The role of K+ and H+ transport systems during glucose‐ and H2O2‐induced cell death in Saccharomyces cerevisiae

Glucose, in the absence of additional nutrients, induces programmed cell death in yeast. This phenomenon is independent of yeast metacaspase (Mca1/Yca1) and of calcineurin, requires ROS production and it is concomitant with loss of cellular K⁺ and vacuolar collapse. K⁺ is a key nutrient protecting the cells and this effect depends on the Trk1 uptake system and is associated with reduced ROS production. Mutants with decreased activity of plasma membrane H⁺‐ATPase are more tolerant to glucose‐induced cell death and exhibit less ROS production. A triple mutant ena1‐4 tok1 nha1, devoid of K⁺ efflux systems, is more tolerant to both glucose‐ and H₂O₂‐induced cell death. We hypothesize that ROS production, activated by glucose and H⁺‐ATPase and inhibited by K⁺ uptake, triggers leakage of K⁺, a process favoured by K⁺ efflux systems. Loss of cytosolic K⁺ probably causes osmotic lysis of vacuoles. The nature of the ROS‐producing system sensitive to K⁺ and H⁺ transport is unknown. Copyright © 2010 John Wiley & Sons, Ltd.     

大豆异黄酮影响类风湿性关节炎模型大鼠FLS凋亡

采用完全弗氏佐剂制备类风湿性关节炎(Rheumatoid arthritis,RA)模型对照大鼠,模型对照大鼠采用大豆异黄酮(Soybean isoflavone,SBI)灌胃治疗后,分离大鼠滑膜组织,原代培养大鼠(Fibroblast like synoviocytes,FLS),real time qPCR分别检测SBI各剂量灌胃治疗对模型对照组大鼠FLS细胞凋亡相关基因和RA相关基因表达的影响。结果发现,经SBI 50 mg/kg体重、100 mg/kg体重和150 mg/kg体重3个剂量灌胃治疗后,细胞凋亡因子caspase 3、促凋亡基因Bax表达显著升高,抗凋亡基因Bcl-2表达显著降低,MMP3和fibronectin表达同样显著降低。SBI可能通过促进FLS凋亡影响模型对照组大鼠滑膜增值和关节炎症。     

Anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates as p53 activators in cervical cancer cells

A library of new anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates were designed, synthesized, and evaluated for their anticancer activity in cervical cancer cells such as HeLa and SiHa that possess low levels of p53. All 24 conjugates showed antiproliferative activity, while some of them exhibit significant cytotoxicity. In assays related to cell-cycle distribution, these conjugates induced G(2) /M arrest in HeLa cells and G(1) cell-cycle arrest in SiHa cells. Immunocytochemistry assays revealed that these compounds cause nuclear translocation of p53, thereby indicating the activation of p53. In cervical cancer cells, the p53 protein is degraded by E6 oncoprotein. Immunoblot and RT-PCR analyses proved the presence of mitochondria-mediated apoptosis with involvement p53 target genes such as BAX, Bcl2, and p21 (CDKI). Moreover, these compounds increased the phosphorylated forms of p53 and provide signals for apoptosis induction. Interestingly, one of the conjugates, (2-phenyl-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-(thiophen-2-ylmethylamino)benzoyl)piperazin-1-yl)methanone, is the most promising candidate in this series and has the potential to be taken up for further detailed studies.     

Immunological Effects of Oenothein B,an Ellagitannin Dimer,on Dendritic Cells

Oenothein B is a unique macrocyclic ellagitannin dimer that has been found in various medicinal plants belonging to Onagraceae, Lythraceae, and Myrtaceae, with diverse biological activities. The immunological effects of tannins in terms of cytokine-release from macrophages and monocytes have been discussed, while the effects on other immunocompetent cells have been the subject of minimal investigation. We evaluated the immunomodulatory effects induced by tannin treatment in human dendritic cells (DCs), which play a critical role in the initial immune response, by measuring the changes in cytokine production, cell differentiation, and cell viability. Oenothein B showed significant down-regulation of the expression of cell surface molecules, CD1a and CD83, suggesting the inhibition of DC differentiation and/or maturation. The suppressive effect on DCs was associated with the induction of apoptosis without the activation of caspase-3/7, 8, and 9, and this was supported by the morphological features indicating significant nuclear condensation. Oenothein B also markedly suppressed the production of inflammatory cytokines, such as IL-1β and IL-6, in a dose-dependent manner. These data may, in part, be able to explain the traditional use of tannin-containing medicinal plants for the treatment of a variety of inflammatory diseases, including inflammatory bowel disease, celiac disease, and rheumatoid arthritis.     

Effect of Antibiotic Amphotericin B Combinations with Selected 1,3,4-Thiadiazole Derivatives on RPTECs in an In Vitro Model

4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol (C1) and 4-[5-(naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl] benzene1,3-diol (NTBD) are representative derivatives of the thiadiazole group, with a high antimycotic potential and minimal toxicity against normal human fibroblast cells. The present study has proved its ability to synergize with the antifungal activity of AmB. The aim of this work was to evaluate the cytotoxic effects of C1 or NTBD, alone or in combination with AmB, on human renal proximal tubule epithelial cells (RPTECs) in vitro. Cell viability was assessed with the MTT assay. Flow cytometry and spectrofluorimetric techniques were used to assess the type of cell death and production of reactive oxygen species (ROS), respectively. The ELISA assay was performed to measure the caspase-2, -3, and -9 activity. ATR-FTIR spectroscopy was used to evaluate biomolecular changes in RPTECs induced by the tested formulas. The combinations of C1/NTBD and AmB did not exert a strong inhibitory effect on the viability/growth of kidney cells, as evidenced by the negligible changes in the apoptotic/necrotic rate and caspase activity, compared to the control cells. Both NTBD and C1 displayed stronger anti-oxidant activity when combined with AmB. The relatively low nephrotoxicity of the thiadiazole derivative combinations and the protective activity against AmB-induced oxidative stress may indicate their potential use in the therapy of fungal infections.     

Dengue Nonstructural Protein 1 Maintains Autophagy through Retarding Caspase-Mediated Cleavage of Beclin-1

Dengue virus (DENV) infection is a significant public health threat in tropical and subtropical regions; however, there is no specific antiviral drug. Accumulated studies have revealed that DENV infection induces several cellular responses, including autophagy and apoptosis. The crosstalk between autophagy and apoptosis is associated with the interactions among components of these two pathways, such as apoptotic caspase-mediated cleavage of autophagy-related proteins. Here, we show that DENV-induced autophagy inhibits early cell apoptosis and hence enhances DENV replication. Later, the apoptotic activities are elevated to suppress autophagy through cleavage of Beclin-1, an essential autophagy-related protein. Inhibition of cleavage of Beclin-1 by a pan-caspase inhibitor, Z-VAD, increases both autophagy and viral replication. Regarding the mechanism, we further found that DENV nonstructural protein 1 (NS1) is able to interact with Beclin-1 during DENV infection. The interaction between Beclin-1 and NS1 attenuates Beclin-1 cleavage and facilitates autophagy to prevent cell apoptosis. Our study suggests a novel mechanism whereby NS1 preserves Beclin-1 for maintaining autophagy to antagonize early cell apoptosis; however, elevated caspases trigger apoptosis by degrading Beclin-1 in the late stage of infection. These findings suggest implications for anti-DENV drug design.     

Expression of caspase-8 and caspase-9 in rat hippocampus during postnatal development

The role of caspases in the regulation of apoptosis of neurons during development is well established. An emerging body of evidence indicates that caspases may also play significant roles which are nonapoptotic. We have demonstrated previously that the executor caspase-3 exhibited a unique pattern of spatiotemporal expression in the postnatal rat hippocampal subregions, and the activation of caspase-3 in different hippocampal neurons appeared to have distinct roles during postnatal development. In the present study, we examined the expressions of initiator caspases in the hippocampus, using immunofluorescent staining for caspase-8 and caspase-9, and Hoechst 33342 staining for nuclear chromatin to assess caspase-8 and -9 expression in the CA1, CA3, and the dentate gyrus (DG) on postnatal days (P) 0, P2, P4, P7, P14, P21, P28, P56. The results indicate that caspase-8 and caspase-9 were expressed in pyramidal neurons of CA1 and CA3 fields, and granular neurons of the DG during development. Caspase-8 was expressed in a general upward trend while caspase-9 showed a slight downward pattern, but still remained at high levels in the adult hippocampus. The expression profiles of caspases-8 and -9 are distinct from that of the apoptotic cells. These data indicate that caspase-8 may be involved not only in the classical apoptotic function, but also in the cell death of necrosis, and in response to different insults and other nonapoptotic functions. Caspase-9 plays a role in apoptosis during postnatal development, but it may have other functions as well.     

Different expression of caspase-3 in rat hippocampal subregions during postnatal development

Recent studies indicate that caspase-3 has distinct characteristics in postmitotic and neuronal progenitor apoptosis. Pyramidal neurons in CA1 and CA3 of the hippocampus become postmitotic during early postnatal development, whereas granule cells in the dentate gyrus (DG) undergo self-renewal throughout life. The distribution of caspase-3 in the hippocampal subfields during postnatal development is largely unknown. We used immunofluorescent staining for two isoforms of caspase-3 (an active 17 kDa isoform and an inactive 35 kDa precursor) and the Hoechst 33342 staining for nuclear chromatin to assess caspase-3 expression in the CA1, CA3, and DG of rat hippocampus during postnatal development. The expression of active caspase-3 reached a peak at P7 in CA1, at P2 in CA3, and then decreased with age. Whereas in DG, active caspase-3 expression increased slightly after P7, and remained at high levels for the rest of the investigated period. Procaspase-3 immunoreactivity was strong at P2 and decreased gradually to a basal plateau by P21 in the three regions examined. In addition, the number of apoptotic cells in the three regions all reached maximum levels at P7, and then decreased with age. These data indicate that there were specific spatio-temporal patterns of expression of active and precursor caspase-3 in the postnatally developing rat hippocampal subregions, and that the activation of caspase-3 in neuronal progenitor cells of DG and that in the postmitotic neurons of CA1 and CA3 may have distinct roles and mechanisms during postnatal development.