OBJECTIVETo mutate human annexin V gene and transform it to Pichia Pastoris for mutant human annexin V expression, so as to be purified as active annexin V with endogenous metal chelating site.

METHODSThe 5' and 3' end of native annexin V gene were mutated by specific primers. The mutant annexin V gene was inserted into pPIC9K and sequenced. The correct plasmid was linearized and transformed into Pichia Pastoris strain GS115 by electroporation. The transformants were selected from MD plates and cultured in BMGY medium and induced with methanol. The culture was centrifuged and the supernatant was analyzed by SDS-PAGE and silver staining. The binding activity of mutant human annexin V from culture supernatant was determined with phosphatidylserine exposed erythrocytes and fluorescein isothiocyanate-annexin V.

RESULTSThe 5' end of native human annexin V gene was fused with GCAGGCGGCTGCGGCCAT coding sequence and 3' end 946-948 site TGT was mutated to AGC. Pichia Pastoris transformants secreted proteins of relative molecular mass 36 000 48 h after methanol induction. The concentration of this protein that inhibited 50% of the binding of fluorescein-annexin V was 4nmol/L.

CONCLUSIONHighly-active recombinant mutant human annexin V with endogenous metal-chelating sites can be expressed in Pichia Pastoris system.

Annexin A5 ; genetics ; Base Sequence ; Humans ; Molecular Sequence Data ; Mutation ; Pichia ; genetics ; RNA, Messenger ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

The aim of this study is to investigate the effect of monoammonium glycyrrhizinate (MAG) on lipopolysaccharide (LPS) -induced acute lung injury (ALI) and its anti-inflammatory mechanism in mice. All male ICR mice were randomly divided into six groups: LPS group; control group; MAG 3, 10, and 30 mg x kg(-1) groups; and dexamethasone (DXM) 5 mg x kg(-1) group. Lung dry weight and wet weight percentage and permeability were detected. Neutrophil infiltration in bronchoalveolar lavage fluid (BALF) and lung tissues was detected by cell count and morphological analysis. The levels of TNF-alpha and IL-10 in lung were detected by ELISA. MPO activity was determined followed the specification. MAG induced a decrease in lung wet weight/dry weight ratio, and significantly decreased in total leucocyte number and neutrophil percentage in the BALF, and MPO activity of lung in a dose-dependent manner. Importantly, It could up-regulate the IL-10 level and down-regulate the TNF-alpha level in the lung tissue of ALI mice. These results suggested that the protective effect of MAG in mice on LPS induced ALI was associated with the regulation of TNF-alpha/IL-10 balance, and MAG maybe a potentially treatment for ALI/ARDS.
Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Bronchoalveolar Lavage Fluid ; cytology ; Glycyrrhizic Acid ; pharmacology ; Interleukin-10 ; metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung ; pathology ; Male ; Mice ; Mice, Inbred ICR ; Neutrophils ; pathology ; Organ Size ; drug effects ; Peroxidase ; metabolism ; Protective Agents ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVENucleic vaccine of pVVP3IL-18HN expressing apoptin gene, Newcastle disease virus HN gene and IL-18 gene were constructed to observe the combinative antitumor effect of the above three genes.

METHODSEukaryotic expression plasmid pVVP3IL-18HN was constructed by inserting apoptin gene and fragment comprising fused IL-18HN gene and IRES promoter into the downstream of CMV promoter of vector pVAX1. The expression of inserted gene was identified by RT-PCR, indirect immunofluorescence and Western-blot. The recombinant plasmid was introduced into Hep-2 cells by liposome, then suppression rate of Hep-2 of different time and different quantity was calculated according to MTT results.

RESULTThe recombinant plasmid of pVVP3IL-18HN suppressed Hep-2 successfully and its suppression rate was up to 61.9% with 20 microg/ml, incubation of 72 hours.

CONCLUSIONThe nucleic vaccine constructed pVVP3IL-18HN had antitumor effect on Hep-2. It may can be used to the therapy and research of laryngeal carcinoma.

Cancer Vaccines ; biosynthesis ; Gene Expression ; Genetic Vectors ; HN Protein ; genetics ; Hep G2 Cells ; Humans ; Interleukin-18 ; genetics ; Laryngeal Neoplasms ; immunology ; prevention & control ; Newcastle disease virus ; immunology ; Plasmids ; Transfection ; Vaccines, DNA ; biosynthesis

OBJECTIVETo investigate antigen-induced changes of leukotriene B(4)(LTB(4))content and LTA(4)-hydrolase mRNA expression in lung tissue and cerebral cortex in sensitized rats.

METHODSThe contents of LTB(4) in lung tissue and cerebral cortex homogenates and LTA(4)-hydrolase mRNA expression after antigen challenge by aerosol were respectively detected by reverse-phase high performance liquid chromatography(RP-HPLC) and semi-quantitative RT-PCR.

RESULTThe LTB(4) levels in lung tissue and cerebral cortex homogenates in asthmatic rats were significantly higher than those in control (P%0.05), and LTA4-hydrolase mRNA expression was also increased in asthmatic group. Dexamethason(DXM, 0.5 mg/kg, i.p.) decreased the LTB(4) content and inhibited the LTA(4)-hydrolase mRNA expression significantly in asthmatic rats(P%0.05).

CONCLUSIONLTB(4) content and LTA(4)-hydrolase mRNA expression in cerebral cortex and lung tissue are increased in asthmatic rats, and there may exist neuroimmunological cross-talking between central nervous system and lung tissue in asthma.

Animals ; Asthma ; metabolism ; Cerebral Cortex ; chemistry ; metabolism ; Epoxide Hydrolases ; genetics ; Female ; Leukotriene B4 ; analysis ; Lung ; chemistry ; metabolism ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo assess the value of an infusion-based separation technique to assist in ultrasound (US)-guided percutaneous radiofrequency ablation (RFA) of liver cancers abutting the liver edge.

METHODSTwenty-four cases of malignant liver tumors abutting the hepatic edge were treated with US-guided puncture accompanied by the assistant infusion technique. The US-guided puncture was made with a 22-G needle through the hepatic tissue and into the abdominal cavity near the target tumor. Infusion of a saline solution was used to separate the liver from any surrounding structures so that percutaneous RFA could be safely performed. Complications, including gastrointestinal injury, hemorrhage and death, were recorded. Technical efficacy and safety were evaluated.

RESULTSAmong the 24 patients, the target tumors were adjacent to the right kidney (n=6), colon (n=6), stomach (n=5), pericardium (n=4), and gall bladder (n=3). Twenty-three patients received a successful radical percutaneous RFA with assistant infusion. The assistant infusion volumes ranged from 80-390 ml and created spaces ranging from 0.8-2.5 cm between the liver and surrounding structures. Five of the cases with tumors adjacent to the stomach or colon received the largest volume infusions. The infusion failed to create a separation space in only one case, due to the presence of an adhesion; as a result, this patient was treated with palliative RFA. The mean hospital stay for all 24 patients was four days after surgery. No severe complications or deaths occurred. At 1-month follow-up, computed tomography images showed that 22 cases had complete ablation, yielding a technical success rate of 95.7% (22/23). No needle track implantation was observed.

CONCLUSIONAssistant infusion for percutaneous radiofrequency ablation creates a protective space between the liver and surrounding structures in patients with liver tumors abutting the liver edge. This safe and effective assistant technique broadens the range of patients available for percutaneous RFA treatment.

Adult ; Aged ; Carcinoma, Hepatocellular ; surgery ; Catheter Ablation ; methods ; Female ; Humans ; Isotonic Solutions ; administration & dosage ; Liver Neoplasms ; surgery ; Male ; Middle Aged ; Treatment Outcome ; Ultrasonography, Interventional

OBJECTIVETo investigate the differential expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and their potential role in the pathogenesis of chronic suppurative otitis media and cholesteatoma.

METHODSNormal canal skin of 30 patients with tympanosclerosis were enrolled as control, 30 cases with chronic suppurative otitis media and 30 patients with cholesteatoma were studied. Real-time PCR, Western blot and Immunohistochemistry were preformed to detect the expression of TLR2/TLR4 in normal canal skin, mucosa and granulation tissue of chronic suppurative otitis media, mucosa, granulation tissue, cholesteatoma epithelium of cholesteatoma, and the differential expression were analyzed.

RESULTS(1) the mRNA and protein expression of TLR2 and TLR4 were detected in all normal canal skin, mucosa and granulation tissue of chronic suppurative otitis media, mucosa, granulation tissue, cholesteatoma epithelium of cholesteatoma. (2) Both mRNA and protein level of TLR2/TLR4 in mucosa of chronic suppurative otitis media and cholesteatoma were higher than those in normal canal skin, but lower in cholesteatoma epithelium, there was no significant difference in mucosa of the two otitis media groups. (3) The mRNA and protein expression of TLR2/TLR4 in granulation tissue of chronic suppurative otitis media and cholesteatoma were significant increased when compared with normal canal skin, and TLR2 expression level was higher in granulation tissue of cholesteatoma than in chronic suppurative otitis media. (4) TLR2/TLR4 positive cells mainly infiltrated in granulations, significantly more than in normal skin, while fewer in the epithelium of cholesteatoma.

CONCLUSIONSDifferential expression of TLR2 and TLR4 in mucosa suggests middle ear is a TLR2/TLR4 participated functional modulation of the innate immune system and also suggests that they may play a different role in the pathophysiology of chronic otitis media and cholesteatoma.

Case-Control Studies ; Cholesteatoma, Middle Ear ; metabolism ; Chronic Disease ; Humans ; Otitis Media, Suppurative ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVEThe aim of this study is to observe the effect of combined amiodarone and antiarrhythmic peptide (AAP10) use on the incidence of induced ventricular arrhythmias in healed myocardial infarction (MI) rabbits.

METHODSTwenty Japanese rabbits underwent thoracotomy without coronary artery ligation (Sham, group A), the middle left circumflex branch were ligated to induce MI in 180 Japanese rabbits. Eight weeks after operation, 124 rabbits survived MI operation and were divided into four groups: control group (group B, n = 31), amiodarone group (group C, n = 31), AAP10 group (group D, n = 31) and amiodarone plus AAP10 group (group E, n = 31). The A and B and D groups were treated with saline 2 ml/d, the C and E groups were treated with 2 ml saline containing amiodarone 100 mg×kg(-1)×d(-1). All rabbits were examined by echocardiogram at 12 weeks after operation, then anesthetized by sodium barbital, the left wedge ventricular preparations were cannulated and artery perfused by Tyrode's solution in vitro in the absence (Group A, B and C) and presence of AAP10 (500 nmol/L, Group D and E). The volume electrocardiogram, QT Interval, QRS interval, effective refractory period (ERP), the T-peak to T-end interval (T(p-e)), and ventricular tachycardia episodes induced by programmed stimulation were recorded. The T(p-e)/QT ratio was calculated. After perfusion, gap junctions protein connexin 43 (Cx43) expression was detected by Western blot and immunofluorescence.

RESULTSThe incidence of induced ventricular tachycardia episodes of group A, B, C, D, E was 0, 62.5%, 26.9%, 40.0%, 22.2% respectively. The incidence of induced ventricular tachycardia episodes of group E was less than group B. The T(p-e)/QT ratio in group B, C, D were greater than in group A. The T(p-e)/QT ratio of group E was less than group B. The myocardial Cx43 in the group B was down-regulated and disorganized compared to group A, up-regulated in group C and E compared to group B, up-regulated in group E compared to group D. The Cx43 in the heart of group D and E were well organized than in group B and C.

CONCLUSIONSThe artery perfused rabbits wedge preparations with healed myocardial infarction with high incidence of induced ventricular tachycardia episodes are good platform for ventricular arrhythmias research. Combined amiodarone and AAP10 use could decrease the T(p-e)/QT ratio and the incidence of induced ventricular tachycardia episodes. Amiodarone and AAP10 have synergistic effects on upregulating Cx43 and preventing ventricular arrhythmias in this rabbit model of healed myocardial infarction.

Amiodarone ; pharmacology ; therapeutic use ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; therapeutic use ; Arrhythmias, Cardiac ; etiology ; Connexin 43 ; metabolism ; Male ; Myocardial Infarction ; drug therapy ; metabolism ; physiopathology ; Oligopeptides ; pharmacology ; therapeutic use ; Rabbits ; Treatment Outcome

A novel series of sorafenib analogs containing 2-picolinyl hydrazide moiety were designed and synthesized. In vitro, most of synthesized compounds have antiproliferation activity on MDA-MB-231, ACHN, HepG2, Mia-PaCa-2 and SW1990 cell lines tested by MTT assay. It is worth noting that the antitumor activities of compounds 2c, 2d and 2f are more potent than that of sorafenib on pancreatic cancer cells Mia-PaCa-2 and SW1990, and the activities of compounds 3f and 3g are 2-3 times than that of sorafenib on human hepatocellular carcinoma HepG2 cell line.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Design ; Drug Screening Assays, Antitumor ; Humans ; Molecular Structure ; Niacinamide ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Phenylurea Compounds ; chemical synthesis ; chemistry ; pharmacology ; Structure-Activity Relationship

OBJECTIVETo assess the value of application of percutaneous radiofrequency ablation (RFA) with artificial hydrothorax for liver cancer in the hepatic dome.

METHODSThirty-two patients with 43 lesions of hepatic malignant tumors in the hepatic dome underwent ultrasound-guided percutaneous radiofrequency ablation (RFA) with artificial hydrothorax. Artificial hydrothorax was created by infusion of saline via an intrathoracically placed 14-G central venous catheter, which was ultrasound-guided percutaneously inserted before RFA, separating the right lung from the hepatic dome. The adverse reaction and therapeutic efficacy were also analyzed.

RESULTSIn the 32 patients with 43 lesions in the hepatic dome (4 tumors in segment IV 21 tumors in segment VII and 18 tumors in segment VIII), 18 lesions of 14 patients were not observed by ultrasound before the operation. Thirty-two patients received the ultrasound-guided placement of intrathoracical catheter, and (1606.3 ± 485.9) ml (1000 - 2500 ml) saline solution was infused successfully. After obtaining an image of the whole tumor, 31 patients received percutaneous RFA therapy on schedule, and 22 patients received percutaneous transdiaphragmatic RFA therapy. One patient with 2 lesions gave up the treatment, because one of his tumors was not detectable by ultrasound. Diaphragmatic muscle hemorrhage was seen in two patients, subcutaneous edema in two patients, and pneumothorax in one patient. All the complications were cured, and no serious complications or related death occurred. 1-month follow-up with contrast-enhanced CT/MRI images showed that 29 patients had complete ablation, and the effective rate of this technique was 93.5% (29/31).

CONCLUSIONSArtificial hydrothorax helps us not only to visualize the whole tumor in the hepatic dome, but also offers a transdiaphragmatic route for therapy. Ultrasound-guided percutaneous RFA with artificial hydrothorax is a feasible, safe, and effective technique for treating liver cancer in the hepatic dome and worthy of being promoted.

Adult ; Aged ; Carcinoma, Hepatocellular ; diagnostic imaging ; surgery ; Catheter Ablation ; methods ; Female ; Follow-Up Studies ; Humans ; Hydrothorax ; Liver Neoplasms ; diagnostic imaging ; surgery ; Male ; Middle Aged ; Sodium Chloride ; Ultrasonography, Interventional

OBJECTIVE: To investigate the epidemiological characteristics, phenotype, genotype, and prognosis of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) in the Chinese population. METHODS: A retrospective analysis was performed for the clinical data of the neonates who underwent screening with high-performance liquid chromatography-tandem mass spectrometry from January 2009 to June 2018 and were diagnosed with MCADD by gene detection. RESULTS: A total of 2 674 835 neonates underwent neonatal screening, among whom 12 were diagnosed with MCADD. Gene detection was performed for 10 neonates with MCADD and found 13 mutation types at 16 mutation sites of the ACADM gene, among which there were 7 reported mutations (p.T150Rfs*4, p.M1V, p.R206C, p.R294T, p.G310R, p.M328V, and p.G362E), 5 novel mutations (p.N194D, p.A324P, p.N366S, c.118+3A>G, and c.387+1del G), and 1 exon 11 deletion; p.T150Rfs*4 was the most common mutation (4/16). The detection rate of mutation sites in the ACADM gene was 80%. No phenotype-genotype correlation was observed. Dietary guidance and symptomatic treatment were given after confirmed diagnosis. No acute metabolic imbalance was observed within 4-82 months of follow-up. All neonates had good prognosis except one who had brain dysplasia. CONCLUSIONS MCADD is relatively rare in southern China, and p.T150Rfs*4 is a common mutation in the Chinese population. Cases with positive screening results should be evaluated by octanoylcarnitine C8 value and gene detection.
Acyl-CoA Dehydrogenase ; deficiency ; Carnitine ; China ; Follow-Up Studies ; Humans ; Infant, Newborn ; Lipid Metabolism, Inborn Errors ; Mutation ; Neonatal Screening ; Retrospective Studies