OBJECTIVETo construct effective RNA-interference plasmids targeting mouse HIF-1alpha gene and testify their effects and specificities in interfering HIF-1alpha expression.

METHODSThree RNA-interference plasmids targeting mouse HIF-1alpha gene, pBS/U6/HIF-1alpha-siRNAI~III, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1alpha in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1alphai-II in SH-SY5Y cell line was further investigated.

RESULTSAll the three RNA-interference plasmids, especially pBS/U6/HIF1alphai-II, showed significant inhibition in HIF-1alpha expression in 293T cell line. pBS/U6/HIF1alphai-II could also inhibit HIF-1alpha expression in SH-SY5Y cell line, in a dose-dependent way.

CONCLUSIONPlasmid pBS/U6/HIF1alphai-II constructed in our study can effectively and specifically inhibit HIF-1alpha expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/HIF1alphai-II plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and ischemia development.

Analysis of Variance ; Animals ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Gene Silencing ; physiology ; Green Fluorescent Proteins ; genetics ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Mice ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; pharmacology ; Transfection ; methods

A new EMA real-time fluorescence PCR method was developed to detect alive Listeria monocytogenes in foods.The specific primers and probe were designed based on the conserved inlA gene.The pretreatment conditions including EMA of different concentrations and irradiating times were optimized.The detection limit and inhibition rate to dead bacteria of this method were confirmed by using direct plating method.The detection specificity was evaluated by using 35 L.monocytogenes strains,25 non-L.monocytogenes strains and 92 non-Listeria strains.Simulation detection experiments were performed on 15 beverage samples and 15 cooked meat samples supplemented separately with inactivated L.monocytogenes,alive L.monocytogenes and Staphylococcus aureus.Results showed that the Ct of EMA real-time fluorescence PCR for alive L.monocytogenes was Ct=38.46-3.30 × log (R2=0.999).The detection limit was 55 cfu per reaction.Inhibition rate of DNA of inactivated strains was over 99.98％.The Ct of 35 L.monocytogenes strains were between 16.21 and 29.38,while 25 non-L.monocytogenes strains and 92 non-Listeria strains had Ct ＞35.The variation coefficient of CT was less than 5％ when the experiments were repeated.Results of 30 simulation samples were consistent with that by using standard method.The test time by using newly developed EMA real-time fluorescence PCR was shortened from 3-5 days to about 10 h.The newly developed EMA real-time fluorescence PCR method for alive L.monocytogenes is rapid,convenient,specific and sensitive and could be applyed in foods inspection.

Malaria is a worldwide epidemic that extensively endangers health of human beings. Before artemisinin was developed to treat with malaria, about 400 million person-time of malaria infections and at least 1 million deaths from malaria were reported in the world every year. Thus malaria has been listed as one of the world's three major death diseases by the WHO. The discovery of artemisinin by Chinese scientists created a novel therapy approach to treat with malaria effectively. Amorpha-4,11-diene oxidase is a plant cytochrome P450 enzymes, i.e. CYP71AV1, which catalyzes each of the three oxidation steps from amorpha-4,11-diene to form artemisinic acid, the intermediate of artemisinin. CYP71AV1 is the key enzyme in artemisinin biosynthesis. By constructing the prokaryotic expression vector pCWOri(+)-CYP71AV1, functional expression and purification of complementary CYP71AV1 were performed. The enzyme activity was monitored by CO differential spectrum assay and the heme-based activity analysis. The preliminary crystallization condition was obtained by crystallization screening. These studies provide basis for resolving the crystal structure of CYP71AV1 and for producing artemisinin in large scale through biosynthetic biology approach, and will provide references for over expression, purification and crystallization of other plant P450 enzymes.

Objective To evaluate the protective rate and the variation of HFRS-IgG on hemorrhagical fever with renal syndrome (HFRS) vaccine.Methods Cluster,random sampling and cross-sectional study were used to assess the protective rate of HFRS vaccination.Level of HFRS-IgG was detected with ELISA in epidemic and non-epidemic areas of HFRS.Results Curve equation was obtained as Yprocective rate=(0.863+0.283/Xvaccination term) × 100％ by protective rate with vaccination term.Protective rates showed a reducing trend,90％ after 7-8 years of vaccination,88％ after 10 years,and 94％ on average.Absorbance (A) value of HFRS-IgG was 4 times higher in persons with vaccination than those without,in the epidemic area.Higher antibody level could be obtained after primary vaccination,but the level of antibody had a 50％ reduction after 5-10 years of vaccination,and a 60％ reduction after 10 years of vaccination.Conclusion HFRS antibody had a 50％ reduction after 5-10 years of vaccination.The protective rate of HFRS vaccination had a 90％ loss,after 7-8 years of vaccination.Booster dose was necessary after 7 years of vaccination.

BACKGROUNDPlantar pressure serves as a key factor for predicting ulceration in the feet of diabetes patients. We designed this study to analyze plantar pressure changes and correlating risk factors in Chinese patients with type 2 diabetes.

METHODSWe recruited 65 patients with type 2 diabetes. They were invited to participate in the second wave 2 years later. The patients completed identical examinations at the baseline point and 2 years later. We obtained maximum force, maximum pressure, impulse, pressure-time integral, and loading rate values from 10 foot regions. We collected data on six history-based variables, six anthropometric variables, and four metabolic variables of the patients.

RESULTSOver the course of the study, significant plantar pressure increases in some forefoot portions were identified (P < 0.05), especially in the second to forth metatarsal heads. Decreases in heel impulse and pressure-time integral levels were also found (P < 0.05). Plantar pressure parameters increased with body mass index (BMI) levels. Hemoglobin A1c (HbA1c) changes were positively correlated with maximum force (β = 0.364, P = 0.001) and maximum pressure (β = 0.366, P = 0.002) changes in the first metatarsal head. Cholesterol changes were positively correlated with impulse changes in the lateral portion of the heel (β = 0.179, P = 0.072) and pressure-time integral changes in the second metatarsal head (β = 0.236, P = 0.020). Ankle-brachial index (ABI) changes were positively correlated with maximum force changes in the first metatarsal head (β = 0.137, P = 0.048). Neuropathy symptom score (NSS) and common peroneal nerve sensory nerve conduction velocity (SCV) changes were positively correlated with some plantar pressure changes. In addition, plantar pressure changes had a correlation with the appearance of infections, blisters (β = 0.244, P = 0.014), and calluses over the course of the study.

CONCLUSIONSWe should pay attention to the BMI, HbA1c, cholesterol, ABI, SCV, and NSS changes in the process of preventing high plantar pressure and ulceration. Some associated precautions may be taken with the appearance of infections, blisters, and calluses.

Adult ; Aged ; Asian Continental Ancestry Group ; Diabetes Mellitus, Type 2 ; physiopathology ; Diabetic Foot ; diagnosis ; physiopathology ; Female ; Foot ; physiopathology ; Humans ; Male ; Middle Aged ; Pressure ; Prospective Studies ; Risk Factors

OBJECTIVETo study the incidence of cases with rash and fever illness (RFIs) after measles vaccine (MV) inoculation.

METHODSDuring 1999 to 2002, 150 RFIs cases reported by the special measles surveillance system in Shandong province, China, were investigated and analyzed epidemiologically.

RESULTS7 674 690 ml MV were distributed during 1999 to 2002 and the annual average incidence of RFIs cases after MV inoculation was 0.20/10 000 ml (0.2 ml per dose). There was significant difference of incidences each year (chi(2) = 10.13, P < 0.05). All RFIs cases were sporadically distributed without epidemiological links. Clinical symptoms showed that 88.67% of the 150 RFIs cases having > 38.5 degrees C fever and 75.33% of all cases appeared typical rash after 4 to 11 days (the medium was 8 days) after MV inoculation. The order of rash onset among RFIs cases was consistent with that of regular measles cases caused by wild virus. 68.67% of the RFIs cases had first MV inoculation and 94.71% were 8 to 12 month-olds. IgM sera antibody test from RFIs cases were rubella negative and 45.65% positive for measles.

CONCLUSIONRFIs due to allergic reaction or measles vaccine virus infection might occur after MV inoculation. There seemed to be a correlation between RFIs incidence and the doses of MV. Measles virus genotype analysis needs to be carried out to confirm if the onset of some RFIs cases is aetiologically associated to MV vaccine virus infection.

Exanthema ; etiology ; virology ; Fever ; etiology ; virology ; Humans ; Measles ; prevention & control ; Measles Vaccine ; adverse effects ; Measles virus ; immunology ; Polymerase Chain Reaction ; Vaccination

OBJECTIVETo screen and identify the peptides that specifically bind to CD13 on monocytes.

METHODSThe phages capable of specific binding to CD13 were screened in the phage-displayed 12-peptide library. The affinity of the selected phages with CD13 was verified with enzyme-linked immunosorbent assay (ELISA). The sequences of the peptides bound to the phages were deduced according to the phage DNA sequences, and the functional peptides aligned using the BLASTP on the Website NCBI were synthesized. To analyze the biological function of the screened peptides, the location of the peptides bound to THP-1 cells was detected using immunofluorescence assay. The blocking effect of WM15 on the peptide binding to THP-1 cells was assessed by immunofluorescence assay.

RESULTSThe phages that specifically bound to CD13 were effectively enriched to approach saturation after 4 rounds of panning. The recovery rate in the fourth round was 30 times that in the first round. Twenty selected phages were verified by ELISA, and the signals of 10 phages were higher than the control. The sequences of the peptides P9 and P7 showed 83% and 100% identity with those of human cytomegalovirus (HCMV) UL38 and UL105, respectively. The peptides bound to the cell membrane of THP-1 cells as shown by immunofluorescence assay. The binding of the peptides P9 and P7 to THP-1 cells was blocked by CD13-specific monoclonal antibody WM15 at different levels.

CONCLUSIONTwo peptides (P7 and P9) that can specifically bind to CD13 have been screened successfully, and these two peptides show specific binding to CD13 on the membrane of THP-1 cells.

Amino Acid Sequence ; Binding, Competitive ; CD13 Antigens ; analysis ; metabolism ; Cell Line ; Humans ; Molecular Sequence Data ; Peptide Library ; Peptides ; metabolism ; Protein Binding

OBJECTIVETo investigate the efficacy, safety and the life quality improvement of uroacitides injection in the treatment for patients with advanced malignant tumors.

METHODSA total of 160 patients with advanced stage cancers were enrolled into this multicenter, open and non-randomized phase II clinical trial, including cancers of the lung (33 cases), liver (45 cases), breast (17 cases), esophagus (11 cases), stomach (18 cases), colon (19 cases), pancreas (3 cases) and kidney (4 cases), and glioma (10 cases). Uroacitides was administrated in a dose of 300 ml daily via the superior vena cava catheter for consecutive 4-8 weeks.

RESULTSOf the 160 patients, 21 dropped out and one patient died during the trial. Efficacy could be evaluated in 138 patients and safety in 160. The total objective response rate (ORR, CR + PR)) and tumor control rate (CR + PR + MR + SD) of the 138 evaluable patients were 5.8% and 65.2%, respectively. Clinical benefit response (CBR) rate was 57.2%. Major adverse effects were grade I - II and reversible nausea/vomiting (21.9%) and pain (6.3%).

CONCLUSIONUroacitides injection is effective in the control for various kinds of advanced cancers with mild, reversible and tolerable adverse effects, and can also improve the patient's quality of life. It is worth being studied further.

Breast Neoplasms ; blood ; drug therapy ; pathology ; CA-19-9 Antigen ; blood ; Carcinoembryonic Antigen ; blood ; Carcinoma, Non-Small-Cell Lung ; blood ; drug therapy ; pathology ; Catheterization, Central Venous ; Colorectal Neoplasms ; blood ; drug therapy ; pathology ; Humans ; Liver Neoplasms ; blood ; drug therapy ; pathology ; Lung Neoplasms ; blood ; drug therapy ; pathology ; Methyltransferases ; administration & dosage ; adverse effects ; antagonists & inhibitors ; therapeutic use ; Nausea ; chemically induced ; Neoplasm Staging ; Peptides ; administration & dosage ; adverse effects ; therapeutic use ; Phenylacetates ; administration & dosage ; adverse effects ; therapeutic use ; Quality of Life ; Remission Induction ; Salvage Therapy ; Treatment Outcome ; Vomiting ; chemically induced ; alpha-Fetoproteins ; metabolism

Objective To access the effects of different doses of recombinant fusion protein of human tumor necrosis factor receptor mutant and Fc fragment [rhTNFR(m):Fc] after a single subcutaneous injection on the plasma concentration of tumor necrosis factor-α (TNF-or) in Chinese healthy volunteers.Methods A total of 56 healthy Chinese volunteers were randomly divided into 6 groups to receive a single injection of 10,20,35,65,75 mg of rhTNFR(m):Fc.The plasma concentrations of TNF-α and total TNF-α were determined at 1 h pre-dose and at 4,48,96,168,216,264,312,384,480 h post-dose.Results After administration of rhTNFR(m):Fc at 0-264 h,the plasma concentrations of free TNF-α and total TNF-α increased significantly in the each group.At 264-480 h post-dose,the concentration of them began to decrease,and at 480 h the concentration of free TNF-α almostly decreased to normal levels.In the dose range of 10-75 mg,the exposure of free TNF-α and total TNF-α (Cmax) had no significant correlation with the dose of rhTNFR (m):Fc.Conclusion After giving the single dose of rhTNFR (m):Fc,there was an increase of free and total TNF-α plasma concentration in Chinese healthy volunteers.As a result,the plasma concentration of free and total TNF-α may not be a suitable pharmacodynamic evaluation index.

Salvia miltiorrhiza is widely used in the treatment of the angina pectoris, coronary heart disease and myocardial infarction. CYP76AH3 is the key P450 enzyme, locating at the branch point of the tanshinone synthesis pathway. The crystal structural study and key amino acid analysis are of great significance for synthetic biology study on tanshinone. But it is always a challenge for scientists to carry out protein purification, crystallization and crystal structural studies on transmembrane type Ⅱ P450 enzymes. In this study, the prokaryotic expression plasmid was generated, and the high-purity target protein was purified. CYP76AH3 was successfully crystallized, and the crystal structure was solved.After docking range was determined by Cavityplus analysis, molecular docking with Discovery Studio was conducted. The docking result indicated that Gly298 and Asp294 had hydrogen bond interaction with the substrate, while Phe479, Leu367 and Leu293 had hydrophobic interaction with the substrate.In addition, the effect of mutations at the key amino acids on the protein structure stability was predicted throung point mutation simulation. This study would provide a target for protein engineering of CYP76AH3 and lay a foundation for the study of synthesis biology on tanshinones.