The specific ScFv gene against botulinum neurotoxin A (BoNTa)was cloned into pPIC9k. Positive integrators were screened by increasing the dose of G418 in culture and expressed in Pichia pastoris GS115. As a result, engineered recombinant clone were obtained. 26 kD product of interest was seen easily in SDS-PAGE. Expression of human ScFv got the highest level 15% of total secreted proteins during 72～84 h after 1% methanol inducing. Purification of ScFv was finished by two steps: gel filter and ion exchange. Competing ELISA showed that recombinant ScFv could compete with antiserum to specific bind BoNTa.

Cardiomyopathies ; diagnosis ; pathology ; Echocardiography ; Heart Ventricles ; pathology ; Humans ; Magnetic Resonance Imaging ; Young Adult

In order to construct a prokaryotic expression vector of human receptor syt II N-fragment and to express recombinant MBP-Syt fusion protein in E.coli and to purify and identify its activity. According to codon preference of E.coli, a DNA fragment encoding human syt II N-fragment was synthesized, and then cloned into prokaryotic vector pMAL-c2x for sequencing. Then the recombinant plasmid pMAL-Syt was introduced into E.coli ER2566 by transformation for expression and the obtained engineered bacteria were induced by IPTG. The fusion protein was purified by amylose resin affinity chromatography and identified by SDS-PAGE and Western blot. The binding activity of the protein was determined by ELISA. It is concluded that MBP-Syt protein is of good binding activity.

Idiopathic thrombocytopenia purpura (ITP) is an autoimmune disease which is characterized by destruction of platelets by macrophages in the reticuloendothelial system. Recent studies suggest that ITP is related to the abnormal activation and apoptosis of T/B cells which lead to failure of immune tolerance. Now it is becoming clear that costimulatory signals are required for full T/B cell activation and assumed to modulate T/B cells responses as well as other aspects of the immune system. This review focuses on the role and state-of-the-art advancements of costimulatory signals in ITP.
Antigens, CD ; immunology ; Antigens, Differentiation ; immunology ; Antigens, Differentiation, T-Lymphocyte ; immunology ; B-Lymphocytes ; immunology ; CD28 Antigens ; immunology ; CTLA-4 Antigen ; Humans ; Immune Tolerance ; Inducible T-Cell Co-Stimulator Protein ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; Receptors, Tumor Necrosis Factor ; immunology ; Signal Transduction ; physiology ; T-Lymphocytes ; immunology ; physiology

ObjectiveTo observe protective effects of agmatine (AGM) on inflammatory response and spleen immune function in mice with trauma.Methods Forty-eight adult male C57BL/6 mice were randomly divided into three groups (n= 16 each), including control group, model group (bilateral femoral fracture and removal of 35% of the total blood volume), and AGM group (trauma/hemorrhage & AGM 200 mg/kg). Eight mice in each group were sacrificed at 3 hours and 24 hours, respectively, after modeling, and blood samples and tissue homogenate of spleen and liver were collected. The contents of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β) in serum and liver tissue were determined with enzyme linked immunosorbent assay (ELISA). Serum aspartate transaminase (AST), alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) were determined with automatic biochemistry analyzer. Spleen proliferation response stimulated with concanavalin A (ConA) was evaluated with methyl thiazolyl tetrazolium colourimetry (MTT).γ-interferon (IFN-γ) and IL-2 releases were determined with ELISA.Results Compared with control group, 3 hours after trauma/hemorrhage, the levels of serum TNF-α, IL-6, and IL-1β in model group were significantly elevated [TNF-α (ng/L): 145.38±31.50 vs. 23.06±11.14, IL-6 (ng/L): 496.94±50.76 vs. 47.13±17.47, IL-1β (ng/L): 321.31±43.02 vs. 29.25±16.24,allP< 0.01]. It was found that AGM treatment could alleviate the increase in serum pro-inflammatory mediators induced by trauma/hemorrhage, such as TNF-α (ng/L:111.56±25.47 vs. 145.38±31.50), IL-6 (ng/L: 412.56±44.33 vs. 496.94±50.76), IL-1β (ng/L: 273.38±45.25 vs. 321.31±43.02,P< 0.05 orP< 0.01). Twenty-four hours after trauma/hemorrhage, serum pro-inflammatory mediators were recovered to the levels in control group. There was no significant difference in TNF-α and IL-6 levels at 3 hours after trauma/hemorrhage among groups. Compared with control group, the expressions of liver TNF-α and IL-6 in model group were increased at 24 hours following trauma [TNF-α (ng/mg): 32.93±4.90 vs. 26.58±2.33, IL-6 (ng/mg): 11.20±1.66 vs. 8.38±0.89,bothP< 0.01]. However, AGM inhibited the level of TNF-α (ng/mg:28.92±3.16 vs. 32.93±4.90) and IL-6 (ng/mg: 9.03±1.28 vs. 11.20±1.66) in the liver as induced by trauma/hemorrhage (P< 0.05 andP< 0.01). At 24 hours after modeling, model group and AGM group had distinctly higher serum AST, ALT, LDH levels than those of control group [AST (U/L): 405.9±31.2, 245.7±22.1 vs. 128.2±15.9; ALT (U/L): 92.1±6.3, 51.6±5.0 vs. 30.1±3.2; LDH (U/L): 606.7±36.3, 478.7±25.3 vs. 384.0±16.6, allP< 0.01]. Nevertheless,the increase in serum AST, ALT and LDH was alleviated in AGM group (allP< 0.01). Meantime, trauma/hemorrhage produced a noticeable depression of proliferation of splenic cells and IFN-γ and IL-2 release stimulated with ConA compared with control group [proliferation rate: (40.97±4.13)% vs. (89.99±7.76)%, IFN-γ(ng/L): 91.6±12.3 vs. 353.2±21.5,IL-2 (ng/L): 53.4±6.4 vs. 91.0±12.2,allP< 0.01]. In contrast, AGM notably restored the capacity of proliferation response of splenic cells [proliferation rate: (74.86±5.75)% vs. (40.97±4.13)%, P< 0.01],enhanced the release of IFN-γ and IL-2 stimulated with ConA [IFN-γ (ng/L): 327.8±23.6 vs. 91.6±12.3, IL-2 (ng/L): 74.8±10.4 vs. 53.4±6.4, bothP< 0.01].Conclusion AGM can dramatically alleviate spleen immunosuppression, excessive inflammation and organ damage induced by trauma/hemorrhage.

Objective To investigate factors with pelvic adhesions and the effect of different degrees pelvic adhesions on fallopian tube recanalization in infertile patients.Methods Total of 527 infertile patients undergoing hysteroscopy and laparoscopic surgery in Affiliated Hospital of Chinese People's Armed Police Forccs Logistics College were studied retrospectively.According to the extent of pelvic adhesions,tubal umbrella adhesions and atresia,377 cases were classified into adhesion groups,including 73 cases in grade Ⅰ,221 cases in grade Ⅱ,75 cases in grade Ⅲ and 8 cases in grade Ⅳ based on adhesion score.The 150 cases with no obvious pelvic adhesion were matched as control group.Among 8 cases with grade Ⅳ ahesion were exluded from ahesion group the relationship between pelvic adhesions and related history,abdominal lesions,tubal patency and the prognosis were studied.Results(1)Related factors:the frequency of pelvic adhesion and more than 7 years of infertility of 23.9％(88/369)in adhesion group were significantly higher than 12.0％(18/150)in control groups.(2)History:compared with the control group(12.7 ％,19/150;28.7％,43/150;11.3％,17/150;12.0％,18/150;17.3％,26/150),patients with pelvic adhesions present more incidence abortion(23.6％,87/369),uterine cavity operation(38.2％,141/369),ectopic pregnancy(20.9％,77/369),pelvic inflammatory disease(25.5％,94/369)and abdominopelvic surgery (31.4％,116/369).(3)Endoscopy exploration:the incidence of hydrosalpinx(24.7％,91/369),tube distorted(15.7％,58/369)and salpingostomy(72.9％,269/369)in adhesion group were higher than those in control group(2.0％,3/150;4.0％,6/150;12.0％,18/150),but relatively lower incidence of pelvic endometriosis lesions(5.7％,21/369)and mesosalpinx cysts(16.3％,60/369)than those in control group(16.0％,24/150;30.0％,45/150).The rate of proximal tubal recanalization(59.5％,91/153)in adhesion group was lower than 75.4％(52/69)in control group.However,the rate of distant tubal recanalization of 84.4％,(281/333)in adhesion group and;13/15 in control group didn't show statistical difference.(4)Prognosis:the rate of ectopic pregnancy of 9.7％(29/299)in adhesion group was significantly higher than 3.1％(4/128)in control group.Among cases with grade Ⅲ adhesion exhibited the highest rate of ectopic pregnancy(13.0％,7/54;OR =4.62,95％ CI:1.29-16.50).(5)Multivariate analysis:it was found that more than two drug abortions(OR =3.29,95％ CI:1.34-8.07),pelvic and (or)abdominal surgery history(OR =2.20,95％ CI:1.35-3.57)and pelvic inflammatory disease history (OR =1.54,95％ CI:1.21-1.97)were risk factors with pelvic adhesions.Conclusion More than or equal to two drug abortion history,pelvic inflammatory disease and pelvic and abdominal surgery damage were important factors for pelvic adhesions of infertility patients,which may decrease the possibility of proximal tubal recanalization and increase ectopic pregnancy risk.

This study tested the effects of the gastrointestinal pulse train electrical stimulation with different parameters and at different locations on the neuronal activities of the lateral hypothalamus area (LHA) in obese rats in order to find the optimal stimulation parameter and location. Eight gastric electrical stimulations (GES) with different parameters were performed and the neuronal activities of gastric-distension responsive (GD-R) neurons in LHA were observed. The effects of stimulations with 8 parameters were compared to find the optimal parameter. Then the optimal parameter was used to perform electrical stimulation at duodenum and ileum, and the effects of the duodenal and ileac stimulation on the GD-R neurons in LHA were compared with the gastric stimulation of optimal parameter. The results showed that GES with the lowest energy parameter (0.3 ms, 3 mA, 20 Hz, 2 s on, 3 s off) activated the least neurons. The effects of GES with other parameters whose pulse width was 0.3 ms were not significantly different from those of the lowest energy parameter. Most gastric stimulations whose pulse width was 3 ms activated more LHA neurons than the smallest energy parameter stimulation, and the effects of those 3 ms gastric stimulations were similar. Accordingly, the lowest energy parameter was recognized as the optimal parameter. The effects of stimulations with the optimal parameter at stomach, duodenum and ileum on the LHA neuronal activities were not different. Collectively, gastrointestinal electrical stimulation (GIES) with relatively large pulse width might have stronger effects to the neuronal activities of GD-R neurons in LHA of obese rats. The effects of the GIES at different locations (stomach, duodenum and ileum) on those neurons are similar, and GES is preferential because of its easy clinical performance and safety.

The effect of phenol concentration on the structure and function of microbial communities,which were cultured in different conditions using coking wastewater biofilm as seeding,was investigated by Biolog and denaturing gradient gel electrophoresis(DGGE)methods.The less number of bands of cultivated sam-ples on the denaturing gradient gel electrophoresis fingerprint of 16S rRNA gene indicated reduction of di-versity after enrichment and cultivation.Some bands on the DGGE gel were significantly influenced by the phenol concentration in medium.The results of Biolog showed that the original biofilm sample had the highest substrate utility capacity as measured by average well color development(AWCD).But low concen-tration of phenol enriched sample S7 showed more diverse activity on the utility of Carboxylic acids.The principal component analysis(PCA)of Biolog data revealed that the metabolic patterns were similar when using the same phenol concentration,although the sample S7 much less similar to other cultivated samples.These results suggested that the enrichment and cultivation with phenol supplemented decreased the diver-sity and also changed the metabolic function of the microbial community.Lower phenol concentration in-creased the microbial community metabolic activity.The phenol degrading capacity of isolates from each samples indicated that the enrichment and cultivation condition had changed the type and property of cul-truable bacteria.Based on these results,we concluded that the different microorganisms will be isolated un-der different cultivation condition.

Objective To explore the role of synaptic plasticity on the formation of visceral hyper- sensitivity induced by acute restraint stress in rats.Methods Twenty male Sprague-Dawley rats were randomly divided into control group and acute restraint stress group(model group).Visceral hypersensi- tivity was made by acute restraint stress for 1 h.The colorectal distension(CRD) with different pressure were performed and the abdominal electromyography(EMG) was recorded.The visceral sensitivity was determined by the frequency of EMG.The ultrastrueture of synapse was observed with transmission electron microscope.The expression of synaptophysin was measured by RT-PCR and Western-blot. Results①The frequency of EMG was significantly correlated with CRD pressure(control group,r=0.992, P=0.008;model group,r=0.978,P=0.022).The frequencies of EMG in model group(at 40,60 and 80 mm Hg) were significantly more than that in control group(P value=0.043,0.024,0.038,respectively).②There were more synaptic vesicles accumulated in presynaptical terminal.The post synaptic density was increased in model group compared to control group.③In the proximal and distant colon,the expressions of rnRNA and protein of synaptophysin were higher in model group (P