Icariin Stimulates hFOB 1.19 Osteoblast Proliferation and Differentiation via OPG/RANKL Mediated by the Estrogen Receptor.

BACKGROUND: Icariin (ICA), one of the main effective components isolated from the traditional Chinese herb Epimedium brevicornu Maxim., has been reported to possess extensive pharmacological actions, including enhanced sexual function, immune regulation, anti-inflammation, and antiosteoporosis. METHODS: Our study was designed to investigate the effect of ICA on cell proliferation and differentiation and the molecular mechanism of OPG/RANKL mediated by the Estrogen Receptor (ER) in hFOB1.19 human osteoblast cells. RESULTS: The experimental results show that ICA can stimulate cell proliferation and increase the activity of Alkaline Phosphatase (ALP), Osteocalcin (BGP) and I Collagen (Col I) and a number of calcified nodules. Furthermore, the mRNA and protein expression of OPG and RANKL and the OPG/ RANKL mRNA and protein expression ratios were upregulated by ICA. The above-mentioned results indicated that the optimal concentration of ICA for stimulating osteogenesis was 50ng/mL. Subsequent mechanistic studies comparing 50ng/mL ICA with an estrogen receptor antagonist demonstrated that the effect of the upregulated expression is connected with the estrogen receptor. In conclusion, ICA can regulate bone formation by promoting cell proliferation and differentiation and upregulating the OPG/RANKL expression ratio by the ER in hFOB1.19 human osteoblast cells.

The role of FTO variants in the susceptibility of polycystic ovary syndrome and in vitro fertilization outcomes in Chinese women.

We investigated the association between single nucleotide polymorphisms (SNPs) in the fat mass and obesity associated (FTO) gene (rs9926289 A/G, rs79206939 A/G, rs9930506 A/G, rs8050136 A/C, and rs1588413 C/T) and polycystic ovary syndrome (PCOS), as well as outcomes of in vitro fertilization (IVF). A case-control study consisting of 147 PCOS patients and 120 healthy controls was conducted. FTO SNPs were genotyped by PCR to determine allelic frequencies, and IVF outcomes were analyzed. The results showed that FTO rs8050136 (p = .025) and rs1588413 (p = .042) were significantly associated with PCOS susceptibility, and women with risk alleles were often found to be obese (p < .05). For SNP rs8050136, women with AA + AC genotypes had higher body mass indexes (BMIs), oral glucose tolerance test/2 h (OGTT) levels and implantation rates but lower follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) day progesterone levels and ovulation numbers (all p < .05) than those with the CC genotype. For SNP rs1588413, women carrying risk alleles exhibited higher BMIs, implantation rate, and levels of luteinizing hormone (LH), estradiol, and OGTT/2 h (all p < .05) compared with those with non-risk genotypes. Therefore, these findings suggest that rs8050136 and rs1588413 are associated with PCOS susceptibility, and that women with risk alleles have less ovulation numbers but higher implantation rates than those with other genotypes.

Curcumin enhances vascular contractility via induction of myocardin in mouse smooth muscle cells.

A variety of cardiovascular diseases is accompanied by the loss of vascular contractility. This study sought to investigate the effects of curcumin, a natural polyphenolic compound present in turmeric, on mouse vascular contractility and the underlying mechanisms. After mice were administered curcumin (100 mg·kg-1·d-1, ig) for 6 weeks, the contractile responses of the thoracic aorta to KCl and phenylephrine were significantly enhanced compared with the control group. Furthermore, the contractility of vascular smooth muscle (SM) was significantly enhanced after incubation in curcumin (25 µmol/L) for 4 days, which was accompanied by upregulated expression of SM marker contractile proteins SM22α and SM α-actin. In cultured vascular smooth muscle cells (VSMCs), curcumin (10, 25, 50 µmol/L) significantly increased the expression of myocardin, a "master regulator" of SM gene expression. Curcumin treatment also significantly increased the levels of caveolin-1 in VSMCs. We found that as a result of the upregulation of caveolin-1, curcumin blocked the activation of notch1 and thereby abolished Notch1-inhibited myocardin expression. Knockdown of caveolin-1 or activation of Notch1 signaling with Jagged1 (2 µg/mL) diminished these effects of curcumin in VSMCs. These findings suggest that curcumin induces the expression of myocardin in mouse smooth muscle cells via a variety of mechanisms, including caveolin-1-mediated inhibition of notch1 activation and Notch1-mediated repression of myocardin expression. This may represent a novel pathway, through which curcumin protects blood vessels via the beneficial regulation of SM contractility.

New Insights into mTOR Signal Pathways in Ovarian-Related Diseases: Polycystic Ovary Syndrome and Ovarian Cancer

mTOR, the mammalian target of rapamycin, is a conserved serine/threonine kinase which belongs to the phosphatidyl-linositol kinase-related kinase (PIKK) family. It has two complexes called mTORC1 and mTORC2. It is well established that mTOR plays important roles in cell growth, proliferation and differentiation. Over-activation of the mTOR pathway is considered to have a relationship with the development of many types of diseases, including polycystic ovary syndrome (PCOS) and ovarian cancer (OC). mTOR pathway inhibitors, such as rapamycin and its derivatives, can directly or indirectly treat or relieve the symptoms of patients suffering from PCOS or OC. Moreover, mTOR inhibitors in combination with other chemical-molecular agents may have extraordinary efficacy. This paper will discuss links between mTOR signaling and PCOS and OC, and explore the mechanisms of mTOR inhibitors in treating these two diseases, with conclusions regarding the most effective therapeutic approaches.

The association between androgen receptor gene CAG polymorphism and polycystic ovary syndrome: a case-control study and meta-analysis.

PURPOSE: Many studies have been carried out to confirm the relationship between androgen receptor gene CAG repeat polymorphism and polycystic ovary syndrome (PCOS), without consistent results. Hence we conducted the current study to research this relationship. METHODS: 224 Chinese Han women with PCOS and 223 in vitro fertilization and embryo transplantation (IVF-ET) infertile women with tubal factor or male infertility served as the controls were recruited in our study. PCR-based assays were applied to genotype the (CAG)n repeat alleles. A meta-analysis including 1,536 PCOS patients and 1,807 controls was conducted to produce a pooled estimate. RESULTS: We observed that the CAG bi-allelic mean lengths were similar in PCOS patients and controls (22.65 ± 2.5 vs. 23.09 ± 2.1, P = 0.116). When CAG bi-allelic were divided into two categories (mean repeats ≤22, >22), the short AR-CAG bi-allelic showed more frequent in PCOS group than in controls (56.25% vs 29.14%, P < 0.001). Further analysis presented that, in PCOS, there was a lower mean CAG repeat lengths in mean bi-allelic lengths (22.3 ± 2.5 vs. 23.9 ± 2.2, P = 0.008) and long bi-allelic lengths (24.3 ± 1.4 vs. 25.9 ± 1.6, P = 0.05) among patients with testosterone less than 0.7 ng/ml compared with those whose testosterone was more than 0.7 ng/ml. Besides, the testosterone were positively correlated with the CAG polymorphism (r = 0.237, P = 0.008), which accorded with our meta-analysis results. CONCLUSIONS: The distribution of AR-CAG allele differed between PCOS patients and controls, and polymorphism of CAG repeat lengths may contribute to hyperandrogenism in PCOS.

Discrimination of A1555G and C1494T point mutations in the mitochondrial 12S rRNA gene by on/off switch.

The objective of this study was to apply the "on/off" switch consisting of 3' phosphorothioate-modified allele specific primers and exo(+) polymerase in single base discrimination of A1555G and C1494T mutations in the highly conserved sites of the mitochondrial 12S rRNA. The two point mutations are the hotspot mutations associated with either aminoglycoside antibiotics induced deafness or inherited nonsyndromic hearing loss. The PCR products of mitochondrial DNA (mtDNA) 12S rRNA gene were inserted into the pMD19-T vector for transformation into Escherichia coli JM109 competent cells for preparing wild-type pMD19-T/mt vector. Inverse PCR was carried out for mtDNA 12S rRNA gene C1494T and A1555G mutagenesis and DpnI endonuclease degradating methylated pMD19-T/mt vector existing in the inverse PCR products was carried out to construct the mutation-type pMD19-T/mtM vector. These constructed vectors were confirmed by DNA sequencing. Allelic specific primers targeting wild-type and mutation-type templates were designed with 3' terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. Amplified by exo(+) polymerase, allelic specific primers perfectly matching wild-type allele were extended while no products were produced from primers targeting point-mutated deafness-related allele. Similarly, allelic specific primers perfectly matching point-mutated deafness-related mutation-type allele were extended and no products were yielded from primers targeting wild-type allele. No specific product was observed in the primer extension reaction mediated by on/off switch in screening the mtDNA 12S rRNA gene harboring either C1494T or A1555G mutation in 40 healthy volunteers tested. These data suggest that the "off switch" mediated by exo(+) polymerase is highly reliable in the diagnosis of monogenic diseases and the novel "on/off" switch has enormous applications in systematic and extended screening of the12S rRNA gene A1555G and C1494T mutations. The established assay can be widely used not only for hearing loss patients but also for normal subjects before the use of aminoglycoside antibiotics.

[Preparation and identification of monoclonal antibody against human thrombomodulin.].

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.

On/off switch mediated by Exo+ polymerases: experimental analysis for its physiological and technological implications.

The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.

[Effect of 3' exonuclease activity of polymerase on extension of phosphorothioate-modified primers].

OBJECTIVE: To determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase. METHODS: Two-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated. RESULTS: Exo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase. CONCLUSION: These data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.