To investigate the function of ALK3 gene, the gene regulation and the signaling pathway related to ventricular septum defect during heart development. The model mice with ALK3 gene knock-out via alpha-MHC-Cre/lox P system were bred. The mRNA expression level of control group was compared with that of experiment group and ALK3 downstream genes were screened using PCR-select cDNA subtraction microarray. The mRNA of control group was extracted from E11.5 normal mouse hearts, and that of experiment group, from E11.5 hearts of mice with alpha-MHC Cre(+/-) ALK3(F/+) genotype. It was found that the mice with ALK3 gene knock-out produced heart defects involving the interventricular septum. The platelet-activating factors acetylhydrolase and the transcription factor Pax-8 and so on, were down-regulated. However, the Protein Tyrosine Kinase (PTK) of Focal Adhesion Kinase (FAK) subfamily and beta subtype protein 14-3-3 were up-regulated in the alpha-MHC Cre(+/-) ALK3(F/-) mice. These data provide support that ALK3 gene played an important role during heart development. The platelet-activating factors acetylhydrolase and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and beta subtype protein 14-3-3 might be regulatory factors in this pathway.
1-Alkyl-2-acetylglycerophosphocholine Esterase ; genetics ; metabolism ; 14-3-3 Proteins ; genetics ; metabolism ; Animals ; Bone Morphogenetic Protein Receptors, Type I ; genetics ; metabolism ; Genotype ; Heart Septal Defects, Ventricular ; genetics ; Mice ; Mice, Knockout ; Oligonucleotide Array Sequence Analysis ; PAX8 Transcription Factor ; Paired Box Transcription Factors ; genetics ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; genetics ; physiology

OBJECTIVETo investigate the changes of gene expression in rat neuron induced by 1.8 GHz radiofrequency electromagnetic fields (RF EMF) to screen for RF EMF-responsive genes and the effect of different exposure times and modes on the gene expression in neuron.

METHODSTotal RNA was extracted immediately and purified from the primary culture of neurons after intermittent exposed or sham-exposed to a frequency of 1.8 GHz RF EMF for 24 hours at an average special absorption rate (SAR) of 2 W/kg. Affymetrix Rat Neurobiology U34 array was applied to investigate the changes of gene expression in rat neuron. Differentially expressed genes (Egr-1, Mbp and Plp) were further confirmed by semi-quantitative revere transcription polymerase chain reaction (RT PCR). The expression levels of Egr-1, Mbp and Plp were observed at different exposure times (6, 24 h) and modes (intermittent and continuous exposure).

RESULTSAmong 1200 candidate genes, 24 up-regulated and 10 down-regulated genes were found by using Affymetrix microarray suite software 5.0 which are associated with multiple cellular functions (cytoskeleton, signal transduction pathway, metabolism, etc.) after functional classification. Under 24 h and 6 h intermittent exposure, Egr-1 and Plp in experiment groups showed statistic significance (P < 0.05) compared with the control groups, while expression of Mbp did not change significantly (P > 0.05). After 24 h continuous exposure, Egr-1 and Mbp in experiment groups showed statistic significance (P < 0.05) compared with the control group, while expression of Plp did not change significantly (P > 0.05). Under the same exposure mode 6 h, expression of all the 3 genes did not change significantly. Different times (6, 24 h) and modes (intermittent and continuous exposure) of exposure exerted remarkable different influences on the expression of Egr-1, Mbp, Plp genes (P < 0.01).

CONCLUSIONThe changes of many genes transcription were involved in the effect of 1.8 GHz RF EMF on rat neurons; Down-regulation of Egr-1 and up-regulation of Mbp, Plp indicated the negative effects of RF EMF on neurons; The effect of RF intermittent exposure on gene expression was more obvious than that of continuous exposure; The effect of 24 h RF exposure (both intermittent and continuous) on gene expression was more obvious than that of 6 h (both intermittent and continuous).

Animals ; Cells, Cultured ; Dose-Response Relationship, Radiation ; Down-Regulation ; radiation effects ; Electromagnetic Fields ; Neurons ; metabolism ; radiation effects ; Rats ; Up-Regulation ; radiation effects

OBJECTIVETo study the effects of GSM 1800 MHz radiofrequency electromagnetic fields (RF EMF) on DNA damage in Chinese hamster lung (CHL) cells.

METHODSThe cells were intermittently exposed or sham-exposed to GSM 1800 MHz RF EMF (5 minutes on/10 minutes off) at a special absorption rate (SAR) of 3.0 W/kg for 1 hour or 24 hours. Meanwhile, cells exposed to 2-acetylaminofluorene, a DNA damage agent, at a final concentration of 20 mg/L for 2 hours were used as positive control. After exposure, cells were fixed by using 4% paraformaldehyde and processed for phosphorylated form of H2AX (gammaH2AX) immunofluorescence measurement. The primary antibody used for immunofluorescence was mouse monoclonal antibody against gammaH2AX and the secondary antibody was fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). The gammaH2AX foci and nuclei were visualized with an Olympus AX70 fluorescent microscope. Image Pro-Plus software was used to count the gammaH2AX foci in each cell. For each exposure condition, at least 50 cells were selected to detect gammaH2AX foci. Cells were classified as positive when more than five foci were detected. The percentage of gammaH2AX foci positive cells was adopted as the index of DNA damage.

RESULTSThe percentage of gammaH2AX foci positive cell of 1800 MHz RF EMF exposure for 24 hours (37.9 +/- 8.6)% or 2-acetylaminofluorene exposure (50.9 +/- 9.4)% was significantly higher compared with the sham-exposure (28.0 +/- 8.4)%. However, there was no significant difference between the sham-exposure and RF EMF exposure for 1 hour (31.8 +/- 8.7)%.

CONCLUSION1800 MHz RF EMF (SAR, 3.0 W/kg) for 24 hours might induce DNA damage in CHL cells.

Animals ; Cells, Cultured ; Cricetinae ; Cricetulus ; DNA Breaks, Double-Stranded ; radiation effects ; DNA Damage ; radiation effects ; Electromagnetic Fields ; adverse effects ; Fibroblasts ; chemistry ; radiation effects ; Radio Waves

OBJECTIVETo investigate the changes of gene expression in rat neurons induced by 1.8 GHz radiofrequency electromagnetic fields (RF EMF) and to screen for the RF EMF-responsive genes.

METHODSNewly-born SD rats in 24 hours were sacrificed to obtain cortex and hippocampus neurons. The cells were divided randomly into two groups: the experiment group (the irradiation group) and the control group (the false irradiation group). In the irradiation group, after twelve days' culture, neurons were exposed to 1.8 GHz RF EMF modulated by 217 Hz at a specific absorption rate (SAR) of 2 W/kg for 24 hours (5 minutes on/10 minutes off) while in the false control group, the neurons were put in the same waveguide as in the irradiation group, but were not exposed to any irradiation. The total RNA was isolated and purified immediately after exposure. The affymetrix rat neurobiology U34 assay was used for detecting the changes in gene expression profile according to the manufacturer's instruction. RF EMF-responsive candidate gene was confirmed by using ribonuclease protection assay (RPA).

RESULTSAmong 1200 candidate genes, the expression levels of 34 genes were up or down regulated. Microtubule associated protein 2 (Map2) gene was selected as the candidate and subjected to further analysis. RPA data clearly revealed that Map2 was statistically significantly up-regulated after neurons were exposed to the RF EMF (P < 0.05).

CONCLUSIONThe modulation of gene expression and function of Map2 as a neuron specific cytoskeleton protein is crucial to maintain the normal framework and function of neurons. The finding that 1.8 GHz RF EMF exposure increases the expression of Map2 might indicate some unknown effects of RF EMF on neurons.

Animals ; Animals, Newborn ; Cell Phone ; Cells, Cultured ; Dose-Response Relationship, Radiation ; Down-Regulation ; Electromagnetic Fields ; Female ; Gene Expression ; radiation effects ; Male ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neurons ; metabolism ; radiation effects ; Radio Waves ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

Objective To investigate the changes of gene expression in rat neurons induced by 1.8 GHz radiofrequency electromagnetic fields (RF EMF) and to screen RF EMF-responsive genes. Methods The rat primary cultured neuronal cells were divided into two groups, the radiation group and control group, from which the total RNA was extracted immediately and purified after intermittently (5min on/10min off) exposed or U34 array was applied to detect the changes of gene expression in rat neurons. Results Among 1200 candidate genes, 24 up-regulated and 10 down-regulated genes which are associated with multiple cellular functions (cytoskeleton, signal transduction pathway, metabolism, etc.) after functional classification were found by using Affymetrix microarray suite software 5.0. Although the changes in gene expression were less than 2 folds, they had statistical significance (P<0.01). Conclusion RF radiation of 1.8GHz induce the changes of many genes transcription in rat neurons, some of which indicate the negative effects of RF radiation on neurons.

OBJECTIVETo observe the effects of ryanodine on rapamycin treated endothelial outgrowth cells (EOCs).

METHODSThe mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, then induced into EOCs and expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were pretreated with or without ryanodine (10 µmol/L) for 1 h, and then treated with or without rapamycin (10 nmol/L) for 24 h. Proliferation was evaluated by CCK8 and migration was measured by Transwell. The protein expression of EOCs was evaluated by immunobloting technique with total eNOS antibody and phospho-eNOS (Thr495) antibody.

RESULTSCompared with control group, the proliferation and migration capacities of EOCs were significantly reduced while the phosphorylation of eNOS (Thr495) protein was significantly upregulated in rapamycin group (P < 0.05), expression of total eNOS was not affected by rapamycin (P > 0.05). Compared with rapamycin group, the proliferation and migration capacities of EOCs were significantly increased and the phosphorylation of eNOS (Thr495) protein was significantly downregulated in ryanodine + rapamycin group (P < 0.05). The proliferation and migration capacities, the phosphorylation of eNOS (Thr495) protein and the expression of total eNOS were not affected by ryanodine alone (P > 0.05).

CONCLUSIONSRapamycin reduced proliferation and migration capacities while upregulated the phosphorylation of eNOS (Thr495) protein of EOCs and these effects could be partly reversed by cotreatment with ryanodine.

Cells, Cultured ; Down-Regulation ; Drug Synergism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Nitric Oxide Synthase Type III ; metabolism ; Phosphorylation ; Ryanodine ; pharmacology ; Sirolimus ; pharmacology

OBJECTIVEConventional deletion of ALK3, also termed as bone morphogenetic protein (BMP) receptor IA, in mice might result in early embryonic lethality. To investigate the function of ALK3 in cardiac development, the cardiac-specific deletion of ALK3 in mice was made by Dr. Schneider, using Cre recombinase driven by the alpha-MHC promoter that Dr. Fukushipe worked out. Such specific deletion of ALK3 caused death in mid-gestation with defects in the trabeculae, interventricular septum, and endocardial cushion. Since ALK3 is not a cardiac-specific gene, it is extremely important to identify ALK3 downstream genes.

METHODSAlpha-MHC Cre+/-, ALK3 F/- and alpha-MHC Cre+/-, ALK3 F/+ embryos were obtained after 20 alpha-MHC Cre+/-, ALK3 +/- mice and 20 ALK3 F/F mice were mating. The ALK3 downstream genes were screened using microarray made in Germany that could identify 25000 genes in mouse. Two populations of mRNA, one derived from the embryonic heart (11.5 days) of alpha-MHC Cre+/-, ALK3 F/- mice, and the other derived from the alpha-MHC Cre+/-, ALK3 F/+ mice, were compared. Cardiac-specific ALK3 downstream genes were identified using real time quantitative RT-PCR and in situ hybridization.

RESULTSThe expression of 12 genes, such as Pax-8 and Hox-3.5 were down-regulated in alpha-MHC Cre+/-, ALK3 F/- mouse heart. The expression of 16 genes including Ras-related protein Rab-5b and EPS-8 protein was up-regulated in the group of alpha-MHC Cre+/-, ALK3 F/-. It was found that the Box protein Pax-8 gene was down-regulated by 7.1 fold (P < 0.001) in the alpha-MHC Cre+/-, ALK3 F/- mice by real time quantitative RT-PCR. It was also revealed that the Box protein Pax-8 gene was expressed stronger in alpha-MHC Cre+/-, ALK3 F/+ than alpha-MHC Cre+/-, ALK3 F/- E11.5 days mouse heart by means of in situ hybridization.

CONCLUSIONThe Box protein Pax-8 gene is an important and cardiac-specific ALK3 downstream gene in the BMP signaling pathway during inter-ventricular septum development.

Animals ; Bone Morphogenetic Protein Receptors ; DNA-Binding Proteins ; genetics ; Down-Regulation ; Female ; Heart ; embryology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Myocardium ; metabolism ; pathology ; Nuclear Proteins ; Oligonucleotide Array Sequence Analysis ; PAX8 Transcription Factor ; Paired Box Transcription Factors ; Receptors, Growth Factor ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; genetics

OBJECTIVETo explore the relationship between physical and biological effects of alternating magnetic field and study the influence of the magnetic field on the reproductive function of murine testes.

METHODSThirty ICR mice were randomized into 5 groups: normal control, X-ray radiation, weak magnetic field (1000 Hz), 1 h strong magnetic field and 2 h strong magnetic field (2000 Hz). The mice were sacrificed at 7 days after the exposure for the analysis of testicular sperm motility, observation of histopathological changes in the testis by HE staining and evaluation of the changes by modified Johnsen grade criteria.

RESULTSThe rates of sperm motility were (42.37 +/- 10.24)% in the normal control group, (39.00 +/- 12.35)% in the X-ray radiation group, (36.00 +/- 17.28)% in the weak magnetic field group, (10.72 +/- 5.67)% in the 1 h strong magnetic field group and (4.44 +/- 2.87)% in the 2 h strong magnetic field group, respectively. Johnsen's scores decreased and the testis damage increased in a dose- and time-dependent manner.

CONCLUSIONMagnetic field, either strong or weak, may damage the testis function by inducing injury to seminiferous tubules and Leydig cells, thickening of the basal membrane, derangement, exfoliation, massive apoptosis and necrosis of spermatogenic cells in the lumen, situation of the epididymis, and consequently the absence of sperm.

Animals ; Electromagnetic Fields ; adverse effects ; Leydig Cells ; pathology ; Male ; Mice ; Mice, Inbred ICR ; Sperm Motility ; Testis ; cytology ; pathology ; radiation effects

Acute Disease ; Adolescent ; Adult ; Aged ; China ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Pesticides ; poisoning ; Poisoning ; epidemiology

OBJECTIVETo investigate the effects of Compound Salvia injection (CSI) on the number and activity of endothelial progenitor cells (EPCs).

METHODMononuclear fraction of human umbilical cord blood was obtained by density gradient centrifugation and plated on fibronectin coated culture dishes. Cells were divided in to five groups: group control, group VEGF, group CSI 50, group CSI 10 and group CSI 2 (supplemented with none cytokine, VEGF 10 ng x mL(-1), CSI 50, 10, 2 microg x mL(-1), respectively). After six days in culture, cell clusters were viewed with an inverted microscope, fluorescence-activated cell sorting (FACS) analysis of PE-CD34 and FITC-VE-Cadherin was performed to detect number of EPCs, adhesion assay was performed by replating cells on fibronectin coated dishes, and then counting adherent cells.

RESULTNumbers of EPCs of group VEGF, group CSI 10 and group CSI 2 were significantly increased as compared with those of group control ( P < 0.01, P < 0.05, P < 0.01, respectively), and numbers of EPCs of group CSI 2 were more than those of group CSI 10 and group V (P < 0.01). Compared with group control, number of EPCs of group CSI 50 was significantly decreased (P < 0.01). Compared with group control, numbers of clusters and adhesive EPCs of group CSI 10 and group CSI 2 were significantly increased, while those of group CSI 50 were significantly decreased.

CONCLUSIONLow concentration CSI can significantly promote EPCs augmentation and enhance its functional activity, while high concentration CSI significantly restrains it.

Blood Cell Count ; Cell Adhesion ; drug effects ; Dose-Response Relationship, Drug ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Endothelium, Vascular ; cytology ; Fetal Blood ; cytology ; Humans ; Injections ; Plants, Medicinal ; chemistry ; Salvia miltiorrhiza ; chemistry ; Stem Cells ; cytology ; drug effects