Objective To provide the experimental basis for the further research of the interacting proteins with Stathmin ,the Stathmin gene Pichia pastoris expression system was constructed ,the expressed Stathmin product was purified and identified .Meth‐ods Stathmin gene was amplified from tumor cell line of SKBR3 by PCR method and cloned into the yeast expression vector pPIC3 .5K .The recombinant vector pPIC3 .5K‐Stathmin was constructed and transformed into Pichia pastoris GS115 .The positive clones were screened by YPD medium containing Geneticin 600 μg/mL .Expression was induced with 0 .5% methanol and expres‐sion products were identified by SDS‐PAGE and Western Blotting .Results DNA sequencing result showed that the gene fragment was consistent with Stathmin gene sequence .pPIC3 .5K‐Stathmin was selected from YPD culture medium containing Geneticin ,and the positive clones were identified by PCR .SDS‐PAGE showed that a 37 × 103 protein band could be seen on the PAGE gel after Coomassie Blue staining ,which was further confirmed and identified as Stathmin protein by Western Blotting .Conclusion Stathmin yeast expression vector is successfully constructed and expressed in Pichia pastoris ,which laid the foundation for the study of inter‐acting proteins with Stathmin ,and for the preparation of the biological treatment drugs of Stahtmin target .

In-vitro assay methods were established to evaluate transactivation and binding activity of compounds on peroxisome proliferator-activated receptor y (PPARγ). Firstly, plasmids were constructed for transactivation assay of PPARγ response element (PPRE) triggered reporter gene expression, and for cell-based binding activity assay of the chimeric receptor, which was fused with PPARγ ligand binding domain (LBD) and yeast transcriptional activator Gal4. Secondly, by using PPARy competitive binding assay based on time resolved-fluorescence resonance energy transfer (TR-FRET), affinities of compounds and drugs to PPARγ were evaluated. In application of these above methods, the PPARγ activating potency and characteristics of different compounds were evaluated, and a novel benzeneselfonamide derivative, ZLJ01, was found to have comparable binding activity and affinity with the well-known PPARy agonist, but lack of PPRE mediated transactivation activity. In preliminary study on in-vitro hypoglycemic activity, ZLJ1 was found to promote insulin-stimulated glucose uptake by liver cells. Therefore, we believe that combining transactivation and binding activity as well as affinity evaluation, the system could be used to screen non-agonist PPARγ ligand as anovel PPARγ modulator
Genes, Reporter ; Hepatocytes ; Hypoglycemic Agents ; chemistry ; Ligands ; PPAR gamma ; agonists ; chemistry ; Plasmids ; Response Elements ; Sulfonamides ; chemistry ; Transcriptional Activation

Adult ; Chondrosarcoma ; metabolism ; pathology ; Collagen Type II ; metabolism ; Diagnosis, Differential ; Femoral Neoplasms ; metabolism ; pathology ; Giant Cells ; pathology ; Humans ; Male ; Osteoclasts ; pathology ; Osteosarcoma ; metabolism ; pathology ; S100 Proteins ; metabolism ; Vimentin ; metabolism

Objective To explore the relationship between the Stathmin gene expression in breast cancer cells MDA-MB-231, MCF-7 and the biological behaviours such as cell growth,adhesion and invasion,and provide experimental basis of breast cancer metastasis for further study.Methods Used RT-PCR and Western Blot methods to detect the Stathmin gene expres-sion levels in MDA-MB-231 and MCF-7 cells,and in the mean while to test the MDA-MB-231 and MCF-7 cell growth,adhe-sion,invasion ability by CCK-8 cell proliferation experiments,cell adhesion experiments,cell invasion experiments,then, analyed the relationship of Stathmin gene expression and cell growth,adhesion,invasion ability.Results Over-expression levels of Stathmin gene were observed both in the MDA-MB-231 and MCF-7 cells (F=10.173,P<0.05),and furthermore, the expression levels of Stathmin gene in MDA-MB-231 cells was higher than in MCF-7 cells (t=4.562,P<0.05).While, the growth,adhesion and invasion ability of the MDA-MB-231 cells was higher than that of MCF-7 cells(P<0.05).Conclu-sion The higher level of Stathmin gene expression,the stronger breast cancer cells had ability of growth,invasion,and ad-hesive.The Stathmin gene expression levels was closely correlated with breast cancer cell invasive.

Aim To investigate the cytotoxicity of XN4 against AML cells, and the underlying mechanisms by which XN4 might induce DNA damage and apoptotic cell death through reactive oxygen species ( ROS ) . Methods The proliferation inhibition ratio of AML cells was measured by MTT. The level of extracellular ROS, DNA damage, cell cycle process and apoptosis were tested by flow cytometry ( FCM ) . Western blot was applied to test the expression of proteins. Results XN4 significantly inhibited the proliferation of HL-60 and KG1α with IC50 ( 2. 79 ± 0. 15 ) μmol · L-1 and (2. 76 ± 0. 20) μmol·L-1 respectively. XN4 signifi-cantly increased the generation of intracellular ROS, followed by inducing DNA damage and activating the ATM-γ-H2AX signaling, which led to increases of cells in the S phases of the cell cycle. Subsequently, XN4 induced apoptotic cell death through activation of caspase-3 and Parp. Moreover, the above effects were all reversed by the ROS scavenger N-acetylcysteine ( NAC ) . Conclusion XN4-induced DNA damage and cell apoptosis in AML cells are mediated via ROS generation.

Single clones of Leptospirillum ferrooxidans were obtained on bilayer solid medium plate by incorporating Het-erotroph Acidiphilium SJH into the underlayer broth medium. The morphologies of the bacteria were investigated using electronic microscope.

Objective To study the curative effect and adverse reaction of Oxcarbazepine(OXC)on treating epilepsy in children.Me-thods One hundred and twenty-nine children with different types of epilepsy were orally given OXC,and the drug dose was added gradually.According to the seizure frequency,the cases were divided into 2 groups.Group A:more than 3 seizures occurred in 3 months prior to to take OXC;group B:more than 3 seizures occurred in 1 year prior to taking OXC.After 5 months and 1 year from beginning to take OXC,the original curative effect of the 2 groups was evaluated,respectively;on the other hand,the adverse reaction and the retention were studied.Results 1.Original effect:the rate of seizure-free was 45.8% and the total curative efficiency was 66.7% in group A(n=47);the rate of seizure-free was 92.3% in group B(n=13);in 60 partial epilepsy children,the rate of seizure-free was 56.7% and the total curative efficiency was 73.3%;Both rates were 62.2% and 75.6% of patients with OXC monotherapy and 40%,66.7% of patients were given OXC in combination with another antiepileptic drugs.2.Drug adverse reaction:24% of patients were found to have adverse reaction and most of the symptoms were light and most transient.3.Tolerability:patients' retention of OXC in 1 year was 72.2%,and in 2 years was 80.0%.Conclusions The antiepileptic effect of OXC is satisfactory and adverse reaction is light and mostly transient,OXC as a new antiepileptic drug is well tolerated as monotherapy and adjunctive therapy and should be used extensively.J Appl Clin Pediatr,2009,24(1):53-55

In temporomandibular disorders (TMD), pain takes place when neuropeptides stimulate synovial tissue to produce several cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, which activate neurons and glia of synovial membrane at the bilaminar regions of temporomandibular joint (TMJ). It has been reported that, after neurogenic differentiation, the synovial mesenchymal stem cells (SMSCs), deriving from TMJ, possess the same cytological features as the neuronal cells. This study examined the ability of substance P (SP) and calcitonin gene-related peptide (CGRP) to stimulate SMSCs and neurogenic SMSCs secreting inflammatory cytokines during TMD, evaluated the mutual effects of inflammatory cytokines and neuropeptides and tested the analgesic effect of hyaluronic acid (HA). The levels of IL-1β, IL-6 and TNF-α in SMSCs and neurogenic SMSCs in the presence of neuropeptides were measured by ELISA. SP and CGRP produced by SMSCs and neurogenic SMSCs were determined by RT-PCR and Western blotting. The results showed that the expression of SP and CGRP was significantly enhanced in the neurogenic SMSCs in response to IL-1β, IL-6 and TNF-α, and the effect was remarkably inhibited by HA. IL-1β, IL-6 and TNF-α, in return, could be enhanced in the neurogenic SMSCs upon stimulation by SP and CGRP. Neuropeptides and inflammatory cytokines might work mutually on the TMD pain. The HA-mediated analgesic effect may be implicated in the inhibition of SP and CGRP expression in neurogenic SMSCs.

In this study, a combination of metabolomics and network pharmacology was used to study the pharmacodynamic substances and mechanism of action of Yiyi Fuzi powder (YYFZ) on rheumatoid arthritis (RA) rats. The animal experiments were conducted in accordance with the requirements of the Experimental Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (approval number: TCM-LAEC2021241). The metabolomic analysis using UPLC-Q-TOF/MS technique identified 22 metabolites, including arachidonic acid, tryptophan, linoleic acid, phenylalanine, as significant biomarkers for the treatment of RA with YYFZ, and they were significantly regressed after YYFZ treatment. The analysis of YYFZ blood components also revealed that 11 blood components, including hypaconitine, benzoylhypaconitine, and deoxyaconitine, may be the components that exert direct pharmacological effects in YYFZ in vivo, and further network pharmacological analysis of blood components obtained that YYFZ may exhibit anti-inflammatory effects through acting on PI3K/Akt signaling pathway, estrogen signaling pathway, vascular endothelial growth factor (VEGF) signaling pathway. The results of this study provide implications for the clinical application of YYFZ.

OBJECTIVETo establish osteoblast model, primary cilla model was removed by chloral hyrate, observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field.

METHODSThree 3-day-old male SD rats weighed between 6 and 9 g were killed, cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non-involved group (control group), 2 mM, 4 mM and 8 mM chloral hydrate group, and cultured in 37 °C, 5% CO2 incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope, and incidence of osteoblast primary cilia was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups, including control group (C), Electromagnetic fields group (EMFs), and EMFs with 4 mM chloral hydrate group. DMEM nutrient solution contained 10%FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12 days' cultivation, formation of mineralization nodes was observed by alizarin red staining.

RESULTSCompared with control group and 2mM chloral hydrate group,4 mM chloral hydrate group could effectively remove osteoblast primary cilla (P<0.01). Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group, the area of ALP and mineralization nodes in EMFs with 4 mM chloral hydrate group were decreased obviously (P<0.01).

CONCLUSION4mM chloral hydrate could effectively remove osteoblast primary cilia. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Culture Techniques ; instrumentation ; methods ; Cells, Cultured ; Chloral Hydrate ; pharmacology ; Cilia ; drug effects ; enzymology ; physiology ; Male ; Osteoblasts ; cytology ; enzymology ; Rats ; Rats, Sprague-Dawley