Follicle stimulating hormone controls granulosa cell glutamine synthesis to regulate ovulation.

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility. Inadequate understanding of the ovulation drivers hinders PCOS intervention. Herein, we report that follicle stimulating hormone (FSH) controls follicular fluid (FF) glutamine levels to determine ovulation. Murine ovulation starts from FF-exposing granulosa cell (GC) apoptosis. FF glutamine, which decreases in pre-ovulation porcine FF, elevates in PCOS patients FF. High-glutamine chow to elevate FF glutamine inhibits mouse GC apoptosis and induces hormonal, metabolic, and morphologic PCOS traits. Mechanistically, follicle-development-driving FSH promotes GC glutamine synthesis to elevate FF glutamine, which maintain follicle wall integrity by inhibiting GC apoptosis through inactivating ASK1-JNK apoptotic pathway. FSH and glutamine inhibit rapture of cultured murine follicles. Glutamine removal or ASK1-JNK pathway activation with metformin or AT-101 reversed PCOS traits in PCOS models that are induced with either glutamine or EsR1-KO. These suggest that glutamine, FSH and ASK1-JNK pathway are targetable to alleviate PCOS.

Decreased expression of hepatocyte nuclear factor 4α (Hnf4α)/microRNA-122 (miR-122) axis in hepatitis B virus-associated hepatocellular carcinoma enhances potential oncogenic GALNT10 protein activity.

MicroRNA-122 (miR-122), a mammalian liver-specific miRNA, has been reported to play crucial roles in the control of diverse aspects of hepatic function and dysfunction, including viral infection and hepatocarcinogenesis. In this study, we explored the clinical significance, transcriptional regulation, and direct target of miR-122 in hepatitis B virus (HBV)-associated hepatocellular carcinoma. Reduced expression of miR-122 in patients with HBV-associated hepatocellular carcinoma was correlated with venous invasion and poor prognosis. Furthermore, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-10 (GALNT10) was identified as a bona fide target of miR-122 in hepatoma cells. Ectopic expression and knockdown studies showed that GALNT10 indeed promotes proliferation and apoptosis resistance of hepatoma cells in a glycosyltransferase-dependent manner. Critically, adverse correlation between miR-122 and GALNT10, a poor prognosticator of clinical outcome, was demonstrated in hepatoma patients. Hepatocyte nuclear factor 4α (Hnf4α), a liver-enriched transcription factor that activates miR-122 gene transcription, was suppressed in HBV-infected hepatoma cells. Chromatin immunoprecipitation assay showed significantly reduced association of Hnf4α with the miR-122 promoter in HBV-infected hepatoma cells. Moreover, GALNT10 was found to intensify O-glycosylation following signal activation of the epidermal growth factor receptor. In addition, in a therapeutic perspective, we proved that GALNT10 silencing increases sensitivity to sorafenib and doxorubicin challenge. In summary, our results reveal a novel Hnf4α/miR-122/GALNT10 regulatory pathway that facilitates EGF miR-122 activation and hepatoma growth in HBV-associated hepatocarcinogenesis.

A novel functionalized silver nanoparticles solid chemosensor for detection of Hg(II) in aqueous media.

We report a simple strategy for the fabrication of a highly selective and sensitive Hg(II) chemosensor based on HMS-Ag composite functionalized rhodamine derivative (R). The prepared chemosensor HMS-Ag-R was characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), UV-vis spectrum and Fourier transform infrared spectroscopy (FT-IR). HMS-Ag-R has both fluorescence and colorimetry performance, and it can realize onsite and real-time detection of Hg(II) with a high sensitivity (0.7 ppb) in aqueous solution. In high concentration of mercury ions, the fluorescence intensity of HMS-Ag-R against Hg(II) sufficiently showed a typical sigmoidal shape. Moreover, HMS-Ag-R presents excellent anti-disturbance ability when exposed to a series of competitive cations such as Ag(I), K(I), Li(I), Na(I), Ba(II), Ca(II), Cd(II), Co(II), Cu(II), Mg(II), Mn(II), Ni(II), Pb(II) and Zn(II). It can be applied to the determination of Hg(II) in aqueous media. The interaction between HMS-Ag-R and Hg(II) could occur in a short time (90 s). Importantly, HMS-Ag-R could be regenerated with tetrapropyl ammonium hydroxide (TPAOH) solution.

ST6Gal-I predicts postoperative clinical outcome for patients with localized clear-cell renal cell carcinoma.

Hyperactivated α2-6-sialylation on N-glycans due to overexpression of the Golgi enzyme ß-galactoside: α2-6- sialyltransferase (ST6Gal-I) often correlates with cancer progression, metastasis, and poor prognosis. This study was aimed to determine the association between ST6Gal-I expression and the risk of recurrence and survival of patients with localized clear-cell renal cell carcinoma (ccRCC) following surgery. We retrospectively enrolled 391 patients (265 in training cohort and 126 in validation cohort) with localized ccRCC underwent nephrectomy at a single center. Tissue microarrays were constructed for immunostaining of ST6Gal-I. Prognostic value and clinical outcomes were evaluated. High ST6Gal-I expression was associated with Fuhrman grade (p<0.001 and p=0.016, respectively) and the University of California Los-Angeles Integrated Staging System (UISS) score (p=0.004 and p=0.017, respectively) in both cohorts. Patients with high ST6Gal-I expression had significantly worse overall survival (OS) (p<0.001 and p<0.001, respectively) and recurrence free survival (RFS) (p<0.001 and p=0.002, respectively) than those with low expression in both cohorts. On multivariate analysis, ST6Gal-I expression remained associated with OS and RFS even after adjusting for the UISS score. Stratified analysis suggested that the association is more pronounced among patients with low and intermediate-risk disease defined by the UISS score. High ST6Gal-I expression is a potential independent adverse predictor of survival and recurrence in ccRCC patients, and the prognostic value is most prominent in those with low and intermediate-risk disease defined by the UISS score.

Estrogen receptor α (ERα) mediates 17ß-estradiol (E2)-activated expression of HBO1.

BACKGROUND: HBO1 (histone acetyltransferase binding to ORC1) is a histone acetyltransferase (HAT) which could exert oncogenic function in breast cancer. However, the biological role and underlying mechanism of HBO1 in breast cancer remains largely unknown. In the current study, we aimed to investigate the role of HBO1 in breast cancer and uncover the underlying molecular mechanism. METHODS: Immunohistochemistry was applied to detect HBO1 protein expression in breast cancer specimens (n=112). The expression of protein level was scored by integral optical density (IOD) for further statistical analyses using SPSS. Real-time PCR was used to simultaneously measure mRNA levels of HBO1. The HBO1 protein expression in breast cancer cells was confirmed by western blot. RESULTS: HBO1 was highly expressed in breast cancer tissues and significantly correlated with estrogen receptor α (ERα) (p<0.001) and progestational hormone (PR) (p=0.002). HBO1 protein level also correlated positively with histology grade in ERα positive tumors (p=0.016) rather than ERα negative tumors. 17ß-estradiol (E2) could upregulate HBO1 gene expression which was significantly inhibited by ICI 182,780 or ERα RNAi. E2-increased HBO1 protein expression was significantly suppressed by treatment with inhibitor of MEK1/2 (U0126) in T47 D and MCF-7 cells. CONCLUSIONS: HBO1 was an important downstream molecule of ERα, and ERK1/2 signaling pathway may involved in the expression of HBO1 increased by E2.

The expression of tumor necrosis factor-alpha (TNF-alpha) by the intrathecal injection of lipopolysaccharide in the rat spinal cord.

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular site of TNF-alpha synthesis is still a matter of controversy. Therefore, we focused our study on TNF-alpha protein synthesis and expression patterns in spinal dorsal horn of naives and rats under intrathecal challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-alpha reached peak at 8 h. Double immunofluorescence revealed that LPS-induced expression of TNF-alpha exclusively located in a subpopulation of microglia, which increased at 8 h in the rat spinal dorsal horn (the injected side). Positive staining of TNF receptor 1 (TNFR1) were also found in microglia. These observations have demonstrated the production of this proinflammatory cytokine by central nerve glia especially microglia. Synthesized TNF-alpha might directly act on microglia via TNFR1, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation.

[The role of hepatic stellate cells SSeCKS expression in liver fibrosis].

OBJECTIVE: To investigate the change and effect of SSeCKS (src suppressed c kinase substrates) in the activation of hepatic stellate cells (HSCs). METHODS: HSCs were isolated from normal rats, the change of SSeCKS mRNA expression on HSCs culture in vitro was determined using real-time PCR, protein level was determined by Western blot and immunofluorescence methods. A rat model of liver fibrosis was established. The expression and location of SSeCKS and alpha-SMA (alpha-smooth muscle actin) in liver tissues were detected by immunofluorescence methods. RESULTS: SSeCKS mRNA expression was low in freshly isolated HSCs cell and the expression increased in activated HSCs in vitro. In liver fibrosis tissue, the number of SSeCKS-positive cells was increased and these cells were distributed along the sinusoids which also contained alpha-SMA positive cells. CONCLUSION: The expression of SSeCKS was increased in activated HSCs in vitro. Therefore, SSeCKS may be involved in the liver inflammation and fibrosis.

Spatiotemporal expression of SSeCKS in injured rat sciatic nerve.

SSeCKS (src suppressed C kinase substrate) functions in the control of cell signaling and cytoskeletal arrangement. It is expressed in brain and spinal cord, but little is known about its expression in peripheral nerves. In this study, in rats, real-time polymerase chain reaction and Western blot analysis showed that expression of SSeCKS in crushed sciatic nerve reached its highest level 6 hr after crushing, whereas in a transection model, SSeCKS peaked at 2 days in the proximal stump and 12 hr in the distal stump. Immunohistochemical analysis demonstrated up-regulation of SSeCKS protein surrounding the crush site and in the two stumps of the transected nerve. In addition, SSeCKS colocalized with growth-associated protein 43 and with S100, which also changed with time after injury. These findings support the idea that SSeCKS participates in the adaptive response to peripheral nerve injury and may be associated with regeneration.

Phosphorylation of extracellular signal-regulated kinases 1/2 predominantly enhanced in the microglia of the rat spinal cord following lipopolysaccharide injection.

The present study was initiated to investigate the role of extracellular signal-regulated kinases (ERK) 1/2 signaling pathway in the early response of spinal cord to systemic inflammation by using Western blotting and immunohistochemical techniques in a rat model intraperitoneally injected with 10 mg/kg of lipopolysaccharide (LPS). The results showed that there was a considerable amount of phosphorylated ERK 1/2 protein in the spinal cord of inflamed animals killed under pentobarbital anesthesia. The result of Western blotting showed that the phosphorylation level of ERK 1/2 in the spinal cord was increased at one hour; then 12 and 24 h after LPS injection the level decreased, while the total ERK 1/2 level seemed unchanged. The phosphorylated ERK 1/2 dominantly existed in the microglia cells of the gray matter of spinal cord, as demonstrated with double immunofluorescent staining 1 h after LPS injection. Collectively, the present results suggest that ERK signal pathway involve the cellular activation in the spinal cord following systemic inflammation, with ERK mainly in microglia. The increase of phosphorylation of ERK 1/2 in microglia of spinal cord after LPS injection implicates that ERK signaling pathway involves intracellular activity of microglia responding to the inflammation.

Developmental regulation of SSeCKS expression in rat brain.

SSeCKS (src suppressed C kinase substrate) was identified as a PKC substrate/PKC-binding protein, which plays a role in mitogenic regulatory activity and has a function in the control of cell signaling and cytoskeletal arrangement. Previous studies showed that expression of SSeCKS mRNA and protein levels were developmentally regulated in rat testis and the molecular might have some effects on the process of spermiogenesis. Here we carried out experiments to investigate the expression of SSeCKS in rat brain. Western blot analysis indicated that SSeCKS could be detected in the whole brain of developing rat embryos and reached its peak at 1 week after birth, while during mature period, its level was decreasing. Regional-distribution analysis showed that the expression pattern of SSeCKS in telencephalon, hippocampus and diencephalons was in accordance with the result from whole brain both in mRNA and protein level. However, in cerebellum, SSeCKS was almost in the same level, and in brainstem, the expression level was higher in 4-week-old rat brain than in 1-week-old one. Immunohistochemistry results showed SSeCKS was in diffused and granule-like distribution. Double immunofluorescence staining showed that it was expressed by some GFAP positive cells. All the results suggested that SSeCKS might affect brain development and further research is needed to have a good understanding of its function and mechanism.

Src suppressed C kinase substrate regulates the lipopolysaccharide-induced TNF-alpha biosynthesis in rat astrocytes.

The protein kinase C (PKC) is known to be a critical component in the signaling cascades that lead to astrocyte-activation. To further understand the mechanism of PKC signaling in astrocyte-activation, we investigated the effect of SSeCKS, a PKC substrate, on LPS-induced cytokine expression in astrocytes by RT-PCR and enzyme-linked immunosorbent assay. Exposure of the cells to LPS induced rapid translocation of SSeCKS to the perinuclear sides, ERK activation and pronounced TNF-alpha production, which can be inhibited by the PKC inhibitor Gö6983. By using siRNA knockdown of SSeCKS expression, LPS-induced signaling events were partly inhibited, including ERK activation, inducible TNF-alpha biosynthesis and secretion. These results suggest that SSeCKS is involved in the LPS-induced TNF-alpha expression in astrocytes mediated by PKC.

Lipopolysaccharide-induced upregulation of tumor necrosis factor-alpha (TNF-alpha) and TNF receptors in rat sciatic nerve.

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular sites of TNF-alpha and TNF receptors synthesis are still a matter of controversy. Therefore, we focused our study on TNF-alpha, TNFR1, and TNFR2 protein synthesis and expression patterns in sciatic nerve of controls and rats under systemic challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-alpha reached peak at 6 h. Double immunofluorescence revealed that LPS-induced expression of TNF-alpha exclusively located in a subpopulation of Schwann cells, endothelial cells, and macrophages, which increased at late time point in the rat sciatic nerve. Positive staining of TNF receptors were also found in Schwann cells and a few endothelial cells. These observations have demonstrated the production of this proinflammatory cytokine by peripheral nerve glia especially Schwann cells. Synthesized TNF-alpha might directly act on peripheral nerve glia via TNF receptors, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation.

[Expression and correlation of Skp2 and p27(kip1) in 92 cases of non-Hodgkin's lymphoma].

OBJECTIVE: To investigate the expression and correlation of Skp2 and p27kipl in non-Hodgkin's lymphoma. METHODS: The expression of Skp2, p27(kip1) and Ki-67 (the proliferation index)were detected in sections of 92 cases of non-Hodgkin's lymphoma (NHL) and 14 cases of reactive lymph nodes by immunohistochemistry and histopathology. The expression of Skp2 and p27(kip1) in 4 NHL cell lines were detected by Western blot. RESULTS: The expression of Skp2 in NHL cases were significantly higher than that in reactive lymph nodes (except the germinal centers), positively correlated with proliferation activity, and an increasing tumor aggressiveness was associated with the increased expression of Skp2. The expression of p27(kip1) protein in NHL cases were significantly lower than that in reactive lymph nodes (except the germinal centers), negatively correlated with proliferation activity, and an increasing tumor aggressiveness was associated with decreased expression of p27(kip1). The statistical analysis indicated that there was no obvious correlation between Skp2 and p27(kip1) expression in NHL tissues. CONCLUSION: The higher expression of Skp2 and lower expression of p27(kip1) in NHL tissues may play a role in the tumorigenesis and development of NHL.

Changes of Src-suppressed C kinase substrate expression in cytokine induced reactive C6 glioma cells.

OBJECTIVE: To investigate effect of tumor necrosis factor-alpha (TNF-alpha) on the Src-suppressed C kinase substrate (SSeCKS) in C6 glioma cells. METHODS: Cultured C6 glioma cells were randomly divided into two groups. In time-dependent group, cells were cultured with TNF-alpha (2 ng/mL) for 0 h, 1 h, 3 h, 6 h, 12 or 24 h, respectively; in dose-dependent group, cells were cultured with TNF-alpha (0 ng/mL, 0.02 ng/mL, 0.2 ng/mL, or 2 ng/mL) for 6 h. The expression of SSeCKS was detected by Realtime PCR and Western blot analysis, and immunocytochemistry was used to investigate SSeCKS's subcellular localization. RESULTS: TNF-alpha induced rapid phosphorylations of protein kinase C (PKC) substrates in C6 glioma cells, and upregulated SSeCKS expression in a time and concentration dependent manner. Immunocytochemistry suggested that SSeCKS was localized in the cyroplasm and the leading end of podosomal extensions in control groups, while TNF-alpha induced translocation of SSeCKS perinuclear. This effect could be partly reversed by PKC inhibitor Ro-31-8220. CONCLUSION: TNF-alpha activates PKC and upregulates SSeCKS expression in C6 glioma cells. These effects are associated with PKC activity, suggesting that SSeCKS plays a role in response to glia activation in PKC mediated pathway.

[Expression, localization and interrelationship of P27kip1 and cyclin D3 in non-Hodgkin's lymphoma].

OBJECTIVE: To investigate the expression, localization and interrelationship of P27(kip1) and cyclin D3 in non-Hodgkin's lymphoma (NHL). METHODS: The expressions of P27(kip1), cyclin D3 and index Ki-67 was detected in 100 NHL and 20 reactive lymph nodes by immunohistochemical technique. The expression and localization of P27(kip1) and cyclin D3 in 3 NHL cell lines were detected by Western blot, double immunolabelling and laser scanning confocal microscopy, respectively. RESULTS: In general the expression of P27(kip1) in NHL was lower than in control group, and was negatively related to the tumor aggressiveness and proliferating activity; the expression of cyclin D3 in NHL was higher than in control group, and was positively related to the tumor aggressiveness and proliferating activity. There was a negative correlation between P27(kip1) and cyclin D3. Nevertheless, anomalous high P27(kip1) expression was found in a few NHL tissues with high expression of cyclin D3 and Ki-67. Overexpression and colocalization of P27(kip1) and cyclin D3 was found in Raji cell line. CONCLUSIONS: Under expression of P27(kip1) and overexpression of cyclin D3 may play a role in the occurrence and development of NHL. Anomalous high P27(kip1) expression and its interaction with cyclin D3 may be another mechanism for tumor genesis of NHL.