[The Establishment and Identification of Stable CHO Cell Line with Canine Transferrin Receptor Expression].

OBJECTIVES: To construct eukaryotic expressing recombinant vector of canine transferrin receptor gene (TfR ), then to transfect Chinese hamster ovary (CHO) cells with the vector for establishment of stable expression of TfR in CHO cell line. METHODS: The full-length TfR fragment was amplified by RT-PCR from the canine cells (walter reed dog cell, WRD) and then inserted into eukaryotic expression vector pCDNA3. After identification with enzyme digestion and sequencing, the recombinant vector was transfected into CHO cells by TransLipid Transfection Reagent. The stable transfected CHO cell line was then established by screening cultures with G418, and the expression of TfR was identified by RT-PCR, Western blot and immunofluorescence, respectively. RESULTS: The eukaryotic expression vector pCDNA3-TfR was constructed successfully by checking with enzyme digestion and sequencing, and the highly expressed canine TfR was observed in CHO cells transfected with pCDNA3-TfR by using RT-PCR, Western blot and immunofluorescence, respectively. The stable CHO cell line with canine TfR expression was established. CONCLUSIONS: The construction of the eukaryotic expression vector pCDNA3-TfR and the establishment of stable CHO cell line with TfR expression provide solid foundation for further experimental studies on the function of TfR.

Evolution of Acoustic Emission and Microcrack in Jointed Coal under Loading and Their Correlations.

Understanding of continuous damage and cracking process of compressed jointed coal is critical to predict its stability. Herein, for jointed coal under uniaxial compression with bedding planes perpendicular (sample A) and parallel (sample B) to the loading direction, an acoustic emission (AE) monitoring device and a computed tomography (CT) scanner were employed to study the damage and cracking process. Furthermore, the correlations between the AE signals and internal fracture parameters were analyzed. Results show that (1) shear and tension cracks are respectively main damage forms in samples A and B; (2) there is a drop valley of variations of AE count fractal dimension (D1) over time before the stress peak, and compared with sample B, the drop valley for sample A are much wider; (3) many fractures between joint planes formed at yielding and postpeak stages, fracture fractal dimensions (D2) first increase slightly with loading and then increase significantly at yielding and postpeak stages; especially D2 of sample A is larger than that of sample B; (4) the accumulative value of D1 (AD1) increases with D2 monotonously, and an expression of porosity or connectivity of the compressed jointed coal was developed, by which the porosity and connectivity of jointed coal could be calculated. The study outcomes could contribute to predict coal stability and the variations of water and gas flowing channels in seam in underground coal mines.

[Detection of TERC gene amplification by fluorescence in-situ hybridization in cervical intraepithelial lesions].

OBJECTIVE: To explore the feasibility and practical value of fluorescence in situ hybridization (FISH) detection of TERC gene amplification in cervical intraepithelial lesions (CIN) and squamous cell carcinoma (SCC). METHODS: Tissue microarray was constructed to cover 150 cases of various cervical conditions, including 24 cases of normal cervical mucosa, 78 cases of CINs (CINI, 25 cases; CINII, 21 cases and CINIII, 32 cases) and 48 cases of SCC. FISH was used to detect TERC gene amplification. RESULTS: TERC gene amplification was detected in 8% (2/25) CINI, 47.6% (10/21) CINII, 71.9% (23/32) CINIII and 87.5% (42/48) SCC. There were significant differences among these groups (P < 0.05). The amplification rates of TERC gene in SCC, CINIII and CINII were significantly higher than those of normal cervical epithelium and CINI (P < 0.05). Significant differences were also observed among CINI and CINII, CINIII and SCC (P < 0.05), and between CINII and SCC (P < 0.05). There were no significant differences between normal cervical epithelium and CINI, CINII and CIN III, and between CINIII and SCC (P > 0.05). FISH detection of amplification of TERC gene in CINI and CINII-III demonstrated the following statistics: sensitivity of 62.3%, specificity of 92.0%, accuracy of 71.8%, positive and negative predictive values of 94.3% and 53.5%, respectively. CONCLUSIONS: FISH detection is a reliable method in detecting TERC gene amplification using paraffin tissue sections. When histological evaluation becomes difficult, TERC amplification detectable by FISH may offer a diagnostic distinction of CINI from CINII. Moreover, TERC amplification may be used as a biomarker in predicting CIN progression to invasive cancer.

Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents 6-hydroxydopamine induced apoptosis in SH-SY5Y cells.

Oxidative stress caused by dopamine may play an important role in the pathogenesis of Parkinson's disease. Salvianolic acid B is an antioxidant derived from the Chinese herb, Salvia miltiorrhiza. In this study, we investigated the neuroprotective effect of salvianolic acid B against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with salvianolic acid B significantly reduced 6-hydroxydopamine-induced generation of reactive oxygen species, and prevented 6-hydroxydopamine-induced increases in intracellular calcium. Our data demonstrated that 6-hydroxydopamine-induced apoptosis was reversed by salvianolic acid B treatment. Salvianolic acid B reduced the 6-hydroxydopamine-induced increase of caspase-3 activity, and reduced cytochrome C translocation into the cytosol from mitochondria. The 6-hydroxydopamine-induced decrease in the Bcl-x/Bax ratio was prevented by salvianolic acid B. Additionally, salvianolic acid B decreased the activation of extracellular signal-regulated kinase and induced the activation of 6-hydroxydopamine-suppressed protein kinase C. These results indicate that the protective function of salvianolic acid B is dependent upon its antioxidative potential. Our results strongly suggest that salvianolic acid B may be effective in treating neurodegenerative diseases associated with oxidative stress.

Protective effect of paeoniflorin on irradiation-induced cell damage involved in modulation of reactive oxygen species and the mitogen-activated protein kinases.

Ionizing radiation can induce DNA damage and cell death by generating reactive oxygen species (ROS). The objective of this study was to investigate the radioprotective effect of paeoniflorin (PF, a main bioactive component in the traditional Chinese herb peony) on irradiated thymocytes and discover the possible mechanisms of protection. We found 60Co gamma-ray irradiation increased cell death and DNA fragmentation in a dose-dependent manner while increasing intracellular ROS. Pretreatment of thymocytes with PF (50-200 microg/ml) reversed this tendency and attenuated irradiation-induced ROS generation. Hydroxyl-scavenging action of PF in vitro was detected through electron spin resonance assay. Several anti-apoptotic characteristics of PF, including the ability to diminish cytosolic Ca2+ concentration, inhibit caspase-3 activation, and upregulate Bcl-2 and downregulate Bax in 4Gy-irradiated thymocytes were determined. Extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 kinase were activated by 4Gy irradiation, whereas its activations were partly blocked by pretreatment of cells with PF. The presence of ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580 decreased cell death in 4Gy-irradiated thymocytes. These results suggest PF protects thymocytes against irradiation-induced cell damage by scavenging ROS and attenuating the activation of the mitogen-activated protein kinases.

Inhibition of hepatocellular carcinoma growth by antisense oligonucleotides to type I insulin-like growth factor receptor in vitro and in an orthotopic model.

AIM: The type I insulin-like growth factor receptor (IGF-IR) is overexpressed in many tumors including human hepatocellular carcinoma (HCC). It is a critical signaling molecule for tumor cell proliferation and survival. In the present study, IGF-IR expression was down-regulated by phosphorothioate antisense oligonucleotides (AS[S]ODN) to evaluate their specific effects on growth of hepatoma cells in vitro and in vivo. METHODS: HepG2 cells were transfected with different doses of AS[S]ODN, sense [S]ODN, mismatch [S]ODN, or Lipofectin for 72 h, and cell proliferation was analyzed by MTS assay. In vivo, an orthotopic transplant model of HCC was established in nude mice, which were then injected with AS[S]ODN, sense [S]ODN, 5-fluorouracil or saline. At the endpoint of treatment, the tumors were excised and evaluated. RESULTS: Compared to sense and mismatched oligonucleotides, AS[S]ODN targeting to IGF-IR mRNA significantly inhibited hepatoma cell lines HepG2 proliferation and IGF-IR expression at both mRNA and protein levels. The in vivo results showed that systemic treatment also resulted in significant inhibition in tumor growth. Tumor growth in mice treated with AS[S]ODN (50 and 75 mg/kg per day) was significantly inhibited (71.81% and 61.74%, respectively) compared to the saline-treated group (P < 0.01) in a dose-dependent manner. The antitumor effect of IGF-IR AS[S]ODN was associated with down-regulation of IGF-IR in tumor xenografts. Furthermore, IGF-IR AS[S]ODN prevented liver recurrence tumor growth and metastasis in the lung, showing a dose-dependent response. The level of serum alpha-fetoprotein in AS[S]ODN-treated groups was also decreased in a dose-dependent manner, and a good correlation was observed between tumor volume and serum alpha-fetoprotein concentration. CONCLUSIONS: These data suggest that IGF-IR AS[S]ODN can effectively and specifically inhibit HCC growth in vitro and in vivo. Blockage of IGF-IR expression could be a promising therapeutic approach for the management of patients with HCC.

Dose-dependent attenuation of intravenous nalbuphine on epidural morphine-induced pruritus and analgesia after cesarean delivery.

Epidural morphine in patient-controlled analgesia regimens controls postoperative pain well but easily induces pruritus and other epidural morphine-related side effects. With 90 pregnant American Society of Anesthesiologists physical status II females scheduled for elective cesarean delivery, the present study was designed to evaluate the efficacy and safety profile of patient-controlled antipruritus (PCP) use of intravenous nalbuphine-based regimens for attenuation of postoperative pruritus and related side effects in combination with epidural morphine patient-controlled analgesia with regard to the quality of postoperative pain management. Patients were randomly assigned to two nalbuphine groups (5 µg/kg/hour, Group N5 or 10 µg/kg/hour, Group N10) and bolus dose of 1.6 µg/kg for PCP or the control (normal saline) group. Comparable visual analog scale scores for rest pain at each measured time interval among the three groups demonstrated that adequate pain relief was offered; however, the cumulative dose of nalbuphine administered to the patients in Group N10 attenuated the analgesic effect of epidural morphine in moving pain at POh24 only. Fewer episodes and milder severity of pruritus were observed in patients in Groups N5 and N10 at all postoperative time intervals. Epidural morphine provided good postoperative pain relief but with incommodious side effects. In addition, intravenous nalbuphine not only attenuated the incidence of pruritus but also decreased total morphine consumption. In conclusion, intravenous administration of low-dose nalbuphine (5 µg/kg/hour) for PCP maintained analgesia produced by epidural morphine and offered low pruritus incidence.

Proteomic analysis of ionizing radiation-induced proteins at the subcellular level.

Irradiation induces a series of liver diseases. However the molecular mechanisms involving in the process of liver diseases induced by irradiation are still unclear. Subcellular proteomics provides a method to understand regional differences in protein expression levels. With accumulating evidence in the literature that new proteins are implicated in radiation response, in the present study, C57BL/6 mice were treated with irradiation, liver cell homogenates were subfractionated by differential ultracentrifugation into nuclei, mitochondria and cytosol, which were subjected to 2-DE to generate the proteomic maps of these fractions. The differentially expressed proteins in the nuclei, mitochondria and cytosol compartment of liver at 24 and 48 h after exposure to 20 Gy irradiation compared to control were identified by MALDI-TOF MS respectively. Total 37 proteins at 24 h and 29 proteins at 48 h were matched with known proteins after database searching in nuclei, mitochondria and cytosol, respectively, among which nine proteins exhibited changes at both time points. Most of these proteins are involved in antioxidant response, energy metabolism, molecular chaperones and inflammatory response. More antioxidant-associated proteins were induced at 48 h than 24 h. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting further validated 2-DE results of two of these proteins. It is feasible that the differential proteins identified in this study have a biological significance and may provided clues for understanding the mechanism of injury in liver induced by irradiation.

Protective effect of (+/-) isoborneol against 6-OHDA-induced apoptosis in SH-SY5Y cells.

Oxidative stress caused by dopamine (DA) may play an important role in the pathogenesis of Parkinson's disease (PD). (+/-) Isoborneol is a monoterpenoid alcohol present in the essential oils of numerous medicinal plants and is a known antioxidant. In this study, we investigated the neuroprotective effect of isoborneol against 6-hydroxydopamine (6-OHDA)-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with isoborneol significantly reduced 6-OHDA-induced generation of reactive oxygen species (ROS) and 6-OHDA-induced increases in intracellular calcium. Furthermore, apoptosis induced by 6-OHDA was reversed by isoborneol treatment. Isoborneol protected against 6-OHDA-induced increases in caspase-3 activity and cytochrome C translocation into the cytosol from mitochondria. Isoborneol prevented 6-OHDA from decreasing the Bax/Bcl-2 ratio. We also observed that isoborneol decreased the activation of c-Jun N-terminal kinase and induced activation of protein kinase C (PKC) which had been suppressed by 6-OHDA. Our results indicate that the protective function of isoborneol is dependent upon its antioxidant potential and strongly suggest that isoborneol may be an effective treatment for neurodegenerative diseases associated with oxidative stress.

beta-sitosterol decreases irradiation-induced thymocyte early damage by regulation of the intracellular redox balance and maintenance of mitochondrial membrane stability.

Both radiation injury and oxidation toxicity occur when cells are exposed to ion irradiation (IR), ultimately leading to apoptosis. This study was designed to determine the effect of beta-sitosterol (BSS) on early cellular damage in irradiated thymocytes and a possible mechanism of effect on irradiation-mediated activation of the apoptotic pathways. Thymocytes were irradiated (6 Gy) with or without BSS. Cell apoptosis and apoptosis-related proteins were evaluated. BSS decreased irradiation-induced cell death and nuclear DNA strand breaks while attenuating intracellular reactive oxygen species (ROS) and increasing the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). BSS decreased the release of cytochrome c from mitochondria to the cytosol and the mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, BSS partially inhibited the radiation-induced increase of cleaved caspase 3 and cleaved PARP, and attenuated the activation of JNK and AP-1. In addition, evidence suggests that ROS generated by irradiation are involved in this course of cell damage. The results indicate that BSS confers a radioprotective effect on thymocytes by regulation of the intracellular redox balance which is carried out via the scavenging of ROS and maintenance of mitochondrial membrane stability.