Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.
Neuroblastomas, an embryonic cancer of the sympathetic nervous system, often occur in young children. Honokiol, a small-molecule polyphenol, has multiple therapeutic effects and pharmacological activities. This study was designed to evaluate whether honokiol could pass through the blood-brain barrier (BBB) and induce death of neuroblastoma cells and its possible mechanisms. Primary cerebral endothelial cells (CECs) prepared from mouse brain capillaries were cultured at a high density for 4 days, and these cells formed compact morphologies and expressed the ZO-1 tight-junction protein. A permeability assay showed that the CEC-constructed barrier obstructed the passing of FITC-dextran. Analyses by high-performance liquid chromatography and the UV spectrum revealed that honokiol could traverse the CEC-built junction barrier and the BBB of ICR mice. Exposure of neuroblastoma neuro-2a cells and NB41A3 cells to honokiolinduced cell shrinkage and decreased cell viability. In parallel, honokiol selectively induced DNA fragmentation and cell apoptosis rather than cell necrosis. Sequential treatment of neuro-2a cells with honokiol increased the expression of the proapoptotic Bax protein and its translocation from the cytoplasm to mitochondria. Honokiol successively decreased the mitochondrial membrane potential but increased the release of cytochrome c from mitochondria. Consequently, honokiol induced cascade activation of caspases-9, -3, and -6. In comparison, reducing caspase-6 activity by Z-VEID-FMK, an inhibitor of caspase-6, simultaneously attenuated honokiol-induced DNA fragmentation and cell apoptosis. Taken together, this study showed that honokiol can pass through the BBB and induce apoptotic insults to neuroblastoma cells through a Bax-mitochondrion-cytochrome c-caspase protease pathway. Therefore, honokiol may be a potential candidate drug for treating brain tumors.
Purpose.: To investigate the protective effects of glucosamine (GlcN) using oxidative stress and rat models of ischemia-reperfusion (I/R) injury and to determine the antiapoptotic and anti-inflammatory mechanisms of GlcN treatment. Methods.: We determined the effects of GlcN and the levels of O-linked N-acetylglucosamine (O-GlcNAc) in in vitro retinal ganglion cells (RGCs) treated with or without H2O2. The survival and apoptosis rates of RGCs were compared after the addition of GlcN, glucose, or O-(2-acetamido-2-deoxy-Dglucopyranosylidene) amino-N-phenylcarbamate (PUGNAc). Retinal I/R injury was induced in Sprague-Dawley rats by elevating the IOP to 110 mm Hg for 60 minutes. An intraperitoneal injection of GlcN (1000 mg/kg) or normal saline was administered in the different groups, including a control group, a GlcN group, an I/R group, a GlcN+I/R group (1000 mg/kg GlcN 24 hours before I/R injury), and an I/R+GlcN group (7-day period of 1000 mg/kg GlcN 24 hours after I/R injury). The rats were killed 7 days after the I/R injury, and the retinas were collected from each rat for thickness measurements. Quantitative analysis of RGC survival was further determined using labeling with FluoroGold. Results.: The GlcN increased levels of O-GlcNAc in a dose-dependent manner in the RGCs treated with or without H2O2. The GlcN resulted in increased cell survival and reduced apoptosis in the RGCs under oxidative stress conditions. In the rat model of I/R injury, GlcN significantly protected against I/R-induced retinal thinning and suppressed the I/R-induced reductions in a- and b-wave amplitudes of the ERG. In terms of RGC survival, significant incremental density of RGCs was found in the I/R+GlcN group compared with the I/R group. Notably, the use of GlcN in the rat retina decreased apoptosis and the formation of reactive oxygen species (ROS) after I/R injury. We also found that mitogen-activated protein kinase signal pathways played a critical role in the GlcN-mediated attenuation of ROS-induced damage in vitro and I/R injury in vivo. Conclusions.: Glucosamine treatment provides multiple levels of retinal protection, including antiapoptotic, anti-inflammatory, and antioxidative benefits. More research on the role of GlcN as a potential agent for the prevention and treatment of glaucoma is warranted.
Purpose A prospective study was performed to evaluate refractive and ocular biometric changes in acute hyperglycemic status in patients with diabetes mellitus. Methods From January to August 2002, 48 eyes of 24 patients with persistent diabetes and a plasma glucose level 17 mmol/L or HbA1c 10.0% on admission were enrolled in this prospective study. Upon admission to Tri-Service General Hospital in Taipei, Taiwan, these patients underwent intensive glycemic control. The basic ophthalmic examinations, including visual acuity, intraocular pressure measurement, slit lamp, and fundus examinations, were conducted. The ocular parameters including refraction, anterior chamber depth, lens thickness, axial length, mean keratometry and thinnest corneal thickness were evaluated by A-mode scan and Orbscan II. Each patient underwent clinical follow-up visits at 1, 2, and 4 weeks after the acute hyperglycemic episode. Results Of the 24 patients, 18 were male and 6 were female. The mean age of the patients was 55 years (range: 38 to 69). Comparing the refractive status on admission and at week 4, the authors found that 8 cases (16 eyes, 33%) showed hyperopia during hyperglycemia (+1.9±0.8 D), but in the other 16 cases (32 eyes, 67%), there were no significant changes. In addition, there were also no significant changes in anterior chamber depth, lens thickness, axial length, thinnest corneal thickness, or mean keratometry in the follow-up period. Conclusions Transitory hyperglycemia produces hyperopia. The alteration in refractive index in the lens may contribute to the hyperopic change, but no change of ocular biometrics in lens or cornea is observed.
Corneal dystrophy of the Bowman layer is typically recognised as Reis–Bucklers corneal dystrophy or Thiel–Behnke corneal dystrophy (TBCD) with different clinical presentations, featuring a progressive autosomal dominant dystrophy characterised by bilateral, symmetrical opacities located in the Bowman layer in early childhood.1 In vivo confocal microscopy (IVCM) may be a useful auxiliary diagnostic tool in the differentiation among the different spectrum of corneal dystrophy. We described three cases of TBCD with R555Q mutation in TGFBI/BIGH3 had distinct presentations in the findings of IVCM.
This study describes one family with history of corneal dystrophy of the Bowman layer (figure 1). Three patients in the same family were diagnosed as having TBCD. All affected individuals received molecular genetic survey and IVCM after giving written informed consent. All medical records and clinical photographs were examined. After adequate anaesthesia and application of a sterile non-applanating lens, the central and peripheral regions of cornea were examined …
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