Tomoko Nomura

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.

β4GalT6 is involved in the synthesis of lactosylceramide with less intensity than β4GalT5

Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, β1,4-galactosyltransferase 6 (B4galt6) cDNA was isolated as a LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of β4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that β4GalT6 plays a role as a LacCer synthase, whereas β4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.

A rapid and simple assay method for UDP-glucose : ceramide glucosyltransferase

We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransferase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction product, [14C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [14C]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn2+, Mg2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Apparent Km values of 8.7 microM for UDP-glucose and 292 microM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.

Hyperbaric Oxygen Exposure Reduces Age-Related Decrease in Oxidative Capacity of the Tibialis Anterior Muscle in Mice

The effects of exposure to hyperbaric oxygen on the oxidative capacity of the skeletal muscles in mice at different ages were investigated. We exposed 5-, 34-, 55-, and 88-week-old mice to 36% oxygen at 950 mmHg for 6 hours per day for 2 weeks. The activities of succinate dehydrogenase (SDH), which is a mitochondrial marker enzyme, of the tibialis anterior muscle in hyperbaric mice were compared with those in age-matched mice under normobaric conditions (21% oxygen at 760 mmHg). Furthermore, the SDH activities of type IIA and type IIB fibers in the muscle were determined using quantitative histochemical analysis. The SDH activity of the muscle in normobaric mice decreased with age. Similar results were observed in both type IIA and type IIB fibers in the muscle. The decrease in the SDH activity of the muscle was reduced in hyperbaric mice at 57 and 90 weeks. The decreased SDH activities of type IIA and type IIB fibers were reduced in hyperbaric mice at 90 weeks and at 57 and 90 weeks, respectively. We conclude that exposure to hyperbaric oxygen used in this study reduces the age-related decrease in the oxidative capacity of skeletal muscles.

Relationships between Transepidermal Water Loss, Cutaneous Microcirculatory Function and Autonomic Nervous Activity

Several studies have shown that a deterioration of skin properties, an impaired cutaneous microcirculatory function and an imbalance of autonomic nervous activity are observed in smokers and in patients with diabetes mellitus or Raynauds phenomenon. These observations suggest that skin properties are associated with cutaneous microcirculatory function and autonomic nervous activity in pathological conditions. However, there is no published evidence to support the concept that these two functions have any relationship with skin properties even in healthy subjects. To investigate the hypothesis that these properties are related, we conducted a survey of healthy adult subjects to investigate the relationships between cutaneous microcirculatory function and autonomic nervous activity and skin properties.

Hyperbaric oxygen improves ultraviolet B irradiation-induced melanin pigmentation and diminishes senile spot size.

Background: The effects of exposure to hyperbaric oxygen on ultraviolet B (UVB) irradiation‐induced melanin pigmentations of skins and on senile spot sizes of faces were investigated.

Dry skin conditions are related to the recovery rate of skin temperature after cold stress rather than to blood flow.

Cutaneous blood flow plays an important role in the thermoregulation, oxygen supply, and nutritional support necessary to maintain the skin. However, there is little evidence for a link between blood flow and skin physiology. Therefore, we conducted surveys of healthy volunteers to determine the relationship(s) between dry skin properties and cutaneous vascular function.

Mild hyperbaric oxygen activates the proliferation of epidermal basal cells in aged mice

The proliferation of epidermal basal cells decreases with age. This study examined the effects of exposure to mild hyperbaric oxygen on the proliferative activity of epidermal basal cells in aged mouse skin. Hairless mice aged 5, 34 and 55 weeks were exposed to mild hyperbaric oxygen at 1266 hPa with 36% oxygen for 6 h/day for 1 or 2 weeks. Skin samples were then collected from the back area to evaluate epidermal thickness and the number and proliferative activity of epidermal basal cells. Exposure to mild hyperbaric oxygen had no effect on the epidermal thickness, irrespective of age, but accelerated the proliferative activity of epidermal basal cells in aged mouse skin.