Akihiko Tawara

Tip60 is regulated by circadian transcription factor clock and is involved in cisplatin resistance.

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

Nipradilol and timolol induce Foxo3a and peroxiredoxin 2 expression and protect trabecular meshwork cells from oxidative stress.

PURPOSE Oxidative stress plays an important role in pathogenesis of glaucoma. The purpose of this study is to investigate the novel effect of antiglaucoma drugs on the expression of antioxidant peroxiredoxins of trabecular meshwork (TM) cells. METHODS The expression of the peroxiredoxin family was investigated using immortalized TM cell lines. Cells were treated with antiglaucoma drugs and analyzed for the expression of peroxiredoxin, and cellular sensitivity to oxidative stress. Furthermore, the effect of antiglaucoma drugs on the molecular regulation of the expression of peroxiredoxin was examined using a reporter assay and siRNA strategy. RESULTS Glaucomatous TM cells highly express peroxiredoxin 2 when compared with normal TM cells. Nipradilol and timolol, but not latanoprost, induce the expression of peroxiredoxin 2 through the activation of the Foxo3a transcription factor. TM cells showed reduced sensitivity to H(2)O(2) when cells were treated with either nipradilol or timolol, but not with latanoprost. In addition, both Foxo3a and PRDX2 expression were enhanced by drug-induced signal transduction through its receptor. CONCLUSIONS These results indicate that both nipradilol and timolol possess a novel mechanism of action and function as potent protective agents against oxidative stress.

Immunohistochemical evaluation of the extracellular matrix in trabecular meshwork in steroid-induced glaucoma

BackgroundTo immunohistochemically examine the localization of the extracellular matrix (ECM) in the trabecular meshwork (TM) in steroid-induced glaucoma (SG) specimens.MethodsWe morphologically and immunohistochemically examined six trabecular tissues from three eyes with SG, two with primary open angle glaucoma (POAG), and one without glaucoma. For the morphological study, the ultra-microtome sections were observed using an electron microscope. For the light microscopic immunohistochemical analyses, frozen sections were stained by the avidin-biotin complex method using anti-type IV collagen, anti-type VI collagen, anti-heparan sulfate proteoglycan (HSPG), anti-fibronectin or anti-myocilin (MYOC) antibody.ResultsThe morphological examinations revealed accumulations of basement membrane-like and fine fibrillar-like materials in the outer TM of SG specimen. Type IV collagen, HSPG and fibronectin antibodies in SG specimens showed a greater degree of staining in the outer TM in comparison to the POAG and non-glaucomatous specimens; in contrast, the other antibodies including the type VI collagen and MYOC, did not.ConclusionsThe localization of ECMs in the TM is different in SG in comparison to that in POAG patients.

The tolerance range of binocular disparity on a 3D display based on the physiological characteristics of ocular accommodation

Abstract This study investigates the permissible value of horizontal binocular disparity when gazing at a three-dimensional (3D) display based on ocular accommodation function. For the closely perceived image on the 3D display screen, the crossed disparity of +1.0° and of +0.5° were given and for the far image, which gave the image far away from the screen, the uncrossed disparities of −1.0° and of −0.5° were taken. The disparity was 0° when the image was displayed on the screen. When the disparity was +1.0° and the stereoscopic image had been perceived, the accommodative response became significantly larger in comparison to that at 0°. An accommodation lead was observed significantly at a disparity +1.0°. The tolerance of binocular disparity on the 3D display based on the physiological aspect of ocular accommodation is thus suggested to be less than +1.0°.

Mutations in the TSPAN12 Gene in Japanese Patients with Familial Exudative Vitreoretinopathy

PURPOSE To search for mutations in the TSPAN12 gene in 90 Japanese probands with familial exudative vitreoretinopathy (FEVR) and their family members and to determine the types and frequencies of the mutations. DESIGN Laboratory investigation and clinical case analyses. METHODS Direct sequencing after polymerase chain reaction of the coding exons of TSPAN12 was performed for 90 probands with FEVR and some of their family members. The clinical signs and symptoms that were characteristic of individuals with TSPAN12 mutations were determined. RESULTS Three families were found to carry 2 mutations in TSPAN12. One of these mutations was a new missense change, L245P, and the other was an already reported nonsense mutation, L140X, in 2 families. Mutations in TSPAN12 accounted for 3% of Japanese FEVR patients and 8% of the FEVR families who did not have mutations in any of the known FEVR genes, FZD4, LRP5, and NDP. The clinical signs and symptoms varied among the patients, but the retinal findings with TSPAN12 mutations were not different from those with mutations in the known FEVR-causing genes. CONCLUSIONS Mutant TSPAN12 is responsible for approximately 3% of FEVR patients in Japan. The results provide further evidence that mutations in TSPAN12 are FEVR causing and that the gene products most likely play a role in the development of retinal vessels.

Identification of Causative Pathogens in Eyes with Bacterial Conjunctivitis by Bacterial Cell Count and Microbiota Analysis

PURPOSE To determine the causative pathogens in eyes with bacterial conjunctivitis. DESIGN Evaluation of diagnostic test or technology. PARTICIPANTS Thirteen eyes diagnosed clinically with bacterial conjunctivitis and 12 eyes with normal conjunctival sac were studied. METHODS The bacterial cell numbers were counted in the samples stained by ethidium bromide (EtBr). The microbiota was determined by the clone library method using polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA (rRNA) gene with universal primers. In addition, examinations of smears and cultures of samples were performed. MAIN OUTCOME MEASURES Bacterial cell numbers determined by the EtBr staining method and microbiota analysis based on 16S rRNA gene of samples from eyes with bacterial conjunctivitis. RESULTS The bacterial cell numbers in the eyes with bacterial conjunctivitis were significantly higher than those in the normal conjunctival sacs. Ten of 13 samples from the eyes with bacterial conjunctivitis had positive PCR results. The remaining 3 samples and all 12 samples from the normal conjunctiva had negative PCR results. In 5 of the 10 PCR-positive samples, the predominant species accounted for 84.5% or more of each clone library. In the remaining 5 samples, the predominant and the second dominant species accounted for 27.4% to 56.3% and 19.0% to 26.8%, respectively, of each clone library. The number of detected species in the clone libraries was between 8 and 20 (average ± standard deviation, 7.5 ± 5.8). Bacteria were detected in 8 of the 10 bacterial conjunctivitis samples and in 5 of the 12 normal samples in the cultures. The number of species detected by cultures was 1 in the eyes with bacterial conjunctivitis and between 1 and 3 (mean ± standard deviation, 1.6 ± 0.9) in the normal conjunctiva. CONCLUSIONS These results showed that the bacterial cell number in a sample is a good method of determining bacterial conjunctivitis. The microbiota analysis detected a diverse group of microbiota in the eyes with bacterial conjunctivitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The combination of bacterial cell count and microbiota analysis is a good method for identifying the causative pathogens in cases of monomicrobial and polymicrobial conjunctivitis.

Angiographic Changes in Iris and Iridocorneal Angle Neovascularization After Intravitreal Bevacizumab Injection

OBJECTIVE To evaluate the effects of the intravitreal (IV) injection of bevacizumab on anterior segment neovascularization using anterior segment angiography. METHODS We observed 1 eye with iris and iridocorneal angle neovascularization and 3 with neovascular glaucoma from 4 patients with diabetic retinopathy in 3 eyes and central retinal vein occlusion in 1 eye. Two healthy eyes from 2 other patients served as control eyes. Three eyes, including 1 normal eye, were examined by iris angiography; the other eyes underwent iridocorneal angle angiography with fluorescein (FA) and indocyanine green (IA) using a Heidelberg Retina Angiograph 2. After angiography, 4 eyes with neovascularization were treated with IV bevacizumab (1.25 mg per 0.05 mL) and underwent angiography once more 4 to 6 days after treatment. RESULTS Iris angiography with indocyanine green revealed many iris vessels, but not dye leaking, in both normal and glaucomatous eyes, and the angiography with fluorescein showed intensive vessel leakage in the iris as well as iridocorneal angle neovascularization, but not in normal eyes. Angle angiography revealed vessel structures with indocyanine green and intensive leakage with fluorescein in the iris and showed iridocorneal angle neovascularization and neovascular glaucoma, whereas no vessel structures appeared with IA or FA in the normal eye. After IV bevacizumab injection in eyes with neovascularization, the vascular structure did not change with IA, but dye leakage remarkably decreased with FA in the iris and angle. However, newly formed vessels in the iris and iridocorneal angle seemed to disappear on slitlamp examination. CONCLUSION Intravitreal injection of bevacizumab effectively reduces vascular permeability, whereas newly formed vessels are still present in the iris and iridocorneal angle.

Trabecular meshwork in neovascular glaucoma eyes after the intravitreal injection of bevacizumab

Neovascular glaucoma (NVG) is a severe consequence of ocular ischaemic disease. The mechanism of intraocular pressure (IOP) elevation is considered to be the increased permeability of the newly formed vessels,1 angle closure by the peripheral anterior synechia and intertrabecular neovascular tissue.2 3 The intravitreal injection of bevacizumab (IVB) was reported to be effective in the regression of new vessels.4 This injection may provide us with sufficient time to treat these patients with retinal photocoagulation. In addition, it may also be used as an adjunctive therapy for a mitomycin C (MMC) trabeculectomy to treat NVG. It remains to be elucidated as to how bevacizumab exerts its effects on the neovascular tissue in NVG. This study was carried out to examine the trabecular meshwork of eyes with NVG following the IVB. A dose of 1.25 mg (0.05 ml) of IVB (Avastin 100 mg/4 ml, Roche, Reinach, Switzerland) was given in the superotemporal quadrant 4 mm posterior to the limbus of the affected eyes of three patients. The underlying diseases …

Neovascularization in the anterior segment of the rabbit eye by experimental anterior ischemia

Abstract.Purpose: To investigate the effects of anterior ischemia accompanied by neither retinal nor choroidal ischemia on the anterior segment of the eye. Methods: Both long posterior ciliary arteries in the right eye of 14 rabbits were directly cauterized with an electric coagulator. The eyes were enucleated 1, 2, 4, 7, 9 or 14 days after cauterization, then fixed with 4% paraformaldehyde. Semi-thin sections were studied by light microscopy. Several sections were stained with Griffoniasimplicifolia lectin, which bound specifically to mammalian vascular endothelium. Other specimens were examined immunohistochemically for vascular endothelial growth factor (VEGF) protein. The tissue specimens of the first postoperative day were studied for expression of VEGF mRNA by in situ hybridization. Results: Atrophy of the iris and ciliary body was seen after the second postoperative day. Corneal neovascularization appeared after 7 days. Neovascularization on the anterior surface of the iris and in the trabecular meshwork was detected after the ninth postoperative day. The proliferative tissues with newly formed vessels obstructed the iridocorneal angle 14 days after the treatment. There was no histological change in either the retina or choroid. Immunohistochemically, VEGF protein was detected in the epithelial and vascular cells of the iris on the first and fourth postoperative day. Expression of VEGF mRNA was detected in the epithelial cells of the ciliary body on the day following the treatment. Conclusions: Anterior segment ischemia, when unaccompanied by retinal ischemia, causes neovascularization in the cornea, iris and trabecular tissue.

A classic temporal optic disc pit showing progression in the corresponding optic nerve fiber and visual field defects.

PurposeWe report a rare case with a classic temporal optic disc pit (ODP) showing the progression of an associated nerve fiber layer defect (NFLD) with the corresponding visual field defect (VFD).MethodsWe describe the patient’s medical records and review the pertinent literature.ResultsA 54-year-old woman had a temporal ODP, which was considered to be congenital, associated with both NFLD expanded to both the upper and lower sides of the horizontal line and corresponding VFD and a small ODP-like excavation at the 6.5 o’clock position on the disc edge with a narrow NFLD OD. During the 5-year follow-up period, both the NFLD and VFD associated with the temporal pit progressed without serous retinal detachment. The small ODP-like excavation located at the 6.5 o’clock position also showed progressive NFLD in the temporal lower quadrant with advanced VFD, which suggested that the excavation might be associated with glaucoma.ConclusionBased on the observation that the VFD occupied the two temporal quadrants with no step, an NFLD with corresponding VFD associated with a classic temporal ODP, although not considered to be related to glaucoma, can progress.