In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.
Amidohydrolases ; genetics ; metabolism ; Bile Acids and Salts ; metabolism ; Escherichia coli ; metabolism ; Gene Expression ; Lactobacillus ; enzymology ; genetics ; Substrate Specificity

Objective To study the relationship between suicidal attitude and childhood trauma in bipolar disorders.Methods 67 patients with bipolar disorder and 101 healthy individuals were assessed with the Questionnaire of Suicidal Attitude (QSA) and Childhood Trauma Questionnaire(CTQ).Results There was no statistical difference between the patients group and healthy control group in the results of suicidal attitude (P＞0.05).The scores of emotional neglect (M =10)and physical neglect (M =9) in patients group were significantly higher than those of the healthy control group(M =8,M =6)(P＜0.05).The attitude to suicidal behavior was negative correlation with emotion neglect(r=-0.181,P＜0.05)in patients group.Conclusion Emotional and physical neglect are the main types of trauma in patients with BD,and may increase the risk of suicide.It is important to survey the patients with BD on childhood trauma,and intervention is necessary.

The influenza A virus matrix protein2 gene(M2)which deleted transmembrane region was amplified by overlap extending PCR,and the multiepitope gene of hemagglutinin(HA)was PCR amplified with seven continuous synthesized segments by designing primer.The two gene segments were separately cloned into pMD18T vector to sequence analysis and prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+M2dHA.The recombinant plasmid was transformed into E.coli BL21(DE3),and the high expression strain was obtained by screening monoclones.The recombinant protein existed as inclusion bodies,which accounted about 45% of the total cellular protein.The inclusion bodies were washed with 1% Triton X100 solution twice,and dissolved in 8 mol/L urea solution.The solution protein was purified by Ni+2 affinity chromatography,and refolded by dilution renaturation,then purified by Q Sepharose FF cation exchange column.The purity of the protein was over 90% by HPLC analysis.The result of Western blot showed it has good antigenicity and specificity.These results strongly supported for the further study of the broadspectrum influenza virus subunit vaccine.

The gene of mutated TNF-?D4 gene was amplified by overlap PCR and cloned into the prokaryotic expressive vector pBV220.TNF-?D4 contains two changes:substitutions of Pro8Arg,Ser9Lys,Asp10Arg,Ile157Phe,Leu29Ser,Arg31Val and a deletion of the N terminal four amino acids.The recombinant vector pBV220-TNF-?D4 was transformated into E.coli strain DH5?,and the high expression strain was obtained by screening monoclones.The level of expression was about 45% of total cell protein.After purification,the purity of fusion protein was above 90% by HPLC and relative ability was 8 ?107.TNF-?D4 was modificated by mPEG-ButyrALD。After purification,the purity of mPEG-TNF-?D4 was above 85% and relative ability was 8.6?107.The in vivo systemic toxicity of mPEG-TNF-?D4,which is indicated by LD50,is lower than that of rhTNF-?.These results strongly supported for the further study and exploitation of TNF-antitumor drug.

AIMTo study changes of function of transmitter glycine in nitrogen narcosis.

METHODSSynaptosomes of rat spinal cord were prepared. Glycine uptake of synaptosomes of rat spinal cord in 0.7 MPa (7ATA) hyperbaric air pressure was observed by the methods of isotope.

RESULTSGlycine uptake slowed down and took a longer period of time to reach saturation in 0.7 MPa (7ATA). The maximum glycine uptake was lessened. Vm was diminished, but Km was increased. Vm rose in 0.7 MPa (7ATA) when corticosterone was added.

CONCLUSIONWhen nitrogen narcosis arose in 0.7 MPa (7ATA), the function of transporters of glycine re-uptake was reduced, the affinity of glycine for transporters subsided. Corticosterone was conductive to the recovery of the function of glycine transporters of high affinity.

Air Pressure ; Animals ; Glycine ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism ; Synaptosomes ; metabolism

OBJECTIVETo explore the effects of arecoline on hepatic insulin resistance in type 2 diabetes rats and to elucidate its possible mechanism.

METHODSForty five Wistar rats were fed with high fructose diet for 12 weeks to induce type 2 diabetic rat model. rats were randomly divided into 5 groups (n = 8): control group, model group and model group were treated with different dose (0, 0.5, 1, 5 mg/kg) of arecoline. After 4 weeks, the fasting blood glucose, blood lipid and insulin level measured , mRNA expression of liver constitutive androstane receptor (CAR), pregnane X receptor (PXR), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected by reverse transcription polymerase chain reaction (RT-PCR), the protein expression of p-AKT and glucose transporter4 (GLUT4) were detected by Western blot.

RESULTS1.5 mg/kg arecoline could significantly decrease the level of fasting blood glucose, blood lipid, blood insulin level and liver G6Pase, PEPCK, IL-6, TNF-alpha mRNA level in type 2 diabetes rats. 1.5 mg/kg arecoline also could significantly increase CAR, PXR mRNA level and p-AKT and GLUT4 protein expression.

CONCLUSIONArecoline improved hepatic insulin resistance in type 2 diabetes rats by increasing the mRNA levels of CAR and PXR leading to the creased glucose metabolism and inflammation related genes expression.

Animals ; Arecoline ; pharmacology ; Diabetes Mellitus, Experimental ; metabolism ; Diabetes Mellitus, Type 2 ; metabolism ; Glucose Transporter Type 4 ; metabolism ; Glucose-6-Phosphatase ; metabolism ; Insulin Resistance ; Interleukin-6 ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Phosphoenolpyruvate Carboxykinase (GTP) ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Wistar ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Receptors, Steroid ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Objective To explore the influences of extracellular matrices (ECM) secreted by ultraviolet B (UVB)-induced senescent fibroblasts on the proliferation of and extracellular signal-regulated kinase (ERK) signaling in HaCaT cells. Methods Fibroblasts were irradiated with UVB of 15 mJ/cm2 once daily for 5 days to induce premature senescence, which was identified by SA-β-gal staining 72 hours after the last irradiation.HaCaT cells were divided into 3 groups and inoculated into plates coated with extracellular matrices secreted by non-senescent (PRE-ECM) or senescent fibroblasts (SIPS-ECM) or into uncoated plates (NON-ECM), fol-lowed by additional culture. U0126, an inhibitor of ERK1/2, was used to treat the HaCaT cells 1 hour before inoculation. Then, MTT assay was carried out to detect the proliferation of HaCaT cells after a 3-day culture,Western blot to assess the phosphorylation of ERK at 0.5, 1, 2 and 4 hours after the inoculation, flow cytometry to analyse cell cycle and apoptosis after 24 hours of culture. Results The most rapid and intense phosphory-lation of ERK was observed in SIPS-ECM group. Inhibiting the activation of ERK pathway with U0126 could completely suppress the promoting effect of ECM from senescent fibroblasts on the proliferation of HaCaT cells.After the blocking of ERK activation, the proportion of HaCaT cells in S and G2/M phase decreased from 37.40%, 41.34% and 43.31% to 29.41%, 36.48% and 39.96%, respectively, in NON-ECM, PRE-ECM and SCIP-ECM group. Conclusion The ECM produced by UVB-induced senescent fibroblasts promote the prolifera-tion of HaCaT cells via inducing the phosphorylation of ERK.

It has been received wide attention for the increasing prevalence of myopia worldwide in recent years.The myopic fundus lesions,which may be associated with the variation level of the thickness of retinal and choroidal membrane in the early stage of myopia,remained the main cause of vision impairment and reduce the quality of life.However,the changes in retinal and choroidal thickness in the progress of myopia and the associated factors are not clear.This article gives a brief review the change in the thickness of retinal and choroidal thickness in progress of myopia and its associated factors,which can provide support for clinical diagnosis,prevention and monitoring of macula/choroidal associated diseases.

Objective To explore the effect of recovery sleep on the executive function after 36 h of total sleep deprivation by event related potential technology.Methods Thirteen healthy male college students participated in two trials. At the first trial normal sleep as control was investigated. At the second trial participants experienced 36 h of sleep deprivation and then accepted 8 h recovery sleep. In each trial six Go/Nogo tests were employed to test the executive control function and the ERP data were recorded. Results There was no statistical difference in behavior and ERP results at each time point as the subjects had normal sleep. After 36 h of sleep deprivation, the behavior results were statistically significant when compared to the baseline. The amplitude and latency of Nogo-N2, Nogo-P3 on Fz electrode, the amplitude and latency of Nogo-P3 on Cz electrode showed statistical significance when compared to the baseline. After 8 h recovery sleep, the average correct reaction time and the Go correct reaction rate had statistical significance compared to 36 h value. The amplitude of Nogo-N2 and Nogo-P3 had no statistical significance compared to the baseline.However,it was of statistical significance[(-6.80 3.95)vs(-3.37 2.63)μV,(10.63±6.62)vs(5.63±5.45)μV,(9.49±7.37)vs(6.08±6.56)μV] compared to 36 h value. The latency of the recovery value of Nogo-N2 and Nogo-P3 was statistically significant[(254.14±15.55)vs(243.08±13.97)ms(382.14±41.07)vs(349.17±30.36)ms,(369.86±26.48)vs(347.48±29.24)ms]compared to the baseline.Conclusion As the time of sleep deprivation is prolonged, the executive function is impaired and the executive function is not completely recovered after 8 h recovery sleep.

To develop an oral drug,ITF gene encoding ITF proein,was expressed in a live delivery vehicle lactococcus lactis.First,the ITF gene was cloned into the prokaryotic expressive vector pNICE:sec.Second,the recombinant vector pNICE:sec-ITF was transformated into Lactococcus lactis strain NZ9000 to express ITF protein.Then the recombinant ITF was induced to express and was identified by SDS-PAGE and Western blot.Rabbits are divided into blank control group,preparation group and therapeutic group which are respectively administrated wih PBS and pNICE:sec-ITF Lactococcus lactis.By grades of ulcer test whether administrated pNICE:sec-ITF Lactococcus lactis protects against HCl-induced gastric injury in rabbits.The results were described as follows.The ITF was amplified and cloned in the vector pNICE:sec successfully.The fusion protein(5.9kDa) was expressed in L.lactis by the induction of the nisin.The quantity of expression accounted for 5% of the total bacterial protein.Western bolt analysis confirmed that fusion protein could be recognized specially by Monoclonal Anti-human TFF3 Antibody.Preparation groups and therapeutic groups do good than control group.Prove that administrated pNICE:sec-ITF Lactococcus lactis is biologically active in an HCl-induced rabbit gastric mucosal injury model.