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Background/Aim: Concomitant proton pump inhibitor (PPI) and immune checkpoint inhibitor (ICPI) were determined as risk factors of acute kidney injury. To identify the type of PPI associated with ICPI-induced nephritis, we used the Japanese Adverse Drug Event Report database. Patients and Methods: ICPIs (nivolumab, pembrolizumab, ipilimumab, atezolizumab, durvalumab, and avelumab) and PPIs (esomeprazole, omeprazole, vonoprazan, rabeprazole, and lansoprazole) were selected as suspected nephritis-inducing drugs. Results: The cases of concomitant use of atezolizumab and rabeprazole, ipilimumab and omeprazole, ipilimumab and lansoprazole, nivolumab and esomeprazole, nivolumab and omeprazole, nivolumab and rabeprazole, nivolumab and lansoprazole, pembrolizumab and esomeprazole, as well as pembrolizumab and lansoprazole had a significantly higher reported odds ratio than monotherapy cases. Conclusion: Male patients or patients using ICPIs and PPIs (excluded vonoprazan) concomitantly should be monitored for renal function after chemotherapy.

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced. Therefore, identification of glomerular TG2 substrates is the first step in elucidating the role of TG2 as a crosslinking enzyme during disease progression. To clarify potential glomerular TG2 substrates, and to establish a procedure for substrate identification, we attempted to identify those molecules using normal mouse glomeruli. Extracts from mouse glomerular and non-glomerular fractions were treated with our established biotin-labeled substrate peptide, which specifically crosslinks to the lysine-donor substrates depending on TG2 activity. Peptide-incorporated proteins were then purified using avidin resin and identified via mass spectrometry. In parallel, we performed the identification using corresponding samples from TG2 knockout mice. Consequently, potential TG2 substrates were separately identified in glomerular and non-glomerular fractions. They were mainly identified as novel TG2 substrates and partly include the well-known substrates. These results potentially provide novel insights into the mechanism underlying IgA nephropathy and may help elucidate the physiological functions of TG2.

Fluorescence in situ hybridization (FISH) is an essential tool for genetic diagnosis in daily pathological work. Almost full automation of FISH can be achieved with the recently released automated SureFISH platform (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA). Its utility has been reported in HER2 amplification of breast and gastric carcinoma and ALK-rearranged lung cancer. Here, we examined the utility of automated SureFISH for the identification of rearrangement signals in translocation-related sarcomas (TRSs), including 11 EWSR1-rearranged and 10 synovial sarcoma cases, compared with non-automated conventional FISH using the same specimens. The percentages of EWSR1 or SS18 split signals were higher in automated SureFISH than in conventional FISH in 13 of the 21 cases. On the other hand, 8 of the 21 cases showed the same or lower percentage of split signals in automated SureFISH. Both FISH approaches detected EWSR1 and SS18 split signals in more than 10% of tumor cells in all cases. The strongest advantage of automated SureFISH is its ability to reduce running time without sacrificing quality. Other advantages include improved signal sharpness with oligo probes and reduced ecological toxicity by avoiding formamide use. Automated SureFISH is an excellent tool for the genetic diagnosis of TRSs and contributes to their rapid definitive diagnosis.

We have reported that excessive nitric oxide (NO), like other reactive oxygen species (ROS), causes a decrease in cytochrome c oxidase (CCO) activity and ATP levels (mitochondrial damage) resulting in lens opacity. In addition, previous reports have shown that oxidative stress caused by ROS enhances amyloid β (Aβ) production in mammalian lenses, and that Aβ1-42 stimulates inducible nitric oxide synthase (iNOS) promoter activity. Based on these reports, we investigated the relationship between NO and Aβ1-42 production in human lens epithelial (HLE) cells. iNOS was induced by the co-incubation of HLE cells with 1000 IU interferon-γ (IFN-γ) and 100ng/ml lipopolysaccharide (LPS) for 48h. This led to enhanced NO release, an increase in the gene expression levels of proteins related to Aβ production, and the cellular accumulation of Aβ1-42. Moreover, both aminoguanidine (AG, a selective inhibitor of iNOS) and diethyldithiocarbamate (DDC, a nuclear factor-kappa B (NFκB) inhibitor) attenuated these changes in IFN-γ and LPS stimulated HLE cells. Based on our finding that Aβ1-42 accumulation is induced by co-incubation of HLE cells with both IFN-γ and LPS, we prepared a HLE cell model with Aβ1-42 accumulation (Aβ-accumulated-HLE cell model) by pre-stimulating cells with IFN-γ and LPS for 48h. Aβ1-42 accumulation caused NO production via iNOS, resulting in an enhancement in the mRNA levels for enzymes necessary for the proteolysis of amyloid precursor protein (APP) to Aβ in HLE cells. In addition, excessive NO produced in response to Aβ1-42 accumulation led to a decrease in CCO activity and ATP levels. Taken together, we hypothesize that excessive NO production in the lens epithelium enhances Aβ1-42 production, and that this enhancement accelerates NO release. The enhancement in NO production in the lens epithelium based on positive feedback (NO-Aβ positive feedback loop, a vicious cycle) may promote the onset of cataracts (lens opacification) via the decrease in CCO activity and ATP levels. These findings provide significant information that can be used to design further studies aimed at developing anti-cataract drugs.

In a study to find ways to prevent the side effects of indomethacin (IMC), we previously reported that magnesium ion (Mg2+) can prevent the onset of IMC-induced gastric mucosa in adjuvant-induced arthritis (AA) rats, a model for rheumatoid arthritis (RA). In this study we investigated whether the co-administration of IMC and Mg2+ prevents the formation and aggravation of intestinal ulcerogenic lesions in AA rats. The single oral administration of an excessive dose of IMC (40 mg/kg) induces hemorrhagic lesions and nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) in the jejunal and ileal mucosa of AA rats, and the extent of the lesions, as well as iNOS and NO levels in AA rats are higher than in normal rats. On the other hand, the co-administration of 200 mg/kg Mg2+ attenuates intestinal ulceration and the elevation in the iNOS and NO levels in AA rats. Further, hemorrhagic lesioning and enhanced iNOS and NO levels in AA rats also result from the repetitive oral administration of 3 mg/kg IMC (therapeutic dose) for 42 d (once a day), and these changes are also prevented by the co-administration of 200 mg/kg Mg2+. In conclusion, the co-administration of Mg2+ suppresses the ulcerogenic response to IMC in the jejunal and ileal mucosa of AA rats, probably by preventing the elevation of iNOS and NO levels in the region.

Previous studies showed an increased prevalence of cataracts in postmenopausal women. In this study, we investigated changes in the levels of calcium ion (Ca(2+)) and interleukin (IL)-18, which are factors in cataract development, in the lenses of ovariectomized (OVX) rats, a model of postmenopausal woman. Although the Ca(2+) content in the blood of OVX rats increased 1 month after ovariectomy and subsequently decreased, the Ca(2+) content in the lenses was unchanged in OVX rats 1-3 months after ovariectomy. The Ca(2+)-ATPase activity in the lenses of OVX rats peaked 1 month after ovariectomy, and the behavior of Ca(2+)-ATPase activity in lenses of OVX rats was similar to that of the Ca(2+) concentration in the blood. It is possible that hypercalcemia increases the Ca(2+) inflow into the lens; however, the enhanced Ca(2+)-ATPase activity prevents the Ca(2+) level from rising. On the other hand, we found that the levels of both IL-18 and interferon (IFN)-γ in the lenses of OVX rats were significantly increased as compared with the lenses of sham (control) rats during the period 1-3 months after surgery. These results suggest that the expression of IFN-γ via IL-18 in the lenses of OVX rats is induced by ovariectomy, and that excessive IL-18 and IFN-γ production in the lenses may be related to cataract development in postmenopausal women. These findings support those of previous studies that assessed lens opacification in postmenopausal women.

Disulfiram (DSF) is a dimer of diethyldithiocarbamate (DDC) that we previously added to a solution of 2-hydroxypropyl-β-cyclodextrin (DSF solution). We found that the instillation of this DSF solution delayed lens opacification in a hereditary cataractous ICR/f rat. In this study, we attempted to design an ophthalmic formulation containing DSF nanoparticles for use as a lens targeted drug delivery system (nano-DSF suspension), and investigated the changes in drug content in the lens after the instillation of DSF solution or nano-DSF suspension. The nano-DSF suspension was prepared by a bead mill method to yield a mean particle size of nano-DSF of 181 nm. Following the instillation of 1.4% DSF solution or the nano-DSF suspension, DDC was detected only in the aqueous humor and lens; in both, the area under the curve (AUC) and mean residence time (MRT) for the nano-DSF suspension were higher than for the DSF solution. In addition, we found that the DDC residence time in the cortex and nucleus of the lens was higher than in the capsule-epithelium. Although DDC was not detected in the cortex and nucleus of lenses following the instillation of the 1.4% DSF solution, the instillation of a 1.4% nano-DSF suspension led to the accumulation of DDC in both areas. In conclusion, it is possible that the instillation of a nano-DSF suspension can supply more DDC into the aqueous humor and lens than a conventional formulation, and these findings provide information significant for the prevention of cataracts and the design of a lens targeted drug delivery system.

It was reported that cilostazol (CLZ) suppressed disruption of the microvasculature in ischemic areas. In this study, we have designed novel injection formulations containing CLZ nanoparticles using 0.5% methylcellulose, 0.2% docusate sodium salt, and mill methods (CLZnano dispersion, particle size 81 ± 59 nm, mean ± S.D.), and investigated their toxicity and usefulness in a cerebral ischemia/reperfusion-induced injury model (MCAO/reperfusion mice). The pharmacokinetics of injections of CLZnano dispersions is similar to that of CLZ solutions prepared with 2-hydroxypropyl-β-cyclodextrin, and no changes in the rate of hemolysis of rabbit red blood cells, a model of cell injury, were observed with CLZnano dispersions. In addition, the intravenous injection of 0.6 mg/kg CLZnano dispersions does not affect the blood pressure and blood flow, and the 0.6 mg/kg CLZnano dispersions ameliorate neurological deficits and ischemic stroke in MCAO/reperfusion mice. It is possible that the CLZnano dispersions will provide effective therapy for ischemic stroke patients, and that injection preparations of lipophilic drugs containing drug nanoparticles expand their therapeutic usage.

Esophageal cancer is one of the most frequent causes of cancer-related deaths worldwide. This is due to its asymptomatic nature or mild nonspecific symptoms. Most patients are diagnosed after appearance of prominent symptoms, and tumors are frequently accompanied by severe infiltration. Therefore, molecular biomarkers for the prognosis of early-stage esophageal cancer are desired. In this study, we examined the prognostic potential of lipase H (LIPH), a recently reported biomarker for lung adenocarcinoma and squamous carcinoma. We found that LIPH mRNA is also frequently upregulated in esophageal adenocarcinoma. Immunohistochemical analysis confirmed LIPH protein expression in various esophageal tumor tissue sections. Interestingly, higher expression of LIPH in esophageal adenocarcinoma showed a positive correlation with longer survival of patients. Our data suggest that LIPH may have prognostic value for esophageal cancer.

The goal in the search for successful therapies for glaucoma is the reduction of intraocular pressure (IOP), and the search for effective eye drops that reduce IOP is a high priority. We previously reported the potential of a 2-hydroxypropyl-β-cyclodextrin (HPβCD) solution containing 0.5% DSF (DSF solution) to provide effective anti-glaucoma treatment in eye drop form. In this study, we designed new ophthalmic formulations containing 0.5% DSF nanoparticles prepared by a bead mill method (DSFnano dispersion; particle size 183 ± 92 nm, mean ± S.D.), and compared the IOP-reducing effects of a DSFnano dispersion with those of a DSF solution. The high stability of the DSFnano dispersion was observed until 7 days after preparation, and the DSFnano dispersion showed high antimicrobial activity against Escherichia coli (ATCC 8739). In transcorneal penetration experiments using rabbit corneas, only diethyldithiocarbamate (DDC) was detected in the aqueous humor, while no DSF was detected. The DDC penetration level (area under the curve, AUC) and corneal residence time (mean residence time, MRT) of the DSFnano dispersion were approximately 1.45- and 1.44-fold higher than those of the DSF, respectively. Moreover, the IOP-reducing effects of the DSFnano dispersion were significantly greater than those of the DSF solution in rabbits (the IOP was enhanced by placing the rabbits in a dark room for 5 h). In addition, DSFnano dispersion are tolerated better by a corneal epithelial cell than DSF solution and commercially available timolol maleate eye drops. It is possible that dispersions containing DSF nanoparticles will provide new possibilities for the effective treatment of glaucoma, and that an ocular drug delivery system using drug nanoparticles may expand their usage as therapy in the ophthalmologic field. These findings provide significant information that can be used to design further studies aimed at developing anti-glaucoma drugs.

Hypercalcemia is often observed in postmenopausal women as well as in patients with primary hyperparathyroidism or malignant tumors. In this study, we investigated the relationship between calcium ion (Ca(2+)) levels in lacrimal fluid and the rate of corneal wound healing in hypercalcemia using ovariectomized (OVX) rat debrided corneal epithelium. We also determined the effects of Ca(2+) levels on cell adhesion, proliferation and viability in a human cornea epithelial cell line (HCE-T). The calcium content in bones of OVX rats decreased after ovariectomy. Moreover, the Ca(2+) content in the blood of OVX rats was increased 1 month after ovariectomy, and decreased. The Ca(2+) content in the lacrimal fluid of OVX rats was also increased after ovariectomy, and then decreased similarly as in blood. Corneal wound healing in OVX rats was delayed in comparison with Sham rats (control rats), and a close relationship was observed between the Ca(2+) levels in lacrimal fluid and the rate of corneal wound healing in Sham and OVX rats (y=-0.7863x+8.785, R=0.78, n=25). In addition, an enhancement in Ca(2+) levels caused a decrease in the viability in HCE-T cells. It is possible that enhanced Ca(2+) levels in lacrimal fluid may cause a decrease in the viability of corneal epithelial cells, resulting in a delay in corneal wound healing. These findings provide significant information that can be used to design further studies aimed at reducing corneal damage of patients with hypercalcemia.

Indomethacin (IMC), a nonsteroidal anti-inflammatory drug, has been used in the treatment of rheumatoid arthritis (RA), although its clinical use has been limited by its systemic side effects that include gastrointestinal lesions. Therefore, the development of IMC formulations that do not cause gastrointestinal lesions is highly anticipated. In this study, we designed new topical formulations containing IMC solid nanoparticles (IMCnano gel ointment), and investigated their pharmacokinetics. In addition, we demonstrate the preventive effects of this topical application of IMC nanoparticles on inflammation in adjuvant-induced arthritis rat (AA rat). The IMCnano gel ointment was prepared using Bead Smash 12 (a bead mill) and additives including 2-hydroxypropyl-β-cyclodextrin, methylcellulose and Carbopol 934; the mean particle size of the IMC nanoparticles was 173 ± 91 nm (means ± S.D.). The application of the IMCnano gel ointment attenuated the increase in paw edema of the hind feet of AA rats in comparison with AA rats treated with gel ointment containing IMC microparticles (IMCmicro gel ointment, particle diameter 17.1 ± 11.6 mm, means ± S.D). In addition, the accumulation of IMC from the IMCnano gel ointment in skin tissue was significantly large than for the IMCmicro gel ointment; however, the plasma IMC concentrations were similar for the IMCmicro and IMCnano gel ointments. Our findings suggest that the dermal application of nanoparticles may enable a medication to be applied without high-systemic drug levels, which could provide efficient and effective therapy that spares patients from unwanted side effects. A formulation of a topical drug delivery system using IMC nanoparticles may provide a delivery option for the clinical treatment of RA.

The accumulation of amyloid β(1-42) peptide (Aβ(1-42)) in retina is implicated in the development of retinal ganglion cell apoptosis and diabetic retinopathy. In this study we demonstrate that spontaneous diabetes mellitus Otsuka Long-Evans Tokushima Fatty (OLETF) rats can be used as an animal model in studies to identify the expression of Aβ in diabetic retinas. In addition, we investigated the relation between glucose level and Aβ production in the retinas of OLETF rats. In the retinas of Long-Evans Tokushima Otsuka (LETO) rats used as normal controls and OLETF rats, no expression of neprilysin (NEP), which degrades Aβ, was detected, and the expression levels of genes associated with Aβ production (amyloid precursor protein, β site APP cleaving enzyme, and presenilin) and Aβ(1-42) levels in the retinas of 60-week-old OLETF rats with diabetes mellitus were significantly higher than in 60-week-old LETO rat retinas. Furthermore, the increase in the expression levels of genes associated with Aβ production was enhanced by administration of glucose (3.0 g/kg; OGT test), and close relations between the retinal Aβ(1-42) level and plasma blood glucose and HbA1c were observed. In conclusion, we have found that Aβ accumulates easily in the retinas of LETO and OLETF rats due to the absence of NEP. In addition, we determined that the accumulation of Aβ(1-42) in the retinas of OLETF rats is promoted by high plasma glucose levels. Therefore OLETF rats may be a suitable model for studies to identify the expression of Aβ in diabetic retinas.

Cilostazol (CLZ) is an anti-platelet agent that is generally used after the onset of cerebral infarction. However, CLZ is a poorly water-soluble drug and a strategy for increasing its bioavailability is required. In the present study, novel oral formulations were designed containing CLZ solid nanoparticles to improve bioavailability. The present study investigated the therapeutic effect of the oral formulations containing CLZ nanoparticles on ischemic stroke using a cerebral ischemia/reperfusion-induced injury model (MCAO/reperfusion mice). The oral formulation containing CLZ nanoparticles (CLZ/Rnano tablet) was prepared using a combination of recrystallization and ball milling with the following ingredients: CLZ, docusate sodium, methylcellulose, 2-hydoxypropyl-β-cyclodextrin, gum arabic, polyvinylpyrrolidone, and mannitol. The particle size after re-dispersion of the CLZ/Rnano tablet was 64±47 nm (mean ± standard deviation). The CLZ areas under the concentration-time curve (AUC) and mean residence time (MRT) in rats that were administered CLZ/Rnano tablets were significantly greater compared with those in rats that were administered CLZ/Rmicro tablets. Results indicated, the AUC after administration of CLZ/Rnano tablets was 3.1-fold higher compared with that after administration of the commercially available CLZ OD tablet. In addition, oral administration with CLZ/Rnano tablets ameliorated neurological deficits caused by ischemic stroke in MCAO/reperfusion mice. It is possible that the oral formulation containing CLZ nanoparticles will be useful for the treatment of patients with ischemic stroke and that these findings will provide significant information that can be used to improve the drug with low bioavailability.

We designed ophthalmic formulations containing dexamethasone-loaded solid nanoparticles (DEXnano dispersion), and investigated corneal permeability and toxicity. 0.1% dexamethasone (DEX) powder (DEX microparticles), 0.026% methyl p-hydroxybenzoate (MP), 0.014% propyl p-hydroxybenzoate (PP), and 0.5% methylcellulose were used, and the DEXnano dispersion was prepared by the bead mill method. The mean particle size of DEXnano dispersion was 78 nm. Antimicrobial activity of the DEXnano dispersion were measured by using Escherichia coli, and the corneal epithelium-debrided rat model and HCE-T cells (immortalized human corneal epithelial cell line) were used to estimate the corneal toxicity. The transcorneal penetration of the DEXnano dispersion were evaluated in the corneas of rabbit. The DEXnano dispersion was found to be highly stable until 14 d after its preparation. Although DEX itself did not exhibit antimicrobial activity, the DEXnano dispersion containing parabens (MP and PP) showed high antimicrobial activity, approximately equal to that of the solution containing parabens without DEX. The corneal penetration rate (Jc) and mean residence time (MRT) of DEX from the DEXnano dispersion were approximately 5.1- and 1.3-fold higher, respectively, than those of a dispersion containing DEX microparticles (mean particle size, 11.3 µm). In addition, no significant difference was found in corneal stimulation between the vehicle and DEXnano dispersion. In conclusion, we successfully prepared high quality dispersion containing DEX solid nanoparticles, and the nanoparticle-based ophthalmic formulation of DEX enhanced the corneal permeability and residence time of the drug. It is possible that DEXnano dispersion will show increased effectiveness in treating ocular inflammation.

Foreign matter sensation and blurred vision following instillation of ophthalmic suspension are often observed, and remaining of solid particle on cornea is related these side effects. In addition, low dispersion stability in the ophthalmic suspension affects the therapeutic effect. In this study, we have attempted to enhance the dissolution rate and stability of commercially available pirenoxine ophthalmic suspension (CA-pirenoxine eye drops), anti-cataract eye drops, by changes in particle size. Methylcellulose, zirconia beads (0.1 mm) and Micro Smash were used to mill the pirenoxine (bead mill method), and the distribution of particle size was changed to approximately 60-900 nm (nanodispersions) from 70 nm-3 μm (CA-pirenoxine eye drops). The dissolution rate of pirenoxine increased by the bead mill, and the dissolution rate constant in pirenoxine nanodispersions was 2.1-fold than that in CA-pirenoxine eye drops. Moreover, the dispersion stability in nanodispersions also significant higher in comparison with the CA-pirenoxine eye drops. The dispersion ratio in CA-pirenoxine eye drops and pirenoxine nanodispersions at 2 d after suspension was 48%, 99%, respectively. In conclusion, we showed that the dissolution rate and dispersion stability of CA-pirenoxine eye drops were enhanced by the bead mill method. These findings provide significant information that can be used in the design of ophthalmic suspension.

Fluorescence in situ hybridization (FISH) is an essential tool for genetic diagnosis in daily pathological work. Almost full automation of FISH can be achieved with the recently released automated SureFISH platform (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA). Its utility has been reported in HER2 amplification of breast and gastric carcinoma and ALK-rearranged lung cancer. Here, we examined the utility of automated SureFISH for the identification of rearrangement signals in translocation-related sarcomas (TRSs), including 11 EWSR1-rearranged and 10 synovial sarcoma cases, compared with non-automated conventional FISH using the same specimens. The percentages of EWSR1 or SS18 split signals were higher in automated SureFISH than in conventional FISH in 13 of the 21 cases. On the other hand, 8 of the 21 cases showed the same or lower percentage of split signals in automated SureFISH. Both FISH approaches detected EWSR1 and SS18 split signals in more than 10% of tumor cells in all cases. The strongest advantage of automated SureFISH is its ability to reduce running time without sacrificing quality. Other advantages include improved signal sharpness with oligo probes and reduced ecological toxicity by avoiding formamide use. Automated SureFISH is an excellent tool for the genetic diagnosis of TRSs and contributes to their rapid definitive diagnosis.

We attempted to design a combination ointment containing solid tranilast nanoparticles and dissolved sericin as a wound-healing drug (TS-combination ointment), and evaluated its usefulness as therapy for wound-healing deficits in streptozotocin-induced diabetic rat (STZ rat) using kinetic analyses as an index. Solid tranilast nanoparticles were prepared by bead mill methods with low-substituted methylcellulose; the mean particle size of the tranilast nanoparticles was 70 nm. The ointment was designed to contain the tranilast nanoparticles plus sericin powder and/or Carbopol® 934. Skin wound healing in STZ rats begins significantly later than in normal rats. Although the skin wound healing rate in STZ rats treated with an ointment containing tranilast nanoparticles was lower than in STZ rats treated with vehicle, the ointment was effective in reducing redness. An ointment containing sericin enhanced the skin-healing rate, but the preventive effect on redness was weak. On the other hand, the combination of tranilast and sericin increased both the skin healing rate and reduction in redness. In conclusion, we have adapted kinetic analyses to skin wound healing in rats, and found these analyses to be useful as an index of wound healing ability by a wound-healing drug. In addition, we show that treatment with the TS-combination ointment enhances the skin wound healing rate and reduces redness. These findings provide information significant to the search for new wound-healing therapies and for the design of wound-healing drugs.

We attempted to develop anti-glaucoma eye drops using 0.5% disulfiram (DSF), 5% 2-hydroxypropyl-β-cyclodextrin, 0.1% hydroxypropylmethylcellulose, and 2% methylcellulose (MC) (DSF eye drops with MC), and tested the ability of a DSF eye drops with MC to reduce intraocular pressure (IOP) in rabbit models.Elevated IOP was induced by the rapid infusion of 5% glucose solution (15 ml/kg of body weight) through the marginal ear vein or by keeping rabbits in the dark for 5 h. IOP and the nitric oxide (NO) level in the aqueous humor were measured with an electronic tonometer and by a microdialysis method, respectively. ΔIOP and ΔNO values were analyzed as the differences in IOP and NO in rabbits instilled with saline or eye drops, respectively.Increased IOP in rabbit models was reduced by the instillation of DSF eye drops with or without MC, and a close relationship was observed between IOP and NO levels in rabbit receiving a rapid infusion of isotonic glucose. We present kinetic parameters [secondary AUC (prolonged drug effect) and secondary MRT (prolonged effective time)] analyzed as the area under the curve (AUC) of ΔIOP or ΔNO versus time using rabbits instilled with eye drops 10, 50, or 90 min prior to the infusion of the isotonic glucose solution. The elevations in IOP and NO level were reduced by the instillation of DSF eye drops with or without MC; the addition of MC increased the secondary AUC and MRT of DSF eye drops.The present study demonstrates that 0.5% DSF eye drops suppress increased IOP in rabbit models, probably by inhibiting the elevation in NO levels. In addition, we propose a kinetic analysis method to predict drug effects and effective time. These findings suggest that a low-substituted MC-based drug delivery system promotes drug effectiveness and effective time.

Several studies have reported that hydrogen peroxide (H2O2) is related to the toxicity of amyloid β (Aβ), and that the accumulation of Aβ in the lenses of humans causes lens opacification. In this study, we investigate the accumulation of Aβ1-42 in the lenses of UPL rats, which then leads to lens opacification. In addition, we demonstrate the effect of disulfiram eye drops (DSF), a potent radical scavenger, on Aβ1-42 accumulation in the lenses of UPL rats. The H2O2 levels in 46- to 60-day-old UPL rat lenses are significantly higher than in normal rats, and the Aβ1-42 levels in 53- and 60-day-old UPL rats are also increased only in lens epithelium containing capsules (capsule-epithelium), not in the lens cortex and nucleus. However, no increases in amyloid precursor protein (APP), β- or γ-secretase mRNA were observed in lenses of the corresponding ages. It has been thought that Aβ1-42 that accumulates in the lenses of UPL rats is actually produced in another tissue containing neuronal cells, such as brain or retina. Aβ1-42 levels in the brain and retina rise with aging, and the levels of APP, β- and γ-secretase mRNA in the retinas of 53-day-old UPL rats with opaque lenses are significantly higher than in 25-day-old UPL rats with transparent lenses. In contrast to the results in retinas, the levels of APP, β- and γ-secretase mRNA in the brains of 25- and 53-day-old UPL rats are similar. On the other hand, in an in vitro study, Aβ1-42 attachment in the lens capsule-epithelium of UPL rats was found to increase in H2O2. In addition, in an in vivo study, the inhibition of H2O2 by DSF was found to attenuate the increase in Aβ1-42 in the lens capsule-epithelium of 60-day-old UPL rats. Taken together, we hypothesize that excessive H2O2 in the lens enhances the attachment of Aβ1-42 in the lens capsule-epithelium of UPL rats, and that the instillation of DSF has the ability to attenuate the attachment of Aβ1-42 by inhibiting H2O2 production in lens. These findings provide significant information that can be used to design further studies aimed at developing anti-cataract drugs.

Lung cancer is one of the most frequent causes of cancer-related death worldwide. However, molecular markers for lung cancer have not been well established. To identify novel genes related to lung cancer development, we surveyed publicly available DNA microarray data on lung cancer tissues. We identified lipase member H (LIPH, also known as mPA-PLA1) as one of the significantly upregulated genes in lung adenocarcinoma. LIPH was expressed in several adenocarcinoma cell lines when they were analyzed by quantitative real-time polymerase chain reaction (qPCR), western blotting, and sandwich enzyme-linked immunosorbent assay (ELISA). Immunohistochemical analysis detected LIPH expression in most of the adenocarcinomas and bronchioloalveolar carcinomas tissue sections obtained from lung cancer patients. LIPH expression was also observed less frequently in the squamous lung cancer tissue samples. Furthermore, LIPH protein was upregulated in the serum of early- and late-phase lung cancer patients when they were analyzed by ELISA. Interestingly, high serum level of LIPH was correlated with better survival in early phase lung cancer patients after surgery. Thus, LIPH may be a novel molecular biomarker for lung cancer, especially for adenocarcinoma and bronchioloalveolar carcinoma.

Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.

It is well known that oxidative stresses induce the production of amyloidβ(Aβ) in the brain, lens, and retina, leading to age-related diseases. In the present study, we investigated the effects of ferulic acid on the Aβlevels in H2O2-stimulated human lens epithelial (HLE) SRA 01/04 cells. Three types of Aβpeptides (Aβ1-40, Aβ1-42, and Aβ1-43) were measured by ELISA, and the levels of mRNA for the expressed proteins related to Aβproduction (APP, BACE1, and PS proteins) and degradation (ADAM10, NEP, and ECE1 proteins) were determined by quantitative real-time RT-PCR. H2O2stimulation augmented gene expression of the proteins related to Aβproduction, resulting in the production of three types of Aβpeptides. Treatment with 0.1 μM ferulic acid attenuated the augmentations of gene expression and production of the proteins related to the secretion of three types of Aβpeptides in the H2O2-stimulated HLE cells. These results provided evidence of antioxidative functions of ferulic acid for lens epithelial cells.

Several epidemiologic studies have found that magnesium ion (Mg²⁺) is related to obesity and type 2 diabetes mellitus. However, there have been almost no reports on the effects of a combination of excessive food intake and Mg²⁺ supplementation on metabolic syndrome and various blood tests values for diabetes mellitus. In this study, we investigated changes in body weight and blood test values for diabetes mellitus of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model for human type 2 diabetes mellitus via metabolic syndrome, under conditions of combined excessive food intake and Mg²⁺ supplementation. The rats received Mg²⁺ supplementation by drinking magnesium water (Mg²⁺; 200 mg/l). No significant differences were observed in the levels of food or water intake between OLETF rats drinking purified water (PW) or magnesium water (MW). Type 2 diabetes mellitus with metabolic syndrome developed at 30 weeks of age, and the body weights and plasma insulin levels of OLETF rats at 60 weeks of age were lower than those of normal rats. The plasma glucose (PG) levels in 38-week-old OLETF rats drinking MW were significantly lower than in those of rats drinking PW, while the body weights and the levels of triglycerides (TG) and insulin of 38-week-old MW-drinking OLETF rats were significantly higher than those of their PW-drinking counterparts. On the other hand, the decreases in body weight and insulin levels in 60-week-old OLETF rats were suppressed by MW supplementation. The present study demonstrates that Mg²⁺ supplementation delays the development of diabetes mellitus in OLETF rats under conditions of excessive food intake. In addition, obesity and high blood TG levels were observed in OLETF rats receiving Mg²⁺ supplementation in conjunction with excessive food intake.

Recent findings have implicated the involvement of interferon-γ (IFN-γ), a part of the Th1 cytokine response, in the retinal inflammation of diabetic patients. In the present study, we investigate whether hyperglycemia relates to the expression of interleukin 18 (IL-18), and leads to the production of IFN-γ in the retinas of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus. Plasma blood glucose, triglyceride and cholesterol levels in 60-week-old OLETF rats, in which the development of diabetes mellitus was observed, were significantly higher than in 60-week-old Long-Evans Tokushima Otsuka (LETO) rats used as normal controls. The expression levels of genes that cause IL-18 activation (IL-18, IL-18 receptor and caspase-1) in OLETF rats were increased at 60 weeks of age, and the levels of IL-18 and IFN-γ in 60-week-old OLETF rat retinas were also higher than in 60-week-old LETO rats. Furthermore, IFN-γ levels increased with increasing IL-18 levels in the retinas of OLETF rats, and a close relationship was observed between the levels of IL-18 and HbA1c. The rapid increase in plasma glucose levels following the oral administration of glucose solution (3.0 g/kg) did not affect the IL-18 and IFN-γ levels in the retinas of LETO rats, whereas the levels in the retinas of OLETF rats increased significantly. In conclusion, the expression of IL-18 is increased in the retinas of OLETF rats, and chronic hyperglycemia may accelerate the release of IL-18 and IFN-γ from inflammatory cells in retinal blood vessel. It is possible that IFN-γ production via IL-18 in the retinas of 60-week-old OLETF rats is caused by hyperglycemia, and plays a role in the inflammation of the OLETF rat retinas.

We investigated the protective effects of sericin on corneal damage due to benzalkonium chloride (BAC) used as a preservative in commercially available timolol maleate eye drops using rat debrided corneal epithelium and a human cornea epithelial cell line (HCE-T). Corneal wounds were monitored using a fundus camera TRC-50X equipped with a digital camera; eye drops were instilled into the rat eyes five times a day after corneal epithelial abrasion. The viability of HCE-T cells was calculated by TetraColor One; and Escherichia coli (ATCC 8739) were used to measure antimicrobial activity. The reducing effects on transcorneal penetration and intraocular pressure (IOP) of the eye drops were determined using rabbits. The corneal wound healing rate and rate constants (kH) as well as cell viability were higher following treatment with 0.005% BAC solution containing 0.1% sericin than in the case of treatment with BAC solution alone; the antimicrobial activity was approximately the same for BAC solutions with and without sericin. In addition, the kH for rat eyes instilled with commercially available timolol maleate eye drops containing 0.1% sericin was significantly higher than that of eyes instilled with timolol maleate eye drops without sericin, and the addition of sericin did not affect the corneal penetration or IOP reducing effect of commercially available timolol maleate eye drops. A preservative system comprising BAC and sericin may provide effective therapy for glaucoma patients requiring long-term anti-glaucoma agents.

It is well known that oxidative stresses induce the production of amyloid β (Aβ) in the brain, lens, and retina, leading to age-related diseases. In the present study, we investigated the effects of ferulic acid on the Aβ levels in H2O2-stimulated human lens epithelial (HLE) SRA 01/04 cells. Three types of Aβ peptides (Aβ1-40, Aβ1-42, and Aβ1-43) were measured by ELISA, and the levels of mRNA for the expressed proteins related to Aβ production (APP, BACE1, and PS proteins) and degradation (ADAM10, NEP, and ECE1 proteins) were determined by quantitative real-time RT-PCR. H2O2 stimulation augmented gene expression of the proteins related to Aβ production, resulting in the production of three types of Aβ peptides. Treatment with 0.1 μM ferulic acid attenuated the augmentations of gene expression and production of the proteins related to the secretion of three types of Aβ peptides in the H2O2-stimulated HLE cells. These results provided evidence of antioxidative functions of ferulic acid for lens epithelial cells.

In the therapy of rheumatoid arthritis, ibuprofen (IBU) is widely used; however, it has been limited the clinical use by its systemic side effect, such as gastrointestinal lesions. Therefore, we prepared topical gel ointment used IBU solid nanoparticles (IBUnano-gel formulation). In addition, we demonstrated their anti-inflammatory effect by using arthritis model rat (adjuvant-induced arthritis rat, AA rat). The gel formulations were prepared using additives (Carbopol 934, 2-hydroxypropyl-β-cyclodextrin and methylcellulose) and bead mill-method. The IBU particle size in the IBUnano-gel formulation was 208 nm. The increase in inflammation of the hind feet of AA rats was attenuated by the treatment with the IBUnano-gel formulation, and preventive effect was higher than that of a gel formulation containing IBUmicroparticles (IBUmicro-gel formulation, mean particle size 85.4 μm); the accumulation and permeability through the skin of IBU from the IBUnano-gel formulation were significantly larger in comparison with the IBUmicro-gel formulation. Further, no gastrointestinal lesions were observed in AA rats following the repetitive administration of the 5% IBUnano-gel formulation (0.30 g) for 42 days (once a day). These results suggest that the dermal application of IBU-nanoparticles provide effective and efficient therapy that spares patients from unwanted side effects.

The dissolution test is a core performance test in pharmaceutical development and quality control of solid drug products. The conventional HPLC dissolution method (batch-sampling method) involves many steps including the filtration, collection and replenishment of sample solutions. We previously reported a dissolution test that involved microdialysis methods (microdialysis-HPLC method) and allowed many steps to be omitted. However, the recovery rate of theophylline by the microdialysis-HPLC method was lower, and the decrease in the flow rate through the dialysis probe caused variation between each tablet. In this study, we have attempted to improve the dissolution test by using a precise micro-controlled roller pump and microfiltering probe (microfiltering-HPLC method). Sustained release preparations of Theodur (100 mg) were used, and the test solutions used were water, buffer at pH 1.2 and pH 6.8, and pH 6.8-buffer containing 0.1-1% polysorbate 80 or sodium lauryl sulfate. In all test solutions, the microfiltering-HPLC method was able to accomplish continuous sampling of sample solutions, and the recovery rate of theophylline was over 90%. The dissolution behavior by the microfiltering-HPLC method tends to reflect the pharmaceutical design in comparison with the batch-sampling method, and the standard deviations by the microfiltering-HPLC are lower than with the batch-sampling method. In addition, the microfiltering-HPLC method allows many steps to be omitted, such as the filtration, collection and replenishment of sample solutions. These findings provide significant information that can be used in the pharmaceutical development and quality assessment of solid drug products.

We compared a monoclonal antibody (SV2-61γ) and a polyclonal antibody (Dako HercepTest) in immunohistochemical assessments of human epidermal growth factor receptor 2 (HER2) expression in 73 samples of advanced gastric cancer. Results were scored as 0 to 3+, and equivocal or discordant (SV2-61γ/Dako HercepTest = 0/2+, 0/3+, 1+/3+ or 2+/3+) cases were subjected to fluorescence in situ hybridization (FISH) analysis. The frequencies of HER2 scores of 2+ or 3+ were 15.1% (11/73) using SV2-61γ and 38.4% (28/73) using Dako HercepTest. All of the equivocal or discordant cases with a HER2 score of 3+ using Dako HercepTest exhibited amplification of the HER2 gene regardless of the HER2 score determined with SV2-61γ. The results of the HER2 tests differed according to the antibodies used for immunohistochemistry that preceded FISH analysis, being 15.1% (11/73) using SV2-61γ and 23.3% (17/73) using Dako HercepTest. Thus, therapeutic decisions might be markedly influenced by the selection of antibody used in the HER2 test.

Benzalkonium chloride (BAC) is known to cause corneal epithelial damage. In this study we investigated the effect of a BAC solution containing a thickening agent, which enhanced residence time in the eyes, on corneal wound healing using in vivo rat model debrided corneal epithelium. 0.5% or 1.0% methylcellulose (MC), carboxymethylcellulose (CMC) and hydroxypropyl-methylcellulose (HPMC) were used as the thickening agent. The levels of corneal wound healing of rat eyes injected with saline were alone approximately 45.0% at 12 h and 93.6% at 24 h after corneal epithelial abrasion, and healing was almost complete at 36 h. The healing rate in the rat eye treated just with MC, CMC and HPMC was higher than that in those injected with saline. In contrast to the treatment result using only this thickening agent, the healing rate in the eye treated with BAC was lower than that in those injected with saline: the corneal wounds in the BAC-treated eye showed approximately 20% healing at 12 h after abrasion. The injection of 0.02% BAC solution containing MC, CMC and HPMC more significantly delayed the healing than did the injection of 0.02% BAC alone. The results show that the in vivo evaluation method for corneal damage using rat debrided corneal epithelium reflects a toxic change depending upon residence time. These findings provide valuable safety and efficacy information for use in the design of eye drops.

We previously found two mechanisms for the dysfunction in Ca(2+) regulation caused by excessive nitric oxide (NO) using the lenses of hereditary cataract model rats: the first is that NO causes a decrease in Adenosine-5'-triphosphate (ATP) level via cytochrome c oxidase (CCO), resulting in a decrease in ATPase function; the second is that NO causes enhanced lipid peroxidation, resulting in the oxidative inhibition of Ca(2+)-ATPase. In this study, we demonstrate the effect of excessive NO on lipid peroxidation and ATP production in human lens using a human lens epithelial cell line, SRA 01/04 (human lens epithelial (HLE) cells).Excessive NO via inducible NO synthase (iNOS) was induced by stimulating cells with a combination of interferon-gamma (1000 IU IFN-γ) and lipopolysaccharide (100 ng/mL LPS). CCO activity was measured using a Mitochondrial Isolation kit and Cytochrome c Oxidase Assay kit, and ATP levels were determined using a Sigma ATP Bioluminescent Assay Kit and a luminometer AB-2200.Cytochrome c oxidase activity and ATP levels were decreased in HLE cells stimulated with IFN-γ and LPS, and aminoguanidine (AG) and diethyldithiocarbamate (DDC) added 6 h before cell collection significantly attenuated these decreases in cells stimulated with the IFN-γ and LPS for 24-30 h. However, the lower CCO activity and ATP levels in HLE cells stimulated with the IFN-γ and LPS for 30 h were not changed by treatment with AG or DDC for 6-12 h, while the CCO activity and ATP levels in HLE cells treated with AG or DDC for 18 were recovered.Excessive NO causes a decrease in CCO activity and ATP levels, and the recovery time for CCO activity is related to exposure time to NO in HLE cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) comprise one of the most frequently used classes of medicines in the world; however, NSAIDs have significant side effects, such as gastroenteropathy, and rheumatoid arthritis patients taking NSAIDs are more susceptible to NSAID-induced gastric lesions as compared to patients with other diseases. In Asian countries, loxoprofen has been used clinically for many years as a standard NSAID. We demonstrate the preventive effect of the co-administration of water containing magnesium ion (magnesium water, 1-200 µg/kg) on the ulcerogenic response to loxoprofen in adjuvant-induced arthritis (AA) rats. Oral administration of loxoprofen (100 mg/kg) caused hemorrhagic lesions in the gastric mucosa of AA rats 14 d after adjuvant injection, and, following loxoprofen administration, the lesion score of AA rats was significantly higher than that of normal rats. The expression of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production in the gastric mucosa of AA rats were also increased by the administration of loxoprofen, and the increase in lesions and NO were prevented by the administration of aminoguanidine, an iNOS inhibitor. The co-administration of magnesium water decreased the ulcerogenic response to loxoprofen in AA rats. In addition, the co-administration of magnesium water attenuated the increase in iNOS mRNA expression and NO production in AA rats receiving loxoprofen. These results suggest that the oral co-administration of magnesium water to AA rats has a potent preventive effect on the ulcerogenic response to loxoprofen, probably by inhibiting the rise in iNOS and NO levels in the gastric mucosa.

Dissolution testing is a core performance test in pharmaceutical development and quality control. The conventional HPLC dissolution method (batch-sampling method) has many steps such as the filtration, collection and replenishment of sample solutions. We previously reported the dissolution test by using microdialysis methods (microdialysis-HPLC method) that can omit many steps. In this study, we investigated whether the microdialysis-HPLC method can be applied to quality assessment for sustained release preparations by a dissolution test. Calcium-channel blockers nifedipine tablets (20 mg) were used, and the test solution used was 0.2 M hydrogen phosphate-citric acid buffer (pH 6.8) with or without 1% sodium lauryl sulfate. In both test solutions, the microdialysis-HPLC method is able to accomplish continuous sampling of sample solutions, and the dissolution behaviors of original nifedipine tablets by the microdialysis-HPLC method were similar to that of the batch-sampling method. In contrast, the dissolution behaviors by the microdialysis-HPLC method were different between original nifedipine tablets and generic products, and the dissolution behaviors in the microdialysis-HPLC method tend to reflect the pharmaceutical design in comparison with the batch-sampling method. In addition, standard deviation in the microdialysis-HPLC method was lower than that of the batch-sampling method. We found that the recovery rate of nifedipine by the microdialysis-HPLC method was increased with the decrease in flow rate through dialysis probe. These findings provide significant information that can be used in pharmaceutical development and quality assessment for original and generic pharmaceutical products, which are sustained release preparations.

To investigate potency of oral disulfiram (DSF) compared with that of dexamethasone (Dexa), on endotoxin-induced uveitis (EIU) in rats. The oral administration with 750 mg/kg DSF suppressed the number of inflammatory cells, protein concentration, and levels of tumor necrosis factor (TNF)-α, Nitric oxide (NO) and prostaglandin (PG) E2 in the aqueous humor and improved the histiologic status of the ocular tissue at 24 hr after lipopolysaccharide (LPS) injection. The anti-inflammatory potency of DSF oral administration was as strong as that observed with 0.5 mg/kg Dexa in the present study. The results suggest that DSF might pave the way for a novel therapeutic agent for the management of uveitis.

We have studied the effect of disulfiram (DSF) solution containing 2-hydroxypropyl-beta-cyclodextrin and hydroxypropylmethylcellulose (DSF eye drops) on intraocular pressure (IOP) in experimentally induced ocular hypertension in rabbits. In both in vitro and in vivo transcorneal penetration experiments using rabbit corneas, only diethyldithiocarbamate (DDC) was detected in the aqueous humor, while DSF was not detected. The amount of DDC penetration for 0.25% DSF eye drops was about 4-fold that for 0.1% DSF eye drops in in vivo transcorneal penetration experiments. The elevation in IOP was induced by the rapid infusion of 5% glucose solution (15 ml/kg of body weight) through the marginal ear vein, and IOP was measured with an electronic tonometer. The induced elevation in IOP was reduced by the instillation of 0.1-0.5% DSF eye drops, and the IOP-reducing effect increased with the increase in DSF concentration in the drops. Nitric oxide (NO) levels increased in the aqueous humor following the infusion of the 5% glucose solution, and this increase was also suppressed by the instillation of DSF eye drops. In conclusion, the present study demonstrates that the instillation of DSF eye drops has an IOP-reducing effect in rabbits with experimentally induced ocular hypertension, probably caused by the suppression of NO production.

To evaluate the pharmacological properties of cilostazol (CLZ), we examined its intraocular pressure (IOP) -lowering effect. CLZ is an inhibitor of Type III phosphodiesterase that increases intracellular cyclic AMP levels by restraining platelet aggregation, and has a potential protective effect against atherosclerosis. We attempted to apply it for use as an anti-glaucoma agent; however, the application of CLZ in the ophthalmic field is limited due to its poor water solubility. We attempted to enhance CLZ solubility using 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD). The solubility of CLZ increased with increasing HPbetaCD concentrations, and 0.05% CLZ was dissolved in 10% HPbetaCD. Moreover, fine particle suspension of 0.5% CLZ in 5% HPbetaCD (soluble CLZ: ca. 0.027%) were prepared using a Microfluidizer, an impact-type emulsifying comminution device. In an in vitro transcorneal penetration experiment through the rabbit cornea, the CLZ penetration rate was dependent on the CLZ content of the solutions and suspensions. When a 0.05% CLZ ophthalmic solution was instilled into a rabbit eye, the absorption rate constant for CLZ into an aqueous humor was 0.0059+/-0.001 min(-1), and the elimination rate constant was 0.048+/-0.024 min(-1). Also CLZ ophthalmic solutions and fine particle suspension were examined to for their ability to reduce enhanced intraocular pressure (IOP) of rabbits in a darkroom. The instillation of 0.05% CLZ ophthalmic solutions and 0.5% CLZ fine particle suspensions into rabbit eyes reduced the enhanced IOP. These results demonstrate that the instillation of CLZ ophthalmic solutions and fine particle suspensions may represent an effective anti-glaucoma formulation.

The area around tidal inlet is potentially dangerous for marine activities because of strong tidal currents and waves together with the complexity of topography. This study intends to collect useful information about the flow field around tidal inlet for prevention of marine accidents. Field observations of waves and currents have been conducted on two sites of Imakirekuchi Inlet at Lake Hamana in Shizuoka Prefecture and Daiichikoguchi Inlet at Saroma Lake in Hokkaido. The continuous 30 days observation of currents at both sites were successfully conducted. By analyzing the observation data, characteristics of temporal- and spatial-variation of currents in and around the inlet have been examined to clarify the relation among tide, current, and topography.

The amygdala is located in the caudal part of the ventral telencephalon. It is composed of many subdivisions and is involved in the control of emotion. It is important to know the mechanisms of amygdalar development in order to analyze the pathogenesis of emotional disorders, but they are still not adequately understood. In the present study the migration, differentiation, and distribution of amygdalar neurons in the mouse embryo were investigated by means of in utero electroporation. Ventricular zone cells in restricted regions, that is, the caudal ganglionic eminence (CGE), the ventral pallium, the lateral pallium, and the diencephalon, were labeled with an expression vector of the enhanced green fluorescent protein (EGFP) gene. Labeling at embryonic day (E)10 revealed that the central nucleus originates from the neuroepithelium in the ganglionic eminence and the labeling at E11 and E12 revealed that the basolateral complex originates from the neuroepithelium of the ventral and lateral pallia. The introduction of the EGFP gene into the neuroepithelium of the third ventricle at E11 showed that the medial nucleus originates, at least in part, from the neuroepithelium of the diencephalon and migrates over the diencephalo-telencephalic boundary. The radial glial arrangement corresponded well with the initial migration of amygdalar neurons, and the radial processes later formed the boundary demarcating the basolateral complex. These findings indicate that the neurons originating from the temporally and spatially restricted neuroepithelium in both the telencephalon and diencephalon migrate and differentiate to form the mosaic of amygdalar subdivisions.