To understand molecular modules related to polyunsaturated fatty acids (PUFA) synthesis and eventually produce PUFA at high efficiency, we developed a protein complex analysis technology in Synechocystis sp. PCC 6803, and applied it to identify possible partner proteins interacting with the key enzymes that catalyze PUFA biosynthesis. We first constructed a recombinant expression of protein of slr1609 encoding the fatty acid activation enzyme, by fusing 3xFLAG tag with the target protein. Then we verified its expression by Western blotting targeting 3xFLAG tag. To maximize purification of Slr1609 protein complex, we optimized the protein expression conditions of Slr1609 in Synechocystis in a 5 L fermenter by monitoring its gene expression using RT-qPCR. The purification of the Slr1609 protein complexes was demonstrated by a Native-PAGE analysis. Finally, LC-MS/MS proteomic analysis allowed identification of the possible partner proteins interacting with Slr1609.
Bacterial Proteins ; chemistry ; Chromatography, Liquid ; Fatty Acid Synthases ; chemistry ; Fatty Acids, Unsaturated ; biosynthesis ; Proteome ; chemistry ; Proteomics ; Synechocystis ; enzymology ; Tandem Mass Spectrometry

Objective To study protective immunity effects of co-immunization with P30 DNA vaccine and protein vaccine. Methods Forty-eight 5-6 weeks old BALB/c female mice were divided into four groups (A,B,C,D), 12 mice of each group. In group A (control group) each mouse was immunized with 100 ?g pcDNA3.1 plasmid DNA by intramuscular (i.m.) for three times at week 0,2 and 4; in group B (P30 protein group) each mouse was immunized (i.m.) with 50 ?g rP30+50 ?g CFA for three times at week 0, 2 and 4; in group C (pcDNA3.1-P30 group) each mouse was immunized with 100 ?g pcDNA3.1-P30 plasmid DNA (i.m.) for three times at week 0, 2 and 4; in group D (P30 DNA+rP30 co-immunization group) each mouse was immunized with 100 ?g pcDNA3.1-P30 plasmid DNA (i.m.) for two times at week 0, 2 and immunized by subcutaneous with 50 ?g rP30+50 ?g CFA at week 4. Each mouse was infected with 100 tachyzoites of Toxoplasma gondii RH strain four weeks later after last immunization. The anti-P30 antibodies were detected with ELISA before the challenge. Results The P30 DNA vaccine was successfully constructed. High titers of anti-P30 antibodies were induced in each mouse immunized with DNA vaccine. The protective trial proved that there was no significant difference between control group and experimental group though the survival time of mouse from experimental group had been prolonged. Conclusion The P30 DNA vaccine could induced high titers of anti- P30 antibodies in immunized mice, and it may be a potential DNA vaccine candidate.

Object To study the microwave assisted procedure for the extraction of baicalin from root of Scutellariae baicalensis Georgi Methods An MSP 100D domestically made microwave sample preparation system with a maximum power of 850 watts was used. The effects of solvents, pressure/temperature of solvents and microwave radiation time on the yield of baicalin were studied by orthogonal experimental design. Results The optimal experimental conditions were extraction at 70% of microwave power with 30 times of 35% ethanol at a constant heating pressure/temperature of 0.15 MPa for 30 s. Conclusion The microwave assisted extraction not only takes a shorter time with better parallel results, but also gave an increased yield of about 10% as compared with ultrasonic extraction.

Objective To investigate the clinical efficacy of levonorgestrel- releasing intrauterine system (LNG-IUS) in the treatment of adenomyosis and the influence of ovarian function. Methods Eighty patients with adenomyosis were followed up for 0, 1, 3, 6, 12 months after treating with LNG-IUS. The menstrual blood volume, dysmenorrheal, size of uteri, CA125 and EMAb level, hepatic function, serum glucose and serum lipids were observed and evaluated. Serum- levels of FSH and LH were tested before insertion and followed up for 6, 12 months after insertion, respectively. Results After inserting LNG-IUS,dysmenorrhea was obviously alleviated, and it was significantly decreased after 6 months. The menstrual blood volume was (200. 0 ±60)ml, (40. 0 ± 15)ml and (28 ±7)ml in 0, 6, 12 months after the insertion.Serum CA125 and positive rate of EMAb also significantly reduced [CA125: (50. 69 ± 10. 00) IU/L vs (18. 60 ±3.55)IU/L;EMAb:80. 0% vs 3. 8%, P ＜0. 05]. The volume of uterus reduced without significant changed (P ＞0. 05). There were little changes in the levels of hepatic function, serum glycosum, serum lipids, FSH and LH at 6 and 12 months after LNG-IUS(P ＞ 0. 05). Conclusions LUG-IUS is an effective and safe therapy for adenomyosis with dysmenorrheal and menorrhagia, and it has no significant effects on ovarian function.

Objective To study the effects of EP4 and EP2 antagonists on the differentiation of Treg/ Th17 cells and disease progression in mice of collagen-induced arthritis (CIA) model.Methods DBA/1 mice wereimmunized subcutaneously twice at the root of the tail with type Ⅱ collagen emulsified in Freund's complete adjuvant.EP2 and EP4 antagonist therapies were intraperitoneally administrated for 14 consecutive days after the second immunization.Clinical signs,histological manifestation,serum interleukin (IL)-17 and quantity of CD4+CD25+Foxp3+ Treg cells were determined.ANOVA and t-test were used for statistical analysis.Results Clinical signs of the disease appeared on day 27 and peaked on day 35 after the first immunization.The quantity of CD4+CD25+Foxp3+ Treg cells in spleens [(1.67±0.15)％] and draining inguinal lymph nodes [(3.30±0.36)％] isolated from CIA mice were significantly lower than those of normal DBA/1 mice [(2.77±0.45)％ and (4.73 ±0.45)％ respectively,P＜0.05].Serum IL-17 level of CIA mice [(27±7) pg/ml] was significantly higher than that of normal DBA/1 mice [(14±4) pg/ml,P＜0.05].Intra-peritoneal injection of EP4 but not EP2 antagonist to CIA mice decreased paw edema and swelling,and alleviated the histological manifestations (1.8±1.0 vs 3.5±0.6,P＜0.05) on day 35 after the first immunization.The percentages of CD4+CD25+Foxp3+ Treg cells in both inguinal lymph nodes [(4.20±0.32)％] and spleens [(2.63±0.40)％] were significantly higher in EP4 antagonist-treated but not EP2 antagonist-treated CIA mice compared with CIA mice group [(3.30±0.36)％ and (1.67±0.15)％ respectively,P＜0.05].The level of serum IL-17 was significantly lower in EP4 antagonist-treated [(15±7) pg/ml] but not EP2 antagonist-treated CIA mice compared with CIA mice group [(27±7) pg/ml,P＜0.05].Conclusion EP4 antagonist therapy alleviates clinical symptoms of CIA,improves the histological manifestations,decreases the serum IL-17 level and increases the percentages of CD4+CD25+Foxp3+ Treg cells in both spleens and draining inguinal lymph nodes,so targeting EP4 receptor may be a new possible therapeutic possibility in the prevention and treatment of rheumatoid arthritis.

Background Glial cell line derived neurotrophic factor (GDNF) is determined to have a neurotrophy effect and promoting effect to the growth of axon.GDNF has been applied in ophthalmology.Research showed that artemin,a new member of GDNF family,has a better function in protection of neuron,but seldom relevant document of distruibution of artemin in retina is found so far.Objective The aim of the present study is to investigate the distribution and expression of artemin in normal rat retinal neuron cells and retinal ganglion cells,and imitate diabetic environment to observe the expression of artemin at the condition of high glucose.Methods Retinal tissue was isolated from clean neonatal SD rats and cultured by expand culture method in DMEM/F12 containing 10% fetal bovine serum.40 mmol/L of glucose was added in medium in the seventh day after culture for 12 hours as experimental group.The expression and location of artemin in retina were tested by real-time PCR and cell immunofluorescence assay.Use of experimental animals followed the Management Regulation of experimental animals of Jiangsu Province.Results Cultured cells showed the typical cell body and processes in the seventh day.Cultured retinal ganglion cells (RGCs) presented the red fluorescence for Thy1.1 antibody,and multiple fluorescence label revealed that RGCs exhibited the green fluorescence for artemin antibody and red fluorescence for Thy1.1 antibody,indicating artemin protein was positively expressed in cultured RGCs.The numbers of positive cells for Thy1.1 antibody was (442±9)/high field in normal culture group and (263±7) /high field in 40mmol/L glucose culture group,showing a significant difference between them (P＜0.05).The expression of artemin mRNA in normal culture group and in 40 mmol/L glucose culture group,was showing a considerably difference between them(P＜0.05).Conclusion Artemin can be expressed in cultured retinal neuron cells and RGCs in rats.High glucose environment down-regulate the expression of artemin.This study proved a new idea for protecting RGCs against damage.

Objective To explore the influence of different training mode on the parenting stress in mothers of children with autism. Methods According to the wishes of parents, 46 children with autism by first diagnosed and their mothers were divided into combination training group (23 cases) and control group (23 cases). The children in combination training group received a family rehabilitation training besides the structured institution-based teaching programme, and the children in control group only received the structured institution-based teaching programme. Comparisons were made between the two groups by the Parenting Stress Index-Short Form score after 1 year teaching. Results Before teaching, the domain score of parental distress, parent-child dysfunctional interaction, difficult child and total score of parenting stress were (37.7 ± 5.6), (35.4 ± 4.1), (38.3 ± 5.4), (111.3 ± 10.5) points in control group, while those were (37.9 ± 5.8), (34.7 ± 4.3), (38.8 ± 4.9), (112.1 ± 9.3) points in combination training group. There was no significant difference between two groups(t=0.155-0.531, all P>0.05). After 1 year teaching, the domain score of parental distress, parent-child dysfunctional interaction, difficult child and total score of parenting stress were (34.0±3.4), (30.7±2.8), (34.3±2.2), (99.0±4.7) points in control group, while those were (34.3±3.7), (28.7±3.1), (31.1±2.4), (94.1±6.8) points in combination training group. The combination training group reported a lower level than control group in the domains score of parent-child dysfunctional interaction, difficult child and total score of parenting stress (t=-2.340,-4.595,-2.890, all P<0.05), while the domain score of parental distress had no significant difference (t= 0.236, P>0.05). Both two groups after 1 year teaching, mothers of children with autism experienced a lower level in all the domains and the total score of parenting stress than before teaching(combination training group: t=3.562-7.925, control group:t=2.534-4.768, all P<0.05). Conclusions Family rehabilitation training combined with institutions could reduce the parenting stress of mothers with autistic children and be worthy of clinical promotion and application.

Objective To evaluate clinical value of MGFA classification and QMG score on pre-dicting late extubation after thymectomy for myasthenia gravis(MG).Methods Total of 61 patients with MG received extended thymectomy from January 2007 to February 2012 were enrolled.Patients were divided into two groups:normal extubation group contained the other 47 patients without pro-longed postoperative mechanical ventilation and delayed extubation group included 14 patients with prolonged postoperative mechanical ventilation.The following factors were evaluated:gender,age, weight,MGFA classification,QMG score,history of steroid hormones or anticholinesterase drugs be-fore operation,the function of liver and kidney before operation,preoperative electrolyte,preoperative hemoglobin content,etc.Receiver operator characteristic curve (ROC)was plotted,and the predictive value, sensitivity and specificity of preoperative MGFA clinical classification and QMG score predicting postoperative ventilation in MG were calculated.Results Fourteen patients(22.95%)de-veloped breathing support after the anaesthetic or endotracheal intubation again in 48 hours.the area under ROC curve(AUC)for preoperative MGFA clinical classification predicting postoperative ventila-tion was 0.723 in MG,it had the sensitivity of 78.5% and specificity of 63.8%.The AUC for QMG score predicting postoperative ventilation was 0.866,the QMG score threshold value of 8.5 had the sensitivity of 78.6% and specificity of 87.2%.Conclusion MGFA classification and QMG score can predict late extubation after thymectomy in patients with myasthenia gravis.

Objective To study the receptors signaling of prostaglandin E2 on the differentiation of regulatory T (Treg) cells and Th17 cells.Methods The expression of prostaglandin E2 receptors (EP1/EP2/EP3/EP4) on the MACS-purified CD4+CD62L+ T (Th0) cells was analyzed by flow cytometry and reverse transcription polymerase chain reaction (RT-PCR).The quantity of CD25+Foxp3+ cells was examined by flow cytometry,the expression of FoxP3 mRNA and RORγt mRNA were detected using real-time RT-PCR,the level of IL-17 in the culture supernatants was detected by enzyme-linked immunosorbent assay (ELISA).ANOVA,LSD-t,Dunnett T3 were used for statistical analysis.Results EP1,EP2,EP3,EP4 were expressed on Th0 cells at different levels,and EP2 [(89.7±9.1)％] had the strongest expression.PGE2 [(3.0± 2.2) ％],EP2 agonist [(4.5± 1.0) ％] and EP4 agonist [(8.8 ±2.5) ％] decreased the quantity of CD25 +Foxp3 + cells compared with the control group [(28.6±6.8)％] (t=7.156,P=0.021; t=6.958,P=0.032; t=5.359,P=0.044).PGE2(0.210±0.020),EP1 agonist (0.833±0.045),EP2 agonist (0.227±0.025) and EP4 agonist (0.450±0.060) decreased the expression of Foxp3 mRNA compared with the control group (1.000) (t=23.817,t=5.026,t=23.313,t=16.581; all P=0.000).PGE2 [(22±6)pg/ml],EP2 agonist [(24±5)pg/ml]and EP4 agonist [(207±19) pg/ml] decreased the secretion of IL-17 compared with the control group [(678±87) pg/ml] (t=14.925,P=0.004; t=14.873,P=0.004; t=10.480,P=0.008).PGE2 (0.141±0.027),EP1 agonist (0.869±0.033),EP2 agonist (0.176±0.029) and EP4 agonist(0.371±0.042) decreased the expression of RORγt mRNA compared with the control group (1.000) (t=34.046,t=5.184,t=32.673,t=24.962,all P=0.000).Conclusion EP1,EP2,EP3,EP4 receptors are expressed on CD4+CD62L+ T (Th0) cells at different levels.Prostaglandin E2 inhibits the differentiation of Treg cells and Th17 cells via the EP2 and EP4 receptors signaling.

Objective To examine the proto_oncoge ne c_f os expression and its distribution in rat medulla oblongata induced by electro_a cupuncture of Neiguan point. Methods Anti_c_fos immunohistochemical method w as applied. Results Fos_like immunoreactive positive neurons mainly located in nucleus tractus solitarius, dorsal motor nucleus of vagus nerve, inferior ol ive nucleus, cuneate nucleus, external cuneate nucleus, and ventrolateral m edulla(VLM). VLM included lateral reticular nucleus, ambiguous nucleus and the s urrou nding reticular structure. Conclusion Electro_acupuncture of Neiguan point c an activate the proprioceptive pathways and the nucleus in the medulla oblongata that is related to visceral informatio n.