Objective To compare the therapeutic effect of inhaled aerosolized and intravenous milrinone,a phosphodiesterase-3 inhibitor in rats with oleic acid-induced acute lung injury (ALI) .Methods Forty male SD rats weighing 300-350 g were randomly divided into 4 groups (n=10 each) : group Ⅰ normal control: group Ⅱ ALI; group Ⅲ milfinone inhalation and group Ⅳ intravenous milrinone.The animals were anesthetized with intraperitoneal 2% pentobarbital 40 mg/kg,tracheostomized and mechanically ventilated (FiO2 30%,VT 10 ml/kg,RR 80 bpm,I:E=1:2).The chest was opened and the heart was exposed.Pulmonary artery was catheterized via fight ventricle.MAP,CVP,airway pressure and pulmonary artery pressure (PAP) were monitored.ALI was induced with 10% oleic acid 2 ml/kg administered through fight external jugular vein in group Ⅱ,Ⅲ and Ⅳ.In control group 0.1% BSA solution 2 ml/kg was administered iv instead of oleic acid.In group Ⅲ at 30 min after oleic acid administration aerosolized milrinone 1 mg/ml was inhaled 4 times at 60 min interval.Each time milrinone was inhaled for 10 min.In group Ⅳ at 30 min after oleic acid administration a bolus of 10 μg/kg milrinone was given iv followed by 10 min milrinone infusion at 1 μ·kg-1·min-1.The same procedure was repeated 4 times at 60 min interval.MAP and PAP were recorded and blood samples were taken from carotid artery and pulmonary artery for blood gas analysis at the 1st,2nd,3rd and 4th treatment.PaO2/FiO2 and Qs/Qt were calculated.The animals were sacrificed by exsanguination after the 4th treatment.The lungs were removed.The left lung was lavaged.Neutrophil count and protein content in broncho-alveolar lavage fluid (BALF) were determined.W/D lung weight ratio and lung myeloperoxidase (MPO) activity were measured.The uhrastructure of the lung was examined with electron microscope.Results The MAP was significantly lower after oleic acid adminstration in group Ⅳ than in other 3 groups.PaO2/FiO2 was significantly decreased and Qs/Qt increased by iv oleic acid in group Ⅱ ,Ⅲ and Ⅳ.PAP was significantly increased after iv oleic acid in group Ⅱ ,Ⅲ and Ⅳ but was significantly lower in group Ⅲ and Ⅳ than in group Ⅱ .The neutrophil count and total protein content in BALF,W/D ratio and lung MPO activity were significantly increased in group Ⅱ ,Ⅲ and Ⅳ as compared with control group(Ⅰ) and were significantly higher in group Ⅳ than in group Ⅲ.The lung damage induced by oleic acid was less serious in group Ⅲ and Ⅳ than in group Ⅱ .Conclusion Inhaled aerosoLized milrinone has better therapeutic effect than intravenous milrinone in rats with oleic acid-induced ALI and is safer.

Objective To explore the activated brain region of acute epilepsy in cat model induced by pentylenetetrazol(FFZ)with manganese enhanced-functional MR imaging(ME-fMRI),and evaluate the application of ME-fMRI on localization of the activated brain.Methods Forty cats were divided into 4 groups by random number table method as epileptic A and B groups as well as control A and B groups. Cats of epileptic groups were injected with PTZ(55 mg/kg)intramuscularly,and those of control groups were injected with the saline at same dose.The behavior change in the epileptic and control group A was observed and electroencephalogram(EEG)was also undertaken.Cats of epileptic and control group B were performed ME-fMRI,and the percentage of the enhanced signal intensity was then calculated.Results After injection with PTZ(55 mg/kg)intramuscularly,epileptic seizure was all evoked,and then EEG recording showed spike-wave and polyspike-wave complexes.The neocortex of cats of epileptic group B was diffusely phanero-enhanced on ME-fMRI.The percent enhancement of signal intensity in cortex of frontal lobe,parietal lobe and occipital lobe was(34.6?5.7)% and that in cortex of temporal lobe with(22.9? 6.5)%,whereas those of control group B with(14.9?4.5)% and(11.6?3.2)% respectively.And there was significant difference between the above different localization of the brain in the two groups (t=-10.43,-5.46 respectively,P

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.
Aminoacyltransferases ; metabolism ; Bacterial Proteins ; metabolism ; Cyclization ; Cysteine Endopeptidases ; metabolism ; Enzymes, Immobilized ; metabolism ; Kinetics ; Peptides ; metabolism ; Peptides, Cyclic ; biosynthesis ; Staphylococcus aureus ; enzymology

Objective To investigate the risk factors affecting the mortality in patients with severe chest trauma (SCT).Methods A total of 777 patients with SCT (AIS≥ 3) treated at Chongqing Emergency Medical Center from January 2006 to April 2009 were involved for retrospective study.Multivariate stepwise logistic regression analysis was used to analyze 15 possible risk factors affecting their mortality.Results The factors affecting mortality in patients with SCT included hemorrhagic shock (X6,B =1.710,OR =1.291,P＜0.01),multiple organ dysfunction syndrome (MODS) (X7,B=3.453,OR =1.028,P＜0.01 ),pulmonary infection ( X9,B =2.396,OR=10.941,P ＜ 0.01 ),abdominal organ injury (X11,B=1.542,OR=1.210,P＜0.01) and thoracic AIS ≥3 (X14,B =0.487,OR =1.622,P＜0.01 ).While the protective factors affecting mortality in patients with SCT contained age ≤60 years old (X1,B =-0.035,OR =0.962,P＜0.05) and GCS≥12 (X13,B=- 0.635,OR=0.530,P＜0.05).Conclusions The age,posttraumatic complications (hemorrhagic shock,MODS,pulmonary infection)and accurate diagnosis and evaluation of trauma severity are the related factors to predict the prognosis.Development of effective treatment measures based on these risk factors plays a key role in the survival rate of patients with SCT.

OBJECTIVE ToobservetheprotectiveeffectandmechanismofCompoundGinkgo biloba(CGB)againstalcohol-inducedliverinjury.METHODS MiceweregivenCGB0.125,0.25and 0.75 g·kg -1 ,Ginkgo biloba extract (GBE)0.1 25 g·kg -1 and bifendate(Bif)0.1 5 g·kg -1 for 8 weeks, respectively.At the end of 4th week the mice were given wine by gavage (56% V/V,0.01 L·kg -1 ), and (56% V/V,0.016 L·kg -1 )at the end of the 8th week.The serum was obtained to measure alanine transaminase (GPT),aspartate aminotransaminase (GOT),mitochondrial aspartate aminotransferase (mGOT)and tumor necrosis factor-α(TNF-α).Liver histopathology was revealed by HE staining.The protein expression of cytochrome P450 (CYP)2E1 ,NF-E2-related factor 2 (Nrf2)and TNF-αin the liverwasanalyzedbyWesternblotting.RESULTS Comparedwithnormalcontrolgroup,theactivitiesof GOT and mGOT were increased in model group (P<0.01 ).Compared with model group,CGB 0.25 and 0.75 g·kg -1 groups and Bif 0.1 5 g·kg -1 group significantly decreased the activity of GOT and mGOT in serum (P<0.05,P<0.01 ),while there was no significant difference between these groups in serum GPT activity (P>0.05).Fatty degeneration and neutrophil infiltration were significantly ameliora-ted in CGB 0.25 and 0.75 g·kg -1 groups.Preliminary mechanism research showed CGB not only increased the protein expression of Nrf2 with a positive dose-effect relationship (r=0.942,P<0.01 ), but reduced the protein expression of hepatic CYP2 E1 and the level of TNF-αin hepatic tissue with a negative dose-effect relationship (r=-0.987,P<0.05;r=-0.940,P<0.05).In addition.The level ofTNF-αwasalsosignificantlydecreasedintheserum(P<0.05,P<0.01).CONCLUSION CGB may protect the liver fro m acute alcoholic injury and the mechanis m may be that it increases the protein expression of Nrf2,restrains the protein expression of hepatic CYP2E1 and TNF-αand reduces the TNF-αlevel in the serum.

Objective To establish the enzyme-linked immunosorbent assay (ELISA) for the detection of serum alcohol dehydrogenase (ADH) antibody and evaluate its role in its diagnosis of autoimmune hepatitis( AIH ). Methods The reactivity between yeast ADH and human anti-ADH serum antibody was tested by Western blot analysis. ELISA was established using yeast ADH. The method was applied in serums of 67 AIH patients,94 primary biliary cirrhosis(PBC) patients, 199 chronic hepatitis B (CHB) patients, 132 chronic hepatitis(CHC) patients, 24 alcohol hepatitis disease(ALD) patients, 99 connective tissue disease(CTD) patients and 31 healthy individuals. The positive rate of ADH antibody in the patients and healthy individuals was measured. The χ2 test was used to compare the positive rates. Results The ELISA method for detecting human anti-ADH serum antibody was established successfully and the optimum reaction conditions were defined. Western blot showed that yeast ADH has cross reactivity with human anti-ADH antibody. The positive rate of anti-ADH antibody in the AIH group [59. 7% ,40/67 ] was higher than that in the normal control group(0,χ2 = 31. 271 ,P <0. 05), PBC group (6. 4% ,χ2 =54. 492,P <0. 05), CHB group( 14. 1% ,χ2 =54. 848,P <0. 05) ,CHC group(21.2% ,χ2 = 29.269,P<0.05), ALl) group ( 25. 0% ,χ2 =8.512,P <0.05)and CTD group ( 43. 4% ,χ2 =4.229, P <0. 05). Conclusions Compared with the PBC, CHB, CHC, ALD and CTD group, the anti-ADH antibody positive rate in the serums of AIH was significantly increased. The antibody may be helpful to the diagnosis of autoimmune hepatitis.

OBJECTIVETo investigate the relationship between lamivudine-resistant mutants of hepatitis B virus (HBV) and serum HBV DNA loading before antiviral therapy.

METHODSThis study involved 106 patients with hepatitis B receiving lamivudine treatment for an average of 32 months (rang 12-48 months). Serum HBV DNA loadings were measured with PCR before and every 4 to 6 months during lamivudine therapy. HBV YMDD mutants were detected using mismatched PCR-restriction fragment length polymorphism (PCR-RFLP) during lamivudine treatment.

RESULTSHBV DNA loading was significantly higher in patients infected with HBV YMDD mutants during lamivudine therapy than those infected with HBV without YMDD mutation.

CONCLUSIONHigh viral loading in hepatitis B patients before treatment is associated with high likeliness of HBV YMDD mutation during lamivudine treatment. HBV DNA loading may be indicative for the occurrence of YMDD mutation during lamivudine therapy.

Antiviral Agents ; pharmacology ; DNA, Viral ; blood ; Drug Resistance, Viral ; genetics ; Female ; Hepatitis B ; blood ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Humans ; Lamivudine ; pharmacology ; Male ; Middle Aged ; Mutation ; Viral Load ; genetics

Objective:To stablish of chemiluminescent Southern blot detection system for examining HBV DNA replication intermediates in HepG2.2.15,and analyse the inhibition of HBV replication with three kind of drugs with different targets.Methods:The HBV DNA replication intermediates were extracted and analyzed by Southern blot with HBV probe,which(pTHBV1047) was labelling with digoxigenin.The results of the hybridization were detected by chemiluminescent,and the condition of hybridization was optimized.After treated with lamivudine,Bay41-4109,?-Galcer in different concentration,the HBV DNA from the HepG2215 cells were detected with the system.Results:the sensitivity of the system was 1pg of pTHBV1047,and HBV specific positive signals was detected with the DNA from HepG2.2.15.The three kinds of drugs can inhibit the HBV replication obviously with chemiluminescent Southern blot detection system,the IC50 were 1.53?mol/L,0.41?mol/L,0.01?mol/L.Conclusion:The HBV replication intermediates from the cell of HepG2.2.15 can reflect the antiviral effect accurately with different targets drugs and this mothod would be used in the study of Chinese-midicine.

Objective To detect plasma interleukins-35 (IL-35 )level in the patients with active tuberculosis complicated with bronchiectasis and to analyze its clinical significance.Methods Peripheral blood of patients with active tuberculosis from depart-ment of Dongguan 6th People′s hospital were collected,assigned to the active tuberculosis complicated with bronchiectasis group and active tuberculosis group.The healthy volunteers served as the control group.The plasma IL-35 level was measured by ELISA, and peripheral blood neutrophils and lymphocytes were detected by hematology analyzer.Results The levels of plasma IL-35 signif-icantly increased in both patients with active tuberculosis complicated with bronchiectasis and patients with active tuberculosis.The level of plasma IL-35 of patients with active tuberculosis complicated with bronchiectasis was significantly higher than that of the patients with active tuberculosis.The absolute value and percentage of peripheral blood neutrophils of patients with active tubercu-losis complicated with bronchiectasis were significantly higher than those of healthy volunteers.However,the percentage of periph-eral blood lymphocytes of patients with active tuberculosis complicated with bronchiectasis was significantly lower than that of healthy volunteers.Pearson correlation analysis showed that the absolute value of peripheral blood neutrophils of patients with ac-tive tuberculosis was positively correlated to the level of plasma IL-35.Conclusion IL-35 may play an important role in the progres-sion of active tuberculosis complicated with bronchiectasis.The determination of IL-35 may be helpful to the diagnosis of patients with active tuberculosis complicated with bronchiectasis.

Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.