In order to improve the thermostability of the Penicillium expansum Lipase (PEL), the lipase encoding genes was mutated by site-directed mutagenesis. A recombinant vector pAO815-ep8-K55R which contain double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K55R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8- K55R can secret double-mutant lipase PEL-ep8-K55R-GS into the medium when it was induced by Methanol. The yield of the double-mutant lipase is 508 u/mL, which is 81% that of the wild type lipase PEL-GS (627 u/mL) and 55% that of random-mutant PEL-ep8-GS (924 u/mL). The specific activity of double-mutant lipase is 2309.1 u/mg, which is similar to random-mutant lipase PEL-ep8-GS and the wild type lipase PEL-GS. The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL-ep8-GS. While the Tm of the double-mutant lipase is 41.0 degrees C, 2.3 degrees C higher than the wild type lipase PEL-GS and 0.8% higher than the random-mutant lipase PEL-ep8-GS, indicating that the double-mutant lipase PEL-ep8-K55R-GS has higher thermostability.
Electroporation ; Enzyme Stability ; Hot Temperature ; Lipase ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutant Proteins ; metabolism ; Penicillium ; enzymology ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics

BACKGROUND:Simvastatin enhanced the expression of bone morphogenetic protein-2(BMP-2),which plays an anabolic role in bone metabolism and osteoblastic lineage differentiation.However,little is known about the molecular mechanism of simvastatin on regulation of bone marrow stromal cells differentiation.OBJECTIVE:To investigated the effect of simvastatin on osteoblastic differentiation of bone marrow stromal cells based on genetics level.METHODS:Bone marrow stromal cells derived from femur and tibia were cultured in different mediums with simvastatin or Vehicle for 7 days Following extraction and purification,mRNA was reverse-transcripted into cDNA.Fluorescence labelina was employed and the samples were then hybridized with oligonucleotide chip to screen the different genes,which were utillzed to analyze osteogenesis-related factors.Alkaline phosphatase and Von Kossa staining were performed at days 14 and 21,respectively.RESULTS AND CONCLUSIONS:At day 14,alkaline phosphatase-positive cells were more in the experimental group than control group.Von Kossa staining demonstrated that simvastatin could promote BMSCs osteoblastic differentiation and mineralization.Comparative analysis showed that 103 genes out of 22 575 rat genes had differential expression (≥2 fold or≤ 0.5 fold),and some genes were related to cell proliferation and ostoeblastic differentiation,including C/EBP δ,Cited,Ascl2,Ptpnl6,Wisp2,Tieg,etc.Simvastatin could induce osteoblastic differentiation of bone marrow stromal cells,involving in many osteogenetic-related genes.

Background and purpose:B-cell translocation gene 1(BTG1) can inhibit cell proliferation, promote cell apoptosis and regulate cell cycle progression and differentiation in a variety of cell types. This study aimed to explore the inlfuence on cell proliferation, apoptosis and cell cycle and its related mechanism of laryngeal cancer Hep - 2 cell lines through BTG1 overexpression byin vitro experiments.Methods:The BTG1 expression plasmids were constructed and transfected into Hep-2. They were divided into experimental group (transfected BTG1 of Hep-2 cells) and control group (transfected empty plasmid of Hep-2 cells). Western blot method was used to identify BTG1 protein expression levels of cells; proliferation activity of cells was detected by MTT assay; lfow cytometry was used to analyze the cell cycle distribution and AnnexinⅤ-FITC/PI cell apoptosis; Western blot was also used to assay cell cycle regulatory protein and apoptosis-related protein expression.Results:The pEGFP-N1-BTG1 plasmid was constructed successfully, and the expression of BTG1 protein was higher in experimental group than that in control group (0.921±0.091vs 0.308±0.047,P<0.05). Compared with the two group of laryngeal cancer Hep-2 cells, the cell growth in experimental group was slowed down and the proliferation was reduced (P<0.05); Cyclin D1 protein expression level was decreased (0.436±0.023vs 0.916±0.092,P<0.05), the proportion of G0/G1 phase cell cycle was increased [(85.1±5.2)%vs (63.8±3.1)%,P<0.05], the proportion of S phase cell was decreased [(8.3±1.1)%vs (23.1±1.5)%, P<0.05], phosphatidylserine ectropion in experimental group was increased, cell early apoptosis was significant [(10.3±1.1)%vs (2.8±0.3)%,P<0.05] and anti-apoptotic protein Bcl-2 expression level was reduced(0.167±0.009vs 0.834±0.084,P<0.05).Conclusion:BTG1 high expression could inhibit the proliferation growth of laryngeal Hep-2 cells and promote its apoptosis, and the possible mechanisms are interrelated with BTG1 involved in cell cycle regulation and causing cell apoptosis.

Objective To assessed the expression of inducible costimulator(ICOS)on peripheral blood and joint fluid CD4,CDS,CD45RO T cells and B cells in rheumatoid arthritis(RA).Methods Expression of ICOS and ICOS/CD45RO on peripheral blood and joint fluid CD4~+CD8~+T cells and ICOS ligand(ICOSL)on CD19 B cells from RA patients and healthy volunteers were determind by three-color flow cytometry.Compar- ision with active and inactive RA,initial and relapsed RA had been done.Results Joint fluid CD4 and CD8 T cells expressing ICOS,ICOS/CD45RO were significantly increased than peripheral blood in RA patients and healthy subjects.Joint fluid B cells expressing ICOSL were significantly reduced than peripheral blood in RA patients.Meanwhile,peripheral blood B cells expressing ICOSL were significantly reduced in active RA than inactive RA patients.Conclusion Hyperexpression of ICOS and ICOS/CD45RO on joint fluid CD4 and CD8 T cells and lowexpression of ICOSL in B cells from RA patients,expecially in active RA may contribute to the local immunopathological roles and joint destructions in the pathogenesis of RA.

Objective To detect levels of IL - 4 and INF - ? in induced sputum dynamically in children with mycnplasma pneamo-niae pneumonia( MPP), and to analyze the function of Th1 /Th2 cell factor immune response in the genesis and development of MPP, so as to evaluate the clinical value of induced sputum method in MPP research. Methods There were 38 cases who were diagnoses as MPP using 3% high osmotic pressure of saline water to ultrasonic atomizing inhalation for inducing sputum. ELISA was used to detect IL-4 and INF-?. Results The content of IL-4 in acute stage was higher than that in convalescence stage in induced sputum of MPP children. Severe stage was higher than mild stage. However, the comparison between acute and convalescence stage didn't have statistics difference in the content of INF-?, neither did the comparison between severe and mild stage. IL- 4/INF- ? in acute stage was higher than that in convalescence stage. Severe stage was higher than mild stage. In convalescence stage, the comparison of INF - ?, IL - 4, IL - 4/INF - ? between the severe and the mild didn' t have statistic significance. Conclusions IL-4 and INF - ? have participated in the monogenesis of MPP. The disequilibria of Th1 /Fh2 is existed in MPP and Th2 reaction predominates. So induced sputum analysis can be a better way to judge the light or heavy press degree of MPP practically, conveniently and sensitively.

Objective To investigate the clinical features and prognostic factors of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) in adults.Methods Clinical data of 40 adult patients with EBV-HLH admitted in the First Affiliated Hospital of Nanjing Medical University during January 2010 and September 2013 were retrospectively analyzed.The patients were divided into fatal group or survival group based on the results of 1-month follow-up.Logistic regression analysis was performed to identify risk factors of early death.Results EBV-HLH in clinic were characterized by fever,hepatosplenomegaly and lymphadenopathy.The incidences of peripheral blood cytopenias in 2 or 3 lines,hepatic dysfunction and hyperferritinemia in EBV-HLH patients were 60.0％ (24/40),95.0％ (38/40) and 94.9％ (37/39),respectively.Hemophagocytic phenomenon in bone marrow was observed in 83.8％ patients (31/37).Soluble CD25 (sCD25) was detected in 12 patients,and all were higher than 2 400 pg/mL.Nineteen patients died 1 month after fiual diagnosis with a fatality rate of 47.5％.Univariate logistic regression analysis indicated that EBV-DNA ＞ 105 copies/mL (OR =4.85,95％ CI:1.14-25.83,P ＜ 0.05),PLT ＜ 50 × 1012/L (OR =6.81,95 ％ CI:1.41-32.83,P ＜ 0.05),total bilirubin ＞ 50 μmol/L (OR =6.91,95％ CI:1.24-38.52,P ＜0.05),albumin ＜ 30 g/L (OR =7.50,95％ CI:1.80-31.28,P ＜ 0.01),serum ferritin ＞5 000 μg/L (OR =8.75,95％ CI:2.03-37.67,P ＜0.01) and polyserositis (OR =5.40,95％ CI:1.18-24.64,P ＜0.05) were risk factors of death within 1 month after final diagnosis,while chemotherapy was a protective factor of early death (OR =0.15,95％ CI:0.03-0.81,P ＜ 0.05).Conclusions EBV-HLH in adult has a poor prognosis.Significant elevated EBV-DNA,total bilirubin or serum ferritin,and notable decrease of peripheral blood platelet count or albumin level may indicate a poor prognosis,while chemotherapy may reduce the risk of early death.

12E7 Antigen ; Antigens, CD ; metabolism ; Bone Neoplasms ; metabolism ; pathology ; surgery ; Cell Adhesion Molecules ; metabolism ; Chondrosarcoma, Mesenchymal ; metabolism ; pathology ; surgery ; Diagnosis, Differential ; Hemangiopericytoma ; pathology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; surgery ; Male ; Middle Aged ; Pneumonectomy ; methods

OBJECTIVETo analyze the masseter inhibitory reflex (MIR) and the recovery cycle of the MIR reflex after magnetic stimulation in normal subjects.

METHODSIn 30 healthy subjects we studied the MIR evoked by single magnetic stimulation in the mental territory. Masseter electromyographic activity, latency and duration of the early silent periods (SP1) and late silent periods (SP2), and SP2 amplitude percent were recorded. Paired stimuli technique was used, conditioning and test stimuli were delivered at different interstimulus intervals (ISI), ie.100, 200, 300, 400, 500, and 600 ms, then the recovery cycle of the MIR was analyzed.

RESULTSLatency of SP1 was 12.1 (11.1, 14.4) ms, and duration of the SP1 was (17.3 ± 2.9) ms. Latency of SP2 was (47.7 ± 6.0) ms, and duration of the SP2 was (39.7 ± 13.3) ms. SP2 amplitude percent was 100.0%. With the paired stimuli technique, SP1 of the inhibitory reflex evoked by the test stimuli was found to be stable at every ISIs, but SP2 of the inhibitory reflex evoked by the test stimuli, instead, varied according to different ISI. With the short ISI, the area of SP2 evoked by the test stimuli was reduced, and with the increase of the ISI, the recovery degree of the area of SP2 evoked by the test stimuli was ascending, at 100 ms the SP2 amplitude percent was 17.1%, at 400 ms it was 93.4%, and it was close to 100% at 600 ms.

CONCLUSIONSThe study indicates that the use of the MIR elicited by the single and paired magnetic stimulation may be useful to examine or quantify some craniofacial diseases.

Adult ; Electric Stimulation ; Female ; Humans ; Magnetics ; Male ; Masseter Muscle ; physiology ; Reaction Time ; physiology ; Reflex ; physiology ; Young Adult

The purpose of this study is to systematically investigate the characteristics of absorption and metabolism of oxymatrine (OMT) using rat intestinal perfusion model. Ultra performance liquid chromatography (UPLC) and high performance liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (HPLC-ESI(+)-Q-TOF-MS) were used to test absorption of OMT in intestine at 100, 200 and 400 µmol · L(-1). The absorption rate and permeability of OMT is not dependent on concentration, but through passive absorption in intestine (P > 0.05). In the rat intestine, the absorbed amount of OMT was significantly different in four sections of the intestine in an order of duodenum > jejunum > ileum > colon (P < 0.05). OMT is metabolized into two metabolites in duodenum and jejunum, and matrine (MT) is the major one.
Alkaloids ; metabolism ; Animals ; Chromatography, High Pressure Liquid ; Intestinal Absorption ; Intestines ; metabolism ; Quinolizines ; metabolism ; Rats ; Spectrometry, Mass, Electrospray Ionization

ObjectiveTo explore the state of misdiagnosis, missed diagnosis and excesseive diagnosis related to cerebral palsy(CP).Methods389 cases were retrospectively analyzed who were misdiagnosed, missed or escessively diagnosed related to CP as the first diagnosis in the inpatient and outpatient department from July 1999 to March 2010.ResultsAmong 389 cases, 156 cases were missed or misdiagnosed as nutritional disease, and 118 cases of other diseases were misdiagnosed as CP, while 115 cases who were normal children was excessively diagnosed as CP. The false diagnosis had relativity with children's age: doctors are more prone to make misdiagonsis when the children's age are smaller, while 293 cases were misdiagnosed before 12 months old (75.3%); 102 cases (65.4%) were misdiagnosed or missed as other diseases before September, 2004, while 87 cases (75.7%) were excessively diagnosed as CP after September, 2004. Frequency of misdiagnosis as CP reduced from 2004, and the proportion dropped from 55.9% to 44.1%.ConclusionIt is very important to master the diagnostic standard of CP. Both sides of the question are important, one side is to make early diagnosis and early treatment in order to achieve the best effect of rehabilitation, and the other side is to prevent misdiagnosis and excessive diagnosis which woud delay illness, or waste medical resources, increase the financial and psychological burden.