Angiopoietin 1 inhibits ocular neovascularization and breakdown of the blood-retinal barrier.

Several retinal and choroidal diseases are potentially treatable by intraocular delivery of genes whose products may counter or neutralize abnormal gene expression that occurs as part of the diseases. However, prior to considering a transgene, it is necessary to thoroughly investigate the effects of its expression in normal and diseased eyes. An efficient way to do this is to combine tissue-specific promoters with inducible promoter systems in transgenic mice. In this study, we used this approach to evaluate the effects of ectopic expression of angiopoietin-1 (Ang1) in normal eyes and those with ocular neovascularization. Adult mice with induced expression of Ang1 ubiquitously, or specifically in the retina, appeared normal and had no identifiable changes in retinal or choroidal blood vessels or in retinal function as assessed by electroretinography. Increased expression of Ang1 in eyes with severe retinal ischemia or in eyes with rupture of Bruch's membrane significantly suppressed the development of retinal or choroidal neovascularization, respectively. This inhibition of ocular neovascularization is particularly interesting and noteworthy, because overexpression of Ang1 in skin stimulates neovascularization. Ang1 also significantly reduced VEGF-induced retinal vascular permeability. These data suggest that intraocular delivery of ang1 has potential for treatment of ocular neovascularization and macular edema.

In vitro inhibitory effects of J-113397 on nociceptin/orphanin FQ-stimulated.

J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) is a recently developed antagonist of the opioid receptor-like 1 (ORL1) receptor. We compared the in vitro functional profile J-113397 on [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTPgammaS) binding to mouse brain with that of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 and naloxone benzoylhydrazone (NalBzoH). J-113397 antagonized nociceptin/orphanin FQ-stimulated [35S]GTPgammaS binding to mouse brain with an IC50 value of 7.6 nM, but had no effect on basal [35S]GTPgammaS binding by itself. [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 partially antagonized nociceptin/orphanin FQ-stimulated [35S]GTPgammaS binding but showed agonistic activity on ORL1 by itself. NalBzoH showed antagonistic activity on ORL1 receptor but had significant agonistic activity on other opioid receptors at lower doses. Schild plot analysis demonstrated competitive antagonism of J-113397 on ORL1 receptor in mouse brain. A [35S]GTPgammaS binding study using ORL1 receptor-deficient mice confirmed the selective antagonism of J-113397 on ORL1 receptor. These data indicate that J-113397 is the most potent and selective antagonist of ORL1 receptor in mouse brain that has yet been reported, and therefore will be a useful tool for characterization of ORL1 receptors in the brain.

A novel and efficient methodology for the C-C bond forming radical cyclization of hydrophobic substrates in water.

[reaction: see text]. The combination of water-soluble radical initiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane] (VA-061), water-soluble chain carrier 1-ethylpiperidine hypophosphite (EPHP), and surfactant cetyltrimethylammonium bromide (CTAB) was found to be the most suitable condition for effective radical cyclization in water for a variety of hydrophobic substrates.

Facile and efficient sulfenylation method using quinone mono-O,S-acetals under mild conditions.

A novel sulfenylation method induced by aromatization of quinone mono-O,S-acetals is described. These sulfenylation reagents readily react with silyl enolethers or electron rich aromatic compounds to give sulfenylation products under mild conditions. In particular, O,S-acetal 2j, which possesses a pentafluorophenylthio function, is the most effective reagent from the standpoint of the adaptability for various substrates.

Regioselective nucleophilic addition of methoxybenzene derivatives to the beta-carbon of p-benzoquinone mono O,S-acetal.

Regioselective nucleophilic addition of electron rich aromatics to the beta-position of acetal carbon of p-benzoquinone mono O,S-acetal was achieved by modifying the acetal moiety.

Cataractogenesis in neonatal Sprague-Dawley rats by N-methyl-N-nitrosourea.

Cataract was induced by a single intraperitoneal injection of 100 mg/kg N-methyl-N-nitrosourea (MNU) to 0-, 5-, 10-, 15-, or 20-day-old male and female Sprague-Dawley rats. In day 0, 5, 10, and 15 MNU-treated rats, mature cataracts were constantly seen 7, 14, 14, and 30 days after dosing, respectively. In the day 20 MNU-treated rats, only subcapsular cataract was seen 30 days after dosing. Therefore, the rats exposed to MNU at an earlier age caused cataract more rapidly and severely. In the day 0 MNU-treated rats, 7-methyldeoxyguanosine DNA adduct was detected in the lens epithelial nuclei 12 hours after MNU dosing, followed by apoptosis, which was confirmed by morphology, by TUNEL signals, and by DNA ladder and peaked 3 days after MNU dosing. In the apoptosis cascade, upregulation of Bax, downregulation of Bcl-2, and increased CPP32 protease (caspase-3) activity were seen 12 hours after MNU dosing. Therefore, the pathogenesis of MNU-induced cataract was associated with DNA adduct formation in the lens epithelial cell nuclei leading to apoptosis by upregulation of Bax protein, downmodulation of Bcl-2 protein, and activation of caspase-3.

In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist.

1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl -1, 3-dihydro-2H-benzimidazol-2-one (J-113397) was found to be the first potent nonpeptidyl ORL1 receptor antagonist (K(i): cloned human ORL1=1.8 nM) with high selectivity over other opioid receptors (K(i): 1000 nM for human mu-opioid receptor, >10,000 nM for human delta-opioid receptor, and 640 nM for human kappa-opioid receptor). In vitro, J-113397 inhibited nociceptin/orphanin FQ-stimulated [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) binding to Chinese Hamster Ovary (CHO) cells expressing ORL1 (CHO-ORL1) with an IC(50) value of 5.3 nM but had no effect on [35S]GTP gamma S binding by itself. Schild plot analysis of the [35S]GTP gamma S binding assay and cAMP assay using CHO-ORL1 indicated competitive antagonism of J-113397 on the ORL1 receptor. In CHO cells expressing mu-, delta- or kappa-opioid receptors, J-113397 had no effects on [35S]GTP gamma S binding up to a concentration of 100 nM, indicating selective antagonism of the compound on the ORL1 receptor. In vivo, J-113397, when administered subcutaneously (s.c.), dose-dependently inhibited hyperalgesia elicited by intracerebroventricular (i.c.v.) administration of nociceptin/orphanin FQ in a tail-flick test with mice. An in vitro binding study using mouse brains indicated that J-113397 possesses high affinity for the mouse ORL1 receptor (K(i): 1.1 nM) as well as the human receptor. In summary, J-113397 is the first potent, selective ORL1 receptor antagonist that may be useful in elucidating the physiological roles of nociceptin/orphanin FQ.

Time-specific occurrence of alopecia in neonatal C57BL mice treated with N-methyl-N-nitrosourea and the therapeutic efficacy of tacrolimus hydrate.

Alopecia was induced in male and female neonatal C57BL mice by a single intraperitoneal injection of 60 mg/kg N-methyl-N-nitrosourea (MNU). MNU administration was most effective in the 8-day-old mice and less effective in the 5-day-old mice (at active and early anagen stages of the first hair cycle, respectively). No alopecia was seen in the day 14 MNU-treated animals (at telogen stage of the first hair cycle). MNU effectively induced hair follicular cell apoptosis at the anagen stage by up-regulation of Bax protein without down-modulation of Bcl-2 protein. In day 8 MNU-treated mice, the immunosuppressive agent 0.01% tacrolimus hydrate (FK506), when topically applied for 5 days from 1 day after MNU treatment (before the occurrence of alopecia), decreased the severity of alopecia. However, it did not stimulate hair growth when applied for 5 days from 20 days of age (after occurrence of alopecia).

Mechanisms of photoreceptor cell apoptosis induced by N-methyl-N-nitrosourea in Sprague-Dawley rats.

A single intraperitoneal injection of 75 mg/kg N-methyl-N-nitrosourea (MNU) was given to 50-day-old female Sprague-Dawley rats and examined sequentially 12 and 24 hours, and 3 and 7 days after MNU treatment. Photoreceptor cell death was evoked in all treated rats. After MNU treatment, 7-methyldeoxyguanosine DNA adduct was detected selectively in photoreceptor cell nuclei at 12 hours, followed by photoreceptor cell apoptosis as confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling signals which peaked at 24 hours and continued until day 7 when several layers of photoreceptor cell nuclei were left. In apoptosis cascade, down-regulation of Bcl-2 was seen at 12 hours and up-regulation of Bax was seen at 24 hours, and caspase family (caspase 3/CPP32, caspase 6/Mch2, and caspase 8/FLICE protease) activities peaked 72 hours after MNU treatment. Therefore MNU-induced photoreceptor cell death was attributed to DNA adduct formation restricted to photoreceptor cell nuclei leading to photoreceptor cell apoptosis by up-regulation of Bax protein, down-modulation of Bcl-2 protein, and activation of caspases 3, 6, and 8.

[Angiographic findings and histological localization of indocyanine green in N-methyl-N-nitrosourea induced retinal degeneration in rats].

PURPOSE: We used N-methyl-N-nitrosourea (MNU) to induce chorioretinopathy as a model of retinitis pigmentosa, and compared the histological localization of indocyanine green (ICG) with ICG angiographic features. METHODS: Brown-Norway pigmented rats received a single intraperitoneal injection of MNU (75 mg/kg body weight). At 3 and 21 days after treatment, we compared ICG angiographic findings with histological localization of ICG in the retina and choroid. Histological localization of ICG was observed with an infrared light microscope. RESULTS: 3 days after treatment, destruction of the photoreceptor cells and photoreceptor segments had developed, and the retinal pigment epithelial cells (RPEs) were also damaged. In ICG angiography, diffuse hyperfluorescence was evident. In histological localization of ICG, RPEs were stained by ICG, and ICG was seen in the sensory retina through the damaged RPEs. At 21 days after treatment, the inner nuclear layer directly contact with the choroid. The photoreceptor cells, RPEs and choriocapillaris had disappeared. In ICG angiography, hypofluorescence was seen in the chorioretinal atrophic area. In histological localization of ICG, there was no ICG in the atrophic area, but ICG leaked from the remaining choriocapillaris into the neighboring sensory retina. CONCLUSION: These results support the precise interpretation of ICG angiographic findings in clinical use.

Molecular cloning of homologs of RAS and RHO1 genes from Cryptococcus neoformans.

We cloned and sequenced homologs of RAS(CnRAS) and RHO1(CnRHO1) genes from Cryptococcus neoformans. The proteins encoded by the CnRAS and CnRHO1 genes contained 216 and 197 amino acids, respectively. The deduced amino acid sequence of the CnRAS gene shared a high degree of sequence identity with the Ras proteins in other fungal species: Coprinus cinereus(76%), Lentinula edodes(74%), Saccharomyces cerevisiae RAS2(72%), and Schizosaccharomyces pombe(68%). The deduced amino acid sequence of the CnRHO1 gene shared a high degree of sequence identity with the Rho1 proteins in other fungal species: Candida albicans(78%), S. pombe(77%) and S. cerevisiae(76%). The deduced proteins contained GTP-binding and GTP-hydrolysis domains, and the prenylation site that are conserved among the small G protein superfamily. The synthetic peptides that contained the C-terminal amino acid sequence of the CnRas and CnRho1 proteins were geranylgeranylated.

Retinal degeneration induced by N-methyl-N-nitrosourea in Syrian golden hamsters.

BACKGROUND: The sequential retinal changes in Syrian golden hamsters induced by N-methyl-N-nitrosourea (MNU) have not been studied. METHODS: Female hamsters received a single intraperitoneal injection of 90 mg/kg MNU at 50 days of age, and the retina was examined light and electron microscopically, immunohistochemically and by the TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method until 20 weeks after the treatment. RESULTS: The retinal changes were as follows: (1) Photoreceptor apoptosis occurred 1 day after the treatment and resulted in photoreceptor loss at day 7. During the degeneration, Müller cell proliferation was conspicuous at day 5. (2) After the photoreceptor cell loss, migration of the pigment epithelial cells in all layers of the retina which were in contact with blood vessels occurred. Due to the Müller cell proliferation, gliosis was prominent at the later stage. CONCLUSIONS: The MNU injection caused photoreceptor apoptosis followed by pigment epithelial cell migration around the blood vessels, accompanied by gliosis. The primary event and the course of this disease closely resemble those of retinitis pigmentosa in humans.

A novel cell-free system for peptide synthesis driven by pyridine.

Previously we demonstrated that ribosomes can synthesize polypeptides in the presence of high concentrations (40-60%) of pyridine without any protein factors. Here we analyze additional ribosomal parameters in 60% pyridine using Escherichia coli ribosomes. Ribosomal subunits once exposed to pyridine failed to re-associate to 70S ribosomes in aqueous buffer systems even in the presence of 20 mM Mg2+, whereas they formed 70S complexes in the presence of 60% pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins located at the protuberances of the large subunit, e. g. L7/L12 and L11 forming the elongation factor-binding domain, were released in the pyridine system. The aminoglycoside neomycin, a strong inhibitor of the ribosomal (factor-independent) translocation reaction, completely blocked poly(Phe) synthesis and translocation activities in the pyridine system, whereas these activities were not affected at all by gypsophilin, a ribotoxin that inhibits factor-dependent translocation. Another inhibitor of the ribosomal translocation, thiostrepton, had no effect concerning the two activities, which is consistent with the fact that this antibiotic requires L11 for its binding to the ribosome. These results suggest that the ribosomes can perform a translocation reaction in the pyridine system, but in a factor-independent (spontaneous) manner.

Time-specific action of N-methyl-N-nitrosourea in the occurrence of retinal dysplasia and retinal degeneration in neonatal mice.

The morphologic response of neonatal mouse retina to the alkylating agent N-methyl-N-nitrosourea (MNU) was examined at different periods of retinal development. A dose of 60 mg/kg N-methyl-N-nitrosourea was injected intraperitoneally to neonatal C57BL mice at 0, 3, 5, 8, 11, 14, 17, and 20 days of age and to C3H mice at 0 days of age, and the retinas were examined sequentially. In the C57BL mice, MNU evoked a time-dependent occurrence of retinal dysplasia and retinal degeneration. With MNU treatment at day 0 and day 3 (the stage of retinal cell proliferation), retinal dysplasia characterized by the progressive disorganization of neuroblasts, which led to the formation of rosettes, was found in the outer neuroblastic/nuclear layer above the normal pigment epithelial cells during days 8-20, but decreased at day 50. The rosettes were surrounded by photoreceptor segments and Müller cell processes, and by photoreceptor nuclei. The MNU response was related to retinal differentiation; following MNU treatment at day 5 or 8 (the stage of retinal cell differentiation) the cells were much less sensitive (i.e. no retinal response was found). However, with MNU treatment at days 11, 14, 17, and 20 (after cellular differentiation), retinal degeneration characterized by selective photoreceptor apoptosis was seen. These results suggest that there is a critical period for the time of MNU administration in the development of mouse retinal lesions. In C3H (rd/rd) mice, MNU treatment at day 0 resulted in retinal degeneration with only slight rosette formation at the peripheral retina.

Factors related to axillary lymph node metastasis in T1 breast carcinoma.

The current study was performed on 38 cases of T1 breast cancers ( 2 cm in greatest diameter) to identify the factors related to recognition of axillary lymph node (AxLN) metastasis. Ten patients (26.3%) had lymph node metastases. Comparing the AxLN positive (+) group with the AxLN negative (-) group revealed that tumor size and hormone receptor status as well as age of the patients were not significantly different. However, the Ki-67 labeling index (22.2 +/- 5. 9% vs. 12.5 +/- 2.8%), the microvessel count (43.8 +/- 12.4/0.785 mm2 vs. 27.0 +/- 8.4/0.785 mm2) and bcl-2+ cases (70% vs. 29%) were significantly higher in the AxLN+ cases. These results suggest that the Ki-67 labeling index, microvessel count and Bcl-2 expression, especially when combined, are useful predictors of AxLN metastases in T1 breast cancers.

Inhibition of tumor growth and metastasis by angiogenesis inhibitor TNP-470 on breast cancer cell lines in vitro and in vivo.

Antitumor and antimetastatic activity of the angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), a semisynthetic analogue of fumagillin, was evaluated in breast cancer cell lines. In an in vitro MTT assay, after 72 hrs continuous exposure to TNP-470, growth inhibition was observed in all seven cell lines of murine (JYG-A, JYG-B, DD-762, and BALB/c-MC) or human (KPL-1, MDA-MB-231, and MKL-F) origin, in which the 50% inhibitory concentrations (IC50) at 72 hrs treatment were 4.6, 4.4, 4.6, 10.1, 35.0, 25.3, and 33.4 micrograms/ml, respectively. In an in vivo assay using JYG-A, JYG-B, KPL-1, and MDA-MB-231 cells by orthotopic (right thoracic mammary fat pad) transplantation in female nude mice, TNP-470 at 30 or 50 mg/kg body weight was injected s.c. every other day from the day of tumor cell inoculation until the end of the experiment. The inhibitory effect on primary tumor growth was obtained in all four cell lines in a dose-dependent manner. In the 50 mg/kg TNP-470-treated group, the reductions in tumor weight of the JYG-A, JYG-B, KPL-1, and MDA-MB-231 cells with respect to the controls were 50%, 30%, 4%, and 49%, respectively. Metastasis was seen in the JYG-A, JYG-B, and KPL-1 cells. The numbers of mice bearing pulmonary metastases of JYG-A and JYG-B cells and regional axillary lymph node metastases of KPL-1 cells were reduced, and TNP-470 at the 50 mg/kg dose to KPL-1 cells significantly reduced lymph node metastases compared with the control. Although the weight gain was retarded in the TNP-470-treated mice, weight loss was not seen. TNP-470 was highly effective in the treatment of breast cancer cells. These results suggest that the clinical use of TNP-470 may be a promising treatment for breast cancer patients.

Morphologic characteristics of N-methyl-N-nitrosourea-induced retinal degeneration in C57BL mice.

Morphologic characteristics of retinal degeneration induced by a single systemic administration of N-methyl-N-nitrosourea (MNU) in mice was investigated. The aim was to characterize the MNU-induced retinal lesions in mice and compare them with human retinitis pigmentosa. A dose of 60 mg/kg body weight MNU, injected intraperitoneally into male and female C57BL mice, evoked progressive retinal degeneration in all treated mice, while control mice remained normal. An early change was photoreceptor apoptosis followed by infiltration of macrophages and swelling of the pigment epithelial cells with phagosomal inclusions for apoptotic photoreceptor cell removal. Loss of the majority of photoreceptor cells occurred within a week. Then, Feulgen-positive corpuscles, indicative of an aggregation of degenerative photoreceptor elements, vitread the outer limiting membrane were surrounded by Müller cell processes, and the duplication of the pigment epithelial cells sclerad the outer limiting membrane were seen 2 and 3 weeks after the treatment. Finally, the Feulgen-positive corpuscles disappeared and Müller cell processes were in direct contact with the continuous lining of the single layer of pigment epithelial cells. As in retinitis pigmentosa in humans, the primary event was loss of photoreceptor cells by apoptosis, but the migration of the pigment epithelial cells within the retina was not seen in the present model.

Keratin expression in normal uterine cervical epithelium and carcinomas of cervical origin.

The immunohistochemical expression of keratins 7, 8, 10, 13, 14, 17, 18 and 19 was examined in formalin-fixed paraffin-embedded tissues of normal uterine cervical epithelium and carcinomas of cervical origin (4 squamous cell carcinoma in situ, 17 squamous cell carcinoma, 9 adenocarcinoma, and 1 adenoid basal carcinoma). A panel of 8 monoclonal antibodies capable of recognizing 8 individual keratin subtypes was employed using microwave oven heating and a labeled streptavidin biotin method. Ectocervical squamous epithelium expressed keratins 14 and 19 in the basal cell layer, and keratins 10 and 13 in the suprabasal cell layer. Endocervical columnar cells were found to express keratins 7, 8, 18 and 19, whereas the reserve cells expressed keratins 7, 8, 14, 17, 18 and 19. Most of the squamous cell carcinomas, both keratinizing and non-keratinizing, as well as the carcinoma in situ revealed a keratin phenotype detected in normal ectocervical squamous cells (keratins 10, 13, 14 and 19) and endocervical subcolumnar reserve cells (keratins 7, 17 and 18). The adenocarcinomas, both endocervical and endometrial type, were positive for keratins 7, 8, 17, 18 and 19. The adenoid basal carcinoma expressed all the keratins examined including the expression of reserve cell keratin. Reserve cell keratins were found mostly in squamous cell carcinomas, adenocarcinomas and adenoid basal carcinoma of cervical origin. Therefore, the keratin expression pattern indicates the origin of a variety of carcinomas of the uterine cervix from a common progenitor, endocervical reserve cells.

Pigmentary degeneration induced by N-methyl-N-nitrosourea and the fate of pigment epithelial cells in the rat retina.

Pigmentary degeneration of the retina was induced by a single intraperitoneal injection of 75 mg/kg of N-methyl-N-nitrosourea (MNU) in female Brown-Norway colored rats at 50 days of age, which were then observed at 24, 48 and 72 h and 7, 21, 35 and 150 days after the treatment. MNU-treated rats showed selective destruction of the photoreceptor cells by an apoptotic mechanism 24 h after the treatment, and the destruction was completed by day 7. During the photoreceptor cell degeneration, proliferation of Müller cells and infiltration of macrophages was prominent 72 h and 21 days after the treatment, respectively. Müller cell proliferation and macrophage infiltration corresponded to degenerative photoreceptor cell phagocytosis, and proliferating Müller cell processes responded to stabilize the damaged retina. Pigment epithelial cell detachment from the Bruch's membrane was seen 72 h after the treatment, and migration within all layers of the retina was seen at day 7 when photoreceptor cells were lost. At 21, 35 and 150 days after the treatment, lack of photoreceptor cells and deposition of pigment epithelial cells within the retina but not in contact to vascular endothelial cells were characteristic. MNU-induced photoreceptor apoptosis followed by Müller cell and macrophage reaction then pigment epithelial cells deposition within the retina partially resembles retinitis pigmentosa in humans.

Fas-independent apoptosis of photoreceptor cells in C3H mice.

The morphogenesis of the photoreceptor cells in the retinas of C3H mice carrying the rd gene and C57BL mice carrying the normal gene was compared, and retinas of the C3H mutants (C3H-lpr/lpr, -lprcg/lprcg, and -lpr/lpr-gld/gld) defective in apoptosis through the Fas system were examined. In the C57BL retina, the inner and outer nuclear layers were separated at 8 days of age, and the photoreceptor inner and outer segments began to grow between 8-11 days after birth with their most rapid growth occurring between 14-17 days of age. In the C3H retina, the development was comparable to that of the C57BL retina at 8 days of age but the reduction in thickness of the outer nuclear and photoreceptor layers was noted at 11 days of age, and the outer nuclear layer became reduced to only a few nuclei in thickness at 14 days, being completely missing or reduced to a single row of cells at 20 days. The degeneration was by an apoptotic mechanism as confirmed morphologically and by the TUNEL method. In all the C3H mutant retinas examined over 24 days of age, the complete depletion of the outer nuclear layer or reduction to a single row comparable to 20-day-old C3H mice was seen. The rd gene action is therefore independent of Fas/Fas ligand-medicated apoptosis.