Hayato Umekawa

Structural study of immunoaffinity-purified DNA polymerase α-DNA primase complex from calf thymus

The DNA polymerase alpha-DNA primase complex was purified over 17,000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 A and a native molecular weight of 250-260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase alpha-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 A in diameter, in agreement with the physiochemical results. The binding of the complex to DNA was also demonstrated.

Tryptophans 286 and 288 in the C-terminal region of protein B23.1 are important for its nucleolar localization.

Nucleolar protein B23 can shuttle between the nucleolus and cytoplasm. However, the mechanism involved in the protein moving and staying in the nucleolus is not fully understood. To identify the nucleolar localization signal sequence of protein B23, we examined the subnuclear location of B23.1 mutant proteins fused with green fluorescent protein in HeLa cells. The results suggested that the two C-terminal tryptophan residues (Trp-286 and Trp-288) of protein B23.1 were important in this phenomenon.

Phosphorylation of caldesmon by protein kinase C

Protein kinase C catalyzes phosphorylation of caldesmon, an F-actin binding protein of smooth muscle, in the presence of Ca2+ and phospholipid. Protein kinase C incorporates about 8 mol of phosphate/mol of chicken gizzard caldesmon. When calmodulin was added in the medium, there was an inhibition of phosphorylation. The fully phosphorylated, but not unphosphorylated, caldesmon inhibited myosin light chain kinase activity. The possibility that protein kinase C plays some role in smooth muscle contractile system through caldesmon, warrants further attention.

Potential Ability of Hot Water Adzuki (Vigna angularis) Extracts to Inhibit the Adhesion, Invasion, and Metastasis of Murine B16 Melanoma Cells

The 40% ethanol eluent of the fraction of hot-water extract from adzuki beans (EtEx.40) adsorbed onto DIAION HP-20 resin has many biological activities, for example, antioxidant, antitumorigenesis, and intestinal α-glucosidase suppressing activities. This study examined the inhibitory effect of EtEx.40 on experimental lung metastasis and the invasion of B16-BL6 melanoma cells. EtEx.40 was found significantly to reduce the number of tumor colonies. It also inhibited the adhesion and migration of B16-BL6 melanoma cells into extracellular matrix components and their invasion into reconstituted basement membrane (matrigel) without affecting cell proliferation in vitro. These in vivo data suggest that EtEx.40 possesses a strong antimetastatic ability, which might be a lead compound in functional food development.

Effects of dietary alpha- or gamma-linolenic acid on levels and fatty acid compositions of serum and hepatic lipids, and activity and mRNA abundance of 3-hydroxy-3-methylglutaryl CoA reductase in rats

The effects of diets containing equal amounts of alpha (alpha)- or gamma (gamma)-linolenic acid on lipid metabolism were compared in rats. Four groups of male Wistar rats were given the diets containing 20% perilla/corn mixed oil or borage oil in the absence (PO- and BO-diets, respectively) or presence (CPO- and CBO-diets) of cholesterol for 20 days. The PO-diet yielded lower serum cholesterol than the BO-diet, although the difference was not observed between the CPO and CBO groups. The PO and CPO groups showed lower high-density lipoprotein cholesterol than the BO and CBO groups, respectively. A similar tendency was observed in serum phospholipids. The CPO-diet gave markedly lower hepatic triglycerides than the CBO-diet. The activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was much lower on the PO-diet than on the BO-diet. mRNA abundance of HMG-CoA reductase was lower in rats on the PO-diet than on the BO-diet, though there was no significant difference between the CPO and CBO groups. The present results indicate that alpha-linolenic acid exhibits a larger hypocholesterolemic effect than gamma-linolenic acid, and it may be displayed mainly through the repression of the activity and mRNA expression of HMG-CoA reductase.

Comparative effects of short- and long-term feeding of safflower oil and perilla oil on lipid metabolism in rats

Diets high in linoleic acid (20% safflower oil contained 77.3% linoleic acid, SO-diet) and alpha-linolenic acid (20% perilla oil contained 58.4% alpha-linolenic acid, PO-diet) were fed to rats for 3, 7, 20, and 50 days, and effects of the diets on lipid metabolism were compared. Levels of serum total cholesterol and phospholipids in the rats fed the PO-diet were markedly lower than those fed the SO-diet after the seventh day. In serum and hepatic phosphatidylcholine and phosphatidylethanolamine, the proportion of n-3 fatty acids showed a greater increase in the PO group than in the SO group in the respective feeding-term. At the third and seventh days after the commencement of feeding the experimental diets, expressions of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA were significantly higher in the SO group than those in the PO group, although the difference was not observed in the longer term. There were no significant differences in the LDL receptor mRNA levels between the two groups through the experimental term, except 3-days feeding. These results indicate that alpha-linolenic acid has a more potent serum cholesterol-lowering ability than linoleic acid both in short and long feeding-terms.

Inhibition of calmodulin function by CV-159, a novel dihydropyridine compound

1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylic acid methyl 6-(5-phenyl-3-pyrazolyloxy)hexyl ester (CV-159), a new compound synthesized from dihydropyridine, was examined for its effect on calmodulin (CaM) function. The concentration of CV-159 producing 50% inhibition of Ca2+/CaM activated myosin light chain kinase (MLC kinase) was 6.2 microM. The apparent Ki value of CV-159 was 0.8 microM for MLC kinase. On the other hand, the concentration of CV-159 producing 50% inhibition of Ca2+/CaM activated cyclic nucleotide phosphodiesterase (Ca2+-PDE) was 0.55 microM. CaM antagonized competitively the CV-159-induced inhibition of activation of both MLC kinase and Ca2+-PDE. Interaction of CV-159 with CaM was also demonstrated by fluorescence studies using dansyl-CaM (5-dimethylaminonaphthalene-1-sulfonylated CaM). CV-159 produced a decrease in fluorescence intensity of dansyl-CaM, in a Ca2+-dependent fashion, and the concentration of this drug producing 50% inhibition of dansyl-CaM fluorescence was 1.2 microM. However, the concentration of nicardipine producing 50% inhibition of MLC kinase exceeded 100 microM. CaM did not antagonize the nicardipine-induced inhibition of Ca2+-PDE. These results suggest that the action of CV-159 is unique in that it inhibits both Ca2+-PDE and MLC kinase, through interaction with calmodulin. CV-159 seems to be a different class of drug from known dihydropyridine compounds.

Comparative effects of safflower oil and perilla oil on serum and hepatic lipid levels, fatty acid compositions of serum and hepatic phospholipids, and hepatic mRNA expressions of 3-hydroxy-3-methylglutaryl CoA reductase, LDL receptor, and cholesterol 7alpha-hydroxylase in young and adult rats

Male Wistar rats, 4 and 33 weeks of age, were fed the diets containing safflower oil (SO-diet, 77.3% linoleic acid) or perilla oil (PO-diet, 58.4% α-linolenic acid) for 7 days. Serum total cholesterol was lower on the PO-diet in both ages. On the other hand, hepatic cholesterol and phospholipids were significantly higher in the PO group than in the SO group of the adult rats. The PO group showed significantly lower 20:4 n-6 but higher 18:2 n-6 in hepatic phosphatidylcholine compared with the SO group in both ages. Hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA was significantly lower on the PO-diet than on the SO-diet irrespective of age. The present results show that α-linolenic acid has a higher hypocholesterolemic ability than linoleic acid in rats irrespective of age and these fatty acids behaved differently in affecting hepatic mRNA expressions of HMG-CoA reductase, LDL receptor, and cholesterol 7α-hydroxylase.

Structural characterization of the calcium binding protein S100 from adipose tissue

A partial amino acid sequence for bovine adipose tissue S100 was elucidated by characterization of peptides generated by cyanogen bromide cleavage. The cyanogen bromide peptides were aligned by homology with the bovine brain S100 beta sequence. The results demonstrate that adipose S100 beta is probably identical to brain S100 beta, and suggest that S100 beta is a conserved protein among tissues of the same species.

Inhibition of eukaryotic DNA polymerase α by persimmon (Diospyros kaki) extract and related polyphenols

The effects of persimmon extract (Diospyros kaki) and related polyphenols on eukaryotic DNA polymerase α were examined. It was found that persimmon extract, epigallocatechin gallate, and epicatechin gallate strongly inhibited the activity of DNA polymerase α purified from calf thymus. Among these polyphenols, persimmon extract had the most potent effect on DNA polymerase α activity and the concentration of persimmon extract producing 50% inhibition of the activity was 0.191 μM. Persimmon extract showed a weaker effect on DNA polymerase β and slightly inhibited primase and DNA polymerase I. The inhibition of DNA polymerase α by persimmon extract was competitive with the template‐primer and noncompetitive with dTTP substrate. The Ki value of DNA polymerase α for persimmon extract was estimated to be 70 nM. Moreover, persimmon extract inhibited [3H]thymidine incorporation of human peripheral lymphocyte cells stimulated by PHA.