Makoto Takehana

Production of Overmodified Type I Procollagen in a Case of Osteogenesis Imperfecta

Collagen synthesis in cultured skin fibroblasts from a patient with osteogenesis imperfecta was studied. Approximately 2 fold accumulation of collagen in the cell layer was found. The slower mobility of proα1 (I) and proα2 (I) as well as α1 and α2 (I) polypeptide on sodium dodecylsulfate‐polyacrylamide gel electrophoresis was detected, indicating that abnormal posttranslational modification could be present in type I procollagen in patient fibroblasts. The degrees of hydroxylation and subsequent glycosylation of lysine residues in the affected collagen were elevated 1.5 and 1.4 fold, respectively. There were no significant changes in the relative content of type III to type I collagen nor the incorporation of mannose into the carboxyterminal propeptide of proα1 (I) and proα2 (I). These results indicate that the patient produces an over‐modified type I procollagen which is responsible for the clinical features and has a collagen abnormality already reported in type II osteogenesis imperfecta.

Quantitative analysis of ascorbic acid permeability of aquaporin 0 in the lens

Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10 min after incubation in medium containing 1000 μM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0. AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner. The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.

Ascorbate free radical reductase activity in vertebrate lenses of certain species

PURPOSEnTo clarify the function of ascorbate free radical (AFR) reductase in the antioxidation system of different vertebrate lenses.nnnMETHODSnThe soluble and insoluble fractions were prepared from bullfrog, guinea pig, rat, rabbit, swine, and bovine lenses, and membrane-bound enzymes in the insoluble fraction were extracted by 0.3% Triton X-100. Ascorbate free radical reductase and diaphorase activities in each fraction were determined.nnnRESULTSnAscorbate free radical reductase activity in the lens soluble fraction was the highest in the bullfrog. That in the guinea pig and rabbit was at the next level. There was only a little activity in rat and swine lenses, and none was detected in the bovine lenses. However, a large species difference in AFR reductase activity was not observed in the 0.3% Triton X-100 extracts. Diaphorase activity was three to nine higher than AFR reductase activity in the soluble fractions of bullfrog, guinea pig, and rabbit. In the 0.3% Triton X-100 extracts of all animal species used, it was very high, 108 to 311 times the AFR reductase activity.nnnCONCLUSIONnThese results indicate that the lens soluble and membrane-bound AFR reductase in the different animals may be individual enzyme molecules and have different antioxidative functions. Because the lenses of bullfrog, guinea pig, and rabbit are known to contain a near-ultraviolet (UV) light-absorbing compound, reduced pyridine nucleotide, at a high concentration, the soluble AFR reductase activity is expected to be high in the vertebrate lenses with a near-UV light filter, to enhance the antiphoto-oxidation capacity of ascorbate.

The Extracellular C-loop Domain Plays an Important Role in the Cell Adhesion Function of Aquaporin 0.

ABSTRACT Purpose: Although aquaporin 0 (AQP0) is a member of the AQP family, it has limited water permeability compared with other members. AQP0 may also have cell adhesion-related functions, but the evidence is still limited. Here, we studied the relationship of AQP0 to cell adhesion and determined the region required for cell adhesion.Methods: L-cell fibroblasts stably expressing AQP0 or AQP1 (L-AQP0 or L-AQP1) were established. One group of cells was stained with CellTracker Red and cultured into a confluent monolayer, whereas the other group was loaded with CellTracker Blue and seeded over the monolayer. To study cell adhesion, the percentages of lower and upper layer cells were measured using flow cytometry. To determine the region of AQP0 required for adhesion, activity was done by pull-down assay using glutathione S-transferase fusion proteins. To study the water permeability, Xenopus laevis oocyte expressing AQP0 wild-type or AQP0 mutated in C-loop was transferred to a hypotonic solution and photographed, and the diameter was measured to calculate the volume.Results: More cells adhered to the lower cells in the L-AQP0 homotypic pair than other pairs such as L-AQP1 homotypic or L-AQP0/L-AQP1 heterotypic pairs. Pull-down assays revealed that AQP0 could bind to itself via the C-loop extracellular domain. Furthermore, we determined that 109Pro and 110Pro in the C-loop were important for cell adhesion. However, mutation of the C-loop in AQP0 did not affect its water permeability.Conclusions: AQP0 is known to bind lipids in the opposing membrane. Our data suggest that this cell-to-cell adhesion occurs not only in the AQP0/liquids but also via AQP0/AQP0 interaction through the C-loop domain. Mutations in the C-loop amino acids did not affect the water permeability of AQP0 but did affect its cell adhesion function. These independent dual functions of AQP0 are important for lens transparency.

Presence of the factor that inhibits collagen synthesis and stimulates cell proliferation in developing embryonic chick skin extracts

The effects were studied of the extracts prepared from 10- and 18-day developing chick skins on collagen synthesis and proliferation of the monolayered fibroblasts established from 10-day skin. Cell proliferation was stimulated by both 10- and 18-day skin extracts at the concentration of 3.5 micrograms/ml. Collagen synthesis was suppressed by the treatment of extracts prepared from both 10- and 18-day skins at the concentration of 3.5 micrograms/ml, to a greater extent in 18-day extract. The results indicate that skin extracts possess factors exhibiting both a stimulating effect on cell proliferation and an inhibitory effect on collagen synthesis.