Elastin expression in cultured human keratinocytes: exon 26A of elastin primary transcript is always included in terminally differentiated keratinocytes.
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Alternative Splicing↗
Biomedical subjects
M Takehana
Publications and source records attributed to M Takehana.
Expression of a small heat shock protein 27 (HSP27) in mouse skin tumors induced by UVB-irradiation.
We investigated the expression of heat shock protein 27 (HSP27) at intermediate stages of a cutaneous tumor induced by UVB-irradiation stress (290-380 nm, max. 312 nm) using an immunostaining method. After 15-20 weeks of chronic exposure to UVB irradiation at a dose of 2 kJ/m2, HSP27 was found in the upper cell layers of bowenoid multilayers of epidermis, in areas of the lesions where normal stratification seems to be conserved. After 25 weeks, HSP27 was weakly expressed in squamous cell carcinoma (SCC). The HSP27 distribution patterns during cutaneous tumor progression resemble that of cytokeratin 10, a differentiation marker in keratinocytes. In SCC, a low degree of HSP27 expression was detected in the well-differentiated carcinomatous areas, but not in the poorly differentiated areas. These results indicate that the level of HSP27 decreases significantly as epithelial carcinoma growth progresses upon UVB-exposure. The expression of HSP27 may be associated with the onset of skin keratinocyte differentiation, but not with progression of SCC.
Animals↗
NADH-dependent dehydroascorbate reductase in the rabbit lens.
The present investigation demonstrates the existence of NADH-dependent dehydroascorbate (DHA) reductase activity in the soluble fraction of the rabbit lens. This DHA reductase was specific for NADH, and its apparent Km values for DHA and NADH were 5.7 mM and 4.0 microM, respectively. In a gel filtration of the lens soluble fraction on a Sephadex G-75 superfine column, the NADH-dependent DHA reductase activity was eluted at the oligomeric betaL1-crystallin fraction, which may also contain lambda-crystallin (a rabbit-specific crystallin). Furthermore, about 80% of protein fractions separated from the betaL1-crystallin fraction by DEAE-cellulose ion-exchange column chromatography exhibited DHA reductase activity. In the SDS-PAGE analysis of the protein fractions with DHA reductase activity, 32-33, 27 and 25 kDa protein subunits were commonly identified. These results suggest that oligomers of beta-crystallin and/or lambda-crystallin subunits may be associated with the DHA reductase activity. The present paper also discusses that the function of the reductase may be to enhance the antiphotoxidation capacity of the lens.
Animals↗
Ascorbate Free Radical Reductase Activity in Vertebrate Lenses of Some Species.
Purpose: To clarify the function of ascorbate free radical (AFR) reductase in the lens antioxidation mechanism, we investigated the difference among species in AFR reductase activity in different vertebrate lenses.Materials and Methods: Soluble and insoluble fractions were prepared from the lenses of frogs, guinea pigs, rats, rabbits, pigs, and calves. AFR reductase and diaphorase activity of each fraction was determined.Results: AFR reductase activity in the lens soluble fraction was the highest in frogs. That of guinea pigs and rabbits was at the next level; there was only a little activity in rats and pigs, and none was detected in calves. Membrane-bound AFR reductase in the lens insoluble fraction was extracted by 0.3% Triton X-100. The membrane-bound enzyme activity was almost at the same level in frogs, rats, rabbits, and calves, and a little higher in guinea pigs and pigs. However, such species-specificity of AFR reductase activity as in the soluble fraction was not observed in 0.3% Triton X-100 extracts. Diaphorase activity was 3 to 9 times as much as AFR reductase activity in the soluble fractions of frogs, guinea pigs, and rabbits, but in 0.3% Triton X-100 extracts of all vertebrate species used, it was very high, 108 to 311 times the AFR reductase activity.Conclusion: These results suggest that the lens soluble and membrane-bound AFR reductases are individual enzyme molecules and have different anti-oxidative functions. The lenses of frogs, guinea pigs, and rabbits contain a near-ultraviolet (UV) light absorbing compound, reduced pyridine nucleotide at a high concentration. Therefore, the soluble AFR reductase activity may be high in the vertebrate lenses with a near-UV light filter, and enhance the antiphotoxidation of ascorbic acid.
Journal Article↗
[Ascorbate free radical reductase activity in vertebrate lenses of some species].
PURPOSE: To clarify the function of ascorbate free radical (AFR) reductase in the lens antioxidation mechanism, we investigated the difference among species in AFR reductase activity in different vertebrate lenses. MATERIALS AND METHODS: Soluble and insoluble fractions were prepared from the lenses of frogs, guinea pigs, rats, rabbits, pigs, and calves. AFR reductase and diaphorase activity of each fraction was determined. RESULTS: AFR reductase activity in the lens soluble fraction was the highest in frogs. That of guinea pigs and rabbits was at the next level; there was only a little activity in rats and pigs, and none was detected in calves. Membrane-bound AFR reductase in the lens insoluble fraction was extracted by 0.3% Triton X-100. The membrane-bound enzyme activity was almost at the same level in frogs, rats, rabbits, and calves, and a little higher in guinea pigs and pigs. However, such species-specificity of AFR reductase activity as in the soluble fraction was not observed in 0.3% Triton X-100 extracts. Diaphorase activity was 3 to 9 times as much as AFR reductase activity in the soluble fractions of frogs, guinea pigs, and rabbits, but in 0.3% Triton X-100 extracts of all vertebrate species used, it was very high, 108 to 311 times the AFR reductase activity. CONCLUSION: These results suggest that the lens soluble and membrane-bound AFR reductases are individual enzyme molecules and have different antioxidative functions. The lenses of frogs, guinea pigs, and rabbits contain a near-ultraviolet (UV) light absorbing compound, reduced pyridine nucleotide at a high concentration. Therefore, the soluble AFR reductase activity may be high in the vertebrate lenses with a near-UV light filter, and enhance the antiphotoxidation of ascorbic acid.
Animals↗
Protective effects of sodium-L-ascorbyl-2 phosphate on the development of UVB-induced damage in cultured mouse skin.
The protective effect of sodium-L-ascorbyl-2 phosphate (As-2P), a stable form of ascorbic acid (AsA), against photodamage induced by a single dose of UVB exposure (290-320 nm, Max 312 nm) was investigated using cultured mouse skin. When the cultured skin was treated with various As-2P concentrations, the cutaneous AsA level increased in proportion to the As-2P concentration. After 3 h of incubation, the AsA level in the cultured skin treated with 2, 20 and 100 mM As-2P increased 1.03-, 2.17- and 6.27-fold, respectively, compared with that of the control skin. These results suggest that As-2P was transported into the cultured mouse skin where it was converted to AsA. After 3 h, the cutaneous AsA level in irradiated (20 kJ/m2) skin was depleted to a half of that in the control skin. However, the level in skin pretreated with 20 mM As-2P was maintained within normal limits, even after 24 h. Pretreatment with 20 mM As-2P significantly prevented such photodamage as sunburn cell formation, DNA fragmentation and lipid peroxidation, which were caused by a single dose of UVB irradiation. These results suggest that the protective effect of 20 mM As-2P on UVB-induced cutaneous damage is due to the maintenance of a normal As level by conversion of As-2P to As in skin tissue.
Animals↗
Expression of sialylated Lewisx gangliosides in cultured lens epithelial cells from rhesus monkey.
Monkey and human lenses contain essentially the same glycosphingolipids, and Lewisx and sialylated Lewisx epitopes are expressed on the terminal structure of neolactotetraosylceramide. However, monolayer cultures of lens epithelial cells from rhesus monkey expressed gangliosides GM3, GD3 and a small amount of GM1, but not sialylated Lewisx epitopes. Eight-week-old cultures on various extracellular matrices resulted in morphological changes in lens epithelial cells. Monolayer of cells cultured on vitronectin or polylysine assembled into aggregates after 4 weeks of culture. Cells cultured on vitronectin expressed sialyl-Lewisx gangliosides and did not exhibit GD3. On collagens, fibronectin and laminin elongated cells were observed in cells cultured for 8 weeks. Thus, the interaction between cells and extracellular matrices influenced morphology and glycosphingolipid composition in lens epithelial cells.
Animals↗
Postadministration protective effect of magnesium-L-ascorbyl-phosphate on the development of UVB-induced cutaneous damage in mice.
The effects of stable vitamin C, magnesium-L-ascorbyl-2-phosphate (MAP), administered after acute and chronic exposure to UVB irradiation were investigated using hairless mice. Intraperitoneal administration of 100 mg/kg of MAP immediately after acute exposure to 15 kJ/m2 of UVB significantly prevented increases of UVB-induced lipid peroxidation in skin and sialic acid in serum, an inflammation marker. Administration of 50 mg/kg of MAP immediately after each exposure significantly delayed skin tumor formation and hyperplasia induced by chronic exposure to 2 kJ/m2 of UVB. Intraperitoneal administration of 200 mg/kg of MAP produced an increase in ascorbic acid (As) levels in the serum, liver and skin within 15 min. Serum As levels quickly returned to normal, but hepatic and cutaneous levels remained elevated before returning to normal after 24 h, suggesting that MAP was converted to As in the serum and in those tissues. Ultraviolet B-induced hydroxyl radical generation in murine skin homogenates was scavenged by As-Na addition, which was directly detected by electron spin resonance (ESR). These results suggest that postadministration of MAP delays progression of skin damage induced by UVB irradiation. It is presumed that MAP, once converted to As, exhibits such inhibitory effects by scavenging hydroxyl and lipid radicals generated as a direct or indirect result of UVB exposure.
Animals↗
Evidence for the involvement of calpain in cataractogenesis in Shumiya cataract rat (SCR).
The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in the nuclear and perinuclear portions at 11-12 weeks of age. It was found that the proteolysis of some crystallins and cytoskeletal proteins is significantly enhanced in cataractous SCR lenses. The calcium concentrations in cataractous lenses rise markedly with age as compared with control lenses and the autolytic product of calpain is also detected in cataractous lenses. In order to provide direct evidence for the involvement of calpain in the proteolytic modification of lens proteins, we developed antibodies exclusively specific to the proteolytic products of some lens proteins produced by the action of calpain and analyzed their degradation during cataractogenesis in SCR by Western blotting and immunohistochemical staining. The results demonstrate that calpain participates in the proteolytic modification of lens proteins, at least alpha-crystallin (A and B chain), betaB1-crystallin, and alpha-fodrin. The proteolytic products formed by the action of calpain on these proteins are detected in cataractous lenses of SCR as young as 8 weeks of age and accumulate with age. It was also found that betaB1-crystallin, originally a soluble protein, is converted to an insoluble form by limited calpain proteolysis. The chaperon-like activity of alpha-crystallin from control lens is markedly reduced by calpain proteolysis in vitro, and alpha-crystallin in opaque lens that has already undergone proteolysis by calpain shows significantly reduced chaperon-like activity. Immunohistochemical studies reveal that the area where the calpain-mediated alpha-crystallin proteolysis is in progress coincides well with the area developing and destined to develop the opacification. These results strongly suggest that calpain may contribute to lens opacification during cataract formation in SCR.
Aging↗
Specific racemization and isomerization of the aspartyl residue of alphaA-crystallin due to UV-B irradiation.
We have reported that the aspartyl (Asp)-151 residue in alphaA-crystallin in human eye lens was inverted to the D-isomer and isomerized to beta-Asp residue with age. We report here that ultraviolet (UV)-B irradiation induces the racemization and isomerization of the Asp-151 residue of alphaA-crystallin from lenses of 6-week-old rats to form D-isomer and beta-Asp residue. Simultaneous racemization and isomerization of the specific Asp residue indicate that the reaction proceeds via formation of a succinimide intermediate. This modification was not observed in UV-A irradiated and normal lenses. UV-B irradiation induced the racemization of only the Asp-151 residue and did not affect the other Asp residues in alphaA-crystallin. On the other hand, the high molecular weight fraction of the lens protein increased upon UV-B irradiation. Modification of the Asp residue would affect the three-dimensional packing array of the lens protein.
Amino Acid Sequence↗
UVB irradiation induces changes in cellular localization and phosphorylation of mouse HSP27.
We investigated the induction, cellular localization and phosphorylation of a low-molecular weight stress protein (heat shock protein 27, HSP27) by UVB (290-320 nm, max. 312 nm) irradiation stress using immunoblot and indirect immunofluorescence analysis in in vivo and in vitro experiments. The HSP27 was constitutively expressed and distributed in the cytoplasmic fraction of Pam 212 cells (mouse keratinocyte line) or dorsal skin. The increase in the cytoplasm HSP27 level induced by UVB irradiation was less than two-fold that in nonirradiated controls. On the other hand, the translocation of HSP27 from cytoplasm to the nucleus or perinuclear area was time- and dose-dependently induced by UVB irradiation. After UVB irradiation, three isoforms having different isoelectric points were detected in nucleic HSP27 by two-dimensional immunoblotting. The most basic isoform was the unphosphorylated type and the two acidic isoforms were phosphorylated, suggesting that HSP27 is phosphorylated in response to UVB irradiation and accumulates in or around the nucleus as a phosphorylated isoform. These results suggest that the translocation and phosphorylation of HSP27 are induced in response to UVB-irradiation stress.
Alkaline Phosphatase↗
Protective effect of magnesium-L-ascorbyl-2 phosphate against skin damage induced by UVB irradiation.
The protective effect of magnesium-L-ascorbyl-2-phosphate (MAP) on cutaneous photodamage such as lipid peroxidation and inflammation induced by ultraviolet B (UVB) exposure (290-320 nm, max. 312 nm) was investigated using hairless mice. When MAP was administered intraperitoneally to mice at a dose of 100 mg of ascorbic acid (AS) per kg body weight base immediately before irradiation (15 kj/m2), the expected increases in thiobarbituric acid reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, were significantly reduced. However, the expected decrease in the level of cutaneous AS was unchanged. Similar results were observed for animals given 100 mg of AS-Na per kg body weight before UVB irradiation. When MAP was administered intracutaneously immediately before irradiation, the expected UVB-induced increases in TBARS and sialic acid were again significantly prevented. Ascorbic acid-Na had a less protective effect than intracutaneous MAP administration. The cutaneous AS level was significantly higher in the MAP-treated mice than in the controls, and the UVB-induced decrease in tissue AS was prevented by intracutaneous MAP administration. These results suggest that MAP protects against UVB irradiation-induced lipid peroxidation and inflammation in cutaneous tissue, regardless of the drug administration route. We found, in an in vitro experiment, that MAP was converted to AS as it crossed the epidermis, but that AS-Na did not pass through the epidermis. Furthermore, MAP was also converted to AS in serum. These results suggest that the protective effect of MAP on UVB-induced cutaneous damage is due to conversion of MAP to AS.
Animals↗
Glutathione isopropyl ester reduces UVB-induced skin damage in hairless mice.
The protective effect of administration of glutathione (GSH) isopropyl ester on photodamage, such as lipid peroxidation, inflammation and tumorigenesis induced by UV exposure (290-400 nm, max. 312 nm), was investigated using hairless mice. Pretreatment with 20 mg/kg GSH isopropyl ester prevented the increases of thiobarbituric acid-reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, which were caused by a single dose (15 kJ/m2) of UV irradiation. The level of epidermal GSH in skins of the GSH ester-treated mice was maintained within normal limits. When mice were exposed to UV at a dose of 2 kJ/m2, three times weekly, skin tumors developed in all of them after 25 weeks. The formation of skin tumors was significantly inhibited by administration of 10 mg/kg GSH ester prior to each UV irradiation for 25 weeks. Moreover, the increases of cutaneous TBARS and serum sialic acid in the tumor-bearing mice were also prevented by continuous pretreatment with GSH ester. Even after 24 weeks, the epidermal GSH content of the pretreated mice was mostly retained compared to nonirradiated mice. However, administration of GSH prior to acute or chronic UV irradiation had no effect on the UV-induced damage. The present results suggest that the protection from photodamage afforded by pretreatment with GSH ester is due to maintenance of a normal GSH level.
Animals↗
Morphological study of age-related changes in Fischer rat lens.
The authors conducted a morphological investigation of age-related changes in the lens of male Fischer rats at 6, 12, 18 and 24 months of age. At 6 months, there were no abnormalities of particular note other than slight swelling of the subcapsular fibers. Moreover, other than a slight decrease in the density of epithelial cells and slightly more extensive fiber swelling, there were no remarkable changes seen in the lens at 12 months. At 18 months of age, posterior invasion by the processes of the cortical lens fibers toward the lens capsule and slight swelling of the anterior cortical fibers were seen in addition to the above-mentioned changes. By 24 months of age, these changes had become more noticeable, with further reduction in the density of epithelial cells, swelling and degeneration of both anterior and posterior fibers and extension of the thin processes of the equatorial fibers toward the capsule. However, the bow configuration at the equator appeared almost normal. These findings indicate that the age-related changes in the Fischer rat lens occurred in the epithelial cells and superficial layer of the cortical fibers, subsequently leading to the loss of clarity in the lens.
Aging↗
Post-translational regulation of type I collagen synthesis by heparin in vascular smooth muscle cells.
Heparin is a potent inhibitor of smooth muscle cell proliferation. It reduced the level of type I collagen by one-half in culture medium of smooth muscle cells, whereas it increased the type I collagen expression 4-fold in the cell layer compartment under the conditions where it inhibited cell proliferation. No significant change in the level of prolyl hydroxylation in the newly synthesized type I procollagen or steady-state levels of pro alpha 1 (I) and pro alpha 2 (I) collagen mRNAs was detected after heparin treatment. Short-pulse and pulse-chase experiments revealed that heparin inhibited the secretion of type I procollagen and stimulated the accumulation of type I collagen in the extracellular space. These results suggest that heparin could modulate type I collagen expression at a posttranslational level. The modulation appeared to be closely related with its inhibitory effect on cell proliferation via two different mechanisms: the inhibition of the secretion of type I procollagen and stabilization of type I collagen in the cell layer. Heparin thus appears to be a multifunctional modulator for cell proliferation and the expression of type I collagen in vascular smooth muscle cells.
Animals↗
Protective effects of glutathione isopropyl ester on the sensitivity of cultured cells to UVB irradiation.
The effect of glutathione (GSH) isopropyl ester on cellular sensitivity to UVB irradiation was investigated in HeLaS3 cells. Pretreatment with 0.1-0.5 mM GSH isopropyl ester for 4 h significantly inhibited the decrease of thymidine (TdR) incorporation caused by UVB irradiation at a dose of 500 J/m2, whereas pretreatment with a high dose (1 mM) had no effect. The colony formation ability of the pretreated cells (0.3 mM) was significantly better than that of cells that received irradiation only. When the cells were treated with GSH isopropyl ester, their intracellular GSH level increased dose-dependently over a 4 h period, suggesting that GSH isopropyl ester was transported into the cells and there converted to GSH. Within 2 min of exposure, the intracellular GSH level depleted rapidly to about 75% of that in non-irradiated normal cells. In contrast, the GSH level in cells pretreated with 0.3 mM GSH isopropyl ester was maintained at the same level as that in normal cells, indicating that the maintenance of intracellular GSH level is due to converted GSH from GSH isopropyl ester. These results clearly show that intracellular GSH is involved in cell protection against photodamage, and that GSH isopropyl ester is a useful antioxidant for protection against photooxidative injury.
Free Radical Scavengers↗
Glycosphingolipids in cultured lens epithelial cells from dog and rhesus monkey.
Vertebrate lens tissues contain several species of acidic and neutral glycosphingolipids in relatively high amounts. However, the epithelia with capsule from dog and rhesus monkey lenses had a simpler composition and lower content of glycosphingolipids than whole lenses. Gangliosides and neutral glycosphingolipids in monolayer cultures of lens epithelial cells were also different from those in whole lenses. Although alpha-galactosyl (Gal alpha 1-3Gal-R) or Lewis(x) (Gal beta 1-4[Fuc alpha 1-3]GlcNAc-R) epitopes were found in glycosphingolipids from whole lenses, they were not detected in those from monolayer cultures of dog and rhesus monkey lens cells. In addition, significant changes in ganglio-series gangliosides were induced in monolayer cultures of both cells, where GM3 and GD3 were predominant. Immunofluorescence study revealed a characteristic distribution of cell surface gangliosides in confluent monolayers. These findings suggest that glycosphingolipid synthesis in lens epithelia is intrinsically different from that in cortical and nuclear fibres, and that the expression of Lewis(x) and alpha-galactosyl epitopes in glycosphingolipids appears to be associated with the differentiation of epithelial cells to fibres.
Animals↗
Identification of nondisulfided pro alpha 1 (IV) chain produced by cultured B16 melanoma cells.
Two collagenous polypeptides with apparent molecular weights of 180 and 500 kDa under a nonreducing condition were found to be produced in the culture medium of B16 mouse melanoma cells. Under a reducing condition, the higher molecular weight polypeptide (500 kDa) migrated at an identical position to the 180 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both polypeptides were found to have an identical primary structure by V8 protease peptide mapping. Cyanogen bromide cleavage patterns, the fragments produced by pepsin digestion, and partial amino acid sequences of the 180 kDa polypeptide were compatible with those of pro alpha 1 (IV) chain. These results indicate that B16 mouse melanoma cells preferentially produce pro alpha 1 (IV) chains consisting of two forms: disulfide- and nondisulfide-bonded forms.
Amino Acid Sequence↗