Takashi Kanamoto

High frequency of open-angle glaucoma in Japanese patients with Alzheimer's disease

The clinical and genetic relationships between Alzheimers disease (AD) and glaucoma remain obscure. The aim of this study was to determine the prevalence of open-angle glaucoma (OAG) in patients with AD and whether the apolipoprotein E (APOE) 4 allele is associated with AD, with or without OAG, in Japanese. The groups consisted of 172 patients with the diagnostic criteria of AD and 176 age-matched controls. Ophthalmic examinations were conducted, and genomic analysis was performed by PCR and digestion of products with an enzyme. OAG was found in 41 (23.8%) of the AD patients, which was a significantly (p = 0.0002) higher prevalence than that in the controls (9.9%). Furthermore, there was no significant difference between intraocular pressures (IOPs) in AD patients with OAG and without OAG. The percentage of AD patients who carried an APOE epsilon4 allele (29.5%) was significantly (p = 0.0007) higher than that of the controls (9.1%). However, the percentage of AD patients with OAG who carried an APOE epsilon4 allele (35.7%) was not significantly different than that of AD patients without OAG (27.7%, p = 0.42). In summary, the prevalence of OAG is high in Japanese patients with AD, suggesting that common factors other than APOE may contribute to the two diseases.

The influence of skeletal maturity on allogenic synovial mesenchymal stem cell-based repair of cartilage in a large animal model.

One of the potential factors that may affect the results of mesenchymal stem cell (MSC)-based therapy is the age of donors and recipients. However, there have been no controlled studies to investigate the influence of skeletal maturity on the MSC-based repair of cartilage. The purpose of this study was to compare the repair quality of damaged articular cartilage treated by a scaffold-free three-dimensional tissue-engineered construct (TEC) derived from synovial MSCs between immature and mature pigs. Synovial MSCs were isolated from immature and mature pigs and the proliferation and chondrogenic differentiation capacities were compared. The TEC derived from the synovial MSCs were then implanted into equivalent chondral defects in the medial femoral condyle of both immature and mature pigs, respectively. The implanted defects were morphologically and biomechanically evaluated at 6 months postoperatively. There was no skeletal maturity-dependent difference in proliferation or chondrogenic differentiation capacity of the porcine synovial MSCs. The TEC derived from synovial MSCs promoted the repair of chondral lesion in both immature and mature pigs without the evidence of immune reaction. The repaired tissue by the TEC also exhibited similar viscoelastic properties to normal cartilage regardless of the skeletal maturity. The results of the present study not only suggest the feasibility of allogenic MSC-based cartilage repair over generations but also may validate the use of immature porcine model as clinically relevant to test the feasibility of synovial MSC-based therapies in chondral lesions.

TGFbeta1/Smad3 counteracts BRCA1-dependent repair of DNA damage.

Inactivation of the BRCA1 gene has been found to confer susceptibility to early-onset familial breast and ovarian cancers. BRCA1 regulates DNA repair, chromatin remodeling and affects gene transcription. Transforming growth factor-β (TGFβ) is a potent regulator of growth, apoptosis and invasiveness of tumor cells, including breast cancer cells. Here we show that Smad3 which is a component of the TGFβ signaling pathway, forms a complex with BRCA1 in vitro and in vivo. The interaction is mediated by the MH1 domain of Smad3 and the C-terminal part of BRCA1. We observed a co-localization of Smad3 and BRCA1 in nuclear complexes. We also found that TGFβ1/Smad3 counteracted BRCA1-dependent repair of DNA double-strand breaks in human breast epithelial cells, as evaluated by BRCA1 nuclear foci formation, single-cell gel electrophoresis and cell survival assays. Thus, TGFβ1/Smad3 suppresses BRCA1-dependent DNA repair in response to a DNA damaging agent.

Cloning and regulation of the vertebrate homologue of lin‐41 that functions as a heterochronic gene in Caenorhabditis elegans

The Caenorhabditis elegans gene lin‐41 is the one of the heterochronic genes that regulate the timing of many developmental events. MicroRNA let‐7 negatively regulates the expression of lin‐41 through RNA‐RNA interaction on its 3′ untranslated region (UTR). Here, we report the isolation of chick and mouse homologues of lin‐41 that encode the RBCC‐NHL family protein and their expression patterns. C. elegans lin‐41 is one of the RBCC‐NHL families and the predicted amino acid sequences of isolated two genes encode the same family proteins. Chick and mouse lin‐41 expression was also observed in developing limb buds, branchial arches, and tail buds. The 3′UTRs of the mouse and chick lin‐41 genes contain multiple let‐7 complementary sites. Using luciferase assay, we showed that lin‐41 expression can be regulated through let‐7 complementary sites. Developmental Dynamics 235:1142–1149, 2006.

MRI analysis of the attachment of the anteromedial and posterolateral bundles of anterior cruciate ligament using coronal oblique images

PurposeThe purpose of this study was to evaluate the course of the anteromedial bundle (AMB) and the posterolateral bundle (PLB) of the anterior cruciate ligament (ACL) with magnetic resonance imaging (MRI) in order to clarify the relationship between the bundles and surrounding anatomic landmarks.MethodsEighty-eight knees with intact ACLs were included in this study. MRI coronal oblique images were obtained with the knee in extension and used to assess the following characteristics of the AMB and PLB: (1) course of the ligament, (2) location of the tibial attachment, and (3) femoral attachment and geometry of the lateral femoral condyle inner wall.ResultsIn terms of the tibial attachment, the AMB was confluent with the apex of the medial intercondylar ridge (MIR) in all cases. Sixty-five PLBs (74%) inserted into the region between the apex and the slope of the MIR. The resident’s ridge was detected in 91% of the knees in the AMB image, whereas the ridge was clearly visualized in only 17% of the knees in the PLB image. A bony eminence was observed at the inner articular margin of the lateral femoral condyle in the PLB image.ConclusionIn terms of the tibial attachment, the AMB was confluent with the apex of the medial intercondylar ridge in all cases. Most of the PLBs attached to the region between the apex and the slope of the MIR. Because the bone tunnel location influences clearance between the grafts and the surrounding tissues, these results should be considered during anatomic double-bundle ACL reconstruction.Level of evidenceStudy of nonconsecutive patients without a universally applied gold standard, Level III.

BRCA2 and Smad3 synergize in regulation of gene transcription

Smad3 is an essential component in the intracellular signaling of transforming growth factor-β (TGFβ), which is a potent inhibitor of tumor cell proliferation. BRCA2 is a tumor suppressor involved in early onset of breast, ovarian and prostate cancer. Both Smad3 and BRCA2 possess transcription activation domains. Here, we show that Smad3 and BRCA2 interact functionally and physically. We found that BRCA2 forms a complex with Smad3 in vitro and in vivo, and that both MH1 and MH2 domains of Smad3 contribute to the interaction. TGFβ1 stimulates interaction of endogenous Smad3 and BRCA2 in non-transfected cells. BRCA2 co-activates Smad3-dependent transcriptional activation of luciferase reporter and expression of plasminogen activator inhibitor-1 (PAI-1). Smad3 increases the transcriptional activity of BRCA2 fused to the DNA-binding domain (DBD) of Gal4, and reciprocally, BRCA2 co-activates DBD-Gal4-Smad3. Thus, our results show that BRCA2 and Smad3 form a complex and synergize in regulation of transcription.

Isolation and characterization of a novel plasma membrane protein, osteoblast induction factor (obif), associated with osteoblast differentiation

BackgroundWhile several cell types are known to contribute to bone formation, the major player is a common bone matrix-secreting cell type, the osteoblast. Chondrocytes, which plays critical roles at several stages of endochondral ossification, and osteoblasts are derived from common precursors, and both intrinsic cues and signals from extrinsic cues play critical roles in the lineage decision of these cell types. Several studies have shown that cell fate commitment within the osteoblast lineage requires sequential, stage-specific signaling to promote osteoblastic differentiation programs. In osteoblastic differentiation, the functional mechanisms of transcriptional regulators have been well elucidated, however the exact roles of extrinsic molecules in osteoblastic differentiation are less clear.ResultsWe identify a novel gene, obif (osteoblast induction factor), encoding a transmembrane protein that is predominantly expressed in osteoblasts. During mouse development, obif is initially observed in the limb bud in a complementary pattern to Sox9 expression. Later in development, obif is highly expressed in osteoblasts at the stage of endochondral ossification. In cell line models, obif is up-regulated during osteoblastic differentiation. Exogenous obif expression stimulates osteoblastic differentiation and obif knockdown inhibits osteoblastic differentiation in preosteblastic MC3T3-E1 cells. In addition, the extracellular domain of obif protein exhibits functions similar to the full-length obif protein in induction of MC3T3-E1 differentiation.ConclusionsOur results suggest that obif plays a role in osteoblastic differentiation by acting as a ligand.

Platelet-derived growth factor receptor alpha is associated with oxidative stress-induced retinal cell death.

PURPOSE The purpose of this study was to investigate the role played by platelet-derived growth factor-α (PDGFRα) in oxidative stress-induced retinal cell death. A previous proteomic study from our laboratory showed that expression of PDGFRα is elevated considerably in the retinas of an animal model of glaucoma-the excitatory amino acid carrier (EAAC) 1-deficient (EAAC1-/-) mouse. METHODS Retinal sites and expression patterns of PDGFRα were determined by immunohistochemistry in the retinas of EAAC1-/- and control CRL:CD1(ICR) mice. A retinal cell line was exposed to hydrogen peroxide, and expression PDGFRα determined by Western blot analysis. Effects of PDGF-AA and PDGFRα-siRNA on hydrogen peroxide-induced retinal cell death were examined. RESULTS PDGFRα was detected in the retinal ganglion cell layer (RGL) of both EAAC1-/- and ICR mice, and was also localized in the internal nuclear layer (INL) of EAAC1-/- mice. While treatment with excess PDGF-AA had no additional effect on retinal cell death, expression of PDGFRα increased with exposure to hydrogen peroxide. Hydrogen peroxide-induced retinal cell death was inhibited by exposure to PDGF-AA via phosphatidylinositol 3 kinase (PI3K); cell death was promoted by PDGFRα-siRNA. CONCLUSIONS PDGFRα is expressed in mouse retina, where it is essential for retinal cell survival under conditions of oxidative stress.

Association study of genetic variants on chromosome 7q31 with susceptibility to normal tension glaucoma in a Japanese population

The caveolin 1 to caveolin 2 (CAV1–CAV2) gene region on chromosome 7q31 has been reported to be associated with susceptibility to primary open angle glaucoma (POAG) and normal tension glaucoma (NTG) in previous studies. We investigated whether genetic variants in the CAV1–CAV2 region are associated with NTG in Japanese patients. Two hundred and ninety-two Japanese patients with NTG and 352 Japanese healthy controls were recruited. We genotyped three single-nucleotide polymorphisms; that is, rs1052990, rs4236601, and rs7795356, in the CAV1–CAV2 gene region and assessed the allelic diversity among cases and controls. The frequency of the minor allele (G) of rs1052990 was significantly decreased in NTG cases compared with controls (P=0.014, OR=0.71), whereas NTG or POAG cases had a significantly higher frequency of the allele than controls in previous studies. Conversely, rs7795356 did not show any significant association with NTG cases, and rs4236601 was monomorphic in the Japanese study population. Our findings did not correspond with previous positive results, suggesting that CAV1–CAV2 variants studied in the present study are not important risk factors for NTG susceptibility in all populations. Further studies are needed to elucidate the possible contribution of the CAV1–CAV2 region to the development of glaucoma.

Evaluation of extraocular muscle enlargement in dysthyroid ophthalmopathy.

PURPOSE To evaluate the extraocular muscle enlargement in dysthyroid opthalmopathy. METHOD Extraocular muscle enlargement was assessed by orbital computed tomography (CT) in 573 patients with dysthyroid ophthalmopathy in order to investigate the frequency and exact location of extraocular muscle enlargement and the clinical features of related ocular symptoms in patients with dysthyroid ophthalmopathy. RESULTS AND CONCLUSION 187 patients (38% of the total) showed evidence of extraocular muscle enlargement on CT images, and this disorder were more frequent in male patients than in female patients. Extraocular muscle enlargement was also more frequently seen in older patients than in younger patients. Enlargement of a single muscle was found in 55% of the 187 patients, with the most frequently affected muscle being the inferior rectus muscle. When multiple muscles were enlarged, the inferior rectus muscle was the most frequently affected, followed by the medial rectus muscle. Of the clinical findings specific to dysthyroid ophthalmopathy examined in the present study, the incidence of exophthaomos, upper lid swelling, superior limbic keratoconjunctivitis, keratitis, diplopia, and dysthyroid optic neuropathy were significantly increased in patients with dysthyroid ophthalmopathy, indicating a close relationship between these findings and the underlying disease, but the incidence of upper lid retraction was not significantly increased.