The Gene Encoding Flavanone 3-Hydroxylase is Expressed Normally in the Pale Yellow Flowers of the Japanese Morning Glory Carrying the speckled Mutation Which Produce Neither Flavonol nor Anthocyanin but Accumulate Chalcone, Aurone and Flavanone

The Japanese morning glory carrying the recessive mutable speckledallele with the dominant speckled-activator bears colorlessflowers with fine and round colored spots distributed over thecorolla whereas the plant without the speckled-activator producespale yellow flowers. Previous chemical analysis has indicatedthat a mutation in the gene for flavanone 3-hydroxylase (F3H)is a likely candidate for the speckled allele. However, theF3HmRNA without sequence alteration accumulates normally inthe pale yellow flowers, indicating that the speckled alleleis neither the F3H gene nor a regulatory gene acting on theF3H gene expression. (Received April 4, 1997; Accepted June 2, 1997)     

Purification and Properties of Prostaglandin H-E Isomerase from the Cytosol of Human Brain: Identification as Anionic Forms of Glutathione S-Transferase

Abstract: Prostaglandin H-E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl-Sephar-ose chromatography, gel filtration on a BioGel P-100 column, GSH-agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were ∼5.4. The enzymes catalyzed selective conversion of prostaglandin H₂ to prostaglandin E₂. The K _m values for prostaglandin H₂ of peaks 1 and 2 were 147 and 308 μ M , respectively, and the V _max values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S -transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H-E isomerase. Prostaglandin H-E isomerase activity of peak 2 enzyme was competitively inhibited by 1-chloro-2,4-dinitrobenzene, a substrate of glutathione S -transferase. These results suggested that prostaglandin H-E isomerases in human brain cytosol were identical with anionic forms of glutathione S -transferase.     

Regulation of retrochalcone biosynthesis: Activity changes of O-methyltransferases in the yeast extract-induced Glycyrrhiza echinata cells

Three O-methyltransferases which catalyze S-adenosyl-L-methionine (SAM)-dependent O-methylation of licodione (LMT), flavone/flavonol (FMT), and caffeic acid (CMT) were separated from the callus culture of Glycyrrhiza echinata, and characteristic differences between their pH optima and Mg²⁺ requirement for activity were demonstrated. The activity of LMT, which is involved in retrochalcone (echinatin) biosynthesis, but not of FMT or CMT, was found to be stimulated when suspension-cultured G. echinata cells were treated with yeast extract (YE), which causes rapid production of echinatin in the cells. Cycloheximide suppressed both the YE-induced echinatin formation and LMT enhancement. The results indicate a selective induction of retrochalcone pathway in Glycyrrhiza cells in response to stress.Abbreviations SAM S-adenosyl-L-methionine - LMT, SAM licodione 2-O-methyltransferase - FMT, SAM flavone/flavonol O-methyltransferase - CMT, SAM caffeate 3-O-methyltransferase - OMT O-methyltransferase - CH cycloheximide - YE yeast extract This paper is Part 47 in the series Studies on Plant Tissue Cultures. For Part 46, see Ayabe S, Iida K, Furuya T (1986) Phytochemistry: in press     

Comparison of the regional distribution of calspectin (nonerythroid spectrin or fodrin), alpha-actinin, vinculin nonerythroid protein 4.1, and calpactin in normal and avian sarcoma virus- or Rous sarcoma virus-induced transformed cells

With fluorescence and interference reflection microscopy (IRM), we compared the regional distribution of calspectin, its interacting proteins (nonerythroid protein 4.1 and calpactin), alpha-actinin, and vinculin in NRK cells and their avian sarcoma virus (ASV)- or temperature-sensitive (ts) Rous sarcoma virus (RSV)-transformed cells. The localization of these cytoskeletal proteins was determined with the specific antibodies. In NRK cells, alpha-actinin and vinculin were concentrated at adhesion plaques. By contrast, calspectin was distributed throughout the cytoplasm, but not concentrated at adhesion plaques. In ASV- and ts RSV-transformed cells, all three cytoskeletal proteins were concentrated at dot structures representing cellular feet. Nonerythroid protein 4.1 and calpactin were diffusely distributed throughout the cytoplasm of NRK cells and their transformed counterparts. In the case of calpactin, a part of this protein was excluded near regions of the terminal ends of stress fibers. These two proteins did not show the restricted location at the dot structures of transformed cells. From these findings, it is apparent that the accumulation of calspectin into dot structures is a specific event for cell transformation induced by the src protein.     

Phospholipase C in human endometrial fibroblasts and its regulation by estrogens

1. Stimulated inositolphospholipid turnover has been proposed as a signal-transducing mechanism in many cell types. It appears to be initiated by stimulation of hydrolysis of inositolphospholipid by a phospholipase C. 2. In human endometrial fibroblasts, estradiol was observed to cause sequential enhancement of [32P]phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), indicating an accelerating effect of estradiol on inositolphospholipid turnover. Specific 32P-radioactivity in the gamma-phosphate of ATP was increased in response to estradiol. Estrone or estriol were without any effects. 3. To investigate possible mechanisms by which estradiol activates a phospholipase C enzyme in the fibroblasts, the plasma membrane fraction isolated from the fibroblasts was exposed to estradiol in the presence of guanosine triphosphate (GTP) to detect inositol trisphosphate (IP3) production. The IP3 production was Ca2+ dependent, a dependency not affected by estradiol. 4. However, ATP decreased the Ca2+ concentration required for IP3 production in a dose-dependent manner; adenosine diphosphate (ADP), cytidine triphosphate (CTP) showed no effects. 5. These findings from cell and cell-free systems might suggest that estradiol stimulates a phospholipase C, as a result of enhancement of intracellular ATP synthesis, but not as a result of a direct effect on the enzyme molecule or direct activation of receptor-phospholipase C unit. 6. This may give us new insight into estrogen-stimulated cellular phenomenon through some mechanisms other than that classically associated with the action of estrogen.     

Random mutagenesis using degenerate oligodeoxyribonucleotides

An efficient method for random mutagenesis was applied to a 75-bp target sequence. The mutational changes in the target region are introduced by the technique of oligodeoxyribonucleotide(oligo)-directed, site-specific mutagenesis using mixtures of degenerate oligos. These are designed in such a way that they carry with a high probability randomly distributed substitutions, which are introduced into the oligos by utilizing appropriate concentrations of all four nucleotide precursors during each chain elongation step. These mixtures of degenerate oligos were hybridized to the appropriate M13-hybrid ss-template and then extended in vitro using PolIk. In order to avoid any bias artificially created by the Escherichia coli mismatch repair system, homoduplex molecules were synthesized in vitro according to the method of Taylor et al. [Nucleic Acids Res. 13 (1985) 8765-8785]. After transformation of the appropriate E. coli host, M13 plaques were randomly analysed by DNA sequencing. Using appropriate preparations of template DNA and oligos we attained mutagenesis efficiencies in the range of 20-50%. The analysis of 85 different mutants revealed that the distribution of the mutations is random and that all expected substitutions occur with about the same probability.     

The KKRKK sequence is involved in heat shock-induced nuclear translocation of the 18-kDa actin-binding protein, cofilin.

The exposure of cultured mammalian cells to elevated temperatures induces the translocation of actin and cofilin into the nuclei and the formation of intranuclear bundles of actin filaments decorated by cofilin (actin/cofilin rods). Cofilin has a stretch of five basic amino acids, KKRKK, which was assumed to be the sequence involved in the heat shock-dependent accumulation of cofilin in nuclei. To examine this possibility, the site-directed mutagenesis technique was employed to alter the KKRKK sequence of cofilin to KTLKK and the mutated cofilin was expressed under the human beta-actin promoter in transfectants of mouse C3H-2K cell line. All the recombinants derived from porcine cofilin cDNA were constructed so as to possess an extra-nonapeptide at their N-termini when expressed; their intracellular distribution could, therefore, be discriminated from that of endogenous cofilin using the indirect immunofluorescence method with polyclonal antibodies directed against the extra-peptide. The results clearly showed that the mutated cofilin possessing KTLKK instead of KKRKK did not translocate into the nuclei in response to heat shock whereas a recombinant cofilin with the unaltered sequence of KKRKK responded to heat shock and formed intranuclear rods together with actin. Although in vitro actin binding experiments showed that KTLKK-cofilin has a weaker affinity to actin filaments than KKRKK-cofilin, KTLKK-cofilin was found to form cytoplasmic actin/cofilin rods when transformants were incubated in NaCl buffer. Furthermore, we have noted that endogenous cofilin present in cells expressing KTLKK-cofilin behaved normally, translocated into nuclei and formed intranuclear actin/cofilin rods upon heat shock. These results suggest that the KKRKK sequence of cofilin functions as a nuclear location signal upon heat shock.