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The purpose of the present study was to elucidate whether premature senescence contributes to the outcome of radiotherapy (RT) and to validate senescence biomarkers in vitro and in vivo. Cultured human cancer cell lines and xenografted mice were exposed to single (SR; 2, 6 or 12 Gy) or fractionated radiation (FR; 3 x 2 Gy or 6 x 2 Gy), and premature senescence was assessed using senescence-associated β-galactosidase (SA-β-Gal) activity, hypophosphorylation of pRb and p21 accumulation. A variety of senescence-associated biomarkers including cathepsin D (CD), the eukaryotic translation elongation factors eEF1A1, eEF1B2, decoy receptor 2 and Dec1 were further validated in vivo or in vitro. We demonstrated the beneficial tumor suppressive role of ionizing radiation (IR)-induced premature senescence in vitro and in vivo. FR inhibited tumor growth via induction of premature senescence as effectively as an equivalent SR dose (≥6 Gy). In addition, CD and eEF1 were valuable biomarkers of cellular senescence in either SR- or RF-exposed carcinoma cells or xenograft mice. Our results suggest that 2 Gy of a conventional RT regime could achieve a better clinical outcome if premature senescence could be increased through an improved understanding of its molecular action mechanism.

The objective of this study was to investigate the effects of the combined RF radiation (837 MHz CDMA plus 1950 MHz WCDMA) signal on levels of intracellular reactive oxygen species (ROS) in neuronal cells. Exposure of the combined RF signal was conducted at specific absorption rate values of 2 W/kg of CDMA plus 2 W/kg of WCDMA for 2 h. Co-exposure to combined RF radiation with either H2O2 or menadione was also performed. The experimental exposure groups were incubator control, sham-exposed, combined RF radiation-exposed with or without either H2O2 or menadione groups. The intracellular ROS level was measured by flow cytometry using the fluorescent probe dichlorofluorescein diacetate. Intracellular ROS levels were not consistently affected by combined RF radiation exposure alone in a time-dependent manner in U87, PC12 or SH-SY5Y cells. In neuronal cells exposed to combined RF radiation with either H2O2 or menadione, intracellular ROS levels showed no statically significant alteration compared with exposure to menadione or H2O2 alone. These findings indicate that neither combined RF radiation alone nor combined RF radiation with menadione or H2O2 influences the intracellular ROS level in neuronal cells such as U87, PC12 or SH-SY5Y.

An 18-year-old male with a Fontan circulation underwent excision of a pheochromocytoma after conversion from laparoscopic surgery. The pneumoperitoneum established for laparoscopic surgery may have adverse effects on the Fontan circulation, because it increases the intra-abdominal pressure (IAP), intra-thoracic pressure, pulmonary vascular resistance, and systemic vascular resistance (SVR), and decreases cardiac preload and cardiac output. Meticulous monitoring is also required during carbon dioxide exsufflation, because a rapid decrease in IAP can provoke hemodynamic deterioration by decreasing venous return and SVR. Furthermore, catecholamines released by the pheochromocytoma can worsen the hemodynamic status of Fontan circulation during surgery. Therefore, sophisticated intraoperative anesthetic care is required during laparoscopic pheochromocytoma excision in patients with a Fontan circulation.

The contributions of superoxide and nitric oxide to ischemia/reperfusion (I/R)-induced neuropathic pain have previously been demonstrated in an animal model that mimics the symptoms of complex regional pain syndrome type I (CRPS I). Targeting peroxynitrite, which is the product of their interaction, may provide effective treatments for I/R-induced neuropathic pain. In this study, the effect of the peroxynitrite decomposition catalyst FeTMPyP [5,10,15,20-tetrakis (N-methyl-4'-pyridyl)porphyrinato iron (III)], administered at doses of 1, 3 and 10 mg/kg via intraperitoneal injection 30 min prior to reperfusion, was evaluated in rats with chronic post-ischemic pain. The pain behavior of the rats was tested with a von Frey filament. Phosphorylation of N-methyl-D-aspartate (NMDA) receptors in the L4/6 section of the spinal cord was measured on the third day following reperfusion by western blotting. The rats treated with 3 or 10 mg/kg FeTMPyP demonstrated significant increases in their paw withdrawal thresholds and decreased levels of phosphorylated NMDA receptor subunit 1 compared with those of the vehicle group (all P<0.001). These findings suggest that nitrosative stress, specifically that associated with peroxynitrite, may be involved in the mechanical allodynia and central sensitization that are associated with CRPS I and may provide a rationale for CRPS I treatment strategies using peroxynitrite decomposition catalysts.

Background: Clinical outcome after unplanned extubation (UE) in patients admitted to the surgical intensive care unit (SICU) has not been fully investigated. In this study we assessed in-hospital mortality of patients with UE and determined whether UE is a predictor of in-hospital mortality. Finally, we sought to identify predictors of reintubation after UE in mechanically ventilated patients in the SICU., Methods: Medical charts of patients (n = 4,407) admitted to the SICU between October 2007 and December 2011 were reviewed retrospectively., Results: Eighty-five episodes of UE occurred in 81 patients. Patients with UE required emergency surgery more frequently and had higher ICU and hospital mortality rates, reintubation rate, and APACHE II scores and longer mechanical ventilation (MV) and ICU stay than patients without UE (P < 0.05 for all associations). Multivariate analysis revealed that reintubation (odds ratio [95 % confidence interval]: 4.14 [2.58-6.67]; P < 0.001), APACHE II scores (1.14 [1.12-1.17]; P < 0.001), emergency surgery (1.73 [1.18-2.53]; P = 0.005), and chronic neurologic disease (2.11 [1.30-3.41]; P = 0.002) were associated with hospital mortality. Reintubation was necessary in 17 patients. On multivariate analysis, a score on the Richmond Agitation-Sedation Scale (RASS, 0.48 [0.31-0.76]; P = 0.001), PaO2/FiO2 ratio (0.99 [0.99-1.00]; P = 0.048), and MV duration before UE (1.46 [1.08-1.98]; P = 0.014) were independently associated with reintubation after UE., Conclusions: Our results indicated that although patients with UE had high in-hospital mortality, UE was not directly associated with in-hospital mortality. Reintubation, chronic neurologic disease, emergency operation, and higher APACHE II score were related to increased in-hospital mortality. A low RASS score, a low PaO2/FiO2 ratio, and long MV duration before UE were related to reintubation after UE.

This study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from 95 cyclic hens aged up to 54-56 weeks and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions.

Background: Recent research has shown that reactive oxygen species (ROS) play a significant role in the development and persistence of neuropathic pain through central sensitization via N-methyl-D-aspartate (NMDA) receptor activation. In the present study, we examined whether the intraperitoneal administration of vitamins C and E alone or together could alleviate mechanical allodynia in a chronic post-ischemia pain (CPIP) rat model., Methods: Vitamins C and E were administered intraperitoneally to 48 male Sprague Dawley rats once per day for 3 days before hindpaw ischemia-reperfusion (I/R) injury was induced. On the third day, the CPIP rat model was produced by inducing ischemia in the left hindpaw by applying an O-ring for 3 h, followed by reperfusion. Three days after reperfusion, hindpaw mechanical allodynia was assessed by measuring the withdrawal response to von Frey filament stimulation. The rats were sacrificed immediately after behavioral testing to determine the phosphorylated NMDA receptor subunit 1 (pNR1) and extracellular-signal-regulated kinases (pERK) levels in the spinal cord., Results: When the antioxidant vitamins C and E were administered intraperitoneally to CPIP rats, I/R injury-induced mechanical allodynia was attenuated, and pNR1 and pERK levels were decreased in the rat spinal cord. Additionally, the co-administration of both vitamins had an increased antiallodynic effect., Conclusions: The reduced phosphorylated NR1 and ERK levels indicate that vitamins C and E inhibit the modulation of spinal cord neuropathic pain processing. Co-administration of vitamins C and E had a greater antiallodynic effect.

Background: Pain on injection of rocuronium is a common clinical problem. We compared the efficacy of lidocaine, ketorolac, and the 2 in combination as pretreatment for the prevention of rocuronium-induced withdrawal movement., Methods: For this prospective, randomized, placebo-controlled, double-blind study a total of 140 patients were randomly allocated to one of 4 treatment groups to receive intravenously placebo (saline), lidocaine (20 mg), ketorolac (10 mg), or both (n = 35 for each group), with venous occlusion. The tourniquet was released after 2 min and anesthesia was performed using 5 mg/kg thiopental sodium followed by 0.6 mg/kg rocuronium. The withdrawal response was graded on a 4-point scale in a double-blind manner., Results: The overall incidence of withdrawal movements after rocuronium was 34.3% with lidocaine (P = 0.001), 40% with ketorolac (P = 0.004), and 8.6% with both (P < 0.001), compared with 74.3% with placebo. There was a significantly lower incidence of withdrawal movements in patients receiving the lidocaine/ketorolac combination than in those receiving lidocaine or ketorolac alone (P = 0.009 and 0.002, respectively). The incidence of moderate to severe withdrawal movements was 14.3% with lidocaine, 17.2% with ketorolac, and 2.9% with lidocaine/ketorolac combination, as compared to 45.7% with the placebo. There was no significant difference in withdrawal movement between the lidocaine group and the ketorolac group., Conclusions: Ketorolac pretreatment had an effect comparable to that of lidocaine in attenuating rocuronium-induced withdrawal movements and the lidocaine/ketorolac combination pretreatment, compared with lidocaine or ketorolac alone, effectively reduced withdrawal movements during rocuronium injection.

Acute hypotension after reperfusion of the liver graft occurs frequently during liver transplantation. A randomized, prospective trial was performed to test the effects of epinephrine and phenylephrine pretreatments for attenuating postreperfusion syndrome (PRS). Ninety-three adult liver recipients were randomly allocated to receive an intravenous bolus of 10 μg of epinephrine, 100 μg of phenylephrine, or normal saline (the control group) at the time of graft reperfusion. The occurrence of PRS, the use of vasoactive drugs, and the postoperative courses were compared. The epinephrine and phenylephrine groups showed PRS less frequently (39% and 48%) than the control group (77%, P = 0.006) as well as higher mean arterial pressures (MAPs) immediately after reperfusion (P < 0.05). An overshoot of MAP was observed in one-third of the pretreated patients with minimal heart rate changes. Only 2 patients in each pretreatment group showed an increase in MAP that was greater than 20% of the baseline value. The intraoperative epinephrine and dopamine requirements were significantly lower in both pretreatment groups. Perioperative laboratory data, postoperative stays, and in-hospital mortality rates were similar for the 3 groups. In conclusion, pretreatment with 10 μg of epinephrine or 100 μg of phenylephrine significantly reduces the occurrence of PRS and vasopressor requirements without immediate or delayed adverse effects in adult liver transplantation., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA-induced silencing complex (RISC), with target p21 mRNA via binding of the stem-loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA-mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA-mediated gene silencing. In addition, these findings indicate that fine-tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence.

Cellular senescence is a physiological program of irreversible growth arrest that is considered to play an important role in tumor suppression. Recent studies demonstrated that senescent cells secrete multiple growth regulatory proteins that could alter the behavior of neighboring cells. In this study, we investigated the effect of secretory proteins from ionizing radiation (IR) induced senescent tumor cells on normal and tumor cells. Conditioned medium (CM) from IR-induced senescent MCF7 cells significantly increased cell proliferation, invasion, migration, and wound healing activity in MCF7 cells and HUVECs. Comparative proteomics analysis revealed 24 differentially secreted protein spots including Raf kinase inhibitor protein (RKIP), α-Enolase, AKAP9, and MARK4, and the findings were confirmed by Western blot analysis of IR-induced senescent cancer cells. We found that RKIP was secreted via the classical pathway, and the transfection of small interfering RNA against RKIP suppressed CM-induced migration in MCF7 cells. Treatment with recombinant human RKIP increased the migratory activity of MCF7 cells. Taken together, our results demonstrate that the senescence-associated secretory protein RKIP could be the principal target to prevent the potential effects of the secretome from IR-induced senescent tumor cells on neighboring cell migration., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The chemical reaction of carbon dioxide absorbent and sevoflurane is known to produce compound A. However, carbon dioxide absorbents are not controlled by the Food and Drug Administration, but are treated as industrial products in some nations. Moreover, carbon dioxide absorbents differ in their capacities to produce compound A, because their chemical compositions differ. In this study, we compared the renal safety between carbon dioxide absorbent products in patients under sevoflurane anesthesia., Methods: Eighty patients with no preexisting renal disease undergoing elective gynecologic surgery were randomly assigned to receive sevoflurane or isoflurane anesthesia with one of four carbon dioxide absorbent products (Sodasorblime®, Sodalyme®, Sodasorb®, Spherasorb®) at the same fresh gas flow of 2 L/min. The renal safety was evaluated by changes of blood urea nitrogen (BUN), creatinine and urine N-acetyl-b-glucoseaminidase (NAG)-creatinine ratio at 24 hours and 72 hours after surgery from preoperative level., Results: There was no significant difference in the renal safety indicators between carbon dioxide absorbents during sevoflurane anesthesia (P > 0.05). However, the BUN and urine NAG-creatinine ratios at 72 hours after surgery were higher in isoflurane anesthesia in some carbon dioxide absorbent groups (P = 0.03 and 0.04, respectively)., Conclusions: We could not find significant differences of renal safety indicators with carbon dioxide absorbents. Although the adverse effect of carbon dioxide absorbents on renal function was not proved, consideration should be given to their contol by the regulation on their efficacy and safety because carbon dioxide absorbents can produce compound A.

Egg formation and embryonic development occur as the yolk passes through the magnum, isthmus, and shell gland of the oviduct before oviposition in hens. The present study identified candidate genes associated with secretory function of the chicken oviduct after ovulation and contributing to egg formation and oviposition. Hens (n = 5 per time point) were euthanized to recover the reproductive tract when the egg was in the magnum (3 h after ovulation) and the shell gland (20 h after ovulation). Total RNA was extracted from each segment of the oviducts and subjected to Affymetrix chicken GeneChip analysis. Quantitative PCR and in situ hybridization analyses of selected genes confirmed the validity of the gene expression patterns detected using microarray analysis. In particular, ACP1, CALB1, CYP26A1, PENK, RCAN1 and SPP1 expression increased significantly in the shell gland between 3 h and 20 h postovulation, whereas only RCNA1 expression increased significantly in the magnum between 3 h and 20 h postovulation. Results of the high-throughput analysis revealed cell-specific and temporal changes in gene expression in the oviduct at 3 h and 20 h postovulation in laying hens provide novel insight into changes at the molecular and cellular levels of candidate genes related to formation of the egg and oviposition.

Bupivacaine inhibits cardiac conduction and contractility. Insulin enhances cardiac repolarization and myocardial contractility. We hypothesizes that insulin therapy would be effective in resuscitating bupivacaine-induced cardiac toxicity in rabbits. Twelve rabbits were tracheally intubated and midline sternotomy was performed under general anesthesia. Cardiovascular collapse (CVC) was induced by an IV bolus injection of bupivacaine 10 mg/kg. The rabbits were treated with either saline (control) or insulin injection, administered as a 2 U/kg bolus. Internal cardiac massage was performed until the return of spontaneous circulation (ROSC) and the time to the return of sinus rhythm (ROSR) was also noted in both groups. Arterial blood pressure, and electrocardiography were continuously monitored for 30 min and plasma bupivacaine concentrations at every 5 min. The ROSC, ROSR and normalization of QRS duration were attained faster in the insulin-treated group than in the control group. At the ROSC, there was a significant difference in bupivacaine concentration between two groups. Insulin facilitates the return of myocardial contractility and conduction from bupivacaine-induced CVC in rabbits. However, recovery of cardiac conduction is dependent mainly on the change of plasma bupivacaine concentrations.

The aim of this study was to determine whether extremely low frequency magnetic fields (ELF-MF) could affect intracellular reactive oxygen species (ROS) levels and antioxidant enzyme activity. After MCF10A human breast epithelial cells were exposed to 1 mT of 60 Hz ELF-MF for 4 hours, intracellular ROS level, superoxide dismutase (SOD) activity, and reduced to oxidized glutathione (GSH/GSSG) ratio were measured. The cells exposed to ELF-MF did not evidence statistically significant changes in the above-mentioned biological parameters as compared to either the incubator controls or sham-exposed cells. By way of contrast, the IR-exposed cells exhibited marked changes in ROS level, SOD activity, and GSH/GSSG ratio. When we assessed morphological changes and senescence-associated beta-galactosidase (SA-β-Gal) activity, only the IR-exposed cells were positive. According to our results, it could be concluded that ELF-MF has no effect on intracellular ROS level, SOD activity, and GSH/GSSG ratio under our exposure condition.

Purpose: This study aimed to compare the incidence and clinical significance of transient versus persistent acute kidney injury (AKI) on acute ST elevation myocardial infarction (STEMI)., Materials and Methods: The study was a retrospective cohort of 855 patients with STEMI. AKI was defined as an increase of ≥0.3 mg/dL in creatinine level at any point during hospital stay. The study population was classified into 5 groups: 1) patients without AKI; 2) patients with mild AKI that was resolved by discharge (creatinine change less than 0.5mg/dL compared with admission creatinine during hospital stay, transient mild AKI); 3) patients with mild AKI that did not resolve by discharge (persistent mild AKI); 4) patients with moderate/severe AKI that was resolved by discharge (creatinine change more than 0.5 mg/dL compared with admission creatinine, transient moderate/severe AKI); 5) patients with moderate/ severe AKI that did not resolve by discharge (persistent moderate/severe AKI). We investigated 1-year all-cause mortality after hospital discharge for the primary outcome of the study. The relation between AKI and 1-year mortality after STEMI was analyzed., Results: AKI occurred in 74 (8.7%) patients during hospital stay. Adjusted hazard ratio for mortality was 3.139 (95% CI 0.764 to 12.897, p=0.113) in patients with transient, mild AKI, and 8.885 (95% CI 2.710 to 29.128, p<0.001) in patients with transient, moderate/severe AKI compared to patients without AKI. Persistent moderate/severe AKI was also independent predictor of 1 year mortality (hazard ratio, 5.885; 95% CI 1.079 to 32.101, p=0.041)., Conclusion: Transient and persistent moderate/severe AKI during acute myocardial infarction is strongly related to 1-year all cause mortality after STEMI.

MicroRNAs (miRNAs) play a critical role in determining the differentiation fate of pluripotent stem cells and germ cells in mammals. However, the mechanism(s) of miRNA-mediated posttranscriptional regulation with regard to lineage specification and differentiation in chick development require further investigation. Therefore, we conducted miRNA expression profiling to explore specific miRNA signatures in undifferentiated blastoderm and primordial germ cells (PGCs). We identified seven miRNAs that are highly expressed in blastoderm and 10 that are highly expressed in PGCs. In this study, miR-302a and miR-456 for blastoderm and miR-181a* for PGCs were analyzed further for their target transcripts and regulatory pathways. Both miR-302a and miR-456 bound directly to the sex-determining region Y box 11 transcript and could act as posttranscriptional coregulators to maintain the undifferentiated state of the chicken blastoderm through the suppression of somatic gene expression and differentiation. Moreover, miR-181a* showed a bifunctional role in PGCs by binding to two different transcripts. miR-181a* inhibited the somatic differentiation of PGCs by silencing homeobox A1 expression. Additionally, miR-181a* prevented PGCs from entering meiosis through the repression of the nuclear receptor subfamily 6, group A, member 1 transcript. Collectively, our data demonstrate that in chickens miRNAs intrinsically regulate the differentiation fate of blastoderms and PGCs and that the specific timing of germ cell meiosis is controlled through miRNA expression.

In the present study, we investigated the effects of gallic acid (GA) (3,4,5-trihydroxybenzoic acid), a polyhydroxyphenolic compound, isolated from Rhus chinensis, on the human monocytic lymphoma cell line U937. In vitro experiments showed that treating U937 cells with various amounts of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA induces apoptosis, we examined the gene expression of p53, nuclear factor κB (NF-κB), and inhibitor of NF-κB (I-κB) after treating the cells with GA and found that expression levels of the genes for p53 and NF-κB increased and that for I-κB decreased. The results obtained from western blotting with U937 cells showed up-regulation of NF-κB protein and down-regulation of proliferating cell nuclear antigen and I-κB protein. These results demonstrate that GA efficiently induces apoptosis in U937 cells and that GA is a potential chemotherapeutic agent against lymphoma.

Interspecific hybrids provide insights into fundamental genetic principles, and may prove useful for biotechnological applications and as tools for the conservation of endangered species. In the present study, interspecies hybrids were generated between the Korean ring-necked pheasant (Phasianus colchicus) and the White Leghorn chicken (Gallus gallus domesticus). We determined whether these hybrids were good recipients for the production of germline chimeric birds. PCR-based species-specific amplification and karyotype analyses showed that the hybrids inherited genetic material from both parents. Evaluation of biological function indicated that the growth rates of hybrids during the exponential phase (body weight/week) were similar to those of the pheasant but not the chicken, and that the incubation period for hatching was significantly different from that of both parents. Primordial germ cells (PGCs) of hybrids reacted with a pheasant PGC-specific antibody and circulated normally in blood vessels. The peak time of hybrid PGC migration was equivalent to that of the pheasant. In late embryonic stages, germ cells were detected by the QCR1 antibody on 15 d male gonads and were normally localized in the seminiferous cords. We examined the migration ability and developmental localization of exogenous PGCs transferred into the blood vessels of 63 h hybrid embryos. Donor-derived PGCs reacted with a donor-specific antibody were detected on 7 d gonads and the seminiferous tubules of hatchlings. Therefore, germ cell transfer into developing embryos of an interspecies hybrid can be efficiently used for the conservation of threatened animals and endangered species, and many biotechnological applications., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We encountered a case of a rupture of an endotracheal tube cuff during robot-assisted thyroid surgery in a 35-year-old male patient. Two hours after commencing surgery, the bellows of the ventilator were not filled and a rupture of the endotracheal tube cuff was suspected. Once the robot-manipulator is engaged, the position of the operating table cannot be altered without removing it from the patient. Reintubation with direct laryngoscopy was performed with difficulty in the narrow space between the patient's head and robot-manipulator without moving the robot away from the patient. The rupture of the endotracheal tube cuff was confirmed by observing air bubbles exiting from the balloon in water. The patient was discharged 3 days after surgery without complications. In robot-assisted thyroid surgery, a preoperative arrangement of the robot away from the patient's head to obtain easy access to the patient is essential for safe anesthetic care.

The production of chimeric birds is an important tool for the investigation of vertebrate development, the conservation of endangered birds, and the development of various biotechnological applications. This study examined whether gamma (γ)-irradiation depletes endogenous primordial germ cells and enhances the efficiency of somatic chimerism in chickens. An optimal irradiation protocol for stage X embryos was determined after irradiation at various doses (0, 100, 300, 500, 600, 700, and 2,000 rad). Exposure to 500 rad of γ-irradiation for 73 s significantly decreased the number of primordial germ cells (P < 0.0001). Somatic chimera hatchlings were then produced by transferring blastodermal cells from a Korean Oge into either an irradiated (at 500 rad) or intact stage X White Leghorn embryo. An analysis of feather color pattern and polymerase chain reaction-based species-specific amplification of various tissues of the hatchlings confirmed chimerism in most organs of the chick produced from the irradiated recipient; a lesser degree of chimerism was observed in the non-irradiated control recipient. In conclusion, the exposure of chick embryos to an optimized dose of γ-irradiation effectively depleted germ cells and yielded greater somatic chimerism than non-irradiated control embryos. This technique can be applied to interspecies reproduction or the production of transgenic birds.

Imperfect base-pairing between microRNA (miRNA) and the 3'-untranslated region of target messenger RNA (mRNA) triggers translational repression of the target mRNA. Here, we provide evidence that human Argonaute 2 targets cap-binding protein (CBP)80/20-bound mRNAs and exon junction complex-bound mRNAs and inhibits nonsense-mediated mRNA decay (NMD), which is restricted tightly to CBP80/20-bound mRNAs. Furthermore, microarray analyses reveal that a subset of cellular transcripts, which are expected to be targeted for NMD, is stabilized by miRNA-mediated gene silencing. The regulation of NMD by miRNAs will shed light on a new post-transcriptional regulation mechanism of gene expression in mammalian cells.

Nuclear receptor subfamily 6, group A, member 1 (NR6A1), also known as germ cell nuclear factor/retinoid receptor-related testis-associated receptor and neuronal cell nuclear factor, is a member of the nuclear orphan receptor superfamily. NR6A1 has been cloned in various species including humans and mice, but it has been scarcely investigated in avian species. In the present study, we cloned the chicken NR6A1 (cNR6A1) from a testis cDNA library. The cloned cNR6A1 sequence was mapped to chromosome 17 and contained an open reading frame of 1.4 kb encoding 445 amino acids. Multiple alignment analysis of the cNR6A1 protein-coding sequence with NR6A1s from humans, mice, boars, rats, zebrafish, and Xenopus showed high degrees of homology, 89%, 90%, 89%, 88%, 83%, and 87%, respectively. Using RNA interference, changes in the expression of pluripotency-, germ cell-, and differentiation-related key genes by silencing of cNR6A1 were validated in chicken blastoderm-derived embryonic stem cells. Among those genes, the relative expression levels of POU5F1, CRIPTO, DAZL, DDX4, BMP15, GSC, and SOX7 changed significantly compared to the control group. We also confirmed that the activity of alkaline phosphatase, known as a pluripotency marker, was maintained by cNR6A1 gene silencing in chicken blastodermal cells. Collectively, our data suggest that cNR6A1 may play an important role during chicken embryonic development and differentiation.

Human transforming growth factor-beta receptor type 2 (TGFbetaR2) mRNA harboring a premature translation termination codon (PTC) generated by frameshift mutation is targeted for nonsense-mediated translational repression (NMTR), rather than nonsense-mediated mRNA decay (NMD). Here we show that exon junction complex (EJC) downstream of a PTC plays an inhibitory role in translation of TGFbetaR2 mRNA. Translational repression by core EJC components occurs after formation of 80S ribosome complex, which is demonstrated using different types of internal ribosome entry sites (IRESes). Our findings implicate EJCs or core EJC components as negative regulators of translation., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Translation of spliced mRNAs is enhanced by exon junction complex (EJC), which is deposited on mRNAs as a result of splicing. Although this phenomenon itself is well known, the underlying molecular mechanism remains poorly understood. Here we show, using siRNAs against Y14 and eIF4AIII and spliced or intronless constructs that contain different types of internal ribosome entry sites (IRESes), that Y14 and eIF4AIII increase translation of spliced mRNAs before and after formation of the 80S ribosome complex, respectively. These results suggest that EJC modulates translation of spliced mRNA at multiple steps.

Although increased liver iron in individuals with chronic hepatitis C virus (HCV) is associated with a poor response to interferon therapy, the underlying molecular mechanisms are poorly understood. In this study, we show that iron enhances the translation initiation mediated by the internal ribosome entry site (IRES) of HCV. We also demonstrate by UV cross-linking analysis that specific cellular proteins bind to HCV 5' untranslated region (5' UTR) in an iron-dependent manner. Notably, p85 and p110 are competed out for their binding to HCV 5' UTR when excess amounts of iron-responsive element (IRE) competitor RNAs are treated. This indicates that at least these two factors are common proteins for binding to HCV 5' UTR and IRE. Our results, taken together, suggest that intracellular iron modulates the iron sensing pathway and HCV IRES-dependent translation by changing the binding affinities of the common cellular factors to IRE and HCV IRES, respectively. As a consequence, the coordinated regulation of gene expression by intracellular iron could provide favorable conditions for HCV proliferation.

Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism; this process removes faulty mRNAs harboring premature termination codons (PTCs). NMD targets newly synthesized mRNAs bound by nuclear cap-binding proteins 80/20 (CBP80/20) and exon junction complex (EJC), the former of which is thought to recruit the ribosome to initiate the pioneer round of translation. After completion of the pioneer round of translation, CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E, which mediates steady-state translation in the cytoplasm. Here, we show that overexpression of eIF4E-T preferentially inhibits cap-dependent steady-state translation, but not the pioneer round of translation. We also demonstrate that overexpression of eIF4E-T or Dcp1a triggers the movement of eIF4E into the processing bodies. These results suggest that the pioneer round of translation differs from steady-state translation in terms of ribosome recruitment.

Small heterodimer partner (SHP) is an atypical member of nuclear receptor superfamily that lacks a DNA binding domain. Here, we show that SHP expression increases during monocytic differentiaton with exposure HL-60 leukemia cells to a 12-O-tetradecanoylphorbol-13-acetate (TPA) response element, whose treatment induced the SHP promoter activity dependent on c-Jun expression, which is well known to be involved in the commitment step in the TPA-induced differentiation of HL-60 leukemia cells. We also show that overexpression and activation signaling of c-Jun increase the SHP promoter activity, suggesting that the level of SHP expression is normally limiting for c-Jun-dependent monocytic differentiation. Electrophoretic mobility shift assays using oligonucleotides derived from the SHP promoter reveal that c-Jun exhibit TPA-induced DNA binding, providing a mechanism for the transcriptional activation of SHP gene expression. It was also found that overexpression of SHP and c-Jun greatly facilitated monocytic differentiation by TPA and surprisingly, that expression of SHP or c-Jun alone was sufficient to make cells differentiate into functionally mature monocytes, but silencing of SHP and c-Jun by RNA interference diminished the TPA-induced monocytic differentiation. Taken together, these works suggest that c-Jun works to activate the expression of SHP genes associated with the cascade regulation of monocytic differentiation.

beta-Catenin is a known regulator of cell-cell adhesion and transcriptional regulation. However, the role of beta-catenin and its regulation in non-adherent cells has not been examined. Therefore, we examined the role and fate of beta-catenin during hematopoietic cell apoptosis using Jurkat T-acute lymphoblastic and U937 acute myeloblastic leukemia cells. The results presented here demonstrate that the treatment of Jurkat cells with the apoptosis inducers anti-Fas, TRAIL, staurosporine, and etoposide induces proteolytic fragments of beta-catenin, as did TRAIL and staurosporine in U937 cells. In Jurkat cells, beta-catenin was cleaved at both the N- and C-terminal after anti-Fas addition. Cleavage of intact beta-catenin was completely inhibited by caspase selective protease inhibitors. There was a clear accumulation of the large proteolytic fragment in Jurkat cells treated with lactacystin or N-acetyl-leucyl leucyl-methioninal (ALLM). These results suggest that both the proteasome and calpain may recognize the large beta-catenin fragment as a substrate for further degradation. Densitometric analysis demonstrated that the loss of intact beta-catenin was more rapid in the cell nucleus (beta-catenin T1/2 of approximately 1.5h in cytoplasm and 0.5h in nucleus). Down-regulation of beta-catenin-associated transcription was an early event in response to anti-Fas. These results suggest that beta-catenin plays a role in promoting Jurkat survival.

Protein-tyrosine phosphatases (PTPs) constitute a family of receptor-like, and cytoplasmic enzymes, which catalyze the dephosphorylation of phosphotyrosine residues in a variety of receptors and signaling molecules. Together with protein tyrosine kinases (PTKs), PTPs are critically involved in regulating many cellular signaling processes. In this study, diverse compounds were screened for PTP inhibition and selectively screened for inhibitors with the end product inhibition properties. Among phosphate analogues and their derivatives for PTP inhibition, Keggin compounds phosphomolybdate (PM) and phosphotungstate (PT) strongly inhibited both PTP-1B and SHP-1, with K(i) values of 0.06-1.2 micromM in the presence of EDTA. Unlike the vanadium compounds, inhibition potencies of PM and PT were not significantly affected by EDTA. PM and PT were potent, competitive inhibitors for PTPs, but relatively poor inhibitors of Ser/Thr phosphatase. Interestingly, PM and PT did not inhibit alkaline phosphatase at all. The crystal structure of PTP-1B in complex with PM, at 2.0 A resolution, reveals that MoO(3), derived from PM by hydrolysis, binds at the active site. The molybdenium atom of the inhibitor is coordinated with six ligands: three oxo-ligands, two apical water molecules and a S atom of the catalytic cysteine residue. In support of the crystallographic finding, we observed that molybdenium oxides (MoO(3), MoO(2), and MoO(2)Cl(2)) inhibited PTP-1B with IC(50) in the range 5-15 micromM.

The effects of the subchronic administration of Panax ginseng extracts were examined on the hepatic cytochrome P450-dependent monooxygenase system of guinea pigs pre-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Panax ginseng extracts were intraperitoneally administered to guinea pigs at 100 mg/kg/day for 14 days from 1 week after a single intraperitoneal injection of 1 microg of TCDD/kg of body weight. TCDD treatment increased the total cytochrome P450 content 2.86-fold, and this was remarkably inhibited by the administration of Panax ginseng extracts. Treatment with ginseng extract alone also decreased the contents of cytochrome P450 by 33%, but both TCDD and ginseng extracts had no effect on cytochrome b(5) content. The administration of TCDD resulted in a 1.73-fold increase in microsomal NADPH-cytochrome P450 reductase activity in the guinea pig liver, and this was significantly inhibited by ginseng extracts, but treatment with ginseng extracts alone had no effect on its activity, and no statistical changes in the activity of NADPH-cytochrome b(5) reductase were observed in guinea pig liver due to TCDD and/or ginseng extract administration. Compared to the control, ECOD activity remarkably (1.76-fold) increased after TCDD administration, but this increase was completely inhibited by treatment with ginseng extract. Treatment with ginseng extract alone resulted in a 50% reduction of ECOD activity. TCDD administration remarkably induced benzphetamine demethylation (BPDM) activity, while ginseng extract also slightly increased the enzyme's activity, but the induction attributed to ginseng extracts was not statistically significant. Even though administration of ginseng extracts slightly inhibited TCDD-induced BPDM activity, the inhibition was not statistically significant. These results indicate that ginseng extract exerts different effect on the induction of P450 isozymes. From these results, we suggest that Panax ginseng extracts may act as an inhibitor of CYP1A rather than that of CYP2B.