Michihiro Hattori

Purification, cDNA Cloning, and Expression of UDP-Gal: Glucosylceramide {beta}-1,4-Galactosyltransferase from Rat Brain

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29.7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N-glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn2+ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse β-1,4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to β-1,4-GlcNAc in glycoproteins.

cDNA cloning and expression of human lactosylceramide synthase

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycolipids in mammals. We have isolated and sequenced the cDNA clone encoding human lactosylceramide synthase. The deduced amino acid sequence of the human lactosylceramide synthase showed 94.2% identity with rat lactosylceramide synthase. Northern blotting analysis revealed that lactosylceramide synthase mRNA was expressed in various tissues, with the highest level in brain and adrenal gland.

Age‐related changes in the three‐dimensional morphological structure of human facial skin

The human face shows morphological changes with age. Although these changes are generally recognized as wrinkle formation, there have been no quantitative studies of three‐dimensional morphological changes with age in each area of the face. The object of the present study, therefore, was to evaluate age‐related three‐dimensional morphological changes in each area of the face.

The relationship between quantitative changes in collagen and formation of wrinkles on hairless mouse skin after chronic UV irradiation.

Female albino hairless mice were irradiated chronically with sub-erythemal doses of UVB radiation. Collagen extracted from the irradiated or non-irradiated dorsal skin of mice was fractionated into neutral salt-soluble (NSC), acid-soluble (ASC) and insoluble fractions (ISC). An age-related exponential decrease in the content and proportion of acid-soluble collagen was found in each group. The contents and the proportions of ASC from irradiated mice were always significantly lower than those from age-matched control animals. Age-related slight decreases were observed in the contents (per fresh weight of tissues) of NSC, ISC and total collagen in the control group but decreases in these collagen contents after UVB irradiation were marked. A dramatic decrease in ASC occurred nearly concomitantly with wrinkle formation in the irradiated mice. The decrease of acid-soluble skin collagen in irradiated mice may play a role in the formation of wrinkles on hairless mouse dorsal skin.

FUNCTIONS OF LIPIDS ON HUMAN SKIN

Abstract In order to clarify the roles of lipids in the water-holding property of stratum corneum, the forearm skin of healthy male volunteers was treated with acetone/ether (1/1) or sodium dodecyl sulfate (5%) for 1-30 min. A prolonged treatment period of 5-30 min produced a chapped and scaly appearance of the stratum corneum without any inflammatory reactions. Under these conditions, there was a marked decrease in the water-holding capacity of the stratum corneum accompanied by a selective loss of stratum corneum lipids such as cholesterol, cholesterol esters, and sphingolipids. Two daily applications of the isolated stratum corneum lipids to experimentally induced dry skins caused a significant increase of conductance, accompanied by a marked improvement in the level of scaling. Meanwhile, the isolated sebaceous lipids exhibited no significant recovery in both the conductance value and the scaling. Out of chroma-tographically separated fractions of the stratum corneum lipids, topical applications of ce...

Quantitative two‐dimensional analysis of facial wrinkles of Japanese women at various ages

To evaluate individual differences in the recognition of facial wrinkles, we asked 40 Japanese female observers to identify wrinkles using transparent sheets over frontal facial photos of four females aged 20, 39, 55 or 75 years. We then measured the number and length of those wrinkles by image analysis. Wrinkles identified by those 40 observers showed aged‐related increases in the standard deviation (SD) values for number and length but age‐related decreases in the coefficient of variation (CV)%. Therefore, to clarify factors affecting the degree of wrinkle detection, wrinkles were identified by two groups of age‐matched male and female observers, by two groups that differed by age, and by two other groups, one of which who felt that there was an improvement in their wrinkles after application of an antiwrinkle agent and another group who did not feel that there was any improvement after the same treatment. Improvement was observed by replica image analysis in all groups. The degree of wrinkles identified was not affected by the age or by the sex of the observer group. However, the group who felt that there was an improvement in their wrinkles after treatment with the antiwrinkle agent identified a significantly higher number of wrinkles than did the group who did not feel that there was an improvement.