Biomedical subjects

T Tsukuda

Publications and source records attributed to T Tsukuda.

30 records · Page 2Linked to original sources

PubMed1989

Isolation of the REC1 gene controlling recombination in Ustilago maydis.

Previous studies on the rec1 mutant of Ustilago maydis revealed a complex phenotype. The mutant was found to be defective in DNA repair, recombination, growth, mutagenesis, and meiosis. We cloned the REC1 gene by complementing the phenotype of the mutant using a genomic DNA library made in a replicating vector and provided proof of identity by one-step gene disruption of the wild-type (wt) REC1 locus with a mutant allele constructed in vitro. The transcript of the REC1 gene was identified as a damage-inducible 1.7-kb RNA. After UV irradiation, the level of the transcript was observed to rise ten- to 15-fold. In the rec1 mutant, the basal level of the 1.7-kb transcript was tenfold lower than in wt. In addition, a second transcript of 1.1 kb was noted in rec1-1 cells in response to DNA damage.

Alleles↗

PubMed1988

Isolation and characterization of an autonomously replicating sequence from Ustilago maydis.

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment. UARS1 contained a cluster of active sequences. This element could be reduced further into three separate subfragments, each of which retained ARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.

Base Sequence↗

PubMed1988

Glucuronidation of 7-hydroxycoumarin in periportal and pericentral regions of the lobule in livers from untreated and 3-methylcholanthrene-treated rats.

Rates of production of 7-hydroxycoumarin glucuronide were measured in specific zones of the liver lobule using micro-light guides placed on periportal and pericentral regions on the surface of livers from untreated and 3-methylcholanthrene-treated rats. Livers were perfused with sulfate-free buffer under normoxic conditions and fluorescence of free 7-hydroxycoumarin was monitored. The formation of nonfluorescent 7-hydroxycoumarin glucuronide was then inhibited completely by perfusion with N2-saturated perfusate containing 20 mM ethanol. The difference between fluorescence readings under normoxic and hypoxic conditions was used to calculate rates of glucuronidation. Maximal rates of glucuronidation (11.9-13.5 mumol/g/hr) did not differ significantly in periportal and pericentral regions in livers from either 3-methylcholanthrene-treated or untreated rats. In all regions of the liver lobule, glucuronidation was half-maximal with about 20 microM 7-hydroxycoumarin. Glucuronosyltransferase assayed in lyophilized tissue sections with saturating concentrations of UDPGA (9 mM) was 2.3-fold greater in pericentral than in periportal areas in livers from untreated rats. In livers from 3-methylcholanthrene-treated rats, activities were similar in periportal and pericentral regions but were 4- to 7-fold higher than values from untreated rats. In addition, glucuronosyltransferase activity assayed in native microsomes with physiological concentrations of UDP-glucuronic acid (UDPGA) (0.4 mM) with UDP-N-acetylglucosamine (0.3 mM) was 2-fold higher in preparations from 3-methylcholanthrene-treated than untreated rats. Thus, 3-methylcholanthrene treatment increased glucuronosyltransferase activity in vitro but did not alter rates of glucuronide formation in periportal and pericentral regions of the liver lobule of intact liver. Infusion of epinephrine (50 nM) into perfused livers from untreated and 3-methylcholanthrene-treated rats increased rates of glucuronidation by about 35%. Since epinephrine probably acts by increasing the supply of the cofactor UDPGA due to increased breakdown of glycogen, it follows that UDPGA supply limits rates of glucuronidation in perfused livers from both untreated and 3-methylcholanthrene-treated rats.

Animals↗

PubMed1984

Calcium-mediated inhibition of glucuronide production by epinephrine in the perfused rat liver.

Rates of production of p-nitrophenyl glucuronide by isolated perfused livers from fed or fasted phenobarbital-treated rats were estimated by monitoring the concentration of glucuronide in the effluent perfusate. Infusion of epinephrine decreased the steady state level of p-nitrophenyl glucuronide by about 39% (half-maximal inhibition at approximately 5 microM). This result was unexpected because epinephrine activated glycogenolysis and elevated hepatic UDP-glucuronic acid contents. The effect of epinephrine can be attributed to its interaction with alpha-adrenergic receptors, since the inhibition of glucuronide production by epinephrine was reversed by an alpha-antagonist (phentolamine) but not by a beta-antagonist, propranolol. Since alpha-adrenergic agonists increase the cytosolic free calcium concentration, we investigated the possibility that the decrease in glucuronide production elicited by epinephrine was mediated by calcium. Removal of calcium from the perfusion fluid diminished the inhibition of glucuronide production by epinephrine, while increasing extracellular calcium from 0 to 150 microM restored the inhibition in a dose-dependent manner. In the presence of extracellular calcium, glucuronide production was inhibited by the addition of the calcium ionophore A23187 or angiotensin II, a hormone which increases cytosolic calcium. Concentrations of ionized calcium comparable to physiological intracellular levels (0.1-2 microM) increased microsomal beta-glucuronidase activity by 50 to 100% but had no effect on microsomal glucuronosyl-transferases . These results indicate that activation of hepatic alpha-adrenergic receptors increases cytosolic calcium which stimulates microsomal beta-glucuronidase activity. This decreases net glucuronide formation by the liver. In support of this hypothesis, rates of glucuronide production were unaffected by epinephrine in perfused livers from beta-glucuronidase-deficient C3H/HeJ mice.

Animals↗

PubMed1983