The present study was undertaken to find out the role of NO on cataractogenesis in the experimenally-induced uveitis(EIU) model. Nitrite and nitrate, stable oxidative products of nitric oxide(NO), were measured in the aqueous humor and the progression of inflammations and lens opacities were evaluated with slit lamp biomicroscope. Immunoperoxidase staining and immunofluorescent staining for inducible NO synthase(iNOS) and peroxynitrite were performed to confirm the site of production of NO and peroxynitrite. The grades of inflammation were peaked at 24 hours inflammation was gradually decreased after 48 hours and lens opacity after 72 hours. These changes returned to the baseline level by one week after LPS injection. Similarly, NO concentration in aqueous humor was peaked at 24 hours. And it was then decreased after 48 hours and returned to the baseline level by one week. These inflammatory signs and lens opacities were significantly decreased in NG-nitro-L-arginine methyl ester(L-NAME) administrated group. Inflammatory cells in anterior chamber, iris, and ciliary body expressed highly iNOS which was coincide with peroxynitrite immunolocalization. Therefore, these results suggest that cataract formation in EIU is related to the NO production in aqueous humor. Furthermore, lipid peroxidation by peroxynitrite is possibly related with cataractogenesis in EIU. But, we need a further evaluation to seek the relationship between cataractogenesis and increased nitric oxide concentration, combined with studies of other biochemical changes in anterior chamber and lens.
Anterior Chamber ; Aqueous Humor ; Cataract* ; Ciliary Body ; Inflammation ; Iris ; Lipid Peroxidation ; Nitric Oxide ; Nitroarginine ; Peroxynitrous Acid ; Uveitis*

The role of nitric oxide (NO) in ocular surface diseases remains unknown. We investigated the conditions leading to increase NO generation in tears and the main sources of ocular surface tissue. We evaluated the possibility of a dual action (cell survival or cell death) depending on the amount of NO. The concentration of nitrite plus nitrate, the stable end-product of NO, was measured in the tears of various ocular surface diseases. We also examined the main source of nitric oxide synthase (NOS) using immunohistochemical staining & Western blot analysis. When cultured human corneal fibroblasts were treated with NO producing donor with or without serum, the viability of cells was studied. We found that sources of NO in ocular surface tissue primarily included corneal epithelium, fibroblasts, endothelium and inflammatory cells. Three forms of NOS (eNOS, bNOS, & iNOS) were expressed in experimentally induced inflammation. Cell death by NO revealed TUNEL positive staining, however in the EM finding, this NO specific cell death was an atypical necrosis showing perinuclear large vacuolization and mitochondrial swelling. In the fibroblasts culture system, the NO donor (SNAP, S-nitroso-N-acetyl-D, L-penicillamine) prevented the death of corneal fibroblasts caused by serum deprivation in a dose dependent manner up to 500 m SNAP, although a higher dose decreased cell viability. This study suggested that NO might act as a double-edged sword in ocular surface disease depending on the degree of inflammatory condition related with NO concentration.
Animal ; Cells, Cultured ; Cornea/metabolism ; Eye Diseases/*physiopathology ; Human ; Nitric Oxide/*metabolism ; Tears/metabolism

We previously reported an identification of a 77-kDa GTP-binding protein that co-purified with the alpha 1-adrenoceptor following ternary complex formation. In the present paper, we report on the purification and characterization of this GTP-binding protein (termed G alpha h5) isolated from pig heart membranes. After solubilization of pig heart membranes with NaCl, G alpha h5 was purified by sequential chromatographies using DEAE-Cellulose, Q-Sepharose, and GTP-agarose columns. The protein displayed high-affinity GTP gamma S binding which is Mg(2+)-dependent and saturable. The relative order of affinity of nucleotide binding by G alpha h5 was GTP > GDP > ITP >> ATP > or = adenyl-5'-yl imidodiphosphate, which was similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, the G alpha h5 demonstrated transglutaminase (TGase) activity that was blocked either by EGTA or GTP gamma S. In support of these observations, the G alpha h5 was recognized by a specific antibody to G alpha h7 or TGase II, indicating a homology with G alpha h (TGase II) family. These results demonstrate that 77-kDa G alpha h5 from pig heart is an alpha 1-adrenoceptor-coupled G alpha h (TGase II) family which has species-specificity in molecular mass.
Animal ; Binding Sites ; Binding, Competitive ; Cross Reactions ; GTP-Binding Proteins/metabolism* ; GTP-Binding Proteins/isolation & purification* ; GTP-Binding Proteins/immunology ; Guanosine 5'-O-(3-Thiotriphosphate)/metabolism ; Molecular Weight ; Myocardium/chemistry* ; Protein-Glutamine gamma-Glutamyltransferase/metabolism ; Receptors, Adrenergic, alpha-1/metabolism ; Swine

This study investigated the effects of proline-serine (PS) and valine-serine (VS) dipeptides on melanogenesis in Mel-Ab cells. Proline-serine and VS significantly inhibited melanin synthesis in a concentration-dependent manner, though neither dipeptide directly inhibited tyrosinase activity in a cell-free system. Both PS and VS down-regulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. In a follow-up study also described here, the effects of these dipeptides on melanogenesis-related signal transduction were quantified. Specifically, PS and VS induced ERK phosphorylation, though they had no effect on phosphorylation of the cAMP response element binding protein (CREB). These data suggest that PS and VS inhibit melanogenesis through ERK phosphorylation and subsequent down-regulation of MITF and tyrosinase. Properties of these dipeptides are compatible with application as skin-whitening agents.
Cell-Free System ; Cyclic AMP Response Element-Binding Protein ; Dipeptides ; Down-Regulation ; Follow-Up Studies ; Melanins ; Microphthalmia-Associated Transcription Factor ; Monophenol Monooxygenase ; Phosphorylation ; Signal Transduction

In this study we investigated the effects of fucoidan on the proliferation of fibroblasts and the reconstruction of a skin equivalent (SE). Fucoidan significantly stimulated the proliferation of CCD-25Sk human fibroblasts and Western blot analysis demonstrated that fucoidan markedly increased the expression of cyclin D1 and decreased the expression of p27. Fucoidan was used to reconstruct SE. Immunohistochemical staining showed that the addition of fucoidan to dermal equivalents increased expression of proliferating cell nuclear antigen (PCNA) and p63. In addition, expression of alpha6-integrin was significantly increased by fucoidan, whereas expression of beta1-integrin, type 1 collagen, elastin, fibronectin did not markedly change. These results suggest that fucoidan has positive effects on epidermal reconstruction and will therefore be beneficial in the reconstruction of SE.
Blotting, Western ; Collagen Type I ; Cyclin D1 ; Elastin ; Fibroblasts ; Fibronectins ; Humans ; Proliferating Cell Nuclear Antigen ; Skin*

Fucoidan, a fucose-rich sulfated polysaccharide derived from brown seaweed in the class Phaeophyceae, has been widely studied for its possible health benefits. However, the potential of fucoidan as a possible treatment for hyperpigmentation is not fully understood. This study investigated the effects of fucoidan on melanogenesis and related signaling pathways using Mel-Ab cells. Fucoidan significantly decreased melanin content. While fucoidan treatment decreased tyrosinase activity, it did not do so directly. Western blot analysis indicated that fucoidan downregulated microphthalmia-associated transcription factor and reduced tyrosinase protein expression. Further investigation showed that fucoidan activated the extracellular signal-regulated kinase (ERK) pathway, suggesting a possible mechanism for the inhibition of melanin synthesis. Treatment with PD98059, a specific ERK inhibitor, resulted in the recovery of melanin production. Taken together, these findings suggest that fucoidan inhibits melanogenesis via ERK phosphorylation.
Blotting, Western ; Hyperpigmentation ; Insurance Benefits ; Melanins ; Microphthalmia-Associated Transcription Factor ; Monophenol Monooxygenase ; Phaeophyta ; Phosphorylation ; Phosphotransferases ; Seaweed

The purpose of this study is to characterize the effects of KHG26792 (3-(naphthalen-2-yl(propoxy) methyl)azetidine hydrochloride), a potential skin whitening agent, on melanin synthesis and identify the underlying mechanism of action. Our data showed that KHG26792 significantly reduced melanin synthesis in a dose-dependent manner. Additionally, KHG26792 downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase, the rate-limiting enzyme in melanogenesis, although tyrosinase was not inhibited directly. KHG26792 activated extracellular signal-regulated kinase (ERK), whereas an ERK pathway inhibitor, PD98059, rescued KHG26792-induced hypopigmentation. These results suggest that KHG26792 decreases melanin production via ERK activation. Moreover, the hypopigmentary effects of KHG26792 were confirmed in a pigmented skin equivalent model using Cervi cornus Colla (deer antler glue), in which the color of the pigmented artificial skin became lighter after treatment with KHG26792. In summary, our findings suggest that KHG26792 is a novel skin whitening agent.
Animals ; Antlers ; Cornus ; Hypopigmentation ; MAP Kinase Signaling System ; Melanins* ; Microphthalmia-Associated Transcription Factor ; Monophenol Monooxygenase ; Phosphotransferases ; Skin Lightening Preparations ; Skin* ; Skin, Artificial