Home About us Contact
|
Home About us Contact | ||
|
|
||
End Labeling (end + labeling)
Kinds of 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] The Effect of Hyperbaric Oxygen Therapy on Erectile Function Recovery in a Rat Cavernous Nerve Injury ModelTHE JOURNAL OF SEXUAL MEDICINE, Issue 3 2008Alexander Müller MD ABSTRACT Introduction., Cavernosal oxygenation appears to be important for preservation of erectile tissue health. Hyperbaric oxygen therapy (HBOT) has been shown to improve tissue oxygenation and has neuromodulatory effects. Aim., This study was designed to define the effects of HBOT on erectile function (EF) and cavernosal tissue in the rat cavernous nerve (CN) injury model. Methods., Four groups of Sprague-Dawley rats were studied: rats with bilateral CN crush, HBOT treated (Crush+/HBOT+); bilateral CN-crush/no HBOT (C+/H,); no crush/no HBOT (C,/H,); and no crush/HBOT (C,/H+). HBOT was delivered daily for 90 minutes at three atmospheres for 10 days commencing the day of CN crush. Main Outcome Measures., Ten days after CN injury, the animals underwent CN stimulation measuring the maximal intracavernosal pressure/mean arterial pressure (ICP/MAP) ratios. Corporal tissue was harvested pre-sacrifice, and immunohistochemically stained for nerve growth factor (NGF), endothelial nitric oxide synthase (eNOS), and cluster of differentiation molecule (CD31). Histologic analysis was performed for Masson's trichrome to assess the smooth muscle,collagen ratio. Terminal deoxynucleotidyl transferase Biotin-dUTP Nick End Labeling assay was used to define apoptotic indices (AIs). Results., The C+/H, group had significantly lower ICP/MAP ratios compared with C,/H, rats, (31% vs. 70%, P < 0.001). C+/H+ rats had significantly higher ICP/MAP ratio recovery compared with the C+/H, group (55% vs. 31%, P = 0.005). NGF and eNOS staining densities were higher in C+/H+ rats compared with C+/H, rats (P < 0.05 and P < 0.001, respectively). No difference was seen in CD31 expression. Staining density for MT displayed a trend toward higher smooth muscle preservation after HBOT. AIs were significantly increased by HBOT (P < 0.05). Conclusion., HBOT following a CN injury improved EF preservation in this model, supporting the cavernosal oxygenation concept as protective mechanism for EF. The effects appear to be mediated via preservation of neurotrophic and endothelial factor expression. Müller A, Tal R, Donohue JF, Akin-Olugbade Y, Kobylarz K, Paduch D, Cutter SC, Mehrara BJ, Scardino PT, and Mulhall JP. The effect of hyperbaric oxygen therapy on erectile function recovery in a rat cavernous nerve injury model. J Sex Med 2008;5:562,570. [source] Increased Apoptosis in Human Amnion is Associated with Labor at TermAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5 2000CHAUR-DONG HSU PROBLEM: To characterize whether increased apoptosis in human amnion was associated with labor at term. METHOD OF STUDY: Human amnion were obtained from term patients with vaginal delivery (n=5) or who underwent elective Cesarean section (C/S) without labor (n=5). Apoptosis was performed by the TUNEL (Terminal dUTP Nuclear End Labeling) assay. All nucleated cells stained with propidium iodide in the amnion epithelial cells were identified in red fluorescence. TUNEL positive apoptotic nuclei were identified in green fluorescence. Five random fields of each specimen were blindly counted by investigators. The percentage of apoptotic nuclei of total nuclei (apoptotic index) was calculated and compared between the two groups (25 microscopic fields for each group, respectively). RESULTS: Patients with term labor had a significantly higher mean apoptotic index in amnion epithelial cells than that with elective C/S without labor (27.3±4.1% versus 3.6±1.6%, P<0.001). CONCLUSIONS: Our data indicate that apoptosis in human amnion is significantly increased and associated with labor at term. [source] Perinatal development of the rat kidney: Apoptosis and epidermal growth factorCONGENITAL ANOMALIES, Issue 3 2003Toshiya Okada ABSTRACT, Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase,mediated d,UTP,biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glomeruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glomeruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney. [source] Ectopic germline cells in embryos of Xenopus laevisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2007Kohji Ikenishi Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23,48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44,47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43,48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages. [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] Differences and relationships of thymidine phosphorylase expression in tumor-associated macrophages and cancer cells in squamous cell carcinoma of the esophagusDISEASES OF THE ESOPHAGUS, Issue 1 2002N. Koide SUMMARY. Thymidine phosphorylase (TP), which has been shown to be identical to platelet-derived endothelial cell growth factor, is expressed in tumor-associated macrophages (TAMs) as well as cancer cells. The aim of this study was to clarify the differences or relationships of TP expression in TAMs and cancer cells in esophageal squamous cell carcinoma (SCC). Tissues samples were taken from 56 patients with esophageal SCC after curative surgery. The expression of TP in TAMs or SCC cells was examined using a monoclonal antibody to TP (clone 654,1). Microvessels in SCC that stained positively for Factor VIII-related antigen were counted (microvessel density, MVD). Macrophages in SCC that stained positively for CD68 antigen were counted (monocytic count). Ki-67 antigen was immunostained with MIB-1, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling was performed, and Ki-67 labeling index (LI) and apoptotic index were calculated. The expression of TP in stromal cells and cancer cells was observed in 43 (76.8%) and 33 patients (58.9%), respectively. There were significant correlations between TP expression in stromal cells (TAMs) as well as in cancer cells and venous invasion, distant metastasis, or MVD. There was a correlation between TP expression in cancer cells and lymph node metastasis, and there were correlations between TP expression in TAMs and monocytic count or Ki-67 LI; however, there was no correlation between TP expression in TAMs and lymph node metastasis. On the other hand, in SCCs with TP expression in both TAMs and cancer cells, higher frequencies of venous invasion and distant metastasis, higher MVD and lower apoptotic index were observed than in other SCCs. The 5-year survival rate in patients with TP expression in both TAMs and cancer cells was poorer than that in patients with TP expression in neither TAMs and cancer cell. In conclusion, these results suggest that co-expression of TP in TAMs and cancer cells is strongly associated with angiogenic promotion and distant metastasis. However, other effects of TP, such as promotion of tumor growth and lymph node metastasis, may be different depending on whether these are expressed in TAMs or cancer cells in esophageal SCCs. Patients with coexpression of TP in TAMs and cancer cells may be associated with a poor prognosis. [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] Matrix metalloproteinase inhibitor reduces apoptosis induction of bone marrow cells in MDS-RAEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2004Kosei Arimura Abstract:,Background and objectives:,We examined the involvement of apoptosis with myelodysplastic syndrome (MDS) accompanied by peripheral cytopenias despite normo-hypercellular bone marrow. Materials and methods:,Bone marrow smears from 31 patients with MDS-refractory anemia (RA) and five normal controls were stained using the in situ end labeling (ISEL) method. Next, the inhibitory effects of a caspase-3 inhibitor, matrix metalloproteinase inhibitor (MMPI), anti-tumor necrosis factor (TNF)- , or anti-Fas antibody upon the apoptosis induction in overnight cultures of bone marrow cells from the patients were examined. Further, TNF- ,, transforming growth factor (TGF)- , and soluble Fas ligand (sFasL) concentrations in culture supernatants of the cells were assessed by enzyme-linked immunosorbent assay (ELISA). Results:,The incidence of ISEL-positive cells among MDS patients was significantly higher than in normal controls (50.8 ± 14.0% vs. 11.3 ± 2.4%; P < 0.0001). A caspase-3 inhibitor reduced significantly the ISEL-positive rates (32.6 ± 15.2% vs. 50.2 ± 16.5%; P < 0.0001). Anti-TNF- , or anti-Fas antibody reduced the ISEL-positive rates significantly (28.2 ± 6.0%, 29.2 ± 5.8%, vs. 44.2 ± 3.4%, P < 0.001, P = 0.001, respectively). KB-R7785 also significantly decreased the ISEL-positive rates (18.0 ± 9.3% vs. 43.6 ± 14.0%; P < 0.0001). The concentration of TNF- , was significantly reduced by KB-R7785 (P < 0.05), whereas that of TGF- , was not. Concentration of sFasL was under detectable level in the present assay system. The derivatives of KB-R7785 that can be administrated orally showed inhibitory effect on apoptosis induction as well. Conclusions:,These findings suggest that MMPIs inhibits the apoptosis induction of MDS bone marrow cells via the inhibition of TNF- , and probably sFasL secretion, and that MMPIs can be used to control the abnormal induction of apoptosis in MDS. [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] Enhanced Expression of Transcription Factor E2F in Helicobacter pylori -infected Gastric MucosaHELICOBACTER, Issue 3 2002Hajime Isomoto Abstract Objective.Helicobacter pylori is implicated in gastric carcinogenesis through increased gastric epithelial cell turnover. In fact, high proportions of proliferating and apoptotic epithelial cells are found in H. pylori -infected gastric mucosa. E2F, a transcription factor, induces coordinated transactivation of a set of genes involved in cell cycle progression. The aim of this study was to investigate the expression of E2F in H. pylori -infected gastric mucosa and examine the correlation between such expression and gastric epithelial cell proliferation and apoptosis. Methods. Twenty-five patients with H. pylori -associated gastritis (HAG) and 13 control subjects negative for H. pylori were examined. E2F expression was studied in situ by Southwestern histochemistry, a method used to localize transcription factors. Labeled double-stranded oligo-DNA with specific consensus sequence for E2F binding sites was reacted with frozen sections from antral biopsy specimens obtained at endoscopy. Gastric epithelial cell proliferation was assessed by immunostaining of proliferating cell nuclear antigen (PCNA), while apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The percentages of epithelial cells with nuclear staining for PCNA and E2F were expressed as a positivity index (PI). The percentage of TUNEL-positive epithelial cells was defined as apoptotic index. Results. E2F was expressed in the nuclei of gastric epithelial cells within gastric pits. E2F PI in H. pylori -infected gastric mucosa was significantly higher than that in noninfected. Expression of E2F correlated well with PCNA-positive epithelial cells. We also demonstrated colocalization of PCNA with E2F expression in the same epithelial cells. Apoptotic index was also high in H. pylori -infected mucosa, and correlated with E2F PI. Conclusion. Our results demonstrated a significant increase in the expression of E2F in H. pylori -infected mucosa, which correlated with both the percentages of PCNA- and TUNEL-positive cells. Our results suggest that enhanced E2F expression in gastric mucosa may be involved in H. pylori -related gastric carcinogenesis through accelerated cell turnover. [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] Spatial Distribution of Bax and Bcl-2 in Osteocytes After Bone Fatigue: Complementary Roles in Bone Remodeling Regulation?,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2002Olivier Verborgt Abstract Osteocyte apoptosis appears to play a key role in the mechanism by which osteoclastic resorption activity targets bone for removal, because osteocyte apoptosis occurs in highly specific association with microdamage and subsequent remodeling after fatigue. However, beyond terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay, little is known about the mechanisms controlling osteocyte apoptosis in vivo. In the current studies, expression of Bax, a proapoptotic gene product, and Bcl-2, an antiapoptotic gene product, was determined in osteocytes of fatigued rat bone using immunocytochemical staining and compared with TUNEL staining patterns. Bax and Bcl-2 were evident in osteocytes by 6 h after loading. Moreover, Bax and Bcl-2 in osteocytes were expressed differently as a function of distance from microdamage sites. The peak of Bax expression and TUNEL+ staining in osteocytes was observed immediately at the microcrack locus, which is where bone resorption occurs in this system; in contrast, Bcl-2 expression, the antiapoptotic signal, reached its greatest level at some distance (1-2 mm) from microcracks. These data suggest that near sites of microinjury in bone, those osteocytes that do not undergo apoptosis are prevented from doing so by active protection mechanisms. Moreover, the zone of apoptotic osteocytes around microcracks was effectively "walled in" by a surrounding halo of surviving osteocytes actively expressing Bcl-2. Thus, the expression pattern of apoptosis-inhibiting gene products by osteocytes surrounding the apoptotic osteocyte at microdamage sites also may provide important signals in the guidance of resorption processes that occur in association with osteocyte apoptosis after fatigue. [source] Matrix Regulation of Skeletal Cell Apoptosis II: Role of Arg-Gly-Asp-Containing PeptidesJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2002Robert L. Perlot Jr. Abstract This investigation was based on the assumption that arg-gly-asp (RGD)-containing peptides are released from the extracellular matrix of bone and cartilage during the remodeling cycle. We asked the question: Can RGD peptides influence skeletal cell viability? Primary human osteoblasts, mouse MC-3T3-E1 cells, and chick chondrocytes were incubated with purified RGD-containing peptides and cell viability was determined. The RGD peptide did not kill osteoblasts, chondrocytes, or MC-3T3-E1 cells. In contrast, RGDS and GRGDSP peptides killed all three cell types. Osteoblast death was quite rapid, occurring within 6 h of treatment. transferase uridyl mediated nick end labeling (TUNEL) and transmission electron microscopy (TEM) analysis indicated that death was mediated by apoptosis. To learn if mitochondria transduced the death signal, cells were treated with RGDS and organelle function was evaluated using a voltage-sensitive fluorescent probe. It was observed that there was no net loss of fluorescence and, hence, it was concluded that mitochondria were not the primary effectors of the apoptotic response. Experiments were performed with enzyme inhibitors to determine the import of the caspase pathway on RGDS-mediated osteoblast apoptosis. Results of these studies, as well as a study conducted using a fluorescent substrate, pointed to caspase 3 mediating the effector stage of the apoptotic process. Finally, using a purified labeled-RGDS peptide, we showed that the molecule was not restricted by the plasma membrane because it was accumulated in the cytosolic compartment. Results of the investigation support the view that resorption of the extracellular matrix generates peptide products that can induce apoptosis of vicinal cells. [source] Loss of Osteocyte Integrity in Association with Microdamage and Bone Remodeling After Fatigue In Vivo,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000Olivier Verborgt Abstract As a result of fatigue, bone sustains microdamage, which is then repaired by bone-remodeling processes. How osteoclastic activity is targeted at the removal of microdamaged regions of bone matrix is unknown. In the current studies, we tested the hypothesis that changes in osteocyte integrity, through the initiation of regulated cell death (apoptosis), are associated with fatigue-related microdamage and bone resorption. Ulnae of adult rats were fatigue-loaded to produce a known degree of matrix damage. Osteocyte integrity was then assessed histomorphometrically from terminal deoxynucleotidyl transferase,mediated deoxyuridine triphosphate,nick end labeling (TUNEL),stained sections to detect cells undergoing DNA fragmentation associated with apoptosis; toluidine blue,stained sections were used for secondary morphological confirmation. Ten days after loading, large numbers of TUNEL-positive osteocytes were found in bone surrounding microcracks and in bone surrounding intracortical resorption spaces (,300% increases over controls, p < 0.005). TUNEL labeling in loaded ulnae at sites distant from microcracks or resorption foci did not differ from that in control bone. Osteocytes in toluidine blue,stained sections showed equivalent trends to TUNEL-stained sections, with significant increases in pyknotic nuclei and empty lacunae associated with microcracks and intracortical resorption spaces. TUNEL-positive osteocytes were observed around bone microdamage by 1 day after loading (p < 0.01 relative to baseline), and their number remained elevated throughout the entire experimental period. Increases in empty lacunae and decreases in normal osteocyte numbers were observed over time as well. These studies show that (1) osteocyte apoptosis is induced by bone fatigue, (2) this apoptosis is localized to regions of bone that contain microcracks, and (3) osteoclastic resorption after fatigue also coincides with regions of osteocyte apoptosis. The strong associations between microdamage, osteocyte apoptosis, and subsequent bone remodeling support the hypothesis that osteocyte apoptosis provides a key part of the activation or signaling mechanisms by which osteoclasts target bone for removal after fatigue-induced matrix injury. [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] Antiapoptotic and antiautophagic effects of glial cell line-derived neurotrophic factor and hepatocyte growth factor after transient middle cerebral artery occlusion in ratsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 10 2010Jingwei Shang Abstract Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser2448 (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to ,-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways. © 2010 Wiley-Liss, Inc. [source] Expressions of nitrotyrosine and TUNEL immunoreactivities in cultured rat spinal cord neurons after exposure to glutamate, nitric oxide, or peroxynitriteJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2001Y. Manabe Abstract Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanism is not fully understood. We examined cell damage of primary culture of 11-day-old rat spinal cord by addition of glutamate, nitric oxide (NO) or peroxynitrite (PN) with detection of nitrotyrosine (NT) or terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immunoreactivity for NT became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 48 hr and positive in the axons of the ventral horn at 24 to 48 hr. TUNEL positive nuclei were found in spinal large motor neurons from 24 hr, and the positive cell number greatly increased at 48 hr in contrast to the vehicle. Pretreatment of cultures with ,-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor antagonist, NO-suppressing agent, and antioxidant protected the immunoreactivity for NT or TUNEL. The present results suggest that both excitotoxic and oxidative stress play an important role in the upregulation of NT nitration and the apoptotic pathway in cultured rat spinal neurons. J. Neurosci. Res. 65:371,377, 2001. © 2001 Wiley-Liss, Inc. [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 Rapid Induction of Apoptosis in Gastrulating Mouse Embryos by Ethanol and Its Prevention by HB-EGFALCOHOLISM, Issue 1 2006Brian A. Kilburn Background: Ethanol exposure during gastrulation and early neurulation induces apoptosis within certain embryonic cell populations, leading to craniofacial and neurological defects. There is currently little information about the initial kinetics of ethanol-induced apoptosis, and interest in the ability of endogenous survival factors to moderate apoptosis is growing. Ethanol alters intracellular signaling, leading to cell death in chick embryos, suggesting that apoptosis could occur rapidly and that signaling pathways activated by survival factors might reduce apoptosis. Methods: Pregnant mice were intubated with 1, 2, or 4 g/kg ethanol on day 7.5 of embryogenesis (E7.5) 1, 3, or 6, hours before harvesting gastrulation-stage embryos. Control animals received maltose/dextran. Blood alcohol concentrations (BAC) were determined by gas chromatography. E7.5 embryos isolated from untreated dams were cultured in vitro for 1 or 3 hr with 0 or 400 mg% ethanol and 0 or 5 nM heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). Apoptosis was quantified using fluorescence microscopy to detect annexin V binding and DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL)] in whole-mount or sectioned embryos. Results: Both annexin V binding and TUNEL were elevated (p<0.05) in embryos exposed in utero to 1 g/kg ethanol for 3 hours, increasing linearly with time and ethanol concentration. Apoptosis increased (p<0.05) in all germ cell layers. Mice treated with 4 g/kg sustained BAC of 400 mg% for nearly 3 hours, significantly increasing apoptosis within the first hour. Cultured embryos exposed to 400 mg% ethanol displayed 2- to 3-fold more TUNEL than vehicle-treated embryos (p<0.05); however, exogenous HB-EGF prevented apoptosis. Conclusions: Ethanol rapidly produced apoptosis in gastrulation-stage embryos, consistent with induction by intracellular signaling. The ethanol-induced apoptotic pathway was blocked by the endogenous survival factor, HB-EGF. Differences in the expression of survival factors within individual embryos could be partly responsible for variations in the teratogenic effects of ethanol among offspring exposed prenatally. [source] 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] Ethanol Exposure Enhances Apoptosis Within the TestesALCOHOLISM, Issue 10 2000Qianlong Zhu Background: Chronic ethanol abuse causes testicular atrophy and male infertility in alcoholic men. It is well known that ethanol exposure disrupts the hypothalamic-pituitary-gonadal axis, adversely affects the secretory function of Sertoli cells, and produces oxidative stress within the testes. It is still not clear what cellular mechanisms are responsible for the morphologic alteration of the testes that results in a reduction of testicular mass as a consequence of ethanol exposure. The hypothesis tested was that ethanol enhances apoptosis of testicular germ cells. Methods: In the experiments of chronic ethanol exposure, male Sprague Dawley® rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) were fed Liber-Decarlie liquid diet for 9 weeks. In the experiments of acute ethanol exposure, a small volume of 20% ethanol solution was administered by intratesticular injection. Both 3,-end labeling of isolated testicular deoxyribonucleic acid (DNA) and labeling of apoptotic cells in situ by the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5,-triphosphate nick end-labeling method were used to determine apoptosis rates within the testes. The expression of proteins involved in apoptosis was assessed by reverse transcription-polymerase chain reaction and by Western blotting. Results: The testes of rats that were fed an ethanol-containing liquid diet had more testicular DNA fragmentation than did those of animals that were fed an isocaloric control diet. Ethanol increased the number of apoptotic spermatogonia as well as spermatocytes. Direct intratesticular injections of ethanol solution enhanced testicular DNA fragmentation, suggesting an increase in apoptosis. Moreover, Fas ligand levels were increased within the testes of rats that were chronically fed ethanol. In vitro, ethanol treatment of cultured Sertoli cells enhanced the production of Fas ligand. In addition, testicular levels of p53 messenger ribonucleic acid were increased in rats that were chronically fed ethanol. Conclusions: All of these observations suggest that ethanol enhances testicular germ cell apoptosis. [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] Cell death and apoptosis-related proteins in muscle biopsies of sporadic amyotrophic lateral sclerosis and polyneuropathyMUSCLE AND NERVE, Issue 8 2001Benedikt G.H. Schoser MD Abstract To investigate disease-related differences of cell death and apoptosis in human denervation atrophy, we studied DNA fragmentation by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) method in 38 biopsies of clinically nonaffected and affected muscles from patients with sporadic amyotrophic lateral sclerosis (sALS), in 13 muscle biopsies from patients with chronic peripheral neuropathies, and in 8 biopsies from control subjects. In addition, expression of apoptosis-related proteins, bax, bcl-2, and Fas, was studied in 20 biopsies of sALS and 10 chronic peripheral neuropathies. We identified DNA cleavage in 10% of myofibers of patients and in up to 1.5% of control samples. In clinically affected muscles of ALS, a larger amount of TUNEL-positive myofibers (mean 10.5 ± 5.9%) was detected, similar to chronic peripheral neuropathies (mean 10.0 ± 7.4%). Atrophic myofibers were immunopositive for bax, bcl-2, and, to a weaker extent, for Fas. However, bax-, bcl-2-, or Fas-positive atrophic myofibers did not reveal consecutive DNA cleavage. Differences between sALS subgroups and chronic peripheral neuropathies were not found. In human denervation atrophy the bcl-2/bax and the FasL/Fas systems are apparently active independently of DNA fragmentation and apoptosis. DNA fragmentation thus displays an additional reaction that is not disease-specific at chronic stages of human denervation processes, probably recapitulating events like skeletal muscle fiber remodeling in embryonic skeletal tissue development. © 2001 John Wiley & Sons, Inc. Muscle Nerve 24: 1083,1089, 2001 [source] Induction of apoptosis in the LNCaP human prostate carcinoma cell line and prostate adenocarcinomas of SV40T antigen transgenic rats by the Bowman,Birk inhibitorPATHOLOGY INTERNATIONAL, Issue 11 2009MingXi Tang The soybean-derived serine protease inhibitor, Bowman,Birk inhibitor (BBI), has been reported as a potent chemoprevention agent against several types of tumors. The present study was undertaken to evaluate the effects of BBI on androgen-sensitive/dependent prostate cancers using a human prostate cancer cell (LNCaP) and the transgenic rats developing adenocarcinoma of the prostate (TRAP) model. Treatment of LNCaP prostate cancer cells with 500 µg/mL BBI resulted in inhibition of viability measured on WST-1 assays, with induction of connexin 43 (C×43) and cleaved caspase-3 protein expression. Feeding of 3% roughly prepared BBI (BBIC) to TRAP from the age 3 weeks to 13 weeks resulted in significant reduction of the relative epithelial areas within the acinus and multiplicity of the adenocarcinomas in the lateral prostate lobes. C×43- and terminal deoxynucleotidyl transferase mediated dUTP-biotin end labeling of fragmented DNA (TUNEL)-positive apoptotic cancer cells were more frequently observed in the lateral prostates treated with BBIC than in the controls. These in vivo and in vitro results suggest that BBI possesses chemopreventive activity associated with induction of C×43 expression and apoptosis. [source] Apoptosis as an independent prognostic indicator in squamous cell carcinoma of the esophagusPATHOLOGY INTERNATIONAL, Issue 7 2001Hiroshi Shibata Apoptosis plays a crucial role in determining net cell proliferation and cell turnover in various tumors. The rate of apoptosis in tumor cells has been reported to be a useful prognostic indicator in colorectal carcinoma. We examined apoptosis in 72 specimens of esophageal squamous cell carcinoma, by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) digoxigenin,nick end labeling (TUNEL) method. We examined correlation of apoptosis with outcome, clinicopathological features, and expression of the apoptosis-related proteins p53 and Bcl-2. The percentage of apoptotic cells, or apoptotic index (AI), ranged from 0.8 to 9.4 (mean: 3.47; SD: 2.02). Overall, 5-year survival of patients with high AI (AI , 5.0; n= 18) tumors was significantly higher than that of patients with low AI tumors (AI < 5.0; n= 58; 76.9% versus 44.9%; P= 0.042). AI did not correlate significantly with the clinicopathological features of patient age and sex, depth of tumor and histological differentiation, lymph node metastasis, lymphatic invasion, or venous invasion. In p53-negative tumors, the AI was significantly higher than in p53-positive tumors. We concluded that AI may be a useful prognostic indicator in esophageal squamous cell carcinoma following curative surgery, and that apoptosis in this tumor is related to relative underexpression of p53 protein. [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] Plasma and liver proteomic analysis of 3Z-3-[(1H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one-induced hepatotoxicity in Wistar ratsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2010Ying Wang Abstract 3Z-3-[(1H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one (Z24), a synthetic anti-angiogenic compound, inhibits the growth and metastasis of certain tumors. Previous works have shown that Z24 induces hepatotoxicity in rodents. We examined the hepatotoxic mechanism of Z24 at the protein level and looked for potential biomarkers. We used 2-DE and MALDI-TOF/TOF MS to analyze alternatively expressed proteins in rat liver and plasma after Z24 administration. We also examined apoptosis in rat liver and measured levels of intramitochondrial ROS and NAD(P)H redox in liver cells. We found that 22 nonredundant proteins in the liver and 11 in the plasma were differentially expressed. These proteins were involved in several important metabolic pathways, including carbohydrate, lipid, amino acid, and energy metabolism, biotransformation, apoptosis, etc. Apoptosis in rat liver was confirmed with the terminal deoxynucleotidyl transferase dUTP-nick end labeling assay. In mitochondria, Z24 increased the ROS and decreased the NAD(P)H levels. Thus, inhibition of carbohydrate aerobic oxidation, fatty acid ,-oxidation, and oxidative phosphorylation is a potential mechanism of Z24-induced hepatotoxicity, resulting in mitochondrial dysfunction and apoptosis-mediated cell death. In addition, fetub protein and argininosuccinate synthase in plasma may be potential biomarkers of Z24-induced hepatotoxicity. [source]
| | |||||||||||||||||||||||||||