Phospholipase D2 activity suppresses hydrogen peroxide-induced apoptosis in PC12 cells

Phospholipase D (PLD) plays an important role as an effector in the membrane lipid-mediated signal transduction. However, the precise physiological functions of PLD are not yet well understood. In this study, we examined the role of PLD activity in hydrogen peroxide (H(2)O(2))-induced apoptosis in rat pheochromocytoma (PC12) cells. Treatment of PC12 cells with H(2)O(2) resulted in induction of apoptosis in these cells, which is accompanied by the activation of PLD. This H(2)O(2)-induced apoptosis was enhanced remarkably when phosphatidic acid production by PLD was selectively inhibited by pretreating the PC12 cells with 1-butanol. Expression of PLD2, but not of PLD1, correlated with increased H(2)O(2)-induced PLD activity in a concentration- and time-dependent manner. Concomitant with PLD activation, the PLD2 activity suppressed H(2)O(2)-induced apoptosis in PC12 cells. Expression of PLD2 lipase-inactive mutant (K758R) had no effect on either PLD activity or apoptosis. PLD2 activity also suppressed H(2)O(2)-induced cleavage and activation of caspase-3. Taken together, the results suggest that PLD2 activity is specifically up-regulated by H(2)O(2) in PC12 cells and that it plays a suppressive role in H(2)O(2)-induced apoptosis.     

Dividing roles of prion protein in staurosporine-mediated apoptosis

Prion protein (PrPC) is a normal cellular glycoprotein that is expressed in almost all tissues including the central nervous system. Much attention has been focused on this protein because conversion of the normal PrPC to the diseased form (PrPSc) plays an essential role in transmissible spongiform encephalopathies such as mad cow disease and Creutzfeldt-Jakob disease. In spite of the extensive effort, the normal physiological function of PrPC remains elusive. Emerging evidence suggests that PrPC plays a protective role against cellular stresses including apoptosis induced by various pro-apoptotic agents such as Bax and staurosporine (STS), however, other reports showed overexpression of PrPC enhances STS-mediated apoptosis. In this study, we took a different approach by depleting endogenous PrPC using specific interfering RNA technique and compared the depleting and overproducing effects of PrPC on STS-induced apoptosis in neuro-2a (N2a) cells. We demonstrate here that down-regulation of PrPC sensitizes N2a cells to STS-induced cytotoxicity and apoptosis. The enhanced apoptosis induced by STS was shown by increased DNA fragmentation, immunoreactivity of Bax, and caspase-3 cleavage. We also showed that overproduction of PrPC had little or no effect on STS-mediated DNA fragmentation in N2a cells but it augments STS-mediated apoptosis in HEK293 cells, suggesting a cell line-specific effect. In addition, the inhibitory effect of PrPC on STS-mediated cellular stress appears to be modulated in part through induction of cell cycle G2 accumulation. Together, our data suggest that physiological level of endogenous PrPC plays a protective role against STS-mediated cellular stress. Loss of this protection could render cells more prone to cellular insults such as STS.     

Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin analogues as inhibitors of tubulin polymerization and apoptosis inducers

A series of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates (8a–r) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated anticancer activity against five human cancer cell lines (lung, colon, renal, prostate and cervical). All the tested compounds showed potent anticancer activity with IC₅₀ values ranging from 0.87 to 16.59 μM. Among them compounds 8n and 8p showed significant anticancer activity in lung cancer cells with IC₅₀ values 0.91 and 0.87 μM, respectively. Flow cytometric analysis revealed that these compounds induced cell cycle arrest in G₂/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay and immunofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, DNA fragmentation analysis also suggested that these compounds induced cell death by apoptosis. Overall, the current study demonstrated that the synthesis of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates as promising anticancer agents with G₂/M cell cycle arrest and apoptotic-inducing activities via targeting tubulin.     

Caspase-8 dependent trail-induced apoptosis in cancer cell lines is inhibited by vitamin C and catalase

TNF-related apoptosis-inducing ligand (TRAIL/ Apo-2L) is a member of the TNF family of apoptosis-inducing proteins that initiates apoptosis in a variety of neoplastic cells while displaying minimal or absent cytotoxicity to most normal cells. Therefore, TRAIL is currently considered a promising target to develop anti-cancer therapies. TRAIL-receptor ligation recruits and activates pro-caspase-8, which in turn activates proteins that mediate disruption of the mitochondrial membranes. These events lead to the nuclear and cytosolic damage characteristic of apoptosis. Here we report that TRAIL-induced apoptosis is mediated by oxidative stress and that vitamin C (ascorbic acid), a potent nutritional antioxidant, protects cancer cell lines from apoptosis induced by TRAIL. Vitamin C impedes the elevation of reactive oxygen species (ROS) levels induced by TRAIL and impairs caspase-8 activation. We found that the removal of hydrogen peroxide by extracellular catalase during TRAIL-induced apoptosis also impairs caspase-8 activation. These data suggest that hydrogen peroxide is produced during TRAIL-receptor ligation, and that the increase of intracellular ROS regulates the activation of caspase-8 during apoptosis. Additionally we propose a mechanism by which cancer cells might resist apoptosis via TRAIL, by the intake of the nutritional antioxidant vitamin C. This work was supported by grants from the National Institutes of Health (CA 30388), the New York State Department of Health (M020113) and the Lebensfeld Foundation.     

Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats

The aim of this study was to investigate the role of apoptosis or necrosis in the development of delayed infarct, and the relationship between the level of XIAP gene, caspase-3 activation and ischemic cell death following transient focal cerebral ischemia. Adult male Sprague-Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 50 min and reperfusion for 0.5, 4, 8, 24 h, 3, 7, 14 days. On TTC-stained coronal sections, delayed infarct was observed to develop in the whole MCA territory, especially in frontoparietal cortex after ischemia. Near total infarct was shown in striatum 24 h after MCAo, while delayed infarct was evident in the cortex. By day 3, the infarct had progressively expanded to the nearly whole area of the frontoparietal cortex. Flow cytometric analysis of Annexin-V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that MCAo dominantly induced necrosis in ischemic core, striatum. Apoptosis contributed to delayed infarct and cell death in the border zone, dorsolateral cortex and hippocampus. The time-course of caspase-3 activation was consistent with the changes of apoptosis and infarct following MCAo. Further RT-PCR experiments indicated that there was a biphasic regulation of XIAP in time- and region-dependent manner after ischemia. In the infarct core (striatum), following a transient and slight increase during 0.5 h to 4 h post-MCAo, expression of XIAP mRNA markedly decreased. On the other hand, a longer and larger upregulation of XIAP was observed at early time points in border zone (0.5 to 8 h, in dorsolateral cortex; 0.5 to 24 h in hippocampus), then the level of XIAP reduced. A negative correlation was observed between apoptosis and regulation of XIAP gene in these regions. Our findings suggest a possible association between expression of XIAP gene, apoptosis and delayed infarct following ischemia.     

The stingless bee honey protects against hydrogen peroxide-induced oxidative damage and lipopolysaccharide-induced inflammation in vitro

Oxidative stress, DNA damage, and unresolved inflammation are the predisposing factors of many chronic and degenerative diseases, including cancer. Stingless bee honey (SBH) is recognized to have high medicinal value by traditional medicine practitioners and has been used to treat various illnesses traditionally. This study aimed to determine the antioxidant, anti-inflammatory, and genoprotective effects of SBH by using in vitro cell culture models. The sugar content, total phenolic content, radical scavenging activity, and ferric reducing antioxidant power (FRAP) of SBH were determined in this study. Then, the protective effect of SBH against hydrogen peroxide (H₂O₂)-induced cell death and DNA damage was studied by using WIL2-NS human lymphoblastoid cell line, while the lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages cell line was used to study the anti-inflammatory effects of SBH. Results from this present study showed that the major sugar contents of SBH were fructose (19.39 + 0.01%) and glucose (14.03 ± 0.03%). Besides, the total phenolic content, the radical scavenging activity, and the FRAP value of SBH were 15.38 ± 0.02 mg GAE/100 g of honey, 34.04 ± 0.21%, and 206.77 + 1.76 μM AAE/100 g honey respectively. Pretreatment with SBH protected WIL2-NS cells from H₂O₂-induced cell death and DNA damage (p < 0.001). Moreover, SBH was also able to attenuate the production of nitric oxide by inhibiting the expression of inducible nitric oxide synthase in LPS-induced RAW 264.7 cells (p < 0.001). In conclusion, SBH is rich in total phenolic content and possesses strong antioxidant, anti-inflammatory, and genoprotective properties. Our current findings suggest that SBH might be useful in the prevention and treatment of many diseases caused by oxidative stress and inflammation assuming the observed effects are also achievable in vivo.     

Dehydrotrametenolic acid selectively inhibits the growth of H-ras transformed rat2 cells and induces apoptosis through caspase-3 pathway

The screening of natural products that preferentially inhibit growth of H-ras transformed rat2 cells vs. rat2 cells was performed to identify H-ras specific growth inhibitor. A lanostane-type triterpene acid, dehydrotrametenolic acid (3beta-hydroxylanosta-7,9(11),24-trien-21-oic acid), was isolated from the sclerotium of Poria cocos (Polyporaceae). Dehydrotrametenolic acid selectively inhibited the growth of H-ras transformed cells with a GI(50) value of 40 microM. FACS analysis indicated that the compound exerted its anti-proliferation effects through cell cycle arrest at G2/M phase and accumulation of sub-G1 population. Dehydrotrametenolic acid-induced apoptosis was further confirmed with chromosomal DNA fragmentation, caspase-3 activation, and degradation of PARP and Lamin A/C degradation. The compound also regulated the expression of H-ras, Akt and Erk, which are the downstream proteins of H-ras signaling pathways. The results suggest that dehydrotrametenolic acid can be a potential anticancer agent against H-ras transformed tumor.     

Facile construction of fused benzimidazole-isoquinolinones that induce cell-cycle arrest and apoptosis in colorectal cancer cells

Colorectal cancer (CRC) is one of the most frequent, malignant gastrointestinal tumors, and strategies and effectiveness of current therapy are limited. A series of benzimidazole-isoquinolinone derivatives (BIDs) was synthesized and screened to identify novel scaffolds for CRC. Of the compounds evaluated, 7g exhibited the most promising anti-cancer properties. Employing two CRC cell lines, SW620 and HT29, 7g was found to suppress growth and proliferation of the cell lines at a concentration of ～20?µM. Treatment followed an increase in G₂/M cell cycle arrest, which was attributed to cyclin B1 and cyclin-dependent kinase 1 (CDK1) signaling deficiencies with simultaneous enhancement in p21 and p53 activity. In addition, mitochondrial-mediated apoptosis was induced in CRC cells. Interestingly, 7g decreased phosphorylated AKT, mTOR and 4E-BP1 levels, while promoting the expression/stability of PTEN. Since PTEN controls input into the PI3K/AKT/mTOR pathway, antiproliferative effects can be attributed to PTEN-mediated tumor suppression. Collectively, these results suggest that BIDs exert antitumor activity in CRC by impairing PI3K/AKT/mTOR signaling. Against a small kinase panel, 7g exhibited low affinity at 5?µM suggesting anticancer properties likely stem through a non-kinase mechanism. Because of the novelty of BIDs, the structure can serve as a lead scaffold to design new CRC therapies.     

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H₂O₂) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H₂O₂ preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H₂O₂ stimulated divergent pathways, with caspases being activated only by H₂O₂. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H₂O₂, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H₂O₂. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.     

Neuronal apoptosis induced by pharmacological concentrations of 3-hydroxykynurenine: characterization and protection by dantrolene and Bcl-2 overexpression

We have studied neurotoxicity induced by pharmacological concentrations of 3-hydroxykynurenine (3-HK), an endogenous toxin implicated in certain neurodegenerative diseases, in cerebellar granule cells, PC12 pheochromocytoma cells, and GT1-7 hypothalamic neurosecretory cells. In all three cell types, the toxicity was induced in a dose-dependent manner by 3-HK at high micromolar concentrations and had features characteristic of apoptosis, including chromatin condensation and internucleosomal DNA cleavage. In cerebellar granule cells, the 3-HK neurotoxicity was unaffected by xanthine oxidase inhibitors but markedly potentiated by superoxide dismutase and its hemelike mimetic, MnTBAP [manganese(III) tetrakis(benzoic acid)porphyrin chloride]. Catalase blocked 3-HK neurotoxicity in the absence and presence of superoxide dismutase or MnTBAP. The formation of H(2)O(2) was demonstrated in PC12 and GT1-7 cells treated with 3-HK, by measuring the increase in the fluorescent product, 2',7'-dichlorofluorescein. In both PC12 and cerebellar granule cells, inhibitors of the neutral amino acid transporter that mediates the uptake of 3-HK failed to block 3-HK toxicity. However, their toxicity was slightly potentiated by the iron chelator, deferoxamine. Taken together, our results suggest that neurotoxicity induced by pharmacological concentrations of 3-HK in these cell types is mediated primarily by H(2)O(2), which is formed most likely by auto-oxidation of 3-HK in extracellular compartments. 3-HK-induced death of PC12 and GT1-7 cells was protected by dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. The protection by dantrolene was associated with a marked increase in the protein level of Bcl-2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl-2 in GT1-7 cells elicited by gene transfection suppressed 3-HK toxicity. Thus, dantrolene may elicit its neuroprotective effects by mechanisms involving up-regulation of the level and function of Bcl-2 protein.     

Synthesis of beta-alanine C60 derivative and its protective effect on hydrogen peroxide-induced apoptosis in rat pheochromocytoma cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the effective ways to prevent the reactive oxygen species (ROS) mediated cellular injury is dietary or pharmaceutical augmentation of some free radical scavenger. Water-soluble amino-fullerene derivative is a novel compound that behaves as a free radical scavenger with excellent biocompatibility. In the present study, we synthesized a novel beta-alanine C(60) derivative. The product was characterized by FT-IR, (1)H NMR, (13)C NMR, LC-MS and elemental analysis. We investigated the protective effect on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death determined by MTT, flow cytometry analysis and PI/Hoechst 33342 staining. Moreover, the scavenging ability of beta-alanine C(60) derivative to reactive oxygen species both in vivo and in vitro of PC12 cells was measured. The results suggest that beta-alanine C(60) derivative has the potential to prevent oxidative stress-induced cell death without evident toxicity. Hence, on the basis of the above-mentioned studies, we can hypothesize that the protective effect of beta-alanine C(60) derivative on H(2)O(2) induced apoptosis is related to their known scavenger activity toward ROS.     

Age-dependent upregulation of p53 and cytochrome c release and susceptibility to apoptosis in skeletal muscle fiber of aged rats: role of carnitine and lipoic acid

Mitochondrial dysfunction has been implicated in the regulation of myofiber loss during aging, possibly by apoptotic pathways. However, the mitochondrial-mediated pathway of apoptosis by cytochrome c in skeletal muscle remains ambiguous. To understand this, we have studied the upstream and downstream events of cytochrome c release, and assessed the efficacy of carnitine and lipoic acid cosupplementation. The results show that elevated levels of cytosolic cytochrome c activate apoptosis in aged rats, and was confirmed further by in vitro caspase-3 assay. Interestingly, the exogenous addition of cytochrome c results in a much higher increase of caspase-3 activity in aged treated rats than age-matched control rats, strongly suggesting that cytochrome c is a limiting factor for caspase-3 activation in the cytosol. Carnitine and lipoic acid supplement decreased apoptosis in aged rats by maintaining mitochondrial membrane integrity and thereby preventing further loss of cytochrome c in vivo. Furthermore, the upregulation of p53 observed in aged rats is attributed to the loss of outer mitochondrial membrane integrity and subsequent release of cytochrome c through BH3-only proteins. In conclusion, the p53-dependent activation of the mitochondrial-cytochrome c pathway of apoptosis in the present study suggests the existence of cross talk between mitochondria and nucleus. However, the exact molecular mechanism remains to be explored. Oral supplements of carnitine and lipoic acid play an antiapoptotic role in aged rat skeletal muscle by protecting mitochondrial membrane integrity.     

Mild thermotolerance induced at 40 °C increases antioxidants and protects HeLa cells against mitochondrial apoptosis induced by hydrogen peroxide: Role of p53

Exposure of cells to mild temperatures (40 °C) induces thermotolerance, which renders cells resistant to subsequent toxic insults. Thermotolerance is usually associated with accumulation of heat shock proteins. This study determines whether mild thermotolerance (40 °C, 3 h) can induce other defense proteins (e.g. antioxidants, anti-apoptosis proteins), and protect HeLa cells against apoptosis triggered by H₂O₂. Protein expression and enzymatic activity of MnSOD and catalase were increased in thermotolerant cells, as well as intracellular glutathione levels and γ-glutamylcysteine synthetase expression. Furthermore, levels of reactive oxygen species (ROS) were increased in thermotolerant cells, which caused mitochondrial membrane hyperpolarisation. Mild thermotolerance inhibited activation of the mitochondrial cascade of apoptosis by H₂O₂. This entailed inhibition of mitochondrial Bax translocation, mitochondrial membrane depolarisation, cytochrome c release, activation of caspases-9/-3 and chromatin condensation. Thermotolerance inhibited H₂O₂-induced caspase-independent apoptosis involving apoptosis-inducing factor, and activation of p53 and increased expression of its target protein PUMA. Thermotolerance induced at mild physiological temperatures protects cells against both caspase-dependent and caspase-independent apoptosis triggered by oxidative stress.     

Role of thiocyanate, bromide and hypobromous acid in hydrogen peroxide-induced apoptosis

We have previously reported that H₂O₂-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H₂O₂-induced apoptosis. Furthermore, thiocyanate inhibits H₂O₂-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H₂O₂-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H₂O₂/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H₂O₂ and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H₂O₂/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.     

Overexpression of fibroblast growth factor‐21 (FGF‐21) protects mesenchymal stem cells against caspase‐dependent apoptosis induced by oxidative stress and inflammation

The clinical application of stem cells offers great promise as a potential avenue for therapeutic use in neurodegenerative diseases. However, cell loss after transplantation remains a major challenge, which currently plagues the field. On the basis of our previous findings that fibroblast growth factor 21 (FGF‐21) protected neurons from glutamate excitotoxicity and that upregulation of FGF‐21 in a rat model of ischemic stroke was associated with neuroprotection, we proposed that overexpression of FGF‐21 protects bone marrow‐derived mesenchymal stem cells (MSCs) from apoptosis. To test this hypothesis, we examined whether the detrimental effects of apoptosis can be mitigated by the transgenic overexpression of FGF‐21 in MSCs. FGF‐21 was transduced into MSCs by lentivirus and its overexpression was confirmed by quantitative polymerase chain reaction. Moreover, FGF‐21 overexpression did not stimulate the expression of other FGF family members, suggesting it does not activate a positive feedback system. The effects of hydrogen peroxide (H₂O₂), tumor necrosis factor‐α (TNF‐α), and staurosporine, known inducers of apoptosis, were evaluated in FGF‐21 overexpressing MSCs and mCherry control MSCs. Caspases 3 and 7 activity was markedly and dose‐dependently increased by all three stimuli in mCherry MSCs. FGF‐21 overexpression robustly suppressed caspase activation induced by H₂O₂ and TNF‐α, but not staurosporine. Moreover, the assessment of apoptotic morphological changes confirmed the protective effects of FGF‐21 overexpression. Taken together, these results provide compelling evidence that FGF‐21 plays a crucial role in protecting MSCs from apoptosis induced by oxidative stress and inflammation and merits further investigation as a strategy for enhancing the therapeutic efficacy of stem cell‐based therapies.     

Hydrogen peroxide-induced apoptosis in pc12 cells and the protective effect of puerarin

In this study, the effect of puerarin on hydrogen peroxide-induced apoptosis in PC12 cells was studied. Exposure of cells to 0.5mM H(2)O(2)may cause significant viability loss and apoptotic rate increase. When c-Myc, Bcl-2 and Bax expression and caspase-3 activity were measured, using Ac-DEVD-AMC as a substrate, the changes in these apoptosis regulatory and effector proteins suggested that the elevation of c-Myc, decrease in Bcl-2:Bax protein ratio, and caspase-3 activation all play a key role in apoptosis. When cells were treated with puerarin prior to 0.5 mM H(2)O(2)treatment, a reduction in viability loss and apoptotic rate was seen. In addition, c-Myc expression decreased and Bcl-2:Bax ratio increased. Puerarin also reduced the H(2)O(2)-induced elevation of caspase-3 activation. These results suggest that puerarin can protect neurons against oxidative stress. It can block apoptosis in its early stages via the regulation of anti- and pro-apoptotic proteins, as well as by the attenuation of caspase-3 activation in H(2)O(2)-induced PC12 cells.     

Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H₂O₂-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H₂O₂-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.     

Extracellular calcium induces activation of Ca(2+)/calmodulin-dependent protein kinase II and mediates spontaneous activation in rat oocytes

Ovulated rat oocytes are activated spontaneously soon after recovery from the oviducts. To investigate the kinetics and mechanism of rat oocyte spontaneous activation (OSA), we investigated the effect of aging in oviducts, hyaluronidase treatment, and extracellular and intracellular calcium, and examined the activity of CaMKII and the effect of its inhibitor on OSA. Oocyte aging in oviducts and hyaluronidase did not affect OSA. However, OSA was significantly decreased in calcium-free medium and in calcium-containing medium containing L-type calcium channel blocker and IP(3)R inhibitor. Moreover, significantly lower OSA was shown with an inhibitor of CaMKII. There was a significant increase of CaMKII activity at 30min after oocyte recovery and constitutively active CaMKII was located near the meiotic spindle in freshly recovered oocytes. Therefore, CaMKII is one of the upstream signals to activate rat oocytes spontaneously after recovery and rat oocytes respond very sensitively to extracellular calcium.     

Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells

Interplay between calcium ions (Ca²⁺) and reactive oxygen species (ROS) delicately controls diverse pathophysiological functions of vascular smooth muscle cells (VSMCs). However, details of the Ca²⁺ and ROS signaling network have been hindered by the absence of a method for dual measurement of Ca²⁺ and ROS. Here, a real-time monitoring system for Ca²⁺ and ROS was established using a genetically encoded hydrogen peroxide indicator, HyPer, and a ratiometric Ca²⁺ indicator, fura-2. For the simultaneous detection of fura-2 and HyPer signals, 540 nm emission filter and 500 nm∼ dichroic beamsplitter were combined with conventional exciters. The wide excitation spectrum of HyPer resulted in marginal cross-contamination with fura-2 signal. However, physiological Ca²⁺ transient and hydrogen peroxide were practically measurable in HyPer-expressing, fura-2-loaded VSMCs. Indeed, distinct Ca²⁺ and ROS signals could be successfully detected in serotonin-stimulated VSMCs. The system established in this study is applicable to studies of crosstalk between Ca²⁺ and ROS. [BMB Reports 2013; 46(12): 600-605]