Terminal Deoxyribonucleotide Transferase-mediated dUTP Nick Research Articles

Aberrant PTEN, PIK3CA, pMAPK, and TP53 expression in human scalp and face angiosarcoma.

Angiosarcoma is a rare, highly aggressive malignant tumor originating from endothelial cells that line the lumen of blood or lymphatic vessels. The molecular mechanisms of scalp and face angiosarcoma still need to be elucidated. This study aimed to investigate the expression of phosphatase and tensin homolog (PTEN), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphorylated mitogen-activated kinase-like protein (pMAPK), and tumor protein p53 (TP53) in scalp and face angiosarcoma and to assess tumor tissue apoptosis.The expression and intracellular distribution of PTEN, PIK3CA, pMAPK, and TP53 proteins in 21 specimens of human scalp and face angiosarcoma and 16 specimens of human benign hemangioma were evaluated using immunohistochemistry. Tumor cell apoptosis was assessed by terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling staining.Significantly lower PTEN but higher PIK3CA, pMAPK, and TP53 immunostaining were detected in the angiosarcoma specimens than in the benign hemangioma specimens(P < .01). The angiosarcoma tissues exhibited significantly higher apoptosis indices than the benign hemangioma tissues (P < .01). The positive expression rates of PIK3CA, pMAPK, and TP53 were correlated with the degree of tumor differentiation in the human scalp and face angiosarcoma.The PI3K, MAPK, and TP53 pathways might be involved in angiosarcoma tumorigenesis in humans and may serve as therapeutic targets for the effective treatment of this malignancy.

Delivery of Arsenic Trioxide by Multifunction Nanoparticles To Improve the Treatment of Hepatocellular Carcinoma

Arsenic trioxide (ATO) is effective in the treatment of hematological malignancies and solid tumors. However, its toxicity and side effects are severe, posing an obstacle in its clinical application. A controlled-release ATO carrier with mitochondrial targeting was constructed in this study. The safety and efficacy in vitro were investigated using a hemolysis test, cytotoxicity, proliferation, migration, apoptosis, and other changes in cell behavior. The safety and efficacy were further evaluated in vivo by hematoxylin-eosin staining, terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling staining, and blood testing in tumor-bearing mice. Immunohistochemically and western blotting experiments were conducted to explore the mechanism of combination therapy of material-based chemotherapy and microwave hyperthermia in vitro. We demonstrated that the nano-zirconia (ZrO2) loading platform may be used to administer the ATO, with local precision-controlled release and mitochondrial targeting. Furthermore, we showed the safety of this approach for delivering high doses of ATO. In addition, we explored this new method in combination with in vitro microwave heat therapy, providing a potentially novel intravenous approach to chemotherapy. We described a new non-invasive treatment that improved the efficacy of ATO chemotherapy against hepatocellular carcinoma through nano-ZrO2 carriers.

Sevoflurane Protects against Intestinal Ischemia-Reperfusion Injury by Activating Peroxisome Proliferator-Activated Receptor Gamma/Nuclear Factor-κB Pathway in Rats

Background: Intestinal ischemia/reperfusion (I/R) injury is a clinical challenge with high morbidity and mortality, whereas the effective therapeutic strategy is limited. Inflammatory reaction plays important roles in I/R-induced intestinal damage and multi-organ dysfunction syndrome. Peroxisome proliferator-activated receptor gamma (PPARγ) has been identified as an endogenous anti-inflammatory regulator by inhibiting nuclear factor-κB (NF-κB) activation. Our previous research has shown that the pretreatment with inhaled anesthetic sevoflurane protects intestinal I/R injury. However, whether the protection induced by sevoflurane is mediated by inhibiting intestinal inflammatory reaction via activation of PPARγ/NF-κB pathway is underdetermined. In this study, we investigated the effects of sevoflurane on intestinal inflammatory reaction during intestinal I/R and the role of PPARγ/NF-κB pathway. Methods: Rat model of intestinal I/R was used in this study. The superior mesenteric artery was clamped for 60 min followed by 120-min reperfusion. Sevoflurane at 0.5 minimum alveolar concentration was inhaled for 30 min before ischemic insult. GW9662, a specific PPARγ antagonist, was injected intraperitoneally before sevoflurane inhalation. Results: Intestinal I/R caused severe intestinal mucosa histopathological injury evaluated by Chiu’s scoring, induced epithelial cell apoptosis evaluated by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling and activation of caspase-3, upregulated serum MOD levels, reduced protein expression of Bcl-2 and PPARγ, increased protein expression of NF-κB P65 and proinflammatory cytokine tumor necrosis factor-α and interleukin-6 in the intestine. Sevoflurane preconditioning significantly ameliorated these changes induced by intestinal I/R. However, GW9662 partly blocked the protective effects induced by sevoflurane. Conclusions: Our results suggest sevoflurane-induced protection against intestinal I/R injury is partly mediated by inhibiting intestinal inflammatory reaction via activation of PPARγ/NF-κB pathway.

Thymoquinone suppresses platelet-derived growth factor-BB-induced vascular smooth muscle cell proliferation, migration and neointimal formation.

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are mainly responsible for vascular occlusion diseases, such as pulmonary arterial hypertension and restenosis. Our previous study demonstrated thymoquinone (TQ) attenuated monocrotaline‐induced pulmonary arterial hypertension. The aim of the present study is to systematically examine inhibitory effects of TQ on platelet‐derived growth factor‐BB (PDGF‐BB)–induced proliferation and migration of VSMCs in vitro and neointimal formation in vivo and elucidate the potential mechanisms. Vascular smooth muscle cells were isolated from the aorta in rats. Cell viability and proliferation were measured in VSMCs using the MTT assay. Cell migration was detected by wound healing assay and Transwell assay. Alpha‐smooth muscle actin (α‐SMA) and Ki‐67‐positive cells were examined by immunofluorescence staining. Reactive oxygen species (ROS) generation and apoptosis were measured by flow cytometry and terminal deoxyribonucleotide transferase–mediated dUTP nick end labelling (TUNEL) staining, respectively. Molecules including the mitochondria‐dependent apoptosis factors, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), PTEN/AKT and mitogen‐activated protein kinases (MAPKs) were determined by Western blot. Neointimal formation was induced by ligation in male Sprague Dawley rats and evaluated by HE staining. Thymoquinone inhibited PDGF‐BB–induced VSMC proliferation and the increase in α‐SMA and Ki‐67‐positive cells. Thymoquinone also induced apoptosis via mitochondria‐dependent apoptosis pathway and p38MAPK. Thymoquinone blocked VSMC migration by inhibiting MMP2. Finally, TQ reversed neointimal formation induced by ligation in rats. Thus, TQ is a potential candidate for the prevention and treatment of occlusive vascular diseases.

Sevoflurane Preconditioning Reduces Intestinal Ischemia-Reperfusion Injury: Role of Protein Kinase C and Mitochondrial ATP-Sensitive Potassium Channel

Ischemic preconditioning (IPC) has been considered to be a potential therapy to reduce ischemia-reperfusion injury (IRI) since the 1980s. Our previous study indicated that sevoflurane preconditioning (SPC) also reduced intestinal IRI in rats. However, whether the protective effect of SPC is similar to IPC and the mechanisms of SPC are unclear. Thus, we compared the efficacy of SPC and IPC against intestinal IRI and the role of protein kinase C (PKC) and mitochondrial ATP-sensitive potassium channel (mKATP) in SPC. A rat model of intestinal IRI was used in this study. The superior mesenteric artery (SMA) was clamped for 60 min followed by 120 min of reperfusion. Rats with IPC underwent three cycles of SMA occlusion for 5 min and reperfusion for 5 min before intestinal ischemia. Rats with SPC inhaled sevoflurane at 0.5 minimum alveolar concentration (MAC) for 30 min before the intestinal ischemic insult. Additionally, the PKC inhibitor Chelerythrine (CHE) or mKATP inhibitor 5-Hydroxydecanoic (5-HD) was injected intraperitoneally before sevoflurane inhalation. Both SPC and IPC ameliorated intestinal IRI-induced histopathological changes, decreased Chiu’s scores, reduced terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive cells in the epithelium, and inhibited the expression of malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α). These protective effects of SPC were similar to those of IPC. Pretreatment with PKC or mKATP inhibitor abolished SPC—induced protective effects by increasing Chiu’s scores, down-regulated the expression of Bcl-2 and activated caspase-3. Our results suggest that pretreatment with 0.5 MAC sevoflurane is as effective as IPC against intestinal IRI. The activation of PKC and mKATP may be involved in the protective mechanisms of SPC.

Sevoflurane protects against intestinal ischemia–reperfusion injury partly by phosphatidylinositol 3 kinases/Akt pathway in rats

Intestinal ischemia-reperfusion injury (IRI) is a clinical challenge with high morbidity and mortality, leading to intestine damage, systemic inflammation, and multiorgan failure. Previous research has shown that the inhaled anesthetic sevoflurane protects various organs from IRI. However, whether sevoflurane protects against intestinal IRI and which application condition is the most effective are not completely clear. Thus, we investigated the effects of sevoflurane on intestinal IRI with sevoflurane given before, during or after intestinal ischemia, and the role of phosphatidylinositol 3 kinases (PI3K)/Akt pathway in these effects. Rat model of intestinal ischemia-reperfusion (IR) was used in this study. The superior mesenteric artery was clamped for 60 minutes followed by 120 minutes of reperfusion. Sevoflurane at 0.25, 0.5, and 1.0 minimum alveolar concentration (MAC) was inhaled for 30 minutes before, during, or after ischemic insult. LY294002, a PI3K inhibitor, was injected intraperitoneally before sevoflurane inhalation. Intestinal IR caused a significant decrease of mean arterial blood pressure, severe intestinal mucosa injury and epithelial cell apoptosis, downregulation of the levels of phospho-Akt and phospho-Bad proteins. Exposure to 0.5 or 1.0 MAC sevoflurane before or after intestinal ischemia or 0.5 MAC during intestinal ischemia significantly ameliorated IR-induced histopathologic changes and decreased Chiu's scores. Pretreatment with 0.5 MAC sevoflurane also inhibited intestinal IR-induced increase of terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling-positive cells and activation of caspase-3 and restored expression of phospho-Akt and phospho-Bad. LY294002 partly blocked the protective effects induced by 0.5 MAC sevoflurane pretreatment. Our results suggest that sevoflurane inhalation at clinical related concentration before, during, or after ischemia protects against IR-induced intestinal injury. The pretreatment-induced protection was partly mediated by inhibiting intestinal mucosal epithelial apoptosis via activation of the PI3K/Akt pathway.

Both JNK and P38 MAPK pathways participate in the protection by dexmedetomidine against isoflurane-induced neuroapoptosis in the hippocampus of neonatal rats.

Dexmedetomidine, a highly selective α2-adrenergic agonist, has been reported to attenuate isoflurane-induced cognitive impairment and neuroapoptosis. However, the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate whether mitogen-activated protein kinase (MAPK) pathway was involved in dexmedetomidine-induced neuroprotection against isoflurane effects. Seven-day-old (P7) neonatal Sprague-Dawley rats were pretreated with various concentrations of dexmedetomidine, and then exposed to 0.75% isoflurane or air for 6h. Terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) was used to detect neuronal apoptosis in their hippocampus. Activated caspase-3, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinases (JNK), p38, phospho-ERK1/2, phospho-JNK and phospho-p38 proteins were detected by Western blotting in the hippocampus at the end of exposure. Also, P7 rats were pretreated with 75 μg/kg dexmedetomidine alone, or given the ERK inhibitor U0126 before dexmedetomidine pretreatment, or pretreated with the p38 MAPK inhibitor SB203580 or JNK inhibitor SP600125 alone, and then exposed to 0.75% isoflurane for 6h. Isoflurane induced significant neuroapoptosis, increased the protein expression of phospho-JNK, phospho-c-Jun, phospho-p38 and phospho-nuclear factor-κB (NF-κB), decreased the level of phospho-ERK1/2 protein and reduced the ratio of Bcl-2/Bax in the hippocampus. Dexmedetomidine pretreatment inhibited isoflurane-induced neuroapoptosis and restored proteins expression of MAPK pathways and the Bcl-2/Bax ratio after isoflurane exposure. Moreover, SB203580 and SP600125 also partly attenuated the isoflurane-induced protein changes. However, U0126 did not reverse dexmedetomidine-induced neuroprotection. Our results indicate that the JNK and p38 pathways, not the ERK pathway are involved in dexmedetomidine-induced neuroprotection against isoflurane effects.

Dexmedetomidine Reduces Isoflurane-Induced Neuroapoptosis Partly by Preserving PI3K/Akt Pathway in the Hippocampus of Neonatal Rats

Prolonged exposure to volatile anesthetics, such as isoflurane and sevoflurane, causes neurodegeneration in the developing animal brains. Recent studies showed that dexmedetomidine, a selective α2-adrenergic agonist, reduced isoflurane-induced cognitive impairment and neuroapoptosis. However, the mechanisms for the effect are not completely clear. Thus, we investigated whether exposure to isoflurane or sevoflurane at an equivalent dose for anesthesia during brain development causes different degrees of neuroapoptosis and whether this neuroapoptosis is reduced by dexmedetomidine via effects on PI3K/Akt pathway that can regulate cell survival. Seven-day-old (P7) neonatal Sprague-Dawley rats were randomly exposed to 0.75% isoflurane, 1.2% sevoflurane or air for 6 h. Activated caspase-3 was detected by immunohistochemistry and Western blotting. Phospho-Akt, phospho-Bad, Akt, Bad and Bcl-xL proteins were detected by Western blotting in the hippocampus at the end of exposure. Also, P7 rats were pretreated with various concentrations of dexmedetomidine alone or together with PI3K inhibitor LY294002, and then exposed to 0.75% isoflurane. Terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 were used to detect neuronal apoptosis in their hippocampus. Isoflurane, not sevoflurane at the equivalent dose, induced significant neuroapoptosis, decreased the levels of phospho-Akt and phospho-Bad proteins, increased the expression of Bad protein and reduced the ratio of Bcl-xL/Bad in the hippocampus. Dexmedetomidine pretreatment dose-dependently inhibited isoflurane-induced neuroapoptosis and restored protein expression of phospho-Akt and Bad as well as the Bcl-xL/Bad ratio induced by isoflurane. Pretreatment with single dose of 75 µg/kg dexmedetomidine provided a protective effect similar to that with three doses of 25 µg/kg dexmedetomidine. Moreover, LY294002, partly inhibited neuroprotection of dexmedetomidine. Our results suggest that dexmedetomidine pretreatment provides neuroprotection against isoflurane-induced neuroapoptosis in the hippocampus of neonatal rats by preserving PI3K/Akt pathway activity.

Acacetin (5,7-dihydroxy-4′-methoxyflavone) exhibits in vitro and in vivo anticancer activity through the suppression of NF-κB/Akt signaling in prostate cancer cells

Acacetin (5,7-dihydroxy-4'-methoxyflavone) is a flavonoid compound with antimutagenic, antiplasmodial, antiperoxidant, anti-inflammatory and anticancer effects. However, the molecular targets and pathways underlying the anticancer effects of acacetin are yet to be elucidated. In this study, we investigated whether acacetin induces apoptosis in the human prostate cancer cell line, DU145. The results of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays revealed that cell viability decreased in a dose- and time-dependent manner in response to acacetin. 4',6-Diamidino-2-phenylindole (DAPI) staining revealed that chromatin condensation significantly increased in a dose-dependent manner. Flow cytometric analysis indicated that acacetin suppressed the viability of DU145 cells by inducing apoptosis. Western blot anlaysis of various markers of signaling pathways revealed that acacetin targets the Akt and nuclear factor (NF)-κB signaling pathways by inhibiting the phosphorylation of IκBα and NF-κB in a dose-dependent manner. Consistent with its ability to induce apoptosis, the acacetin-mediated inhibition of the pro-survival pathway, Akt, and of the NF-κB pathway was accompanied by a marked reduction in the levels of the NF-κB‑regulated anti-apoptotic proteins, Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP), as well as of the proliferative protein, cyclooxygenase (COX)-2. We further evaluated the effects of acacetin on prostate cancer using mice subcutaneously injected with DU145 prostate cancer cells. The acacetin-treated nude mice bearing DU145 tumor xenografts exhibited significantly reduced tumor size and weight, due to the effects of acacetin on cancer cell apoptosis, as determined by terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assay. Our findings suggest that acacetin exerts antitumor effects by targeting the Akt/NF-κB signaling pathway. Rurther investigations on this flavonoid are warranted to evaluate its potential use in the prevention and therapy of prostate cancer.

Ginsenoside Rb1 prevents interleukin-1 beta induced inflammation and apoptosis in human articular chondrocytes

PurposeOsteoarthritis (OA) is an age-related joint disease that is characterised by the degeneration of articular chondrocytes. Ginsenosides, the most important pharmacological ingredients of ginseng, have been proven to provide effective therapy for neurodegenerative diseases and can inhibit cell apoptosis. We investigated whether ginsenoside Rb1 can modulate inflammation and apoptosis in human chondrocytes.MethodsChondrocytes were isolated from OA patients undergoing total knee replacement surgery. Apoptosis was assessed by TUNEL (terminal deoxyribonucleotide transferasemediated dUTP nick end-labelling)-positive staining. Levels of PGE2 and NO2- were detected by ELISA. Gene expression levels were measured for type II collagen (Col2A1), aggrecan, MMP-13, COX-2, iNOS, caspase-3, and PARP.ResultsThe results showed that TUNEL-positive staining chondrocytes were decreased by Rb1 compared with IL-1β. Both 10 or 100 μg/ml Rb1 inhibited the effect of IL-1β on chondrocytes by decreasing levels of PGE2, NO2-, MMP-13, COX-2, iNOS, caspase-3 and PARP and increasing aggrecan and Col2A1 gene expression levels, to block IL-1β-induced cell inflammation and apoptosis.ConclusionsThe results suggest that Rb1 possesses potential anti-inflammatory and anti-apoptotic properties in human chondrocytes, possibly by binding to oestrogen receptors to exert its pharmacological effects.

Effect of ecdysterone on heteroptopic heart transplantation in rats

Objective To investigate the protective effects of ecdysterone(EDS) on the allograft heart transplant model of rats. Methods Fifty healthy Sprayue-Dawley(SD) rats were divided into donors and recipients randomly. Hearts were harvested and placed heterotopically into allogenic recipients. All animals were divided into three groups: sham-operation group(only opening and closing the abdomen, n=10), EDS group(injected intraperitoneally with 20 mg/(kg · day) of EDS, n = 10), and control group (injected intraperitoneally with normal saline, n = 10). The pathological changes of myocardial tissue were analyzed by light microscopy and transmission electron microscopy and the levels of myocardial enzymes(GOT, LDH, CPK), SOD, ET-1, NO, MDA in serum were measured. Tissue samples underwent the detection of apoptotic cell death by in situ terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling for apoptotic index(AI) and also by immunohistochemistry method to study the expressions of Bcl-2 and Bax. Results Pathological examination and transmission electron microscope observation showed greater myocardium damage in the control group. EDS group showed a decrease in the amount of myocardial enzymes, MDA, ET-1 and an increase in the levels of SOD, NO, compared to the control group. The AI of EDS group became lower than that of control group, meanwhile the EDS group increased the expression of Bcl-2 and decreased the expression of Bax. Conclusion EDS has protective effects on heteroptopic heart transplantation, which provides a new idea for myocardial protection in heart transplantation. However, the mechanism of its protective effect needs further research.

Annexin 5 overexpression increased articular chondrocyte apoptosis induced by basic calcium phosphate crystals

Objectives:Basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbapatite (CA) and hydroxyapatite (HA)) are associated with severe forms of osteoarthritis. In advanced osteoarthritis, cartilage shows chondrocyte apoptosis, overexpression of annexin...

HIV‐1 protease inhibitor induces growth arrest and apoptosis of human prostate cancer LNCaP cells in vitro and in vivo in conjunction with blockade of androgen receptor STAT3 and AKT signaling

This study found that the HIV-1 protease inhibitor nelfinavir (NFV) induced growth arrest and apoptosis of human prostate cancer cells (LNCaP, DU145 and PC-3 cells), as measured by MTT and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays, respectively, on the third day of culture. In addition, NFV blocked androgen receptor (AR) signaling in association with downregulation of nuclear levels of AR in LNCaP cells as measured by reporter assay and western blot analysis. As expected, NFV downregulated the level of the AR target molecule prostate specific antigen in these cells. Moreover, NFV disrupted STAT3 signaling; protease inhibitors blocked interleukin-6-induced phosphorylation of STAT3 and inhibited STAT3 DNA binding activity in LNCaP and DU145 cells, as measured by western blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. Furthermore, NFV blocked AKT signaling in prostate cancer cells as measured by kinase assay with glycogen synthase kinase-3alpha/beta as a substrate. Importantly, NFV inhibited the proliferation of LNCaP cells presented as tumor xenografts in BALB/c nude mice without side-effects. Taken together, NFV inhibited the proliferation of prostate cancer cells in conjunction with blockade of signaling by AR, STAT3, and AKT, suggesting that this family of compounds might be useful for the treatment of individuals with prostate cancer.

Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling of the mycelium in submerged cultures of Streptomyces antibioticus ETH7451

Viability stain and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling (TUNEL) have been applied to submerged cultures of Streptomyces antibioticus ETH7451, the last technique after a suitable permeabilization treatment. Areas of dead mycelium can be clearly delineated by the viability stain within the network of hyphae which forms the mycelial masses characteristic of the submerged cultures. In addition, the TUNEL reaction shows that DNA fragmentation accompanies the death processes in the mycelium. These techniques permit the investigation of the influence of the medium and nutritional conditions on the viability of the cells. This has relevant biotechnological implications for the study of these important filamentous bacteria in the industrial fermentation processes. These techniques also allow a straighforward analysis of the physical and chemical reagents which provoke damage in Streptomyces DNA.

Oxidative Preconditioning and Apoptosis in L-cells: ROLES OF PROTEIN KINASE B AND MITOGEN-ACTIVATED PROTEIN KINASES

Oxidative stress can cause significant cell death by apoptosis. We performed studies in L-cells to explore whether prior exposure to oxidative stress ("oxidative preconditioning") can protect the cell against the apoptotic consequences of subsequent oxidative insults and to establish the mediators in the preconditioning signaling cascade. Cells were preconditioned with three 5-min exposures to H(2)O(2), followed by 10-h recovery and subsequent exposure to 600 microm H(2)O(2) for 10 h. A single 10-h exposure to H(2)O(2) induced substantial apoptotic cell death (approximately 90%), as determined by enzyme-linked immunosorbent assay, TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling), and Annexin V methods, but apoptosis was largely prevented in preconditioned cells. The degree of cytoprotection depended on the strength of preconditioning or H(2)O(2) concentration (20 approximately 600 microm). Transient increases in mitogen-activated protein kinase (MAPK), p38, and JNK/SAPK activities and sustained protein kinase B (Akt) activation, accompanied by drastically reduced caspase 3 activity, were seen after preconditioning. The expression levels of these kinases were unaltered. Inhibitors of p38 (SB203580) and phosphoinositide 3-kinase (PI3K, LY294002) pathways abolished the protection provided by preconditioning. We conclude that oxidative preconditioning protects cells against apoptosis and that this effect involves MAPK and PI3K/Akt pathways. This system may be important in regulating apoptotic cell death in development and disease states.

Inhibition of extracellular signal-regulated kinase enhances Ischemia/Reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart.

Three major mammalian mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal protein kinase (JNK), have been identified in the cardiomyocyte, but their respective roles in the heart are not well understood. The present study explored their functions and cross talk in ischemia/reoxygenation (I/R)-induced cardiac apoptosis. Exposing rat neonatal cardiomyocytes to ischemia resulted in a rapid and transient activation of ERK, p38, and JNK. On reoxygenation, further activation of all 3 mitogen-activated protein kinases was noted; peak activities increased (fold) by 5.5, 5.2, and 6.2, respectively. Visual inspection of myocytes exposed to I/R identified 18.6% of the cells as showing morphological features of apoptosis, which was further confirmed by DNA ladder and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL). Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of I/R-induced ERK activation, whereas p38 and JNK activities were increased by 70.3% and 55.0%, respectively. In addition, the number of apoptotic cells was increased to 33.4%. With pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, a p38 and JNK2 inhibitor, I/R+PD98059-induced apoptotic cells were reduced by 42.8% and 63.3%, respectively. Hearts isolated from rats treated with PD98059 and subjected to global ischemia (30 minutes)/reoxygenation (1 hour) showed a diminished functional recovery compared with the vehicle group. Coadministration of SB203580 attenuated the detrimental effects of PD98059 and significantly improved cardiac functional recovery. The data taken together suggest that ERK plays a protective role, whereas p38 and JNK mediate apoptosis in cardiomyocytes subjected to I/R, and the dynamic balance of their activities is critical in determining cardiomyocyte fate subsequent to reperfusional injury.

Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling.

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3'-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3'-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3'-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.