Recent evidences show that peroxisome proliferator-activated receptor c (PPARc) is involved in the modulation of the amyloid-b (Ab) cascade causing Alzheimer’s disease (AD) and treatment with PPARc agonists protects against AD pathology. However, the function of PPARc steady-state activity in Ab cascade and AD pathology remains unclear. In this study, an antagonist of PPARc, GW9662, was injected into the fourth ventricle of APP/PS1 transgenic mice to inhibit PPARc activity in cerebellum. The results show that inhibition of PPARc significantly induced Ab levels in cerebellum and caused cerebellar motor dysfunction in APP/PS1 transgenic mice. Moreover, GW9662 treatment markedly decreased the cerebellar levels of insulin-degrading enzyme (IDE), which is responsible for the cellular degradation of Ab. Since cerebellum is spared from significant Ab accumulation and neurotoxicity in AD patients and animal models, these findings suggest a crucial role of PPARc steady-state activity in protection of cerebellum against AD pathology.

The effects of two polyphenols, chebulinic acid and tellimagrandin I, on DNA strand breaks mediated by H2O2/Cu(Ⅱ), hydroquinone (HQ)/Cu(Ⅱ) and H2O2/Fe(Ⅱ) in pBR322 plasmid DNA and genomic DNA of cultured MRC-5 human embryo lung fibroblasts were examined. The results demonstrated that chebulinic acid and tellimagrandin I obviously inhibited HQ/Cu(Ⅱ)-and H2O2/Cu(Ⅱ)-mediated pBR322 DNA strand breaks. When MRC-5 cells were treated with HQ/Cu(Ⅱ), the presence of chebulinic acid or tellimagrandin I inhibited HQ/Cu(Ⅱ)-mediated double strand breaks of genomic DNA. The presence of chebulinic acid or tellimagrandin I did not affect the H2O2-and HQ-mediated reduction of Cu(Ⅱ) to Cu(I). Both polyphenols could slightly inhibit H2O2/Fe(Ⅱ)-mediated plasmid DNA strand break at the lower concentration (1-10 lM), but potentiate the DNA strand break at the higher concentration (over 50 lM). These results demonstrated that chebulinic acid and tellimagrandin I possessed antioxidant action in certain conditions and exerted prooxidant action on DNA strand breaks in other conditions.

The present study established a model of RyR2 knockdown cardiomyocytes and elucidated the role of RyR2 in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR2 expression. Reduction of RyR2 expression was documented by RT-PCR, western blot, and immunofluorescence. Ca2+ signals were investigated by measuring the relative intracellular Ca2+ concentration, spontaneous Ca2+ oscillations, caffeine-induced Ca2+ release, and L-type Ca2+ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca2+ oscillations were observed, and the baseline [Ca2+]i remained at the low level. Exposure to 3 mMaconitine increased the frequency and decreased the amplitude of Ca2+ oscillations; the baseline [Ca2+]i and the level of caffeine-induced Ca2+ release were increased but the L-type Ca2+ currents were inhibited after application of 3 mM aconitine for 5min. In RyR2 knockdown cardiomyocytes, the steady and periodic spontaneous Ca2+ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca2+]i level; the level of caffeine-induced Ca2+ release was increased but L-type Ca2+ currents were inhibited. Alterations of RyR2 are important consequences of aconitine-stimulation and activation of RyR2 appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca2+ leakage through the sarcoplasmic reticulum RyR2 channel.

Aconitine is an effective ingredient in Aconite tuber, an important traditional Chinese medicine. Aconitine is also known to be a highly toxic diterpenoid alkaloid with arrhythmogenic effects. In the present study, we have characterized the properties of arrhythmic cytotoxicity and explored the possible mechanisms of aconitine-induced cardiomyocytes. Results show that aconitine induces significant abnormity in the spontaneous beating rate, amplitude of spontaneous oscillations and the relative intracellular Ca2+ concentration. Also, mRNA transcription levels and protein expressions of SR Ca2+ release channel RyR2 and sarcolemmal NCX were elevated in aconitine-induced cardiomyocytes. However, co-treatment with ruthenium red (RR), a RyR channel inhibitor, could reverse the aconitineinduced abnormity in intracellular Ca2+ signals. These results demonstrate that disruption of intracellular Ca2+ homeostasis in the cardiac excitation–contraction coupling (EC coupling) is a crucial mechanism of arrhythmic cytotoxicity in aconitine-induced cardiomyocytes. Moreover, certain inhibitors appear to play an important role in the detoxification of aconitine-induced Ca2+-dependent arrhythmias.

To elucidate the mechanism of nacre biomineralization, the mantle of Pinctada fucata (P. fucata) from the South China Sea was used. Using the mantle cDNA library and the ESTs we have cloned through suppression subtractive hybridization (SSH), ten novel genes including PFMG1 were obtained through nested PCR. Bioinformative results showed that PFMG1 had a high homology (40%) with Onchocerca VolVulus calcium-binding protein CBP-1 and had two EF-hand calcium-binding domains from the 81st to the 93rd amino acid and from the 98th to the 133rd amino acid in the deduced amino acid sequence. The results of multitissue RT-PCR and in situ hybridization demonstrated the high expression of PFMG1 in the mantle of P. fucata and confirmed the SSH method. The results of GST-PFMG1 on CaCO3 crystallization showed significant effects on nucleation and precipitation of CaCO3. PFMG1 was cloned into the pcDNA.3.1/myc-HisA vector and was subsequently transfected into MC3T3-E1 cells. RT-PCR revealed upregulation of the marker genes related to cell growth, differentiation, and mineralization, and BMP-2, osterix, and osteopontin were upregulated as a result. This research work suggests that PFMG1 plays an important role in the nacre biomineralization, and the SSH method can pave the way for the bulk cloning and characterization of new genes involved in biomineralization in P. fucata and may accelerate research on the mechanism of pearl formation.

Tellimagrandin I and chebulinic acid, two hydrolysable tannins, have been shown to exert anti-tumor properties. Dysfunctional gap junctional communication (GJIC) has been recognized as being involved in carcinogenesis. The human cervical carcinoma HeLa cells have been reported to be deficient in functional GJIC. In present study, we investigated whether tellimagrandin I and chebulinic acid might restore functional GJIC in HeLa cells. Both compounds could inhibit the growth of HeLa cells. Either Lucifer yellow transfer assay or calcein transfer assay demonstrated that tellimagrandin I improved GJIC in HeLa cells while chebulinic acid showed no effect on GJIC. The GJIC enhancement by tellimagrandin I occurred along with an increase of Cx43 gene expression atmRNAand protein levels. Exposure to tellimagrandin I also led to inhibition of proliferation and anchorage-independent growth of HeLa cells. In addition, tellimagrandin I decreased the percentage of cells in the G0/G1 and G2/M phases coinciding with an increase in the percentage of cells in the S phase. The accumulation of cells in S phase was coupled with a decreased expression of cyclin A that was critical to the progression of S phase. These results suggested that restoring GJIC might be one explanation for tellimagrandin I antitumor effects, whereas chebulinic acid exerted antitumor action through other pathways.

Methyl protodioscin is a furostanol bisglycoside with antitumor properties. The present study investigated its effects on human chronic myelogenous leukemia K562 cells. Cell cycle analysis showed that methyl protodioscin caused distinct G2/M arrest, with the appearance of polyploidy population. The levels of cyclin B1 decreased, whereas Cdc2 kept at a steady level. Subsequent apoptosis after G2/M blockage was demonstrated through DNA fragmentation and the annexin V staining assay. Methyl protodioscin induced a biphasic alteration (i.e. an early hyperpolarization, followed by depolarization) in mitochondrial membrane potential of K562 cells. The transient decline of intracellular Ca2C concentration was observed at early stage. The generation of reactive oxygen species was also detected. The anti-apoptotic Bcl-xL transiently increased and then decreased. And the pro-apoptotic Bax was markedly up-regulated. Taken together, these data demonstrated that methyl protodioscin inhibits K562 cell proliferation via G2/M arrest and apoptosis, with mitochondrial hyperpolarization and the disruption of Ca2C homeostasis playing important roles.

A novel human gene full-length cDNA sequence-TSARG2 was identified from a human testis cDNA library using the SRG2 gene (GenBank Accession No. AF395083), which was significantly up-regulated in cryptorchidism, as an electronic probe. TSARG2 was 1223 bp in length. The putative protein encoded by this gene was 305 amino acids with a theoretical molecular weight of 34,751 and isoelectric point of 9.85. The sequence shared no significant homology with any known protein in databases except SRG2. Northern blot analysis revealed that 1.7 kb TSARG2 transcript was detected selectively in human testis. Furthermore, results of in situ hybridization assay confirmed that TSARG2 was expressed in seminiferous tubules, more precisely in spermatogonia and spermatocyte. No mutation was found by PCR-SSCP in 122 cases of azoospermia, severe oligzoospermia, and cryptorchidism. The green fluorescence produced by pEGFP-C1/TSARG2 was detected on the nucleus of COS7 cells after 24 h post-transfection. The pcDNA3.1())/TSARG2 plasmid was constructed and introduced into MCF7 cells by liposome transfection. TSARG2 can accelerate MCF7 cells to traverse the S-phase and enter the G2-phase compared with the control without transfection of TSARG2, which suggested that this gene plays an important role in the development of cryptorchid testis and is a testis-specific apoptosis candidate oncogene.

Tellimagrandin I is a hydrolysable tannin compound widely present in plants. In this study, the effect of tellimagrandin I on chemically induced erythroid and megakaryocytic differentiation was investigated using K562 cells as differentiation model. It was found that tellimagrandin I not only inhibited the hemoglobin synthesis in butyric acid (BA)-and hemin-induced K562 cells with IC50 of 3 and 40μM, respectively, but also inhibited other erythroid differentiation marker including acetylcholinesterase (AChE) and glycophorin A (GPA) in BA-induced K562 cells. Tellimagrandin I also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of CD61 protein, a megakaryocytic marker. RT-PCR analysis showed that tellimagrandin I decreased the expression of erythroid genes (γ-globin and porphobilinogen deaminase (PBGD)) and related transcription factors (GATA-1 and NF-E2) in BA-induced K562 cells, whereas tellimagrandin I induced the overexpresison of GATA-2 transcription factor that played negative regulation on erythroid differentiation. These results indicated that tellimagrandin I had inhibitory effects on erythroid and megakaryocytic differentiation, which suggested that tannins like tellimagrandin I might influence the anti-tumor efficiency of some drugs and the hematopoiesis processes.

Tannins are a group of widely distributed plant polyphenols, some of which are beneficial to health because of their chemopreventive activities. In the present study, we investigated the effects and action mechanisms of woodfordin I, a macrocyclic ellagitannin dimer, on human chronic myelogenous leukemia (CML) K562 cells. The results showed that woodfordin I was able to suppress the proliferation and induce apoptosis in K562 cells. Apoptosis was evaluated by cytomorphology, internucleosomal DNA fragmentation, and externalization of phosphatidylserine. Woodfordin I treatment caused a rapid and sustained loss of mitochondrial transmembrane potential (MMP), transient generation of reactive oxygen species (ROS), transient elevation of intracellular Ca2+ concentration, and cytosolic accumulation of cytochrome c. The activation of caspase-9 and 3, but not caspase-8, was also demonstrated, indicating that the apoptotic signaling triggered by woodfordin I was mediated through the intrinsic mitochondria-dependent pathway. Western blot and immunofluorescence analysis revealed that the antiapoptotic Bcl-2 and Bcl-xL levels were downregulated, together with the pro-apoptotic Bax protein. Significantly, woodfordin I-induced apoptosis was associated with a decline in the levels of c-Abl, Bcr-Abl, and cellular protein tyrosine phosphorylation. Considering the consequence of all the events in the process of woodfordin I-induced apoptosis, the mitochondrial dysfunction is directly responsible for the pro-apoptotic effects on K562 cells. Furthermore, because CML is a malignancy of pleuripotent hematopoietic cells caused by the dysregulated tyrosine kinase activity of Bcr-Abl, these findings suggest that woodfordin I may be a potential lead compound against CML.