Deoxyribonucleotidyl Transferase-mediated dUTP Nick-end Labeling Research Articles

A Novel Allele Encoding 7-Hydroxymethyl Chlorophyll a Reductase Confers Bacterial Blight Resistance in Rice.

Rice spotted leaf mutants are helpful to investigate programmed cell death (PCD) and defense response pathways in plants. Using a map-based cloning strategy, we characterized novel rice spotted leaf mutation splHM143 that encodes a 7-hydroxymethyl chlorophyll a reductase (OsHCAR). The wild-type (WT) allele could rescue the mutant phenotype, as evidenced by complementation analysis. OsHCAR was constitutively expressed at all rice tissues tested and its expression products localized to chloroplasts. The mutant exhibited PCD and leaf senescence with increased H2O2 (hydrogen peroxide) accumulation, increased of ROS (reactive oxygen species) scavenging enzymes activities and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling) -positive nuclei, upregulation of PCD related genes, decreased chlorophyll (Chl) contents, downregulation of photosynthesis-related genes, and upregulation of senescence-associated genes. Besides, the mutant exhibited enhanced bacterial blight resistance with significant upregulation of defense response genes. Knockout lines of OsHCAR exhibited spotted leaf phenotype, cell death, leaf senescence, and showed increased resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) coupled with upregulation of five pathogenesis-related marker genes. The overexpression of OsHCAR resulted in increased susceptibility to Xoo with decreased expression of pathogenesis-related marker genes. Altogether, our findings revealed that OsHCAR is involved in regulating cell death and defense response against bacterial blight pathogen in rice.

Salidroside induces apoptosis and autophagy in gastric cancer cells via regulation of mitogen-activated protein kinases signaling pathway

Background: Salidroside, an active ingredient of Rhodiola rosea, exhibits antiproliferative effect in gastric cancer (GC) cells. However, the involvement of salidroside in apoptosis and autophagy of GC cells has not been elucidated. Materials and Methods: Cell viability of BGC-823 cells was assessed by CCK-8 assay, and apoptosis was analyzed with terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay and flow cytometry. The apoptosis- and autophagy-associated proteins and mitogen-activated protein kinases (MAPKs) were determined by Western blot analysis. Autophagy was evaluated by green fluorescent protein-fused LC3 punctate formation. Results: Salidroside inhibited proliferation and promoted apoptosis and autophagy of BGC-823 cells in a dose-dependent manner. In addition, salidroside enhanced the phosphorylation of p38 MAPK, ERK1/2, and JNK. Furthermore, inhibition of MAPKs significantly abolished the effects of salidroside in BGC-823 cells. Conclusion: Salidroside induced apoptosis and autophagy of BGC-823 cells via activation of MAPK signaling pathway.

<p><strong>Antileishmanial apoptotic activity of <em>Nigella sativa</em> L. essential oil and thymoquinone triggers on <em>Leishmania tropica</em></strong></p>

Nigella sativa L., commonly called Black cumin, is well-known in folk medicine and numerous studies have shown its various pharmacological activities. In this study, we estimated the cytotoxic effects of N. sativa essential oil (NEO) and its major bioactive component Thymoquinone (TQ) on Leishmania tropica promastigotes that cause cutaneous leishmanias, and also observed the programmed cell death features. The extraction of NEO was done by hydro-distillation and analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). Antileishmanial activity of NEO and TQ was determined using 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) assay and the obtained results are expressed as 50% inhibitory concentration (IC 50 ). The leishmanicidal activity of NEO and TQ was mediated via apoptosis as evidenced by in situ labelling of DNA fragments using terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell cycle arrest at sub G 0 /G 1 phase. The IC 50 values were 5 µg/mL and 1.3 µg/mL, respectively. We noted a significant increase in DNA fragmentation in treated parasites with IC 50 of both NEO and TQ as well as a cell cycle arrest. These results revealed that NEO and TQ possess potential antileishmanial activity that mediated high possibility by programmed cell death.

Effects of sorafenib on fibroblast-like synoviocyte apoptosis in rats with adjuvant arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by synovial inflammation and hyperplasia resulting from an imbalance between the proliferation and apoptosis of fibroblast-like synoviocytes (FLSs). Our previous study found that sorafenib had inhibitory effects in rats with adjuvant arthritis (AA). The present study investigated the role of sorafenib in the induction of AA FLS apoptosis in vitro. FLSs obtained from AA rats were cultured in vitro and identified. Cell apoptosis was detected using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling methods. Real-time PCR and Western blotting assays were used to quantify the expression levels of Fas, Caspase-3, Mcl-1, NF-κB and C-jun gene products in AA FLSs. Our data revealed that sorafenib (4 μmol/L) induced apoptosis in AA FLSs, and flow cytometry analysis showed that AA FLSs treated with sorafenib (4 μmol/L) in vitro accumulated in early and late apoptosis. There were significant increases in the expression levels of Fas, Caspase-3 and Mcl-1, and significant decreases in NF-κB and C-jun expression in AA FLSs treated with sorafenib. In summary, these results demonstrate that sorafenib promotes AA FLS apoptosis, which may be related to the upregulation of Fas and Caspase-3 and downregulation of NF-κB and C-jun. All of these findings suggest that sorafenib exerts an inhibitory effect on AA rats in vivo via AA FLS apoptotic induction, which has potential therapeutic implications for RA.

DNA damaging effect of paclitaxel in the epididymal sperms as a chemotherapeutic agent and possible remedies to prevent this effect: A study on reproductive potential of male cancer patients of reproductive age

Cancer is a major public health problem, young cancer patients therefore undergo chemotherapy, and most of them may lose their fertility. DNA damage level provides important clues about the quality and reproductive potential of spermatozoa. In this study, we evaluated the levels of both DNA fragmentation and abnormal DNA integrity in the epididymal sperms of New Zealand rabbit (Oryctolagus cuniculus) after cryopreservation using the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay and the toluidine blue (TB) staining methods and assessed the effects of paclitaxel, resveratrol, l-glutamine (LG), and basal medium eagle (BME) solution on DNA damage. Paclitaxel induced the levels of both DNA damages in the sperms, but resveratrol ameliorated this effect. LG and BME supplementation to the extender prevented the sperm samples from DNA fragmentation after cryopreservation. Chemotherapy drugs containing paclitaxel can cause the sperm DNA to be damaged, and hence adversely affect the fertility of male cancer patients of reproductive age. The administration of resveratrol together with paclitaxel may ameliorate the DNA damage inducing effect of paclitaxel. Sperm banking and cryopreservation with the appropriate cryoprotectants such as LG and BME prior to cancer treatment can also be suggested to all male cancer patients of reproductive age facing cancer treatment for fertility preservation.

LQB-118, an orally active pterocarpanquinone, induces selective oxidative stress and apoptosis in Leishmania amazonensis

The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing. Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2',7'-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨm) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨm after 24 h of incubation with 2.5 μM (18.7%), 5 μM (63.7%) or 10 μM (70.7%) LQB-118. A sub-G0/G1 cell cycle phenotype was observed in 21%-83% of the promastigotes incubated with 1.25-10 μM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5-10 μM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 μM treatment. Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.

Evaluation of reactive oxygen species (ROS) and DNA integrity assessment in cases of idiopathic male infertility

Background Studies suggest that reactive oxygen species (ROS) attack the integrity of DNA in the sperm nucleus by causing base modifications, DNA strand breaks, and chromatin cross-linking. Sperm DNA damage analysis may reveal hidden sperm DNA abnormalities in infertile men with normal standard semen analysis values who were diagnosed with idiopathic infertility. Aim of the work We explored the levels of ROS and their correlation with sperm DNA damage in patients with idiopathic male infertility. Patients and methods A total of 93 men were included in this study. Among them, 68 presented to our Andrology outpatient clinic with idiopathic infertility and were selected, and 25 were healthy fertile men, who were assigned to the control group. Both groups were subjected to the following laboratory investigations: semen analysis including peroxidase test, measurement of ROS levels by chemiluminescence assay, and sperm DNA damage assessment by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Results There were no significant differences in the semen parameters between idiopathic infertile men and controls. However, ROS and sperm DNA damage levels were significantly higher in idiopathic infertile men compared with controls ( P ͳ 0.001). In addition, the results showed a strong positive correlation between ROS levels and the percentage of sperm DNA damage ( P ͳ 0.001). Conclusion Traditional semen analysis does not reveal seminal defects at the molecular level that might be induced by ROS. Therefore, our results suggest that standard semen analysis should be coupled with measurement of ROS and assessment of DNA integrity in cases of idiopathic male infertility as even sperms with normal morphology and motility may harbor DNA damage.

PTX3 Controls Activation of Matrix Metalloproteinase 1 and Apoptosis in Conjunctivochalasis Fibroblasts

Conjunctivochalasis (CCh) is an age-related inflammatory ocular surface disease manifesting redundant, loose conjunctiva folds. The pathogenic role of Pentraxin 3 (PTX3) in controlling upregulation of matrix metalloproteinase 1 (MMP-1) and MMP-3 in CCh remains undefined. Cytolocation of PTX3 and apoptosis were compared by immunostaining and terminal deoxyribonucleotidyl transferase-mediated FITC-linked dUTP nick-end DNA labeling (TUNEL) assay between normal and CCh specimens containing the conjunctiva and the Tenon. Second to third cultures of normal and CCh fibroblasts were treated with or without Aprotinin, Batimastat, or N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), followed by transfection with or without PTX3 siRNA, and TNF-α or IL-1β. Cell lysates and culture media were collected to assess apoptosis measured by the Cell Death Detection ELISA and expression of PTX3, MMP-1, and MMP-3 transcripts and proteins by quantitative RT-PCR and Western blot, respectively. PTX3 immunostaining was negative in normal specimens, but strongly positive in the subconjunctival stroma of CCh specimens. More apoptotic cells were found in CCh samples than in normal specimens. Expression of PTX3 transcripts and protein was not constitutive in resting normal fibroblasts but was in resting CCh fibroblasts and was upregulated by IL-1β in both cell lysates and culture media of both fibroblasts. PTX3 siRNA further upregulated MMP-1 and MMP-3 transcripts in resting normal fibroblasts, but synergistically with IL-1β upregulated the expression of MMP-1 and MMP-3 transcripts only in CCh fibroblasts, with activation of MMP-1 more so than MMP-3. PTX3 siRNA knockdown also promoted cell death characterized by apoptosis and necrosis, and such cell death could be rescued by inhibitors against serine proteinase, MMP1, or MMP3. Perturbation of PTX3 expression might partake in apoptosis and pathogenesis of CCh by upregulating expression of MMP-1 and MMP-3, and activation of MMP-1 and MMP-3.

Blockade of acid-sensing ion channels protects articular chondrocytes from acid-induced apoptotic injury

Acid-sensing ion channels (ASICs) are members of the degenerin/epithelial sodium channel (DEG/ENaC) protein superfamily and play a critical role in acid-induced cell injury. In this study, we examined whether drugs such as amiloride that block ASICs could attenuate acid-induced apoptotic injury to articular chondrocytes. Articular chondrocytes were isolated from Sprague-Dawley rats, and their phenotype was determined by toluidine blue and immunocytochemical staining. Articular chondrocyte viability assay was performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Apoptosis of chondrocytes was observed by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling method as well as propidium iodide labeling methods. Intracellular calcium ([Ca(2+)](i)) was analyzed by a Ca(2+)-imaging method. In addition, the expression levels of calpain and calcineurin in articular chondrocytes were examined by real-time PCR and immunocytochemical staining. The activity of caspase-3 was evaluated by spectrophotometric assays. Positive staining for glycosaminoglycan and collagen II was seen in articular chondrocytes. Blocking acid-sensing ion channels significantly decreased the cell death percentage and increased cell viability following acid exposure. After pretreated with amiloride, acid-induced [Ca(2+)](i) rises were reduced. Amiloride also inhibited calpain and calcineurin expression levels in acid-induced chondrocytes, and inhibited caspase-3 activity. The data presented in this study provided some experimental evidence that blocking ASICs could protect acid-induced apoptotic injury to chondrocytes.

Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, the comet assay, the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation. Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay.

Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation

An embryo's ability to grow and implant can be improved by selection of a normal spermatozoon with a vacuole-free head. However, large vacuoles in spermatozoa have yet to be fully characterized. The present study aimed to determine whether these vacuoles are of nuclear, membrane and/or acrosomal origin. We studied 15 infertile patients with differing sperm profiles. For each sperm sample, we used high-magnification (×10 000) contrast microscopy to select and assess 30 normal 'top' spermatozoa and 30 spermatozoa with a large sperm-head vacuole (≥ 25% of the head's cross-sectional area). We subsequently analysed the spermatozoa's degree of chromatin condensation (aniline blue staining), DNA fragmentation (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay) and chromosome content (fluorescence in situ hybridization X,Y,18). Atomic force microscopy enabled us to map the plasma sperm membrane in detail. Three-dimensional deconvolution microscopy enabled us to reconstruct images of the nucleus and acrosome in 'top' and 'vacuolated' spermatozoa. We studied a total of 450 'top' spermatozoa and 450 vacuolated spermatozoa. The rate of non-condensed chromatin was higher for 'vacuolated' spermatozoa than for 'top' spermatozoa (36.2 ± 1.9 versus 7.6 ± 1.3%, respectively; P < 0.0001). 'Top' and 'vacuolated' spermatozoa did not differ significantly in terms of DNA fragmentation (0.7 ± 0.4 versus 1.3 ± 0.4% respectively; P = 0.25) or aneuploidy (1.1 ± 0.5 versus 2.2 ± 0.7% respectively; P = 0.21). The majority of aneuploid spermatozoa (9 out of 15) lacked chromatin condensation. In all vacuolated spermatozoa, the acrosome was intact, the plasma membrane was sunken but intact and the large vacuole was identified as an abnormal, 'thumbprint'-like nuclear concavity covered by acrosomal and plasmic membranes. The large vacuole appears to be a nuclear 'thumbprint' linked to failure of chromatin condensation.

Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Granule-associated perforin and granzymes (gzms) are key effector molecules of cytotoxic T lymphocytes (Tc cells) and natural killer cells and play a critical role in the control of intracellular pathogens and cancer. Based on the notion that many gzms, including A, B, C, K, H, and M exhibit cytotoxic activity in vitro, all gzms are believed to serve a similar function in vivo. However, more recent evidence supports the concept that gzms are not unidimensional but, rather, possess non-cytotoxic potential, including stimulation of pro-inflammatory cytokines and anti-viral activities. The present study shows that isolated mouse gzmB cleaves the actin-severing mouse protein, cytoplasmic gelsolin (c-gelsolin) in vitro. However, when delivered to intact target cells by ex vivo immune Tc cells, gzmB mediates c-gelsolin proteolysis via activation of caspases 3/7. The NH(2)-terminal c-gelsolin fragment generated by either gzmB or caspase 3 in vitro constitutively severs actin filaments without destroying the target cells. The observation that gzmB secreted by Tc cells initiates a caspase cascade that disintegrates the actin cytoskeleton in target cells suggests that this intracellular process may contribute to anti-viral host defense.

Apoptosis-like death in Leishmania donovani promastigotes induced by diospyrin and its ethanolamine derivative

Previous studies have demonstrated that diospyrin ( 1), a quinonoid plant product, can inhibit the growth of Leishmania donovani parasites. Here, several derivatives of 1 were evaluated by the MTT assay and it was observed that the ethanolamine analogue ( 10) exhibited maximum cytotoxicity [50% inhibitory concentration (IC 50) = 2.9 μM] against L. donovani promastigotes. Subsequently, the mode of cell death in promastigotes was investigated through externalisation of membrane-associated phosphatidylserine, mitochondrial membrane depolarisation, DNA laddering and in situ labelling of DNA fragmentation by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) methods. Whilst both 1 and 10 were found to induce apoptosis-like death in promastigotes, the effect of 10 was evidently stronger even at a lower concentration. Hence, the ethanolamine derivative ( 10) of diospyrin ( 1) may be a prospective ‘lead’ for the development of novel cytotoxic agents inducing apoptosis in L. donovani parasites.

Activation of Akt/GSK-3β signaling pathway is involved in intermedin1–53 protection against myocardial apoptosis induced by ischemia/reperfusion

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. We investigated the cardioprotective mechanism of IMD(1-53) in the in vivo rat model of myocardial ischemia/reperfusion (I/R) injury and in vitro primary neonatal cardiomyocyte model of hypoxia/reoxygenation (H/R). Myocardial infarct size was measured by 2,3,5-triphenyl tetrazolium chloride staining. Cardiomyocyte viability was determined by trypan blue staining, cell injury by lactate dehydrogenase (LDH) leakage, and cardiomyocyte apoptosis by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay, Hoechst staining, gel electrophoresis and caspase 3 activity. The translocation of mitochondrial cytochrome c of myocardia and expression of apoptosis-related factors Bcl-2 and Bax, phosphorylated Akt and phosphorylated GSK-3beta were determined by western blot analysis. IMD(1-53) (20 nmol/kg) limited the myocardial infarct size in rats with I/R; the infarct size was decreased by 54%, the apoptotic index by 30%, and caspase 3 activity by 32%; and the translocation of cytochrome c from mitochondria to cytosol was attenuated. IMD(1-53) increased the mRNA and protein expression of Bcl-2 and ratio of Bcl-2 to Bax by 81 and 261%, respectively. IMD(1-53) (1 x 10(-7) mol/L) inhibited the H/R effect in cardiomyocytes by reducing cell death by 43% and LDH leakage by 16%; diminishing cellular apoptosis; decreasing caspase 3 activity by 50%; and increasing the phosphorylated Akt and GSK-3beta by 41 and 90%, respectively. The cytoprotection of IMD(1-53) was abolished with LY294002, a PI3K inhibitor. In conclusion, IMD(1-53) exerts cardioprotective effect against myocardial I/R injury through the activation of the Akt/GSK-3beta signaling pathway to inhibit mitochondria-mediated myocardial apoptosis.

Oncolytic activity of Sindbis virus in human oral squamous carcinoma cells

Background:Sindbis virus (SIN) infection causes no or only mild symptoms (fever, rash, and arthralgia) in humans. However, SIN has a strong cytopathic effect (CPE) on various cancer cells. This study focuses on the oncolytic activity of SIN AR399 on oral cancer cells compared with reovirus, a well-known oncolytic virus that targets cancer cells.Methods:We analysed the cytotoxicity and growth of SIN in 13 oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, H-1, Sa-3, KON, KOSC-2, OK-92, HO-1-N1, SCC-4, SAT, SKN-3) and normal human oral keratinocytes (NHOKs).Results:Sindbis virus infection induced CPE in 12 OSCC cell lines at a low multiplicity of infection (MOI) of 0.01, but not in the OSCC cell line, HSC-4 or NHOKs. Sindbis viral growth was not observed in NHOKs, whereas high SIN growth was observed in all OSCC cell lines, including HCS-4. The cytotoxicity and growth of SIN was the same as reovirus at an MOI of 20 in 12 OSCC cell lines. The CPE was shown, by terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labelling assays, to be apoptotic cell death. Furthermore, quantitative RT-PCR of mRNA in HSC-3 and HSC-4 cells after SIN infection showed that activation of caspases, cytochrome c, and IκBα was associated with SIN-induced apoptosis.Conclusion:As a replication-competent oncolytic virus, SIN may be a useful therapeutic modality for oral cancers.

Activation of Akt/GSK-3β signaling pathway is involved in intermedin1-53 protection against myocardial apoptosis induced by ischemia/reperfusion

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. We investigated the cardioprotective mechanism of IMD(1-53) in the in vivo rat model of myocardial ischemia/reperfusion (I/R) injury and in vitro primary neonatal cardiomyocyte model of hypoxia/reoxygenation (H/R). Myocardial infarct size was measured by 2,3,5-triphenyl tetrazolium chloride staining. Cardiomyocyte viability was determined by trypan blue staining, cell injury by lactate dehydrogenase (LDH) leakage, and cardiomyocyte apoptosis by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay, Hoechst staining, gel electrophoresis and caspase 3 activity. The translocation of mitochondrial cytochrome c of myocardia and expression of apoptosis-related factors Bcl-2 and Bax, phosphorylated Akt and phosphorylated GSK-3beta were determined by western blot analysis. IMD(1-53) (20 nmol/kg) limited the myocardial infarct size in rats with I/R; the infarct size was decreased by 54%, the apoptotic index by 30%, and caspase 3 activity by 32%; and the translocation of cytochrome c from mitochondria to cytosol was attenuated. IMD(1-53) increased the mRNA and protein expression of Bcl-2 and ratio of Bcl-2 to Bax by 81 and 261%, respectively. IMD(1-53) (1 x 10(-7) mol/L) inhibited the H/R effect in cardiomyocytes by reducing cell death by 43% and LDH leakage by 16%; diminishing cellular apoptosis; decreasing caspase 3 activity by 50%; and increasing the phosphorylated Akt and GSK-3beta by 41 and 90%, respectively. The cytoprotection of IMD(1-53) was abolished with LY294002, a PI3K inhibitor. In conclusion, IMD(1-53) exerts cardioprotective effect against myocardial I/R injury through the activation of the Akt/GSK-3beta signaling pathway to inhibit mitochondria-mediated myocardial apoptosis.

COOH-Terminal Truncated HBV X Protein Plays Key Role in Hepatocarcinogenesis

X protein (HBx), a product of hepatitis B virus, has been closely associated with the development of hepatocellular carcinoma (HCC). Based on observations that the COOH-terminal truncated HBx was frequently detected in HCC, the aim of this study is to evaluate the function of COOH-terminal truncated HBx in hepatocarcinogenesis. Expression pattern of HBx was analyzed by immunohistochemistry on tissue microarray containing 194 pairs of HCCs and their matched nontumor liver tissues. MIHA and HepG2 cells transfected with full-length (X2) and COOH-terminal truncated HBx (X1) were tested for their ability to grow in soft agar and form tumors in vivo. Proliferation and apoptosis were assessed using 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt and terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assays, respectively. To gain additional insight, the expression profile of HepG2-X2 and HepG2-X1 were compared using cDNA microarray. COOH-terminal truncated HBx was frequently detected in HCCs (79.3%, n = 111), and our in vitro and in vivo studies showed that the truncated rather than the full-length HBx could effectively transform immortalized liver cell line MIHA. Interestingly, expression profiling revealed differential expression of key genes implicated in the control of cell cycle and apoptosis. These findings strongly suggest that the COOH-terminal truncated HBx plays a critical role in the HCC carcinogenesis via the activation of cell proliferation.

Mechanism of Fibroblast‐Like Synoviocyte Apoptosis Induced by Recombinant Human Endostatin in Rats with Adjuvant Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by pronounced synovial hyperplasia, in which there may be an imbalance between the growth and death of fibroblast-like synoviocytes (FLS). The present study was undertaken to examine the effect of recombinant human endostatin (rhEndostatin) on FLS apoptosis in experimental RA. Adjuvant arthritis (AA) was induced in male Sprague Dawley (SD) rats. Using cultured AA FLS obtained from these rats, the apoptosis process was measured by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) as well as Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling methods. In addition, the expression levels of the Fas, c-jun, NFkappaB, and caspase-3 gene products in synovial tissues were quantified by quantitative real-time polymerase chain reaction (qPCR) and/or Western blotting assays. Our data revealed that rhEndostatin induced apoptosis in AA FLS. The number and signal density of TUNEL-positive cells were significantly increased in rats treated with rhEndostatin (2.5 mg/kg). The percentage of Annexin V-FITC-positive cells was 6.67% after treatment with rhEndostatin at 25 microg/mL for 48 hr, compared with only 3.32% among untreated control cells. There were significant increases in Fas mRNA, c-jun mRNA, c-Jun protein, and caspase-3 (p20) protein in AA synovial tissues treated with rhEndostatin (2.5 mg/kg), whereas no significant difference in NFkappaB expression was detected between treated and untreated tissues. These findings indicate that rhEndostatin has a therapeutic effect on RA by inducing FLS apoptosis, which is strongly associated with increased expression of Fas, c-jun, and caspase-3, but not NFkappaB.

Correlation between DNA damage and sperm parameters: a prospective study of 1,633 patients

To investigate DNA fragmentation by using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling in relation to World Health Organization parameters and computer-aided sperm analysis (CASA) in sperm to determine the possibility of obtaining a correlation among CASA parameters, sperm morphology, and DNA fragmentation. Sperm analysis according to World Health Organization parameters, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) for sperm DNA fragmentation, and CASA for sperm movement. Prospective study. All the patients were under clinical management, consulting for hypofertility at a fertility center in France. One thousand six hundred thirty-three men who were referred for infertility investigation, including a complete sperm analysis. Sperm analysis and DNA damage testing. Sperm morphology, DNA fragmentation, and movement characteristics. One third of the patients had a TUNEL rate of >30%. Analysis of the 21 semen parameters tested revealed that 7 of them were significantly correlated with the TUNEL results. World Health Organization sperm parameters and DNA damage are complementary, rather than strongly linked. This should be considered to more fully understand the paternal contribution in assisted reproductive technologies failures.

E2F1 Overexpression Correlates with Thymidylate Synthase and Survivin Gene Expressions and Tumor Proliferation in Non–Small-Cell Lung Cancer

We investigated the clinical significance of E2F1 gene expression in relation to its target genes, thymidylate synthase (TS) and Survivin, in case of non-small-cell lung cancer (NSCLC). One hundred twenty-seven cases of resected NSCLC were analyzed. Quantitative reverse transcription-PCR was done to evaluate the gene expression of E2F1, TS, and Survivin. Immunohistochemistry was done to investigate the protein expression of E2F1, TS, and Survivin. The Ki-67 proliferation index and the apoptotic index using the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling method were also evaluated. E2F1 gene expression significantly correlated with the Ki-67 proliferation index (r = 0.487; P < 0.0001), although no correlation was observed between E2F1 gene expression and the apoptotic index. With regard to E2F1 target genes, E2F1 gene expression significantly correlated with TS gene expression (r = 0.709; P < 0.0001) and Survivin gene expression (r = 0.403; P < 0.0001). The overall survival rate was significantly lower in patients with high-E2F1 tumors than in those with low-E2F1 tumors (P = 0.0027), especially among patients with stage II to III NSCLCs (P = 0.0188). A Cox regression analysis showed that the E2F1 status was a significant prognostic factor for NSCLC patients (hazard ratio, 2.052; P = 0.0261). The present study revealed that E2F1 gene expression correlates with TS and Survivin gene expressions and tumor proliferation. During the progression of NSCLC, E2F1 overexpression could produce more aggressive tumors with a high proliferation rate and chemoresistance.