Takeshi Hara

Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos.

BACKGROUNDnIn preimplantation mouse embryos, the first cell fate specification to the trophectoderm or inner cell mass occurs by the early blastocyst stage. The cell fate is controlled by cell position-dependent Hippo signaling, although the mechanisms underlying position-dependent Hippo signaling are unknown.nnnRESULTSnWe show that a combination of cell polarity and cell-cell adhesion establishes position-dependent Hippo signaling, where the outer and inner cells are polar and nonpolar, respectively. The junction-associated proteins angiomotin (Amot) and angiomotin-like 2 (Amotl2) are essential for Hippo pathway activation and appropriate cell fate specification. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs), and cell-cell adhesion activates the Hippo pathway. In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, thereby suppressing Hippo signaling. The N-terminal domain of Amot is required for actin binding, Nf2/Merlin-mediated association with the E-cadherin complex, and interaction with Lats protein kinase. In AJs, S176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding activity, thereby stabilizing the Amot-Lats interaction to activate the Hippo pathway.nnnCONCLUSIONSnWe propose that the phosphorylation of S176 in Amot is a critical step for activation of the Hippo pathway in AJs and that cell polarity disconnects the Hippo pathway from cell-cell adhesion by sequestering Amot from AJs. This mechanism converts positional information into differential Hippo signaling, thereby leading to differential cell fates.

VEGFR-2-specific ligand VEGF-E induces non-edematous hyper-vascularization in mice

VEGF family members play important roles in angiogenesis and vascular permeability. VEGF-A-transgenic mice showed an increased vascularization with edema due to hyper-vascular permeability and subcutaneous hemorrhage as side effects. VEGF-A binds and activates two receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). To dissect the signals of these two receptors, we generated transgenic mice overexpressing either the VEGFR-2-specific ligand VEGF-E(NZ-7) or VEGFR-1-specific ligand PlGF-II under the control of the Keratin-14 promoter. VEGF-E-mice showed a significant increase in vascularization (about 10-fold compared to control mice) in subcutaneous tissues, whereas PlGF-mice showed only a 2-3-fold increase. Interestingly, VEGF-E-mice did not show any clear edematous lesions or hemorrhagic spots on the skin. Microscopically, VEGF-E-induced capillary networks have a well organized structure with the recruitment of pericytes. These results indicate that VEGF-E is a new angiogenic agent with less side effects for clinical usage.

Long-term follow-up of initial 5-fluorouracil trabeculectomy in primary open-angle glaucoma in Japanese patients.

PurposeTo evaluate the long-term efficacy and safety of initial trabeculectomy with subconjunctival 5-fluorouracil in Japanese patients with primary open-angle glaucoma. Subjects and MethodsClinical records of 117 eyes from 117 patients with records of up to 14 years were retrospectively analyzed using the appropriate statistical methods. ResultsThe mean follow-up period was 6.2 ± 3.6 years. Criteria for successful intraocular pressure control and success probability by Kaplan-Meier method at 12 years were intraocular pressure < 21 mm Hg without medication plus a 30% or more reduction in intraocular pressure, 50.4 ± 5.2% (SE) 38.2 ± 5.3%; and intraocular pressure < 16 mm Hg without medication plus a 30% or more reduction in intraocular pressure, 45.9 ± 5.1% (36.5 ± 5.2%). Cox multiple regression analysis showed that a younger age was associated with a higher success probability (P <0.01). Eyes with lower postoperative intraocular pressure tended to show more positive value of the MD slope (P = 0.0669), whereas 7% of successfully treated eyes showed a negative postoperative MD slope (P < 0.1). The probabilities for developing bleb leaks and bleb-related infections in eyes with a functioning bleb were 28.9 ± 12.5% and 13.0 ± 10.4% at 12 years. ConclusionTrabeculectomy with 5-fluorouracil as an initial surgery in Japanese patients with primary open-angle glaucoma was effective for long-term control of glaucoma. However, the probability of late-onset bleb-related complications was not low in eyes with a functioning bleb, stressing the importance of constant care regarding bleb status.

Mouse Epidermal Keratinocytes in Three-Dimensional Organotypic Coculture with Dermal Fibroblasts Form a Stratified Sheet Resembling Skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1α and 1β in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.

Conditions for balance between lower normal pressure control and hypotony in mitomycin trabeculectomy

Abstractu2002· Background: Striving for low to ultra-low postoperative intraocular pressure (IOP) through filtering surgery usually increases the incidence of prolonged hypotony. Early postoperative prognostic indicators for IOP control to lower normal level and prolonged hypotony in trabeculectomy with mitomycin C were examined to determine conditions required to obtain an optimum balance between them.u2002· Methods: Records of initial trabeculectomy with mitomycin C (0.4 mg/ml, 3 min) in 59 consecutively operated Japanese patients with primary open-angle glaucoma who had used multiple medications before operation were retrospectively analyzed. IOP control to lower normal level was defined as having failed if IOP could not be maintained below 15 mm Hg without medication. Prolonged hypotony was defined as postoperative IOP below 5u2005mm Hg for more than 2 months, excluding the first month. The Cox proportional harzards model was applied to identify early postoperative factors contributing to the two surgical outcomes.u2002· Results: Among the factors studied, only the mean IOP on postoperative days 9–14 was significantly correlated with the surgical outcome. A receiver operating characteristic plot suggested that mean IOP of 8 mm Hg in this period would give an optimum balance between the two.u2002· Conclusion: IOP of 8 mm Hg 9–14 days after surgery may be advisable in patients with preoperative multiple medications in whom IOP control to lower normal level is attempted with mitomycin C trabeculectomy. Knowledge of this may helpful in deciding when laser lysis of sutures is indicated.

Fungal Proteolytic Enzymes: Part I. Isolation of two Kinds of Acid-proteases Excreted by Aspergillus niger var. macrosporus

Two kinds of proteolytic enzyme, tentatively named acid protease A and B which showed a single peak on electrophoresis individually, were isolated from the crude enzyme powder obtained from the broth filtrate cultured with Asper gillus niger var. macrosporus. Acid protease B is similar too the fungal acid protease previously reported, bccause the enzyme exhibits optimum activity on milk casein at about pH 2.6 and 55°C when the incubation was done at pH 2.6. Acid protease A is a new proteolytic enzyme, because the enzyme exhibits optimum activity on milk casein at about 2.0 and 70°C or 60°C when the incubation was done at pH 2.6 or 1.5 respectively.

Increased iris pigmentation after use of latanoprost in Japanese brown eyes

PURPOSEnTo evaluate the incidence of the increased iris pigmentation in Japanese with topical latanoprost.nnnMATERIAL AND METHODSnOne hundred and twelve patients who had homogeneous brown irises were studied. Age of the patients was 23 to 80(average 63.8 +/- 10.4) and follow-up periods were 6 to 12 (10.3 +/- 1.4) months. Slit lamp examination was carefully performed before use and at every month after use of latanoprost. Photographs were taken when necessary. The Kaplan-Meier life table method was used to evaluate the incidence of the increased iris pigmentation.nnnRESULTnIris pigment increase was observed in 47 patients. The incidence of the increased iris pigmentation was calculated as 10.1% at 3 months, 26.4% at 6 months, 50.1% at 9 months and 51.6% at 12 months after use. Pigmentation progressed as a granular concentric or sunrise pattern or a subtype of these. Some pigmentation occurred at one month after use, and progressed monthly. No patients noted their own iris color change, but were told by their relatives. Iris color changes of brown iris were considered to be less strong than in the hazel(green or blue/gray or yellow combined with brown) eye.

Diazepines. III. Synthesis of 4H-Pyrrolo[1,2-a] thieno[3,2-f] [1,4] diazepines†

The synthesis of 4H-pyrrolo[1,2-a]thieno[3,2-f] [1,4]diazepines (8) is described. Phthal-imidomethylfurans 1 were treated with bromine-methanol to give the dihydrofurans 2, which were hydrolyzed and then liydrogenated over Raney nickel or with zinc-acetic acid to afford the 1,4-diketones 5. Condensation of 2-amino-3-benzoylthiophenes 6 with 5 gave 3-benzoyl-2-pyrrolylthiopenes 7. The removal of the phthaloyl group from 7 with hydrazine hydrate and ring closure to the diazepine ring yielded the new heterocycles 8.

Ocular Tissue Concentrations of Mitomycin C With Variable Dose and Duration of Application Time in Rabbits

We measured mitomycin C (MMC) concentrations in ocular tissues in rabbits with variable dose (0.1, 0.2, or 0.4 mg) and duration of application time (1, 3, or 5 minutes) of MMC using high-performance liquid chromatography. Mitomycin C concentrations at the administered site after single subconjunctival application of MMC and after irrigation showed significant correlation with dose and duration of time of application. By multiple regression analysis, MMC concentrations (microg/g) at the conjunctiva were described as -6.73 + 67.4 x Dose (mg) + 1.66 x Time (minutes) (R2 0.65); at the sclera, -1.85 + 38.2 x Dose + 0.927 x Time (R2 0.63); at the cornea, -0.727 + 8.44 x Dose (R2 0.46). With a 0.2-mg MMC dose, in all three application times (1, 3, or 5 minutes), MMC concentrations in the conjunctiva at the administered quadrant were three times higher than in the neighboring quadrants and 6 to 7 times higher than in the opposite quadrant. In the sclera, MMC concentrations were 3.5 times higher than in the neighboring sites and over 8 to 9 times higher than in the opposite site. In the cornea, MMC concentrations were 2 to 3 times higher than in the neighboring sites and opposite site. In the iris-ciliary body, MMC concentrations were 0.61 microg/g at the administered site with 0.2 mg for 3-minute application, 2 times higher than in neighboring sites, and 2 times higher than in opposite sites.

Surgical revision of failed filtering bleb with mitomycin C

The revision of a failed filtering bleb using mitomycin C was performed in 40 eyes (28 eyes with primary open angle glaucoma and 12 eyes with secondary open angle glaucoma) in which the previous trabeculectomy using 5-fluorouracil had failed to control intraocular pressure (IOP). Presurgical IOP ranged from 21 approximately 36 mm Hg (average: 24.7 mmHg) with the maximum tolerable antiglaucoma medication. The follow-up period was at least 2 years (24 approximately 60, mean: 39.8 months). Mitomycin C (0.04%, 5 ml, 5 min) was applied under the subconjunctival space. After rinsing, the wall of the scarred bleb was incised in cases with a localized bleb, or the margin of the scleral flap was incised in cases without a filtering bleb. The conjunctival wound was closed with 10-0 Nylon sutures. IOP control under 21 mmHg was achieved in 53.4% without medication and in 60.2% with or without medication at 60 months. After repeating the same procedure, IOP control was obtained in 82.2% with or without medication at 60 months. The success rate in secondary open angle glaucoma was less than in primary open angle glaucoma. Complications such as flattening of the anterior chamber requiring reformation (8%), choroidal detachment (8%), hyphema (5%), and hypotonic maculopathy (5%) were less than after trabeculectomy but conjunctival wound separation (5%) occurred little more frequently.