Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates.

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.

The antitumor effect of hyperthermia combined with fluorouracil and its analogues.

The effectiveness of 5-fluorouracil (5-FU), FT-207 and FT-207 + uracil in combination with two repetitions of 43 degrees C hyperthermia in the treatment of the Meth-A-Fibrosarcoma and Sarcoma-180 was examined in vivo in BALB/c mice. The antitumor effect was evaluated in terms of inhibition of tumor growth by measuring the tumor for 7 days. The 5-FU concentration in each tumor was also monitored. Hyperthermia combined with FT-207 or FT-207 + uracil showed a synergistic effect for the inhibition of growth of both tumors which was not observed with 5-FU. There were no significant differences in the intratumoral concentration of 5-FU in unheated or heated Sarcoma-180 for any drug treatment after the first hyperthermia treatment, except for significant decreases in the group given 5-FU with the first hyperthermia treatment. After the second hyperthermia treatment, significant decreases in the concentration of 5-FU and FT-207 + uracil were observed. In the Meth-A-Fibrosarcoma, the intratumoral concentration of 5-FU decreased significantly in the group given 5-FU and increased significantly in the group given FT-207 + uracil after the first hyperthermia treatment, while there were significant decreases in 5-FU, FT-207 and FT-207 + uracil administered with the second hyperthermia treatment. Hyperthermia combined with FT-207 or FT-207 + uracil is considered to be effective.

[Pharmacokinetic studies on continuous intraportal 5-fluorouracil infusion].

Pharmacokinetics of 5-FU administered by continuous intraportal infusion were studied by infusing 5 mg/kg, 10 mg/kg, 20 mg/kg, and 40 mg/kg of 5-FU, respectively, into the ileocecal vein for an hour. Blood samples were collected from portal vein, femoral vein and liver, and small intestine specimens were obtained at proper intervals. The rabbits were sacrificed at 120 minutes from the start of 5-FU infusion. The results were as follows: Extremely high concentrations of 5-FU in the portal vein, femoral vein and liver tissue were observed in the 5-FU group infused with 40 mg/kg. This phenomenon was suggested to rely on the effect by which the 5-FU catabolic enzyme, dihydrouracil dehydrogenase, was saturated with a large inflow of 5-FU. FdUMP concentration of the liver was lower than that of the small intestine in all groups. These results suggest that a large dose of 5-FU infusion is effective to increase the FdUMP concentration in the liver with continuous intraportal 5-FU infusion.

Transfer of antibodies to kittens from mother cats chronically infected with Toxoplasma gondii.

By indirect immunofluorescence assay, anti-Toxoplasma gondii antibody levels were examined in fetuses and kittens born from chronically infected cats. Titer of anti-T. gondii IgG in sera of kittens born from infected cats was significantly high on the seventh day post-birth, and decreased to a serologically non-detectable level at 8-12 weeks post-birth under continuous suckling of maternal milk. Littermates nursed by a non-infected cat showed a faster rate of IgG antibody depletion. In sera of fetuses obtained from infected cats, anti-T. gondii IgG titer was lower than that of offspring born from infected cats. Anti-T. gondii IgM titer was non-detectable in sera of all kittens and fetuses. Kittens born from infected cats inoculated with T. gondii oocysts on Day 35 after birth shed oocysts and showed a transient increase of anti-T. gondii IgM titer. Findings in this study suggest that anti-T. gondii antibody IgG in kittens is transferred mainly via colostrum and the kittens that receive maternal anti-T. gondii antibodies develop inadequate resistance to T. gondii infection.

[The effect of cepharanthin on adjuvant chemotherapy induced bone marrow suppression in patients with breast cancer].

We studied the effect of Cepharanthin on bone marrow suppression induced by adjuvant chemotherapy in primary breast cancer patients in our hospital. The 36 patients with breast cancer were divided into two groups. Both groups were administered of MMC 20 mg intravenously on the day of radical operation. One group (23 patients) was administered of Cepharanthin 60 mg every day, but the other (13 patients) group was not administered. In the leukocyte count at 3 weeks after post operation, 82.6% (19 out of 23) of the patients who were administered cepharanthin kept more than 70% of pre-operation's leukocyte. On the other hand, only 46.1% (6 out of 13) of the patients who were not administered Cepharanthin kept more than 70% of pre-operation's leukocyte. The number of thrombocyte was not decreased in both groups. Side effects by Cepharanthin were not observed in this study.