Hypoxia is a general characteristic of most solid malignancies and intimately related to cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor-1alpha (HIF-1alpha) that elicits transcriptional activity through recruitment P300 coactivator. Targeting the interaction of HIF- alpha and P300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis. Here, a screening assay was developed for inhibitors targeting the interaction between HIF-1alpha and P300. The nucleotide sequence of human HIF-1alpha and P300 were cloned into pBIND and pACT vectors, named pBIND-HIF1alpha and pACT-P300. The interaction of HIF-1alpha and P300 was identified in HEK293 cell using mammalian two-hybrid system. And compound chetomin decreased their interaction in this mammalian two-hybrid system. We further verified HIF-1 inhibition effect of chetomin in U251-HRE cells. Therefore, we established a screening assay combined HIF-1alpha and P300 mammalian two-hybrid system and U251-HRE reporter assay for HIF-1 selective inhibitors.
Cell Hypoxia ; Disulfides ; pharmacology ; Drug Screening Assays, Antitumor ; E1A-Associated p300 Protein ; antagonists & inhibitors ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; antagonists & inhibitors ; Indole Alkaloids ; pharmacology ; Two-Hybrid System Techniques

Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseII clinical trials.The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHI and HindII, and cloned into pET43.1a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP(with signal peptide), respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing with1mmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.

Objective The purpose of this study was to develop a high-throughput micro-plate radiobinding assay (RBA) of glutamic acid decarboxylase antibody (GAD-Ab) and to evaluate its clinical application. Methods 35labeled GAD65 antigen was incubated with sera for 24 h on a 96-well plate, and then transferred to the Millipore plate coated with protein A, which was washed with 4℃ PBS buffer, and then counted by a liquid scintillation counter. The GAD-Ab results were expressed by WHO standard unit (U/ml). A total of 224 healthy controls, 162 patients with type 1 diabetes mellitus(T1DM) and 210 patients with newly diagnosed type 2 diabetes (T2DM) were recruited. A total of 119 TI DM and healthy cases with gradually changing GAD-Ab levels were selected to compare the consistency of micro-plate RBA with conventional radioligand assay (RLA). Blood samples were obtained from the peripheral vein and finger tip in 32 healthy controls, 35 T1DM and 24 T2DM patients, and tested with micro-plate RBA and then compared with the conventional RLA to investigate the reliability of finger tip sampling. Linear correlation,student's t-test, variance analysis and receiver operating characteristic (ROC) curve were performed using SPSS 11.5. Results (1) The optimized conditions of micro-plate RBA included 2 μl serum incubated with3 ×104 counts/min 35S-GAD for 24 h under slow vibration, antigen-antibody compounds washed 10 times by 4℃ PBS buffer, and radioactivity counted with Optiphase Supermix scintillation liquid. (2)The intra-batch CV of the micro-plate RBA was 3.8%- 10.2%, and the inter-batch CV was 5.6%- 11.9%. The linearity analysis showed a good correlation when the GAD-Ab in serum samples ranged from 40.3 to 664 U/ml and the detection limit of measurement was 3.6 U/ml. The results from Diabetes Autoantibody Standardization Program (DASP) 2005 showed that the sensitivity and specificity for GAD-Ab were 78% (39 positive among 50 new-onset T1DM) and 98% (2 positive among 100 healthy controls). The results of GAD-Ab obtained with micro-plate RBA and RLA were closely correlated (r=0.915,P<0.001) with a high concordance level of 97.5% and a Kappa value of 0.95. (3)TI DM and T2DM patients showed higher positive rates for GAD-Ab than the healthy controls(46.9% and 5.2% vs 0.89% ,X2=123.5 and 10. 1 ,P <0.001 and <0.01, respectively). (4)The consistency of GAD-Ab measurement with RBA using finger tip blood and RLA measurement using venous blood was 96.7% (r =0.946,P <0.001, Kappa value: 0.905). Conclusions The micro-plate RBA of GAD-Ab has high sensitivity, specificity and reproducibility, and can be measured with finger tip blood sampling. It might be a better alternative for clinical practice.

OBJECTIVETo investigate the applications of percutaneous screw fixation for the treatment of pelvic fractures and its related surgical considerations.

METHODSFrom June 2010 to June 2012,19 patients with pelvic fractures were treated with percutaneous hollow screws. There were 13 males and 6 females, with an average age of 41 years (ranged from 22 to 58 years). Fractures were caused by traffic accidents in 11 cases, by falling down from high place in 8 cases. Based on the Tile classification, there were 15 cases of Tile C type and 4 case of Tile B type. The indexes such as screw inserting time, intraoperative blood loss, complications, functional recovery and reduction conditions were observed. Fixation methods included sacroiliac screws, cannulated screw fixation of the pubic ramus and cannulated screw fixation of the pubic symphysis separation.

RESULTSAnatomical reduction achieved in 7 cases, satisfactory reduction 11 cases, and unsatisfactory reduction 1 case. Union time of fracture union ranged from 8 to 12 weeks (mean 10 weeks). Wound infection,ununion of fracture and nerve injuries were not found. According to the Majeed standards, 12 patients obtained an excellent results, 6 good and 1 fair.

CONCLUSIONPercutaneous screw fixation for the treatment of pelvic fractures under fluoroscopy has several advantages such as less trauma, less blood loss, fewer rates of complications, reliable fixation and no blood transfusion, which can reconstruct the stability of the pelvic ring, but it needs adequate preoperative preparation and high requirements for the surgeon.

Adult ; Bone Screws ; Female ; Fracture Fixation, Internal ; Fractures, Bone ; diagnostic imaging ; surgery ; Humans ; Male ; Middle Aged ; Pelvic Bones ; diagnostic imaging ; injuries ; surgery ; Radiography ; Young Adult

Adult ; Comet Assay ; DNA Damage ; drug effects ; Female ; Humans ; Lipid Peroxidation ; drug effects ; Male ; Peracetic Acid ; toxicity

Currently there is considerable interest among oncologists to find anticancer drugs in Chinese herbal medicine (CHM). In the past, clinical data showed that some herbs possessed anticancer properties, but western scientists have doubted the scientific validity of CHM due to the lack of scientific evidence from their perspective. Recently there have been encouraging results, from a western perspective, in the cancer research field regarding the anticancer effects of CHM. Experiments showed that CHM played its anticancer role by inducing apoptosis and differentiation, enhancing the immune system, inhibiting angiogenesis, reversing multidrug resistance (MDR), etc. Clinical trials demonstrated that CHM could improve survival, increase tumor response, improve quality of life, or reduce chemotherapy toxicity, although much remained to be determined regarding the objective effects of CHM in human in the context of clinical trials. Interestingly, both laboratory experiments and clinical trials have demonstrated that when combined with chemotherapy, CHM could raise the efficacy level and lower toxic reactions. These facts raised the feasibility of the combination of herbal medicines and chemotherapy, although much remained to be investigated in this area.
Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Clinical Trials as Topic ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Humans

Animals ; DNA, Fungal ; Phosphorylation ; RNA Polymerase II ; Sequence Homology, Nucleic Acid ; T-Box Domain Proteins ; genetics

OBJECTIVETo study the clinical and pathological features of drug-induced liver injury (DILI).

METHODSLiver specimens were obtained through needle biopsies from 100 patients with DILI. The histological preparations of the specimens were stained with haematoxylin eosin, several histochemistry methods, and immunohistochemistry stains. The pathological changes of the livers were analyzed together with the patients's clinical data. The patients were divided into two groups, an acute DILI group (n=39) and a chronic DILI group (n=61), based on their clinical courses and histological changes in their livers. In the chronic DILI group, the clinical courses were longer than 6 months and/or fibrosis or cirrhosis occurred in their liver tissues.

RESULTSAmong our cases the leading cause of DILI was Chinese herb medicine, accounting for 21% of the 100 cases; steroids induced cases were 11% of the total. 78% of the patients presented elevated serum transaminases and/or jaundice. The degree of transaminases elevation and the frequency of jaundice happening in the acute group were significantly higher than those in the chronic group (P less than 0.05). The histopathological liver changes in these DILI cases included: (1) necrosis commonly occurred in acinar zone 3, (2) abundant neutrophil and/or eosinophil infiltrations, (3) hepatocytic and/or canalicular cholestasis with little or no inflammation, (4) microvesicular steatosis mixed with macrovesicular steatosis, and (5) presentation of epitheloid cell granuloma. There were no significant differences in liver histopathology between the acute and the chronic DILI groups, except that the fibrosis and the ductular proliferation were different.

CONCLUSIONDILI has become a notable liver disease in mainland China, and the use of Chinese herbal medicine must be improved, standardized and regulated more closely.

Adolescent ; Adult ; Aged ; Chemical and Drug Induced Liver Injury ; pathology ; Child ; Child, Preschool ; Female ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Young Adult

Objective To explore whether the cellular apoptosis susceptibility (CAS) protein could serve as a pathologic marker for HCC diagnosis and the roles of CAS expression in HBV infection associated HCC.Methods The expression of CAS protein in HCC and its paracarcinoma tissues,non-tumor liver cirrhosis and hepatitis tissues were detected by immunohistochemistry.Meanwhile,HBsAg,HBcAg and HBV DNA in HCC tissues with HBV infection were examined by immunohistochemistry and in situ hybridization respectively.Results The expression of CAS protein was significantly higher in HCC than in its paracarcinomas tissues ( P ＜ 0.01 ),a.nd higher in paracarcinomas tissues than in non-tumor liver cirrhosis and hepatitis tissues ( P ＜ 0.01 ).Poorly differentiated tumors immunochemically stained stronger than moderately or well differentiated( P ＜0.01 ).CAS protein expression was significantly higher in HBV-infected HCC tissues than that of in non-HBV infection ( P ＜ 0.01 ).Meanwhile,in HBV-infected HCC tissues,the staining intensity score of CAS protein in HBV DNA positive HCC tissues was significantly higher than HBV DNA negative tissues ( P ＜ 0.05 ).Conclusions Higher expression of CAS protein is found in HCC tissues,and the intensity of CAS protein expression is related closely to tumor differentiation.We suggested that CAS protein might serve as a marker for HCC diagnosis and differentiation estimation,and deduced that CAS might play an important role in the initiation of HBV infection associated HCC through upregulating expression of CAS.

UNLABELLEDTo study the androgen dependence of the neurotransmitter, calcitonin gene-related peptide (CGRP) in rat penis.

METHODSForty-four Sprague-Dawley rats were randomly divided into Group A (intact controls), Group B (castrated) and Group C (gavaged with finasteride 4.5 mg.kg(-1).day(-1)). Four and ten weeks later respectively, half of rats in each group were anaesthetized. Blood samples were taken for the measurement of serum testosterone and dihydrotestosterone (DHT) by means of radioimmunoassay. Penile samples were harvested for the investigation of calcitonin gene related peptide (CGRP)-immunoreactive nerve fibers with immunohistochemistry. The computer-assisted imaging analysis system was applied to calculate the area proportion of the CGRP-positive nerve fibers (CGRP-PNF) in each group.

RESULTS1) Both 4 and 10 weeks later, testosterone and DHT levels in Group B decreased significantly compared with those in Group A, (P <0.05, P <0.01, respectively); DHT level in Group C was also significantly decreased in comparison with that in Group A for both 4- and 10- week animals (P <0.05); 2) There was no significant differences in area proportion of CGRP-PNF among Groups A, B and C 4 weeks after treatments (P >0.05); However, 10 weeks later, the proportion of CGRP-PNF in Groups B and C was significantly less than that in Group A (P <0.01); 3) The proportion of CGRP-PNF of 4-week animals in Groups B and C was significantly higher than that of 10-week animals (P <0.05).

CONCLUSIONThe expression of neurotransmitter, CGRP may depend on androgens, including testosterone and DHT in rat penis.

Androgens ; metabolism ; Animals ; Calcitonin Gene-Related Peptide ; metabolism ; Dihydrotestosterone ; blood ; Gene Expression Regulation ; Immunohistochemistry ; Male ; Penis ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone ; blood