In order to study the application of glial cell line-derived neurotrophic factor(GDNF)in clinic,gene mutation,fusion protein expression in E.coli and purification methods have been used to obtain the fragments of GDNF,GDNF(△N39),GDNF(△N39)-R9.Using primary cultured dopaminergic neurons and PC12 cells with transfected with GFR?1 and Ret to observe their biological function and cytotoxicity.Using B-Endo3 cells and Transwell method to analyze their delivery across the cellular membrane and blood brain barrier.The results show that GDNF(△N39)-R9 has the same neurotrophic function with wild GDNF and nearly no cytotoxicity to dopaminergic neurons and PC12-GFR?1-Ret cells and can get through effectually the cellular membrane and simulacrum of blood brain barrier with matrigel and B-Endo3.

Aim Vascular endothelial cell growth inhibitor(VEGI) is a recently discovered novel member of the TNF superfamily,which is expressed predominantly in endothelial cells.As an endothelial cell-specific negative regulator of angiogenesis,the relationship between structure and function of VEGI is not understood at present.Methods In order to explore the functional key amino acids of VEGI,four mutants of VEGI(E45→R,G47→A,Y111→F,Y111→T) were construced by site-directed mutagenesis,and recombinant proteins were generated from E.coli.Four mutant proteins behaved similar to the wild type VEGI in various physico-chemical assays.The proliferation of HUVEC and chick choriallantic membrane assay were performed to study the activity of four mutants.Results The mutant E45→R significantly decreased the biological activity,and the mutant G47→A caused a slight drop on activity,but the mutants Y111→F,Y111→T almost completely abolished biological activity.Conclusion It suggests that Y111 is an important residue in biological activity,which may play a direct role in receptor recognition.Moreover,the tyrosine ring and hydroxy group of the amino acid are important determinant of biological activity.Additionally,E45 also plays an important role in biological activity of VEGI.

Objective To understand the status of Toxoplasma gondii(TOX)infection in pregnant women in Qingdao area. Methods Antibody capture ELISA was used to detect the TOX-IgM,IgG and TOX-DNA in 1 341 pregnant women and 201 infer-tility,miscarriage,stillbirth pregnant women from June 2011 to July 2013. Results Among 201 abnormal pregnant women,43 cases were TOX antibody positive,accounting for 22.39%,and among the normal pregnant women,84 cases were TOX antibody positive,accounting for 6.26%,and there was a significant difference(χ2 =10.60,P<0.05). The TOX positive rate of 20-29 years old women was higher than that of the women aged over 30 years(χ2=21.9,P<0.05). The TOX antibody positive rates of families with pets and families without pets were 26.63%and 8.77%respectively(χ2=10.93,P<0.05). The TOX antibody posi-tive rates were 10.94%(22/201)in unemployed women,3.98%(8/201)in farmers,3.98%(8/201)in staff,and 3.48%(7/201)in workers. Conclusions The TOX infection rate in abnormal pregnant women is significantly higher than that in normal pregnancy women;therefore,the health education should be strengthened. In addition,in young women,especially 20-29 years old women, and some special occupation women,the control measures should be enhanced.

OBJECTIVETo investigate the theoretical model of the three-dimensional structure of mosquitocidal Cry30Ca2 and its molecular docking with N-acetylgalactosamine.

METHODSThe theoretical model of Cry30Ca2 was predicted by homology modeling on the structure of the Cry4Ba. Docking studies were performed to investigate the interaction of Cry30Ca2 with N-acetylgalactosamine on the putative receptor.

RESULTSCry30Ca2 toxin is a rather compact molecule composed of three distinct domains and has approximate overall dimensions of 95 by 75 by 60Å. Domain II is a helix bundle, Domain II consists of three antiparallel β-sheets, Domain III is composed of two β-sheets that adopt a β-sandwich fold. Residue 321Ile in loop1, residues 342Gln 343Thr and 345Gln in loop2, residue 393Tyr in loop3 of Cry30Ca2 are responsible for the interactions with GalNAc via 7 hydrogen bonds, 6 of them were related to the oxygen atoms of hydroxyls of the ligand, and one to the nitrogen of the ligand.

CONCLUSIONThe 3D structure of Cry30Ca2 resembles the previously reported Cry toxin structures but shows still some distinctions. Several residues in the loops of the apex of domain II are responsible for the interactions with N-acetylgalactosamine.

Acetylgalactosamine ; chemistry ; Amino Acid Sequence ; Animals ; Bacterial Proteins ; chemistry ; pharmacology ; Catalytic Domain ; Culicidae ; drug effects ; Endotoxins ; chemistry ; pharmacology ; Hemolysin Proteins ; chemistry ; pharmacology ; Insecticides ; chemistry ; pharmacology ; Models, Molecular ; Molecular Sequence Data ; Protein Conformation

OBJECTIVETo explore activity laws of mitochondrial complex II in patients of deficiency-cold syndrome (DCS) and deficiency-heat syndrome (DHS) under various ambient temperatures.

METHODSSubjects were recruited by questionnaire and expert diagnosis from grade 1 - 3 undergraduates at Henan College of Traditional Chinese Medicine in November 2012, and assigned to a normal control group, the DCS group, and the DHS group, 20 in each group. Their venous blood samples were collected at two different temperature conditions. Activities of mitochondrial complex II were measured by spectrophotometry.

RESULTS(1) Comparison of mitochondrial complex It under various ambient temperatures: Compared with room temperature in the same group, activity values were all increased in the normal control group at cold temperature with significant difference (P <0.05), but there was no significant difference in the DCS group and the DHS group (P >0. 05). Compared with the normal control group, activity values of complex H were reduced in the DCS group at cold and room temperatures with significant difference (P <0.05). Compared with the DCS group, activity values of complex It were increased in the DHS group with significant difference (P <0. 05). (2) Changes of adjustment rates: Compared with room temperature, the adjustment rate all rose at cold temperature in the normal control group and the DHS group with significant difference (P <0.05), but with no significant difference found in the DCS group (P >0. 05). Compared with the normal control group at the same temperature, the adjustment rate in the DHS group and the DCS group was all reduced at cold and room temperatures with significant difference (P <0. 05). There were no significant difference in the adjustment rate between the DHS group and the DCS group (P > 0. 05).

CONCLUSIONSEnvironment temperature can affect the activity of mitochondrial complex II with different influence degrees on different syndrome types of people, but its change trend are basically identical.

Cold Temperature ; Electron Transport Complex II ; metabolism ; Hot Temperature ; Humans ; Medicine, Chinese Traditional ; Syndrome ; Temperature

Near-infrared (NIR) was used as rapid analysis method to identify the sulfur fumigated Puerariae Lobatae Radix. NIR spectra of the cross-sectional and longitudinal selection of samples were acquired. Principal component analysis was conducted. The samples were randomly selected. The different pretreatment methods were compared. Discriminant models were established for every type of spectra to calculate the recognition rate. The orthogonal test and nonparametric test were used to test data normality. The result showed that absorbance values of different sections were different due to the different structure, and the raw spectra were analyzed by PCA method. The result founded that the cumulative contribution rate was arrived at 99.2% while the PC numbers were arrived at 3. The pretreatment method based on the MSC + 1D + Savitzky-Golay was the best to establish the model. For the 50 models constructed with cross-section and longitudinal spectra and total spectra, the recognition rate were (94.4 ± 0.66)%, (94.4 ± 0.66)%, (95.3 ± 0.65)%, respectively, and no difference was observed. The NIR method could be used to identify the sulfur fumigated Puerariae Lobatae Radix.
Chemistry, Pharmaceutical ; methods ; Discriminant Analysis ; Drugs, Chinese Herbal ; chemistry ; Fumigation ; Pueraria ; chemistry ; Spectroscopy, Near-Infrared ; Sulfur ; chemistry

Polyamidoamine-polyethylene glycol (PAMAM-PEG) copolymers were synthesized using IPDI as coupling reagent by two-step method. The copolymers were characterized by IR spectrum and 1H NMR spectrum, and the PEG conjugating ratios of the copolymers were calculated equal to 10% and 30% separately. MTT assay indicated that after PEGylation a lower cytotoxicity of the copolymers could be found, and with increasing PEG conjugating ratio the cytotoxicity decreased obviously. Agarose gel retardation assay demonstrated that PAMAM-PEG copolymers could be combined with DNA and PAMAM-PEG/DNA complexes were prepared by self-assembly. DLS measurement showed that when N/P > or = 50, the particle size of copolymer/ gene complexes was in a range of 150-200 nm, and the zeta potential was in a range of 10-25 mV. In vitro gene transfection illustrated that when N/P < or = 50, the gene transfection efficiency of PAMAM-PEG copolymers was a little less than that of PAMAM-G5, but the transfection efficiency can be raised by increasing N/P ratio or transfection time. Considering both cytotoxicity and transfection efficiency aspects PAMAM-PEG-13 was more effect than PAMAM-PEG-39 in PEGylation.

OBJECTIVETo explore clinical characteristics and treatment of posterior Pilon fracture.

METHODSFrom January 2011 to January 2013,18 patients with posterior Pilon fracures were treated. Among them, 13 were male and 5 were female, aged from 22 to 63 years old, with an average age of 46. All the patients were closed fractures. Open reduction and internal fixation were performed after swelling subsided, lateral malleolous and posterior Pilon fracture were exposured through lateral approach on healthy side, plates were used to fixed,screws or small plates were used to fix the posterior prominence of medial malleolus after changed to supine position. AOFAS scoring were applied to evaulate clinical effects.

RESULTSAll patients were followed up with an average of 22(ranged, 12 to 48)months. All patients obtained satisfactory reset except one patient. All factures were recovered well with an average healing of 11 weeks. According to AOFAS score at the final following up, 7 cases were excellent,2 cases were moderate, and the total score was 86.8±9.2.

CONCLUSIONPosterior Pilon fracture is not rare in clinical, its mechanism of injury, traumatic anatomy, surgical procedure and prognosis are different from that of classical ankle fracture and Pilon fracture.

Adult ; Ankle Injuries ; surgery ; Female ; Fracture Fixation, Internal ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Tibial Fractures ; surgery ; Treatment Outcome ; Young Adult

Near-infrared (NIR) diffuse reflectance spectroscopy was used to analyze the impact of multi-class particle size of Chinese material medica (CMM) based on the spectral characteristics in overtone and combination region. Several types of CMM (60, 80, 100,120 mesh) were subjected to NIR spectra analysis. Spectral reproducibility was examined after sample repackage. The result showed that the effects of particle size on the NIR spectra were different according to different bands, in the combination region and first combination-overtone region. Spectroscopy intensity was proportional to the particle size and influence of particle size was greater as the wavelength increased. While in the second combination-overtone region, it was inversely proportional to particle size. To the sampling loading error, the result indicated that when the mesh number was larger than 60 mesh, the error was small. The appropriate particle size was clarified to guarantee the accuracy and reliability of NIR diffuse reflectance spectroscopy in CMM.
Drugs, Chinese Herbal ; analysis ; Materia Medica ; analysis ; Particle Size ; Spectroscopy, Near-Infrared ; methods

In order to investigate the ability of PEG-PEI copolymers as gene carriers for delivery of VEGF165. A series of PEG-PEI copolymers with different PEG grafting was prepared and the cytotoxicity was evaluated. Simultaneously,the VEGF165 gene segment with HindⅢ and BamHⅠ site was obtained by PCR, which was cloned into pEGFP-C1. PEG-PEI/ pEGFP-VEGF165 complexes were formed by self-assembly and transfected HUVEc. Transfection efficiency was evaluated by measuring the percentage of cells expressing green fluorecensce protein. The VEGF expression was detected by ELISA, RT-PCR, and the effect of transfection on growth of endothelial cell was evaluated by MTT. The results suggested that the formation of PEG-PEI copolymers could help to reduce the cytotoxicity of PEI. After transfection, the strong expression of green fluorescence protein was observed by fluorescence microscopy. The transfection efficiency was influenced by the number of PEG side chains and N/P ratio. Of all copolymers tested, the transfection efficiency of PEG-PEI(5-25-1) at N/P = 30 reached a maximum, which was much higher than that of PEI. The expression of VEGF protein and mRNA increased significantly, and HUVEc proliferation was accelerated after transfection.These results indicates PEG-PEI copolymers can be used as effective gene carriers for delivery of pEGFP-VEGF165 gene.