Fumiya Kurosaki

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p‐coumaroyl‐CoA and three C2‐units from malonyl‐CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p‐coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross‐reaction between CHS and STS, i.e. resveratrol production by CHS (2.7–4.2% of naringenin) and naringenin production by STS (1.4–2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.

Involvement of plasma membrane-located calmodulin in the response decay of cyclic nucleotide-gated cation channel of cultured carrot cells

Increase in cytoplasmic cyclic AMP concentration stimulates Ca2+ influx through the cyclic AMP‐gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca2+ influx. Cyclic AMP evoked discharge of Ca2+ from inside‐out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 μM of free Ca2+, while Ca2+ lower than 0.1 μM did not affect the Ca2+‐release. The Ca2+ flux across plasma membrane was restored from this Ca2+‐induced inhibition by the addition of calmodulin inhibitors or anti‐calmodulin. These results suggest that Ca2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide‐gated Ca2+ channel.

Cancer chemopreventive effects of constituents of Caesalpinia ferrea and related compounds.

The anti-tumor promoting effects of fruits of Caesalpinia ferrea MART. (Leguminosae) were tested by the in vitro Epstein-Barr virus early antigen (EBV-EA) activation assay, and its active constituents were identified as gallic acid (1) and methyl gallate (2). A total of 49 related compounds of 1 and 2 were analysed for the effects by this assay, and the structure activity relationships have been proposed. Three acetophenone derivatives, 2,6-dihydroxyacetophenone (48), 2,3,4-trihydroxyacetophenone (50) and 2,4,6-trihydroxy- acetophenone (51) were found to show potent inhibitory activity.

Role of inward K+ channel located at carrot plasma membrane in signal cross‐talking of cAMP with Ca2+ cascade

Treatment of cultured carrot cells with dibutyryl cAMP or forskolin resulted in the appreciable decrease in extracellular K+ concentration. This decrease was found to be transient and the concentration of the ion in the culture medium restored to the original level within few minutes. The cAMP‐induced decrease in K+ level in the medium was almost completely inhibited when carrot cells were incubated in the presence of K+ channel blockers, CsCl and tetraethylammonium chloride. Appreciable amounts of 45Ca2+ were discharged from 45Ca2+‐loaded inside‐out vesicles of carrot plasma membrane by the stimulation with cAMP, however, the release of the ion was significantly inhibited in the presence of the K+ channel blockers. The release of 45Ca2+ from the vesicles was also observed when K+ current was evoked with an ionophore, valinomycin, even in the absence of cAMP. These results suggest that the gating of some of the inward K+ channels located at plasma membrane of cultured carrot cells is controlled by cytoplasmic concentration of cAMP and the inward K+ current across the plasma membrane induced by the nucleotide elicits Ca2+ influx into the cells possibly by the activation of voltage‐dependent Ca2+ channels.

Cancer chemopreventive effects of a Brazilian folk medicine, Juca, on in vivo two-stage skin carcinogenesis.

Gallic acid (1) and methyl gallate (2) were isolated from Juca, a Brazilian folk medicine, fruits of Caesalpinia ferrea MART (Leguminosae), decreased significantly the average number of papillomas per mouse in the experiment of the promoting effects of 12-O-tetra- decanoylphorbol-13-acetate (TPA) on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene (DMBA).

6-hydroxymellein synthetase as a multifunctional enzyme complex in elicitor-treated carrot root extract

Synthetic activity of a polyketide compound 6‐hydroxymellein was induced in elicitor‐treated carrot root tissues. The activity was significantly inhibited by an antiserum raised against the acyl carrier protein (ACP) of fatty acid synthetase, suggesting that the enzyme(s) for 6‐hydroxymellein synthesis require(s) a functional unit similar to ACP. However, the synthetic activity was not stimulated by the addition of ACP purified from Escherichia coli and was not lost even after fractionation by gel‐filtration chromatography. The active fraction obtained by gel‐filtration (136 kDa) was subjected to immunoblot analysis, and a 128 kDa polypeptide in the fraction was found to cross‐react with anti‐ACP serum. These observations suggest that the biosynthesis of 6‐hydroxymellein in carrot cells is catalyzed by an enzyme consisting of a single peptide chain.

Carrot Phytoalexin Alters the Membrane Permeability of Candida albicans and Multilamellar Liposomes

The biochemical basis for the antimicrobial effect of the carrot phytoalexin 6-methoxymellein (6-MM) was examined. At fungistatic concentrations 6-MM retarded the ability of Candida albicans to incorporate radioactive thymidine, uridine and leucine into biopolymers. When C. albicans was incubated with 6-MM, 260-nm-absorbing materials and 3H-labelled compounds leaked from the cells. The inhibitory effects of 6-MM on cell growth and membrane functions were, however, reduced as the concentration of divalent metal cations added to the medium was increased. 6-MM interacted with multilamellar liposomes constituted from phosphatidylcholine, cholesterol and dicetyl phosphate, or from phosphatidylcholine only, resulting in the release of glucose trapped in these liposomes. These results suggest that 6-MM exerts its toxic effects on susceptible cells as a result of its interaction with their membranes and disturbance of membrane-associated functions.

A methyltransferase for synthesis of the phytoalexin 6-methoxymellein in carrot cells

Carrot (Daucus carota L.) roots treated with 2‐chloroethylphosphonic acid or uronide elicitor accumulated the phytoalexin 6‐methoxymellein. The extracts of these cells catalyzed methylation of 6‐hydroxymellein to 6‐methoxymellein with S‐adenosyl‐L‐methionine as a methyl donor. Activity of the O‐methyltransferase was not found in fresh carrot roots but increased when they were treated with either type of elicitor in parallel with the increase in 6‐methoxymellein content. The enzyme did not methylate 6‐methoxymellein, 3,4‐dehydro‐6‐methoxymellein, and some coumarin derivatives, though 3,4‐dehydro‐6‐hydroxymellein accepted methyl moiety at the same rate as in 6‐hydroxymellein.

Involvement of GTP-binding protein in the induction of phytoalexin biosynthesis in cultured carrot cells

Biosynthetic activity of carrot phytoalexin 6-methoxymellen was induced in cell suspension culture by the treatment with oligogalacturonide elicitor; however, the elicitor-induced activity appreciably reduced in the presence of suramin, a potent inhibitor of GTP-binding proteins. In contrast, addition of G-protein activators, such as mastoparan or GTP-gamma-S, to carrot cell culture triggered 6-methoxymellein production even in the absence of uronide elicitor. An appreciable GTPase activity was found in purified plasma membrane of cultured carrot cells, and the hydrolytic activity was significantly increased by the addition of elicitor. Carrot plasma membrane was capable of associating with GTP-gamma-S, and the binding ability was markedly increased in the presence of elicitor. However, the binding activity markedly decreased when the membrane preparation was pre-incubated with GTP but not with ATP. These observations strongly suggest that a certain GTP-binding protein located at plasma membrane of cultured carrot cells plays an important role in the oligogalacturonide elicitor-induced 6-methoxymellein production.

Calmodulin-dependency of a Ca2+ -pump at the plasma membrane of cultured carrot cells

Abstract Activity of a Ca 2+ -translocating ATPase which functions as a Ca 2+ -pump was found to be associated with a highly purified plasma membrane fraction of cultured carrot cells. Different classes of calmodulin antagonists — trifluoperazine and W-7 — appreciably inhibited ATP-driven uptake of Ca 2+ into the sealed vesicles of the membrane. The incorporation of Ca 2+ was also markedly inhibited in the presence of anti-calmodulin IgG. This translocating activity was significantly decreased when the vesicles were washed with EGTA-containing buffer; however, it was restored to almost the control level upon the addition of exogenous calmodulin. These results suggest that the Ca 2+ -pumping ATPase at the plasma membrane of cultured carrot cells is regulated by calmodulin, and the modulator protein associates with the enzyme in a Ca 2+ -dependent manner.