Shigeko Fujimoto Sakata

A novel gene family defined by human dihydropyrimidinase and three related proteins with differential tissue distribution

We have isolated cDNA clones encoding dihydropyrimidinase (DHPase) from human liver and its three homologues from human fetal brain. The deduced amino acid (aa) sequence of human DHPase showed 90% identity with that of rat DHPase, and the three homologues showed 57-59% aa identity with human DHPase, and 74-77% aa identity with each other. We tentatively termed these homologues human DHPase related protein (DRP)-1, DRP-2 and DRP-3. Human DRP-2 showed 98% aa identity with chicken CRMP-62 (collapsin response mediator protein of relative molecular mass of 62 kDa) which is involved in neuronal growth cone collapse. Human DRP-3 showed 94-100% aa identity with two partial peptide sequences of rat TOAD-64 (turned on after division, 64 kDa) which is specifically expressed in postmitotic neurons. Human DHPase and DRPs showed a lower degree of aa sequence identity with Bacillus stearothermophilus hydantoinase (39-42%) and Caenorhabditis elegans unc-33 (32-34%). Thus we describe a novel gene family which displays differential tissue distribution: i.e., human DHPase, in liver and kidney; human DRP-1, in brain; human DRP-2, ubiquitously expressed except for liver; human DRP-3, mainly in heart and skeletal muscle.

Molecular cloning and sequencing of a cDNA encoding dihydropyrimidinase from the rat liver

A cDNA encoding dihydropyrimidinase has been isolated from a rat cDNA library. The N-terminal and an internal amino acid sequences were determined, and PCR primers were designed based on these sequences. Using a cDNA fragment amplified by RT-PCR with these primers, three cDNA clones were isolated from a rat liver library. The clone with the longest insert of 2129 bp contained a 1557 bp open reading frame encoding a polypeptide of 519 residues with a molecular mass of 56,832 Da.

Crocetin, a Carotenoid from Gardenia jasminoides Ellis, Protects against Hypertension and Cerebral Thrombogenesis in Stroke-prone Spontaneously Hypertensive Rats

Crocetin is a natural carotenoid dicarboxylic acid that is found in the fruit of Gardenia jasminoides Ellis (Cape Jasmine) and in the stamen and pistil of Crocus sativus L. (saffron). It is used worldwide as an important spice, food colorant, and herbal medicine. In the current investigation, we have examined the cardiovascular effects of crocetin using stroke‐prone spontaneously hypertensive rats (SHRSPs). Male SHRSPs (6 weeks old) were classified into three groups: a control group and two crocetin groups (25 and 50 mg/kg/day). The animals were given crocetin for 3 weeks. Body weights in each group were not significantly different during the treatment period, but the increase in systolic blood pressures observed with age was significantly moderated by crocetin. Thrombogenesis, assessed using a He‐Ne laser technique in pial vessels, was significantly decreased. Antioxidant activity, assessed by measuring urinary 8‐hydroxy‐2′‐deoxyguanosine levels, together with urinary nitric oxide (NO) metabolite levels, was increased significantly after treatment. Acetylcholine‐induced vasodilation was measured using the aorta and indicated that endothelial function was significantly improved by crocetin. These results strongly suggest that the antihypertensive and antithrombotic effects of crocetin were related to an increase in bioavailable NO, possibly mediated by decreased inactivation of NO by reactive oxygen species. Copyright

Purification and expression of a processing protease on β-alanine-oxoglutarate aminotransferase from rat liver mitochondria

GABA[arrow beta]AlaAT convertase is an endopeptidase that processes brain‐type 4‐aminobutyrate aminotransferase (GABA AT; EC 2.6.1.19) to liver‐type β‐alanine‐oxoglutarate aminotransferase (β‐AlaAT I) in rats. Its molecular mass was 180 kDa as determined by gel filtration. A subunit molecular mass of 97 652 Da was measured using MALDI‐TOF MS. The N‐terminal sequence of the purified GABA[arrow beta]AlaAT convertase was SRVEVSKVLILGSGGLSIGQAGEFDYSGSQAV‐ and was identical to residues 418–449 of carbamoyl‐phosphate synthetase I (CPS I; EC 1.2.1.27) purified from rat liver. The subunit molecular mass and the N‐terminal amino acid sequence suggested that GABA[arrow beta]AlaAT convertase was the 418–1305 peptide of CPS I. An expression vector containing the coding region of the 418–1305 peptide of rat CPS I was transfected into NIH3T3 cells and the extract of the cells showed GABA[arrow beta]AlaAT convertase activity.