases, patients with high expression of CTGF (≥ 30%) showed higher overall survival compared with those with low expression (< 30%) (P = 0.004). CONCLUSIONS The current study revealed a correlation between the histologic grade of chondrosarcoma and prognosis, and the concomitant association between CTGF immunostaining and tumor grade and prognosis. Therefore, immunohistochemical staining with CTGF is a useful procedure for assessing the tumor grade and clinical course in patients with chondrosarcoma. Cancer 2000;89:1466–73. © 2000 American Cancer Society.

Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 function in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs24 effectively promoted the proliferation of HCS-2/8 cells and RGC cells in a dose-dependent manner and also dose dependently increased proteoglycan synthesis in these cells. In addition, these stimulatory effects of CTGF/Hcs24 were neutralized by the addition of anti-CTGF antibodies. Furthermore, the recombinant CTGF/Hcs24 effectively increased alkaline phosphatase activity in RGC cells in culture. Moreover, RT-PCR analysis revealed that the recombinant CTGF/Hcs24 stimulated gene expression of aggrecan and collagen types II and X in RGC cells in culture. These results indicate that CTGF/Hcs24 directly promotes the proliferation and differentiation of chondrocytes.

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring β -galactosidase gene ( LacZ ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (Ax1CALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Ax1w) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl- β - D -galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the Ax1CALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint using the adenovirus vector is feasible as an effective in vivo method.

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies. (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process.

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3′-untranslated region (3′-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3′-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3′-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3′-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3′-UTR.

A relation between oral lichen planus (OLP) and oral prosthetic metals has been reported because OLP may improve after their removal. However, the criteria for evaluating their involvement in the clinical and histopathological signs of OLP have not been established.We compared the clinical and histological features of 18 cases of OLP that improved after removal of dental metals with those of 18 cases that did not improve clinically after removal. All patients were positive to dental metals on patch testing and had OLP refractory to conventional therapy. Improved cases of OLP were characterized by unilateral involvement of the buccal mucosa, showing a reticular pattern with the formation of erosions and ulcers.Histologically, most cases had remarkable humoral degeneration of the basal cell layer, lymphocytic invasion of the lamina propria, and spongiosis.

Previously, we cloned an mRNA predominantly expressed in hypertrophic chondrocytes by differential display-PCR from a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) that is identical to that of connective tissue growth factor (CTGF). In the present study, we investigated the roles of CTGF in the proliferation and migration of vascular endothelial cells using its antisense oligonucleotide and antisense RNA, because angiogenesis into the hypertrophic zone of cartilage occurs at the final step of endochondral ossification. Immunohistochemical and immunofluorescence techniques revealed that not only hypertrophic chondrocytes but also endothelial cells in the cost-chondral junctions of mouse ribs were stained with an anti-CTGF antibody in vivo. Northern blot analysis revealed that CTGF was strongly expressed in chondrocytic cells as well as bovine aorta endothelial (BAE) cells in culture, but not in other types of cells such as osteoblastic cells. Its expression in BAE cells was greater in the growing phase than in the confluent phase. When one-half of a monolayer of a confluent culture of BAE cells had been peeled off, only the cells proliferating and extending into the vacant area were stained with the anti-CTGF antibody. The addition of an antisense oligonucleotide inhibited the proliferation and extension of the BAE cells into the vacant area. The antisense oligonucleotide also inhibited the proliferation of BAE cells in the rapidly proliferating phase. In a Boyden chamber assay, pretreatment with the antisense oligonucleotide markedly inhibited the migration of BAE cells. Furthermore, the abilities to proliferate and migrate of BAE cells, which were stably transfected with expression vectors that generate the antisense RNA of CTGF cDNA, were markedly lower than those of the control. These findings suggest that endogenous CTGF expression is involved in the proliferation and migration of BAE cells.