Animals ; Brain Neoplasms ; genetics ; pathology ; Cell Differentiation ; Cell Transformation, Neoplastic ; genetics ; Glioma ; genetics ; pathology ; Humans ; Neoplastic Stem Cells ; pathology ; Neural Stem Cells ; pathology ; Neuroglia ; pathology ; Neurons ; pathology

Central composite design (CCD) is one of the most commonly used design methods in response surface optimization and has been widely applied in the field of pharmaceutics to optimize preparations. On the 20th anniversary of the introduction of CCD into China, the paper reviews its application in domestic pharmaceutical researches. Based on the brief introduction of basic principle and operation steps of CCD, the mistakes emerging in the application of CCD are summarized, including conceptual confusion with Box-Behnken design and face-centered CCD as well as wrong designs. Besides, the issues concerning the selection of factors and responses are discussed. The article is helpful for researchers to comprehensively understand the CCD and facilitates the rational application of this method.

The synthesized full-length hGLP-1 gene was cloned into pET-32a(+) to get the recombinant plasmid pET32-GLP-1, which could express a fusion protein containing thioredox, hexahistidine, and rhGLP-1 consecutively. The recombinant plasmid containing hGLP-1 was transformed into E.coli BL21 (DE3) and expressed by IPTG induction. The fusion protein was purified from lysates with Ni?IDA His?Bind affinity chromatography. rhGLP-1 with the purity of 90% was achieved after enterokinase digestion, Ni?IDA His?Bind affinity chromatography again, then was concentrated by ultrafiltration. The purified rhGLP-1 showed a single band on IEF gel with an isoelectric point between pH5.2 and pH5.85. ESI mass spectrometry showed that the molecular weight was 3355.0kDa as expected. rhGLP-1 was digested with trypsin followed by mass analysis and the peptide mapping produced two expected fragments with the molecular weights of 2097.7kDa and 1005.5kDa, respectively. The purified rhGLP-1 also showed obvious biological activity for both lowering plasma glucose and stimulating insulin secretion in mice.

Objective To investigate the expression and significance of Smad 7,Smurf 1 and Smurf 2 in basal cell carcinoma and squamous cell carcinoma.Methods Biopsy specimens were resected from 14 patients with basal cell carcinoma,19 patients with squamous cell carcinoma and 30 normal controls.Quanti- tative real-time PCR and immunohistochemical techniques were utilized to assess the expression of Smad 7, Smurf 1 and Smurf 2 in these specimens.Results The gray scale for staining of Smad 7,Smurf 1 and Smurf 2 was 166.61?7.11,166.08?8.71,and 166.25?8.15 respectively in basal cell carcinoma,161.66?5.52,166.84?9.27,and 169.98?9.48 respectively in squamous cell carcinoma.The expression levels of Smad 7,Smurf 1 and Smurf 2 were all significantly increased in basal cell carcinoma and squamous cell car- cinoma in comparison with normal controls.Conclusions The over-expression of Smad 7,Smurf 1 and Smurf 2 may interfere with transforming growth factor?signaling transduction pathway through several links,therefore prevent the inhibitory effect of transforming growth factor?on epidermal proliferation,and accelerate the abnormal proliferation in above epidermal tumors.

UNLABELLEDTo investigate the regulatory effects of epimedium flavonoids (EF) on adrenocortical regeneration in rats with inhibited hypothalamic-pituitary-adrenal (HPA) axis.

METHODSCell distribution in cell cycle and cell apoptotic rate were measured with PI stain and flow-cytometry; apoptosis cells were showed by in situ terminal-deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate-fluorescene nick end labeling assay (TUNEL), and the genome-wide gene mRNA expression was detected by oligonucleotide microarrays.

RESULTSCompared to the normal control, adrenal cells isolated from the HPA axis inhibited model group were arrested in Go/GI phase, and showed a higher apoptotic rate (P < 0.05). After treated with EF, cells in G0/G1 phase decreased and those in G2/M phase increased (P < 0.01), and the elevated apoptotic rate reduced significantly (P < 0.05). TUNEL assay showed the number of apoptotic cells per section was 4.67 1.53 in the normal control group, 70.67 +/- 9.29 in the model group, and 18.67 +/- 7.64 in the EF-treated group respectively (n=3). Gene expressions in adrenal were mostly restrained in the model group, including 7 cytocycle promoting genes, including V-ras, V-jun, etc., while after treatment with EF, 6 cytocycle promoting genes, 1 anti-apoptotic gene, and genes that closely related with adrenocortical regeneration as IGF-II and FGF7 and their receptors, as well as 7 steroid biosynthesis participated genes were all up-regulated. Conclusion EF can accelerate adrenocortical cell proliferation, inhibit its apoptosis, and promote steroid biosynthesis so as to enhance adrenocortical regeneration in HPA axis inhibited rats, which may contribute to the beneficial effects of EF in protecting adrenocortical function during glucocorticoid withdrawal.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Epimedium ; chemistry ; Flavonoids ; pharmacology ; Gene Expression ; Gene Expression Profiling ; Glucocorticoids ; adverse effects ; Hypothalamo-Hypophyseal System ; drug effects ; Male ; Oligonucleotide Array Sequence Analysis ; Pituitary-Adrenal System ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Substance Withdrawal Syndrome ; prevention & control

Today, the understanding of the sequence and structure of biologically relevant targets is growing rapidly and researchers from many disciplines, physics and computational science in particular, are making significant contributions to modern biology and drug discovery. However, it remains challenging to rationally design small molecular ligands with desired biological characteristics based on the structural information of the drug targets, which demands more accurate calculation of ligand binding free-energy. With the rapid advances in computer power and extensive efforts in algorithm development, physics-based computational chemistry approaches have played more important roles in structure-based drug design. Here we reviewed the newly developed computational chemistry methods in structure-based drug design as well as the elegant applications, including binding-site druggability assessment, large scale virtual screening of chemical database, and lead compound optimization. Importantly, here we address the current bottlenecks and propose practical solutions.
Computational Biology ; Drug Design ; Drug Discovery ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Quantitative Structure-Activity Relationship

OBJECTIVETo investigate the mechanism of senescence delay of human diploid fibroblast (2BS) and protecting telomere length by epimedium flavonoids (EF).

METHODSThe drug sera of EF were used to treat the 2BS. The population doublings of 2BS cells were observed, the mRNA expression of p16 gene were determined by fluorescence real-time quantitative RT-PCR, the telomerase activation of 2BS cells were determined by TRAP-Hyb, the total retinoblastoma (Rb) and phosphorated Rb protein content were detected by ELISA, the telomere length of 2BS cells were determined by telomere restriction fragment (TRF) Southern blot assay.

RESULTSEF could significantly extend the population doublings of 2BS cells, the expression of p16 mRNA was decreased and the content of phosphorated Rb protein were increased by EF. The telomere lengthening of 2BS cells were improved by EF, but the telomerase was not activated.

CONCLUSIONIn senescence human fibroblasts 2BS cells, p16 gene mRNA expression increased, content of phosphorated Rb protein decreased and the telomere length of 2BS shortened, EF might delay the aging of cells through inhibiting the p16 gene expression, promoting the production of phosphorated Rb protein and to protect the length of telomere, but not activating the telomerase.

Animals ; Cells, Cultured ; Cellular Senescence ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Epimedium ; chemistry ; Fibroblasts ; cytology ; Flavonoids ; pharmacology ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Retinoblastoma Protein ; metabolism ; Telomerase ; biosynthesis ; Telomere ; genetics ; metabolism ; Transduction, Genetic

Objective To evaluate hemodynamic changes in liver treated by transjugular intrahepatic portosystemic stent-shunt(TIPSS)with hepatic computed tomography(CT)perfusion,Doppler ultrasound and portal vein pressure measurement,as well as the correlation among these methods.Methods Hepatic CT perfusion was performed in 9 cirrhotic patients one week before TIPSS and 72 hours after TIPSS. Intraoperative portal vein pressure was measured before and after portosystemic shunt establish.The follow- up hepatic CT perfusion were carried out in 3 cases at 3 months and 6 months postoperatively.The hemodynamic surveillance by Doppler ultrasound were performed in 48 hours and 3 months after TIPSS for 9 cases,and in 6 months after TIPSS for 6 cases.Two cases underwent venography and portal vein pressure measurement in 6 months after TIPSS treatment.Results The mean of portal vein perfusion(PVP),total hepatic blood flow(THBF),hepatic perfusion index(HPI)and portal vein free pressure(PVFP)before TIPSSwere(0.92?0.18)ml?min~(-1)?ml~(-1),(1.28?0.17)ml?min~(-1)?ml~(-1),(28?8)%,and (23.92?0.86)mmHg,respectively.In 72 hours after TIPSS,the mean of PVP,THBF,HPI and PVFP were(0.21?0.15)ml?min~(-1)?ml~(-1),(0.74?0.18)ml?min~(-1)?ml~(-1),(74 +13)%,and (12.62?1.54)mm Hg,respectively.After treatment,the mean of PVP was(0.49?0.05)ml?min~(-1)? ml~(-1)at 3 months and(0.57?0.03)ml?min~(-1)?ml~(-1)at 6 months,respectively.There was negative correlation between PVP and PVFP before TIPSS(r=0.678,P0.05).Moreover,a signifieant correlation was found between the degree of portal vein pressure decrease and portal vein perfusion decrease(r=0.867,P

Objective： The current stady was designed to investigate the reconstitution of T cell receptor repertoire in the patients with chronic Graft-versus-host disease (cGVHD). Methods： The TCR repertoire in peripheral blood mononuclear cells from 5 cGVHD patients was examined after PCR amplification of 24 Vβ gene subfamilies. Results： Only 2－8 Vβ subfamily T cells were found in the samples from these patients, and there were different demonstrations in different patients. We found Vβ T cells proliferated in 4 patients. Conclusion： The TCR repertoire complexities was abnormal in all patients,Vβ may be associated with cGVHD, and the method may be demonstrated the reconstitution of T cell after transplantation.

OBJECTIVETo study the inhibition effect of celastrol on neovascularization.

METHODSThe effect of celastrol on the in vitro proliferation of endothelial cell of vessel (ECV) was examined by MTT assay. The effect of celastrol on endothelial cell migration, tube formation on Matrigel and Chick chorioallantoic membrane angiogenesis was also examined. Matrigel plug assay was used to evaluate the effect of celastrol on angiogenesis in vivo.

RESULTSThe proliferation of ECV was inhibited significantly by celastrol with IC(50) being 1.33 microg/ml. Celastrol inhibited endothelial cell migration and tube formation in a dose-dependent manner. Celastrol also inhibited angiogenesis both in Matrigel plug of mouse model and in chick chorioallantoic membranes.

CONCLUSIONCelastrol, which can inhibit angiogenesis, could be developed as an antiangiogenic drug.

Angiogenesis Inhibitors ; pharmacology ; Animals ; Endothelial Cells ; drug effects ; Mice ; Mice, Inbred BALB C ; Triterpenes ; pharmacology