To clone, express and primarily use human autoantigen Sm D1 in methylotrophic yeast Pichia Pastoris. The gene Sm D1 was cloned by PCR.The PCR product was inserted into the vector pPIC9k. The recombinant plasmid pPIC9k- Sm D1 was transformed into yeast SMD1168 by electroporation. The positive clones were screened in MD plates. The high copy number transformants were rapidly selected by using G418 and were induced by methanol. Supernatants after induction were analyzed by SDS-PAGE and im-munodot. The PCR product was showed about 360 bp in size which was in accordance with predicted. The pPIC9k-Sm D1 showed the same seqencing result with GenBank’s report and restriction enzyme analysis confirmed our prediction. The pPIC9k-Sm D1 positive clone produced an about 16 kD protein which had natural immunogenicity of human autoantigen Sm D1 by SDS-PAGE and immunodot. The sensitivity and specificity of immunodot were 96% and 100%, respectively. The agreement between immunodot and im-munoblot was 98%. Successfully cloning and high-level expression of human autoantigen Sm D1 in methy-lotrophic yeast Pichia pastoris laid a foundation for further research work.

BACKGROUND: Nuclear factor kappa B (NF-κB) is possibly related to regulation of various cell signals that are derived from aqueous uveoscleral outflow pathway.OBJECTIVE: To explore effect of travoprost on the expression of NF-κB and inhibitor-κB (I-κB) in human ciliary muscle cells cultured in vitro. DESIGN, TIME AND SETTING: A contrast study, which was performed in the Laboratory of Zhongshan Ophthalmology Center from March 2005 to November 2006.MATERIALS: Eyeballs were obtained from the youth who died due to other diseases except eye disease no more than one hour. The relatives voluntarily provided the informed consent.METHODS: Travoprost (1 μmol/L) was added in human ciliary muscle cell culture medium, and then the samples were divided into four groups according to culture time, including 0-hour (control group), 6-hour, 12-hour, and 24-hour experimental groups. MAIN OUTCOME MEASURES: Expression of mRNA and protein of NF-κB p65 and I-κBα in the four groups by using real-time RT-PCR, immunofluorescence relative quantitative analysis and enzyme linked immunosorbent assay (ELISA) techniques. RESULTS: As compared to control group, mRNA expression of NF-κB p65 in the 6-hour, 12-hour, and 24-hour experimental groups was decreased (F=17.068, P=0.001); while mRNA expression of I-κBα was not changed remarkably in the 6-hour and 12-hour experimental groups (P > 0.05), but the expression was significantly higher than that in the 24-hour experimental group (F=32.742, P=0.000). Immunofluorescence relative quantitative analysis showed that the fluorescence intensity of NF-κB p65 in the 6-hour, 12-hour, and 24-hour experimental groups were weaker than that in the 0-hour control group (F=17.216, P=0.000); additionally, as compared to 0-hour control group, fluorescence intensity of I-κBα in the 6-hour experimental group was not changed remarkably (P=0.134), that in the 12-hour experimental group was weakened (P=0.032), and that in the 24-hour experimental group was strengthened (F=17.346, P=0.001). ELISA revealed that expression of phosphorylated NF-κB p65 was decreased gradually by the time of being induced by travoprost (F=15.4, P=0.001). CONCLUSION: Travoprost can down-regulate mRNA expression of NF-κB p65, inhibit nuclear translocation, and up-regulate mRNA expression of I-κBα in human ciliary muscle cells.

Objective To obtain recombinant human Smith D1 (Sm D1) antigen and establish detecting assay.Methods Human Smith D1 antigen was synthesized by PCR using human Leukemic cDNA. The prokaryotic expression vector pGEX-ST-Sm D1 was constructed and transformed into E.coli.BL21 cell.Protein expressed under the induction of IPTG.We established DIGFA for detecting anti-Sm D1 antibodies with purified Sm D1 antigens.Results Sequence and restriction analysis revealed Sm D1 gene was cloned in frame into pGEX-5T,SDS-PAGE profile showed a clear protein band with a relative molecular weight of 39 000 and western blotting indicated that the expressed product specifically reacted to polyclonal anti-human Sm D1 genes.There was no significant difference between DIGFA and IB.The agreement between DIGFA and IB was 91.7% as calculated by Kappa statistical method.The sensitivity and specificity of DIGFA were 100% and 83.3% repectively.Conclusions Human Sm D1 gene is successfully cloned、 expressed and purification.The DIGFA,using purified Sm D1 antigens,is as good as IB,rather simpler, more rapid and reliable assay.

Nicotinic acetylcholine receptors (nAChRs) belong to ligand-gated ion channel receptors, of which α7 nAChR subtype is widely distributed in the cerebral cortex, thalamus, hippocampus, and also identified in microglia, macrophages, bone marrow cells, etc. Previous studies revealed that α7 nAChR is closely related to the function of the cholinergic anti-inflammatory pathway, and is a vital target for drug development of Alzheimer's disease and schizophrenia. The establishment of a stable α7 nAChR in vitro drug screening system is crucial for the efficient screening of novel drugs targeting this target. Recombinant expression of different subtypes of nAChRs on Xenopus laevis oocyte membranes and current detected by two-electrode voltage clamp (TEVC) is an advanced and complex model for novel drug screening. Molecular chaperones can assist the assembly of some nAChR subunits to form functional receptors, providing a stable expression model for the screening of compounds targeting this receptor. In this study, a molecular chaperone gene of α7 nAChR, transmembrane protein 35A (Tmem35a), was isolated and cloned from rats. We constructed the recombinant expression vector and obtained the cRNA of Tmem35a by in vitro transcription technique. Two cRNAs (Tmem35a and α7) were mixed and injected into X. laevis oocytes for expression. Then, the effects of this molecular chaperone on the current expression and pharmacological properties of α7 nAChR were evaluated by the TEVC. The results revealed that TMEM35A, also known as novel acetylcholine receptor chaperone (NACHO) could effectively increase the expression of α7 nAChR protein on oocyte membranes, and the amount of α7 nAChR protein was increased about 1-fold. The peak current induced by agonist acetylcholine (ACh) was increased about 10-fold. After injection of Tmem35a cRNA, the median effect concentration (EC₅₀) value of α7 nAChR to agonist ACh is 228.5 μmol·L^-1, which shows almost no difference from native α7 nAChR (EC₅₀: 223.3 μmol·L^-1), indicating the preservation of the normal properties of α7 nAChR. The results of this investigation indicate that the molecular chaperone NACHO effectively assists the heterologous expression of α7 nAChR in X. laevis oocytes, which provides a model for screening the potency of lead compounds targeting α7 nAChR. All animal experiments in this study were reviewed and approved by the Ethics Committee of Guangxi University (approval number: GXU-2023-0249).

N-type voltage-gated calcium (Ca²⁺) channels (N-type VGCC, Ca_V2.2) mediate Ca²⁺ influx in response to action potential at the presynaptic terminal, and play an important role in synaptogenesis, neurotransmitter release and nociceptive signal transduction. It is a new target for the development of drugs for the treatment of neuralgia (chronic pain) and other major diseases. Due to the difficulty of calcium channel expression in vitro and the detection of channel current, there is a great lack of new drug screening models. In this study, we established and optimized the electrophysiological drug screening model using Xenopus laevis oocytes for the recombinant expression of Ca_V2.2 in vitro (this study were reviewed and approved by the Ethics Committee of Guangxi University, approval number: GXU-2023-0249). Firstly, the linear plasmids encoding cDNA of major subunit α1B and auxiliary subunits α2δ1 and β3 of rat Ca_V2.2 were used as templates for in vitro transcription to generate their related mRNA (cRNA), after which three kinds of cRNA were injected into Xenopus laevis oocytes at the mass ratio of 2∶1∶1 for expression. The two-electrode voltage clamp (TEVC) technique was used to detect the inward current produced by Ca_V2.2. At the same time, the expression conditions of Ca_V2.2 were optimized, and its gating function was characterized from the aspects of channel activation and inactivation. The results showed that 3-5 days after cRNA microinjection, stable Ca_V2.2-mediated barium ion (Ba²⁺) currents were successfully detected. The interference of endogenous potassium channels and Ca²⁺-activated chloride channels can be eliminated by tetraethylammonium hydroxide (TEAOH) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA-AM) treatment. The maximum potential for Ca_V2.2 activation is 0 mV, and the current reverses to be outward when the membrane potential is greater than +50 mV. By fitting the steady-state activation and inactivation curves, the half-maximal activation potential and half-maximal inactivation potential of Ca_V2.2 are identified as -15.9 and -60.2 mV. In this study, a stable Ca_V2.2 expression system was established based on Xenopus laevis oocytes. The in vitro expression system can provide a new way for the screening of Ca_V2.2 active compounds or lead drugs.

Objective To comment the severity of severe hand,foot and mouth disease(HFMD)by pediatric risk of mortality score(PRISM),and assess the performance of PRISM in predicting mortality or complication probability in HFMD.Methods Four hundred and twenty-four severe HFMD pediatric patients were recruited in the study from 1th Jan 2010 to 31th June 2013.Information on the outcome and the varia-bles required to calculate PRISM score were collected.The logistic regression model developed in the learning sample was evaluated in the test sample by calculating the area under the receiver operating characteristic (ROC)curve to assess discrimination pneumorrhagia and death.Calibration across deciles of risk was evalua-ted using the Hosmer-Lemeshow goodness-of-fit χ2 test.Results The area under the ROC curve were 0.87 (95%CI 0.80~0.94 )for PRISM in predicting pneumorrhagia probability.The area under the ROC curve were 0.87(95%CI 0.80~0.95)for PRISM in predicting mortality probability.The PRISM in observed and expected pneumorrhagia did not demonstrate good calibration at ten mortality risk intervals (χ2 =36.66, P<0.001 ).The PRISM in observed and expected mortality did not demonstrate good calibration at ten mortali-ty risk intervals(χ2 =41.11,P<0.001).Conclusion The PRISM score is demonstrated good discrimination of pneumorrhagia and death in HFMD pediatric patients,but the performance of calibration is not good.

Objective To investigate the prevalence,genotype and epidemiology of plasmid- mediated AmpC enzyme of Escherichia coli and Klebsiella pneumoniae.Methods A total of 67 clinical isolates of nonrepetitive cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae collected by Fuzhou General Hospital and Fujian Provincial Hospital during a period of Sept.2004 to Mar.2005 were detected by three-dimensional extract test for AmpC enzyme,and PCR for AmpC enzyme and other ?-lactamase gene amplification and DNA sequencing were carried out for genotype of ?-lactamase.Plasmid transformation experiment was used to study the transfer of cefoxitin resistance.The homology of the isolates was determined by ERIC-PCR fingerprinting.Results At two hospitals in Fuzhou,the prevalence of plasmid-mediated AmpC enzyme among cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae were 16.7% and 10.5%, 8.0% and 0,respectively.Two isolates of Klebsiella pneumoniae produced DHA-1 plasmid-mediated AmpC enzyme,and 4 isolates of Escherichia cob and one strain of Escherichia coli produced CMY-2 and CMY-22 plasmid-mediated AmpC enzyme respectively.Furthermore,5 strains of Escherichia coli with CMY AmpC enzyme were also found simuhaneously to produce TEM-144,CTX-M-27,CTX-M-14 and TEM-1 ?-lactamase respectively.Three strains of Escherichia coli and one isolate of Klebsiella pneumoniae could transfer cefoxitin resistance to acceptant bacillus.ERIC-PCR fingerprinting reveals 2 strains of Klebsiella pneumoniae came from same clone,but 5 strains of Escherichia coli came from different clones.Conclusions The clinical isolates of Klebsiella pneumoniae producing DHA-1 plasmid-mediated AmpC enzyme and Escherichia coli producing CMY-2,CMY-22 plasmid-mediated AmpC enzyme are found in Fuzhou.CMY-22 AmpC enzyme and TEM-144 ?-lactamase are the first reported in the world,GenBank accession number: DO256079,DO256080

Objective:To investigate the effect of astragaloside IV (AS-IV) on the secretion of stromal cell-derived factor-1α (SDF-1α) and CXC chemokine receptor 4 (CXCR4) by high glucose injured human umbilical vein endothelial cells (HUVECs), so as to lay a foundation for further study on AS-IV improving angiogenesis by regulating SDF-1 α/CXCR4 axis of endothelial cells.Methods:HUVECs were isolated and cultured from the umbilical vein of full-term healthy newborns and identified by von Willebrand factor (vWF) combined with 4-diamino-2-phenylindole (DAPI) nuclear staining. The obtained HUVECs was cultured in EGM-2 medium with 30 mmol/L glucose for 120 h to obtain high glucose damaged HUVECs. After intervention with different concentration gradients (25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L) AS-IV for 72 hours, the contents of SDF-1α and CXCR4 were detected by enzyme linked immunosorbent assay (ELISA) method to determine the best concentration of AS-IV. The supernatant of damaged HUVECs were collected at 6, 12, 24, 48 and 72 hours after intervention with the best concentration of AS-IV, and the contents of SDF-1α and CXCR4 were detected by ELISA method to determine the best action time of AS-IV. The damaged HUVECs was randomly divided into experimental group and control group, and the blank group was set up at the same time. The experimental group was treated with the best concentration of AS-IV and the best time, the control group and the blank group were treated with the same volume of phosphate buffered saline (PBS) solution, and the contents of SDF-1α and CXCR4 in each group were detected by ELISA method.Results:The vWF factor on the cell membrane was green fluorescence, and the nucleus was blue after DAPI staining. When the fusion image showed green fluorescence, HUVECs were identified by blue fluorescence. The expression of SDF-1α in damaged HUVECs was the best when treated with AS-IV of 100 mg/L for 24 hours (1 642.87 pg/ml), and the expression of CXCR4 in damaged HUVECs was the best when treated with AS-IV of 50 mg/L for 48 hours (8.44 ng/ml). Compared with the control group, the contents of SDF-1α and CXCR4 in the experimental group were significantly increased, and the difference was statistically significant ( P<0.05). While the contents of SDF-1α and CXCR4 in the experiment group were slightly less than those in the blank group and there was no statistically significant difference ( P>0.05). Conclusions:AS-IV can promote the expression of SDF-1α and CXCR4 in HUVECs damaged by high glucose to return to normal physiological level, so as to play the role of vascular repair and neovascularization.

BACKGROUND:Travoprost can increase human ciliary muscle cell interspace, decrease uveoscleral outflow resistance and then decrease intraocular pressure. But whether this action pathway is conducted by enhancing the expression of matrix metalloproteinase (MMP) in the ciliary muscle cells remains unclear.OBJECTIVE:To observe the effect of travoprost on the expression of MMP-2 in the human ciliary muscle cells.DESIGN:Controlled observation analysis.SETTING:Zhongshan Ophthalmic Center,Sun Yat-sen University.MATERIALS:This study was Carried out in the Zhongshan Ophthalmic Center,Sun Yat-sen University between August 2005 and April 2006.Donor was from the unilateral eyeball of a youth patient,who was dead within one hour,had no any disease (informed consent was obtained from the relatives) in the Zhongshan Ophthalmic Center, Sun Yat-sen University.Rabbit anti.human MMP-2 polyclonal antibody (Boster Bioengineering Co.,Ltd,Wuhan),and travoprost (86610F,0.004% solution,ALCON company.USA) were used in this study.METHODS: Experimental intervention: 1μmol/L travoprost was added into bovine serum-free medium of human ciliary muscle cell, serving as experimental group,and meanwhile,the cells which were not interfered by drugs were taken as control group.In the experimental group,cells were harvested 6, 12,and 24 hours after travoprost being added.Experimental evaluation:MMP-2 gene and protein expressions in the human ciliary muscle cells in each group were detected by RT-PCR and ELISA methods.The activity of MMP-2 in each group was detected by Zymography technique.MAIN OUTCOME MEASURES:MMP-2 mRNA expression in the human ciliary muscle cell,MMP-2 protein expression and MMP-2 activity in the extracellular fluid.RESULTS:①In the experimental group, at 6,12 and 24 hours after travoprost being added,the relative expression of MMP-2 mRNA was gradually increased (F=236.959,P＜0.01).②In the experimental group,at 6,12 and 24 hours after travoprost being added,MMP-2 protein was also gradually increased with time (F=38.110,P＜0.01).③Zymography technique detection showed that in the experimental group,at 6,12 and 24 hours after travoprost adding,MMP-2 activity was gradually enhanced with time (F=74.348,P＜0.01).CONCLUSION:After human ciliary muscle cell cultured in vitro being subjected to the intervention of travoprost.MMP-2 expression is gradually increased with action time of travoprost, and meanwhile MMP-2 activity is also gradually enhanced.

Background and purpose: Astrocytoma is the most common neuroepithelial neoplasm, and its grading has profound effect on its treatment and prognosis. To investigate the application of stepwise discriminant analysis in grading astrocytomas, this study developed two models of stepwise discriminant analysis according to relevant factors of astrocytoma. Methods: From January 2008 to April 2009, 111 primary astrocytoma patients were enrolled. Each patient was scored based on location, signal intensity on T1WI, signal intensity on T2WI, enhancement, edema, border, cyst or solidness, and mass effect of their magnetic resonance images. With their age score of grading, Fisher stepwise discriminant analysis and the Logistic discdminant were used. The results from the two models were then evaluated and compared. Results: According to Fisher stepwise diseriminant analysis, the predictive accuracy was 87.7% with 80.0% sensitivity, 91.5% specificity and 0.942 area of ROC curve. However, the predictive accuracy of Logistic discriminant analysis was 84.9% with 80.0% sensitivity, 86.8% specificity and 0.940 area of ROC curve. There were no statistically significant differences in terms of accuracy (P=0.250) and areas under ROC curve (Z=0.433, P=0.665) between the two models. Conclusion: Two stepwise discriminant analysis models are meaningful to predict the grading of astrocytoms, and the application of Fisher stepwise discriminant analysis is simpler than the Logistic discriminant analysis.