Establishment of cell-based screening system for compound regulating the stability of retinoic acid receptors / 上海交通大学学报（医学版）

Objective·To establish a cell-based screening system for identification of compounds with activity in regulating retinoic acid receptor (RARα) stability. Methods·The modified pMSCV plasmid constructs, named as RARα-EGFP-IRES-DsRed, consists of enhanced green fluorescent protein (EGFP) fusing to RARα and red fluorescent protein (DsRed) as internal references incorporating the internal ribosome entry site (IRES) as interval sequence. The RARα-EGFP-IRES-DsRed plasmid was stably transfected into NB4 cells which were named as NB4-pMGIR-RARα. Fluorescence signals of EGFP and DsRed indirectly reflecting the expression of RARα, were detected by flow cytometry in cells that were treated with all-trans retinoic acid, sodium valproate, cytarabine, lenalidomide, etoposide, montelukast and gambogic acid, respectively. Effects of these compounds on the expression of RARα protein were further examined by Western blotting. Results·A double fluorescence reporter system for screening compounds that can increase the stability of RARα protein was successfully established, and sodium valproate was identified as a potent compound to promote the stability of RARα. Conclusion·The double fluorescence reporter system can be used to screen compounds regulating the stability of RARα protein, which can be further used to identify compounds regulating the stability of other proteins.

Biological effects and mechanism of adenanthin on multiple myeloma cells / 上海交通大学学报（医学版）

Objective·To explore the biologic effect and mechanism of adenanthin (Aden) on multiple myeloma (MM) cells. Methods·MM cells, H929 and U266 were treated with various dose of Aden for different time, and the density and viability of MM cells were detected by trypan blue exclusion assay. After H929 and U266 cells were treated with various dose of Aden for 24 hours, cell growth inhibition was examined by CCK8 assay, and cell apoptosis was examined by AnnexinV-APC/PI staining assay. Apoptosis related proteins, NF-κB signaling pathway associated proteins and the NF-κB regulated proteins were detected by Western blotting. The effect of Aden on the thermal stability of IKKβ protein was determined by CETSA assay. Results·Trypan blue exclusion results showed that Aden inhibited cell growth and reduced cell viability in concentration and time dependent manners. U266 was more sensitive than H929 when exposed to the same concentration of Aden. The CCK8 results showed that Aden inhibited the growth of H929 and U266 cells in a concentration dependent manner. Flow cytometry results suggested that Aden induced a low apoptosis rate of MM cells. Moreover, cleavage of caspase3 and PARP were detected in U266 cells but not in H929 cells. CETSA assay indicated that Aden decreased the thermal stability of IKKβ. Expression of p-p65 and p-IκBα proteins decreased in MM cells treated with Aden. Conclusion·Aden significantly inhibits MM cell proliferation by inhibiting NF-κB activation through interacting with IKKβ. Aden has little effect on apoptosis of MM cells.

Effects of sleep deprivation on polysomnography and executive function in patients with depression / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Sleep deprivation (SD) has been used in treatment of depression disorder, and could effectively improve the patients' depressive symptoms.The aim of the study was to explore the effects of SD on electroencephalographic (EEG) and executive function changes in patients with depression.</p><p><b>METHODS</b>Eighteen depression patients (DPs) and 21 healthy controls (HCs) were enrolled in the present study. The whole night polysomnography (PSG) was recorded by Neurofax-1518K (Nihon Kohden, Japan) system before and after 36 hours of SD. The level of subjects' depression state was assessed by Visual Analogue Scale (VAS), and the executive function was assessed by Wisconsin Card Sorting Test (WCST).</p><p><b>RESULTS</b>Significantly decreased sleep latency (SL; before SD: (31.8 ± 11.1) minutes, after SD: (8.8 ± 5.2) minutes, P < 0.01) and REM sleep latency (RL; before SD: (79.8 ± 13.5) minutes, after SD: (62.9 ± 10.2) minutes, P < 0.01) were found after SD PSG in depression patients. Decreased Stage 1 (S1; before SD: (11.7 ± 2.9)%, after SD: (7.3 ± 1.1)%, P < 0.01) and Stage 2 (S2, before SD: (53.8 ± 15.5)%, after SD: (42.3 ± 14.7)%, P < 0.05) of non-rapid eye movement (NREM) sleep, and increased Stage 3 (S3, before SD: (11.8 ± 5.5)%, after SD: (23.6 ± 5.8)%, P < 0.01) and Stage 4 (S4, before SD: (8.8 ± 3.3)%, after SD: (27.4 ± 4.8)%, P < 0.01) NREM sleep were also found. After SD, the depression level in patients decreased from 6.7 ± 2.1 to 2.9 ± 0.7 (P < 0.01). In WCST, the patients showed significantly decreased Response errors (Re, before SD: 22.3 ± 2.4, after SD: 18.3 ± 2.7, P < 0.01) and Response preservative errors (Rpe, before SD: 11.6 ± 3.6, after SD: 9.3 ± 2.9, P < 0.05). Depression patients' RE (t = 2.17, P < 0.05) and Rpe (t = 2.96, P < 0.01) also decreased significantly compared to healthy controls.</p><p><b>CONCLUSION</b>SD can improve depression symptom and executive function in depression patients.</p>

Effects of As(2)O(3) on BCR-ABL protein level and signal transduction in CML cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of As(2)O(3) on BCR-ABL protein level and signal transduction in chronic myeloid leukemia (CML) cells.</p><p><b>METHODS</b>Immunoprecipitation, protein tyrosine kinase (PTK) activity assay, real-time Taqman quantitative PCR and Western blot were used.</p><p><b>RESULTS</b>As(2)O(3) downregulated BCR-ABL protein and STAT1 protein of CML mononuclear cells in the concentrations of 1.0 approximately 2.0 micro mol/L and 0.5 approximately 2.0 micro mol/L after 48 h exposure, respectively. However, p27 protein level was not affected. The PTK activity of BCR-ABL protein was also mildly decreased in CML monouclear cells at 60 h. The bcr-abl mRNA level remained unchanged.</p><p><b>CONCLUSION</b>As(2)O(3) downregulats BCR-ABL protein, STAT1 protein, BCR-ABL signal transduction and PTK activity in CML cells.</p>

Effects of MIP-1β and TGF-β antibody on the expansion of CD34+ cells from cord blood in stroma-contact culture system / 第三军医大学学报

Objective　To study the effects of MIP-1β and TGF-β antibody on CD34+ cells from cord blood in stroma-contact culture system. Methods　Immunomagnetic selected CD34+ cells were inoculated onto the pre-established irradiated human stroma layer. MIP-1β, TGF-β antibody (20 μg/ml) and MIP-1β+anti-TGF-β (5 μg/ml) were added on day 0 and day 5. On day 5 and day 10, cell counting, hematopoietic progenitor cells count were made by semi-solid culture and CD34+ cells were assayed by FACS. Results　On day 5, no significant difference of total cell number was observed as compared with the control group (P>0.05), but the number of CD34+cells, CFC, and HPP-CFC in TGF-β antibody and MIP-1β+ TGF-β antibody groups was significantly higher than that in the control (P<0.05). On day 10, the number of total cell, CD34+ cells, CFC, and HPP-CFC in TGF-β antibody and MIP-1β+ TGF-β antibody groups was significantly higher than that in the control (P<0.05). No significant difference was observed between groups MIP-1β and control either on day 5 or day 10 (P>0.05). Conclusion　MIP-1β has no significant effect on the CD34+ cells as compared with the control while the CD34+ cells can be expanded 1-3 folds with TGF-β antibody (20 μg/ml) or MIP-1β+ TGF-β antibody (5 μg/ml) in 10 days. There are synergic interactions between MIP-1β and TGF-β antibody.