Roles of N-acetylglucosaminyltransferase III in epithelial-to-mesenchymal transition induced by transforming growth factor β1 (TGF-β1) in epithelial cell lines

The epithelial-to-mesenchymal transition (EMT) plays crucial roles in embryonic development, wound healing, tissue repair, and cancer progression. Results of this study show how transforming growth factor β1 (TGF-β1) down-regulates expression of N-acetylglucosaminyltransferase III (GnT-III) during EMT-like changes. Treatment with TGF-β1 resulted in a decrease in E-cadherin expression and GnT-III expression, as well as its product, the bisected N-glycans, which was confirmed by erythro-agglutinating phytohemagglutinin lectin blot and HPLC analysis in human MCF-10A and mouse GE11 cells. In contrast with GnT-III, the expression of N-acetylglucosaminyltransferase V was slightly enhanced by TGF-β1 treatment. Changes in the N-glycan patterns on α3β1 integrin, one of the target proteins for GnT-III, were also confirmed by lectin blot analysis. To understand the roles of GnT-III expression in EMT-like changes, the MCF-10A cell was stably transfected with GnT-III. It is of particular interest that overexpression of GnT-III influenced EMT-like changes induced by TGF-β1, which was confirmed by cell morphological changes of phase contrast, immunochemical staining patterns of E-cadherin, and actin. In addition, GnT-III modified E-cadherin, which served to prolong E-cadherin turnover on the cell surface examined by biotinylation and pulse-chase experiments. GnT-III expression consistently inhibited β-catenin translocation from cell-cell contact into the cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppressed TGF-β1-induced cell motility. Taken together, these observations are the first to clearly demonstrate that GnT-III affects cell properties, which in turn influence EMT-like changes, and to explain a molecular mechanism for the inhibitory effects of GnT-III on cancer metastasis.     

Fluorescence correlation spectroscopy to monitor Kai protein-based circadian oscillations in real time

Dynamic protein-protein interactions play an essential role in cellular regulatory systems. The cyanobacterial circadian clock is an oscillatory system that can be reconstituted in vitro by mixing ATP and three clock proteins: KaiA, KaiB, and KaiC. Association and dissociation of KaiB from KaiC-containing complexes are critical to circadian phosphorylation and dephosphorylation of KaiC. We developed an automated and noninvasive method to monitor dynamic complex formation in real time using confocal fluorescence correlation spectroscopy (FCS) and uniformly labeled KaiB as a probe. A nanomolar concentration of the labeled KaiB for FCS measurement did not interfere with the oscillatory system but behaved similarly to the wild-type one during the measurement period (>5 days). The fluorescent probe was stable against repeated laser exposure. As an application, we show that this detection system allowed analysis of the dynamics of both long term circadian oscillations and short term responses to temperature changes (～10 min) in the same sample. This suggested that a phase shift of the clock with a high temperature pulse occurred just after the stimulus through dissociation of KaiB from the KaiC complex. This monitoring method should improve our understanding of the mechanisms underlying this cellular circadian oscillator and provide a means to assess dynamic protein interactions in biological systems characterized by rates similar to those observed with the Kai proteins.     

Dietary zinc status reversibly alters both the feeding behaviors of the rats and gene expression patterns in diencephalon

Nutritional status influences feeding behaviors, food preferences, and taste sensations. For example, zinc-deficient rats have been reported to show reduced and cyclic food intake patterns with increased preferences for NaCl. Although some impairments of the central nervous and endocrine systems have been speculated to be involved in these phenomena, the effects of short-term zinc deficiency on the brain have not been well examined to date. In this study, we performed a comprehensive analysis of the gene expression patterns in the rat diencephalon, which is a portion of the brain that includes the hypothalamus and thalamus, after short-term zinc deficiency and also during zinc recovery. The rats showed reduced and cyclic food intake patterns with increased salt preferences after a 10-day dietary zinc deficiency. A comparative analysis of their diencephalons using cDNA microarrays revealed that approximately 1% of the genes expressed in the diencephalons showed significantly altered expression levels. On the other hand, a 6-day zinc supplementation following the deprivation allowed for the recovery to initial food intake behaviors and salt preferences. The expression levels of most of the genes that had been altered by exposure to zinc deficient conditions were also recovered. These results show that feeding behaviors, taste preferences and gene expression patterns in the diencephalon respond quickly to changing zinc levels.     

Design, synthesis, and biological evaluation of 4-arylmethyl-1-phenylpyrazole and 4-aryloxy-1-phenylpyrazole derivatives as novel androgen receptor antagonists

A series of 4-arylmethyl-1-phenylpyrazole and 4-aryloxy-1-phenylpyrazole compounds B were designed, synthesized, and evaluated for their potential as new-generation androgen receptor (AR) antagonists therapeutically effective against castration-resistant prostate cancer (CRPC). Introduction of a bulky amide substituent (R(2)) to the terminal aryl ring of the 4-arylmethyl group favored the reduction of agonistic activity and improved the pharmacokinetic (PK) properties. Similarly, introduction of a bulky substituent in the 4-aryloxy derivatives also resulted in improved PK properties. Compounds 28 h and 44b exhibited potent antitumor effects against a CRPC model of LNCaP-hr cell line in a mouse xenograft model. On the contrary, bicalutamide showed only partial suppression of tumor growth. These results suggest that the novel pyrazole derivatives are new-generation AR antagonists, different from the 'first-generation' antagonists such as bicalutamide in a CRPC treatment model.     

How do nitrogen and phosphorus deficiencies affect strigolactone production and exudation?

Plants exude strigolactones (SLs) to attract symbiotic arbuscular mycorrhizal fungi in the rhizosphere. Previous studies have demonstrated that phosphorus (P) deficiency, but not nitrogen (N) deficiency, significantly promotes SL exudation in red clover, while in sorghum not only P deficiency but also N deficiency enhances SL exudation. There are differences between plant species in SL exudation under P- and N-deficient conditions, which may possibly be related to differences between legumes and non-legumes. To investigate this possibility in detail, the effects of N and P deficiencies on SL exudation were examined in Fabaceae (alfalfa and Chinese milk vetch), Asteraceae (marigold and lettuce), Solanaceae (tomato), and Poaceae (wheat) plants. In alfalfa as expected, and unexpectedly in tomato, only P deficiency promoted SL exudation. In contrast, in Chinese milk vetch, a leguminous plant, and in the other non-leguminous plants examined, N deficiency as well as P deficiency enhanced SL exudation. Distinct reductions in shoot P levels were observed in plants grown under N deficiency, except for tomato, in which shoot P level was increased by N starvation, suggesting that the P status of the shoot regulates SL exudation. There seems to be a correlation between shoot P levels and SL exudation across the species/families investigated.     

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.     

Dehydration tolerance in apple seedlings is affected by an inhibitor of ABA 8'-hydroxylase CYP707A

The effects of an abscisic acid (ABA) 8′-hydroxylase inhibitor (Abz-F1) on ABA catabolism, stomatal aperture, and water potential were examined in apple seedlings under dehydration and rehydration conditions. In this study, 9-cis-epoxycarotenoid dioxigenase (MdNCED) and ABA 8′-hydroxylase (MdCYP707A) genes were isolated and their expressions were investigated under dehydration and rehydration conditions. The stomatal aperture decreased up to 4 h after spraying with Abz-F1 and the stomatal aperture in the Abz-F1-treated leaves was generally lower than that in the untreated control-leaves during the dehydration condition. Although the water potential in untreated control-leaves decreased with the progress of dehydration, it was maintained at a higher level in the Abz-F1 treated-leaves than in the untreated control-leaves during dehydration. Endogenous ABA concentrations increased with dehydration in both the Abz-F1 treated- and untreated-control-leaves, but the ABA levels in the Abz-F1 treated-leaves were higher than those in the untreated control-leaves throughout dehydration. In contrast, the phaseic acid (PA) concentrations in the Abz-F1 treated-leaves were lower than those in the untreated control-leaves during dehydration. The expressions of MdNCEDs in the Abz-F1 treated-leaves were lower than those in the untreated control-leaves regardless of the higher endogenous ABA concentrations. Moreover, the expressions of MdCYP707As in the Abz-F1 treated-leaves were also lower than those in the untreated control-leaves. Higher 50% effective concentrations (EC₅₀) and ascorbic acid concentrations were observed in the Abz-F1 treated-leaves, which show that the oxidative damage under dehydration may be reduced by Abz-F1 application.These results suggest that prompt stomata closure is required for survival under dehydration, and Abz-F1 application may therefore be of practical use. The increase of endogenous ABA, which induced prompt stomata closure in Abz-F1 treated-leaves may depend on inhibition of the expression of MdCYP707As. Furthermore, the results showed the close relationship between MdNCEDs and MdCYP707As on ABA catabolism.     

Prostaglandin E₂ increases both osteoblastic and osteoclastic activity in the scales and participates in calcium metabolism in goldfish

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E? (PGE?) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE?(10?? and 10?? M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE? (10?? to 10?? M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE? (10(-7) to 10?? M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE? treated-scales. Thus, PGE? acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE?-injencted goldfish increased significantly. We conclude that, in teleosts, PGE? activates both osteoblasts and osteoclasts and participates in calcium metabolism.     

Consumption of Pueraria Flower Extract Reduces Body Mass Index via a Decrease in the Visceral Fat Area in Obese Humans

In Japan, kudzu is a familiar plant, well-known as an ingredient in the Japanese-style confections kudzu-kiri and kudzu-mochi. In this study, we focused on the flower of kudzu (Pueraria thomsonii) and conducted a clinical trial to investigate the effects of Pueraria thomsonii flower extract (PFE) on obesity using obese Japanese males and females (BMI ≥ 25 kg/m(2)). Eighty-one obese subjects were randomly divided into three groups and consumed test food containing 300 mg of PFE, 200 mg of PFE, and a placebo over 12 weeks. The results indicate that PFE intake reduces BMI and decreases, the visceral fat area, but not the subcutaneous fat area. In addition, the decrease in visceral fat area showed no sexual dimorphism. Consequently, we propose that PFE intake expresses its BMI reduction effects via a decrease in visceral fat area.     

Intrinsic kinase activity and SQ/TQ domain of Chk2 kinase as well as N-terminal domain of Wip1 phosphatase are required for regulation of Chk2 by Wip1

The anti-oncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell cycle checkpoint regulation. Recently, we have shown that Chk2 associates with the oncogenic Wip1 (PPM1D) phosphatase and that Wip1 acts as a negative regulator of Chk2 during DNA damage response by dephosphorylating phosphorylated Thr-68 in activated Chk2 (Fujimoto, H., Onishi, N., Kato, N., Takekawa, M., Xu, X. Z., Kosugi, A., Kondo, T., Imamura, M., Oishi, I., Yoda, A., and Minami, Y. (2006) Cell Death Differ. 13, 1170-1180). Here, we performed structure-function analyses of Chk2 and Wip1 by using a series of deletion or amino acid-substituted mutant proteins of Chk2 and Wip1. We show that nuclear localization of both Chk2 and Wip1 is required for their association in cultured cells and that the serine-glutamine (SQ)/threonine-glutamine (TQ) domain of Chk2, containing Thr-68, and the N-terminal domain of Wip1, comprising about 100 amino acids, are necessary and sufficient for the association of both molecules. However, it was found that an intrinsic kinase activity of Chk2, but not phosphatase activity of Wip1, is required for the association of fulllength Chk2 and Wip1. Interestingly, we also show that the mutant Wip1 proteins, bearing the N-terminal domain of Wip1 alone or lacking an intrinsic phosphatase activity, exhibit dominant negative effects on the functions of the wild-type Wip1, i.e. ectopic expression of either of these Wip1 mutants inhibits dephosphorylation of Thr-68 in Chk2 by Wip1 and anti-apoptotic function of Wip1. These results provide a molecular basis for developing novel anti-cancer drugs, targeting oncogenic Wip1 phosphatase.