To explore the regulation of eIF4E, we screened the protein interacting with eIF4E from human cDNA library by using yeast two-hybrid system. Several clones interacting with eIF4E were identified. One of them was homologous with HUWE1 (HECT, UBA and WWE domain containing 1, also named as ARF-BP1, HECTH9 or HUWE1). Cell co-immunoprecipitation showed that eIF4E could bind to HUWE1 in mammalian cells. We also found that HUWE1 bearing the HECT domain is necessary for its association with eIF4E.
Animals ; Eukaryotic Initiation Factor-4E ; metabolism ; Humans ; Ubiquitin-Protein Ligases ; metabolism

Histocytochemistry ; methods ; Humans ; Pathology, Clinical ; methods ; Specimen Handling ; methods ; Staining and Labeling ; methods

Objective To investigate the inhibitory effects of overexpression of PTEN on renal epithelial-mesenchymal trans-differentiation induced by TGF-β1,and the signaling transduction mechanism.Methods HKC cells were transfected with GFP-PTEN via lipofectAMINE2000.The efficiency of transfection was detected by fluorescence microscope.The expression of PTEN protein and mRNA in the translected cells was detected by Western blot and RT-PCR respectively.The experiment was divided into four groups:normal group,TGF-β1 stimulation group,GFP-PTEN+TGF-β1 group and empty vector+TGF-β1 group.The expression of E-cadherin,a-SMA,Akt and p-Akt was detected by Western blot.Results Most ceils transfected with GFP-PTEN expressed GFP.The expression of PTEN protein and mRNA was strongly increased when HKC cells were transfected with GFP-PTEN(all P<0.05).In both TGF-β1 stimulation group and empty vector+TGF-β1 group,the expression level of E-cadherin was lower(all P<0.05),while that of p-Akt and a-SMA was higher than in normal group(both P<0.05).The expression level of p-Akt and a-SMA in GFP-PTEN+TGF-β1 group was Iower(both P<0.05),while that of E-cadherin was higher than in TGF-β1 stimulation group and empty vector+TGF-β1 group(both P<0.05).The expression of Akt was similar in the four groups.Conclusion Overexpression of PTEN can inhibit renal epithelial-mesenehymal trans-differentiation induced by TGF-β1 through suppressing the activation of PI3K/Akt signal pathway.

BACKGROUND: After neural precursor cells (NPCs) induced from embryonic stem cells (ESCs) have been grafted into the brain, it would still keep some potency of proliferation and differentiation, strong plasticity and integration into the host neural tissues, which would help to observe the therapeutic effect of PD.OBJECTIVE: To observe the differentiation of mouse ESCs into NPCs and the therapeutic effect of NPCs after being transplanted on the behavior of Parkinson disease (PD) rats.DESIGN: Randomly and controlled animal experiment.SETTING: Staff Room of physiology and Staff Room of Neurobiology, Depayment of Basic Medical Sciences, the Third Military Medical University of Chinese PLA.MATERIALS: Totally 50 healthy adult Wistar rats were chosen and randomly divided into experimental group (n=45) and control group (n=5).METHODS: ① 5 μ L (2 g/L)6-hydroxydopamine (6-OHDA) was injected into substantia nigra pars compacta and ventral tegmental area two points in the experimental group to prepare PD rats, and normal saline with the dosage of 5 μL per point was injected into the rats in the control group.Behavioral test began at 1 week after operation to measure successful rate of model establishing, once a week for 7 consecutive weeks. ② Totally 20 successful PD rat models were chosen to perform corpus striatum NPCs with the dosage of 2 μL [the count of cell suspension was (5-8)×106/μL].The other 5 rats were given 2 μL normal saline at corpus striatum as normal saline control group.MAIN OUTCOME MEASURES: ① Successful rate of PD model. ②Effect of NPCs transplantation on the rotation times of PD models. ③ Distribution of transplanted NPCs in vivo, and survival and differentiation.RESULTS: ①6 weeks later, totally 33 of 45 rats in the experimental group achieve the standard of PD model . ② About 85% of mouse ESCs were differentiated into Nestin-positive NPCs 5 days after the embryoid bodies formed in the bacterial dishes and cultured in the N2 serum-free medium. ③The rotation times of the PD rats was significantly decreased after the intracerebral transplantation of NPCs as compared with normal control group. Most of the NPCs grafted into striatum of PD rats were survived, and some were differentiated into TH-positive neurons.CONCLUSION: The mouse ESCs-derived NPCs could be transplanted into striatum of PD rats, and then differentiated into TH-positive neurons,which leads to the obvious decrease of rotation times.

Objective To study the increased radiosensitivity of tumor cells by triterpene acids of loquat leaf (TAL) and mechanism.Methods C6 and MG63 cells were pretreated by TAL,and then were exposed to X-rays at 1,2,3,5,7 Gy.Clonogenic assay was used to evaluate those tumor cells survival fraction (SF) to calculate the sensitization enhancement ratios (SER) of TAL.The cellular micronuclei ratios of tumor cells were analyzed by micronuclei assay.Additionally,the changes of Rad51 and XRCC4 levels were observed by RT-PCR and Western blot.Results D0 values were decreased to 1.31 and 2.85 Gy in C6 and MG63 cells by TAL pretreatment respectively.The SER value of the effect of TAL on C6 and MG63 cells was 1.73 and 2.04,respectively.There was a statistical difference in the cellular micronuclei ratios between tumor cells with TAL above 2 Gy and those without TAL (C6:t =-8.372--2.476,P ＜0.05 ; MG63:t =-4.03--2.557,P ＜ 0.05).TAL attenuated the expression of XRCC4 at transcriptional and translational level,but not for Rad51,a key gene in homologous recombination repair (HRR).Conclusions TAL pretreatment could increase the lethal effect of X-rays on tumor cells in vitro.The mechanism might be involved in the inhibition of non-homologous end joining (NHEJ).

Objective To determine the impact of fluorine and aluminum,and both action combined on the number of rat osteoclasts and bone resorption cultured in vitro and to explore its mechanisms.Methods The osteoclasts and bone marrow stromal cells (BMSCs) isolated from long bone of new born rats were cultured,respectively,in TC199 medium (containing 10％ fetal bovine serum) with fluoride,aluminum and fluoride combined with aluminum.The osteoclasts were inoculated in 96-well culture plate and ivory slice,BMSCs in 6-well culture plate,and culture medium was changed after 2 hours incubation.The cells were divided into control group,fluoride group,aluminum group and fluoride combined with aluminum group; the doses of sodium fluoride were 0,1.0 × 10-4,0,1.0 × 10-4 mol/L and the doses of aluminum chloride were 0,0,1.0 × 10-5,1.0 × 10-5 mol/L,respectively.Tartrate-resistant acid phosphatase (TRAP) staining positive cells were counted under light microscope after TRAP staining on the 5th day and the pit formed in ivory slices were measured by histomorphometry after staining with toludine blue.The expression of osteoprotegerin(OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) was detected by real-time fluorescence quantitative PCR in BMSCs after 8 h treatment.Results ① Fluoride,aluminum and the interactive effects of fluoride and aluminum all had impact on the numbers of osteoclasts (F =7.15,6.56 and 7.98,respectively,all P ＜ 0.05).The numbers of osteoclasts in fluoride group,aluminum group and fluoride combined with aluminum group[(136.9 ± 22.99),(135.4 ± 23.5),(163.0 ± 24.4) per well] were higher than that in the control group[(92.5 ± 22.1) per well,all P ＜ 0.05].② Fluoride,aluminum and the interactive effects of fluoride and aluminum all had impact on the resorption pit area on ivory slices(F =10.47,12.64,14.29,respectively,all P ＜ 0.05).The resorption pit area on ivory slices in fluoride group,aluminum group and fluoride combined with aluminum group[(0.242 ± 0.031),(0.293 ± 0.026),(0.333 ± 0.016)mm2 per slice] was higher than that in the control group [(0.088 ± 0.030)mm2 per slice,all P ＜ 0.05].③Fluoride,aluminum and the interactive effects of fluoride and aluminum all had impact on the expression ratios of RANKL/OPG in BMSCs (F =8.15,15.38,23.59,respectively,all P ＜ 0.05).The expression ratios of RANKL/OPG in BMSCs in fluoride group,aluminum group and fluoride combined with aluminum group [(193.98 ± 137.93)％,(326.11 ± 176.78)％,(599.84 ± 275.82)％] were higher than that in the control group[(100.00 ± 56.02)％,all P ＜ 0.05].Conclusions Both fluoride and aluminum can cause increase in the number of osteoclasts in vitro and promote cell differentiation and bone resorption activity,which may be related to increased expression ratio of RANKL/OPG mRNA in BMSCs.The stimulating effects of fluoride on osteoclasts differentiation and bone resorption is enhanced by aluminum.

Objective To determine whether arsenic has estrogen-like effects,the cell proliferation was measured iil human eervical cancer line(HeLa)in vitro.Methods The HeLa cells were grown in improved RPMI 1640 supplemented respectively with β-estradiol(E2,1 nmol/L),Arsenic trioxide(As2O3,0.5,1.0,5.0 μmol/L),ICI (500 nmol/L),E2(1 nmol/L)+ICI(500 nmol/L),As2O3(1.0 μmol/L)+ICI(500 nmol/L)and control.The growth morphology of HeLa cell was observed under microscope after 72 h.The method of M1Tr was used to study the cell proliferation after 24.48 and 72 h.The technique of flow eytometry was used to measure cell cycle after 48 h. Results HeLa cells in E2 and 0.5 μmoL/L As2O3 treatment were more better growth in morphology than control group.Percentage of HeLa cells proliferation at 24,48,72 h in E2 and 0.5 μmol/L As2O3 treatment were 6.35%, 11.56%,38.33%and 6.35%,8.50%,20.26%respectively.The proliferation effect of HeLa cells was similar in two treatments.The proliferation of HeLa cells were inhibited in other treatments.Compared with control[(41.68± 1.05)%],HeLa cells were promoted go to S phases in E2[(55.72±2.31)%]and 0.5 μmol/L As2O3[(47.82± 1.41)%]treatment.But in other treatments HeLa cells were hold back to S phases.Compared with control,there was a significant differenee(P＜0.05)of cell percentage in S phases in 5.0 μmol/L As2O3[(21.11±4.99)%]and ICI[(20.16±4.76)%]treatments.Conclusion Small amounts of As2O3 impose estrogen.1ike effects and stimulate the proliferation of HeLa cells.

Objective To determine the effects of fluoride on osteoclasts's quantity and bone resorption function in vitro and its mechanisms. Methods The osteoclasts and bone marrow stromal cells(BMSCs) isolated from long bone of new born rats were cultured respectively in TC199 medium (containing 10% fetal bovine serum) with fluoride. The osteoclasts were inoculated in 96-well culture plate and ivory slice, BMSCs were inoculated in 6- well culture plate, respectively, medium were changed after 2 hours incubation. They were divided into control group, low-dose fluoride, medium-dose fluoride and high-dose fluoride groups, the doses of sodium fluoride were 0,2.5 × 10-5,5.0 × 10-5,10.0 × 10-5 mol/L, respectively. Tartrate-resistant acid phosphatase(TRAP) staining positive cells were counted under light microscope after TRAP staining on the 2nd and the 5th day and the pit formed in ivory slices were measured by histomorphometry after staining with toludine blue. The expression of receptor activator of NK-κβ ligand(RANKL) and osteoprotegerin(OPC) was detected by real-time fluorescence quantitative (337.5 ± 70.5), (447.5 ± 43.4), (472.9 ± 34.8), (475.3 ± 24.3)/well in the control group, the low-dose, mediumdose and high-dose fluoride groups, respectively. The differences were statistically significant between these groups and the control group (all P < 0.05). After in vitro culture for 5 days, the numbers of osteoclasts were (92.5 ± 22.1), (123.0 ± 26.4), (135.5 ± 22.2), (136.9 ± 23.0) per well in the control group, the low-dose, medium-dose and high-dose fluoride groups, respectively. The differences were statistically significant between these groups and the (0.088 ± 0.030), (0.100 ± 0.018), (0.152 ± 0.015), (0.242 ± 0.031 )mm2 per piece in the control group, the lowdose, medium-dose and high-dose fluoride groups, respectively. The values of medium-dose and high-dose fluoride BMSCs in the control group, the low-dose, medium-dose and high-dose fluoride groups were 100.00 ± 56.02, 144.95 ± 97.21,223.25 ± 184.48,193.98 ± 137.93, respectively. The values of medium-dose and high-dose fluoride groups were significantly higher than that of control group (all P < 0.05). Conclusions Fluoride can cause increase in the number of osteoclasts in vitro and promote their cell differentiation and bone resorption activity, which may be related to increased expression ratio of RANKL/OPG mRNA in BMSCs.

AIM: To investigate the significance of optical coherence tomography ( OCT ) and fundus fluorescein angiography ( FFA) in patients with high myopia macular degeneration.
●METHODS:A total of 62 cases ( 104 eyes ) of high myopia macular degeneration patients with OCT and FFA data from Department of Ophthalmology in Jan. 2014 to Sep. 2015 were retrospectively analyzed.
● RESULTS: Highly myopic macular degeneration patients with FFA type, OCT classification had significant correlation (r=0. 599, P<0. 001). Type of OCT in patients with high myopia macular degeneration, with BCVA, diopter, axial length, the thickness at the central fovea measured value difference has statistically significant ( P<0. 05). The higher OCT classification, the lower BCVA, the smaller center of foveal thickness and diopter value and the longer axial length in patients. ln degrees of myopia macular degeneration patients with FFA type, the patients with BCVA, diopter, eye axis length determination of value differences were statistically significant (P<0. 05). Foveal thickness difference of no statistically significant (P>0. 05). The higher FFA type, the smaller ametropia degree, BCVA values, the longer ocular axial length. FFA type patients with foveal thickness determination values were not statistically significant (P>0. 05).
● CONCLUSION: High myopic macular degeneration patients with OCT and FFA results have a certain degree of correlation. However, it′s benefit to combine both of them for further diagnosed and treatment of patients.

AIM: To observe the survive, differentiation and therapeutic effect of neural precursor cells (NPCs) differentiated from mouse embryonic stem cells (ESc) when transplanted in the frontal cortex of Alzheimer's disease (AD) rats. METHODS: NPCs were induced from mouse ESc with serum-free methods. The differentiation of transplanted NPCs was observed with immunohistochemistry methods and memory of rats was evaluated with Morris water maze test. RESULTS: About 85% of mouse ESc were differentiated into NPCs 5 days after the embryoid bodies cultured in the N2 medium. 4 and 6 weeks after transplantation, the memory impairment of AD rats was relieved, most of the grafted NPCs were kept undifferentiated and proliferated in clone shape, neuron-like long processes was observed. CONCLUSIONS: The NPCs derived from ESc survive and differentiate into neurons after grafted into the frontal cortex of AD rats, which produces therapeutic effects on AD.