ERK activation induced by selenium treatment significantly downregulates beta/gamma-secretase activity and Tau phosphorylation in the transgenic rat overexpressing human selenoprotein M.

Selenium reportedly contribute to the modulation process of protein phosphorylation to regulate various cellular functions including growth, differentiation, proliferation and development. The aim of this study was to investigate whether selenium and Selenoprotein M (SelM) affects the mechanism of Alzheimer's disease. To achieve this, we determined the change of the MAPK pathway, secretase activity, and Tau phosphorylation in the transgenic rat overexpressing human selenoprotein M. Based on these results, we concluded that, i) CMV/GFP-hSelM Tg rats showed a high activity level of antioxidant enzyme in the brain tissues, ii) in response to selenium treatment, the ERK signaling pathway was significantly increased in Tg rats, but did not change in wild-type rats, iii) the activation of the ERK pathway by selenium treatment and SelM overexpression induced the inhibition of the alpha/gamma-secretase activity related to the protection of Abeta-42 production, iv) the activation of the ERK pathway by selenium treatment and SelM overexpression inhibited the phosphorylation in several sites of Tau protein. Therefore, these results provide strong evidence that selenium treatment and SelM activate the ERK pathway to attenuate alpha/gamma-secretase-mediated proteolysis and Tau phosphorylation to protect brain function.

Genistein inhibits rat aortic smooth muscle cell proliferation through the induction of p27kip1.

Diet is one of the most important factors that influence the risks for cardiovascular diseases. Genistein, an isoflavone found in soy, may benefit the cardiovascular system. Here, we investigated the effect of genistein on platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic smooth muscle cells (RASMCs). Genistein significantly inhibited 25 ng/ml PDGF-BB-induced RASMC proliferation and [3H]-thymidine incorporation into DNA at 10, 20, and 40 microM. In accordance with these findings, genistein blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Western blot analysis showed that genistein not only inhibited phosphorylation of retinoblastoma protein (pRb) and expression of cyclin E, cyclin-dependent kinase (CDK) 2, and proliferating cell nuclear antigen (PCNA) protein, but also inhibited downregulation of cyclin-dependent kinase inhibitor (CKI) p27kip1. However, genistein did not affect p21cip1, CDK4, and cyclin D1 expression or early signal transduction through PDGF beta-receptor, extracellular signal-regulated kinases 1/2 (ERK1/2), Akt, and phospholipase C (PLC) gamma1 phosphorylation. These results suggest that genistein inhibits PDGF-BB-induced RASMC proliferation via G0/G1 arrest in association with induction of p27kip1, which may contribute to the beneficial effects of genistein on the cardiovascular system.

YSK2821, a newly synthesized indoledione derivative, inhibits cell proliferation and cell cycle progression via the cell cycle-related proteins by regulating phosphatidylinositol-3 kinase cascade in vascular smooth muscle cells.

Indoledione derivatives have pronounced biological effects, i.e., cytotoxic activities against cancer cell lines and antifungal and antibacterial activities. The present study was designed to investigate the effects of YSK2821, a newly synthesized indoledione derivative, on platelet-derived growth factor (PDGF-BB)-induced vascular smooth muscle cell (VSMC) proliferation, as well as the molecular mechanisms of the anti-proliferative effects of YSK2821 in VSMCs. We found that YSK2821 caused the accumulation of cells in the G1 phase of the cell cycle and inhibited [3H]-thymidine incorporation. We demonstrated that YSK2821 remarkably decreased Akt kinase phosphorylation as the mechanism by which YSK2821 suppressed cell signal transduction events in VSMC proliferation. Furthermore, in terms of the effects of YSK2821 on cell cycle-related proteins, YSK2821 enhanced the expression of the cyclin-dependent protein kinase (CDK) inhibitor p27 and down-regulated CDK2 and cyclin E expression, but did not affect CDK4 and cyclin D1 expression. YSK2821 also inhibited the phosphorylation of Rb, a key regulator in the cell cycle. These results indicate that YSK2821, a newly synthesized indoledione derivative, may inhibit VSMC proliferation via a phosphatidylinositol (PI)-3 kinase-dependent pathway, and thus shed light on a novel role for YSK2821 as a potential preventive regulator of cardiovascular disease.

Asymmetric synthesis of cyclopropyl-fused 2'-C-methylcarbanucleosides as potential anti-HCV agents.

Novel 2'-C-methyl-cyclopropyl-fused carbocyclic nucleosides as potential anti-HCV agents were stereoselectively synthesized, utilizing regioselective cleavage of the isopropylidene group and cyclic sulfate chemistry as key steps.

Differential effect of 7,12-dimethylbenz[a]anthracene on human and mouse CYP1B1 from livers of castrated transgenic mice.

Humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and human cytochrome P450 1B1 (CYP1B1) (hCYP1B1) have been created by this group. The aims of this study was to determine if 7,12-dimethylbenz[a]anthracene (DMBA) functions as testosterone or doxycycline in its ability to induce or reduce expression of hCYP1B1 or endogenous mouse CYP1B1 (mCYP1B1). This was tested in the livers by treating castrated transgenic males and hCYP1B1/luciferase-transfected cells with DMBA. Herein, DMBA-treated group exhibited (i) gradual reduction of hCYP1B1 expression at the transcript, protein, and activity levels but gradually induced its transcript level during DMBA release; (ii) gradual reduction of hCYP1B1 at the transcript and protein levels, as in the case of doxycycline or testosterone; (iii) gradual induction of mCYP1B1 expression at the transcript and protein levels but gradually reduced its transcript level during DMBA release. In parallel, DMBA-treated transfected cells exhibited gradual increase in luciferase activity in a time-and dose-dependent manner. Thus, castrated transgenic males or in vitro system could be useful as models for the detection of polycyclic aromatic hydrocarbons (PAHs) or environmental toxicants by measuring either hCYP1B1 or mCYP1B1 expressions.

Tau and GSK3beta dephosphorylations are required for regulating Pin1 phosphorylation.

Pin1 binds mitotically phosphorylated Thr231-Pro232 and Thr212-Pro213 sites on tau, and a Pin1 deficiency in mice leads to tau hyperphosphorylation. The aim of this study was to determine if the dephosphorylation or inhibition of tau and GSK3beta phosphorylation induces the Pin1 phosphorylation. To test this, human SK-N-MC cells were stably transfected with a fusion gene containing neuron-specific enolase (NSE)-controlled APPsw gene(NSE/APPsw), to induce Abeta-42. The stable transfectants were then transiently transfected with NSE/Splice, lacking human tau (NSE/Splice), or NSE/hTau, containing human tau, into the cells. The NSE/Splice- and NSE/hTau-cells were then treated with lithium. We concluded that (i) there was more C99-beta APP accumulation than C83-betaAPP in APPsw-tansfectant and thereby promoted Abeta-42 production in transfectants. (ii) the inhibition of tau and GSK3beta phosphorylations correlated with increase in Pin1 activation in NSE/hTau- cells. Thus, these observations suggest that Pin1 might have an inhibitive role in phosphorylating tau and GSK3beta for protecting against Alzheimer's disease.

Stereoselective synthesis of 3-hydroxymethyl-D-cyclopentenone, the versatile intermediate for the synthesis of carbocyclic nucleosides.

The preparative and stereoselective synthesis (45- 50% overall yields, >50 g scale) of the key carbasugars 7a-d was achieved from D-ribose via stereoselective Grignard reaction and oxidative rearrangement as key reactions.

NSE-controlled carboxyl-terminus of APP gene over-expressing in transgenic mice induces altered expressions in behavior, Abeta-42, and GSK3beta binding proteins.

The amyloid protein precursor (APP) is cleaved in its intramembranous domain by gamma-secrease to generate amyloid beta and a free carboxyl-terminal intracellular fragment. The carboxyl-terminal of 105 amino acids of APP (APP-C105) plays a crucial role in the neuropathology of Alzheimer's disease (AD), but it is incompletely understand how APP-C105 overexpression interacts and regulates the brain function and Abeta-42 levels, and whether or not it is associated with the expressions of GSK3beta-binding proteins. To test this, transgenic mice expressing NSE-controlled APP-C105 were produced and tested for their above phenotypes. A behavioral deficit was observed in the 9 months old transgenic mice, and western blot indicated that there was a predominant expression of APP-C105 in transgenic brains compared with those of non-transgenic brains. In parallel, APP-C105 overexpression resulted in the modulation of the Abeta-42 level, gamma-secretase activity, GSK3beta-binding proteins including PS1, tau, and beta-catenin in the brains of the transgenic mice relative to the non-transgenic mice. Thus, altered expressions of these neuropathological phenotypes in APP-C105 transgenic mice could be useful targets in developing new therapeutic treatments.

An in vivo bioassay for detecting antiandrogens using humanized transgenic mice coexpressing the tetracycline-controlled transactivator and human CYP1B1 gene.

The typical strategy used in analysis of antiandrogens involves the morphological changes of a marker in castrated rats Hershberger assay for the prostate, seminal vesicle, levator ani plus bulbocavernosus muscles (LABC), Cowper's gland, and glans penis. However, there are disadvantages to this approach, such as the time required, and the results may not correspond to those in actual human exposure. To evaluate its ability for detecting antiandrogens, in vivo the dose effect of di-(2-ethylhexyl) phthalate (DEHP) and time effect of five antiandrogens, DEHP, di-n-butyl phthalate (DBP), diethyl phthalate (DEP), linuron (3-(4-dichlorophenyl)-methoxy-1-methylurea), and 2,4'-DDE (1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethylene), were investigated using humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and the human cytochrome P450 (CYP) enzyme CYP1B1 (hCYP1B1). Adult transgenic males were treated with each of the five antiandrogens, and their tTA-driven hCYP1B1 expressions analyzed by real-time polymerase chain reaction (PCR) and/or Western blot and for O-debenzylation activity. Herein, the treatments of adult males with the five antiandrogens were shown to affect the increased levels of tTA-driven hCYP1B1 expression in both dose-dependent and repeated experiments. Thus, this novel in vivo bioassay, using humanized transgenic mice, is useful for measuring antiandrogens, and is a means to a more relevant bioassay relating to actual human exposure.

Carboxyl-terminus of the amyloid protein precursor and ERbeta are required for estrogenic effect in activating mitogen-activated protein kinase.

Estrogen influences the processing of the amyloid beta precursor protein (APP) in the pathogenesis of Alzheimer's disease, and this effect is mediated by estrogen receptors (ERs) in activating mitogen-activated protein kinase (MAPK)-signaling pathway. To test whether the estrogenic effect on both carboxyl-terminal amino acid fragment (C-terminal) of APP (APP-C105)- and ERbeta-mediated MAPK activation in in vitro, two hybrid genes containing each human ERbeta and APP-C105 gene fused to the neuron-specific enolase (NSE) promoter were constructed and were transfected to the neuronal SK-N-MC cells. Western blot shows that the activation of JNK-signaling pathway, but not p38 and ERK, is dependent on ERbeta through estrogen treatment and APP-C105 is also mediated through estrogen in activating MAPK-signaling pathway. The results suggest that ERbeta and APP-C105 derived from APP are necessary for estrogenic effect in activating MAPK-signaling pathway.

Aberrant expressions of pathogenic phenotype in Alzheimer's diseased transgenic mice carrying NSE-controlled APPsw.

Mutations in the APP gene lead to enhanced cleavage by the beta- and gamma-secretase, and increased Abeta formation, which are tightly associated with Alzheimer's disease (AD)-like neuropathological changes. To examine whether depositions of Abeta by APP mutations are increased, and if this is associated with potential pathogenic phenotypes, the APPsw was expressed in a transgenic line under the control of the neuron-specific enolase (NSE) promoter. A behavioral dysfunction was shown at 12 months, and intensive staining bands, with APP and Abeta-42 antibodies, were visible in the brains of transgenic mice. Of the MAPK family, both JNK and p38 were activated in the brains of transgenic mice, whereas there was no significant activation of the ERK. In parallel, tau phosphorylation was also enhanced in the transgenic relative to the control mice. Moreover, the Cox-2 levels, from Western blot and immunostaining, were increased in the brains of the transgenic line. Furthermore, there were significant caspase-3- and TUNEL-stained nuclei in the transgenic line compared to the age-matched control mice. Thus, these results suggest that NSE-controlled APPsw transgenic mice appear to be a more relevant model in neuropathological phenotypes of AD, and thus could be useful in developing new therapeutic treatments for targeting the aberrant phenotypes that appear in these mice.

Synthesis of halogenated 9-(dihydroxycyclopent-4'-enyl) adenines and their inhibitory activities against S-adenosylhomocysteine hydrolase.

Novel halovinyl analogues of neplanocin A without 4'-hydroxymethyl group were easily synthesized starting from D-ribose via cyclopentenone 5 as a key intermediate and their inhibitory activity against SAH hydrolase was assayed.

Differential expression of proteins of caspases and Bcl-2 families in the brain of mice.

Apoptosis is an important process in the variety of different biological system including cell death and embryonic development. Inappropriate apoptosis is implicated in many human diseases such as Alzheimer's disease. Central component of the machinery of apoptosis program in neurons of patients with Alzheimer's disease includes proteins of caspases and Bcl-2 families. We examined whether endogenous protein levels of caspases and Bcl-2 families are expressed in a differential manner during the embryonic and postnatal development of BDF1 strain. Here, all four proteins with caspases-3, -9, Bcl-2 and Bax were highly expressed between embryonic day 19 and 1 week age of early postnatal development, but thereafter the expression dramatically declined. These patterns are needed to compare the proteins in the brains of APPsw-transgenic mice that are expected to be expressed highly in the brain of adult mice. Thus, the results are useful to understand fundamentally the mechanisms of the apoptotic changes during the embryonic and postnatal development of Alzheimer's model mice.

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens.

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.