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)     

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.     

Identification of N-[p-(2-benzimidazolyl)phenyl]maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis

Na+,K+-ATPase from pig kidney was specifically modified with a sulfhydryl fluorescent reagent, N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM), by pretreatment of N-ethylmaleimide. The preparation thus obtained retained 100% of initial Na+,K+-ATPase activity and contained 1 BIPM residue/alpha-chain, and it showed almost 2-fold larger fluorescence changes accompanying ATP hydrolysis than the previous preparations which retained 60% of initial activity and contained 3-4 BIPM residues/alpha-chain (Taniguchi, K., Suzuki, K., and Iida, S. (1982) J. Biol. Chem. 257, 10659-10667). Extensive trypsin (Sigma type I) treatment of the new preparation produced mainly two different fluorescent peptide peaks in both ion-exchange and reverse-phase chromatography. Amino acid sequence analysis of both peptides showed that they had the same common sequence, Ser-Tyr-X-Pro-Gly-Met-Gly-Val, except that the larger one contained Ala-Leu next to the Val residue. From the comparison of the amino acid sequence deduced from cDNA from sheep kidney (Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Nature 316, 691-695), X was shown to correspond to Cys-964 of the alpha-chain in Na+,K+-ATPase. The data suggest that the microenvironment of the BIPM residue covalently bound to the sulfhydryl group of Cys-964 changes accompanying sequential appearance of reaction intermediates of Na+,K+-ATPase.     

Bacteriophage P1 derivatives unaffected in their growth by a large inversion or by IS insertions at various locations

Several plaque-forming phage P1 derivatives carrying DNA rearrangements associated with IS elements are described. They have IS1, IS3 and IS5 inserted in four distinct locations, all of which are non-essential regions for phage P1 propagation. One derivative carries a genome segment, inverted relative to the one in the P1 wild-type genome, between two inverted copies of IS1. The inverted DNA segment spans about 23 kb of the 90 kb long P1 genome and it includes the invertible C segment. This phage is as viable as an isomeric P1 which carries the relevant segment in its original orientation. These results are discussed with regard to the genome organization of phage P1.     

Gamma-MSH and its related peptides do not augment ACTH-induced steroidogenesis in isolated rat adrenal cells

In a sensitive ACTH bioassay system using isolated rat adrenal cells, we tested the effect of gamma-MSH related peptides on ACTH-induced steroidogenesis. Peptides, including synthetic gamma1-, gamma2-, gamma3- and Lys-gamma3-MSH, exerted no effect in augmenting ACTH-induced steroidogenesis. None of the 16 kilodalton fragment of ACTH/beta-lipotropin precursor and its cleaved fragment had such an activity. The results are in contrast with previous reports concerning ACTH-potentiating activity of gamma-MSH related peptides and, therefore, indicate the necessity of further investigation of the principle involved in this unique biological activity.