P<0.05, respectively). Delayed treatment with magnolol (100 mg/kg) also protected against ischemic brain damage and improved neurobehavioral scores, even when administered up to 4 hrs post-insult . Additionally, magnolol (0.1 µM) effectively attenuated the rises of intracellular Ca2+ levels, [Ca2+](i), in cultured neurons exposed to glutamate. Consequently, magnolol (0.1–1 µM) significantly attenuated glutamate-induced cytotoxicity and cell swelling (P<0.05). Thus, magnolol offers neuroprotection against permanent focal cerebral ischemia with a therapeutic window of 4 hrs. This neuroprotection may be, partly, mediated by its ability to limit the glutamate-induced excitotoxicity.Neuroprotective efficacy of magnolol, 5,5′-dially-2,2′-dihydroxydiphenyl, was investigated in a model of stroke and cultured neurons exposed to glutamate-induced excitotoxicity. Rats were subjected to permanent middle cerebral artery occlusion (pMCAO). Magnolol or vehicle was administered intraperitoneally, at 1 hr pre-insult or 1–6 hrs post-insult. Brain infarction was measured upon sacrifice. Relative to controls, animals pre-treated with magnolol (50–200 mg/kg) had significant infarct volume reductions by 30.9–37.8% and improved neurobehavioral outcomes ( Additionally, we investigated neuroprotective efficacy of magnolol in rats subjected to permanent focal cerebral ischemia.In the study, we evaluated the protective effects of magnolol against cell damage and swelling as well as increased inflow of [Ca50 value was 129.3±0.1 µM  induced abrupt rises of [Ca2+](i) levels up to ≈1000 nM. Magnolol, however, exhibited a hormetic inhibitory response. Only magnolol at 0.1 µM, but not at 0.01 and 1 µM, effectively inhibited the increased [Ca2+](i) inflow over time . The other physiological parameters were kept within normal limits and did not differ significantly among experiment groups, except that high-dose (200 mg/kg) magnolol-treated animals had arterial pCO2 retention along with reduced heart rate and arterial pH  died prior to completing the protocol following pMCAO and were excluded: 4 (8.2%) were in the vehicle-treated groups and 5 (5.4%) were in the magnolol-treated groups. Following the ischemic onset, the ipsilateral LCBP declined to 14–22% and 32–38% of the baseline data in the ischemic core and penumbral areas, respectively. The LCBP was not significantly different among experiment groups, and was independent of magnolol treatments  when compared with controls (n = 15). Infarction lesions were reduced by 30.9, 33.8, 37.8 and 35.3%, in animals treated with magnolol at 50 mg/kg, 100 mg/kg, 150 mg/kg, and 200 mg/kg, respectively , 100 mg/kg (n = 8), 150 mg/kg (n = 9), or 200 mg/kg (n = 12), but not at 25 mg/kg (n = 13), 1 hr before the ischemic onset, showed significant infarct size reductions , and effectively reduced post-ischemic body weight loss, when administered up to 2 hrs post-insult (P<0.05), but did not affect post-ischemic motor scores  resulted in significant infarct volume reductions when administrated within 4 hrs after the ischemic onset P<0.05; . Relativ P>0.05; . The phy2+](i) inflow and neuronal swelling. This neuroprotection cannot be accounted for by changes in glucose, hemodilution (as measured by blood hematocrit), or differences in mean arterial blood pressure, since these were not significantly different when compared between vehicle-injected and magnolol-treated animals. The changed physiologic parameters were decreases in arterial pH and heart rate, associated with a rise in pCO2, seen in the animals treated with magnolol at 200 mg/kg. These findings suggested that magnolol (200 mg/kg) might have induced a cardiopulmonary suppression, probably due to its centrally-acting muscular relaxant effect Our results indicated that magnolol (50–200 mg/kg) reduced infarct volumes and improved neurobehavioral outcomes in rats subjected to permanent focal cerebral ischemia. Additionally, we found that magnolol (100 mg/kg) was effective in reducing brain infarction and improving neurobehavioral outcomes even when administrated up to 4 hrs post-insult. Moreover, we demonstrated that magnolol not only effectively attenuated both glutamate- and NMDA-induced neurotoxicity, but also reduced the glutamate-induced increases in the [Ca2+](i) remains to be elucidated in vitro dosing response might not represent the trend of dosing response observed in vivoExactly by which mechanisms in the glutamate-stimulated cultured neurons the dose-responsive regimen seen with magnolol for cell swelling inhibition was inconsistent with the “U-shaped” hormetic response observed for inhibiting the rises of (i) were measured on a single cell fluorimeter 2PO4, 0.4; Na2HPO4, 1.25; CaCl2, 2.2; MgSO4, 2; glucose, 10; HEPES, 10, pH 7.3) for 30 min, followed by incubation in dye-free standard buffer for 30 min and, then, the addition of vehicle or magnolol  for 20 min and the exposure of glutamate (300 µM). During experiments, standard buffer was replaced by low Mg2+ saline . The glass coverslip was placed into the stage chamber of an Olympus IX71 inverted microscope, equipped with a 75 W xenon illumination system, a cooled charge-couple device (CCD) camera  coupled to an image intensifier , a Lambda 10-2 filter-wheel and shutter  and a computerized image analyzer . The cells were alternatively illuminated with the light of 340 and 380 nm wavelengths and the emitted light was passed through a 510 nm barrier filter. The 340 and 380 nm images were captured at 6 second intervals and the ratio signals (340 nm excited image/380 nm excited image) were processed and examined for real changes in [Ca2+](i). Approximately 10 neurons in each microscopic field were individually measured. The [Ca2+](i) level was calculated by using the equation: [Ca2+](i)  =  Kd×(Fo/Fs)×[(R−Rmin)/(Rmax−R)] where Kd is the dissociation constant for fura -2 in the cytosol (225 nM), and Fo/Fs is the fluorescence emitted at 380 nm excitation at minimum Ca2+ level divided by the same emission fluorescence at the fura-saturated concentration 2+ and the fura-saturated Ca2+ concentrations, respectively. We used the Fura-2 Calcium Imaging Calibration Kit  to detect the Kd level under conditions. Measurements of Fo and Rmin were performed in nominally Ca2+-free isotonic solution containing 10 mM EGTA. Cells were then superfused with isotonic solution containing 1 µM thapsigargin, 10 µM ionomycin and 10 mM Ca2+ to evaluate Fs and Rmax.The level of [CaThe glutamate (300 µM)-induced neuronal morphologic changes were measured by time-lapse imaging techniques in a microscope equipped with a thermo-controllable heating stage, differential interference contrast (DIC) lens and an image analyzer (MCID Elite) by the method described previously 2O/30% O2. During surgery, body temperature was maintained at 37±0.5°C using a thermostatically controlled heating blanket and rectal probe . The right femoral artery was cannulated for measuring arterial blood gases, glucose, hematocrit and blood pressure Male Sprague-Dawley rats, weighting 220–270 g, were supplied by the University Laboratory Animal Center, and were allowed free access to food and water before and after surgery. Animals were anesthetized with 1–2% halothane in 70% N2, Vasamedics, St. Paul, MN) Focal cerebral ischemia was employed by permanent occlusion of the proximal right middle cerebral artery (pMCAO) with a 4-0 nylon suture occluder, as described previously In the first series of experiments, animals were received either magnolol  or vehicle , 1 hr pre-insult, to test the neuroprotective dose response. An additional set of rats, received magnolol  or vehicle  at 1, 2, 4 or 6 hrs post-insult, was used to evaluate the therapeutic window of opportunity.Neurologic and body weight measurements were conducted by an investigator unaware of treatment protocol at 24 hrs post-insult 3). Brain Infarct volumes were expressed as a percentage of the contralateral hemisphere volume Sacrifice was performed at 24 hrs post-insult by decapitation under anesthesia. The brain was cut into 2 mm coronal sections using a rat brain matrix  and stained according to standard 2, 3, 5-triphenyltetrazolium chloride (TTC) method t test was used to evaluate the response to a change in conditions, and unpaired Students’ t test/one-way analysis of variance (one-way ANOVA) with Fisher’s protected least significant difference (LSD) posthoc comparison was used to evaluate differences between groups. Neurobehavioral scores were analyzed by the Kruskal-Wallis/Mann-Whitney U test. P<0.05 was selected for statistical significance.All data were expressed as the mean±standard deviation (S.D.). Paired Students’


Studies have indicated expression of KCNQ channels in airway smooth muscle (ASM), a tissue that is predominantly regulated by muscarinic receptor signaling. Therefore, we investigated the function of KCNQ channels in rodent ASM and their interplay with Gq/11-coupled M3 muscarinic receptors. Perforated-patch clamp of dissociated ASM cells detected a K+ current inhibited by the KCNQ antagonist, XE991, and augmented by the specific agonist, flupirtine. KCNQ channels begin to activate at voltages near resting potentials for ASM cells, and indeed XE991 depolarized resting membrane potentials. Muscarinic receptor activation inhibited KCNQ current weakly (~20%) at concentrations half-maximal for contractions. Thus, we were surprised to see that KCNQ had no affect on membrane voltage or muscle contractility following muscarinic activation. Further, M3 receptor-specific antagonist J104129 fumarate alone did not reveal KCNQ effects on muscarinic evoked depolarization or contractility. However, a role for KCNQ channels was revealed when BK-K+ channel activities are reduced. While KCNQ channels do control resting potentials, they appear to play a redundant role with BK calcium-activated K+ channels during ASM muscarinic signaling. In contrast to effect of antagonist, we observe that KCNQ agonist flupirtine caused a significant hyperpolarization and reduced contraction in vitro irrespective of muscarinic activation. Using non-invasive whole animal plethysmography, the clinically approved KCNQ agonist retigabine caused a transient reduction in indexes of airway resistance in both wild type and BK β1 knockout (KO) mice treated with the muscarinic agonist. These findings indicate that KCNQ channels can be recruited via agonists to oppose muscarinic evoked contractions and may be of therapeutic value as bronchodilators.KCNQ (K K+ channel agonists may be useful as bronchodilators for asthma since ASM hyperpolarization by K+ channel openers can partly relax ASM  K+ channels  K+ channels in control of contraction of various smooth muscle cell types  following cleavage by phospholipase C (PLC); in addition, stimulation of a number of other PLC-coupled receptors also modulate KCNQ channels via multiple intracellular mechanisms  acts in those cells, at least in part, by modulation of KCNQ channel activity, however, this hypothesis remains to be tested. Moreover, a number of KCNQ openers have been developed that were first targeted as anti-epileptics . We use KCNQ channel pharmacology to evaluate the functional consequences of KCNQ currents on voltage and contraction of rodent ASM. We also utilize BK channel β1 knockout (KO) mice to understand the complementary role of BK and KCNQ channels in ASM.Here, we address the role of KCNQ channels in control of membrane voltage and contractions of ASM. Whereas KCNQ channels have been shown to moderate muscarinic agonist-evoked contractions of ASM  and maintained as homozygous lines. Control animals used in these studies were 2–3 month old C57BL/6J mice strain from Jackson Labs, or 2–3 week old rats (Sprague Dawley) from Charles River Labs. For tracheal constriction studies we used previously published protocols , at 37°C. Resting tension was continuously readjusted to 10 mN for 1 h and then challenged with 67 mM K+ PSS twice or more until reproducible contraction responses were achieved. Subsequent experimental challenges with drugs were normalized to the constriction response to the 67 mM K+ PSS solution. In 67 mM K+ PSS, the K+ reversal potential is depolarized and therefore K+ currents are unlikely to play a role in controlling membrane potential and contraction tone. For experiments that involve two muscarinic challenges, we found that the second challenge does not show significant fatigue . On average, the second challenge shows less than 1% reduction in response from that of first challenge for WT and β1 KO trachea, respectively. Normal PSS used was (mM) 119 NaCl, 4.7 KCl, 2.0 CaCl2, 1.0 KH2PO4, 1.17 MgSO4, 18 NaHCO3, 0.026 EDTA, 11 glucose, and 12.5 sucrose. The pH of the solution was adjusted to 7.35 by a 95% O2 - 5% CO2 mixture. The 67 mM K+ PSS utilized reduced sodium (56.7 mM NaCl) to maintain proper osmolarity. “Low K+” PSS solution was used to measure contractions in relatively hyperpolarized conditions. Low K+ PSS consisted of 1.0 mM K+ derived from the KH2PO4 in the PSS solution, with no added KCl. Normal PSS has a total 5.7 mM K+ derived from 4.7 mM KCl plus 1.0 mM KH2PO4. All other ingredients were unchanged.The BK channel β2+-free HEPES-buffered Krebs solution . After addition of 2.5 U/ml papain (Worthington), 1 mg/ml BSA fraction V, and 1 mg/ml dithiothreitol, TSM tissue was agitated at 37°C on a shaking platform (250 moves/min) for 20 min. Tissue was washed once with the Ca2+-free Krebs solution and digested with 12.5 U/ml of type VII collagenase  for 10 min on a rocking platform at 37°C. Digested pieces of tissue were washed three times in Ca2+-free Krebs-BSA solution by centrifugation (750 g for 2 min). The tissue was then triturated up to 5 min to disburse single tracheal myocytes. TSM cells were stored on ice in Ca2+-free Krebs-BSA solution and used the same day. A small (50 μ l) aliquot of the solution containing isolated tracheal myocytes was placed in an open 1.0 ml perfusion chamber mounted on the stage of an inverted microscope. The TSM cells were allowed to adhere to the glass bottom of the chamber for 20 min and then were perfused (2 ml/min) with Krebs solution.Tracheas were isolated as described above. The dorsal muscle layer was cut away from the hyaline cartilage rings and minced into ~1-mm pieces in Cam) and ionic currents of TSM cells were measured using perforated-patch whole cell recordings. Pipettes were pulled from borosilicate glass capillaries  using a Flaming/Brown micropipette puller P-97  and had resistances of 2–4 MΩ when filled with internal solution and measured in standard bath solution. Membrane current was measured with pipette and membrane capacitance cancellation, sampled at 100 μ s, and filtered at 2.9 kHz using an EPC-10 amplifier, and PATCHMASTER software . In all experiments, the perforated-patch method of recording was used with amphotericin B (600 ng/ml) in the pipette  according to previous procedures Lomask, . Eight t Lomask, , was meat-test. In the cases when more than two experimental groups were compared, a One-Way ANOVA was applied to determine variability among groups. A post-hoc Tukey HSD test was used to compare individual groups. For comparison of two groups with multiple factors, we used a Two-Way ANOVA to distinguish if the groups were different. The effects were deemed significant when a P < 0.05 was obtained. The results are expressed as the means ± standard error of means where applicable.Igor 5 , KaleidaGraph 4.1.1 (Synergy Software), and Excel 2007 (Microsoft Corp.) were used for statistical analyses. Significance was determined with paired or unpaired t levels of target genes normalized to that of β-actin.Tracheal muscle was dissected and pooled from either five wild type or five β1 KO mice. Rat tracheas were analyzed from four individual animals and expression values reflect mean data. The muscle RNA was extracted using Ambion Purelink RNA kit. One microgram of RNA was used for random primer, reverse transcriptase reactions using Applied Biosystems Reverse Transcriptase kit. 1/10 of the cDNA synthesis was used for SYBR green-based PCR amplification of cDNA using the forward and reverse primers listed in Table http://neuromab.ucdavis.edu/catalog.cfm). Guinea pig anti-KCNQ3 (1:1000) antibodies have been successfully utilized for staining of brain sections . The whole lungs were then fixed overnight in 4% paraformaldehyde/PBS, and washed in 20% sucrose/PBS for a second day. Frozen sections were cut in a sliding microtome at 25 micron thickness and immunostained using the free-floating section technique. Primary antibodies used were Neuromab monoclonal antibodies against KCNQ1 , KCNQ2 , and KCNQ4  and a non-commercial antibody against KCNQ3  that has been used successfully in the past  show greatest expression of KCNQ1. However, KCNQ5 is somewhat greater than KCNQ4 expression . We observed relatively strong staining of KCNQ1 and KCNQ4 proteins in the smooth muscle layer lining the airway  voltage commands and quantified the KCNQ current as that present during the last 400 ms of each sweep . Consistent with previous work, flupirtine roughly doubled the KCNQ current at potentials positive to −40 mV , and with flupirtine, they were −36 ± 1.4 mV and 9.1 ± 0.3 mV (n = 26), respectively, consistent with the biophysical and pharmacological behavior of KCNQ channels. Since the qRT-PCR data indicated subtle differences in the expression profile of KCNQ1–5 subunits across species, we also investigated KCNQ currents in mouse ASM, and saw a similar effect of flupirtine and XE991 on the currents attributed to KCNQ channels , which was increased to 16.8 ± 2.7 pA (n = 26) by flupirtine, but reduced to 0.5 ± 0.2 pA (n = 23) by XE991. In mouse ASM cells, the values were 6.4 ± 2.0 pA (n = 7), 13.2 ± 3.2 (n = 5) and 1.2 ± 0.6 pA (n = 4), respectively. Similarly, flupirtine shifted the V1/2 from −26 ± 2 mV, slope factor 9 ± 0.5 (n = 7) to −36 ± 1.7 mV, slope factor 8.9 ± 0.6 (n = 7). Thus, both rat and mouse ASM cells display KCNQ current of similar current amplitudes and pharmacological sensitivities.KCNQ currents were assayed in acutely dissociated tracheal muscle cells using perforated, whole-cell patch clamp recordings. The bath solution included the BK channel blocker paxilline (1 μ M) to enrich for the KCNQ current fraction. To estimate KCNQ currents in these experiments, we used two approaches. The first exploits the much slower kinetics of KCNQ current activation, compared to other Kp Figure . Indeed,p Figure . This cop Figure . At potep Figure , consist Figures , and shi Figures  , and 54 ± 6% (n = 5), respectively  and XE991 depolarized them by 4.7 ± 1.1 mV, relative to control (n = 6). The significant effect of flupirtine and XE991 is consistent with the significant KCNQ channel conductance  whereas there was no significant depolarization induced by XE991 in the presence of CCh. This suggests that although KCNQ channels control resting voltage, they may not be necessary for membrane repolarization during muscarinic-evoked contractions.We next tested whether the KCNQ channels also affect membrane voltage under mAChR stimulation. Given that KCNQ channel currents are largely active in the presence of CCh at its EC2 and M3 mAChR stimulation is the predominant mechanism mediating contraction of airway  . These results are consistent with KCNQ channels being required for control of membrane voltage at rest, but having a redundant role with BK channels during muscarinic signaling. On the other hand, the KCNQ agonist, flupirtine, hyperpolarized the membrane voltage under all circumstances , 112 ± 4% , and 135 ± 7% (n = 12) in control, flupertine and XE991, respectively. For mouse trachea, the percent of high-K+ contraction was 131 ± 3% (n = 3), 108 ± 2% , and 133 ± 4% (n = 3) in control, flupertine and XE991, respectively. The significant effect of the KCNQ-channel opener for both rat and mouse trachea suggests that at potentials where both KCNQ K+ and L-type voltage-gated calcium channels (VGCCs) channels are activated, M-channel openers can moderate ASM contraction. As a further test, when we used a low (1 mM) K+ external solution to hyperpolarize trachea membrane voltage negative to the voltage threshold for activation of L-type Ca2+ channels, there was no effect of flupirtine on the CCh-induced contraction , and in flupirtine, it was 71 ± 4% (n = 3). This is consistent with a specific effect of flupirtine on membrane voltage via KCNQ channels. In contrast to the effect of flupirtine, XE991 had no effect on CCh-induced contractions  at 3 Hz and 20 Hz, respectively, and in the presence of XE991, it was 22.7 ± 2.9% and 42.5 ± 3.9% (n = 5), respectively.The lack of effect of XE991 on CCh-induced contraction may be explained by a robust BK channel current that is sufficient to limit depolarization. To test this hypothesis, we compared the effect of XE991 on contractility of WT mice to mice with a genetically ablated BK channel β1 subunit. β1 KO mice have increased tracheal constrictions due to reduced activation of BK channels  and BK channel β1 KO mice to understand how muscarinic Gq-coupled signaling or redundancies with BK channels, respectively affect the role of KCNQ channels in ASM. We performed these tests at 0.5 μ M CCh, a concentration that produces half-maximal contractions, and at which there was no effect of XE991 both in WT and KO trachea  in the presence or absence of XE991, respectively, whereas in KO trachea, they were 94 ± 10.8% and 149 ± 4.9% , respectively. These results suggest that when BK channels are fully active, KCNQ channels may be functionally redundant in limiting contractions from membrane depolarization. However, when BK channel function is perturbed, as when the β1 subunit is lacking, KCNQ channels are essential in limiting depolarizations, which likely increase airway contraction via influx of Ca2+ through L-type VGCCs. These data further indicate that the lack of significant effect of XE991 at higher CCh concentrations in the KO trachea  in breathing mechanics that is correlated to increases of airway resistance . This contrasts with sympathetic neurons that show 80–90% KCNQ current inhibition at 10 μ M muscarinic agonist  on voltage or contractions during muscarinic signaling. Rather, the data indicate that functional redundancy with BK channels reduces the contribution of KCNQ channels during ACh-evoked contractions. At low muscarinic agonist concentrations, the BK β1 KO was sufficient to reveal effects of XE991. At higher muscarinic agonist concentrations the combination of β1 KO and M3 receptor blockade revealed a significant effect of KCNQ channels on membrane voltage  membrane potentials. However, muscarinic activation eliminated the effect of KCNQ blockade on membrane potential. While KCNQ agonist flupirtine relaxed muscarinic-evoked contractions, KCNQ blockade did not enhance it. This suggests that, although KCNQ channels can be pharmacologically recruited to relax airway, they do not contribute to relaxation during muscarinic-evoked contractions. In part, one might expect that the mechanism would be due to inhibition by signaling downstream of muscarinic receptor activation and that the muscarinic-evoked depolarization of ASM could be due to muscarinic inhibition of KCNQ channels. However, voltage clamp recordings indicate muscarinic agonist only weakly inhibits KCNQ channels (~19% inhibition at 0.5 μ M CCh, 46% at 10 μ M CCh Figure , approxi Figures  and cont Figures . Thus, win vitro constriction study supports a supplementary role for KCNQ channels in control of airway contractility when BK K+ channel activation is perturbed. This is further supported by in vivo experiments showing that RTG reduces the PenH index. Although the whole animal plethysmography may also reflect changes in breathing independent of airway resistance (Glaab et al., The + channel in lower ASM, whereas human lung may be more reliant on KCNQ channels. Further studies should elucidate the relative contribution of these two channel types in lower airway of rodents.This study in rodents support past studies showing KCNQ channel expression in ASM of guinea pig and human tissues (Brueggemann et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The mRNA expression was analyzed by quantitative polymerase chain reaction (qPCR). Enzyme-linked immunosorbent assay (ELISA) was used to confirm protein synthesis. The cell growth inhibition rate in the combination group was significantly higher compared with those in the asiaticoside and rapamycin groups for human aortic fibroblasts  and human aortic smooth muscle cells . However, for human coronary artery endothelial cells (HCAECs), the cell growth inhibition rates in the combination, asiaticoside and rapamycin groups were 11.09±1.17, 26.22±4.24 and 34.80±2.80%, respectively (P<0.05), as detected by MTT assay. The qPCR assay showed that in the combination group the level of von Willebrand factor (vWF) mRNA was downregulated, while platelet endothelial cell adhesion molecule (PECAM-1) and endothelial nitric oxide synthase (eNOS) mRNAs were upregulated in HCAECs compared with the rapamycin group (P<0.05). Transforming growth factor (TGF)-β1 and TIMP1 mRNAs were downregulated while Smad7 and matrix metalloproteinase 1 (MMP1) mRNAs were upregulated in HAFs compared with the rapamycin and AT groups (P<0.05). The ELISA showed that the type I collagen level was significantly reduced in HASMCs and HAFs (P<0.05). The data suggest that asiaticoside combined with rapamycin may be effective in the reduction of ISR.The aim of this study was to investigate and characterize the efficacy and mechanism of action of asiaticoside in combination with rapamycin in the inhibition of in-stent restenosis (ISR). The effects of asiaticoside combined with rapamycin on cell proliferation  In-stent restenosis (ISR) following vascular intervention affects the long-term curative effect markedly . AlthougCentella asiatica, a plant of the Umbelliferae family, which has been used for the treatment of hypertrophic scars for numerous years and is an ingredient in Chinese traditional herbal medicines. Previous studies have demonstrated that asiaticoside has a variety of biological effects, including anti-inflammatory  belongs to a family of cytokines with a variety of functions relating to fibrosis, growth, differentiation and apoptosis . TGF-β iposition .TGF-β1 promotes synthesis of the ECM by upregulating the α2 (type I) collagen gene, which results in an increase in the synthesis of type I collagen in fibroblasts; the increased ECM contributes to artery remodeling. The Smad signaling pathway is the primary signaling pathway for TGF-β. Among the Smad family, Smad7 is a general antagonist of the TGF-β family. Smad7 regulates TGF-β signaling via a negative feedback loop and mediates the crosstalk between TGF-β and other signaling pathways . Matrix ® II were purchased from Invitrogen Life Technologies . SYBR® Premix Ex Taq™ II  was obtained from Takara Bio, Inc. . ABI PRISM® 7900HT Sequence Detection system was purchased from Invitrogen Life Technologies and the enzyme-linked immunosorbent assay (ELISA) kit for collagen type I was purchased from Shanghai BlueGene Biotech Co., Ltd. .Bare metal stents (BMSs) were purchased from Shanghai MicroPort Medical (Group) Co., Ltd. , asiaticoside was purchased from Guangxi Changzhou Natural Pharmaceutical Co., Ltd., , rapamycin was obtained from Shanghai Gene Biotechnology company , Shandon Excelsior ES™ Tissue Processor was purchased from Thermo Fisher Scientific Inc.  and the EXAKT 310 CP Basic cutting system was purchased from Exakt Technologies, Inc. . Human aortic fibroblasts (HAFs), human aortic smooth muscle cells (HASMCs) and human coronary artery endothelial cells (HCAECs) were purchased from ScienCell Research Laboratories . Trypsin 0.25% (w/v), 0.53 mM EDTA, endothelial cell medium, fibroblast medium, smooth muscle cell medium, 3--2,5-diphenyltetrazolium bromide (MTT), TRIzol and SuperScriptPrimary HCAECs were cultured in EC growth medium (EBM-2) containing 5% fetal bovine serum (FBS) and 1% endothelial cell growth supplement  and 5 ml of penicillin/streptomycin solution , antibiotics and antimycotics in an incubator with 5% carbon dioxide at 37°C. Primary HASMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 1 mmol/l L-glutamine in an incubator with 5% carbon dioxide at 37°C. Primary HAFs were cultured in fibroblast medium (FM) containing 20% FBS, 1% fibroblast growth supplement (FGS) and 5 ml 1% penicillin/streptomycin solution (P/S) in an incubator with 5% carbon dioxide at 37°C. All assays were performed on cells at 80–100% confluence, between passages 1 and 2, and were repeated at least 3 times.−12, 1×10−13, 1×10−14, 1×10−15 mol/l) and the rapamycin group was treated with various concentrations of rapamycin . The combination group was treated with asiaticoside and rapamycin, in which the rapamycin concentration was 10−9 mol/l and asiaticoside was used in various concentrations . For qPCR and ELISA analysis, the blank group was treated with 1% dimethyl sulfoxide (DMSO), the asiaticoside group was treated with 10−5 mol/l asiaticoside and the rapamycin group was treated with 10−9 mol/l rapamycin. The combination group was treated with 10−5 mol/l asiaticoside and 10−9 mol/l rapamycin. Following treatment with various drugs, the cells were incubated for 48 h at 37°C. The supernatants were harvested and centrifuged for 15 min at 10,656 × g, and then removed and stored at −20°C for ELISA. The cells were harvested for qPCR.The cells were seeded in 96-well plates with 8,000 cells per well and treated with asiaticoside, rapamycin or both drugs (24 wells for each group) and incubated with 5% carbon dioxide at 37°C for 24 h. For cell viability analysis, the blank group was treated with 1% dimethyl sulfoxide (DMSO), the asiaticoside group was treated with various concentrations of asiaticoside (1×10Cell viability was detected using an MTT assay. MTT (5 mg/ml) was added to each well. The cells were incubated for one hour and then made soluble with cytolysis solution . Absorbance was determined at 570 nm by spectrophotometry.® II. For HCAECs, the vWF, PECAM-1 and eNOS mRNAs were detected. For HAFs, the TGF-β1, Smad7, type I collagen, TIMP1 and MMP1 mRNAs were detected. The primer sequences are listed in ® Premix Ex Taq™ II  was used. The PCR reaction was carried out with the ABI PRISM® 7900HT Sequence Detection system. The samples were analyzed in duplicate. β-actin was used as an internal control. PCR products were separated by electrophoresis in a 2% agarose gel. Densitometry values representing gene expression were first normalized to β-actin expression .Briefly, total RNA was isolated using TRIzol according to the manufacturer’s instructions. Reverse transcription-generated cDNA was obtained using SuperscriptThe culture supernatants were collected and stored at −20°C. For HASMCs and HAFs, the type I collagen level was determined using an ELISA kit for collagen type I.The data were processed using SPSS software . The results are presented as the mean ± standard error of the mean (SEM). The differences among experimental groups were compared by one-way analysis of variance (ANOVA), and two sets of isolated sample data were checked using a Student’s t-test. P<0.05 was considered to indicate a statistically significant result.Compared with the blank group, asiaticoside was able to markedly inhibit the proliferation of HASMCs and HAFs (P<0.01). Compared with the asiaticoside and rapamycin groups, the combination group showed a greater inhibition of HASMCs and HAFs. In HASMCs, the inhibitory rates were 33.12±1.35, 26.21±7.59 and 28.27±4.92%, respectively (P<0.05) and in HAFs, they were 63.50±3.83, 53.06±8.10 and 60.34±4.93%, respectively (P<0.05). These results showed a certain synergism between asiaticoside and rapamycin in HASMCs and HAFs. By contrast, the combination group showed a weaker inhibition of HCAECs compared with that observed in the single drug groups; the inhibitory rates were 11.09±1.17, 26.22±4.24 and 34.80±2.80%, respectively (P<0.05). We suggest that asiaticoside may antagonize the inhibitory effect of rapamycin on vascular ECs .The levels of type I collagen, TGF-β1, Smad7, MMP1 and TIMP1 are shown in As shown in in vivo and in vitro. Asiaticoside combined with rapamycin exerted greater effects than asiaticoside or rapamycin alone. Asiaticoside has a good synergism with rapamycin to inhibit vascular smooth muscle cells (VSMSs) and fibroblasts, while it is also antagonistic to ECs, which may protect the vascular endothelium. The qPCR and ELISA results showed that the combination therapy induced the downregulation of vWF, type I collagen, TGF-β1 and TIMP1, and the upregulation of PECAM-1, eNOS, Smad7 and MMP1. This suggests that the combination therapy may function via the TGF-β pathway.The results indicate that asiaticoside is likely to be effective at reducing ISR ISR is a process involving several pathological pathways, in which VSMC and fibroblast proliferation, neointimal formation, negative remodeling of the artery and epithelialization delay play important roles. Rapamycin-eluting stents (RESs) are widely used to treat severe stenosis of the coronary artery. As an immunosuppressant, rapamycin binds to the cytosolic receptor FKBP12, then inhibits mammalian target of rapamycin (mTOR), which leads to inhibition of the down-regulation of the cyclin-dependent kinase inhibitor p27kip1, thereby inhibiting VSMC proliferation and migration . Howeveret al have demonstrated that asiaticoside inhibits scar fibroblast growth via the Smad signal pathway. The Smad7 protein and mRNA levels were reported to be increased in asiaticoside-treated fibroblasts, compared with control fibroblasts (et al observed that asiaticoside enhanced the expression of type I collagen in human periodontal ligament cells (Our study shows that asiaticoside has a synergism with rapamycin in VSMCs and fibroblasts, which results in a greater increase of cell growth inhibition rate than using a single drug. The combination effects are achieved via several mechanisms. Asiaticoside may inhibit the proliferation of VSMCs and fibroblasts, which is consistent with other studies ,20,21. Aroblasts ,21. It int cells , which cnt cells . Our reset al (In ECs, asiaticoside shows significant activity as a rapamycin antagonist, therefore, the inhibition of cell proliferation in the combination group is lower than that in the rapamycin group. There are few studies concerning the effect of asiaticoside on ECs. Zhou et al  establis


Embryonal tumor with multilayered rosettes  is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current treatment protocols and its distinct biology. Amplification of the miRNA cluster at 19q13.42 has been identified previously as a genetic hallmark for ETMR, but an immunohistochemistry-based assay for clinical routine diagnostics [such as INI-1 for atypical teratoid rhabdoid tumor (AT/RT)] is still lacking. In this study, we screened for an ETMR-specific marker using a gene-expression profiling dataset of more than 1,400 brain tumors and identified LIN28A as a highly specific marker for ETMR. The encoded protein binds small RNA and has been implicated in stem cell pluripotency, metabolism and tumorigenesis. Using an LIN28A specific antibody, we carried out immunohistochemical analysis of LIN28A in more than 800 childhood brain-tumor samples and confirmed its high specificity for ETMR. Strong LIN28A immunoexpression was found in all 37 ETMR samples tested, whereas focal reactivity was only present in a small (6/50) proportion of AT/RT samples. All other pediatric brain tumors were completely LIN28A-negative. In summary, we established LIN28A immunohistochemistry as a highly sensitive and specific, rapid, inexpensive diagnostic tool for routine pathological verification of ETMR. Embryonal tumor with multilayered rosettes (ETMR), also previously reported as embryonal tumor with abundant neuropil and true rosettes (ETANTR) is a rare, albeit likely underdiagnosed embryonal CNS neoplasm with fewer than 100 cases reported in the literature since its initial description in 2000 , 5, 19. n = 1,404). We selected the most specifically expressed gene, namely LIN28A, and subsequently tested its diagnostic specificity for ETMR with a high-quality commercial antibody on a large cohort (n = 816) of paraffin-embedded brain tumors. Our results show that LIN28A expression can be used as a specific diagnostic biomarker to detect ETMR.However, the requirement of non-commercial custom made BAC probes for FISH testing renders this assay virtually inaccessible to nearly every pathology laboratory around the globe. In contrast, the use of immunohistochemistry is universally utilized in neuropathology centers everywhere. As such, ETMR is a highly attractive target for developing tumor-specific immunohistochemistry. For this purpose, we herein performed a screen for ETMR-specific genes based on gene expression profiles collected for a large cohort of various brain tumors (n = 7), pilocytic astrocytoma , ependymoma , astrocytoma grade II , oligodendroglioma grade II , oligoastrocytoma grade II , anaplastic astrocytoma grade III , anaplastic oligodendroglioma grade III , anaplastic oligoastrocytoma grade III , glioblastoma grade IV , diffuse infiltrating pontine glioma , CNS PNET (n = 43), atypical teratoid rhabdoid tumor , medulloblastoma , and ETMR (n = 13). All gene-expression profiles were generated by Affymetrix U133plus 2.0 arrays using RNA isolated from frozen tumors. For comparison we used the publicly available gene expression profiles of 353 normal CNS and non-CNS tissues [http://r2.amc.nl). For immunohistochemical (IHC) analysis of LIN28A, a polyclonal antibody  was applied to a partly overlapping  cohort of 816 malignant pediatric brain tumors constructed on tissue microarrays ; EPIII (n = 223); GBM (n = 131); AT/RT (n = 50); CNS PNET (n = 41); ETMR (n = 37). All cases, except for a few ETMR cases contributed by collaborators, were obtained from the NN Burdenko Neuropathological Institute . IHC was performed with an automated stainer  using antigen-retrieval protocol CC1 and a working antibody dilution of 1:100 with incubation at 37 °C for 32 min. In parallel, FISH analysis for the 19q13.42 locus was performed for all tumors as previously described [To find ETMR specific marker genes, we used 849 publicly available gene expression profiles , 21, 23 AII; n = , oligodeescribed .Table 1DLIN28A, IGF2BP1, CRYBA1, HIC2, PRPF31, SALL4, SHISA3, HMGA2, LOC440416, PRTG, LOC253842, DNMT3B, CRABP1, C2ORF29, MRPS9, FGFBP3, SP8, and HES5) were highly upregulated in all ETMR samples. However, only one gene, LIN28A, was highly upregulated in ETMR but not in any other brain tumor  [let-7 repression and consecutive upregulation or stabilization of let-7 targets, such as MYC(N), RAS, CDK6 and HMGA2 [LIN28A and those of MYCN, NRAS, CDK6, and HMGA2 in the 13 ETMRs  . A potennd HMGA2 . Of noteLIN28A mRNA expression levels contained extensive areas of LIN28A immunonegative neuropil, whereas the four tumors with high LIN28A mRNA expression were composed predominantly of LIN28A immunopositive, histologically primitive small cell areas.To test whether LIN28A expression could be used as a sensitive and specific diagnostic marker for ETMR, we performed IHC screening of a large cohort of 816 malignant pediatric brain tumors Table . Thirty-p < 0.001).Among the other tumor entities, focal cytoplasmic immunoexpression of LIN28A was only found in single cell collections of six AT/RT cases (12 %). Each of these six tumors had obvious losses of nuclear INI1 protein expression, in line with their initial histopathological diagnosis Fig. . In contLIN28A gene expression in ETMR samples correlated closely with the intratumoral heterogeneity observed immunohistochemically. Focal positivity of LIN28A in a small subset of AT/RTs is not considered problematic when taking the simultaneous loss of INI1 expression into account, with the latter representing a fairly specific molecular marker for AT/RT. On the other hand, LIN28A immunoexpression has also been detected recently in CNS germ-cell tumors with diffuse staining patterns found in germinomas, embryonal carcinomas and yolk sac tumors but being only focal in immature teratomas [Therefore, this study represents a report of LIN28A as a highly specific and sensitive diagnostic marker for the distinct pathological verification of ETMR and, correspondingly, as a new important diagnostic tool to be routinely applied in all malignant childhood brain tumors where ETMR may be considered a possible diagnosis. Variability of Our current data also confirmed that ETMR is a unique clinico-pathologic entity from a molecular perspective, exhibiting 19q13.42 amplification and high levels of LIN28A expression accompanied by particularly aggressive clinical behavior. It will now be important to understand the biological consequence of overexpression of such universal and direct miRNA translation regulators as LIN28A and LIN28B in ETMR, taking into account the prototypic amplification of the oncogenic miRNA cluster at 19q13.42 in these tumors.


Echinacea ssp. represent a profitable market sector for herbal medicines thanks to different functional features. Alkamides and polyacetylenes, phenols like caffeic acid and its derivatives, polysaccharides and glycoproteins are the main bioactive compounds of Echinacea spp. This study aimed at investigating the capacity of selected lactic acid bacteria to enhance the antimicrobial, antioxidant and immune-modulatory features of E. purpurea with the prospect of its application as functional food, dietary supplement or pharmaceutical preparation.Extracts and products  from Echinacea purpurea suspension  in distilled water, containing 0.4% (wt/vol) yeast extract, was fermented with Lactobacillus plantarum POM1, 1MR20 or C2, previously selected from plant materials. Chemically acidified suspension, without bacterial inoculum, was used as the control to investigate functional features. Echinacea suspension fermented with Lb. plantarum C2 exhibited a marked antimicrobial activity towards Gram-positive and -negative bacteria. Compared to control, the water-soluble extract from Echinacea suspension fermented with Lactobacillus plantarum 1MR20 showed twice time higher radical scavenging activity on DPPH. Almost the same was found for the inhibition of oleic acid peroxidation. The methanol extract from Echinacea suspension had inherent antioxidant features but the activity of extract from the sample fermented with strain 1MR20 was the highest. The antioxidant activities were confirmed on Balb 3T3 mouse fibroblasts. Lactobacillus plantarum C2 and 1MR20 were used in association to ferment Echinacea suspension, and the water-soluble extract was subjected to ultra-filtration and purification through RP-FPLC. The antioxidant activity was distributed in a large number of fractions and proportional to the peptide concentration. The antimicrobial activity was detected only in one fraction, further subjected to nano-LC-ESI-MS/MS. A mixture of eight peptides was identified, corresponding to fragments of plantaricins PlnH or PlnG. Treatments with fermented Echinacea suspension exerted immune-modulatory effects on Caco-2 cells. The fermentation with Lb. plantarum 1MR20 or with the association between strains C2 and 1MR20 had the highest effect on the expression of TNF-α gene.E. purpurea subjected to lactic acid fermentation could be suitable for novel applications as functional food dietary supplements or pharmaceutical preparations. Echinacea belongs to the Asteraceae family and comprises a group of perennial prairie wildflowers, which are native to central grasslands of North America. The cultivation is mainly extended throughout United States, Canada and Europe . Echinacea angustifolia DC, Echinacea pallida (Nutt.) and, especially, Echinacea purpurea (L.) Moench }; where y is the growth expressed as log CFU/g/h or the acidification rate expressed as dpH/dt (units of pH/h) at the time t; k is the initial level of the dependent variable to be modelled (log CFU/g or pH units); A is the cell density or pH (units) variation (between inoculation and the stationary phase); μmax or Vmax is the maximum growth rate expressed as Δ log CFU/ml h or the maximum acidification rate expressed as dpH/h, respectively; λ is the length of the lag phase measured in hours. The experimental data were modelled by the non-linear regression procedure of the Statistica 8.0 software .Kinetics of growth and acidification were determined and modelled in agreement with the Gompertz equation, as modified by Zwietering et al. : y= k + Aliquots (1 ml) of ES were centrifuged at 10,000 × g for 10 min. The supernatant  was filtered through a Millex-HA 0.22-mm pore size filter . The concentration of glucose, fructose and sucrose was determined through HPLC analysis, using an ÄKTA Purifier system  equipped with a Spherisorb column  and a Perkin Elmer 200a refractive index detector. Elution was at 32°C with a flow rate of 1 ml/min, using acetonitrile 80% as the mobile phase .2SO4 as the mobile phase [Organic acids were determined by HPLC, using an ÄKTA Purifier system  equipped with an Aminex HPX-87H column  and a UV detector operating at 210 nm. Elution was at 60°C with a flow rate of 0.6 ml/min, using 10 mM Hle phase . Peaks wle phase .To determine the gross composition samples were previously freeze-dried. Dry matter was assessed by an infrared moisture analyzer MAC 110/NP . Total nitrogen was measured by the Kjeldahl method no. 920.87 , using ag for 20 min. ME were transferred into test tubes, purged with nitrogen stream and stored at ca. 4°C before analysis. The concentration of total phenols was determined as described by Slinkard and Singleton [The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of ES was determined both on methanol extract (ME) and WSE. Five milliliters of ES were mixed with 50 ml of 80% methanol to get ME. The mixture was purged with nitrogen stream for 30 min, under stirring condition, and centrifuged at 4,600 × ingleton . It was ingleton . The reaThe scavenging effect of freeze dried WSE on DPPH˙ free radical was measured according to the method of Shimada et al. , with soThe antioxidant activity of ME and WSE was also measured according to the method of Osawa and Namiki , with so2, 37°C), using Dulbecco’s Modified Eagle Medium (DMEM), which was supplemented with 10% (w/v) calf bovine serum (CBS), 1% penicillin /streptomycin  mixture, and 1% non-essential amino acid solution (NEAA). The culture medium was renewed every two days and after four passages the cultures were used for viability assays. Cell viability was measured using the MTT -2,5-diphenyltetrazolium bromide) method [4 cells/well and incubated for 24 h. Subsequently, cells were treated with re-suspended freeze dried ES and incubated for further 16 h. The concentration of ES in the reaction mixture varied from 1, 10, 50, 100, 250, 500 and 1000 μg/ml. A negative control, without addition of ES, was used. α-Tocopherol (250 and 500 μg/ml ) was used as the positive control. Following removal of CBS, cells were exposed to 150 μM hydrogen peroxide for 2 h. For each well, 100 μl of MTT , dissolved in DMEM, were added and incubated  in the dark for 3 h. Finally, 100 μl of dimethyl sulphoxide (DMSO) were added to dissolve purple formazan product. The solution was shacked in the dark for 15 min at room temperature. The absorbance of the solution was read at 570 nm in a microplate reader . Each experiment was carried out in triplicate. Data were expressed as the mean percentage of viable cells compared to the control culture, without oxidative stress.Mouse fibroblasts  were cultured under humidified atmosphere (5% CO) method . The capEscherichia coli DSM 30083 and Enterobacter aerogenes DSM 30053 grown on Luria Bertani (LB) broth (Oxoid) at 37°C, Enterococcus durans DSM 20633 and Yersinia enterocolitica DSM 4780 grown on Brain Hearth Infusion (BHI) (Oxoid) at 37°C, Weissella confusa DSM 20196 and Leuconostoc lactis DSM 20202 grown on MRS broth (Oxoid) at 30°C, and Propionibacterium jensenii DSM 20535 grown in sodium-lactate broth at 37°C, which belong to the Culture Collection of the Leibniz Institute DSMZ , and Lactobacillus sakei SAL1 grown on MRS broth at 30°C, Bacillus megaterium F6 grown on LB broth at 37°C, Candida krusei DSM 3433 grown on Yeast Extract Peptone Dextrose (YEPD) broth (Oxoid) at 25°C, and Penicillium roqueforti DPPMAF1 grown on Potato Dextrose Agar (PDA) (Oxoid) at 25°C, which belong to the Culture Collection of the Department of Soil, Plant and Food Sciences , were used to assay the antimicrobial activity.P. roqueforti DPPMAF1, the hyphal radial growth inhibition assay was used, as previously described by Coda et al. [8 CFU/ml) were harvested by centrifugation (8000 × g for 10 min), washed twice with 10 mM phosphate buffer, pH 7.0, and adjusted to 104 CFU/ml. The sterile 96-well microtiter plate was used (Greiner Labortechnik). Fifty microliters of each cell suspension were mixed with 50 μl of each fraction, and 100 μl of each specific culture broth were added. The estimated peptide concentration of each fraction ranged from 5 to 2000 μg/ml. Control wells contained all the components except for the peptide fraction, which was replaced with distilled water (positive control) or with chloramphenicol (100 μg/ml) (negative control). Microplates were incubated at 25, 30 or 37°C depending on the strain, and growth was monitored over 24 h by measuring the optical density of the culture at 620 nm using a microplate reader . The Minimum Inhibitory Concentration (MIC) corresponded to the lowest concentration of the peptide fraction needed to fully inhibit the bacterial growth. When growth was inhibited, cells were recovered from microplates, washed twice with 10 mM phosphate buffer, pH 7.0, and incubated into fresh medium to allow the recovery of growth. All assays were carried out in triplicate.The well-diffusion assay  was useda et al. . After ta et al. . Only ba× g for 60 min.WSE (ca. 400 μl) was subjected to fractionation by ultra-filtration , using membrane sizes of 50, 30, 10 and 5 kDa cut-off . Centrifugation was at 10,000 3CN) (solvent B). The initial conditions were flow 0.8 ml/min, column temperature 30°C and solvent B 12%; after 1 min, the concentration of B was linearly increased to 90% in 35 min. The absorbance between 210 and 400 nm was acquired.Polyphenols were analyzed using a multi-solvents delivery system controller 600 , equipped with a PDA 996 (Waters) and a Synergi Hydro 80Ǻ column, 5 μm particle size, 250 × 4.6 mm . Separation was carried out using a binary gradient of 10% (v/v) formic acid in water (solvent A) and acetonitrile , using a Resource RPC column and an ÄKTA FPLC equipment, with the UV detector operating at 214 nm . Aliquots of WSE, containing ca. 1 mg/ml of peptides, were added to 0.05% (v/v) trifluoroacetic acid (TFA) and centrifuged at 10,000 × Partially purified fractions from WSE, which had the highest antimicrobial activity, were subjected to sequential protein hydrolysis by digestive enzymes, according to the method of Pasini et al. . Freeze Fractions of WSE with the highest antimicrobial activity were subjected to further purification through RP-HPLC, using an ÄKTA Purifier apparatus . The centers of the peaks were collected, freeze dried and used for mass spectrometry analysis. Identification of peptides was carried out by nano-Liquid Cromatography-Electrospray Ionisation-Mass Spectra/Mass Spectra (nano-LC-ESI-MS/MS), using a Finningan LCQ Deca XP Max ion trap mass spectrometer (ThermoElectron) through the nano-ESI interface. According to manufacturer's instrument settings for nano-LC-ESI-MSMS analyses, MS spectra were automatically taken by Xcalibur software (ThermoElectron), in positive ion mode. MS/MS spectra were processed using the software BioWorks 3.2 (ThermoElectron) generating peaklists suitable for database searches. Peptides were identified using MS/MS ion search of Mascot search engine  and NCBInr protein database . For identification of peptides the following parameters were considered: enzyme: "none"; instrument type: "ESI-trap"; peptide mass tolerance: ± 0.1% and fragment mass tolerance: ± 0.5 Da. Results from peptide identification were subjected to a manual evaluation, as described by Chen et al. , and the2 culture flasks at 37°C with 5% CO2. The culture medium was replaced three times a week. Cell viability was measured according to the MTT assay (see elsewhere). After 24 h seeding on 96 well plate, 80% confluent Caco-2 cells were exposed to various concentrations  of freeze dried ES. The control was the basal medium. Plates were incubated at 37°C, 5% CO2, for 24, 48 and 72 h. After each treatment, the medium was aspirated and replaced with 100 μl per well of MTT solution. MTT was dissolved (5 mg/ml) in FBS and diluted 1:10 in the cell culture medium without phenol red. After 3 h of incubation, the basal medium was aspirated and 100 μl per well of DMSO were added to dissolve purple formazan product. The solution was shacked in the dark for 15 min at room temperature. The absorbance of the solutions was read at 570 nm in a microplate reader . Each experiment was carried out in triplicate. Data were expressed as the mean percentage of viable cells compared to the culture in basal medium.Human colon adenocarcinoma Caco-2 cells (ICLC HTL97023) supplied by the National Institute for Cancer Research  were routinely cultured in Eagle’s minimum essential medium (EMEM), with Earle's balanced salt solution (EMEM-EBSS), and supplemented with 10% (wt/vol) heat inactivated fetal bovine serum (FBS), 1% (wt/vol) NEAA, penicillin /streptomycin  and 1% L-glutamin . Cells were maintained in 25 cmTNF-α from Caco-2 cells was investigated through RT-PCR. When ca. 80% confluence was reached, Caco-2 cells were harvested with trypsin/EDTA, seeded, at the density of 1 × 106 cells per well, into 12-well  plates and incubated at 37°C, 5% CO2, for 24 h. Cells in EMEM medium and EMEM with lipopolysaccharide (LPS) (5 μg/ml) were used as the controls. Freeze dried ES at the concentrations of 1, 10, and 50 μg/ml was added to 80% confluent Caco-2 cells with LPS (5 μg/ml), and incubated at 37°C for 16, 24 and 48 h. For quantitative real-time PCR (RT-PCR), total RNA from Caco-2 cells was extracted using Tri Reagent (Sigma Aldrich), as described by Chomczynski and Mackey [TNF- α) and Hs999999_m1  were used for Taqman gene expression assays. Human GAPDH was the housekeeping gene. PCR amplifications were carried out using 40 ng of cDNA on a 20 μl of total volume. The mixture reaction contained 10 μl of 2× TaqMan Universal PCR Master Mix, 1 μl of 20× TaqMan gene expression assay, 5 μl of water and 4 μl of cDNA. PCR conditions were as follows: 50°C for 2 min  and 95°C for 10 min, followed by 40 amplification cycles . Analyses were carried out in triplicate. The average value of target gene was normalized using GAPDH gene and the relative quantification of the levels of gene expression was determined by comparing the Δ cycle threshold (ΔCt) value [After treatment with ES, the expression of d Mackey . The cDNt) value . ResultsP<0.05, using the statistical software, Statistica for Windows (Statistica7.0 per Windows). Student’s t-test was used for MTT assay (GraphPAD 6.0 per Windows).Data were subjected to one-way ANOVA; pair-comparison of treatment means was achieved by Tukey’s procedure at The authors declare that they have no competing interests.CGR carried out purification and identification of bioactive compounds and elaboration of the data; RC carried out the experimental design of the work, microbiological analyses, and elaboration of the data; DSM, and PF performed fermentations and microbiological determinations; DP, BM, and GG carried out ex-vivo assays and related experimental procedures; VMP carried out the chemical analyses; RDC and MG were the supervisors and the coordinators of the research units. All authors read and approved the final manuscript.


TP53, TP63, and TP73 genes comprise the p53 family. Each gene produces protein isoforms through multiple mechanisms including extensive alternative mRNA splicing. Accumulating evidence shows that these isoforms play a critical role in the regulation of many biological processes in normal cells. Their abnormal expression contributes to tumorigenesis and has a profound effect on tumor response to curative therapy. This paper is an overview of isoform diversity in the p53 family and its role in cancer. Alternative splicing allows a single gene to express multiple protein variants. It is estimated that 92–95% of human multiexon genes undergo alternative splicing , 2. Abno The p53 protein family is comprised of three transcription factors: p53, p63, and p73. Phylogenetic analysis revealed that this family originated from a p63/73-like ancestral gene early in metazoan evolution . The p53p53, p63, and p73 genes are located on chromosomes 17p13.1, 3q27-29, and 1p36.2-3, respectively. These genes encode proteins with similar domain structures and significant amino acid sequence homology in the transactivation, DNA-binding and oligomerization domains . The hig The founding member of the p53 family, the p53 protein, had been discovered more than three decades ago , 13. Forin silico provided evidence for the existence of a third putative promoter in the first intron of human TP73 gene [TP73 gene regulates TAp73 isoforms and isoforms, which lack the TA domain: ΔEx2p73, ΔEx2/3p73, and ΔN′p73. The latter isoforms are missing either exon 2 (ΔEx2p73) or both exon 2 and 3 (ΔEx2/3p73) or contain an additional exon 3′ (ΔN′p73) [ Transcriptions of p53, p63, and p73 genes are regulated by similar mechanisms. It is controlled by two promoters: P1 and P2, where P2 is an alternative intragenic promoter . One stuP73 gene . TherefoP73 gene , 16. TA (ΔN′p73) , 18. Oth(ΔN′p73) , 20. Add(ΔN′p73) . α, β, γ), and nine for p73  [δ, ε) and p53  were reported [α, β, and γ. Little is known about the functions of other isoforms. The combination of alternative splicing at the 5′ and 3′ ends, alternative initiation of translation and alternative promoter usage can significantly increase protein diversity. For example, N-terminal variants  can be produced in α, β, and γ “flavors” [ Additional diversity of p53, p63, and p73 transcripts is generated by alternative splicing at the 3′ end of the transcripts . These s and η1) –25. Latereported –28. Howeflavors” , 21. The p53, TAp63, and TAp73 share significant functional resemblance. They can induce cell cycle arrest, apoptosis, or cellular senescence. This similarity can be explained, at least in part, by transactivation of the same transcriptional targets. Genome-wide analyses found an overlap of the transcription profiles of p53, TAp73, and TAp63, though unique targets were identified as well. Analyses using chromatin immunoprecipitation, reporter, and gel-shift assays found that TAp73 and TAp63 interact with p53-responsive elements. γ and TAp73β are similar to that of p53α [α and TAp63α have an additional domain at the COOH-terminus that is not found in p53. This domain, termed SAM or Sterile Alpha Motif, is responsible for protein-protein interactions and is found in a diverse range of proteins that are involved in developmental regulation. It is also implicated in transcriptional repression [ The transactivation and apoptotic potential of p53, TAp73, and TAp63 vary greatly depending on the isoform. TAp63 of p53α . Other i of p53α , 23, 30.pression . Beta anpression , 33. ΔN isoforms function as dominant-negative inhibitors of TA counterparts . Promote ΔN isoforms of p53 and p73 are regulated by a negative feedback loop mechanism. Analogous mechanism was not described for p63 despite its significant similarity to p73. In a nutshell, TA isoforms are able to induce transcription of ΔN isoforms by activating P2 promoters. The induced ΔN isoforms, in turn, inhibit TA isoforms. A good example of these interactions is an induction of Δ133p53 by p53 –38. SimiAlthough many aspects of p53 biology have been thoroughly investigated, the role and regulation of p53 isoforms remain not well understood.  Recent studies suggested that Δ133p53 isoform may play an oncogenic role. Mice overexpressing the Δ122p53 isoform (murine homolog of human Δ133p53) show reduced apoptosis, increased cell proliferation and develop a wide-spectrum of aggressive tumors including lymphoma, osteosarcoma, and other malignant and benign tumors . Anotherα mRNA was also found in renal cell carcinoma [ Several studies reported an elevated expression of Δ133p53 in tumors . In breaarcinoma . In coloarcinoma . Interesarcinoma . In addiarcinoma .β and p53γ isoforms were also reported in different types of cancers  are primarily detected in colon and breast cancers [ε isoform was only expressed in leukemic cells and completely absent in mature myeloid cells [ An increased expression of TAp73 isoforms was also found in tumors, although its role remains unclear . Several cancers , 23. Thiid cells . It is cα inhibits oncogene-induced cellular senescence and cooperates with Ras to promote tumor-initiating stem-like proliferation [−/− null mice showed striking developmental defects demonstrating a critical role of p63 in epithelial development [+/- heterozygous mice were shown to be susceptible to tumor development [Similar to p73, mutations in the p63 gene are rare in human cancers , 138. Seferation . Analysielopment , 142. p6elopment . Howeverelopment , 145. β-dependent cell migration, invasion, and metastasis [ ΔNp63 is a predominant isoform expressed in most epithelial cells. Overexpression of ΔNp63 is found in cancers of nasopharyngeal, head and neck, urinary tract, lung, and ovarian tumors and correlated with poor outcome , 146–149tastasis . This sutastasis , 152. Extastasis , 154.  TAp63 isoforms induce cellular senescence and inhibit cell proliferation –157. TAp Clearly, additional studies are needed to understand the complex regulation of p63 isoforms.TP53 and TP73 gene promoters [Interactions between members of the p53 family and their isoforms have a profound effect on tumorigenesis and anticancer drug response. Perhaps, the most studied are interactions between ΔN and TA isoforms. Inhibition of TAp73 by ΔNp63 has been shown to negatively affect the response to platinum-based chemotherapy in head and neck squamous cell carcinomas and a subset of breast tumors , 154. Inromoters –163. In romoters –166.β inhibits p53-dependent apoptosis in primary sympathetic neurons [β induces cell cycle arrest and apoptosis [ Current analyses suggest that the function of a particular isoform needs to be investigated in the context of expression of other isoforms. For example, ΔNp73 neurons , but whepoptosis .  An interesting observation has been made in mouse embryonic fibroblasts, where the combined loss of p73 and p63 results in the failure of p53 to induce apoptosis in response to DNA damage . More reThe p53 family plays a pivotal role in the control of many critical cellular functions. In recent years, it has been revealed that all members of the p53 family are expressed as a diverse variety of isoforms. We only just started to uncover the mechanisms that regulate this diversity. A number of studies also provided the first glimpses of their functional significance. Clearly, isoforms add a new level of functional regulation to many critical biological processes including cell death, proliferation, cell cycle control, and tumorigenesis. Depending on the isoform expressed, the role of a gene can dramatically change from a tumor suppressor to an oncogene. It is also clear that p53, p73, and p63 isoforms tightly interact. A better understanding of this interacting network and its regulation holds the key to future therapeutic benefits.


Background. Licorice has long been used to treat many ailments including cardiovascular disorders in China. Recent studies have shown that the cardiac actions of licorice can be attributed to its active component, glycyrrhetinic acid (GA). However, the mechanism of action remains poorly understood. Aim. The effects of GA on the delayed rectifier potassium current (IK), the rapidly activating (IKr) and slowly activating (IKs) components of IK, and the HERG K+ channel expressed in HEK-293 cells were investigated. Materials and Methods. Single ventricular myocytes were isolated from guinea pig myocardium using enzymolysis. The wild type HERG gene was stably expressed in HEK293 cells. Whole-cell patch clamping was used to record IK  and the HERG K+ current. Results. GA  inhibited IK  and the HERG K+ current in a concentration-dependent manner. Conclusion. GA significantly inhibited the potassium currents in a dose- and voltage-dependent manner, suggesting that it exerts its antiarrhythmic action through the prolongation of APD and ERP owing to the inhibition of IK  and HERG K+ channel. Cardiac arrhythmias are associated with significant morbidity and mortality in developed and developing countries. Although nonpharmacologic approaches, ablative therapy, and implantable defibrillators, for example, are being used more and more commonly as complementary and alternative interventions for the treatment of cardiac arrhythmias, drug therapy was traditionally the mainstay of arrhythmia treatment. Improved understanding of the cardiomyocyte ion channels holds the promise of identifying novel targets for the treatment of cardiac arrhythmias. Furthermore, the limited efficacy and the potency of provoking life-threatening arrhythmias of present drugs have generated interest in finding new antiarrhythmic agents . + current (IK), the major outward potassium current responsible for ventricular repolarization, can be divided into at least two different components, rapidly activating (IKr) and slowly activating (IKs). The human ether-à-go-go-related gene (HERG) encodes the α-subunit for the rapid delayed rectifier potassium channel (IKr) in cardiac myocytes. IKr and IKs are pivotal in cardiac repolarization, especially in the later phases of the action potential [+ channel modulators are being used clinically at present for the unwanted adverse effects [Potassium channels constitute the most abundant family of ion channels involved in cardiac physiological or pathophysiological processes, disruptions of which could give rise to prolongation of the action potential and potentiate occurrence of lethal arrhythmias subsequently , 3. In totential , 5. Alth effects , 6. As aGlycyrrhiza radix is a commonly prescribed herb to prevent palpitations in Chinese traditional medicine for about 2000 years, derived from the dried roots and rhizomes of Glycyrrhiza uralensis, G. glabra, and G. inflata. Glycyrrhizin, the major constituent of G. glabra, is a glycoside, which occurs as an admixture of sodium, potassium and calcium salts [β-D-glucuronidase-containing intestinal bacteria to yield two molecules of D-glucuronic acid and the aglycone glycyrrhetinic acid (GA), a pentacyclic triterpene [β-D-glucuronidase to the 3-monoglucuronide of glycyrrhetinic acid. This metabolite is excreted via the bile into the intestine, where it is transformed by bacteria into GA, which can be reabsorbed, causing a pronounced delay in terminal plasma clearance [um salts . Orally iterpene . If intrlearance .Glycyrrhiza uralensis as a pharmacological remedy dates back far into the past [Glycyrrhiza radix and its pharmacological activities are comparatively well understood and clear.The use of the past . Variousthe past , 12, antthe past , 13–15. INa. These findings might help to elucidate the traditional use of licorice in therapy for cardiovascular disorders [β-GA has significant potential for development as a novel antiarrhythmic agent and for treating myocardial ischemia by preferentially blocking the INa,L [Recently, wide-ranging studies have provided evidence that GA is cardioprotective; this action involves different pathways. In rat cardiac mitochondria , GA was isorders . It has e INa,L . HoweverIK  in guinea pig ventricular myocytes. We also extended our study to investigate the effects of GA on the human ether-à-go-go-related gene (HERG) K+ channel current expressed in HEK-293 cells. Our study could provide theoretical support for developing the significant potential of GA as a novel antiarrhythmic agent.In this study, we investigated the effects of GA on the 2ATP, MgATP, CdCl2, L-Glutamic acid, Taurine, and EGTA were purchased from Sigma Co. Other reagents were of analytical reagent grade. Male guinea pigs weighing 300–350 g were provided by the Vital river Company, Certificate no.: SCXK (Jing) 2006-0009. Glycyrrhetinic acid ((GA) molecular weight 470.6, purity > 99%) was obtained from the Chinese Biological Product Assay Institute . The white powder was dissolved in dimethylsulfoxide (DMSO) as stock. The percentage of DMSO in the final solution was less than 0.1%. Collagenase II was obtained from Worthington Biochemical Corporation . Protease XIV, Na2+-free Tyrode's solution (mM) contained NaCl 137, KCl 5.4, NaH2PO4 1.2, MgCl2 1.2, HEPES 10, glucose 10, and Taurine 10 (adjusted to pH 7.36–7.38 with NaOH).Ca2PO4 20, Taurine 20, MgCl2 3.0, KOH 70, EGTA 0.5, HEPES 10, and glucose 10 (adjusted to pH 7.25 with KOH).Kraft-Brühe (KB) solution (mM) contained L-glutamic acid 50, KCl 40, KHIK  recordings from ventricular myocytes, the control bath solution contained (mM) NaCl 135, KCl 5.4, CaCl2 1.0, CdCl2 0.2, NaH2PO4 0.33, MgCl2 1.0, HEPES 5, and glucose 5 (adjusted to pH 7.36–7.38 with NaOH). Drugs were added to the bath solution. Pipettes had tip resistances of 1.5–2 MΩ when filled with a solution containing (mM) KCl 140, MgCl2 1, HEPES 5, EGTA 10, and Na2ATP 2 (adjusted to pH 7.25 with KOH).For 2 1, HEPES 10, EGTA 10, and MgATP 5 (adjusted to pH 7.25 with KOH). The external solution contained (mM) NaCl 136, KCl 5.4, CaCl2 1, MgCl2 1, HEPES 10, and glucose 10 (adjusted to pH 7.36–7.38 with NaOH).To record HERG activity in the HEK 293 cells, the internal solution contained (mM) KCl 130, MgCl2+-free Tyrode's solution and then perfused in the Langendorff apparatus at 37°C. Perfusion with Ca2+-free Tyrode's solution for 5 min was followed first by 25 ± 5 min perfusion with low Ca2+ (0.1 mmol/L) Tyrode's solution containing 0.04–0.06 g/L collagenase and 0.5–0.8 g/L BSA and then 5 min perfusion with collagenase-free Tyrode's solution containing 0.5–0.8 g/L BSA. The heart was then minced and the cells were filtered through 200 μm nylon mesh, resuspended in Kraft-Brühe (KB) solution, and stored at room temperature (22–25°C) until use.Adult guinea pigs were fully anesthetized by intraperitoneal injection of urethane (40 mg/kg). The hearts were quickly removed and mounted on a Langendorff column, and cardiac myocytes were isolated as follows. The heart was dissected and rinsed in cold oxygenated Ca2 and plated on a glass culture dish 2-3 days before electrophysiological experiments. The HEK293 cells that stably expressed the wild type HERG gene were kindly provided by Professor Xiaoyan Liu . Dulbecco's Modified Eagle's Medium  supplemented with 10% (v/v) fetal bovine serum  and 1 mg/mL geneticin , the cultures were passed every 3–5 days by use of a brief trypsin treatment. The cells were maintained at 37°C in 5% COAll currents were recorded using the conventional whole-cell patch-clamp technique. Borosilicate glass electrodes had tip resistances of 1–3 MΩ when filled with the pipette solution. All experiments were performed at room temperature (22-23°C) using an Axopatch 200B amplifier . Adequate series resistances (less than five times the pipette resistances) were usually attained within 10 min after the gigaohm seal was formed. Measurements were taken using an Axopatch 200B amplifier (Axon Instruments). The current signals were filtered via a 4 kHz, 4-pole low-pass filter and digitized with an AD-DA converter  for subsequent analysis using pCLAMP 10.0 software.t-tests were used for statistical comparisons when appropriate, and differences were considered significant at P < 0.05.The data were analyzed with the use of Data Processing System (Version 7.05). All data are expressed as means ± SEM. Paired Student's IK using guinea pig ventricular myocytes. IK were recorded by applying voltage pulses ranging from −10 to +80 mV for 5 s from the holding potential of −40 mV, and the repolarization potential was maintained at a constant −30 mV for the IK,tail analysis. μM GA. Under control conditions, the depolarizing steps activated time-dependent outward currents. The amplitude of the outward currents measured at the end of the pulse (IK) increased with increasingly positive voltage steps. The current-voltage relationships for IK,step and IK,tail obtained at various concentrations of GA are shown in Figures IK,step and IK,tail decreased dose dependently . Meanwhile, the IK,tail evoked after repolarization to −30 mV was 1.57 ± 0.11 pA/pF under control conditions and decreased to 1.24 ± 0.12 pA/pF, 0.67 ± 0.15 pA/pF and 0.48 ± 0.06 pA/pF in the presence of 1, 5 and 10 μM GA, respectively . These results show that GA dose-dependently blocked IK,step and IK,tail.We first tested the effect of GA on  Figures . The IKIKr and IKs. Repolarization to 0 mV revealed IKs as a deactivating IKs,tail, and subsequent repolarization to −40 mV resulted in deactivation of IKr. We confirmed that 2 μM E-4031, a selective blocker of IKr, blocked the rapid component of the delayed rectifier K+ current but had no effect on IKs . The IKr,tail evoked after repolarization to −40 mV was 1.57 ± 0.11 pA/pF under control conditions and decreased to 0.57 ± 0.06 pA/pF, 0.29 ± 0.02 pA/pF and 0.21 ± 0.05 pA/pF in the presence of 1, 5 and 10 μM GA, respectively . This result shows that GA blocks the rapid and slow components of delayed rectifier K+ current.To investigate the effects of GA on the rapid and slow components of the delayed rectifier currents in guinea-pig ventricular myocytes, we used a voltage clamp protocol designed to separate the currents electrophysiologically. The holding potential was maintained at −40 mV. Our results revealed that depolarization to +60 mV activated both  on IKs . As showμM) was washed into the bath for 10 min, with the cell kept at the holding potential, before current recording commenced in the presence of the drug  during the test potentials and the tail current (Itail) after a test pulse to 0 mV . The fractional block of Istep was 20.46 ± 2.7% in the 1 mM group (P < 0.05), 29.93 ± 3.1% in the 5 mM group (P < 0.05), and 56.38 ± 4.2% in the 10 mM group (P < 0.05); the corresponding fractional blocks of Itail were 19.26 ± 2.5% (P < 0.05), 36.57 ± 3.0% (P < 0.05), and 55.29 ± 3.6% (P < 0.05), respectively . Thus, 5 μM GA had little effect on the voltage dependence of activation  (n = 5). From −120 to −20 mV, the time constants of inactivation were significantly lower in the presence of 5 μM GA  than in the absence of the drug.To measure inactivation, a special protocol was used that inactivated the channel at a holding potential of 40 mV, recovered the channel from inactivation at various potentials from −120 to 20 mV in 10 mV steps, and measured the resulting peak outward current at constant 20 mV as a measure of steady-state inactivation . Mean da, n = 5) . The tim, n = 5) ; n = 5. μM GA   . I-V protocol shown in μM GA did not change the deactivation rate significantly   .+ channels play a pivotal role in maintaining normal cardiac electrical activity. They regulate the resting membrane potential and excitability, participate in repolarization, and determine the shape and duration of cardiac action potential. A malfunction of the K+ channels due to either gene mutations or drug blockade alters not only cardiomyocyte excitability but also the electrical balance of depolarization and repolarization, which causes a long or short QT interval in the electrocardiogram (ECG) and underlies different types of cardiac arrhythmia [+ channels are important targets for antiarrhythmic drugs. Cardiac Krhythmia , 22. TheIKr, IKs) in guinea pig ventricular myocytes. The cardiac ion channel gene products that are targets for GA are unknown. However, previous studies have demonstrated that the peak and late INa, studied in the Xenopus oocytes expressing either human NaV1.5 or mutant NaV1.5-ΔKPQ of the α-subunit channel, were strongly reduced by GA [In the present study, we have firstly provided the evidence that the antiarrhythmic ionic mechanism of GA is related to the inhibition of potassium currents  may result in changes of currents and APD, even inducing arrhythmias. The inhibition of t effect  could let effect  proposedac death . Proarrhlated to –35 (a) e M cells . Thus, t M cells . It has  blocker .INa [IKr (HERG) and IKs but also inhibits ICa-L. The effect of GA on multiple channels might make it a promising antiarrhythmic that can lead the cardiac cell to restore normal sinus rhythm and prevent further arrhythmia. Further basic and clinical studies will be needed to explore whether GA has proarrhythmic actions. Control of cardiac electrical activity is well organized by an array of ion channels activated with a delicate balance between inward and outward ion currents . Upon reINa , 20. In IK  and the HERG potassium channel expressed in HEK293 cells. The results indicate that the antiarrhythmic activity and prolonged action potential of GA could be due to the blocking of IK  and the HERG channel. It is thought that a good antiarrhythmic drug should affect multiple channels and be able to restore normal sinus rhythm and keep patients free from further episodes of arrhythmia. GA not only blocks the HERG channel, IK , but also inhibits INa, so the results reveal that it has significant potential for development as a novel antiarrhythmic agent, particularly targeting the genesis of arrhythmias. Therefore, our findings might help to elucidate the traditional use of licorice in the treatment of cardiovascular disorders. Nonetheless, further evaluation of the therapeutic potential of GA is warranted. In conclusion, our study demonstrated that GA has an inhibitory effect on


To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20–80 μm control oocytes at three imprinted genes, Snrpn, Peg3, and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition,individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3, or Peg1 in oocytes from Esr2-deficient females, and no perturbation in Snrpn or Peg3de novo methylation in oocytes from Gja4-null females. However, Gja4 deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development.Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation, and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt  BWS is an overgrowth disorder that is caused by imprinting defects that result in a gain of maternal methylation at the t 1) ICR . SRS is  domains . AS is aRPN) ICR . SporadiRPN) ICR . For AS,RPN) ICR . This raDevelopment of healthy oocytes is dependent on interactions between the growing oocyte and surrounding follicular cells . OocytesEsr2) gene are subfertile, producing fewer oocytes following superovulation, as well as litters with fewer pups  augments the actions of follicle stimulating hormone (FSH). In the ovary, ERβ is expressed primarily in granulosa cells and at low levels in the oocyte . Female wer pups . Attenuaimpaired . Mechaniimpaired . ERβ defGja4) gene causes arrested folliculogenesis at the early antral stage, impaired oocyte development and meiotic competency, and premature luteinization of the follicles . These channels are essential for communication between neighboring cells . In the ollicles .Esr2–/– and Gja4–/– genetic models to interfere specifically with endocrine signaling and gap junctional communication, compromising fertility. We hypothesized that inhibition of the ERβ pathway and/or oocyte–granulosa cell gap junctional communication would lead to perturbations in imprinted methylation acquisition. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20–80 μm diameter control oocytes at three imprinted genes, Snrpn, Peg3, and Peg1 . Similar to previous studies  at 10, 14, 21, and 28 days postpartum (dpp), and placed in Waymouth MB 752/1 medium (Invitrogen) supplemented with 10% fetal bovine serum . For furGja4–/– female mice (C57BL/6 background) at 21 and 28 dpp, and placed in Waymouth MB 752/1 medium (Invitrogen) supplemented with 10% fetal bovine serum. Gja4-null oocytes were retrieved through the same collection method as control oocytes and placed in 30 μl M2 medium (Sigma) for further analysis.Ovaries were removed from Esr2–/– females (C57BL/6 background) at 28 dpp and placed in a 100-mm cell culture dish containing 15 ml ice-cold M199 medium (Sigma) supplemented with 1 mg/ml BSA (Invitrogen) and 2.5 g/ml gentamicin  was used to compare the Gja4 deficient to control oocytes. A p-value of <0.05 was taken to be statistically significant.For each imprinted gene, significant difference of CpG methylation percentage was determined by a two-tailed Mann–Whitney In female mammals, imprinted DNA methylation has been shown to arise during follicle growth from the primary to the antral stage in correlation with oocyte diameter , with gede novo methylation acquisition at the Snrpn ICR showed mean methylation levels of 8.7% in 20–40 μm, 12.6% in 40–45 μm, 9.3% in 45–50 μm, 39.3% in 50–55 μm, 82.7% in 55–60 μm, 97.0% in 60–65 μm, 82.8% in 65–70 μm, 93.8% in 70–75 μm, and 98.0% in 75–80 μm oocytes . Likewise, mean methylation levels at the Peg3 DMR were 1.6% in 20–40 μm, 11.2% in 40–45 μm, 16.1% in 45–50 μm, 22.9% in 50–55 μm, 47.5% in 55–60 μm, 51.7% in 60–65 μm, 82.6% in 65–70 μm, 85% in 70–75 μm, and 94.0% in 75–80 μm oocytes . For the Peg1 DMR, mean methylation levels were 4.3% in 20–40 μm, 4.7% in 40–45 μm, 12.2% in 45–50 μm, 15.9% in 50–55 μm, 45.5% in 55–60 μm, 51.6% in 60–65 μm, 91.0% in 65–70 μm, 92.0% in 70–75 μm, and 93.2% in 75–80 μm oocytes . Thus, we observed that each gene had its own acquisition kinetics. DNA methylation acquisition began first for Snrpn at ~50 μm and was near completion at >60 μm. Next was Peg3, where DNA methylation acquisition was initiated at ~45 μm and nearly complete at >65 μm, which was followed by Peg1, where DNA methylation acquisition began at ~55 μm and was near completion by >70 μm. Snrpn had the shortest acquisition interval while Peg3 had the longest.Analysis of Esr2 produce a reduced number of maturing oocytes, but those that do mature appear to not be developmentally compromised . For the Peg3 DMR, mean methylation was 100.0% in 65–70 μm, 99.4% in 70–75 μm, and 98.7% in 75–80 μm oocytes . For the Peg1 DMR, mean methylation levels were 96.5% for 65–70 μm, 95.1% for 70–75 μm, and 96.5% for 75–80 μm oocytes . Thus, oocytes from Esr2-null females had comparable DNA methylation levels to control oocytes, indicating that imprint DNA methylation acquisition was unaffected by Esr2 deficiency.Ovaries deficient in promised . ConsistGja4-null 21 dpp females, which ranged in diameter sizes from 35 to 55 μm and from 28 dpp females, which ranged in size from 50 to 60.5 μm. The maximum diameter obtained was 60.5 μm, consistent with previous studies . No significant difference was observed in methylation levels between Gja4-null and control oocytes. Analysis at the Peg3 DMR showed mean methylation levels of 2.7% for 35–40 μm, 74.0% in 40–45 μm, 28.0% for 45–50 μm, 50.7% for 50–55 μm, and 55.3% for 55–60 μm oocytes . No significant difference was observed in methylation levels between Gja4-null and control oocytes. For the Peg1 DMR, mean methylation levels were 1.8% in 35–40 μm, 2.8% in 40–45 μm, 9.7% in 45–50 μm, 14.3% in 50–55 μm, and 19.1% in 55–60 μm oocytes . Statistical analysis of Peg1 showed a significant difference in methylation acquisition between control and Gja4-deficient oocytes (P = 0.0006). Because Gja4-null oocytes stop growing and are eventually lost from the follicles, it could not be determined whether this is a delay or a disruption in Peg1 DNA methylation acquisition.At the de novo methylation acquisition. Individual oocytes from Esr2- and Gja4-deficient mice were assessed for DNA methylation establishment at Snrpn, Peg3, and Peg1. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3, or Peg1 in oocytes from Esr2-deficient females, and no perturbation in Snrpn or Peg3de novo methylation in oocytes from Gja4-null females. However, Gja4 deficiency resulted in a loss or delay in methylation acquisition at Peg1. One possible explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine whether subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis has an effect post-fertilization on imprint maintenance in the preimplantation embryo.Growth and maturation of oocytes within follicles requires bidirectional signaling and exchange of nutrients, metabolites, and second messengers through gap junctions between the oocyte and granulosa cells . AberranSnrpn,Peg3, and Peg1 in individual oocytes. We observed that each gene has its own size-dependent acquisition kinetics. Snrpn had the shortest acquisition interval with denovo methylation beginning at ~50 μm and near completion at >60 μm. Peg3 had the earliest and longest acquisition interval. DNA methylation acquisition was initiated at ~45 μm and was nearly complete at >65 μm. Peg1 had the latest acquisition of de novo methylation, beginning at ~55 μm and near completion by >70 μm. Previous studies reported similar findings using pooled oocytes where methylation level increased with days postpartum, follicular stage or with oocyte diameter/size, and initiation of acquisition was gene-specific . Secondly, for oocytes within each diameter range , a subset of oocytes had high methylation percentages  while others had low methylation percentages . Finally, scatter plots show two distinct cohorts within the same range of diameter measurements. For example, Peg1 control oocytes between 55 and 65 μm grouped into 0–40% methylation and 75–100% methylation .Our study is the first to investigate imprint methylation acquisition of specific . Oocyte-and Peg1 . Our datGja4-deficient oocytes ceased development and did not achieve mature size, our analyses indicated that they were not compromised in their ability to catalyze DNA methylation as de novo DNA methylation was initiated for the Snrpn and Peg3 imprinted genes. The failure to initiate Peg1 methylation acquisition may simply be due to the fact that oocytes lacking CX37 never reach the size necessary for de novo methylation to commence at late-acquiring loci. However, control oocytes of comparable size (55–60 μm) displayed initiation of de novoPeg1 methylation. This suggests that Peg1 methylation acquisition was lost or delayed in mutant oocytes. Alternatively, CX37-null oocytes may have reduced stores of methyl donors or other metabolites required for DNA methylation that would normally be transported from granulosa cells to the oocyte via gap junctions. If this is the case, then there must have been sufficient availability of methyl donors in mutant oocytes for Snrpn and Peg3 de novo methylation, but oocytes lacking junctional coupling with the granulosa cells may have exhausted their methyl donors during oocyte growth, preventing de novo methylation at late-acquiring genes like Peg1. To investigate the requirement for methyl donors during follicle development, Snrpn ICR and the Peg3 DMR. However, a reduced level of DNA methylation was found at the Peg1 DMR. This provides support for the argument that gap junctional communication provides important metabolites for DNA methylation acquisition. To better understand the mechanism leading to loss or delayed methylation acquisition, further studies are required to assess the level of methyl donors, amount of S-adenosylmethionine, and ability to carry out global and gene-specific methylation in 55–60 μm CX37-null or CX37-depleted oocytes. Furthermore, methylation studies should be carried out using F1 females. For Peg1 CX37 oocytes between 45 and 60 μm, oocytes possessed 0–53% methylation. DNA methylation acquisition was likely initiated on the maternal Peg1 allele in some oocytes, while other oocytes lacked methylation on both parental alleles. Thus, loss or delayed Peg1 methylation acquisition may preferentially lead to a failure of the paternal allele to become methylated. Further studies are required to investigate this potential grandpaternal effect.While Peg1 may also be more susceptible to perturbation by assisted reproductive technologies. Loss of Peg1/PEG1 methylation was observed in mouse oocytes following in vitro maturation (Peg1 to perturbation relates to its late acquisition of methylation or whether a different epigenetic regulatory mechanism(s) operates at this gene. Superovulation also caused imprinting errors in the mouse preimplantation embryo (Peg1 methylation acquisition but may also lead to inadequate stores of maternal products, including those from granulosa cells, that may disrupt imprint maintenance at Peg1 as well as at Snrpn and Peg3 during preimplantation development. Extending studies to preimplantation embryos generated from fertilized ERβ-deficient and CX37-depleted oocytes will be required to determine their effects on imprint maintenance. In addition, further studies are required to determine whether assisted reproductive technologies, such as in vitro oocyte maturation and superovulation, lead to aberrant endocrine and paracrine signaling as well as granulosa cell–oocyte gap junctional communication.turation , and humturation . Furthern embryo , althougn embryo . We hypoIt is important to understand granulosa cell–oocyte communication as technological advances move forward. Procedures such as slow-freezing cryopreservation and ultra-fast vitrification of oocyte-enclosed follicles, which employ cryoprotectants and very low temperatures, may permanently or temporally disrupt actin- or tubulin-rich projections that extend from granulosa cells through the zona pellucida to the oocyte . Slow-frContinued studies in animal models and in humans are required to understand the molecular mechanisms regulating genomic imprinting acquisition and maintenance as well as how impaired fertility and assisted reproductive technologies induce epigenetic changes and disease.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.