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)     

Determinants of Slow Walking Speed in Ambulatory Patients Undergoing Maintenance Hemodialysis

Walking ability is significantly lower in hemodialysis patients compared to healthy people. Decreased walking ability characterized by slow walking speed is associated with adverse clinical events, but determinants of decreased walking speed in hemodialysis patients are unknown. The purpose of this study was to identify factors associated with slow walking speed in ambulatory hemodialysis patients. Subjects were 122 outpatients (64 men, 58 women; mean age, 68 years) undergoing hemodialysis. Clinical characteristics including comorbidities, motor function (strength, flexibility, and balance), and maximum walking speed (MWS) were measured and compared across sex-specific tertiles of MWS. Univariate and multivariate logistic regression analyses were performed to examine whether clinical characteristics and motor function could discriminate between the lowest, middle, and highest tertiles of MWS. Significant and common factors that discriminated the lowest and highest tertiles of MWS from other categories were presence of cardiac disease (lowest: odds ratio [OR] = 3.33, 95% confidence interval [CI] = 1.26–8.83, P<0.05; highest: OR = 2.84, 95% CI = 1.18–6.84, P<0.05), leg strength (OR = 0.62, 95% CI = 0.40–0.95, P<0.05; OR = 0.57, 95% CI = 0.39–0.82, P<0.01), and standing balance (OR = 0.76, 95% CI = 0.63–0.92, P<0.01; OR = 0.81, 95% CI = 0.68–0.97, P<0.05). History of fracture (OR = 3.35, 95% CI = 1.08–10.38; P<0.05) was a significant factor only in the lowest tertile. Cardiac disease, history of fracture, decreased leg strength, and poor standing balance were independently associated with slow walking speed in ambulatory hemodialysis patients. These findings provide useful data for planning effective therapeutic regimens to prevent decreases in walking ability in ambulatory hemodialysis patients.     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

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.     

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.