Tomio Okada

The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts

The effects of ultraviolet A (UVA) radiation and reactive oxygen species (ROS), generated with a xanthine and xanthine oxidase (XOD) system, on collagen enzymatic degradation involving the matrix metalloproteinase (MMP) and its tissue inhibitor of metalloproteinase (TIMP) were investigated using cultured human dermal fibroblasts. Total RNA was isolated and subjected to Northern blot analysis using cDNA clones for human interstitial collagenase (MMP-1), 72-kDa type IV collagenase (MMP-2) and TIMP-2. UVA irradiation resulted in an increase in MMP-1 mRNA up to 2.3-fold, but did not stimulate MMP-2 or TIMP-2 mRNA expression. In contrast, ROS induced by the xanthine and XOD system resulted in a dose-related increase in the level of MMP-2 mRNA up to 2.1-fold and a decrease in the level of TIMP-2 mRNA by 49% in the same fibroblasts. Catalase, used as scavenger, essentially prevented the ROS-induced alterations in MMP-2 and TIMP-2 mRNA levels. These results suggest that ROS produced in the dermis may contribute to biological changes in the connective tissue matrix observed in photoaging skin by accelerating the MMP-2-related matrix degradation system.

The effect of reactive oxygen species on the biosynthesis of collagen and glycosaminoglycans in cultured human dermal fibroblasts

The purpose of this study was to evaluate the possibility that the biological changes observed in connective tissue matrix components of photoaging skin may be induced by an alteration of biosynthesis in fibroblasts damaged by reactive oxygen species (ROS). We investigated the effect of ROS induced by xanthine and the xanthine oxidase system on the biosynthesis of connective tissue matrix components, collagen and glucosaminoglycans (GAGs) in cultured human dermal fibroblasts. ROS decreased collagen production and increased GAGs synthesis. Interestingly, these changes were consistent with the biological alterations of connective tissue matrix components observed in photoaging skin. Moreover, catalase and alpha-tocopherol completely prevented the ROS-induced alterations of collagen and GAGs biosynthesis, whereas superoxide dismutase had no effect on the ROS-induced changes. These results suggest that ROS may be one of the factors which cause the biological changes of connective tissue matrix components observed in photoaging skin.

Effect of Reactive Oxygen Species on the Elastin mRNA Expression in Cultured Human Dermal Fibroblasts

The effects of ultraviolet A (UVA) and reactive oxygen species (ROS), generated by a xanthine and xanthine oxidase (XOD) system, on the mRNA expression of elastin, were investigated using cultured human dermal fibroblasts. Total RNA was isolated and subjected to Northern blot analysis using synthesized 530 base cDNA probe for elastin with primers derived from exon 10 and 1 of human elastin. UVA irradiation did not affect elastin mRNA expression. In contrast, ROS resulted in a dose-related increase in the level of elastin mRNA up to 1.8-fold in cultured human dermal fibroblasts. Catalase, used as scavenger, essentially prevented the ROS induced alterations in elastin mRNA levels. These results suggest that ROS produced in the dermis may contribute to elastin deposition observed in photoaging skin.

Developmental expression of C3 receptor on murine epidermal Langerhans cells during ontogeny

SummaryThe developmental expression of C3 receptor, an important surface marker of murine epidermal Langerhans cells (LCs), was quantitatively studied using an immunohistochemical technique on epidermal sheets and then compared with developmental expression of Ia antigen and membrane ATPase. Anti-Mac-1 monoclonal antibody associated with CR3 was used for detecting C3 receptor and proved positive for LCs by immunoelectron microscopy. Mac-1 positive (Mac-1+) cells showed quite a different distribution from those of ATPase+ and Ia+ cells. Almost the same number of Mac-1+ and ATPase+ cells were present during the embryonic period. The number of Mac-1+ cells gradually decreased from day 1 to day 5 of postnatal life, after which they increased again. Using the doublelabeling technique on epidermal sheets at day 1 of postnatal life, it was shown that Ia+ cells possessed membrane ATPase activity and some Mac-1+ cells expressed Ia antigen. On days 4 and 7 of postnatal life all Mac-1+ cells expressed Ia antigen. These findings suggest that Mac-1 antigen observed during the embryonic period gradually fades after birth and is re-expressed after day 5 of postnatal life.

Abnormalities in Behavior, Learning Ability, and the Cholinergic System Induced by Long-Term Ultraviolet A Irradiation of Mice

Behavioral abnormalities, jumping reaction, increase in spontaneous activity abnormal violence, and lethargy were observed in long-term ultraviolet A (UVA)-irradiated hairy male Crj:CD-1 mice. The learning ability of 6- and 12-months UVA-irradiated mice was significantly reduced compared to un-irradiated age-matched mice. Acetylcholine levels, acetylcholinesterase and choline acetyltransferase activities in the whole brains were decreased in both of 6- and 12-month irradiated mice. Only 1 of 6 mice irradiated for 12 months was histologically observed to have a drastic loss of bilateral hippocampal pyramidal cells in the CA1 field of Ammons horn.

Effect of Harb Extract on Cross-linking of Collagen.

Wrinkle formation is associated with alteration in the extracellular matrix, collagen and elastic fibers in the dermis of the skin. Cross-linking of collagen is a typical alteration and increases with aging. Glycation (nonenzymatic glycosylation) is the so called Maillard reaction and is a posttranslational modification of proteins resulting from reactions of glucose with amino groups in protein. Collagen cross-linking is also caused by the Maillard reaction.In this study, 10 harb extracts used in the prescriptions for wrinkle removal in ancient Chinese medical literature, were selected and studied for their capacity to collagen cross-linking formation induced by the Maillard reaction. Bukuryou and Byakuren were the most effective inhibitors and Byakuren showed the same effect with aminoguanidine as a positive control. Byakuren also inhibited the formation of fluorescent materials due to the Maillard reaction. These findings may possibly be related to control of the advanced stage of the Maillard reaction.

Incidence and Regional Differences of Parakeratotic Cells on Human Facial Skin

The purpose of this study was to investigate the incidence of parakeratotic stratum corne um cells on the cheek skin surface from 335 healthy subjects and the changes of them accor ding to their ages. We also studied the quantitative levels of parakeratotic cells in various regions of face. Stratum corneum cells were collected by tape-stripping method and observ ed with hematoxyrin-eosin stain after formalin fixation. In our results, parakeratotic cells were observed in 68.4% of the total subjects, 64.4% of them were women and 72.7% of them were men. They were observed at about 80% in second and third decades of women and decreased as the age advanced. On the other hand, they were observed at about 80% after the first decade in men. Quantity of parakeratotic cells was the highest in upper cheek and followed by the forehead and nose. They were seldom observed below the cheek and chin. It was suggested that the parakeratotic cells were appeared in the stratum corneum cells in face, because our facial skin is always exposed to many external stimulations such as ultraviolet rays and so on and turn over of epidermis was accelerated. Moreover, we strongly suggested that sebum is one of the factor causing parakeratosis. Because parakeratotic cells were observed more frequently in the region where sebum level was high and most of them were observed around the pore of the hairs. Appearance of parakeratotic cells on skin surface implied the loss of skin barrier function and was important problem to the beauty and healthiness of skin.