OBJECTIVETo investigate the effects of naloxone on glutamate release in combined oxygen-glucose deprivation of primary cultured human embryo neurons.

METHODSThe primary cultured embryonic human cortical neurons were demonstrated by immunocytochemical stain of neural filament (NF). The neurons were randomly allocated into control group, hypoxic group, and experimental group. The experimental group was further divided into three subgroups pretreated with different concentrations of naloxone (0.25, 5, 10 microg/ml). The neurons of hypoxic group and experimental group were deprived both oxygen and glucose for 1 hours followed by 24 hours of reoxygenation. Meanwhile, we used 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, high performance liquid chromatography (HPLC), and biological analysis to study the survival rate of neurons and the changes of extracellular glutamate and lactate dehydrogenase (LDH) levels after 24 hours of reoxygenation.

RESULTSOne hour of oxygen-glucose deprivation followed by 24 hours of reoxygenation was associated with a large increase in extracellular LDH and glutamate and a significant decrease of cell vitality (P < 0.01). Naloxone exerted a concentration-dependent protection against neuronal injury provoked by combined oxygen-glucose deprivation. After reoxygenation, the extracellular concentrations of glutamate gradually decreased (P < 0.05, P < 0.01, respectively) and cell vitality increased (P < 0.01) with increase of the concentration of naloxone compared with control group. All of them returned to control level when naloxone was up to 10 microg/ml (P > 0.05).

CONCLUSIONNaloxone protects neurons from hypoxic injury by inhibiting the release of glutamate and therefore alleviating the exciting toxicity.

Cell Hypoxia ; Cells, Cultured ; Cerebral Cortex ; cytology ; Embryo, Mammalian ; Glutamic Acid ; metabolism ; Humans ; Naloxone ; pharmacology ; Neurons ; drug effects ; metabolism ; Neuroprotective Agents ; pharmacology

AIMTo study the mechanism of butylated hydroxyanisole-induced neural differentiation of fetal liver cells in vitro.

METHODS14.5-day-old mouse fetal liver-derived cells were cultured, and were induced by 200 micromol/L butylated hydroxyanisole (BHA) combined with PI3K inhibitor LY294002 (20 micromol/L), and then were incubated in serum-free medium. Expression of genes in treated or untreated cells were assayed by Western blotting or RT-PCR.

RESULTSThere was low level of neurofilament-L (NF-L) and brain factor-1 (BF-1) but no neurofilament-H (NF-H) and tyrosine hydroxylase (TH) in fetal liver cells. BHA promoted significantly expression of neuron-specific NF-L, NF-H, BF-1, and TH in fetal liver cells. NF-L mRNA increased 5.8 fold, NF-H mRNA 8.0 fold, BF-1 mRNA 2.68 fold, and TH mRNA 30 fold, respectively (all P < 0.01 vs untreated cells). NF-L protein increased 11.29 fold, NF-H 5.5 fold, BF-1 2.53 fold, TH 4.76 fold. Moreover, expression of these BHA-induced genes were inhibited by PI3K inhibitor LY294002.

CONCLUSIONBHA induced neural differentiation of fetal liver cells through PI3K.

Animals ; Butylated Hydroxyanisole ; pharmacology ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Hepatocytes ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Phosphatidylinositol 3-Kinases ; metabolism

OBJECTIVETo investigate the effect of maslinic acid preconditioning against injuries of rat cortical neurons induced by oxygen-glucose deprivation (OGD).

METHODSThe cortical neurons were isolated from Sprague-Dawley rat embryos at 15-17 days of gestation for primary culture. The cortical neurons were incubated with different concentrations (0.1, 1, and 10 micro;mol/L) of maslinic acid prior to OGD. The cell damage and viability were evaluated for lactate dehydrogenase (LDH) leakage and using MTT assay, respectively, and the expression of Bax protein was detected using Western blotting.

RESULTSOGD significantly increased LDH release rate and decreased the viability of the cells. After preconditioning with maslinic acid (1 and 10 micro;mol/L), LDH leakage rate was decreased and cell the viability increased in cells exposed to OGD. Western blotting showed that Bax expression in the cells decreased as maslinic acid concentrations increased.

CONCLUSIONPretreatment with maslinic acid can protect cultured embryonic rat cortical neurons against OGD-induced injury possibly in relation to decreased expression of Bax.

Animals ; Apoptosis ; Cell Hypoxia ; Cells, Cultured ; Cerebral Cortex ; cytology ; drug effects ; Embryo, Mammalian ; Glucose ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Neurons ; drug effects ; metabolism ; Oxygen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Triterpenes ; pharmacology ; bcl-2-Associated X Protein ; metabolism

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.
Biological Markers/metabolism ; Cell Differentiation/*drug effects ; Cell Line ; Cell Lineage/*drug effects ; Cells, Cultured ; Down-Regulation/drug effects ; Embryo, Mammalian/cytology/drug effects/metabolism ; Embryonic Stem Cells/*cytology/*drug effects/enzymology ; Endoderm/*cytology/drug effects ; Enzyme Activation/drug effects ; Free Radical Scavengers/pharmacology ; Humans ; Mesoderm/*cytology/drug effects ; Mitogen-Activated Protein Kinases/metabolism ; Pluripotent Stem Cells/cytology/metabolism ; Reactive Oxygen Species/metabolism/*pharmacology ; Up-Regulation/drug effects

AIMTo investigate the regulatory effects of various anti-inflammatory drugs on both endogenous and TNFalpha-induced NF-kappaB activation as well as the relative biological activity.

METHODSHEK293 cells were cultured in 96-well plate and 6-well plate, treated with meloxicam, indomethacin, dexamethasone and hydrocortisone, without or with 10 ng.mL(-1) TNFalpha for 24 hours. Then cell proliferation was measured by MTT and cell apoptosis was analyzed by pI stain-flow cytometry. HEK293/ kappaB-luc cells transfected stably with pElam-kappaB-luc vector, were cultured in 96-well plate and treated as above. Equal amounts of cell lysates were tested for luciferase activity which represents NF-kappaB activation.

RESULTSEndogenous NF-kappaB activation was present in HEK293 cells and its level can be increased about 2 times by 10 ng.mL(-1) TNFalpha-induction. Dexamethasone (1 x 10(-8) mol.L(-1)) and meloxicam (1 x 10(-7) - 1 x 10(-6) mol.L(-1)) can decrease both endogenous and TNFalpha-induced NF-kappaB activation. Hydrocortisone (1 x 10(-9) mol.L(-1)) increases endogenous NF-kappaB activation but decreases TNFalpha-induced one significantly. No influence of indomethacin on endogenous NF-kappaB activation was observed. However, its influence on TNFalpha-induced NF-kappaB activation is needed for further study. Cell apoptosis was observed after treatment with TNFalpha and 1 x 10(-8), 1 x 10(-6) mol.L(-1) dexamethasone and 1 x 10(-7) mol.L(-1) indomethacin, or only with dexamethasone. No significant effect of these anti-inflammatory drugs on cell proliferation was observed.

CONCLUSIONVarious anti-inflammatory drugs differ in their ability to regulate NF-kappaB activation in HEK293 cells, which indicates that NF-kappaB activation might be a potential useful target to study mechanism and for drug screening.

Anti-Inflammatory Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Dexamethasone ; pharmacology ; Embryo, Mammalian ; Humans ; Hydrocortisone ; pharmacology ; Indomethacin ; pharmacology ; Kidney ; cytology ; metabolism ; NF-kappa B ; metabolism ; Thiazines ; pharmacology ; Thiazoles ; pharmacology

OBJECTIVETo investigate the role of mitogen activated protein kinases (MAPKs) signaling pathways in the regulation of benzo(a)pyrene (B(a)P)-induced c-Jun activation in human embryo lung fibroblasts (HELFs).

METHODSHELFs were cultured with 2.0 micromol/L B(a)P for various time (0, 3, 6, 12, 24 h) or with various concentration of B(a)P (0.0, 0.5, 1.0, 2.0 micromol/L) for 12 h. Western blot was performed to examine the effect of B(a)P on c-Jun activation. The dominant negative mutants of p38, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) were applied to establish stable transfectant, and to detect the relationship of MAPK signal molecules and c-Jun activation in B (a) P-treated cells.

RESULTSB(a)P treatment resulted in a marked activation of c-Jun in time-dependent manner with a peak at 12 h (the densitometric ratios of phosphorylated c-Jun Ser63, Ser73 to actin were 20.1, 15.2 times for control respectively) and in dose-dependent manner. However, there was no evident change on total c-Jun expression in B(a)P-treated HELFs. Moreover, B(a)P-induced activation of c-Jun was inhibited by stable expression of dominant negative mutants of JNK or ERK, but not by dominant negative mutant of p38.

CONCLUSIONJNK and ERK signaling pathways, but not p38 pathway regulate B(a)P-induced c-Jun activation in HELFs.

Benzo(a)pyrene ; pharmacology ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-jun ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo study the relationship between targeting vector structure and homologous recombination rate and investigate whether the mouse p16(INK4a) plays a role in tumor suppression.

METHODSA conditional targeting vector with 2.0 kb EcoR I/Xba I fragment as short arm and 5.9 kb SpeI/NotI fragment as long arm was built. Of the 2 direct locus crossing- over(loxPs) in the vector, one was inserted at 240 bp upstream of the initiate code of p16(INK4a) exon 1a and the other at 1633 bp downstream of the initiate code. Both exon 1a and the selection marker Neo will be deleted in targeted cells when mediated by Cre. After linearlization and purification, t he targeting vector was introduced into ES cells through electroporation.

RESULTSTwenty-four G418- and gancyclovir-resistant ES cell colonies were picked out and one of them was confirmed as positive by Southern hybridization.

CONCLUSIONTargeting vectors with 2 TK genes flanking the homologous arms are likely to produce good result of homologous recombination.

Animals ; Anti-Bacterial Agents ; pharmacology ; Antiviral Agents ; pharmacology ; Base Sequence ; Cell Division ; drug effects ; genetics ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Drug Resistance ; genetics ; Embryo, Mammalian ; cytology ; drug effects ; metabolism ; Exons ; genetics ; Ganciclovir ; pharmacology ; Genetic Vectors ; genetics ; Gentamicins ; pharmacology ; Mice ; Molecular Sequence Data ; Recombination, Genetic ; Stem Cells ; cytology ; drug effects ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Transfection

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.
Acetylation ; Animals ; Blastocyst ; cytology ; Cell Nucleus ; metabolism ; Cinnamates ; pharmacology ; Embryo, Mammalian ; drug effects ; metabolism ; Embryonic Development ; drug effects ; Epigenesis, Genetic ; Female ; Gene Expression ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; In Vitro Techniques ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Nuclear Transfer Techniques ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Swine

OBJECTIVEIn order to explore the effects of lead on the growth and development of cultured hippocampal neural cells and on the expression of Oct-2, the II subtype POU domain protein.

METHODSExperiment cell model was established using primary culture of hippocampal neural cells from SD rat embryos. Target cells were exposed to lead acetate in the different concentrations, i.e. 10(-1), 10(0), 10(1), 10(2), 10(3) micromol/L, while the control group was given the same quantity of the culture medium. The immunohistochemistry method was utilized to detect the expressions of Neurofilament (NF) and Glial Fibrillary Acidic Protein (GFAP), the markers for neuron and astrocyte, respectively, and the expression of Oct-2 as well.

RESULTSThe results showed that 10 micromol/L lead acetate treatment caused diminishing of neuronal cell body and the decreases of both axon lengths and inter-cellular connections. In addition, 1 micromol/L lead acetate significantly increased the number of GFAP-positive cells compared with the control group (P < 0.05). By image analysis system, 1 micromol/L lead acetate treatment was found to induce a statistically significant increase of the positive area rate concerning Oct-2 expression in hippocampal neurons and astrocytes, while both positive area rate and integral density of light of Oct-2 expression were found to increase markedly in the groups treated by 10 micromol/L lead acetate (P < 0.01).

CONCLUSIONSLead acetate treatment may contribute to the inhibitions of both growth and differentiation of hippocampus neurons, and to the stimulation of glial cell hyperplasia simultaneously. In addition, the CNS impairments caused by lead is partly correlated with the enhancement of Oct-2 expression.

Animals ; Astrocytes ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; biosynthesis ; genetics ; Dose-Response Relationship, Drug ; Embryo, Mammalian ; Female ; Glial Fibrillary Acidic Protein ; biosynthesis ; genetics ; Hippocampus ; cytology ; metabolism ; Lead ; toxicity ; Neurofilament Proteins ; biosynthesis ; genetics ; Neurons ; cytology ; metabolism ; Octamer Transcription Factor-2 ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; biosynthesis ; genetics

OBJECTIVETo investigate the regulatory effects of nerve growth factor (NGF) on basal and capsaicin-induced release of neuropeptide substance P (SP) in primary cultured embryonic rat dorsal root ganglion (DRG) neurons.

METHODSDRGs were dissected from 15-day-old embryonic Wistar rats. DRG neurons were dissociated and cultured, and then exposed to different concentrations of NGF (10 ng/mL, 30 ng/mL, or 100 ng/mL) for 72 h. The neurons cultured in media without NGF served as control. RT-PCR were used for detecting the mRNAs of SP and vanilloid receptor 1 (VR1) in the DRG neurons. The SP basal and capsaicin (100 nmol/L)-induced release in the culture were measured by radioimmunoassay (RIA).

RESULTSSP mRNA and VR1 mRNA expression increased in primary cultured DRG neurons in a dose-dependent manner of NGF. Both basal release and capsaicin-evoked release of SP increased in NGF-treated DRG neurons compared with in control group. The capsaicin-evoked release of SP also increased in a dose-dependent manner of NGF.

CONCLUSIONNGF may promote both basal release and capsaicin-evoked release of SP. NGF might increase the sensitivity of nociceptors by increasing the SP mRNA or VR1 mRNA.

Analgesics, Non-Narcotic ; pharmacology ; Animals ; Capsaicin ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Embryo, Mammalian ; Ganglia, Spinal ; cytology ; Gene Expression Regulation ; drug effects ; Nerve Growth Factor ; pharmacology ; Neurons ; drug effects ; RNA, Messenger ; metabolism ; Radioimmunoassay ; methods ; Rats ; Rats, Wistar ; Substance P ; genetics ; metabolism