Chronic/cyclic neutropenia, leukocyte adhesion deficiency syndrome, Papillon-Lefèvre syndrome, and Chédiak-Higashi syndrome are associated with severe periodontitis, suggesting the importance of neutrophils in the maintenance of periodontal health. Various Toll-like receptor (TLR) ligands are known to stimulate neutrophil function, including FcR-mediated phagocytosis. In the present study, the effect of TLR2 activation on the non-opsonic phagocytosis of oral bacteria and concomitant production of reactive oxygen species (ROS) by human neutrophils was evaluated. Neutrophils isolated from peripheral blood were incubated with Streptococcus sanguinis or Porphyromonas gingivalis in the presence of various concentrations of Pam3CSK4, a synthetic TLR2 ligand, and analyzed for phagocytosis and ROS production by flow cytometry and chemiluminescence, respectively. Pam3CSK4 significantly increased the phagocytosis of both bacterial species in a dose-dependent manner. However, the enhancing effect was greater for S. sanguinis than for P. gingivalis. Pam3CSK4 alone induced ROS production in neutrophils and also increased concomitant ROS production induced by bacteria. Interestingly, incubation with P. gingivalis and Pam3CSK4 decreased the amounts of ROS, as compared to Pam3CSK4 alone, indicating the possibility that P. gingivalis survives within neutrophils. However, neutrophils efficiently killed phagocytosed bacteria of both species despite the absence of Pam3CSK4. Although P. gingivalis is poorly phagocytosed even by the TLR2-activated neutrophils, TLR2 activation of neutrophils may help to reduce the colonization of P. gingivalis by efficiently eliminating S. sanguinis , an early colonizer, in subgingival biofilm.

Growing evidence suggests that mitochondrial reactive oxygen species (ROS) are involved in various pain states. This study was performed to investigate whether ROS-induced changes in neuronal excitability in trigeminal subnucleus caudalis are related to ROS generation in mitochondria. Confocal scanning laser microscopy was used to measure ROS-induced fluorescence intensity in live rat trigeminal caudalis slices. The ROS level increased during the perfusion of malate, a mitochondrial substrate, after loading of 2′,7′-dichlorofluorescin diacetate (H2DCF-DA), an indicator of the intracellular ROS; the ROS level recovered to the control condition after washout. When pre-treated with phenyl N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL), malate-induced increase of ROS level was suppressed. To identify the direct relation between elevated ROS levels and mitochondria, we applied the malate after double-loading of H2DCF-DA and chloromethyl-X-rosamine (CMXRos; MitoTracker Red), which is a mitochondria- specific fluorescent probe. As a result, increase of both intracellular ROS and mitochondrial ROS were observed simultaneously. This study demonstrated that elevated ROS in trigeminal subnucleus caudalis neuron can be induced through mitochondrial-ROS pathway, primarily by the leakage of ROS from the mitochondrial electron transport chain.

Luteolysis is a cyclical regression of the corpus luteum in many non-primate mammalian species. Prostaglandin F2α (PGF2α) from the uterus and ovary induces functional and structural luteolysis in bovine. The action of PGF2α is mediated by PGF2α receptor located on the luteal steroidogenic and endothelial cell membranes. PGF2α plays an important role in regulating nitric oxide production in endothelial cells of the bovine corpus luteum. Nitric oxide production and nitric oxide synthase activity are stimulated and induced by PGF2α in luteal endothelial cells. Moreover, the reactive oxygen species inhibits progesterone secretion in bovine luteal cells and induces apoptosis. Thus, the interaction between PGF2α and reactive oxygen species provides important aspects in physiology of the corpus luteum forfunctional and structural luteolysis.

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions (O2˙-) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of O2˙- and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP (10μM) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-β-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.

Antimicrobial actions of reactive oxygen/nitrogen species (ROS/RNS) derived from products of NADPH oxidase and inducible nitric oxide (NO) synthase in host phagocytes inactivate various bacterial macromolecules. To cope with these cytotoxic radicals, pathogenic bacteria have evolved to conserve systems necessary for detoxifying ROS/RNS and repairing damages caused by their actions. In response to these stresses, bacteria also induce expression of molecular chaperones to aid in ameliorating protein misfolding. In this study, we explored the function of a newly identified chaperone Spy, that is localized exclusively in the periplasm when bacteria exposed to conditions causing spheroplast formation, in the resistance of Salmonella Typhimurium to ROS/RNS. A spy deletion mutant was constructed in S. Typhimurium by a PCR-mediated method of one-step gene inactivation with λ Red recombinase, and subjected to ROS/RNS stresses. The spy mutant Salmonella showed a modest decrease in growth rate in NO-producing cultures, and no detectable difference of growth rate in H2O2 containing cultures, compared with that of wild type Salmonella. Quantitative RT-PCR analysis showed that spy mRNA levels were similar regardless of both stresses, but were increased considerably in Salmonella mutants lacking the flavohemoglobin Hmp, which are incapable of NO detoxification, and lacking an alternative sigma factor RpoS, conferring hypersusceptibility to H2O2. Results demonstrate that Spy expression can be induced under extreme conditions of both stresses, and suggest that the protein may have supportive roles in maintaining proteostasis in the periplasm where various chaperones may act in concert with Spy, thereby protecting bacteria against toxicities of ROS/RNS.

The present study was conducted to examine the effect of antioxidant treatment during parthenogenetic activation procedure on the reactive oxygen species (ROS) levels and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by a combination of electric stimulus and 2 mM 6- dimethylaminopurine (6-DAMP) before in vitro culture. During the activation period, oocytes were treated with 50 μM β-mercaptoethanol (β-ME), 100 μM L-ascorbic acid (Vit. C) or 100 μM L-glutathione (GSH). To examine the ROS level, porcine parthenogenetic embryos were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) dye 20 h after culture, examined under a fluorescence microscope, and the fluorescence intensity (pixels) were analyzed in each embryo. The parthenogenetic embryos were cultured for 6 days to evaluate the in vitro development. The apoptosis was measured by TUNEL assay. The H2O2 levels of parthenogenetic embryos were significantly lower in antioxidant treatment groups (26.9±1.6～29.1±1.3 pixels/embryo, p<0.05) compared to control (33.2±1.7 pixels/embryo). The development rate to the blastocyst stage was increased in antioxidant treatment groups (32.0～32.5%) compared to control (26.9%, p<0.05), although, there was no difference in apoptosis among groups. The result suggests that antioxidant treatment during parthenogenetic activation procedure can inhibit the ROS generation and enhance the in vitro development of porcine parthenogenetic embryos.

This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. H2O2 levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in 10 μM 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. H2O2 levels of parthenogenetic embryos were significantly lower in 25 μM β- mercaptoethanol (β-ME), 50 μM L-ascorbic acid (Vit. C), and 50 μM L-glutathione (GSH) treatment groups than each control group (24.0±1.5 vs 39.0±1.1, 29.7±1.0 vs 37.0±1.2, and 32.9±0.8 vs 36.3±0.8 pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison (33.6±0.9～35.2±1.1 pixels/embryo). Thus, an antioxidant of 50 μM Vit. C was selected for SCNT. H2O2 levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure (26.4±1.1 pixels/embryo, p<0.05) than the treatment group during IVM (29.9±1.1 pixels/embryo) and non-treated control (34.3±1.0 pixels/embryo). Moreover, H2O2 level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.

Recent studies indicate that reactive oxygen species (ROS) are critically involved in persistent pain primarily through spinal mechanisms, and that mitochondria are the main source of ROS in the spinal dorsal horn. To investigate whether mitochondrial ROS can induce changes in mem¬brane excitability on spinal substantia gelatonosa (SG) neurons, we examined the effects of mitochondrial electron transport complex (ETC) substrates and inhibitors on the membrane potential of SG neurons in spinal slices. Application of ETC inhibitors, rotenone or antimycin A, resulted in a slowly developing and slight membrane depolarization in SG neurons. Also, application of both malate, a complex I substrate, and succinate, a complex II substrate, caused reversible membrane depolarization and enhanced firing activity. Changes in membrane potential after malate exposure were more prominent than succinate exposure. When slices were pretreated with ROS scavengers such as phenyl-N-tert-buthylnitrone (PBN), catalase and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), malate-induced depolarization was significantly decreased. Intracellular calcium above 100 µM increased malate-induced depolarization, witch was suppressed by cyclosporin A, a mitochondrial permeability transition (MPT) inhibitor. These results suggest that enhanced production of spinal mitochondrial ROS can induce nociception through central sensitization.

The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the H2O2 or ˙OH radical levels were measured. In vitro fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The H2O2 and ˙OH radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes (p<0.05). During early in vitro culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos (p<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

The present study was conducted to examine the reactive oxygen species (ROS) generation levels and subsequent DNA damage in the bovine cultured somatic cells. Bovine ear skin cells were classified by serum starvation, confluence and cycling cells. Cells were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) or 10 μM hydroxyphenyl fluorescein (HPF) dye to measure the H2O2 or ˙OH radical levels. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each cell. H2O2 and ˙OH radical levels of cultured somatic cells were high in confluence group (7.1±0.7 and 8.4±0.4 pixels/cell, respectively) and significantly low in serum starvation group (4.9±0.4 and 7.0±0.4 pixels/cell, respectively, p<0.05). Comet tail lengths of serum starvation (148.3±5.7 μm) and confluence (151.1±5.0 μm) groups were found to be significantly (p<0.05) increased in comparison to that of cycling group (137.1±7.5 μm). These results suggest that the culture type of donor cells can affect the ROS generation, which leads the DNA fragmentation of the cells.

The study aim is to investigate the free radicals scavenging and spermatogenic potentials, as well as to analyze any reproductive toxicity of ethanolic extract of Mucuna prureins (M. pruriens) Linn. in spermatozoa, under different dosages in normal male rat. Normal rats were randomly selected and suspension of the extract was administered orally at the dosages of 150, 200 and 250 mg/kg body weight of the different groups of male rats (n=6) once in a day for 60 days and grouped as group II, III and IV respectively. Saline treated rats served as control -group I. On the 60th day the animals were sacrificed and the epididymal sperm were subjected to various analyses like level of ROS production, LPO, enzymatic and non enzymatic antioxidant, morphology, morphometry, chromosomal integrity and DNA damage. Results showed significant reduction in ROS production and peroxidation and significant increase in both enzymic and non-enzymic antioxidants in all concentration treated groups when compared with control. Results from all the drug treated groups showed good sperm morphology, increased sperm count and motility. There was no DNA damage and showed normal chromosomal integrity even in 250 mg/kg dose. When compared with control all the three extract treated groups showed increased ROS scavenging activity. However, group II (200 mg/kg) showed significant changes in all the parameters. From the present study it was confirmed that the M. pruriens has potential to improve the sperm qualitatively and quantitatively through scavenging the excess ROS with any adverse side effects. These observations suggest that ethanolic seed extract of M. pruriens may serve as anti-oxidant that can exploit to treat the oxidative stress mediated male factor infertility.

The present study was conducted to examine the reactive oxygen species (ROS) generation levels in porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by the combination of electric stimulus and 6‐ DMAP before in vitro culture. Porcine oocytes and parthenogenetic embryos were stained in 10 μM dichlorohydrofluorescein diacetate (DCF) or 10 μM hydroxyphenyl fluorescein (HPF) dye each for 30 min at 39℃. The fluorescent emissions from the samples were recoded as JPEG file and the intensity of fluorescence in oocytes and embryos were analyzed. H2O2 and ˙OH radical levels of porcine oocytes were reduced immediately after electric stimulation. However, H2O2 and ˙OH radical levels of parthenogenetic embryos were increased with time elapsed after electric stimulation from 0 h to 3 h and after DMAP culture. During in vitro culture, H2O2 and ˙OH radical levels were gradually increased from the one‐cell stage to the two‐ and four‐cell stages. The result of the present study suggests that the ROS was not increased by electric pulse in porcine embryos. Rather than it seems to be associated with the stage of development and the culture condition.

The electron transport chain (ETC) delivers electrons from many substrates to reduce molecular oxygen to water. ETC accomplishes the stepwise transfer of electrons through series of protein complexes conferring oxidation‐reduction reactions with concomitant transport of p roton across membrane, g enerating a proton g radient which leads ATP s ynthesis b y F0F1ATPase. Bacterial ETC initiates with oxidation of NADH by NADH dehydrogenase complex (complex I). Therefore, damage of complex I leads to insufficient function of ETC and accumulation of NADH inside the cell. Contribution of ETC activity and its consequent changes of NADH levels to bacterial damage response against reactive oxygen and nitrogen species (ROS/RNS) has been poorly understood. In this study, by constructing ndh mutant Salmonella lacking complex I NADH dehydrogenase 2, we evaluated the effect of ETC deficiency to bacterial resistance against ROS and RNS. The growth of ndh mutant Salmonella is impaired in the culture media containing hydrogen peroxide, but rather accelerates in the media containing nitric oxide donors. Data suggest that redox potential of NADH accumulated inside the cell by ETC blockage may affect inversely to bacterial resistance against reactive oxygen species and reactive nitrogen species.

Plasminogen activators (PAs) are serine protease that cleave plasminogen to form the active protease plasmin and may participate in mammalian fertilization. Although correlations have been reported between reactive oxygen species (ROS) and sperm function, the relationship between PA activity and ROS is unknown. We determined the effects of ROS on sperm function and PA activities in boar spermatozoa preincubated under the X-XO system. When spermatozoa were treated with the X+XO group, a significant increase (p<0.05) was observed in the percentage of acrosome reacted spermatozoa compared with that of the control group. However, when antioxidants were added to the medium with X+XO, the rate of acrosome reaction tended to decrease. Also, a significantly lower percentage of acrosome reacted spermatozoa was observed in the X+XO+catalase group at 6 hr of incubation compared with that of X+XO group. The density of malondialdehyde (MDA) was higher in the X+XO group than in different treatment groups. In another experiment, incubation of spermatozoa in medium with X+XO was associated with a significant (p<0.05) increase in activity of tPA-PAI and tPA compared with the control group. Antioxidants decreased the increased activity of tPA-PAI and tPA by preincubation in the X-XO system. Also, a significantly lower (p<0.05) activities of tPA-PAI and tPA were observed in the X+XO+catalase group compared with the X+XO group. No significant differences, however, were observed in the activity of uPA. These results suggest that the increase of acrosome reaction by the X-XO system resulted in increase of PAs activity in the sperm incubation medium.

Plasminogen activators (PAs) are serine proteases that convert plasminogen to plasmin. The PA/plasmin system has been associated with a number of physiological processes such as fibrinolysis, ovulation and fertilization. Although correlations have been reported between reactive oxygen species (ROS) and oocyte maturation, the relationship between PA activity and ROS is unknown. The present study was undertaken to determine the effects of cumulus cells on PA activity in matured porcine oocytes under xanthine (X)-xanthine oxidase (XO) system. When oocytes were matured under the X-XO system, the proportion of oocytes remaining GV stage was higher (p<0.05) in oocytes without cumulus cells. The incidence of degenerated oocytes was higher (p<0.05) in the X+XO ( and ) than in the control group ( and ). The proportion of TUNEL-positive oocytes and activity of caspase-3 were higher (p<0.05) in cumulus-free oocytes and oocytes exposed to ROS. Tissue-type plasminogen activator-plasminogen activator inhibitor (tPA-PAI) and tissue-type plasminogen activator (tPA) activity were detected in oocytes that were separated from cumulus-oocytes complexs (COCs) at 44 h of maturation culture, and only tPA was produced in oocytes that were denuded before the onset of maturation culture. On the other hand, the activities of PA were increased (p<0.05) when oocytes were cultured under the X-XO system. The higher activity of tPA was observed in denuded oocytes (DOs) underwent apoptotic changes by oxidative stress. In COCs, however, tPA-PAI as well as tPA activity was detected and apoptotic changes such as DNA cleavage or caspase-3 activation were not observed. These results suggest that tP A may be relevant to apoptotic cell death in porcine oocytes by oxidative stress.

Cordycepin (3’-deoxyadenosin), a polyadenylation specific inhibitor, is the main functional component in Cordyceps militaris which is one of the top three famous traditional Chinese medicine. It has been shown to possess many pharmacological activities including immunologically stimulating, anti-cancer, anti-bacterial, and anti-virus, anti-infection effects. However, its anti-cancer molecular mechanisms are poorly understood. In this study, the apoptotic effects by cordycepin were investigates in human leukemia cells. Treatment of cordycepin significantly inhibited cells growth in a concentrationdependent manner by inducing apoptosis, as evidenced by morphological change and apoptotic cell death such as formation of apoptotic bodies, DNA fragmentation and increased populations of sub-G1. Induction of apoptosis by cordycepin was associated with modulation of Bcl-2 and inhibitor of apoptosis proteins (IAP) family expression. Cordycepin also increased reactive oxygen species (ROS) generation, activation of casepase-3, caspase-8, caspase-9, cleavage of poly(ADP-ribose) polymerase (PARP), β-catenin and phospholipase C (PLC)-γ1 protein. The quenching of ROS generation by N-acetyl-L-cysteine administration, a scavenger of ROS, reversed the cordycepin-induced apoptosis effects. Theresults suggested that cordycepin may be a potential chemotherapeutic agent for the treatment of leukemia patients [This work was supported by Blue-Bio Industry RIC at Dong-Eui University as a RIC (08-06-07) program of ITEP under Ministry of Knowledge Economy].