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Iron is an essential trace element for normal body’s functions. The restriction of iron availability directly limits erythropoiesis. The objective of this experiment was to compare the bioavailability of iron nanoparticles (Fe-NPs) with iron-microparticles (Fe-MPs) in anemic mice. There were four experimental groups including normal control group, iron-deficiency anemia (IDA) group, Fe-NPs group, and Fe-MPs group. Animals in normal group fed on an adequate iron-containing diet (45 ppm Fe). Meanwhile, the animals in the other three groups fed on a low Fe diet (4.5 ppm Fe) for 7 weeks. Double deionized water was supplied as drinking water ad libitum. After feeding for 3 weeks with the low Fe diet, the animals in Fe-NPs and Fe-MPs groups were orally treated with Fe-NPs or Fe-MPs at the daily dose of 40 mg/kg for 4 weeks. IDA group showed a markedly decreased red blood cell (RBC) count, hematocrit (Hct), and hemoglobin (Hb) values compared with normal group throughout experimental periods. Treatments of Fe-NPs or Fe-MPs for 4 weeks restored the decreased RBC count and hematological values similar to normal values. Fe-NPs group showed the faster restoration in the values than Fe-MPs as time passes. The iron contents of the upper small intestine in Fe-NPs and Fe-MPs groups were higher than normal group at weeks 2 and 4. Hepatic iron contents and lipid peroxidation were significantly increased by Fe-NPs and Fe-MPs treatments compared with IDA group as time increase. The iron contents in liver and ferritin deposits in spleen were identified in Fe-NPs and Fe-MPs groups similarly to normal group. These results indicate that oral administration of both Fe-NPs and Fe-MPs can recover anemia and Fe-NPs is more absorbable and available in the body than Fe-MPs.

Magnolia bark contains several compounds such as magnolol, honokiol, 4-O-methylhonokiol, obovatol, and other neolignan compounds. These compounds have been reported to have various beneficial effects in various diseases. There is sufficient possibility that ethanol extract of Magnolia officinalis is more effective in amyloidogenesis via synergism of these ingredients. Neuroinflammation has been known to play a critical role in the pathogenesis of Alzheimer's disease (AD). We investigated whether the ethanol extract of M. officinalis (10 mg/ kg in 0.05% ethanol) prevents memory dysfunction and amyloidogenesis in AD mouse model by intraperitoneal lipopolysaccharide (LPS, 250 µg/ kg/day for seven times) injection. We found that ethanol extract of M. officinalis prevented LPS-induced memory deficiency as well as inhibited the LPS-induced elevation of inflammatory proteins, such as inducible nitric oxide synthase and cyclooxygenase 2, and activation of astrocytes and microglia. In particular, administration of M. officinalis ethanol extract inhibited LPS-induced amyloidogenesis, which resulted in the inhibition of amyloid precursor protein, beta-site amyloid-precursor-protein-cleaving enzyme 1 and C99. Thus, this study shows that ethanol extract of M. officinalis prevents LPS-induced memory impairment as well as amyloidogenesis via inhibition of neuroinflammation and suggests that ethanol extract of M. officinalis might be a useful intervention for neuroinflammation-associated diseases such as AD. Copyright © 2012 John Wiley & Sons, Ltd.

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.

A series of novel 4- O -methylhonokiol analogs were synthesized in light of revealing structure–activity relationship for inhibitory effect of COX-2 enzyme. The key strategy of the molecular design was oriented towards modification of the potential metabolic soft spots (e.g., phenol and olefin) or by altering the polar surface area via incorporating heterocycles such as isoxazole and triazole. Most of all exhibited the inhibitory effects on COX-2 and PGF 1 production but not macrophage NO production. Especially, aryl carbamates 10 and 11 exhibited more potent inhibitory activity against COX-2 and PGF 1 production.

The efficiency to produce offspring by somatic cell nuclear transfer (SCNT) is low. It has been showed that treatment of donor cells with Xenopus oocyte extract increased live births in ovine and handmade cloned embryo development in pigs. Scriptaid treatment after oocyte activation is another approach to improve SCNT efficiency. The present study was carried out to investigate (a) the effects of treatment of donor cells with Xenopus egg extract on donor cell DNA methylation at days 0 and 4 with two digitonin permeabilization concentrations (10 and 15 μg/mL), (b) the effects of treatment of donor cells with Xenopus egg extract on early development of cloned embryos, and (c) the effects of combined treatments, treating donor cells with extract before nuclear transfer and treatment of cloned embryos with scriptaid after oocyte activation, on embryo development. Compared to the control, a decrease of DNA methylation in donor cells was observed at 2.5 h after extract treatment. However, this effect was not observed after the cells were cultured for four more days. More embryos developed into blastocysts in the Xenopus egg extract-treated group than in the control (13.4±1.9% vs. 9.1±1.9%, p=0.01). Furthermore, scriptaid treatment of cloned embryos further increased the frequency of development to blastocyst, compared to the control reconstructed with the same extract-treated cells (22.5±0.9% vs. 15.3±0.9%, p

A series of carbamic acid 1-phenyl-3-(4-phenyl-piperazine-1-yl)-propyl ester derivatives were synthesized through discovery strategies for balancing target-based in vitro screening and phenotypic in vivo screening. All the newly synthesized compounds were screened for their analgesic activities and compared with standard drug morphine. Among them, compound 44r, a potent analgesic agent that has favorable pharmacokinetic properties in rats and most importantly, has a wide safety margin. We demonstrated with in vitro and in vivo functional assays that its analgesic activity might be through 5-HT2A antagonism to some extent. Hence, it is concluded that there is ample scope for further study in developing compound 44r as a good lead candidate for an analgesic agent.

In this study, we investigated the effect of methanolic extract isolated from the root of Lycoris aurea (LA) on the growth of cancer cells and the tube formation activity of endothelial cells. Various cancer cells were treated with LA at doses of 0.3, 1, 3, 10 or 30 μg/ml and LA significantly suppressed the growth of several cancer cell lines, including ACHN, HCT-15, K-562, MCF-7, PC-3 and SK-OV-3, in a dose-dependent manner. We also found that LA induced cell cycle arrest at G2/M phase in ACHN renal cell adenocarcinoma cells. Further study demonstrated that LA concentration-dependently inhibited the tube formation, which is a widely used in vitro model of reorganization stage of angiogenesis, in human umbilical vein endothelial cells. Collectively, these results show that LA inhibits the growth of cancer cells and tube formation of endothelial cells and the growth-inhibitory effect of LA might be mediated, at least in part, by blocking cell cycle progression.

Concise and practical synthesis of 4-O-methylhonokiol was achieved in 34% overall yield. The key features of our synthesis include chemoselective ortho-mono bromination of phenol as well as biaryl formation via Suzuki–Miyaura cross-coupling, in which bromophenol was reacted with potassium aryltrifluoroborate using Pd(OAc)2 and RuPhos under microwave conditions.

Maintenance of adequate telomere length in developing cells is the most important concern to preserve the integrity of the genome. The length of telomere is strictly regulated by numerous telomere-binding proteins and/or interacting factors. Even though the expression of telomerase in the male reproductive tract has been characterized, developmental expressional profiling of telomerase and other telomere-associated proteins has not been determined in detail. The present study was attempted to examine expression patterns of catalytic subunit (Tert) and RNA component (Terc) of telomerase and two telomerase associated factors, telomerase associated protein 1 (Tep1) and TERF1 (TRF1) interacting nuclear factor 2 (Tinf2) in the testis and seminal vesicle of male rat during postnatal development. The real-time PCR analysis was utilized to quantify mRNA expression of molecules. The abundance of Tep1 mRNA in the testis and seminal vesicle was the highest at 5 months of age. Expressional fluctuation of Tinf2 during postnatal development was found in the testis, while expression of Tinf2 in the seminal vesicle was gradually increased until 5 months of age and then significantly decreased later. mRNA level of Tert gene in the testis was significantly increased at the adult and the elder, while the highest expression of Tert gene in the seminal vesicle was found at 5 months of age. Expression of Terc transcript in the testis and seminal vesicle was the highest at 5 months of age, followed by significant reduction at 1 and 2 years of ages. Such differential gene expression of telomere-associated factors and telomerase components in different male reproductive tissues during postnatal development indicates that maintenance of telomere length would be regulated in tissue- and/or age-specific manners. (Key words : Telomerase, Testis, Seminal vesicle, Development, Telomerase-associated molecule)

GnRH neurons are hypothalamic neurons that secrete gonadotropin-releasing hormone (GnRH) which stimulates the release of gonadotropins, one of the crucial hormones for sexual development, fertility and maturation. A mathematical model was built to help elucidate the mechanisms underlying electrical bursting and synchronous [Ca 2+ ] transients in GnRH neurons ( Lee et al., 2010 ). The model predicted that bursting in GnRH neurons (at least of the short-bursting type) requires the existence of a [Ca 2+ ]-dependent slow after-hyperpolarisation current (s I AHP-UCL ), and this predicted current was found experimentally. GnRH behaviour under a wide range of conditions (inhibition of Na + channels, IP 3 receptors, [Ca 2+ ]-dependent K + channels, or Ca 2+ pumps, or in the presence of zero extracellular [Ca 2+ ]) is successfully reproduced by the model. In this paper, a simplified version of the previous model, with the same qualitative behaviour, is constructed and studied using timescale separation techniques and bifurcation analysis.

To develop a therapeutic agent for obesity-related metabolic disorders, a mixture of dietary components was prepared, including grape extract, green tea extract and l-carnitine (RGTC), and its effects on obesity, hyperlipidemia and non-alcoholic fatty liver disease examined. The RGTC dramatically inhibited the high-fat diet (HFD)-induced increase in body weight and fat in C57BL/6 mice, whereas food consumption was not affected by RGTC treatment. The RGTC also concentration-dependently suppressed the HFD-induced increase in plasma lipids, such as low-density lipoprotein cholesterol and triglycerides. In addition, increases in liver weight and liver steatosis were returned to normal by RGTC treatment in HFD-fed C57BL/6 mice. The plasma levels of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase were also significantly down-regulated by RGTC treatment. These results suggest that RGTC suppressed HFD-induced obesity, hyperlipidemia and non-alcoholic fatty liver disease, suggesting that RGTC supplementation might be a promising adjuvant therapy for the treatment of these metabolic disorders.

Histone deacetylases (HDACs) are involved in post-translational modification and gene expression. Cancer cells recruited amounts of HDACs for their survival by epi-genetic down regulation of tumor suppressor genes. HDACs have been the promising targets for treatment of cancer, and many HDAC inhibitors have been investigated nowadays. In previous study, we synthesized δ-lactam core HDAC inhibitors which showed potent HDAC inhibitory activities as well as cancer cell growth inhibitory activities. Through QSAR study of the δ-lactam based inhibitors, the smaller core is suggested as more active than larger one because it fits better in narrow hydrophobic tunnel of the active pocket of HDAC enzyme. The smaller γ-lactam core HDAC inhibitors were designed and synthesized for biological and property optimization. Phenyl, naphthyl and thiophenyl groups were introduced as the cap groups. Hydrophobic and bulky cap groups increase potency of HDAC inhibition because of hydrophobic interaction between HDAC and inhibitors. In overall, γ-lactam based HDAC inhibitors showed more potent than δ-lactam analogues.

Melanin, the main component of skin color, is synthesized in the melanosomes of melanocytes through a physiological process called melanogenesis and then transferred to neighboring keratinocytes. Melanin absorbs high-energy UV radiation and transforms it into the thermal energy. In this way, melanin plays a protective role against UV-induced DNA damage. However, abnormal skin pigmentation is a major dermatological concern. Abnormal production of melanin causes hypopigmentation or hyperpigmentation such as vitiligo, melasma, age spot, and freckle. In addition, it has been reported that alteration in melanin production is observed in melanoma and Parkinson’s disease. Although key factors including melanocortin 1 receptor (MC1R), Microphthalmia associated transcription factor (MITF) and tyrosinase have been found, the molecular mechanism of melanogenesis is not yet fully understood. Potent chemical regulators for melanogenesis could be a useful tool to elucidate the complex mechanisms. In particular, the regulation of dyschromias is of growing interest, and further options for the treatment of hyperpigmentation are in great demand across Asia. Such a demand has led to the discovery of a variety of natural or synthetic chemicals to regulate melanin biosynthesis (Figure 1). Most of the reported melanogenesis inhibitors decrease tyrosinase activity, and a few small molecules such as arbutin and kojic acid are available on the market as skin lightening agents. However, arbutin has shown insufficient activity and kojic acid has toxicity issue. We have reported cyclohepta[d]isoxazole derivatives as novel melanogenesis inhibitors that decrease the expression of tyrosinase. Investigation of the Structure-activity relationship (SAR) has been explored through modification of regions B and C, which led to the identification of potent melanogensis inhibitors in our previous study (Figure 1). In order to explore and further extend the SAR of this series of melanogenesis inhibitors, a variety of the ring contracted derivatives were synthesized. Herein, we describe the synthesis and melanogenesis activity of 4,5,6,7-tetrahydrobenzo[d]isoxazole derivatives. Compounds 4a-j were synthesized by amide coupling of arylpiperazines with 4,5,6,7-tetrahydrobenzo[d]isoxazole-3carboxylic acid chloride in the presence of triethylamine in dry THF. The acid chlorides 3a-b were obtained by reaction of commercially available 4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxylic acid with oxalyl chloride and a catalytic amount of DMF in dichloromethane (Scheme 1). O-alkylation of 4f with iodoethane gave compound 5 (Scheme 2). Melanogenesis activity of melanin synthesis inhibition is shown in Table 1. The para-methoxy derivative 4b exhibited slightly greater potency than the phenol 4a. However, cytotoxicity was observed at 10 M. A shift of the methoxy group on the aryl group from the para to ortho position (4c) greatly reduced activity. The introduction of trifluoromethyl at para position (4d) resulted in almost complete loss of potency at even 10 μM. The para-fluoro substituent (4j) did not improve activity. Next, we synthesized compounds removing tert-butyl group in region A. Phenol 4f retained activity at 1 μM but showed cytotoxicity at 10 μM. Figure 1. Representative melanin biosynthesis inhibitors and a validated hit compound (MMS1001) as a down-regulator of tyrosinase expression. Scheme 1. (a) oxalyl chloride, DMF (cat.), CH2Cl2, rt, 2 h; (b) arylpiperazines, Et3N, THF, rt, 1 h, 60-79% for 2 steps.

The hypoxia inducible factor 1 (HIF-1) is a heterodimeric transcription factor which is an important regulator of growing tumor’s response to hypoxia. It was reported that HIF-1 acti-vates genes that allow the cancer ce lls to survive and grow in the hostile hypoxic tumor environment. Furthermore, the activity of HIF-1 has been shown to be central to regulation of apoptosis and cell survival, cell adhesion and extracellular matrix turn-over, cytoskeletal stru cture, cell motility, epithelial homeostasis, vascular tonus, eythropoiesis and iron metabolism, metabolic homeostasis, and pH regulation.HIF-1 is a heterodimeric protein that consists of a constitu-tively expressing nuclear-located β-subunit, also termed aryl hydrocarbon nuclear translocator (ARNT) and a O

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

The novel δ-lactam based HDAC inhibitor, KBH-A118 (3) shows a good HDAC enzyme and cancer cell growth inhibitory activities but has undesirable pharmacokinetics profiles because of instability in mouse liver microsome. To improve metabolic stability, various analogues were prepared with substituents on aromatic ring of cap group and various chain lengths between the cap group and δ-lactam core. The newly prepared analogues showed moderate to potent in vitro activities. Among them six compounds (8a, 8e, 8j, 8n, 8t, and 8v) were evaluated on mouse liver microsome assay and it turned out that the microsomal stabilities were dependent on lipophilicity and the number of the rotatable bonds. Finally, the animal pharmacokinetic profiles of 8e displayed improving oral exposure and oral bioavailability.

Panel speakers often adopts the vibration actuator attached to a plate center to excite the whole panel. When a thin rectangular plate is excited by a point force, the generated bending wave is reflected quickly from the edges, so the plate is governed by the reverberant field. Because many vibrational modes participate even in the low frequencies, the radiated sound spectrum is involved with many peaks and troughs resulting a poor sound quality. To minimize the modal participation, the vibration is rendered to be confined and in-phase within a circular area enclosing the actuator point, and the vibration is being suppressed outside of it. An additional multi-layer actuator array surrounding the speaker zone is employed to control the vibration, thus fulfilling the rendered field. The study aim is now to obtain an appropriate gain of the actuators by solving the inverse problem consisting of the transfer matrix between field points and control actuators. The effect of the number of arrays is tested for the radius of 0.05–0.2 m. The control result reveals that the signal-to-noise ratio in the speaker and baffle zone is improved 8–11 dB by the three-layer array than by the single-layer array with the same size.Panel speakers often adopts the vibration actuator attached to a plate center to excite the whole panel. When a thin rectangular plate is excited by a point force, the generated bending wave is reflected quickly from the edges, so the plate is governed by the reverberant field. Because many vibrational modes participate even in the low frequencies, the radiated sound spectrum is involved with many peaks and troughs resulting a poor sound quality. To minimize the modal participation, the vibration is rendered to be confined and in-phase within a circular area enclosing the actuator point, and the vibration is being suppressed outside of it. An additional multi-layer actuator array surrounding the speaker zone is employed to control the vibration, thus fulfilling the rendered field. The study aim is now to obtain an appropriate gain of the actuators by solving the inverse problem consisting of the transfer matrix between field points and control actuators. The effect of the number of arrays is tested for th...

Chroman derivatives exhibited potent inhibitory activity of NF-kappaB. For SAR, the chroman scaffold was modified with an indoline moiety. A series of indoline-2-carboxylic acid N-(substituted)phenylamide derivatives were synthesized to explore their inhibitory activities of NF-kappaB and they were also evaluated for cytotoxicity against various cancer cell lines. Since intermediates with Boc showed outstanding results, various substituents in place of the Boc group were introduced additionally and these compounds were also evaluated for SAR.

In this letter, we propose a throughput-efficient type-II hybrid automatic reQuest (HARQ) scheme considering quality of service (QoS) to transmitted packets. The proposed algorithm determines the amount of redundancy bits for the (re)transmissions of a HARQ packet while satisfying the given packet outage probability. The throughput performance of the suggested type-II HARQ scheme with low density parity check (LDPC) code is evaluated under both the changing channel conditions and the maximum number of retransmission determined by the allowed packet delay. Result shows that the proposed algorithm achieves higher throughput than the typical type-II HARQ scheme.

Studies undertaken in many species indicate that kisspeptin-Gpr54 signaling is essential for the activation of gonadotropin-releasing hormone (GnRH) neurons to bring about puberty. Investigations in transgenic mouse models, in particular, have highlighted the importance of kisspeptin signaling at the level of the GnRH neuron itself in this process. This review aims to highlight current understanding of the neurobiological mechanisms underlying the kisspeptin activation of postnatal GnRH neurons. The three key features of the kisspeptin-Gpr54-GnRH neuron axis leading up to puberty are (i) the expression of adult-like levels of Gpr54 mRNA in GnRH neurons well in advance of puberty, (ii) a modest increase in the electrical response of GnRH neurons to Gpr54 activation across postnatal development and (iii), the "sudden" appearance of kisspeptin fibers surrounding GnRH neuron cell bodies/proximal dendrites just prior to puberty onset. These kisspeptin fibers are likely to originate from the kisspeptin population located in the rostral periventricular region of the third ventricle (RP3V). Together, available data suggest that the key step in the kisspeptin control of puberty lies in the control of kisspeptin synthesis within RP3V kisspeptin neurons that innervate GnRH neurons. This has recently been shown to be dependent upon circulating estradiol concentrations. As such, we propose that RP3V kisspeptin neurons represent a critical estradiol-dependent amplification mechanism brought into play relatively late in pubertal development to activate GnRH neurons and complete the process of puberty onset. Subsequently, in the adult female, this same circuitry is used to activate GnRH neurons to generate the cyclical preovulatory GnRH/LH surge.

Neuronal activity in the paraventricular nucleus (PVN) is known to be elevated in rats with heart failure. However, the type of neurons involved and the underlying synaptic mechanisms remain unknown. Here we examined spontaneous firing activity and synaptic currents in presympathetic PVN neurons in rats with myocardial infarction (MI), using slice patch clamp combined with the retrograde labeling technique. In PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM), MI induced a significant increase in basal firing rate (1.79 to 3.02 Hz, P < 0.05) and a reduction in the frequency of spontaneous ( P < 0.05) and miniature ( P < 0.01) inhibitory postsynaptic currents (IPSCs). In addition, MI induced an increase in the paired-pulse ratio of evoked IPSCs ( P < 0.05). Bicuculline, a GABAA receptor antagonist, increased the firing rate of PVN-RVLM neurons in sham-operated (1.21 to 2.74 Hz, P < 0.05) but not MI ( P > 0.05) rats. In contrast, in PVN neurons projecting to the intermediolateral horn of the spinal cord (PVN-IML), MI did not induce any significant changes in the basal firing rate and the properties of spontaneous and miniature IPSCs. The properties of spontaneous excitatory postsynaptic currents (EPSCs) were not altered in either neuron group. In conclusion, our results indicate that MI induces an elevation of firing activity in PVN-RVLM but not in PVN-IML neurons and that the elevated firing rate is largely due to a decrease in GABA release. These results provide evidence for a novel target-selective synaptic plasticity in the PVN that is associated with the sympathetic hyperactivity commonly seen in heart failure.

The gonadotropin-releasing hormone (GnRH) neurons represent the key output cells of the neuronal network controlling fertility. Intracellular calcium ion concentration ([Ca(2+)](i)) is likely to be a key signaling tool used by GnRH neurons to regulate and co-ordinate multiple cell processes. This review examines the dynamics and control of [Ca(2+)](i) in GT1 cells, embryonic GnRH neurons in the nasal placode culture, and adult GnRH neurons in the acute brain slice preparation. GnRH neurons at all stages of development display spontaneous [Ca(2+)](i) transients driven, primarily, by their burst firing. However, the intracellular mechanisms generating [Ca(2+)](i) transients, and the control of [Ca(2+)](i) by neurotransmitters, varies markedly across the different developmental stages. The functional roles of [Ca(2+)](i) transients are beginning to be unraveled with one key action being that of regulating the dynamics of GnRH neuron burst firing.

The amino acid γ-aminobutyric acid (GABA) is thought to play a key role in shaping the activity of the GnRH neurons throughout embryonic and postnatal life. However, the physiological roles of direct GABA inputs to GnRH neurons remain unknown. Using a Cre-LoxP strategy, we generated a targeted mouse line, in which all (98 ± 1%) GnRH neurons had the γ2-subunit of the GABAA receptor deleted. Electrophysiological recordings of GABAA-mediated postsynaptic currents from green fluorescent protein-tagged GnRH neurons with the γ2-subunit knocked out (GnRH γ2 KO) showed that the amplitude and frequency of GABAA postsynaptic currents were reduced by 70% (P < 0.01) and 77% (P < 0.05), respectively, and that the response to exogenous GABA was reduced by 90% (P < 0.01). Evaluation of male and female GnRH γ2 KO mice revealed completely normal fecundity, estrous cycles, and puberty onset. Further investigation with gonadectomy and different steroid replacement regimens showed normal basal levels of LH in both sexes, and a normal estradiol-evoked positive feedback mechanism in females. However, the increment in LH after gonadectomy in GnRH γ2 KO female mice was double that of controls (P < 0.05) and also more potently suppressed by 17-β-estradiol (P < 0.05). A similar but nonsignificant trend was observed in GnRH γ2 KO male mice. Together, these findings show that 70–90% reductions in the normal levels of GABAA receptor activity at the GnRH neuron appear to impact upon the estrogen negative feedback mechanism but are, nevertheless, compatible with normal fertility in mice.

Retroviral gene transfer technology is frequently used to establish stable transgenic cell lines. However, no studies to date have evaluated antioxidant defense systems in cells infected with retroviral particles. In the present study, we examined the effects of retroviral infection on antioxidant defense systems using H4IIE cells infected with a retrovirus that overexpresses green fluorescent protein (retro-H4IIE cells). Total oxyradical scavenging capacity and glutathione (GSH), malondialdehyde, and peroxide levels were not significantly altered in retro-H4IIE cells; however, retro-H4IIE cells showed a higher resistance against cytotoxicity, GSH depletion, and malondialdehyde elevation under H(2)O(2)-induced oxidative stress conditions. Immunoblot analysis showed that alpha-class GSH S-transferase (GST) was increased 2.5-fold in retro-H4IIE cells as compared with H4IIE cells; however, catalase, GSH peroxidase-1, peroxiredoxin-1, and thioredoxin-1 remained unaltered or slightly decreased. l-Buthionine-(S,R)-sulfoximine, a GSH synthesis inhibitor, and 1-chloro-2,4-dinitrobenzene, a GST substrate and competitive inhibitor, decreased the difference in H(2)O(2) responses between the two cell types. These results support the hypothesis that the resistance of retro-H4IIE cells to H(2)O(2) can be attributed to an increase in alpha-class GST expression, as levels of GSH and GSH peroxidase-1 were not altered. The present study suggests that antioxidant enzyme expression may change during the establishment of stable transformed cell lines using retroviral techniques.

A lipid-soluble ginseng extract possesses an anticancer activity against advanced human lung tumors in an established murine xenograft model without any sign of toxicity. The extract also shows a potent anti-proliferative activity against human cancer cell lines, leading to expansion of its preclinical applications to specific human tumor types.

Tumor necrosis factor alpha (TNF-alpha) is a major inflammatory cytokine that plays an important role in the development of various inflammatory diseases. TNF-alpha has been considered as a potential therapeutic target for the treatment of chronic inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. In this study, we report that cyclopropyl-{4-[4-(4-fluorophenyl)-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl}amine (DBM1285) is a novel inhibitor of TNF-alpha production. DBM1285 concentration-dependently inhibited lipopolysaccharide (LPS)-induced TNF-alpha secretion in various cells of macrophage/monocyte lineage, including mouse bone marrow macrophages, THP-1 cells, and RAW 264.7 cells. However, LPS-induced mRNA expression of TNF-alpha was not affected by DBM1285 in these cells. Further studies demonstrated that the inhibitory effect of DBM1285 on TNF-alpha production might be mediated by post-transcriptional regulation through the modulation of the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2 (MK2) signaling pathway. We also confirmed that DBM1285 directly inhibits p38 MAPK enzymatic activity. In vivo administration of DBM1285 inhibited LPS-induced increase in the plasma level of TNF-alpha in mice. Whole-blood in vivo target inhibition assay also revealed that DBM1285 attenuates p38 MAPK activity after oral administration in mice. Moreover, DBM1285 suppressed zymosan-induced inflammation and adjuvant-induced arthritis in murine models. Collectively, these results suggest that DBM1285 inhibits TNF-alpha production, at least in part, by blocking the p38 MAPK/MK2 pathway. Furthermore, in vivo results suggest that DBM1285 might be a possible therapeutic candidate for the treatment of TNF-alpha-related chronic inflammatory diseases.

Lipid-soluble ginseng extract was prepared by n-hexane extraction of red ginseng. BALB/c-nu mice were inoculated with human lung cancer (NCI-H460) cells to establish a human tumor xenograft model in nude mice, and the lipid-soluble ginseng extract was orally administered. The tumor inhibitory rates of the lipid-soluble ginseng extract at doses of 0.1, 0.3, and 1.0 g/kg/day were 18.9% (P

DNA methylation is one of the principal epigenetic modifications playing an essential role in regulating gene expression. The TET family (1-3) is implicated in initiating the demethylation process by converting 5-methylcytosine (5mC) to 5-hydroxymethyl cytosine (5hmC) during embryogenesis. Previous studies in mice suggest that TET1 is required for pluripotency and maintenance of embryonic stem cells by managing their epigenetic marks, specifically DNA methylation. This raises the possibility that TET1 is capable of establishing distinct epigenetic marks during embryo development, thus regulating pluripotency-related genes. However, this has not been demonstrated in any species. Previously we have demonstrated that the level of TET1 (mRNA and protein) was high in porcine blastocysts. In this study, we generated TET1 knockout embryos and analysed expression patterns of pluripotency-related genes in blastocysts to study the role of TET1 in maintaining pluripotency during porcine embryo development. The CRISPR/Cas9 system was applied to disrupt the TET1 gene during embryogenesis. Three single-guide RNAs (sgRNAs) were designed based on our previous cloning of the TET1 gene. In vitro-synthesised Cas9 mRNA (20 ng µL−1) and sgRNAs (10 ng µL−1 each) were injected into the cytoplasm of zygotes after IVF. A total of 605 zygotes were used for microinjection and subsequently 54 blastocysts were formed. As a control, 89 IVF blastocysts were developed from 240 embryos. Nine to ten blastocysts per group were collected on Day 7 to analyse gene expression patterns of TET family and pluripotency-related genes using quantitative RT-PCR. Three biological and 3 experimental replications were used. Differences in the gene expression were evaluated by ANOVA. As expected, there was a 2-fold decrease in the transcript level of TET1 in TET1-knockout blastocysts compared with that in control IVF blastocysts (P

Introduction User-Generated Content (UGC) IS A BURGEONING social phenomenon being watched in today's world with keen interest. UGC is an online new-media content created by users rather than by conventional media such as broadcasters. A typical example would be Flickr, an online photo-sharing site with 37 million images, to which its 1.2 million members add up to 200,000 images daily. UGC is shifting the paradigm of Internet use away from the one-way propagation of media content by companies towards the creation and sharing of media content by and among ordinary users. The mobile phone is an especially important means of promoting user generation and exchange of media content. Many mobile phones now have built-in digital cameras and inherent network connectivity. These features have greatly facilitated the creation and sharing of media content. For instance, users can immediately upload photos on Flickr with their mobile phones as well as access Flickr and browse other people's media content. However, the constraints of a typical mobile phone---its small display and limited number of buttons---make use of mobile UGC services challenging. Only a few studies have investigated hardware or software alternatives to address these problems. This article presents a new user interface (UI) for mobile phones, one that makes use of UGC services easier and more efficient. The new interface has two key characteristics: one pertaining to content structure, and another to content visualization. More specifically, the new UI employs two major mobile Web 2.0 technologies, the tag and the tag cloud, and multi-display buttons increase the display size and flexibility of individual buttons. The interface is dedicated specifically towards supporting exploratory browsing within mobile UGC services, because users of such services are likely to focus on exploratory browsing and serendipitous discovery and be more inclined toward entertainment rather than utility. Here, we describe the new interface and investigate whether it aids in enhancing exploratory browsing within mobile UGC services.

We propose the Combined Symbol-based Closed-Loop Orthogonal Space-Time Block Code (CS-CL-OSTBC) for four transmit antennas. In the multiple antenna systems, the CS-CL-OSTBC not only achieves full rate and full diversity with linear maximum-likelihood detection but also obtains higher feedback gain than existing CL-OSTBCs due to more efficient utilization of channel feedback information. In the proposed scheme, all the complex-valued channel coefficients are rotated to positive real values with exact channel phase feedback information. As a result, the channel gain can be expressed as the square of the sum of all positive real values and can obtain the maximum value without any loss. Simulation results on bit error rate performance show that the CS-CL-OSTBC outperforms existing CL-OSTBCs for various modulation schemes.

Hierarchical constellations offer a different property of robustness to the multiple bits that construct a symbol according to channel errors. We apply the characteristics of hierarchical constellations to a multi-user cellular system that has limited modulation levels, in order to improve cell capacity. We propose an adaptive resource allocation scheme based on the hierarchical constellation in which a symbol is shared by multiple users and each bit in a symbol is allocated adaptively according to the channel condition of each user. The numerical results show that the proposed resource allocation scheme provides mobile users with higher modulation levels so that the cell capacity is improved.

Novel disubstituted adamantyl derivatives were synthesized and evaluated in a P-glycoprotein dependent multidrug resistance cancer cell line. The hit to lead optimization provided potent MDR reversal agents. Some potent adamantyl derivatives were more than 10-fold more potent than verapamil without considerable intrinsic cytotoxicity. The 3-trifluorophenyl derivative 14f did not affect the metabolism of CYP450 3A4, whereas most of MDR revertants had a weak inhibitory effect.

In this study, we investigated the anti-tumor activity of KBH-A42 [N-hydroxy-3-(2-oxo-1-(3-phenylpropyl)-1,2,5,6-tetrahydropyridin-3-yl)propanamide], a novel synthetic histone deacetylase (HDAC) inhibitor. KBH-A42 inhibited a variety of HDAC isoforms in enzyme assays and suppressed growth of various cancer cell lines. Among the cell lines examined, colon cancer cells, including SW620, SW480 and HCT-15, were the cell types most sensitive to KBH-A42. KBH-A42 inhibition of cancer cell growth was comparable to or stronger than that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor approved by the FDA to treat cutaneous T cell lymphomas. In SW620 cells, KBH-A42 increased the acetylation of histones, mediated cell cycle arrest (G1 arrest at low doses and G2 arrest at high doses), and induced apoptosis. The cell cycle arrest and apoptosis induced by KBH-A42 might be mediated through up-regulation of p21(Waf1) and activation of caspases, respectively. In addition, KBH-A42 inhibited SW620 tumor growth in a human tumor xenograft model. Taken together, our results indicate that KBH-A42 exerts an anti-tumor activity in vitro and in vivo and is a promising therapeutic candidate to treat human cancers.

Hypoxia-inducible factor (HIF)-1 is a therapeutic target in solid tumors. We report the novel benzimidazole analogue AC1-004, obtained from a chemical library using an HRE-dependent cell-based assay in colorectal carcinoma HCT-116 cells. The accumulation of hypoxia-induced HIF-1alpha was inhibited by compound AC1-004 in various cancer cells, including HCT-116, MDA-MB435, SK-HEP1, and Caki-1. Further, AC1-004 down-regulated VEGF and EPO, target genes of HIF-1, and inhibited in vitro tube formation of HUVEC, suggesting its potential inhibitory activity on angiogenesis. Importantly, AC1-004 was found to regulate the stability of HIF-1alpha through the Hsp90-Akt pathway, leading to the degradation of HIF-1alpha. An in vivo antitumor study demonstrated that AC1-004 reduced tumor size significantly (i.e., by 58.6%), without severe side effects. These results suggest the benzimidazole analogue AC1-004 is a novel HIF inhibitor that targets HIF-1alpha via the Hsp90-Akt pathway, and that it can be used as a new lead in developing anticancer drugs.

Glabridin, a flavonoid present in licorice root, is known to have antiinflammatory and cardiovascular protective activities. The present study reports an inhibitory effect of glabridin on microglial activation. Glabridin dose-dependently attenuated lipopolysaccharide (LPS)-induced production of inflammatory mediators, including nitric oxide, tumor necrosis factor-alpha and interleukin-1beta, in BV-2 cells, a murine microglia cell line. Moreover, mRNA expression of these inflammatory mediators was also suppressed by glabridin in LPS-stimulated BV-2 cells. Further study demonstrated that glabridin inhibited LPS-induced DNA binding activity of NF-kappaB and AP-1 in BV-2 cells. Collectively, the results presented in this report demonstrate that glabridin inhibits the production of inflammatory mediators in BV-2 cells and this is mediated, at least in part, by blocking NF-kappaB and AP-1 activation. The results suggest that glabridin might be a potential therapeutic agent for the treatment of neuroinflammatory and neurodegenerative diseases.

The pharmacokinetics and metabolism of 2'-benzoyloxycinnamaldehyde (BCA) was characterized in male Sprague-Dawley rats as part of the preclinical evaluations for developing this compound as an antitumour agent. BCA was not detected in the plasma following either intravenous or oral dose, whereas its putative metabolites 2'-hydroxycinnamaldehyde (HCA) and o-coumaric acid were present at considerable levels. In separate pharmacokinetics studies, HCA exhibited a high systemic clearance and a large volume of distribution, whereas both pharmacokinetic parameters were much lower for o-coumaric acid. The terminal half-life of both metabolites was approximately 2 h. BCA was converted rapidly to HCA in rat serum, liver microsomes and cytosol in vitro; HCA was subsequently converted to o-coumaric acid in a quantitative manner only in the liver cytosol. In addition, the formation of o-coumaric acid was inhibited significantly by menadione, a specific inhibitor for aldehyde oxidase. Taken collectively, the results suggest that the rapid systemic clearance of HCA is likely due mainly to hepatic clearance occurring from aldehyde oxidase-catalysed biotransformation to o- coumaric acid. In conclusion, the present work demonstrates that the anticancer drug candidate BCA is highly likely to work as its active metabolite HCA in the body.

Cervical cancer is a major cause of cancer mortality in women worldwide and is an important public health problem for adult women in developing countries. Despite aggressive treatment with surgery and chemoradiation, the outcomes for cervical cancer patients remain poor. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against human cervical cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The resulting populations of CIK cells comprised 95% CD3(+), 3% CD3(-)CD56(+), 35% CD3(+)CD56(+), 11% CD4(+)

Silymarin has been known to inhibit chemical-induced irritant contact dermatitis. In the present study, we report that topical application of silymarin suppresses dust mite extract (DPE)-induced atopic dermatitis (AD) in NC/Nga mice. Repeated topical application of ears with DPE caused AD-like skin lesions in NC/Nga mice. However, silymarin reduced AD-like skin lesions in these mice, resulting in decreased ear swelling and leukocyte infiltration into the ear. Moreover, our results showed that mast cell infiltration into the ear was suppressed by silymarin treatment in DPE-treated NC/Nga mice. Silymarin also reduced plasma level of IL-4 and IgE in these mice. Further study demonstrated that the mRNA expression of IL-4 was increased and that of IFN-gamma was decreased by DPE treatment in the ears of NC/Nga mice. However, DPE-induced changes in IL-4 and IFN-gamma mRNA expression were reversed by silymarin. DPE-induced increase in TNF-alpha mRNA expression was also suppressed by silymarin treatment. The results presented in this report suggest that silymarin might be beneficial for the treatment of AD.

PC-9S (N-Ethanol-2-mirystyl-3-oxo-stearamide) is a synthetic ceramide and has been known to be effective in atopic and psoriatic patients. K112PC-5 (2-Acetyl-N-(1,3-dihydroxyisopropyl)-tetradecanamide) is a novel ceramide derivative of PC-9S. In the present study, we examined the effect of K112PC-5 on macrophage and T lymphocyte function in primary macrophages and splenocytes, respectively, as well as the effect of topical application of K112PC-5 on skin inflammation and atopic dermatitis (AD) in mouse models. K112PC-5 inhibited lipopolysaccharide-induced nitrite generation in mouse peritoneal macrophages in a dose-dependent manner. However, K112PC-5 did not affect concanavalin A-induced proliferation, interleukin (IL)-2 secretion and IL-4 secretion in mouse splenocytes. In addition, K112PC-5 significantly suppressed the increase in phorbol ester-induced ear thickness in BALB/c mice. Further study demonstrated that topical application of K112PC-5 also inhibited AD induced by extracts of dust mites, Dermatophagoides pteronyssinus and Dermatophagoides farinae, in NC/Nga mice. Taken together, these results showed that K112PC-5 exerted an anti-inflammatory effect both in vitro and in vivo and proved to be beneficial in an animal model of AD. Our results suggest that K112PC-5 might be beneficial as a topical agent for the treatment of AD.

The present study reports the antiinflammatory activity of a methanol extract isolated from the stem bark of Magnolia kobus (MK). MK potently inhibited lipopolysaccharide (LPS)-induced production of nitric oxide and interleukin-1β (IL-1β) in RAW 264.7 cells, a murine macrophage-like cell line. The secretion of tumor necrosis factor-α (TNF-α) was also suppressed in LPS-stimulated RAW 264.7 cells although the magnitude of inhibition was weaker than that of nitric oxide and IL-1β. The mRNA expressions of inducible nitric oxide synthase (iNOS), IL-1β and TNF-α were also suppressed by MK in LPS-stimulated RAW 264.7 cells. Further study demonstrated that LPS-induced DNA binding of AP-1 and phosphorylation of c-jun N-terminal kinase (JNK) were inhibited by MK treatment in RAW 264.7 cells, whereas phosphorylation of p38 mitogen-activated protein kinase was unaffected. Moreover, topical application of MK suppressed ear swelling in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation model. Collectively, these results suggest that MK exerts antiinflammatory effects in vitro and in vivo and this might be mediated, at least in part, by blocking AP-1 and JNK activation. Copyright © 2008 John Wiley & Sons, Ltd.

Somatic cell nuclear transfer (SCNT) still retains important limitations. Impaired epigenetic reprogramming is considered responsible for altered gene expression and developmental failure in SCNT-derived embryos. After nuclear transfer the donor cell nucleus undergoes extensive changes in gene expression that involve epigenetic modifications and chromatin remodeling. We hypothesized that SNF2-type ATP-dependent chromatin factors contribute to epigenetic reprogramming and the relative amount of these factors in the donor cell affects developmental potential of the reconstructed embryos. In order to test this hypothesis, we assessed the relative amount of SNF2-type ATPases (Brahma, Brg1, SNF2H, SNF2L, CHD3, and CHD5) in three different donor cells as well as in porcine metaphase II oocytes. We performed SCNT with fetal fibroblast cells, olfactory bulb (OB) progenitor cells, and porcine skin originating sphere stem cells (PSOS). We found that OB-NT embryos and PSOS-NT embryos resulted in a higher morulae/blastocysts ratio as compared to fibroblast-NT embryos (23.53%, 16.98%, and 11.63%, respectively; P

Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with natural killer cell phenotypes and functions. In this study, the anti-tumor activity of CIK cells against hepatocellular carcinoma was evaluated in vitro and in vivo. In the presence of anti-CD3 antibody and IL-2 for 14 days, human peripheral blood mononuclear cell population changed to heterogeneous CIK cell population, which comprised 96% CD3(+), 3% CD3( inverted exclamation mark(c))CD56(+), 32% CD3(+)CD56(+), 11% CD4(+), 75% CD8(+), and 30% CD8(+)CD56(+). CIK cells produced significant amounts of IFN-gamma and TNF-alpha; however, produced only slight amounts of IL-2, IL-4, and IL-5. At an effector-target cell ratio of 30:1, CIK cells destroyed 33% of SNU-354 human hepatocellular carcinoma cells, which was determined by the (51)Cr-release assay. In addition, a dose of 1x10(6) CIK cells per mouse inhibited 60% of SNU-354 tumor growth in irradiated nude mice. This study suggests that CIK cells may be used as an adoptive immunotherapy for patients with hepatocellular carcinoma.

We synthesized a series of delta-lactam-based HDAC inhibitors that were identified with various degrees of anti-inflammatory and cell growth inhibitory activities. Compounds possessing significant HDAC inhibitory activity exhibited both anti-inflammatory and cell growth inhibitory activities as well as significant tumor growth inhibition in the in vivo tumor xenograft experiments. Besides, these compounds demonstrated anti-inflammatory properties in vitro via suppression of the production of the proinflammatory cytokine TNF-alpha and nitric oxide by LPS-stimulated RAW264.7 cells.

In the present study, we report the inhibitory effect of equol on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in murine macrophages. In vivo administration of equol (i.p.) attenuated NO production by peritoneal adherent cells isolated from lipopolysaccharide (LPS)-treated mice. Equol dose-dependently inhibited the LPS-induced production of NO in isolated peritoneal adherent cells and RAW 264.7 cells. The mRNA expression of iNOS was also blocked by equol in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced activation of Akt was suppressed by equol in RAW 264.7 cells while the activation of ERK, SAPK/JNK and p38 MAP kinase was not affected. Equol also blocked LPS-induced NF-kappaB activation. Moreover, the LPS-induced NO production and NF-kappaB activation was inhibited by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase/Akt pathway, in RAW 264.7 cells. These results suggest that equol might inhibit NO production and iNOS gene expression, at least in part, by blocking Akt activation and subsequent down-regulation of NF-kappaB activity.

This study was conducted to investigate the presence of lamin A/C in porcine nuclear transfer embryos and to determine whether lamin A/C can serve as a potential marker for nuclear reprogramming. First, lamin A/C was studied in oocytes and embryos produced by fertilization or parthenogenetic oocyte activation. We found that lamin A/C was present in the nuclear lamina of oocytes at the germinal vesicle stage while it was absent in mature oocytes. Lamin A/C was detected throughout preimplantation development in both in vivo-derived and parthenogenetic embryos. Incubation of the activated oocytes in the presence of alpha-amanitin (an inhibitor of RNA polymerase II), or cycloheximide (a protein synthesis inhibitor) did not perturb lamin A/C assembly, indicating that the assembly resulted from solubilized lamins dispersed in the cytoplasm. In nuclear transfer embryos, the lamin A/C signal that had previously been identified in fibroblast nuclei disappeared soon after fusion. It became detectable again after the formation of the pronucleus-like structure, and all nuclear transfer embryos displayed lamin A/C staining during early development. Olfactory bulb progenitor cells lacked lamin A/C; however, when such cells were fused with enucleated oocytes, the newly formed nuclear envelopes stained positive for lamin A/C. These findings suggest that recipient oocytes remodel the donor nuclei using type A lamins dispersed in the ooplasm. The results also indicate that lamin A/C is present in the nuclear envelope of pig oocytes and early embryos and unlike in some other species, its presence after nuclear transfer is not an indicator of erroneous reprogramming.

A concise and expeditious approach to the total synthesis of broussonone A, a p-quinol natural compound, has been developed. The key features of the synthesis include the Grubbs II catalyst mediated cross metathesis of two aromatic subunits, and a chemoselective oxidative dearomatizationin the presence of two phenol moieties. Especially, optimization associated with the CM reaction of ortho-alkoxystyrenes was also studied, which are known to be ineffective for Ru-catalyzed metathesis reactions under conventional reaction conditions because ortho-alkoxy group could coordinate to the ruthenium center, resulting in the potential complication of catalyst inhibition.

Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro–fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SAHA (10 μM) had the highest level of blastocyst development at 43.9%, and all treatments except 10 μM ISAHA had the same percentage of blastocyst development as Scriptaid (p

Artificial oocyte activation is a critical step during somatic cell nuclear transfer (SCNT). Most of current activation protocols focus on inducing an increase in the intracellular free Ca2+ concentration of the oocyte. Here, we have utilized a zinc chelator, TPEN, to enhance the efficiency of oocyte activation during SCNT. TPEN treatment of matured pig oocytes resulted in the reduction of available Zn2+ in pig oocytes; however, the cytosolic Ca2+ concentration in the oocytes was not affected by the TPEN treatment. When various concentrations (100 – 250 μM) and incubation durations (45 min – 2.5 hours) of TPEN were used to activate oocytes, the efficiency of oocyte activation was not different from conventional activation methods. When oocytes that were activated by conventional activation methods were incubated with a lower concentration of TPEN (5 – 10 μM), a significant increase in embryos developing to the blastocyst stage was observed. In addition, when oocytes receiving a small Ca2+ stimulus were further activated by higher concentration of TPEN (100 – 200 μM), a significant increase in the frequency of blastocyst formation was observed, compared to a conventional activation method. This result indicated that TPEN can be a main reagent in oocyte activation. No increase in the cytosolic Ca2+ level was detected when oocytes were exposed to various concentrations of TPEN, indicating the ability of TPEN to induce oocyte activation is independent of an intracellular Ca2+ increase. We were able to produce clones through SCNT using the TPEN-assisted activation procedure, and the piglets produced through the process did not show any signs of abnormality. In this study, we have developed an efficient way to utilize TPEN to increase the developmental potential of cloned embryos.

Background Recent advancements in gene editing techniques have increased in number and utility. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high efficiency of gene editing. We have previously produced cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFNs) in combination with donor DNA containing a total homology length of 1600 bp (800-bp homology on each arm). Our next aim was to introduce the targeted disruption of alpha-1,3-galactosyltransferase (GGTA1) in the CMAH KO genetic background and evaluate the effect of donor DNA homology length on meganuclease-mediated gene targeting. Methods Zinc-finger nucleases from a previous CMAH KO experiment were used as a proof of concept to identify a correlation between the length of donor DNA homology and targeting efficiency. Based on those results, experiments were designed to use transcription activator-like effector nucleases (TALENs) to generate bi-allelically modified GGTA1 cells using donor DNAs carrying various lengths of homology. Donor DNA was designed to symmetrically flank the predicted cleavage sites in CMAH and GGTA1 for both ZFN and TALEN cleavage sites, respectively. For both genes, the length of total homology ranged from 60 to 1799 bp. Sialyltransferase gene expression profiles were evaluated in CMAH and GGTA1 double KO pig cells and were compared to wild-type and CMAH KO cells. Results Introduction of donor DNA with ZFNs demonstrated that small amounts of homology (60 bp) could facilitate homology-directed repair during ZFN-mediated targeting of CMAH; however, donor DNA with longer amounts of homology resulted in a higher frequency of homology-directed repair. For the GGTA1 KO experiments that used TALENs and donor DNA, donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (TALENs alone, no donor DNA present) to a maximum of 3% (TALENs and donor DNA with total homology of 1799 bp). Inclusion of homologous donor DNA in TALEN-mediated gene targeting facilitated a higher incidence of bi-allelically modified cells. Using the generated cells, we were able to demonstrate the lack of GGTA1 expression and the decrease in gene expression sialyltransferase-related genes. Conclusions The approach of using donor DNA in conjunction with a meganuclease can be used to increase the efficiency of gene targeting. The gene editing methods can be applied to other genes as well as other mammalian systems. Additionally, gene expression analysis further confirms that the CMAH/GGTA1 double KO pigs can be a valuable source for the study of pig-to-human xenotransplantation.

Drug-induced liver injury (DILI) via metabolic activation by drug-metabolizing enzymes, especially cytochrome P450 (CYP), is a major cause of drug failure and drug withdrawal. In this study, an in vitro model using HepG2 cells in combination with human liver microsomes was developed for the prediction of DILI. The cytotoxicity of cyclophosphamide, a model drug for bioactivation, was augmented in HepG2 cells cultured with microsomes in a manner dependent on exposure time, microsomal protein concentration, and NADPH. Experiments using pan- or isoform-selective CYP inhibitors showed that CYP2B6 and CYP3A4 are responsible for the bioactivation of cyclophosphamide. In a metabolite identification study employing LC-ESI-QTrap and LC-ESI-QTOF, cyclophosphamide metabolites including phosphoramide mustard, a toxic metabolite, were detected in HepG2 cells cultured with microsomes, but not without microsomes. The cytotoxic effects of acetaminophen and diclofenac were also potentiated by microsomes. The potentiation of acetaminophen cytotoxicity was dependent on CYP-dependent metabolism, and the augmentation of diclofenac cytotoxicity was not mediated by either CYP- or UDP-glucuronosyltransferase-dependent metabolism. The cytotoxic effects of leflunomide, nefazodone, and bakuchiol were attenuated by microsomes. The detoxication of leflunomide by microsomes was attributed to mainly CYP3A4-dependent metabolism. The protective effect of microsomes against nefazodone cytotoxicity was dependent on both CYP-mediated metabolism and nonspecific protein binding. Nonspecific protein binding but not CYP-dependent metabolism played a critical role in the attenuation of bakuchiol cytotoxicity. The present study suggests that HepG2 cells cultured with human liver microsomes can be a reliable model in which to predict DILI via bioactivation by drug metabolizing enzymes.