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

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

One hundred Pseudomonas aeruginosa quorum-sensing-related genes were selected and their primers were synthesized. The fragments of specific sequences which are related QS genes were amplified by PCR. These verified sequences were inserted into the vector pMD-18T for sequencing. These DNA fragments were dotted onto glass slides to make cDNA microarray. Hybridization was performed with cy3/cy5-dCTP labeled probes. The scanning data of early stationary phase and mid-logarithmic phase indicated that 9 genes were up-regulated and 6 genes were down-regulated. Undergoing the different medicines,we took tobramycin as an example to compare the expression diversity. The results confirm that the QS cDNA chip is useful,and may contribute to better understand the mechanism of quorum-sensing,and can help us find the new targets for restraining the growth of Pseudomonas aeruginosa.

Objective To evaluate the influential factors of iron acquisition during Francisella tularensis LVS infection of mouse macrophages.Methods F.tularensis LVS expressing green fluorescent protein was used to infect murine macrophage J774A.1 cells.Transferrin receptor 1(Tfr1)was detected with mono-antibody and visualized with a goat-anti mouse IgG conjugated to Alexa 594.The expression profile of 5 iron metabolism related genes of J774A.1 murine macrophages uninfected or infected with F.tularensis LVS was determined with real-time PCR.Immunoblot analysis was used to compare the Tfr1 expression of live Francisella infected macrophage with dead bacteria.Tfr1 knock-off in J774A.1 cells was performed with siRNA.The transfected cells were infected with Francisella for immunoblotting and microscopy and infection assay.Results It was revealed that the live vaccine strain of F.tularensis induced the expression of Tfr1 in host macrophages.Gene expression analysis indicated that F.tularensis LVS drove an active iron acquisition program with induction of Tfr1 and iron regulatory proteins(Irp1 and Irp2).It was shown by Western-blotting that the siRNA-Tfrc-1 could knock off about 75% of Tfr1 in J774A.1 cells.It was determined by infection assay that,Tfr1 was knocked off,the bacteria number at 1h infection with Francisella was not different from that of control(F=1.06,P=0.326 5),while it was decreased significantly after 24h of infection(F=24.12,P=0.000 6).Conclusions It is demonstrated that upregulation of the Tfr1 may be mediated by post-transcriptional regulation during early infection,but sustained later through increased expression of Irp 1 and Irp 2.Increased expression of Tfr1 expands the intracellular iron pool through transferrin-mediated delivery and may thus be readily available for uptaking by Francisella.Knocking off the expression of Tfr1 does not affect bacterial invasion.Francisella,however,may fail to proliferate in macrophages in which the expression of transferrin receptor has been suppressed.

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.

Objective To evaluate the effectiveness of treating glioma in combination of two kinds of drugs by comparing the size of tumors and the survival time of different groups in rat glioma after targeted blood‐brain barrier (BBB) disruption by focused ultrasound under MRI‐guide. Methods The stereotaxis instruments and the 10 μl gas‐tight syringes were used to inject gliosarcoma cells into the targeted area of the brain in 40 male Sprague‐Dawley rats. The glioma‐bearing rats models were established. Rats were divided into 4 groups to receive different treatment :(1) no treatment (control, n = 8), (2) IV Avastin (Avastin only, n =10), (3) IV liposomal doxorubicin (DOX only, n =10), (4) IV Avastin and liposomal doxorubicin (Avastin+DOX, n =10). The SonoVue microbubbles and DOX or Avastin were injected into the tail vein respectively on the 12th day after implantation. The tumor size was measured by MRI on immediacy, once a week after targeted BBB disruption by focused ultrasound, and the life span in rat glioma was recorded. Results The average survival time of different groups in rat glioma was as follows :no treatment(17 ± 4)d, Avastin(20 ± 4)d, DOX(25 ± 5)d, DOX+ Avastin(40 ± 5)d. The tumor size after post‐treatment of different groups in rat glioma was as follows :no treatment(5 7.0 ± 4 3.0)mm, Avastin(4 3.0 ± 2 5.0)mm, DOX(4 1.2 ± 3 1.0)mm, DOX + Avastin(2. 20 ± 1. 30)mm. There was significant increased in average survival time and decreased in tumor size after a combination treatment DOX+ Avastin compared with other groups( P < 0 0.1). Conclusions The microbubble blasting by MRI‐guided focused ultrasound enhances the local tissue permeability and promotes the drug delivery of chemotherapy and anti‐angiogenesis locally in glioma‐bearing rats. Especially, the combination of two kinds of drugs has a synergism efficacy that may reduce tumor growth and increase survival time significantly after BBB disruption.