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 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 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

Huanshao capsule is widely used in irregular menstruation and has achieved a good effect. Huanshao capsule can promote gonad development in mice, significantly improve the ovarian index in mice, increase estrogen level and reduce FSH level in rats, inhibit the pain response induced by oxytocin and estrogen, inhibit writhing reaction induced by acetic acid pain in mice. Due to the complexity of traditional Chinese medical formula, the pharmacological mechanism of the treatment on the irregular menstruation of the Huanshao capsule is unclear. In this study, the internet-based computation platform (www.tcmip.cn）was used to explore the molecular mechanism of Huanshao capsule on the menstrual. The aim of this study was to find the molecular mechanism of Huanshao capsule in treating menstrual. In the study of the molecular mechanism of Huanshao capsule in the treatment of menstrual by using the internet-based computation platform, Huanshao capsule maybe treat the menstrual by the pathway of endocrine system, GnRH signal transduction pathway, estrogen signal transduction pathway, oxytocin signaling pathway, thyroid hormone signaling pathway, VEGF signaling pathway, FCεRI signaling pathway and purine metabolism and nucleotide metabolism. The early pharmacological study confirmed Huanshao capsule could increase the serum estradiol level and decrease follicle stimulating hormone level and the traditional Chinese medicine pharmacology coincide with the prediction result of internet-based computation platform which roles as the pathway of GnRH signaling pathway and estrogen signal transduction pathway. Other pathway needs further experimental verification.

The risk of plague epidemics and relapse of various types of plague foci persists in Inner Mongolia Autonomous Region. For Marmota sibirica plague foci, the animal plague has not been found but antibody has been detected positive. Nowadays, Marmota sibirica has been increasing in population and distribution in China. In bordering countries Mongolia and Russia, the animal plague has been continuously prevalent. For Spermophilus dauricus plague foci, the animal plague has been taken place now and then. Compared to the above foci, the animal plague is most prevalent in Meriones unguiculatus plague foci and frequently spread to humans. Due to higher strain virulence and historical disaster in Marmota sibirica plague foci and Spermophilus dauricus plague foci, plague prevention and control should be strengthened on these foci. In addition to routine surveillance, epidemic dynamics need to be further monitored in these two foci, in order to prevent their relapse and spread to humans.
Animals ; China/epidemiology* ; Epidemics ; Humans ; Plague/prevention & control* ; Prevalence ; Sciuridae ; Yersinia pestis