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dUTP Nick End Labeling (dutp + nick_end_labeling)
Selected AbstractsSimultaneously multi-parameter determination of hematonosis cell apoptosis by two-photon and confocal laser scanning microscopyJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2004Yi Wang Abstract Flow cytometry (FCM), TdT-mediated dUTP Nick End Labeling (TUNEL), and DNA ladder are conventional methods to detect apoptosis of drug-treated cells. However, the assumption of cell number restricts their applications in clinics. In this paper, we report a cell-saving imaging method for quick identification of the hematonosis cell apoptosis induced by chemotherapeutic drugs. By the combination of two-photon and confocal microscopy, three main apoptosis parameters (the change of nuclear morphology, collapse of mitochondrial membrane potential, and increase of intracellular calcium) were recorded simultaneously for single As2O3 -induced Molt-4 cells. The results are highly in accordance with those produced by classical flow cytometry. This work suggests that this new imaging method would be promising in the quick identification of hematonosis cell apoptosis. J. Clin. Lab. Anal. 18:271,275, 2004. © 2004 Wiley-Liss, Inc. [source] Busulfan-induced central polydactyly, syndactyly and cleft hand or foot: A common mechanism of disruption leads to divergent phenotypesDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2007Takuji Naruse The prevalence of clinical phenotypes that exhibit combinations of central polydactyly, syndactyly, or cleft hand or foot is higher than would be expected for random independent mutations. We have previously demonstrated that maternal ingestion of a chemotherapeutic agent, busulfan, at embryonic day 11 (E11) induces these defects in various combinations in rat embryo limbs. In an effort to determine the mechanism by which busulfan disrupts digital development, we examined cell death by Nile Blue staining and TdT-mediated dUTP nick end labeling (TUNEL) assays; we also carried out whole mount in situ hybridization for fibroblast growth factor-8 (Fgf8), bone morphogenetic protein-4 (Bmp4), and sonic hedgehog (Shh) to examine developmental pathways linked to these defects. In busulfan-treated embryos, diffuse cell death was evident in both ectoderm and mesoderm, peaking at E13. The increased cell death leads to regression of Fgf8 in the apical ectodermal ridge (AER) and Bmp4 and Shh in the underlying mesoderm. The subsequent pattern of interdigital apoptosis and cartilage condensation was variably disrupted. These results suggest that busulfan manifests its teratogenic effects by inducing cell death of both ectoderm and mesoderm, with an associated reduction in tissue and a disruption in the generation of patterning molecules during critical periods of digit specification. [source] Protective effects of naloxone in two-hit seizure modelEPILEPSIA, Issue 3 2010Lu Yang Summary Purpose:, Early life status epilepticus (SE) could enhance the vulnerability of the immature brain to a second SE in adulthood (two-hit seizure model). Naloxone has been proved to possess inflammation inhibitory effects in nervous system. This study was designed to evaluate the dose-dependent protective effects of naloxone in kainic acid (KA),induced two-hit seizure model. Methods:, After KA-induced SE at postnatal day 15 (P15), Sprague-Dawley rats were infused with either saline or different doses (1.92, 3.84, 5.76, and 7.68 mg/kg) of naloxone continuously for 12 h. De novo synthesis of cytokines (interleukin-1, [IL-1,], S100B) was assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) at 12 h after P15 SE. Glial activation states were analyzed by western blotting of glial markers (glial fibrillary acidic protein [GFAP], S100B, Iba1) both at 12 h after P15 SE and at P45. After a second SE at P45, cognitive deteriorations were evaluated by Morris water tests and neuron injuries were evaluated by TdT-mediated dUTP nick end labeling (TUNEL) assays. Results:, Naloxone reduced IL-1, synthesis and microglial activation most potently at a dose of 3.84 mg/kg. Attenuation of S100B synthesis and astrocyte activation were achieved most dramatically by naloxone at a dose of 5.76 mg/kg, which is equal to the most powerful dose in ameliorating cognitive injuries and neuron apoptosis after second SE. Conclusions:, Naloxone treatment immediately after early life SE could dose-dependently reduce cytokine production, glial activation, and further lower the vulnerability of immature brains to a second hit in adulthood. [source] Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2009Wen Ru Yu Abstract Activation of the Fas receptor has been recently linked to apoptotic cell death after spinal cord injury (SCI). Although it is generally considered that Fas activation mediates apoptosis predominantly through the extrinsic pathway, we hypothesized that intrinsic mitochondrial signaling could be involved in the underlying mechanism of Fas-induced apoptosis after SCI. In the present study, we utilized the FejotaTM clip compression model of SCI at T5,6 in C57BL/6 Fas-deficient (lpr) and wild-type mice. Complementary studies were conducted using an in vitro model of trauma or a Fas-activating antibody to induce apoptosis in primary neuronal,glial mixed spinal cord cultures. After in vivo SCI, lpr mice, in comparison with wild-type mice, exhibited reduced numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells at the lesion, reduced expression of truncation of Bid (tBid), apoptosis-inducing factor, activated caspase-9 and activated caspase-3, and increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. After in vitro neurotrauma or the induction of Fas signaling by the Jo2 activating antibody, lpr spinal cord cultures showed an increased proportion of cells retaining mitochondrial membrane integrity and a reduction of tBid expression, caspase-9 and caspase-3 activation, and TUNEL-positive cells as compared to wild-type spinal cord cultures. The neutralization of Fas ligand (FasL) protected against traumatically induced or Fas-mediated caspase-3 activation and the loss of mitochondrial membrane potential and tBid expression in wild-type spinal cord cultures. However, in lpr spinal cord cultures, FasL neutralization had no protective effects. In summary, these data provide direct evidence for the induction of intrinsic mitochondrial signaling pathways following Fas activation after SCI. [source] Human neural stem cell transplantation attenuates apoptosis and improves neurological functions after cerebral ischemia in ratsACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 9 2009P. ZHANG Background: Neuroprotection is a major therapeutic approach for ischemic brain injury. We investigated the neuroprotective effects induced by transplantation of human embryonic neural stem cells (NSCs) into the cortical penumbra 24 h after focal cerebral ischemia. Methods: NSCs were prepared from human embryonic brains obtained at 8 weeks of gestation. Focal cerebral ischemia was induced in adult rats by permanent occlusion of the middle cerebral artery. Animals were randomly divided into two groups: NSCs-grafted group and medium-grafted group (control). Infarct size was assessed 28 days after transplantation by hematoxylin and eosin staining. Neurological severity scores were evaluated before ischemia and at 1, 7, 14, and 28 days after transplantation. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemical analysis of Bcl-2 and Bax were performed at 7, 14, and 28 days after transplantation. Results: Physiological parameters of the two groups were comparable, but not significantly different. NSC transplantation significantly improved neurological function (P<0.05) but did not reduce the infarct size significantly (P>0.05). Compared with the control, NSC transplantation significantly reduced the number of TUNEL- and Bax-positive cells in the penumbra at 7 days. Interestingly, the number of Bcl-2-positive cells in the penumbra after NSC transplantation was significantly higher than that after medium transplantation (P<0.05). Conclusions: The results indicate that NSC transplantation has anti-apoptotic activity and can improve the neurological function; these effects are mediated by the up-regulation of Bcl-2 expression in the penumbra. [source] Apoptosis and Cardiopulmonary BypassJOURNAL OF CARDIAC SURGERY, Issue 2 2007M.S., Miljenko Kova Apoptotic index (AI) obtained with in situ terminal deoxynucleotidyl transferase-labeled dUTP nick end labeling (TUNEL) method and Bak protein expression were compared. Patients and Methods: Twenty consecutive patients who underwent coronary artery bypass surgery, myocardial samples from the right atrium were taken in three stages: before cannulation (the first sample group), after declamping (the second sample group), and 20 minutes after reperfusion (the third sample group). The percentage of apoptotic cells was determined by TUNEL method. Expression of Bak protein was immunohistochemically analyzed. Intermittent ischemia and moderate hypothermia were used as methods of myocardial management during surgery. A statistical analysis was performed by using the Friedman ANOVA analysis of variances, the Kendall coefficient of concordance and the Wilcoxon matched pair test. Results: In the first sample group mean value of Bak expression was 2.61 ± 2.18, compared with AI 5.38 ± 3.58, after declamping (the second sample group) the mean value of Bak expression was 4.31 ± 2.68 while AI was 7.63 ± 4.38 and after 20 minutes of reperfusion in the third sample group mean value of Bak expression was 8.89 ± 4.45, while AI was 15.6 ± 8.45. When compared by using Wilcoxon matched pair test two methods significantly correlated, p > 0.0001. Conclusion: The positive correlation between AI obtained by TUNEL method and expression of Bak protein may suggest that apoptosis is activated mainly through mitochondrial activation pathway in ischemic reperfusion injury. The results suggest that ischemic reperfusion injury increases the AI in the right atrial tissue. If so, immunohistochemical expression of Bak protein could be used as a marker of myocardial ischemia induced injury. [source] Identity of TUNEL-positive cells in the oral buccal epithelium of normal mucosa and lichen lesionsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 5 2004Andreas Karatsaidis Background:,In situ detection of DNA fragmentation by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end labeling (TUNEL) is a widely used technique to identify apoptotic cells in the terminal phases of cell death. Several studies have shown that there are statistically increased numbers of TUNEL+ cells within the epithelium of oral lichen (OL). It was suggested that this indicates an increased rate of apoptosis among basal and suprabasal keratinocytes in OL epithelium. The aim of this study was to identify the TUNEL+ cells in the epithelium of erythematous (ERY) OL and normal oral mucosa (NOM). Methods:, Sections of biopsies from NOM and ERY OL were processed for TUNEL combined with immunostaining for pan-cytokeratin or for cell markers specifically expressed by different leukocytes. Results:, In NOM, TUNEL+ keratinocytes were almost exclusively seen in the outermost epithelial layers. This labeling was absent in ERY OL. In the basal and lower spinous layers, more TUNEL+ cell nuclei were seen in ERY OL as compared with NOM, in accordance with previous studies. The present observations show, however, that only very few of these cells were keratinocytes, but rather were CD4+ lymphocytes and CD68+ macrophages. There was no difference between the numbers of TUNEL+ keratinocytes in basal and lower spinous layers in ERY OL and NOM epithelium. No intraepithelial CD8+ lymphocytes, Langerhans cells, or mast cells were found to be TUNEL+. Conclusion:, The findings indicate that the pathologic changes in ERY OL epithelium cannot be explained by increased prevalence of terminal keratinocyte cell death identified by TUNEL. [source] Comparison of apoptosis and apoptosis-related gene products between extranodal oral B-cell lymphoma and maxillofacial nodal B,-,cell lymphomaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 3 2001Hong-fang Yin Abstract: Twenty-seven cases of primary extranodal oral B-cell lymphoma and 22 cases of primary maxillofacial nodal B-cell lymphoma were investigated for the presence of apoptotic cells and the expression of apoptosis-related gene products by terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) and immunohistochemistry. The majority of extranodal oral diffuse large B-cell lymphomas (17/25, 68%) and maxillofacial nodal diffuse large B-cell lymphomas (14/16, 88%) contained no or less than 10% apoptotic cells. Whereas the majority of extranodal oral diffuse large B-cell lymphomas (18/25, 72%) and maxillofacial nodal diffuse large B-cell lymphomas (13/16, 81%) contained more than 10% of Ki-67-positive cells. Bcl-2-, Bax-, p53- and Ki-67-positive rates were higher in maxillofacial nodal diffuse large B-cell lymphomas than in extranodal oral diffuse large B-cell lymphomas, but only Bax (,2 test, 0.01 Inhibition of Caspases In Vivo Protects the Rat Liver Against Alcohol-Induced Sensitization to Bacterial LipopolysaccharideALCOHOLISM, Issue 6 2001Ion V. Deaciuc Background: The mechanisms of liver sensitization by alcohol to Gram-negative bacterial lipopolysaccharide (LPS) remain elusive. The purpose of this study was two-fold: (1) to test the hypothesis that alcohol-enhanced liver apoptosis may be a sensitizing mechanism for LPS and (2) to further characterize the liver apoptotic response to alcohol. Methods: Rats were fed a high-fat, alcohol-containing liquid diet for 14 weeks, treated with LPS (1.0 mg/kg of body weight, intravenously) or saline, followed by injection of a pan-caspase inhibitor {IDN1965;N -[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino-4-oxo-5-fluoropentanoic acid; 10 mg/kg of body weight, intraperitoneally} or vehicle, and killed. The following parameters were assessed: plasma aspartate: 2-oxoglutarate aminotransferase activity (AST); liver histology and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) response; caspase-3, ,8, and ,9 activity; and mRNA and protein expression for two apoptosis-signaling molecules: Fas receptor and Fas ligand; and three apoptosis adaptors: Bax, Bcl-XL, and Bcl-2. Results: Alcohol-feeding-induced liver steatosis, slightly increased caspases' activity, the number of TUNEL-positive nuclei, and facilitated the LPS necrotic effect without affecting mRNA expression of apoptosis signals and adaptors. LPS induced a significant increase in AST and the number of TUNEL-positive nuclei, both effects being more pronounced in alcohol-treated rats. LPS produced hepatic necrosis only in alcohol-treated rats. LPS effects were associated with up-regulation of mRNA expression for both apoptosis adaptors and signaling molecules. IDN1965 administration 3 hr after LPS injection strongly inhibited caspases' activity, particularly that of caspase-3. IDN1965 also abolished the increase in TUNEL-positive nuclei, reversed the effect of LPS on plasma AST in alcohol-treated rats, and prevented LPS-induced necrosis. Conclusions: (1) Alcohol-enhanced liver apoptosis may not involve regulatory steps at the transcriptional level. LPS-induced liver apoptosis seems to involve transcriptional regulation of several apoptosis adaptors. Therefore, alcohol and LPS may enhance liver apoptosis through different mechanisms. (2) Alcohol-enhanced liver apoptosis precedes and may facilitate the hepatic effects of LPS. LPS superimposed on alcohol further elevates the rate of apoptosis in the liver. This may exceed the phagocytosing capacity of the liver so that all the apoptotic cells are not phagocytosed, but rather die of necrosis. [source] Roles of Endothelial Cell Migration and Apoptosis in Vascular Remodeling during Development of the Central Nervous SystemMICROCIRCULATION, Issue 5 2000SUZANNE HUGHES ABSTRACT Objective: To examine the roles of apoptosis, macrophages, and endothelial cell migration in vascular remodeling during development of the central nervous system. Methods: The terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) technique was combined with Griffonia simplicifolia isolectin B4 histochemistry to detect apoptotic endothelial cells in retinal whole-mount preparations derived from rats at various stages of postnatal development as well as from rat pups exposed to hyperoxia. Macrophages were detected by immunohistochemistry with the monoclonal antibody ED1, and proliferating endothelial cells were identified by incorporation of bromodeoxyuridine. Results: Only small numbers of TUNEL-positive endothelial cells were detected during normal development of the retinal vasculature, with the apoptotic cell density in the inner plexus peaking during the first postnatal week and decreasing markedly during the second week, at a time when vessel retraction was widespread. Neither apoptotic endothelial cells nor macrophages were apparent at sites of initiation of vessel retraction. TUNEL-positive endothelial cells were observed in vessels destined to remain. Hyperoxia induced excessive vessel withdrawal, resulting in the generation of isolated vascular segments containing apoptotic endothelial cells. A topographical analysis showed low numbers of proliferating endothelial cells at sites of angiogenesis whereas vascular proliferation was increased in the adjacent inner plexus. Conclusions: Endothelial cell apoptosis and macrophages do not initiate vessel retraction, but rather contribute to the removal of redundant cells throughout the vasculature. We suggest that vessel retraction is mediated by endothelial cell migration and that endothelial cells derived from retracting vascular segments are redeployed in the formation of new vessels. Only when retraction results in compromised circulation and redeployment is not possible, does endothelial cell apoptosis occur. [source] Lack of apoptosis in patients with progressive external ophthalmoplegia and mutated adenine nucleotide translocator-1 geneMUSCLE AND NERVE, Issue 2 2002Gigliola Fagiolari PhD Abstract Adenine nucleotide translocator-1 (ANT-1), encoded by chromosome 4 (4q34-35 locus), is a component of the mitochondrial permeability transition pores that are involved in apoptotic mechanisms. We studied muscle biopsies from seven individuals with autosomal dominant progressive external ophthalmoplegia caused by ANT-1 mutations. We found no instance of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) positivity nor significant expression of apoptosis-related proteins. Furthermore, there was no morphological evidence of apoptosis at the ultrastructural level. Thus, degeneration of muscle in this disorder is nonapoptotic. © 2002 Wiley Periodicals, Inc. Muscle Nerve 26: 265,269, 2002 [source] Coptis japonica root extract induces apoptosis through caspase3 activation in SNU-668 human gastric cancer cellsPHYTOTHERAPY RESEARCH, Issue 3 2005H. J. Park Abstract Apoptosis-modulating approaches offer an attractive opportunity for therapeutic use for many tumors. We investigated the effects of the roots of Coptis japonica var. dissecta (Ranunculaceae) on human gastric cancer cells, SNU-668. The cytotoxicity of Coptis japonica at 100 µg[sol ]ml (methanol extract) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was 13.89 ± 1.91% of control value. Considering the features by 4,6-diamidino-2-phenylindole (DAPI) staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, it was confirmed that the death of SNU-668 cells was due to apoptosis. In the apoptosis-regulating genes, BCL2 expression was diminished out, whereas BAX and CASP3 expressions were increased, compared with control. Furthermore, the activity of caspase3 was significantly increased by Coptis japonica treatment. These results suggest that Coptis japonica could induce apoptotic anticancer effect through caspase3 activation on SNU-668 human gastric cancer cells. Copyright © 2005 John Wiley & Sons, Ltd. [source] In vitro targeted photodynamic therapy with a pyropheophorbide-a conjugated inhibitor of prostate-specific membrane antigenTHE PROSTATE, Issue 6 2009Tiancheng Liu Abstract BACKGROUND The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on PS-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. METHODS Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. RESULTS Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 hr by HOE33342/PI double staining, becoming more intense by 4 hr. Evidence for the apoptotic caspase cascade being activated was based on the appearance of poly-ADP-ribose polymerase (PARP) p85 fragment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detected DNA fragmentation 16 hr post-PDT, confirming apoptotic events. CONCLUSIONS Cell permeability by HOE33342/PI double staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. Prostate 69:585,594, 2009. © 2009 Wiley-Liss, Inc. [source] Pathology is alleviated by doxycycline in a laminin-,2,null model of congenital muscular dystrophyANNALS OF NEUROLOGY, Issue 1 2009Mahasweta Girgenrath PhD Objective Congenital muscular dystrophy type 1A is an autosomal recessive disease that is caused by loss-of-function mutations in the laminin-,2 gene, and results in motor nerve and skeletal muscle dysfunction. In a previous study, we used genetic modifications to show that inappropriate induction of apoptosis was a significant contributor to pathogenesis in a laminin-,2,deficient mouse model of congenital muscular dystrophy type 1A. To identify a possible pharmacological therapy for laminin-,2 deficiency, we designed this study to determine whether treatment with minocycline or doxycycline, which are tetracycline derivatives reported to have antiapoptotic effects in mammals, would significantly increase lifespan and improve neuromuscular function in laminin-,2,deficient mice. Methods Mice that were homozygous for a targeted, inactivating mutation of the laminin-,2 gene were placed into control, minocycline-treated, or doxycycline-treated groups. Drug treatment began within 2 weeks of birth, and the progression of disease was followed over time using behavioral, growth, histological, and molecular assays. Results We found that treatment with either minocycline or doxycycline increased the median lifespan of laminin-,2,null mice from approximately 32 days to approximately 70 days. Furthermore, doxycycline improved postnatal growth rate and delayed the onset of hind-limb paralysis. Doxycycline-treated laminin-,2,deficient muscles had increased Akt phosphorylation, decreased inflammation, and decreased levels of Bax protein, terminal deoxynucleotidyltransferase,mediated dUTP nick end labeling,positive myonuclei, and activated caspase-3. Interpretation Doxycycline or other drugs with similar functional profiles may be a possible route to improving neuromuscular dysfunction caused by laminin-,2-deficiency. Ann Neurol 2008 [source]
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