Withanolide A is a biologically active secondary metabolite occuring in roots and leaves of Withania somnifera. In the present study, adventitious roots from leaf explants of W. somnifera were induced for the production of withanolide-A by Agrobacterium tumefaciens strain C58C1 to obtain hair roots. Hair roots induction rate reached 30%. The withanolide A was determined by HPLC in different hair roots lines and different parts of W. somnifera. The average content of withanolide A in all hair roots lines were 1.96 times as high as that in wild-plant, the concentration of withanolide A in hair roots (1.783 mg x g(-1) dry weight) were 1.51 times as high as the roots of wild W. somnifera (1.180 mg x g(-1) dry weight), respectively. It is possible to obtain withanolide A from hair roots culture of W. somnifera.
Agrobacterium tumefaciens ; physiology ; Plant Extracts ; analysis ; biosynthesis ; Plant Roots ; chemistry ; growth & development ; metabolism ; microbiology ; Withania ; chemistry ; growth & development ; metabolism ; microbiology ; Withanolides ; analysis ; metabolism

Eukaryotic expression plasmid pCI-neo-mdr1 which contains human multidrug resistance gene 1(mdr1),was constructed and transferred into human hepatocarcinoma HepG2 cells by use of liposome. G418 was used to screen the cells successfully with mdr1 and the selected cells was named HepG2/mdr1 Morphological and biological properties of HepG2/mdr1 cells were observed. The results show that the constructed HepG2/mdr1 cell line was high efficient and stationary in the expression of mdr1. The work was valuable and desirable for the establishment of multidrug resistant cell models,and for the study of MDR in human hepatoma. Furthermore,the work also provided a perfect model for the research of relationship between insulin resistance and MDR in hepatocarcinoma cells.

Objective To study the activity of type 2 Iodothyronine deiodinase(D2)and the expressions of myelin basic protein(MBP)and synapsinⅠin the brain tissue of young rats fed on a diet with different levels of iodine.Methods Wistar rats were fed on a diet with different doses of KIO_3 for 3 months and then mated randomly.The serum TH and the brain D2 activity were measured in 28 days old pups.The protein expressions of MBP and SynapsinⅠin their brains were determined by immunohistochemistry staining.Results Compared with normal iodine group(NI),the serum TH levels of low iodine group(LI) were lower,while those of iodine excess groups were gradually decreased with their increase of iodine intake,especially in 100-fold high iodine group(100 HI),TT_4 and FT_4 were significantly decreased(P0.05).The immunohistochemistry staining showed weakly positive reactivity of MBP in corpus callosum and stronger of synapsin I in hippocampus CA3 in LI group compared with NI. The similar alterations were also found in all iodine excess groups with their increase of iodine intake.But MBP reactivity was stronger in 100 HI rats than the LI ones.Conclusion Iodine deficiency and iodine excess can cause hypothyroidism in degrees in the young rats,more severe hypothyroid and retarded myelin sheath and synapses can be caused in iodine deficiency compared with iodine excess.

Objective To study effect of excessive iodine intake on sodium-iodide symporter(NIS)mRNA and protein expression of breast in lactating rats.Methods60 Wistar rats,having been weaned for one month,were randomly divided into three groups according to their body weights,I.e,①normal iodine(NI,30 rats);②ten fold high iodine(10 HI,15 rats);③one hundred fold high iodine(100 HI,15 rats).Eating food containing iodine of 300μg/L and drinking water of iodine at 5,1845,20 295μg/L,respectively.After fed for 3 months,the rats mated and had offspring,and urine and milk iodine of lactating rats were determined by As-Ce-catalytic spectrophotometric method.Their marmnary glands were sampled at lactation day 10.Then NIS mRNA expression by RT-PCR was determined and NIS protein by immunohistochemistry(SABC)was observed.Results The urine iodine of 10 HI group(3597.5μg/L)and 100HI group(25 404.3μg/L)increased obviously compared with that of NI group(344.7μg/L).The milk iodine of 10HI group(27.1×103μg/L)and 100HI group(191.0×1μg/L)was higher than that of NI group(6.0×103μg/L),but the increased fold of milk iodine was not paralleled with that of urine iodine.Difference of NIS mRNA expression was significant(F=24.19,P＜0.01)among the groups,and the NIS mRNA expression in 10HI(1.250±0.034)and 100HI(1.272±0.039)group were less than that in NI (1.532±0.044)group(P＜0.01).The breast NIS mRNA expression in lactating rats(1.532±0.044)was significantly higher than that in unlactating rats(0.879±0.018,P＜0.01).With the increasing iodine uptake,NIS protein expression decreased.Conclusions The NIS mRNA and protein in rat breasts is down-regulated by excessive iodine intake.So increasing extent of milk iodine concentration is inhibited,which is important to prevent off-spring from getting excessive iodine intake from parental generation.

Objective To explore the feasibility of iodized Nang(bread) prepared with iodized salt and non-iodized rock salt as vehicle of iodine. Methods Two kinds of Nang, each of 10 respectively, were grilled with 30 g iodized salt water and non-iodized rock salt water mixed with 2 kg flour by the local cooker, then put inside of Nang oven using traditional methods of grilled Nang in Xinjiang. The samples were collected from different parts of Nang, including the layers facing oven wall and the fire, as well as inside of Nang. The method for determination of iodine in foodstuff by dry ashing As ~Ⅲ-Ce~(4+) catalytic spectrephotometry was used to determine iodine concentration in Nang. Results Iodine content in iodized and non-iodized Nang was (0.654 ± 0.076)mg/kg and (0.075 ± 0.022)mg/kg, respectively. In addition, Iodine content in two kinds of Nang was significantly different and iodine content of Nang with iodized salt was much higher than that with non-iodized rock salt(t = 13.520, P <0.01 ). Iodine content in two kinds of Nang from the layers facing oven wall and the fire, as well as inside of Nang were (0.700 ± 0.100), (0.064 ± 0.029)mg/kg; (0.647 ± 0.076), (0.070 ± 0.019)mg/kg; (0.659 ± 0.073), (0.073 ±0.030)mg/kg, respectively. Iodine content in two kinds of Nang of the same parts was significantly different(t =3.826,4.201,4.103, all P < 0.01 ). There was no significant difference of iodine content in different parts of the same kind of Nang(F = 0.220,0.190, all P > 0.05). Conclusions Grilled Nang with iodized salt contains sufficient iodine, and the iodine content of the same kind of Nang in different parts has no significant difference. Our studydemonstrated that Nang is a vehicle available for iodine fortification since Nang is very popular food for local population in Xinjiang.

Obej ctive Abnormal proliferation of T cells plays an important role in the development of autoimmune diseases. The article aimed to study the inhibitory effect of small molecule compound BD691 on T cell proliferation and its mechanism. Methods Human peripheral blood T-lymphocytes were isolated and purified by the immunomagnetic microbeads,then T cells were ac-tivated with anti-CD3/CD28 mAbs or alloantigen.The inhibitory effect of BD691 on activated T cell proliferation, the cytotoxic effect BD891 on resting T cells and the expression of activated T cells marker CD25 were measured by flow cytometry.Furthermore, ELISA was used to detect the secretion of cytokines associated with T cell differentiation. Results BD691 significantly inhibited the prolif-eration of T cells being stimulated by anti-CD3/CD28 mAb or alloantigen in a dose-dependent manner, and IC50 values are (8.5 ± 1.5)μmol/L and (7.2 ±1.3)μmol/L, respectively.However, BD691 had no obvious cytotoxic effects on resting T cells and periph-eral blood mononuclear cells, even at a high concentration ( up to 100μmol/L) .In T cells which were not activated by anti-CD3/CD28 mAb, the percentage of CD25+T cells is only 1.6%of the total cells, while the number increased to 68% after activating treatment.Mean-while, in T cells which were activated by 0, 3.3, 10, 30μmol/L BD691, no obvious change of CD25 expression were observed, while immunosuppressant FK506 (0.1μmol/L) significantly decreased the expression of CD25 +T cells (14.9%).In unactivated T cells, 95.6%cells were at G0/G1 phase, while after activation, the percentage of cells at G0/G1 phase reduced to 57.7%.In addition, BD691 inhibited the secretion of IFN-γ, IL-6 and IL-17 in activated T cells, but had no effects on the secretion of IL-2, IL-4 and IL-10. Co nclusion BD691 exerts no effects on T cell activation, but it inhibits T cell proliferation by inducing T cell cycling arrest at G0/G1 phase.Moreover, BD691 inhibits the secretion of key cytokines (such as IFN-γ, IL-6, IL-17) closely related to the differ-entiation of Th1 and Th17 cells.The results suggest that BD 691 is a potential lead compound to develop a new immunosuppressant for the inhibition of abnormal proliferation and differentiation of T cells.

AIM:To study the telomere maintenance mechanism in mesenchymal stem calls(MSCs).METHODS:MSCs were isolated from healthy human bone marrow by their adherence to plastic and then were checked with CD14-FITC,CD45-FITC,CD44-FITC,HLA-DR-FITC,CD34-PE,CD29-PE and CD166-PE.Telomere length and ECTR DNA in MSCs were detected by Southern blotting.The localization of TRF1 and promyelocytic leukemia(PML)in MSCs were detected with immunofluorescence staining.TRAP protocol was performed to detect the telomerase activity in MSCs and MSCs-derived adipocytes.Western blotting and TRAP protocol were applied to measure telomerase activity of MSCs,which were synchronized by serum starvation and aphidicolin treatment.RESULTS:The telomere in length seemed shorter and relatively more homogeneous in MSCs and HeLa cells than that in WI-38-2RA cells.TRF1 did not concide with PML nuclear body in MSCs and HeLa cells while it exclusively did in WI-38-2RA cells.ECTR DNA was negative in MSCs and HeLa cells but positive in WI-38-2RA cells.Telomerase was negative in MSCs but it was positive in MSCs-derived adipocytes detected by TRAP.Moreover,a cell cycle-dependent expression profile of telomerase was found in MSCs when they were synchronized by serum starvation and aphidicolin treatment.Untreated MSCs expressed very low level of telomerase probed by Western blotting with 2C4 mAb,but the telomerase level had significantly increased when these cells were trapped in S phase.CONCLUSION:The telomere of MSCs is maintained by telomerase pathway instead of alternative lengthing of telomere(ALT)and the level of telomerase expression is associated with cell cycle stage.[

Objective To investigate the immediate effects of lower limb with open chain weight-adding on joint position sense and gait symmetry in stroke patients. Methods From January, 2016 to January, 2017, 39 stroke patients were included. Their joint position sense and gait symmetry were compared before and after weight-adding. The joint position senses of active reproduction of active positioning (ARAP) and passive reproduction of passive positioning (PRPP) were assessed during lower limb straight leg raise. The gait symmetry was also as-sessed and three indexes were recorded including the symmetry of foot rotation angle, step length and percentage of single leg support phase. Results After weight-adding, the position sense of PRPP did not improve (t=0.832, P=0.832), nor of the symmetry of foot rotation an-gle (t=-0.704, P=0.483) and percentage of single leg support phase (t=0.381, P=0.702);the position sense of ARAP improved (t=3.158, P=0.011), as well as the symmetry of step length (t=2.022, P=0.041). Conclusion The lower limb with open chain weight-adding could im-prove the active joint position sense and symmetry of step length.

Objective:To investigate the clinical efficacy of laparoscopic resection of retrorectal cystic lesions.Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 58 patients undergoing laparoscopic resection of retrorectal cystic lesions in the Peking Union Medical College Hospital, Chinese Academy of Medical Sciences from August 2012 to August 2019 were collected. There were 5 males and 53 females, aged from 15 to 70 years, with a median age of 38 years. All the 58 patients underwent laparoscopic resection of retrorectal cystic lesions and the combined operation through the transsacral approach was chosen according to the patient condition. Observation indicators: (1) surgical situations; (2) postoperative situations; (3) postoperative histopathological examination; (4) follow-up. Patients were followed up regularly using outpatient examination once every 6 months during the first postoperative year and once every 12 months after the first postoperative year. The recurrence of cysts was evaluated by computed tomography or magnetic resonance imaging examinations during the follow-up up to August 2020. Measurement data with normal distribution were represented as Mean± SD and measurement data with skewed distribution were described as M(range). Count data were described as absolute numbers. Results:(1) Surgical situations: of the 58 patients, 54 cases underwent laparoscopic resection of retrorectal cystic lesions and 4 cases underwent laparoscopic resection of retrorectal cystic lesions combined with the transsacral approach operation. One of the 58 patients who had a huge cyst surrounding the rectum underwent transverse colostomy after repairing the damage of separated posterior wall of rectum. Two cases underwent preventive transverse colostomy because the external rectal wall heat injury could not be excluded after separation of the tight adhesion between cyst and rectum. The operation time and volume of intraoperative blood loss were (123±56)minutes, 20 mL(range, 5?500 mL) of 54 cases who underwent laparoscopic resection of retrorectal cystic lesions and (232±38)minutes, 90 mL(range, 30?800 mL) of 4 cases who underwent laparoscopic resection of retrorectal cystic lesions combined with the transsacral approach operation, respectively. (2) Postoperative situations: 7 of the 58 patients had complica-tions. Of the 7 patients, 2 cases had postoperative rectal fistula and were cured after the treatment of transverse colostomy combined with pelvic drainage, 2 cases had postoperative urinary tract infection and were relieved after anti-infection treatment, 2 cases had urinary retention after removal of catheter and were recovered after 3 weeks of re-indwelling catheter, and 1 case had poor incision healing of transsacral and was healed after wound dressing change. The duration of postoperative hospital stay of the 58 patients was (7±4)days. (3) Postoperative histopathological examination: results of the postoperative histopathological examination showed that there were 26 of 58 patients with epidermoid cyst, 20 patients with teratoma (2 cases with mature teratoma accompanied by mucinous adenocarcinoma and 1 case with mature teratoma accompanied by neuroendocrine carcinoma), 10 patients with dermoid cyst, and 2 patients with tailgut cyst. (4) Follow-up: 57 of the 58 patients were followed up for 2-85 months, with a median follow-up time of 51 months. Of the 57 patients who were followed up, 1 patient was diagnosed with buttock subcutaneous cyst at postoperative 8 months and treated with local excision, 1 patient was diagnosed with a small presacral cyst recurrence by pelvic magnetic resonance imaging at postoperative 6 months and continued follow-up as the cyst without obvious enlargement, and the other 55 patients had no cyst recurrence.Conclusion:The laparoscopic resection of retrorectal cystic lesions is safe and feasible.

BACKGROUNDThe role of epigenetics in gene expression regulation and development significantly enhances our understanding of carcinogenesis. All the tumor related genes may be the target of epigenetical or genetic regulation. We selected some epigenetically regulated genes for cDNA array analysis and observed variability in the expression of the DICER1 gene in distinct stages of gastric cancer. The aim of this study was to assess the correlation between the expression of DICER1, an epigenetically regulated gene, and gastric cancer.

METHODSTo detect the expression of 506 tumor-associated genes, including DICER1, in the matched cancerous mucosa, pre-malignant lesion (adjacent mucosa), non-cancerous gastric mucosa and distant lymphocyte metastatic lesion in 3 cases of gastric cancers using cDNA array. DICER1 mRNA expression and DICER1 protein expression were further analyzed by Real-time PCR and Western blot in 32 cases of progressive gastric cancer. DICER1 protein expression was also detected in 33 early and 30 progressive gastric cancers by the immunohistochemistry (IHC) method.

RESULTSIn 3 cases of gastric cancer cDNA array showed dramatically decreased expression of DICER1 in pre-malignant lesion, cancerous mucosa and distant lymphocyte metastatic lesions compared with matched noncancerous gastric mucosa, pre-malignant lesion and cancerous mucosa. Real-time PCR results showed that the expression level of DICER1 mRNA in gastric cancer was significantly down-regulated compared to normal gastric tissue (P < 0.05). The IHC assay also showed that the expression of DICER1 was significantly decreased in progressive gastric cancer. Among the 63 cases of gastric cancers, 13/33 early (39.4%) and 19/30 (63.3%) progressive cancers showed negative expression of DICER1 (50.8%). The difference in expression of DICER1 between early and progressive gastric cancers was significant (P < 0.01). The result of Western blotting showed that DICER1 protein was down-regulated significantly in advanced gastric cancer (P < 0.05).

CONCLUSIONSDICER1 expression is decreased during the progression of gastric cancer, especially in progressive gastric cancers, which indicating DICER1 may play an important role in the development of cancer and the epigenetical regulation involved.

Blotting, Western ; DEAD-box RNA Helicases ; analysis ; genetics ; physiology ; Endoribonucleases ; analysis ; genetics ; physiology ; Epigenesis, Genetic ; Humans ; Immunohistochemistry ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction ; Ribonuclease III ; Stomach Neoplasms ; chemistry ; etiology ; genetics