It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.
Alkaline Phosphatase/metabolism* ; Animals ; Electromagnetic Fields ; Osteoblasts/metabolism* ; Osteogenesis/genetics* ; Rats ; TRPP Cation Channels/physiology*

The expression of sperm-specific genes is necessary in spermiogenesis, and the genetic polymorphism of these genes may impair spermatogenesis, leading to male infertility. Mamy investigations have been conducted on the polymorphism of some candidate genes that are specifically expressed in spermiogenesis, the relation of genetic polymorphism with male infertility and its possible mechanism. Further attempts have been made at the explanation of the causes of infertility from the genetic angle.
Alkaline Phosphatase ; genetics ; DNA-Binding Proteins ; genetics ; Gene Expression ; Humans ; Infertility, Male ; genetics ; Male ; Polymorphism, Genetic ; Spermatogenesis ; genetics ; Spermatozoa ; metabolism

The relationship among the substituted amino acids, the 3D structure simulated on PC through CPHmodels Server ( http://www.cbs.dtu. dk/services/CPHmodels/) and the thermostable performance of 4 thermostable alkaline phosphatase(TAP) mutants selected from a clone bank of more than 200 mutants were analyzed to explore the mechanism of thermostability change. These mutants are TAP(A410T) (A410-->T), TAP(P396S) (P396-->S), TAP2(N100S T320-->I) and TAP4(N100-->S P396-->S A410 -->V P490-->S). TAP and the mutants' thermostable performance was evaluated by measuring the highest tolerable temperature (T1/2) and the optimal reaction temperature (Topt). The 3D structure neighboring the substituted amino acids was simulated by Swiss-PDBViewer to observe the relationship between the structure change and the thermostable performance of TAP and its mutants. The results displayed that all these amino acid substitutions except the T320-->I mutant brought about only a little local change on TAP's 3D structure and very little effect on their optimal reaction temperature, but a significant decrease (nearly 10 degrees C) on their highest tolerable temperature. However, the T320-->I mutation due to close to TAP's active sites did bring about a significant descendents of the mutant in both the highest tolerable temperature and the optimal reaction temperature. Thus, it seems to be able to conclude that most of the amino acid substitutions, no matter where they locate and what structure change they may make, can cause TAP's highest tolerable temperature reduced significantly. What's more, if the mutation occurring near or in the active sites, it can also cause TAP's optimal reaction temperature reduced significantly at the same time.
Alkaline Phosphatase ; chemistry ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; genetics ; physiology ; Mutation ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Temperature

OBJECTIVETo investigate the mineralization capacity of periodontal ligament stem cells (PDLC) by determining the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN) and matrix extracellular phosphoglycoprotein (MEPE) and to explore the potential of MEPE as a differentiation marker for PDLC, and its possible function in PDLC osteogenic differentiation.

METHODSPDLC were digested and cultured by a solution containing collagenase type I and dispase. PDLC were preceded to osteogenic induction for 7, 14 and 21 days respectively, and the cells before induction served as controls. Mineralization nodules and the expression of OCN in PDLC were investigated by alizarin red and immunohistochemistry respectively. The expressions of ALP, OCN and MEPE mRNA were investigated by quantitative real-time RT-PCR analysis. Statistical analysis was performed to compare the differences of mRNA expression levels among cell samples collected at different time points.

RESULTSThe mRNA expressions of ALP, OCN and MEPE in PDLC before induction were 72, 1.1 and 534 respectively, but increased time-dependently in the induction cultures. The mRNA expressions of ALP, OCN and MEPE were 78, 9.56 and 629.6 on day 7; 290, 133 and 638.3 on day 14; 1108, 925 and 2261.1 on day 21 respectively. The relative mRNA levels of OCN, ALP on day 14 and 21, MEPE on day 21 were significantly higher than control group (P < 0.05).

CONCLUSIONSPDLC showed analogously temporal expression of ALP, OCN and MEPE mRNA while differentiating into cementoblast/osteoblast-like cells in vitro. MEPE may play a regulatory role in PDLC osteogenic differentiation, and may be a potential osteogenic differentiation marker along with ALP and OCN.

Alkaline Phosphatase ; genetics ; metabolism ; Cell Differentiation ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Glycoproteins ; genetics ; metabolism ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; genetics ; metabolism ; Periodontal Ligament ; cytology ; metabolism ; Phosphoproteins ; genetics ; metabolism ; RNA, Messenger ; genetics

PURPOSE: The aim of the present study was to evaluate the clinical characteristics of the primary Epstein-Barr virus (EBV) hepatitis with elevation of both serum alkaline phosphatase (ALP) and gamma-glutamyltransferase (gamma-GT) levels in children. MATERIALS AND METHODS: A retrospective study was performed by reviewing of the medical records of 36 patients who were diagnosed with primary EBV hepatitis. The patients were divided into 2 groups: patients with elevated serum ALP and gamma-GT levels (group 1) and patients without (group 2). RESULTS: The classic features of infectious mononucleosis (fever, pharyngitis and/or tonsillitis, and cervical lymphadenitis) were seen in 20 (57.1%) of group 1 patients and 18 (50.0%) of group 2 patients. Hepatitis with elevated serum ALP and gamma-GT levels were present in 14 (38.9%) of the all patients. Of these patients, Jaundice occurred in only 2 (5.6%). The mean levels of aspartate aminotransferase and alanine aminotransferase (ALT) as well as the number of patients with ALT greater than 400 IU/L were significantly different between the groups (177 IU/L vs. 94 IU/L, 418 IU/L vs. 115 IU/L, and 50.0% vs. 13.6%; p=0.001, p=0.001, p=0.026, respectively). The mean duration of elevated serum ALT levels was 17.5 days in group 1 and 9.0 days in group 2 (p=0.013). All patients recovered fully without any chronic or serious complications. CONCLUSION: Primary EBV hepatitis with predominant biochemical abnormalities of the elevation of ALP and gamma-GT is frequent and mostly anicteric. This may represent a benign disease, but a delay in recovery of liver function as well.
Alkaline Phosphatase/genetics/*metabolism ; Child ; Child, Preschool ; Female ; Hepatitis/*enzymology/*pathology/virology ; Herpesvirus 4, Human/*pathogenicity ; Humans ; Infant ; Male ; gamma-Glutamyltransferase/genetics/*metabolism

OBJECTIVE: To investigate the effect of adiponectin on the osteoblast differentiation and its signal transduction. METHODS: Adipopnectin receptor (AdipoR) was detected by immunoblot analysis. Alkaline phosphatase (ALP) activity was measured by enzyme-linked immunosorbent assay. Osteocalcin was measured by a specific radioimmunoassay kit, and the extent of mineralized matrix was determined. RNA interference was used to down-regulate the expression of AdipoR1 in human osteoblasts, and the effect of adiponectin on osteoblast differentiation was investigated. RESULTS: Only AdipoR1 protein was detected in human osteoblasts. Adiponectin could promote osteoblast differentiation, and result in a dose-dependent increase in ALP activity, osteocalcin secretion, and an increase in mineralized nodules. Suppression of AdipoR1 with siRNA could abolish the adiponectin induced ALP expression. Adiponectin could induce the activation of p38 and JNK, but not ERK1/2 in osteoblasts, and the pretreatment of osteoblasts with the p38 inhibitor (SB203580) could block the adiponectin-induced ALP activity. CONCLUSION Adiponectin can induce human osteoblast differentiation via AdipoR1/p38 pathway.
Adiponectin ; pharmacology ; Alkaline Phosphatase ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; analysis ; RNA, Small Interfering ; genetics ; Receptors, Adiponectin ; biosynthesis ; Signal Transduction

AIMTo establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor beta subtype.

METHODSA recombinant vector pTAL-ERE-SEAP was constructed by inserting a synthetic sequence composed of five estrogen responsive elements in front of promoter of pTAL-SEAP vector. pTAL-ERE-SEAP was then transfected into human embryonic kidney (HEK293) cells. G418 (200 microg x mL(-1)) was added to select positive clones that can be induced by E2 to express reporter gene SEAP. The speciality was tested by several ligands of relative nuclear receptors of the same family. The stability of the model, the time-effect relationship, the dose-response relationship, and the immunocytochemistry staining of ERbeta expression after transfection were observed. 2 622 compounds were screened by using this model.

RESULTSStably transfected clones were obtained. The expression levels of reporter gene SEAP of positive clones was induced by E2 in a dose-response and time-effect relationship manners. The Z' factor value was 0.7. The expression levels of dexamethasone and other ligands were low. The result of immunocytochemistry staining showed the expression of ERbeta. E2 had no proliferating effects on stably transfected clones.

CONCLUSIONStably transfected positive clones transfected with recombinant vector pTAL-ERE-SEAP were obtained. The positive clones may be used to screen for agonists of estrogen receptor beta subtype by measurement of luminescent value of expressed SEAP in wells of microlitre plate.

Alkaline Phosphatase ; genetics ; metabolism ; Cell Line ; Estradiol ; pharmacology ; Estrogen Receptor beta ; agonists ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; Humans ; Immunohistochemistry ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Response Elements ; genetics ; Transfection

Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation.
Adipose Tissue/*cytology ; Alkaline Phosphatase/metabolism ; Biological Markers/metabolism ; Bone Matrix/metabolism ; Calcification, Physiologic/genetics ; *Cell Differentiation/genetics ; Core Binding Factor Alpha 1 Subunit/metabolism ; *Electromagnetic Fields ; Humans ; *Osteogenesis/genetics ; Reproducibility of Results ; Stem Cells/*cytology/enzymology/metabolism

OBJECTIVETo evaluate the effect of orthodontic light force on the expression of Asporin, bone morphogenetic proteins-2 (BMP-2) and alkaline phosphatase (ALP) after auto-transplantation.

METHODSThirty-two maxillary and mandibular incisors in four 13 month-old male Beagle dogs were auto-transplanted to the other side of the same jaw. The teeth were all endodontically treated and divided into four groups, control (group 1) and three experimental groups (groups 2, 3 and 4).In control group, the teeth were unloaded. In the other three experimental groups, continuous force was applied in the 1st week (group 2), 2nd week (group 3) and 4th week (group 4) after auto-transplantation, respectively. The dogs were sacrificed in the 8th week. The mRNA expressions of Asporin,BMP-2 and ALP were examined by real time PCR. The expression of periodontal ligament associated protein-1 (PLAP-1) was examined by Western blotting. The data were statistically analyzed using SPSS 17.0 and one-way analysis of variance (ANOVA).

RESULTSIn group 3, the expression of Asporin mRNA (2.047 ± 0.281) was higher than that in the other three groups, while the expression of BMP-2 (1.358 ± 0.095) was lower than that in group 2 and control group (P < 0.05). The expression of PLAP-1 (1.054 ± 0.113) in group 3 was higher than other groups, while significant difference was found between any two groups.

CONCLUSIONSOrthodontic force could stimulate the expression of Asporin and PLAP-1. The orthodontic force applied in the 2nd week after the tooth auto-transplantation, the expression of Asporin and PLAP-1 reached the highest level.

Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Dogs ; Extracellular Matrix Proteins ; genetics ; metabolism ; Incisor ; metabolism ; transplantation ; Male ; Mandible ; Maxilla ; Orthodontic Extrusion ; RNA, Messenger ; metabolism ; Transplantation, Autologous

BACKGROUND: Congenital scoliosis (CS) is a complex spinal malformation of unknown etiology with abnormal bone metabolism. Fibroblast growth factor 23 (FGF23), secreted by osteoblasts and osteocytes, can inhibit bone formation and mineralization. This research aims to investigate the relationship between CS and FGF23. METHODS: We collected peripheral blood from two pairs of identical twins for methylation sequencing of the target region. FGF23 mRNA levels in the peripheral blood of CS patients and age-matched controls were measured. Receiver operator characteristic (ROC) curve analyses were conducted to evaluate the specificity and sensitivity of FGF23. The expression levels of FGF23 and its downstream factors fibroblast growth factor receptor 3 (FGFr3)/tissue non-specific alkaline phosphatase (TNAP)/osteopontin (OPN) in primary osteoblasts from CS patients (CS-Ob) and controls (CT-Ob) were detected. In addition, the osteogenic abilities of FGF23-knockdown or FGF23-overexpressing Ob were examined. RESULTS: DNA methylation of the FGF23 gene in CS patients was decreased compared to that of their identical twins, accompanied by increased mRNA levels. CS patients had increased peripheral blood FGF23 mRNA levels and decreased computed tomography (CT) values compared with controls. The FGF23 mRNA levels were negatively correlated with the CT value of the spine, and ROCs of FGF23 mRNA levels showed high sensitivity and specificity for CS. Additionally, significantly increased levels of FGF23, FGFr3, OPN, impaired osteogenic mineralization and lower TNAP levels were observed in CS-Ob. Moreover, FGF23 overexpression in CT-Ob increased FGFr3 and OPN levels and decreased TNAP levels, while FGF23 knockdown induced downregulation of FGFr3 and OPN but upregulation of TNAP in CS-Ob. Mineralization of CS-Ob was rescued after FGF23 knockdown. CONCLUSIONS Our results suggested increased peripheral blood FGF23 levels, decreased bone mineral density in CS patients, and a good predictive ability of CS by peripheral blood FGF23 levels. FGF23 may contribute to osteopenia in CS patients through FGFr3/TNAP / OPN pathway.
Humans ; Osteopontin/genetics* ; Alkaline Phosphatase/metabolism* ; Receptor, Fibroblast Growth Factor, Type 3/metabolism* ; Scoliosis/genetics* ; Osteoblasts/metabolism* ; Calcinosis ; RNA, Messenger/metabolism* ; Bone Diseases, Metabolic/metabolism* ; Fibroblast Growth Factors/genetics*