Suppressive effect of polysaccharides from the edible and medicinal mushrooms,Lentinus edodes and Agaricus blazei,on the expression of cytochrome P450s in mice

To investigate the effects of lentinan from Lentinas edodes and polysaccharides from Agaricus blazei (ABPS) on the expression of cytochrome P450s (CYPs), lentinan (10 mg/kg/day) or ABPS (200 mg/kg/day) was administered to female BALB/c mice four times every other day by intraperitoneal injection. Lentinan and ABPS suppressed both the constitutive and 3-methylcholanthrene-induced CYP1A expression and ethoxyresorufin-O-deethylation activity in the liver.     

Luteolin,a flavone,does not suppress postprandial glucose absorption through an inhibition of alpha-glucosidase action

In order to clarify the postprandial glucose suppression via alpha-glucosidase (AGH) inhibitory action by natural compounds, flavonoids were examined in this study. Among the flavonoids (luteolin, kaempferol, chrysin, and galangin), luteolin showed the potent maltase inhibitory activity with the IC50 of 2.3 mM, while less inhibitions were observed against sucrase. In addition, the effects of maltase inhibition by flavonoids were observed in the descending order of potency of luteolin > kaempferol > chrysin > galangin. Apparently, the AGH inhibition power greatly increased with the replacement of hydroxyl groups at 3' and 4'-position of the B-ring. However, the inhibitory power of luteolin was poorer than a therapeutic drug (acarbose: IC50; 430 nM). As a result of a single oral administration of maltose or sucrose (2 g/kg) in SD rats, no significant change in blood glucose level with the doses of 100 and 200 mg/kg of luteolin was observed. These findings strongly suggested that luteolin given at less than 200 mg/kg did not possess the ability to suppress the glucose production from carbohydrates through the inhibition of AGH action in the gut.     

Signification of the sexualizing substance produced by the sexualized planarians

Asexual worms of an exclusively fissiparous strain (the OH strain) of the planarian Dugesia ryukyuensis keep developing hermaphroditic reproductive organs and eventually undergo sexual reproduction instead of asexual reproduction, namely fission, if they are fed with sexually mature worms of an exclusively oviparous planarian, Bdellocephala brunnea, suggesting that the sexually mature worms has a sexualizing substance(s). The fully sexualized worms no longer need the feeding on sexual worms to maintain the sexuality. Here, we demonstrate that the sexualized worms produce enough of their own sexualizing substance similar to that contained in B. brunnea. In case of surgical ablation of the sexualized worms, the fragments with sexual organs regenerate to become sexual, while those without sexual organs, namely head fragments, regenerate to return to the asexual state. The asexual regenerants from the sexualized worms are also fully sexualized by being fed with B. brunnea. Additionally, it was reported that head region in sexually mature worms lacks the putative sexualizing substance necessary for complete sexualization (Sakurai, 1981). These results suggest that the fragments without sexual organ lack enough of an amount of the putative sexualizing substance and the sexuality is maintained by the sexualizing substance contained in the sexualized worms.     

Seasonal changes in the sexualization of the planarian Dugesia ryukyuensis

Asexual individuals in a fissiparous clone of the planarian Dugesia ryukyuensis develop hermaphroditic sexual organs and eventually undergo sexual reproduction instead of asexual reproduction if they are fed with the adults of Bdellocephala brunnea, an oviparous planaria. The experimental sexualization means that the adults of B. brunnea contain a putative sexualizing substance(s), which is the first candidate for the chemical(s) responsible for switching from asexual to sexual reproduction in metazoans. In the present study, the feeding experiment over two consecutive years revealed that the experimental sexualization has seasonal changes. In summer, the asexual individuals were not fully sexualized, though they developed a pair of ovaries. The developing ovaries degenerate if the feeding is stopped. On the contrary, in winter, they developed all the sexual organs. The sexual organs keep developing even if the feeding is stopped after a certain critical point named the point-of-no-return. It was demonstrated that the extreme difference of the sexualization was attributed to the seasonal change of the quality and/or quantity of the sexualizing substance contained in B. brunnea, as well as the minor change of the susceptibility to the sexualizing substance in the asexual individuals. On the other hand, the histological research of B. brunnea revealed that the degree of the maturation of the sexual organs varied extremely through a year. Taking these results into account, we suggest that the production of the sexualizing substance has no direct relation to any particular mature sexual organs.     

Analysis of gene expression profile in p130(Cas)-deficient fibroblasts

p130(Cas) (Cas) is a docking protein that becomes tyrosine phosphorylated in v-Src- or v-Crk-transformed cells and in integrin-stimulated cells. Cas -/- fibroblasts show defects in stress fiber formation, cell spreading, cell migration, and transformation by activated Src. To further characterize the role of Cas in signaling, we compared the expression profile in Cas -/- fibroblasts with that in Cas-re-expressing fibroblasts using the microarray methods. In Cas -/- fibroblasts, the expression of heme oxygenase 1 and caveolin-1 was reduced, but the expression of procollagen 1 alpha 1, procollagen 3 alpha 1, procollagen 11 alpha 1, elastin, periostin, TSC-36, and MARCKS was enhanced. The domains in Cas necessary for the change varied among these genes. Activated Src reduced the expression of most of these genes both in Cas -/- and in Cas +/+ fibroblasts. These results suggest the existence of signaling pathways that emanate from Cas to gene expression.     

Upward shift of the pH optimum of Acremonium ascorbate oxidase

A gene encoding a thermostable Acremonium ascorbate oxidase (ASOM) was randomly mutated to generate mutant enzymes with altered pH optima. One of the mutants, which exhibited a significantly higher activity in the pH range 4.5-7 compared to ASOM, had a Gln183Arg substitution in the region corresponding to SBR1, one of the substrate binding regions of the zucchini enzyme. The other mutant with almost the same pH profile as Gln183Arg had a Thr527Ala substitution near the type 3 copper center and became more sensitive to azide than ASOM. Site-directed mutagenesis in the substrate binding regions with reference to the amino acid sequences of plant enzymes led to isolation of mutants shifted upward in the pH optimum; Val193Pro and Val193Pro/Pro190Ile increased the pH optimum by 1 and 0.5 units, respectively, while retaining the near-wild-type thermostability and azide sensitivity. The homology model of ASOM constructed from the zucchini enzyme coordinates suggested that replacement of Val193 by Pro could disturb the ion pair networks among Arg309, Glu192, Arg194 and Glu311. This perturbation could affect either the molecular recognition between the substrate and ASOM or the electron transfer from the substrate to the type 1 copper center, leading to the alkaline shift of the catalytic activity of the mutant enzyme. The other mutations, Val193Pro/Pro190Ile, could also induce similar structural perturbations involving the ion pair networks.     

Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV

Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly-L-Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase alpha, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 microM CaM was about 10 microg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly-L-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly-L-Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr44. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.