Objective To explore variance and resource of intracellular free Ca2+ and extracellular arachidonic acid (AA) in different types of passage cells during the invasion of T. gondii. Methods The variance and resource of extracellular AA and intraceUular free Caz+ of Vero and J774A. 1 cells during the invasion of T. gondii were detected by multi-purpose scintillation counter and laser scanning confocal microscope. Data were analyzed using Chi-square test and t test. Results The intracellular free Ca2+ levels in J774A. 1 and Vero cells were both increased after T. gondii infection. The maximal changes of fluorescence intensity were (1 219.7±58.4)% (P＜0.01) and (356.3±23.6)% (P＜0.05), respectively.The increase of intracellular Ca2+ level in Vero cell was mostly from the release of intracellular Ca2+ store.And the Ca2+ increase in J774A. 1 cell was from both the release of intracellular Ca2+ store and extracellular Ca2+ influx. Extracellular AA levels were significantly increased in both Vero and J774A. 1 cells after T.gondii infection 5.02 and 8. 44 times respectively (t= 3. 124, t = 3. 852, P＜0.01). The AA elevations could be significantly inhibited by phospholipase A2 (PLA2) inhibitor pretreating T. gondii. Conclusions The phospholipase C of phagocytic host cell and PLA2 of T. gondii are activated by T. gondii infection,which results in the increase of intracellular free Ca2+ and extraeellular AA level. Combined actions of Ca2+and AA play a major role in the invasion of T. gondii to host cell. While only PLA2 of T. gondii may be activated in nonphagoeytic host cell.

The aim of the study is to investigate the effects of concentration, intestinal segments, pH, inhibitors of proteins (P-gp), Na(+)-dependent glucose transporter (SGLT1) on the intestinal absorption of berberine, and to compare intestinal absorption of berberine in combinations. With phenol red as the indicator, in situ single pass intestinal perfusion (SPIP) model was used and intestinal absorption of pure berberine at concentrations of 36.70, 46.17 and 92.33 microg x mL(-1), simulated system of HLJDT (mixture of berberine, baicalin and geniposide), HLJDT with the concentration of berberine 92.33 microg x mL(-1) in perfusion solution of different intestinal segments (duodenum, jejunum, ileum, and colon) were determined by HPLC in combination with diode array detection (DAD). The results indicated that Ka values ofberberine at different concentrations had little significant difference among that obtained after perfusing via duodenum, jejunum, ileum and colon indicating that the absorption of berberine was mainly the passive diffusion. It was also suggested that SGLT1 and P-gp might exert some effects on the absorption of berberine. Ka and Peff values of berberine in a mixture of pure compounds and HLJDT for different intestine segments of rat showed an increasing tendency and was significantly different (P < 0.05) indicating that berberine in a mixture of pure compounds and HLJDT was assimilated better in small intestine. These results indicate that the intestinal absorption of berberine may be affected by compatibility of compounds. Additionally, berberine has wide absorption window and better absorption in colon.

Objective To investigate the correlation between PaCO2 and cerebral blood flow (CBF), cerebral metabolism of oxygen (CMRO2), glucose (CMRglu) and lactate (CMRlact) and intracranial pressure during intracranial surgery. Methods Twenty ASA Ⅰ - Ⅱ patients (6 male, 6 female), aged between 26-54yr, weighing (65 ? 11) kg scheduled for elective intracranial surgery were studied. The patients were unpremedicated. Before general anesthesia radial artery was cannulated and a catheter was inserted into internal jugular vein and advanced cranially until jugular bulb. Lumber puncture was performed at L3-4 and a catheter was inserted into subarachnoid space for 3 cm, for pressure monitoring and CSF sampling. Anesthesia was induced with diazepam 10mg, fentanyl 3-4?g?kg-1, propofol 2mg?kg-1 and vecuronium 0.08mg?kg-1 iv. The patients were mechanically ventilated with a mixture of oxygen and argon (O2 : argon = 3 : 1) after tracheal intubation. Anesthesia was maintained with sevoflurane and intermittent iv boluses of vecuronium. Arterial and cerebral venous blood gases, glucose and lactate levels, CBF, ICP and CSF level of lactate were determined before anesthesia when patients were awake(Ⅰ) and during anesthesia when PETCO2 = 40, 30, 20 mm Hg (Ⅱ,Ⅲ,Ⅳ). CBF was measured by modified Kety-Schmidt inert gas saturation technique with argon. CMRO2 and CMRglu were calculated based on the difference in their arterial -cerebral venous blood levels. Results At PETCO2 20mm Hg (Ⅳ) CBF decreased by 57.75% and CMRO2 by 58.70% as compared with the baseline; CMRglu decreased by 46.93% as compared with the baseline. There was no significant change in lactate level, jugular venous blood O2 saturation and pH. ICP decreased from (22.14 ? 7.88)mm Hg( Ⅰ) to (17.57?5.03)mm Hg( Ⅱ ),(13.43?4.89)mm Hg(Ⅲ) and (10.00? 2.31)mm Hg(Ⅳ) and the differences were significant. All measurements were done when MAP and HR were stable. PET CO2 was (10? 2) mm Hg lower than PaCO2 . Conclusions Cerebral blood flow, cerebral oxygen and glucose metabolism and intracranial pressure change with changes in PET CO2 . Cerebral vascular reactivity to CO2 is not impaired by 1.3 MAC sevoflurane. Mild hypocapnia is necessary during neurosurgery.

Objective To explore the change of cytoskeleton and the variance of Ca2+ in cultured cells during the invasion of Toxoplasma gondii. Methods Tachyzoites suspensions were gathered by routine method and used to infect phagocytic cells(J774A.1)and non-phagocytic cells (HUVEC). The ability of T. gondii invading into the cells and the influence of cytoskeleton inhibitor,colchicine and cytochalasin D,were observed by microscopy. The rearrangement of cytoskeleton of cells was observed by fluoromicroscopy. By using laser scanning confocal microscope,the variance of Ca2+ in J774A.1 and HUVEC was detected. Results Ca2+ increased greatly in J774A.1 during the invasion of T.gondii(P0.05). The microfilaments of J774A.1 were agglomerated during the invasion of T. gondii. Cytoskeleton inhibitor,cytochalasin D(P0.05)during the invasion and cytoskeleton was not changed. Cytochalasin D and colchicine showed little effect on the infection rate of HUVEC. Conclusion The concentration of Ca2+ increases greatly and cytoskeleton(mainly the microfilament)has been rearranged in phagocytic cell during the invasion of T. gondii,while both of them show no significant change in non-phagocytic cell.

PurposeTo investigate the accuracy of18F-FDG PET/CT in diagnosing mediastinal and hilar lymph node metastasis of lung cancer and in guiding surgery. Materials and Methods Seventy-eight pathology-proven lung cancer patients underwent 18F-FDG PET/CT scanning and surgery. Histology was used as gold standard to evaluate the diagnosis of mediastinal and hilar lymph node metastasis.18F-FDG PET/CT was also compared to chest CT.Results The pathological examination confirmed metastasis lymph nodes in 105 out of 231 excised lymph nodes in 18 patients. No lymph node metastasis was found in mediastinum in the other 60 patients with lymph node staging of pN0. Lymph node staging was pN1 in 5 patients, pN2 in 11 patients, and pN3 in 2 patients. In all 78 cases, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of18F-FDG PET/CT were 80.9%, 94.7%, 91.0%, 85.0%, 93.1%; while 61.1%, 71.7%, 69.2%, 39.2%, 86.0% on chest CT. These were statistically different (χ2=4.325,P<0.05).Conclusion PET/CT is superior to plain chest CT examination as an accurate and efficient method in evaluating lymph nodes staging of lung cancer.

Objective To investigate the efficacy,safety,economy evaluation of CYP3A5 * 3 gene polymorphism in providing individualized administration for the use of tacrolimus (Tac) in renal transplantation recipients.Method Pyrophosphate sequencing method was used to determine the CYP3A5 * 3 genotype of renal transplant patients in the first day after surgery.Computer-generated random numbers were used to assign 60 patients into experiment group or control group.Both groups of patients were routinely given the initial dose of Tac (4.0 mg/day) in the first day after surgery.The patients in the experiment group were given different doses of Tac based on the different CYP3A5 * 3 genotypes at the third day after surgery [for AA,AG:0.12 mg/(kg day),and for GG:0.06 mg/(kg day)].The patients in the control group were given different dosages of Tac according to drug concentration.The patients were followed up for 12 months,and different parameters were compared between two groups.A decision tree model was populated with data from the study and used to economics evaluation.Result At day 5 after the transplantation,significantly more patients in the experiment group were within the Tac target C0 range [90％ (27/30)] as compared to the control group [46.67％ (14/30) (P＜0.05).At this time point,the median Tac C0was 5.08 [(2.5-8.7) μg/ L] in the experiment group vs.5.29 [(1.3-13.6) μg/L] in the control group (P＜0.05).When C0/ D was analyzed according to CYP3A5 * 3 genotype,we found the mean C0/D in the both groups with CY3A5 * 3/* 3 ＞CYP3A5 * 3/* 1 ＞ CYP3A5 * 1/* 1.It was noted that the time to achieve target Tas was (4.40 ± 0.97) in the experiment group,vs.(7.57 ± 3.42) in the control group.In total,the number of daily dose modifications was 11 in the experiment group and 49 in the control group in two weeks after transplantation (P＜0.05).Renal function at day 14 after transplantation and adverse events during 12 months of follow-up were similar in both groups.In total,10 adverse events were reported in the experiment group and 11 in the control group (P＞0.05).The results of costeffectiveness analysis showed that the cumulative costs and effects in the experiment group were ￥ 38 067 and 0.90 quality-adjusted life years gained,and those in the control group were ￥38 681 and 0.87 quality-adjusted life years gained,respectively.In the base case analyses,experiment group was more cost-effective.Conclusion Individualized adjustment of Tac doses for patients according to recipients different CYP3A5 * 3 genotypes is beneficial for reaching target concentration as soon as possible and more cost-effective.But the demonstration of the clinical relevance of this approach was not achieved.Higher methodological quality,and larger sample size study are still needed.

objective To determine the existence of virulence-associated invA gene in different genospeeies of Leptospira interrogans reference strains in China.and to understand the alterations of invA gene transcription and expression of L.interrogans strain Lai before or after infecting cells.Methods PCR was applied to detect the invA gene of four L.interrogans strains belonging to four different genospecies and L.biflexa strain Patoc Ⅰ.The entire invA genes from the L.interrogans strains were cloned and then sequenced.The prokaryotic expression system of invA gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was constructed.Using Ni-NTA affinity chromatography,the target recombinant protein rInvA was extracted and purified.Rabbits were immunized with rInvA to obtain antiserum and the titer of antiserum was determined by immunodiffusion test.A model of L interrogans strain Lai infecting human embryo kidney cell line HEK293 was established to detect the alterations of invA gene transcription and expression of the leptospiral strain before or after infecting the host cells by real-time fluorescent quantitative RTPCR and western blot assay.Results All the four tested L.interrogans strains had invA gene whereas L.biflexa strain Patoc Ⅰ not.The similarity of nucleotide and putative amino acid sequences of invA genes from the four L.interrogans strains belonging four different genospecies were 99.33%-100%and 98.66%-100%,respectively.The constructed prokaryotic expression system could efficiently express rInvA and the immunodiffusion titer of rabbit anti-rInvA serum was 1:16.After L.interrogans strain Lai infecting HEK293 cells for 30 min or above,the microbe could adhere the surface of the cells.On the 30 min after the infection,the mRNA level of invA gene of L.interrogans strain Lai was remarkably upregulated,and on the 45 min after infection the mRNA level presented a peak value and then graduated decreased.On the 45 min or 60 min after L.interrogans strain Lai infecting HEK293 cells,InvA protein could be detectable but before infection or after infection for over 90 min the InvA protein expression was negative.Conclusion The invA gene is a unique gene for pathogenic L.interrogans.The invA gene of L interrogans has characteristics of cell-touched expression and transient expression,which may have a close correlation with adhering and invading host cells.

BACKGROUND: Surgery is the common therapy for pterygium, and there are several surgical management techniques.OBJECTIVE: To clinically assess the effect of pterygium retro-resection followed by the transposition of conjunctival autograft rich in stem cells.DESIGN: Follow-up of the cases.SETTING: Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University.PARTICIPANTS: Fifty patients (60 eyes) with pterygium, who were treated in the Fourth Affiliated Hospital of China Medical University from May 2003 to May 2006, were selected. All patients agreed to receive the treatment and participate in the follow-up. The trial was permitted by the Hospital Ethics Committee.METHODS: The head of pterygium was separated from cornea, including the conjunctiva and the underlying proliferating tissue towards lacrimal caruncle until plica semilunaris. The pterygium was totally removed. The adjacent healthy conjunctiva harboring stem cells was transposed to cover the naked sclera. The patients were evaluated following the operation.MAIN OUTCOME MEASURES: ①Epithelization of cornea and conjunctiva; ②reoccurrence of pterygium.RESULTS: ①The epithelium of cornea and conjunctiva in all cases (60 eyes) healed within 1-2 days after the operation. ②The patients were followed for 8-16 months after the sutures were removed. Out of the total of 60 eyes, 26 were followed for 8-12 months and 34 for 13-16 months. The average length of observation was 12 months. Fifty-eight eyes healed completely, and reoccurrence took place in 2 cases.CONCLUSION: Pterygium resection followed by the transposition of adjacent conjunctival autograft harboring stem cells is easy to perform and effective to reduce the recurrence of the lesion.

Objective To determine the function of toxic protein VapC in toxin/antitoxin system of Leptospira species and the cytotoxicity to host cells of the toxic protein.Methods Using genomic DNA of pathogenic L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai as the template,several PCRs were performed to amplify entire vapB,vapC and vapBC genes.Subsequently,the prokaryotic expression systems of vapB,vapC and vapBC genes were constructed.Expression of the target recombinant proteins rVapB and rVapC was detected by SDS-PAGE and the expressed rVapB and rVapC were extracted by NiNTA affinity chromatography.Activity of rVapB and rVapC to lyse the DNAs or RNAs from L.interrogans strain Lai and THP-1 cells were then determined.The changes of transcription and expression of vapB and vapC genes of L.interrogans strain Lai before and after infection of THP-1 cells were detected by real-time fluorescent quantitative RT-PCR and Western blot assay.The eukaryotic expression vectors of the vapB and vapC genes were generated for transfection of host cells and CCK-8 agent was used to detect the effect of leptospiral VapB and VapC proteins on activity of host cells.Results The nucleotide and putative amino acid sequences of the cloned vapB and vapC genes were completely identical with the reported corresponding genes.The constructed prokaryotic expression systems could express rVapB and rVapC,respectively.rVapC displayed RNase avtivity but did not lyse DNA.When L.interrogans strain Lai infected THP-1 cells,the transcription and expression of vapB and vapC genes were upregulated and partial VapC protein was secreted from the leptospiral cells.The mass mortality was observed in HEK293 human renal tubular epithelial ceils containing the vapC gene through transfection.Conclusion VapC protein of L.interrogans strain Lai is a RNase and is secreted during infection of host cells with obvious cytotoxicity.

ObjectiveTo determine the diversity of outer membrane protein expression of Leptospira interrogans during infection of human THP-1 monocytes,and protein antigen candidates for developing leptospiral genetically engineering vaccines.MethodsThe outer membrane proteins(OMPs) of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai before or after infection of human THP-1 monocyte line were extracted using Triton X-114 method.The OMPs extracts were separated by two-dimensional gel electrophoresis(2-DGE) and then detected using silver staining method.Four up-regulated and four down-regulated OMPs were selected for hydrolysis by trypsin and then identified by using liquid chromatography-tandem mass(LC-MS/MS) method.The transmembrane regions and signal peptides in the eight target OMPs were analyzed using bioinformatic software.Several real-time fluorescent quantitative RT-PCRs were performed to determine the mRNA level changes of the eight target genes of L.interrogans strain Lai before or after infection of THP-1 cells.The prokaryotic expression systems of target genes were generated and the immunoprutection of target recombinant proteins was determined using leptospire-infected guinea pigs model.ResultsAfter a 60 min infection of THP-1 cells,four OMPs( Loa22,GroEL,F0F1 ATP synthase alpha- and beta-subunits) of L interrogans strain Lai were noticeably up-regulated ( P＜0.05 ),while the other four OMPs( FlaB2,LigB,OmpA family protein and OmpA) were significantly down-regulated (P＜0.05).The results of real-time fluorescent quantitative RT-PCRs were coincident with those confirmed by the 2-DGE phus silver staining.The bioinformatic analysis indicated that among the eight OMPs,OmpA and OmpA family protein belonged transmembrane proteins,while the rest had no membrane structure.Loa22,LigB and OmpA family protein contained a signal peptide in their sequences.200 μg of the recombinant proteins rLoa22 and rGroEL presented 75.0％ immunoprotective rates in the infected guinea pigs.Conclusion An obvious change of the OMP expression map of L.interrogans strain Lai occurs during infection of host cells.The up-regulated leptospiral OM Ps during infection,especially for the GroEL and Loa22 proteins,can be used as the immunogen candidates for developing leptospiral genetically engineering vaccines.