The role of MAPK and FAS death receptor pathways in testicular germ cell apoptosis induced by lead

The aim of the present study is to investigate gene expression involved in the signal pathway of MAPK and death signal receptor pathway of FAS in lead- induced apoptosis of testicular germ cells. First, cell viabilities were determined by MTT assay. Second, using single cell gel-electrophoresis test （comet assay） and TUNEL staining technique, apoptotic rate and cell apoptosis localization of testicular germ cells were measured in mice treated with 0.15%, 0.3%, and 0.6% lead, respectively. Third, the immunolocalization of K-ras, c-fos, Fas, and active caspase-3 proteins was determined by immunohistochemistry. Finally, changes in the translational levels of K-ras, c-fos, Fas, and active caspase-3 were further detected by western blot analysis. Our results showed that lead could significantly induce testicular germ cell apoptosis in a dose-dependent manner （P 〈 0.01）. The mechanisms were closely related to the increased expressions of K-ras, c-fos, Fas, and active caspase-3 in apoptotic germ cells. In conclusion, K-ras/c-fos and Fas/caspase-3 death signafing receptor pathways were involved in the lead-induced apoptosis of the testicular germ cells in mice.     

Fucoxanthin induces growth arrest and apoptosis in human bladder cancer T24 cells by up-regulation of p21 and down-regulation of mortalin

Fucoxanthin, a natural carotenoid, has been reported to have anti-cancer activity in human colon cancer cells, human prostate cancer cells, human leukemia cells, and human epithelial cervical cancer cells. This study was undertaken to evaluate the molecular mechanisms of fuco- xanthin against human bladder cancer T24 cell line. MTT analysis results showed that 5 and 10 ixM fucoxanthin inhibited the proliferation of T24 cells in a dose- and time- dependent manner accompanied by the growth arrest at Go/G1 phase of cell cycle, which is mediated by the up-regu- lation of p21, a cyclin-dependent kinase （CDK）-inhibitory protein and the down-regulation of CDK-2, CDK-4, cyclin D1, and cyclin E. In addition, 20 and 40 μM fucoxanthin induced apoptosis of T24 cells by the abrogation of morta- lin-p53 complex and the reactivation of nuclear mutant- type p53, which also had tumor suppressor function as wild-type p53. All these results demonstrated that the anti- cancer activity of fucoxanthin on T24 cells was associated with cell cycle arrest at Go/G1 phase by up-regulation of p21 at low doses and apoptosis via decrease in the expres- sion level of mortalin, which is a stress regulator and a mem- ber of heat shock protein 70, followed by up-regulation of cleaved caspase-3 at high doses.     

A static pressure sensitive receptor APJ promote H9c2 cardiomyocyte hypertrophy via PI3K-autophagy pathway

This study is designed to investigate whether APJ receptor acts as a sensor in static pressure-induced cardiomyocyte hypertrophy and to investigate the mechanism of PI3K- autophagy pathway. The left ventricular hypertrophy rat model was established by coarctation of abdominal aorta. H9c2 rat cardiomyocytes were cultured in the presence of static pressure which was given by a custom-made pressure in- cubator. The results revealed that the expression of apelin/ APJ system, PI3K, Akt and their phosphorylation were sig- nificantly increased in the operation group. Static pressure up-regulated the APJ expression, PI3K phosphorylation, Akt phosphorylation, LC3-Ⅱ/Ⅰ and beclin-1 expression in cardio- myocytes. APJ shRNA pGPU6/Neo-rat-399, PI3K inhibitor LY294002, Akt inhibitor 1701-1 blocked the up-regulation of APJ, PI3K phosphorylation, Akt phosphorylation, LC3-H/I and beclin-1 expression, respectively. Moreover, static pres- sure increased the diameter, volume, protein content of cells, and these could be reversed when the cells were treated with pGPU6/Neo-rat-399, LY294002, and autop- hagy inhibitor 3-methyladenine, respectively. These results suggested that static pressure up-regulates APJ expression to promote cardiomyocyte hypertrophy by a PI3K-autop- hagy pathway.     

Kazrin F is involved in apoptosis and interacts with BAX and ARC

Kazrin has recently been identified as a functional protein that is involved in cell-cell junctions and in signal transduction. Here, we identified a new isoform, Kazrin F, which is 518 aa in length and has 97 aa unique at the N-terminus. Knockdown of Kazrin F using siRNA caused cell apoptosis and a marked decrease in cell viability measured by MTT and TUNEL assays. Co-immunoprecipitation analysis revealed that Kazrin F interacts with ARC （apoptosis repressor with caspase recruitment domain） and Bax （Bcl-2-associated X protein）. Co-localization of Kazrin F with ARC and Bax in the cytoplasm was determined by immunofluorescence analysis. These results suggested that Kazrin F might play an important role in regulating cellular apoptosis by interacting with ARC and Bax.     

Hydrogen peroxide induces the activation of the phospholipase C-γ1 survival pathway in PC12 cells： protective role in apoptosis

It has been reported that phospholipase C-γ1 （PLC-γ1） plays an important protective role in hydrogen peroxide （H2O2）-induced pheochromocytoma （PC） 12 cells death. However, most studies have used high doses of H2O2 and the downstream targets of PLC-γ1 activation remain to be identified. The present study was designed to examine the roles of PLC-γ1 signaling pathway in the apoptosis of PC12 cells induced by low dose of H2O2, as well as the downstream factors involved in this pathway. Low-dose treatment of H2O2 resulted in PLC-γ1 tyrosine phosphorylation in a time-dependent manner and H2O2 killed the PC12 cells by inducing necrosis. In contrast, pretreatment of PCI2 cells with U73122, a specific inhibitor of PLC, markedly increased the percentage of dead cells. The mode of cell death was converted to apoptosis as determined by Hoechst/PI nuclear staining and fluorescence microscopy. Western blot analysis demonstrated that the expression of Bcl-2 protein and the activation of pro-caspase-3 were not significantly affected by low dose of H2O2 alone. However, after pretreatment with U73122, Bcl-2 protein expression was dramatically decreased and the activation of pro-caspase-3 was significantly increased. We concluded that PLC-γ1 plays an important protective role in H2O2-induced PC12 cells death. Bcl-2 and caspase-3 probably participate in the signaling pathway as downstream factors.     

Regulation of membrane band 3 Tyr-phosphorylation by proteolysis of p72^syk and possible involvement in senescence process

Erythrocyte senescence is characterized by exposure of cell surface epitopes on cell membrane proteins leading to immune mediated removal of red blood cells. One mechanism for antigen formation is tyrosine phosphorylation （Tyr-P） of the transmembrane protein band 3 by Syk kinase. Our aim was to test the hypothesis that proteolytic activation of Syk kinase by conversion from 72 kDa （p72^syk） to the 36 kDa （p36^syk） isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of 32p uptake into the cytoplasmic domain of band 3 after addition of p72^syk or p36syk. Effect of prephosphorylation of erythrocyte membrane band 3 protein by p36^syk on p72^syk-mediated phosphorylation and the effect of addition of a protease inhibitor （leupeptin） on p72s^yk-mediated phosphorylation were studied by autoradiographic visualization of 32p uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36^Syk had a higher band 3 tyrosine phosphorylating activity compared with p72^syk. Pre-phosphorylation with p36^syk or p72^syk increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72^syk but not p36^syk band 3 tyrosine phosphorylating activity significantly. Both soluble and membrane skeletal fractions of band 3 protein were equally tyrosine phosphorylated by each Syk isoform. In conclusion, we confirmed the hypothesis that proteolytic cleavage of p72^syk is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton.     

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex,the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.     

The role of autophagy in sensitizing malignant glioma cells to radiation therapy

Malignant gliomas represent the majority of primary brain tumors. The current standard treatments for malignant gliomas include surgical resection, radiation therapy, and chemotherapy. Radiotherapy, a standard adjuvant therapy, confers some survival advantages, but resistance of the glioma cells to the efficacy of radiation limits the success of the treatment. The mechanisms underlying glioma cell radioresistance have remained elusive. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Recent studies suggest that autophagy may be important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy. Also, autophagy is a novel response of glioma cells to ionizing radiation. Autophagic cell death is considered programmed cell death type II, whereas apoptosis is programmed cell death type I. These two types of cell death are predominantly distinctive, but many studies demonstrate a cross-talk between them. Whether autophagy in cancer cells causes death or protects cells is controversial. The regulatory pathways of autophagy share several molecules. PI3K/Akt/mTOR, DNA-PK, tumor suppressor genes, mitochondrial damage, and lysosome may play important roles in radiation-induced autophagy in glioma cells. Recently, a highly tumorigenic glioma tumor subpopulation, termed cancer stem cell or tumor-initiating cell, has been shown to promote therapeutic resistance. This review summarizes the main mediators associated with radiation-induced autophagy in malignant glioma cells and discusses the implications of the cancer stem cell hypothesis for the development of future therapies for brain tumors.     

Antioxidant activity of mangostin in cell-free system and its effect on K562 leukemia cell line in photodynamic therapy

Mangostin （MAG）, a kind of xanthone widely used in diet and medicine, has antioxidant, anti-inflammatory, antimicrobial, and anticancer activities. On account of its antioxidant activity, MAG might protect cancer cells from free radical damage in photodynamic therapy （PDT） during which reactive oxygen species production was stimulated leading to irreversible tumor cell injury. In this study, the antioxidant activity of MAG was investigated and the influence of MAG on K562 cells in 5-aminolevulinic acid （ALA）-based PDT is demon- strated. The results showed that MAG could scavenge hydroxyl radical, superoxide anion, and hydrogen per- oxide and inhibit the formation of malondialdehyde （MDA）, but increase the amounts of singlet oxygen in cell-free systems. MAG inhibits cell proliferation and enhances cell apoptosis, lipid peroxidation, and DNA damage in ALA-PDT on K562 cells. NAN3, a singlet oxygen quencher, suppresses the MAG-induced cell apoptosis, lipid peroxidation, and DNA damage. In con- clusion, MAG enhances the PDT-induced cytotoxicity in K562 cells and singlet oxygen was involved in this process. These results implied that the effect of antioxidants on PDT might be determined by its sensitization ability to singlet oxygen.     

Nucleotide Variation in the NCED3 Region of Arabidopsis thaliana and its Association Study with Abscisic Acid Content under Drought Stress

Drought tolerance is a comprehensive quantitative trait that is being understood further at the molecular genetic level. Abscisic acid （ABA） is the main drought-induced hormone that regulates the expression of many genes related to drought responses. 9-cis-epoxycarotenoid dioxygenase （NCED3） is thought to be a key enzyme in ABA biosynthesis. In this paper, we measured the ABA content increase under drought stress, and sequenced and compared the sequence of AtNCED3 among 22 Arabidopsis thaliana accessions. The results showed that the fold of ABA content increase under drought stress was highly variable among these accessions. High density single nucleotide polymorphism （SNP） and insertion/deletion （indel） were found in the AtNCED3 region, on average one SNP per 87.4 bp and one indel per 502 bp. Nucleotide diversity was significantly lower in the coding region than that in non-coding regions. The results of an association study with ANOVA analysis suggested that the 274th site （P←→S） and the 327th site （P←→R） amino acid variations might be the cause of ABA content increase of 163av accession under drought stress.     

A glucocorticoid amplifies IL-2-induced selective expansion of CD4^＋CD25^＋FOXP3^＋ regulatory T cells in vivo and suppresses graft-versus-host disease after allogeneic lymphocyte transplantation

Regulatory T （Treg） cells are a subpopulation of T cells that not only prevent autoimmunity, but also control a wide range of T cell-dependent immune responses. Glucocorticoid treatment （dexamethasone, or Dex） has been reported to amplify IL-2-mediated selective in vivo expansion of Treg cells. We simultaneously administered Dex and IL-2 to the donor in a murine allogeneic lymphocyte transplantation model to expand functional suppressive CD4＋CD25＋FOXP3＋ T cells in the graft and to raise the regulatory T cell/effector T cell （Treg/ Teff） ratio to prevent graft-versus-host disease （GVHD）. After combined treatment of the donor with Dex （5 mg/kg/day） and IL-2 （300,000 IU/mouse/day） for 3 days, grafts were subjected to flow cytometric analysis, and transplantation was carried out from male C57BL/6 mice to female BALB/c mice aged 8-12 weeks. Results showed that short-term simultaneous administration of Dex and IL-2 markedly expanded functional suppressive CD4＋CD25＋FOXP3＋ T cells in the murine spleen. In this murine allogeneic transplantation model, the grafts from donors with Dex and IL-2 pre-treatment led to a longer survival time for the recipients than for the control group （median survival time 〉 60 day vs. 12 day, P = 0.0002）. The ratio of Treg/Teff also increased remarkably （0.43 ±0.15 vs. 0.14 ± 0.01, P = 0.01）. This study demonstrated that co-stimulation with Dex and IL-2 selectively expanded functional CD4＋CD25＋FOXP3＋ T cells in vivo, and that grafts from donors pre-treated with Dex and IL-2 led to longer survival time and greater suppression of GVHD after allogeneic transplantation. Thus, GVHD can be suppressed by the specific expansion of regulatory T cells with Dex and IL-2 in graft donors.     

Macropinocytosis contributes to the macrophage foam cell formation in RAW264.7 cells

The key event in the atherosclerosis development is the lipids uptake by macrophage and the formation of foam cell in subendothelial arterial space. Besides the uptake of modified low-density lipoprotein （LDL） by scavenger receptor-mediated endocytosis, macrophages possess constitutive macropinocytosis, which is capable of taking up a large quantity of solute. Macrophage foam cell formation could be induced in RAW264.7 cells by increasing the serum concentration in the culture medium. Foam cell formation induced by serum could be blocked by phosphoinositide 3-kinase inhibitor, LY294002 or wortmannin, which inhibited macropinocytosis but not receptor-mediated endocytosis. Further analysis indicated that macropinocytosis took place at the gangliosides-enriched membrane area. Cholesterol depletion by β-methylcyclodextrin-blocked macropinocytosis without affecting scavenger receptormediated endocytosis of modified LDLs. These results suggested that macropinocytosis might be one of the important mechanisms for lipid uptake in macrophage. And it made significant contribution to the lipid accumulation and foam cell formation.     

Comparative Detection of Calcium Fluctuations in Single Female Sex Cells of Tobacco to Distinguish Calcium Signals Triggered by in vitro Fertilization

Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca^2＋-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca^2＋ in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca^2＋ signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 μM Fluo-3 for 30 min at 30 ℃. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca^2＋]cyt of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.     

Mammalian cell display for rapid screening scFv antibody therapy

Human antibodies are beginning to draw attention for use in immune gene therapy. The efficient generation of effective therapeutic monoclonal antibodies suitable for the treatment of cancers and infectious diseases would be enormously valuable. Antibody display methods are increasingly used to screen human monoclonal antibodies. Here we report the construction of a mammalian cell display method derived from a naive antibody repertoire, for which human single- chain variable fragments （scFv） have been transiently dis- played on 293T cell surfaces based on a pDisplay vector. The sizes of the current pDisplay-scFv antibody repertoires have been estimated to be 0.74 × 10^7. An immunoblot assay confirmed the expression of the scFv antibody library. The subceilular distribution of ErbB3-scFv expression plasmid facilitated the display of ErbB3 scFv on the cell membrane surface and the efficiency of the display was evaluated by fluorescence-activated cell sorting. This method of mamma- lian cell display was verified by successfully screening ErbB3 scFv candidates. A published scFv control was used to confirm the feasibility of the ErbB3 scFv screening process. Three ErbB3 scFv candidates were produced and they were found to have affinity similar to the published scFv candidate. Thus, the present screening system provided an optimal alternative for rapid acquisition of a novel candi- date scFv sequence to target genes with high affinity in vitro.     

Potentiation of （-）-epigallocatechin-3-gaUate-induced apoptosis by bortezomib. in multiple myeloma cells

The green tea constituent, （-）-epigallocatechin-3- gallate （EGCG）, has chemopreventive and anticancer effects. This is partially because of the selective ability of EGCG to induce apoptosis and death in cancer cells without affecting normal cells. In the present study, the activity of EGCG against the myeloma cell line, KM3, was examined. Our results demonstrated, for the first time, that the treatment of the KM3 cell line with EGCG inhibits cell proliferation and induces apoptosis, and there is a synergistic effect when EGCG and borte- zomib are combined. Further experiments showed that this effect involves the NF-KB pathway. EGCG inhibits the expression of the P65 mRNA and P65/pP65 protein, meanwhile it downregulates pIKBα expression and upregulates IKBα expression. EGCG also activates caspase-3, -8, cleaved caspase-9, and poly-ADP-ribose polymerase （PARP） and subsequent apoptosis. These findings provided experimental evidence for efficacy of EGCG alone or in combination with bortezomib in multiple myeloma therapy.     

Mobilization and Acquisition of Sparingly Soluble P-Sources by Brassica Cultivars under P-Starved Environment I. Differential Growth Response,P-Efficiency Characteristics and P-Remobilization

Phosphorus （P） starvation is highly notorious for limiting plant growth around the globe. To combat P-starvation, plants constantly sense the changes in their environment, and elicit an elegant myriad of plastic responses and rescue strategies to enhance P-solublization and acquisition from bound soil P-forms. Relative growth responses, P-solublization and P- acquisition ability of 14 diverse Brassica cultivars grown with sparingly soluble P-sources （Rock-P （RP） and Ca3（PO4）2 （TCP）） were evaluated in a solution culture experiment. Cultivars showed considerable genetic diversity in terms of biomass accumulation, concentration and contents of P and Ca in shoots and roots, P-stress factor （PSF） and P use efficiency. Cultivars showed variable P-stress tolerance, and cultivars depicting low PSF and high P-efficiency values were better adaptable to P-starvation. In experiment 2, after initial feeding on optimum nutrition for 12 d after transplanting （DAT）, class-I （low P-tolerant （Oscar and Con-II）） and class-II （low P-sensitive （Gold Rush and RL-18）） cultivars were exposed to P-free environment for 25 d. All of the cultivars remobilized P from above ground parts to their roots during growth in P-free environment, the magnitude of which was variable in tested cultivars. P-concentrations （[P]s） at 37 DAT were higher in developing compared with developed leaves. Translocation of absorbed P from metabolically inactive to active sites in P- stressed plants may have helped class-I cultivars to establish a better rooting system, which provided a basis for enhanced P-utilization efficiency （PUE） and tolerance against P-stress. By supplying TCP and RP spatially separated from other nutrients in split root study, class-I cultivars were still able to mobilize RP and TCP more efficiently compared with class-II cultivars. To compare the growth behavior under P-stress, cultivars were grown in pots for 41 d after sowing, using a soil low in P （NaHCO3-extractable P = 3.97 mg/kg, M